{ "PMC4850273": { "annotations": [ { "sid": 0, "sent": "Molecular Dissection of Xyloglucan Recognition in a Prominent Human Gut Symbiont", "section": "TITLE", "ner": [ [ 24, 34, "Xyloglucan", "chemical" ], [ 62, 67, "Human", "species" ] ] }, { "sid": 1, "sent": "Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as \u201cdietary fiber.\u201d However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis.", "section": "ABSTRACT", "ner": [ [ 0, 31, "Polysaccharide utilization loci", "gene" ], [ 33, 36, "PUL", "gene" ], [ 69, 74, "human", "species" ], [ 79, 92, "Bacteroidetes", "taxonomy_domain" ], [ 161, 174, "carbohydrates", "chemical" ], [ 241, 244, "PUL", "gene" ], [ 339, 344, "human", "species" ], [ 345, 354, "bacterial", "taxonomy_domain" ] ] }, { "sid": 2, "sent": "In particular, the molecular basis of complex polysaccharide recognition, an essential prerequisite to hydrolysis by cell surface glycosidases and subsequent metabolism, is generally poorly understood.", "section": "ABSTRACT", "ner": [ [ 38, 60, "complex polysaccharide", "chemical" ], [ 130, 142, "glycosidases", "protein_type" ] ] }, { "sid": 3, "sent": "Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide.", "section": "ABSTRACT", "ner": [ [ 21, 82, "biochemical, structural, and reverse genetic characterization", "experimental_method" ], [ 97, 133, "cell surface glycan-binding proteins", "protein_type" ], [ 135, 140, "SGBPs", "protein_type" ], [ 155, 183, "xyloglucan utilization locus", "gene" ], [ 185, 190, "XyGUL", "gene" ], [ 197, 215, "Bacteroides ovatus", "species" ], [ 266, 275, "vegetable", "taxonomy_domain" ], [ 276, 290, "polysaccharide", "chemical" ] ] }, { "sid": 4, "sent": "Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide.", "section": "ABSTRACT", "ner": [ [ 0, 20, "Biochemical analysis", "experimental_method" ], [ 40, 72, "outer membrane-anchored proteins", "protein_type" ], [ 130, 140, "xyloglucan", "chemical" ], [ 142, 145, "XyG", "chemical" ], [ 147, 161, "polysaccharide", "chemical" ] ] }, { "sid": 5, "sent": "The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the \u03b2-glucan backbone.", "section": "ABSTRACT", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 25, 31, "SGBP-A", "protein" ], [ 35, 39, "SusD", "protein" ], [ 56, 61, "bound", "protein_state" ], [ 62, 65, "XyG", "chemical" ], [ 66, 85, "tetradecasaccharide", "chemical" ], [ 106, 135, "carbohydrate-binding platform", "site" ], [ 180, 188, "\u03b2-glucan", "chemical" ] ] }, { "sid": 6, "sent": "The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain.", "section": "ABSTRACT", "ner": [ [ 12, 25, "tetra-modular", "structure_element" ], [ 26, 35, "structure", "evidence" ], [ 39, 45, "SGBP-B", "protein" ], [ 62, 82, "tandem Ig-like folds", "structure_element" ], [ 89, 92, "XyG", "chemical" ], [ 124, 141, "C-terminal domain", "structure_element" ] ] }, { "sid": 7, "sent": "Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B.\u00a0ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes.", "section": "ABSTRACT", "ner": [ [ 27, 37, "affinities", "evidence" ], [ 42, 45, "XyG", "chemical" ], [ 47, 71, "reverse-genetic analysis", "experimental_method" ], [ 85, 91, "SGBP-B", "protein" ], [ 146, 162, "oligosaccharides", "chemical" ], [ 188, 194, "SGBP-A", "protein" ], [ 221, 233, "carbohydrate", "chemical" ], [ 264, 267, "XyG", "chemical" ], [ 307, 313, "SGBP-A", "protein" ], [ 318, 324, "SGBP-B", "protein" ], [ 366, 378, "carbohydrate", "chemical" ], [ 390, 399, "B.\u00a0ovatus", "species" ], [ 429, 438, "vegetable", "taxonomy_domain" ], [ 439, 450, "xyloglucans", "chemical" ], [ 471, 484, "Bacteroidetes", "taxonomy_domain" ] ] }, { "sid": 8, "sent": "The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute \u201cdietary fiber.\u201d Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism.", "section": "ABSTRACT", "ner": [ [ 4, 17, "Bacteroidetes", "taxonomy_domain" ], [ 31, 39, "bacteria", "taxonomy_domain" ], [ 47, 52, "human", "species" ], [ 103, 126, "complex polysaccharides", "chemical" ], [ 267, 280, "Bacteroidetes", "taxonomy_domain" ], [ 299, 304, "plant", "taxonomy_domain" ], [ 315, 330, "polysaccharides", "chemical" ] ] }, { "sid": 9, "sent": "Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide.", "section": "ABSTRACT", "ner": [ [ 27, 77, "biochemical, crystallographic, and genetic insight", "experimental_method" ], [ 91, 122, "surface glycan-binding proteins", "protein_type" ], [ 140, 158, "Bacteroides ovatus", "species" ], [ 159, 187, "xyloglucan utilization locus", "gene" ], [ 189, 194, "XyGUL", "gene" ], [ 245, 254, "vegetable", "taxonomy_domain" ], [ 255, 269, "polysaccharide", "chemical" ] ] }, { "sid": 10, "sent": "Our combined analysis illuminates new fundamental aspects of complex polysaccharide recognition, cleavage, and import at the Bacteroidetes cell surface that may facilitate the development of prebiotics to target this phylum of gut bacteria.", "section": "ABSTRACT", "ner": [ [ 61, 83, "complex polysaccharide", "chemical" ], [ 125, 138, "Bacteroidetes", "taxonomy_domain" ], [ 231, 239, "bacteria", "taxonomy_domain" ] ] }, { "sid": 11, "sent": "The human gut microbiota influences the course of human development and health, playing key roles in immune stimulation, intestinal cell proliferation, and metabolic balance.", "section": "INTRO", "ner": [ [ 4, 9, "human", "species" ], [ 14, 24, "microbiota", "taxonomy_domain" ], [ 50, 55, "human", "species" ] ] }, { "sid": 12, "sent": "This microbial community is largely bacterial, with the Bacteroidetes, Firmicutes, and Actinobacteria comprising the dominant phyla.", "section": "INTRO", "ner": [ [ 5, 14, "microbial", "taxonomy_domain" ], [ 36, 45, "bacterial", "taxonomy_domain" ], [ 56, 69, "Bacteroidetes", "taxonomy_domain" ], [ 71, 81, "Firmicutes", "taxonomy_domain" ], [ 87, 101, "Actinobacteria", "taxonomy_domain" ] ] }, { "sid": 13, "sent": "The ability to acquire energy from carbohydrates of dietary or host origin is central to the adaptation of human gut bacterial species to their niche.", "section": "INTRO", "ner": [ [ 35, 48, "carbohydrates", "chemical" ], [ 107, 112, "human", "species" ], [ 117, 126, "bacterial", "taxonomy_domain" ] ] }, { "sid": 14, "sent": "More importantly, this makes diet a tractable way to manipulate the abundance and metabolic output of the microbiota toward improved human health.", "section": "INTRO", "ner": [ [ 106, 116, "microbiota", "taxonomy_domain" ], [ 133, 138, "human", "species" ] ] }, { "sid": 15, "sent": "However, there is a paucity of data regarding how the vast array of complex carbohydrate structures are selectively recognized and imported by members of the microbiota, a critical process that enables these organisms to thrive in the competitive gut environment.", "section": "INTRO", "ner": [ [ 68, 88, "complex carbohydrate", "chemical" ], [ 158, 168, "microbiota", "taxonomy_domain" ] ] }, { "sid": 16, "sent": "The human gut bacteria Bacteroidetes share a profound capacity for dietary glycan degradation, with many species containing >250 predicted carbohydrate-active enzymes (CAZymes), compared to 50 to 100 within many Firmicutes and only 17 in the human genome devoted toward carbohydrate utilization.", "section": "INTRO", "ner": [ [ 4, 9, "human", "species" ], [ 14, 22, "bacteria", "taxonomy_domain" ], [ 23, 36, "Bacteroidetes", "taxonomy_domain" ], [ 75, 81, "glycan", "chemical" ], [ 212, 222, "Firmicutes", "taxonomy_domain" ], [ 242, 247, "human", "species" ] ] }, { "sid": 17, "sent": "A remarkable feature of the Bacteroidetes is the packaging of genes for carbohydrate catabolism into discrete polysaccharide utilization loci (PUL), which are transcriptionally regulated by specific substrate signatures.", "section": "INTRO", "ner": [ [ 28, 41, "Bacteroidetes", "taxonomy_domain" ], [ 110, 141, "polysaccharide utilization loci", "gene" ], [ 143, 146, "PUL", "gene" ] ] }, { "sid": 18, "sent": "The archetypal PUL-encoded system is the starch utilization system (Sus) (Fig.\u00a01B) of Bacteroides thetaiotaomicron.", "section": "INTRO", "ner": [ [ 15, 18, "PUL", "gene" ], [ 41, 66, "starch utilization system", "complex_assembly" ], [ 68, 71, "Sus", "complex_assembly" ], [ 86, 114, "Bacteroides thetaiotaomicron", "species" ] ] }, { "sid": 19, "sent": "The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm.", "section": "INTRO", "ner": [ [ 4, 7, "Sus", "complex_assembly" ], [ 19, 33, "lipid-anchored", "protein_state" ], [ 50, 62, "endo-amylase", "protein_type" ], [ 64, 68, "SusG", "protein" ], [ 72, 98, "TonB-dependent transporter", "protein_type" ], [ 100, 104, "TBDT", "protein_type" ], [ 107, 111, "SusC", "protein" ], [ 127, 143, "oligosaccharides", "chemical" ], [ 175, 197, "starch-binding protein", "protein_type" ], [ 199, 203, "SusD", "protein" ], [ 220, 253, "carbohydrate-binding lipoproteins", "protein_type" ], [ 255, 259, "SusE", "protein" ], [ 264, 268, "SusF", "protein" ], [ 290, 306, "exo-glucosidases", "protein_type" ], [ 308, 312, "SusA", "protein" ], [ 317, 321, "SusB", "protein" ], [ 338, 345, "glucose", "chemical" ] ] }, { "sid": 20, "sent": "The importance of PUL as a successful evolutionary strategy is underscored by the observation that Bacteroidetes such as B.\u00a0thetaiotaomicron and Bacteroides ovatus devote ~18% of their genomes to these systems.", "section": "INTRO", "ner": [ [ 18, 21, "PUL", "gene" ], [ 99, 112, "Bacteroidetes", "taxonomy_domain" ], [ 121, 140, "B.\u00a0thetaiotaomicron", "species" ], [ 145, 163, "Bacteroides ovatus", "species" ] ] }, { "sid": 21, "sent": "Moving beyond seminal genomic and transcriptomic analyses, the current state-of-the-art PUL characterization involves combined reverse-genetic, biochemical, and structural studies to illuminate the molecular details of PUL function.", "section": "INTRO", "ner": [ [ 88, 91, "PUL", "gene" ], [ 127, 179, "reverse-genetic, biochemical, and structural studies", "experimental_method" ], [ 219, 222, "PUL", "gene" ] ] }, { "sid": 22, "sent": "Xyloglucan and the Bacteroides ovatus xyloglucan utilization locus (XyGUL). (A) Representative structures of common xyloglucans using the Consortium for Functional Glycomics Symbol Nomenclature (http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml).", "section": "FIG", "ner": [ [ 0, 10, "Xyloglucan", "chemical" ], [ 19, 37, "Bacteroides ovatus", "species" ], [ 38, 66, "xyloglucan utilization locus", "gene" ], [ 68, 73, "XyGUL", "gene" ], [ 95, 105, "structures", "evidence" ], [ 116, 127, "xyloglucans", "chemical" ] ] }, { "sid": 23, "sent": "Cleavage sites for BoXyGUL glycosidases (GHs) are indicated for solanaceous xyloglucan. (B) BtSus and BoXyGUL. (C) Localization of BoXyGUL-encoded proteins in cellular membranes and concerted modes of action in the degradation of xyloglucans to monosaccharides.", "section": "FIG", "ner": [ [ 19, 26, "BoXyGUL", "gene" ], [ 27, 39, "glycosidases", "protein_type" ], [ 41, 44, "GHs", "protein_type" ], [ 64, 75, "solanaceous", "taxonomy_domain" ], [ 76, 86, "xyloglucan", "chemical" ], [ 92, 97, "BtSus", "gene" ], [ 102, 109, "BoXyGUL", "gene" ], [ 131, 138, "BoXyGUL", "gene" ], [ 230, 241, "xyloglucans", "chemical" ] ] }, { "sid": 24, "sent": "The location of SGBP-A/B is presented in this work; the location of GH5 has been empirically determined, and the enzymes have been placed based upon their predicted cellular location.", "section": "FIG", "ner": [ [ 16, 22, "SGBP-A", "protein" ], [ 23, 24, "B", "protein" ], [ 68, 71, "GH5", "protein" ] ] }, { "sid": 25, "sent": "We recently reported the detailed molecular characterization of a PUL that confers the ability of the human gut commensal B.\u00a0ovatus ATCC 8483 to grow on a prominent family of plant cell wall glycans, the xyloglucans (XyG).", "section": "INTRO", "ner": [ [ 66, 69, "PUL", "gene" ], [ 102, 107, "human", "species" ], [ 122, 141, "B.\u00a0ovatus ATCC 8483", "species" ], [ 175, 180, "plant", "taxonomy_domain" ], [ 191, 198, "glycans", "chemical" ], [ 204, 215, "xyloglucans", "chemical" ], [ 217, 220, "XyG", "chemical" ] ] }, { "sid": 26, "sent": "XyG variants (Fig.\u00a01A) constitute up to 25% of the dry weight of common vegetables.", "section": "INTRO", "ner": [ [ 0, 3, "XyG", "chemical" ], [ 72, 82, "vegetables", "taxonomy_domain" ] ] }, { "sid": 27, "sent": "Analogous to the Sus locus, the xyloglucan utilization locus (XyGUL) encodes a cohort of carbohydrate-binding, -hydrolyzing, and -importing proteins (Fig.\u00a01B and C).", "section": "INTRO", "ner": [ [ 17, 26, "Sus locus", "gene" ], [ 32, 60, "xyloglucan utilization locus", "gene" ], [ 62, 67, "XyGUL", "gene" ], [ 89, 148, "carbohydrate-binding, -hydrolyzing, and -importing proteins", "protein_type" ] ] }, { "sid": 28, "sent": "The number of glycoside hydrolases (GHs) encoded by the XyGUL is, however, more expansive than that by the Sus locus (Fig.\u00a01B), which reflects the greater complexity of glycosidic linkages found in XyG vis-\u00e0-vis starch.", "section": "INTRO", "ner": [ [ 14, 34, "glycoside hydrolases", "protein_type" ], [ 36, 39, "GHs", "protein_type" ], [ 56, 61, "XyGUL", "gene" ], [ 107, 116, "Sus locus", "gene" ], [ 198, 201, "XyG", "chemical" ], [ 212, 218, "starch", "chemical" ] ] }, { "sid": 29, "sent": "Whereas our previous study focused on the characterization of the linkage specificity of these GHs, a key outstanding question regarding this locus is how XyG recognition is mediated at the cell surface.", "section": "INTRO", "ner": [ [ 95, 98, "GHs", "protein_type" ], [ 155, 158, "XyG", "chemical" ] ] }, { "sid": 30, "sent": "In the archetypal starch utilization system of B.\u00a0thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide.", "section": "INTRO", "ner": [ [ 18, 43, "starch utilization system", "complex_assembly" ], [ 47, 66, "B.\u00a0thetaiotaomicron", "species" ], [ 133, 153, "starch-binding sites", "site" ], [ 177, 208, "surface glycan-binding proteins", "protein_type" ], [ 210, 215, "SGBPs", "protein_type" ], [ 233, 240, "amylase", "protein_type" ], [ 241, 245, "SusG", "protein" ], [ 258, 262, "SusD", "protein" ], [ 275, 279, "SusE", "protein" ], [ 298, 302, "SusF", "protein" ], [ 370, 374, "SusD", "protein" ], [ 402, 408, "starch", "chemical" ], [ 436, 440, "SusE", "protein" ], [ 442, 446, "SusF", "protein" ], [ 452, 456, "SusG", "protein" ], [ 457, 470, "binding sites", "site" ], [ 508, 522, "polysaccharide", "chemical" ] ] }, { "sid": 31, "sent": "Bacteroidetes PUL ubiquitously encode homologs of SusC and SusD, as well as proteins whose genes are immediately downstream of susD, akin to susE/F, and these are typically annotated as \u201cputative lipoproteins\u201d.", "section": "INTRO", "ner": [ [ 0, 13, "Bacteroidetes", "taxonomy_domain" ], [ 14, 17, "PUL", "gene" ], [ 50, 54, "SusC", "protein" ], [ 59, 63, "SusD", "protein" ], [ 127, 131, "susD", "gene" ], [ 141, 147, "susE/F", "gene" ], [ 187, 195, "putative", "protein_state" ], [ 196, 208, "lipoproteins", "protein_type" ] ] }, { "sid": 32, "sent": "The genes coding for these proteins, sometimes referred to as \u201csusE/F positioned,\u201d display products with a wide variation in amino acid sequence and which have little or no homology to other PUL-encoded proteins or known carbohydrate-binding proteins.", "section": "INTRO", "ner": [ [ 63, 69, "susE/F", "gene" ], [ 191, 194, "PUL", "gene" ], [ 221, 250, "carbohydrate-binding proteins", "protein_type" ] ] }, { "sid": 33, "sent": "As the Sus SGBPs remain the only structurally characterized cohort to date, we therefore wondered whether such glycan binding and function are extended to other PUL that target more complex and heterogeneous polysaccharides, such as XyG.", "section": "INTRO", "ner": [ [ 7, 10, "Sus", "complex_assembly" ], [ 11, 16, "SGBPs", "protein_type" ], [ 111, 117, "glycan", "chemical" ], [ 161, 164, "PUL", "gene" ], [ 208, 223, "polysaccharides", "chemical" ], [ 233, 236, "XyG", "chemical" ] ] }, { "sid": 34, "sent": "We describe here the detailed functional and structural characterization of the noncatalytic SGBPs encoded by Bacova_02651 and Bacova_02650 of the XyGUL, here referred to as SGBP-A and SGBP-B, to elucidate their molecular roles in carbohydrate acquisition in vivo.", "section": "INTRO", "ner": [ [ 30, 72, "functional and structural characterization", "experimental_method" ], [ 80, 92, "noncatalytic", "protein_state" ], [ 93, 98, "SGBPs", "protein_type" ], [ 110, 122, "Bacova_02651", "gene" ], [ 127, 139, "Bacova_02650", "gene" ], [ 147, 152, "XyGUL", "gene" ], [ 174, 180, "SGBP-A", "protein" ], [ 185, 191, "SGBP-B", "protein" ] ] }, { "sid": 35, "sent": "Combined biochemical, structural, and reverse-genetic approaches clearly illuminate the distinct, yet complementary, functions that these two proteins play in XyG recognition as it impacts the physiology of B.\u00a0ovatus.", "section": "INTRO", "ner": [ [ 9, 64, "biochemical, structural, and reverse-genetic approaches", "experimental_method" ], [ 159, 162, "XyG", "chemical" ], [ 207, 216, "B.\u00a0ovatus", "species" ] ] }, { "sid": 36, "sent": "These data extend our current understanding of the Sus-like glycan uptake paradigm within the Bacteroidetes and reveals how the complex dietary polysaccharide xyloglucan is recognized at the cell surface.", "section": "INTRO", "ner": [ [ 60, 66, "glycan", "chemical" ], [ 94, 107, "Bacteroidetes", "taxonomy_domain" ], [ 144, 158, "polysaccharide", "chemical" ], [ 159, 169, "xyloglucan", "chemical" ] ] }, { "sid": 37, "sent": "SGBP-A and SGBP-B are cell-surface-localized, xyloglucan-specific binding proteins.", "section": "RESULTS", "ner": [ [ 0, 6, "SGBP-A", "protein" ], [ 11, 17, "SGBP-B", "protein" ], [ 22, 82, "cell-surface-localized, xyloglucan-specific binding proteins", "protein_type" ] ] }, { "sid": 38, "sent": "SGBP-A, encoded by the XyGUL locus tag Bacova_02651 (Fig.\u00a01B), shares 26% amino acid sequence identity (40% similarity) with its homolog, B.\u00a0thetaiotaomicron SusD, and similar homology with the SusD-like proteins encoded within syntenic XyGUL identified in our earlier work.", "section": "RESULTS", "ner": [ [ 0, 6, "SGBP-A", "protein" ], [ 23, 28, "XyGUL", "gene" ], [ 39, 51, "Bacova_02651", "gene" ], [ 138, 157, "B.\u00a0thetaiotaomicron", "species" ], [ 158, 162, "SusD", "protein" ], [ 194, 212, "SusD-like proteins", "protein_type" ], [ 237, 242, "XyGUL", "gene" ] ] }, { "sid": 39, "sent": "In contrast, SGBP-B, encoded by locus tag Bacova_02650, displays little sequence similarity to the products of similarly positioned genes in syntenic XyGUL nor to any other gene product among the diversity of Bacteroidetes PUL.", "section": "RESULTS", "ner": [ [ 13, 19, "SGBP-B", "protein" ], [ 42, 54, "Bacova_02650", "gene" ], [ 150, 155, "XyGUL", "gene" ], [ 209, 222, "Bacteroidetes", "taxonomy_domain" ], [ 223, 226, "PUL", "gene" ] ] }, { "sid": 40, "sent": "Whereas sequence similarity among SusC/SusD homolog pairs often serves as a hallmark for PUL identification, the sequence similarities of downstream genes encoding SGBPs are generally too low to allow reliable bioinformatic classification of their products into protein families, let alone prediction of function.", "section": "RESULTS", "ner": [ [ 34, 38, "SusC", "protein" ], [ 39, 43, "SusD", "protein" ], [ 89, 92, "PUL", "gene" ], [ 164, 169, "SGBPs", "protein_type" ] ] }, { "sid": 41, "sent": "Hence, there is a critical need for the elucidation of detailed structure-function relationships among PUL SGBPs, in light of the manifold glycan structures in nature.", "section": "RESULTS", "ner": [ [ 103, 106, "PUL", "gene" ], [ 107, 112, "SGBPs", "protein_type" ], [ 139, 145, "glycan", "chemical" ] ] }, { "sid": 42, "sent": "Immunofluorescence of formaldehyde-fixed, nonpermeabilized cells grown in minimal medium with XyG as the sole carbon source to induce XyGUL expression, reveals that both SGBP-A and SGBP-B are presented on the cell surface by N-terminal lipidation, as predicted by signal peptide analysis with SignalP (Fig.\u00a02).", "section": "RESULTS", "ner": [ [ 0, 18, "Immunofluorescence", "experimental_method" ], [ 94, 97, "XyG", "chemical" ], [ 134, 139, "XyGUL", "gene" ], [ 170, 176, "SGBP-A", "protein" ], [ 181, 187, "SGBP-B", "protein" ], [ 236, 246, "lipidation", "ptm" ] ] }, { "sid": 43, "sent": "Here, the SGBPs very likely work in concert with the cell-surface-localized endo-xyloglucanase B.\u00a0ovatus GH5 (BoGH5) to recruit and cleave XyG for subsequent periplasmic import via the SusC-like TBDT of the XyGUL (Fig.\u00a01B and C).", "section": "RESULTS", "ner": [ [ 10, 15, "SGBPs", "protein_type" ], [ 53, 94, "cell-surface-localized endo-xyloglucanase", "protein_type" ], [ 95, 104, "B.\u00a0ovatus", "species" ], [ 105, 108, "GH5", "protein" ], [ 110, 115, "BoGH5", "protein" ], [ 139, 142, "XyG", "chemical" ], [ 185, 199, "SusC-like TBDT", "protein_type" ], [ 207, 212, "XyGUL", "gene" ] ] }, { "sid": 44, "sent": "SGBP-A and SGBP-B visualized by immunofluorescence.", "section": "FIG", "ner": [ [ 0, 6, "SGBP-A", "protein" ], [ 11, 17, "SGBP-B", "protein" ], [ 32, 50, "immunofluorescence", "experimental_method" ] ] }, { "sid": 45, "sent": "Formalin-fixed, nonpermeabilized B.\u00a0ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B.\u00a0ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B.\u00a0ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of \u0394SGBP-B cells labeled with anti-SGBP-B antibodies.", "section": "FIG", "ner": [ [ 33, 42, "B.\u00a0ovatus", "species" ], [ 83, 86, "XyG", "chemical" ], [ 128, 134, "SGBP-A", "protein" ], [ 138, 144, "SGBP-B", "protein" ], [ 210, 217, "Overlay", "experimental_method" ], [ 221, 249, "bright-field and FITC images", "evidence" ], [ 253, 262, "B.\u00a0ovatus", "species" ], [ 299, 306, "Overlay", "experimental_method" ], [ 310, 338, "bright-field and FITC images", "evidence" ], [ 342, 351, "B.\u00a0ovatus", "species" ], [ 388, 406, "Bright-field image", "evidence" ], [ 410, 417, "\u0394SGBP-B", "mutant" ] ] }, { "sid": 46, "sent": "(D) FITC images of \u0394SGBP-B cells labeled with anti-SGBP-B antibodies.", "section": "FIG", "ner": [ [ 4, 15, "FITC images", "evidence" ], [ 19, 26, "\u0394SGBP-B", "mutant" ] ] }, { "sid": 47, "sent": "Cells lacking SGBP-A (\u0394SGBP-A) do not grow on XyG and therefore could not be tested in parallel.", "section": "FIG", "ner": [ [ 6, 13, "lacking", "protein_state" ], [ 14, 20, "SGBP-A", "protein" ], [ 22, 29, "\u0394SGBP-A", "mutant" ], [ 46, 49, "XyG", "chemical" ] ] }, { "sid": 48, "sent": "In our initial study focused on the functional characterization of the glycoside hydrolases of the XyGUL, we reported preliminary affinity PAGE and isothermal titration calorimetry (ITC) data indicating that both SGBP-A and SGBP-B are competent xyloglucan-binding proteins (affinity constant [Ka] values of 3.74 \u00d7 105\u00a0M\u22121 and 4.98 \u00d7 104\u00a0M\u22121, respectively [23]).", "section": "RESULTS", "ner": [ [ 71, 91, "glycoside hydrolases", "protein_type" ], [ 99, 104, "XyGUL", "gene" ], [ 130, 143, "affinity PAGE", "experimental_method" ], [ 148, 180, "isothermal titration calorimetry", "experimental_method" ], [ 182, 185, "ITC", "experimental_method" ], [ 213, 219, "SGBP-A", "protein" ], [ 224, 230, "SGBP-B", "protein" ], [ 245, 272, "xyloglucan-binding proteins", "protein_type" ], [ 274, 291, "affinity constant", "evidence" ], [ 293, 295, "Ka", "evidence" ] ] }, { "sid": 49, "sent": "Additional affinity PAGE analysis (Fig.\u00a03) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a \u03b2(1 \u2192 4)-glucan] and limited affinity for mixed-linkage \u03b2(1\u21923)/\u03b2(1\u21924)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition.", "section": "RESULTS", "ner": [ [ 11, 24, "affinity PAGE", "experimental_method" ], [ 61, 67, "SGBP-A", "protein" ], [ 143, 165, "hydroxyethyl cellulose", "chemical" ], [ 167, 170, "HEC", "chemical" ], [ 174, 189, "\u03b2(1 \u2192 4)-glucan", "chemical" ], [ 216, 250, "mixed-linkage \u03b2(1\u21923)/\u03b2(1\u21924)-glucan", "chemical" ], [ 252, 255, "MLG", "chemical" ], [ 261, 272, "glucomannan", "chemical" ], [ 274, 276, "GM", "chemical" ], [ 284, 292, "glucosyl", "chemical" ], [ 297, 305, "mannosyl", "chemical" ], [ 360, 374, "polysaccharide", "chemical" ] ] }, { "sid": 50, "sent": "In contrast, SGBP-B bound to HEC more weakly than SGBP-A and did not bind to MLG or GM.", "section": "RESULTS", "ner": [ [ 13, 19, "SGBP-B", "protein" ], [ 29, 32, "HEC", "chemical" ], [ 50, 56, "SGBP-A", "protein" ], [ 77, 80, "MLG", "chemical" ], [ 84, 86, "GM", "chemical" ] ] }, { "sid": 51, "sent": "Neither SGBP recognized galactomannan (GGM), starch, carboxymethylcellulose, or mucin (see Fig.\u00a0S1 in the supplemental material).", "section": "RESULTS", "ner": [ [ 8, 12, "SGBP", "protein_type" ], [ 24, 37, "galactomannan", "chemical" ], [ 39, 42, "GGM", "chemical" ], [ 45, 51, "starch", "chemical" ], [ 53, 75, "carboxymethylcellulose", "chemical" ], [ 80, 85, "mucin", "chemical" ] ] }, { "sid": 52, "sent": "Together, these results highlight the high specificities of SGBP-A and SGBP-B for XyG, which is concordant with their association with XyG-specific GHs in the XyGUL, as well as transcriptomic analysis indicating that B.\u00a0ovatus has discrete PUL for MLG, GM, and GGM (11).", "section": "RESULTS", "ner": [ [ 60, 66, "SGBP-A", "protein" ], [ 71, 77, "SGBP-B", "protein" ], [ 82, 85, "XyG", "chemical" ], [ 135, 151, "XyG-specific GHs", "protein_type" ], [ 159, 164, "XyGUL", "gene" ], [ 217, 226, "B.\u00a0ovatus", "species" ], [ 240, 243, "PUL", "gene" ], [ 248, 251, "MLG", "chemical" ], [ 253, 255, "GM", "chemical" ], [ 261, 264, "GGM", "chemical" ] ] }, { "sid": 53, "sent": "Notably, the absence of carbohydrate-binding modules in the GHs encoded by the XyGUL implies that noncatalytic recognition of xyloglucan is mediated entirely by SGBP-A and -B.", "section": "RESULTS", "ner": [ [ 24, 52, "carbohydrate-binding modules", "site" ], [ 60, 63, "GHs", "protein_type" ], [ 79, 84, "XyGUL", "gene" ], [ 126, 136, "xyloglucan", "chemical" ], [ 161, 167, "SGBP-A", "protein" ], [ 172, 174, "-B", "protein" ] ] }, { "sid": 54, "sent": "SGBP-A and SGBP-B preferentially bind xyloglucan.", "section": "FIG", "ner": [ [ 0, 6, "SGBP-A", "protein" ], [ 11, 17, "SGBP-B", "protein" ], [ 38, 48, "xyloglucan", "chemical" ] ] }, { "sid": 55, "sent": "Affinity electrophoresis (10% acrylamide) of SGBP-A and SGBP-B with BSA as a control protein.", "section": "FIG", "ner": [ [ 0, 24, "Affinity electrophoresis", "experimental_method" ], [ 45, 51, "SGBP-A", "protein" ], [ 56, 62, "SGBP-B", "protein" ], [ 68, 71, "BSA", "protein" ] ] }, { "sid": 56, "sent": "All samples were loaded on the same gel next to the BSA controls; thin black lines indicate where intervening lanes were removed from the final image for both space and clarity.", "section": "FIG", "ner": [ [ 52, 55, "BSA", "protein" ] ] }, { "sid": 57, "sent": "The percentage of polysaccharide incorporated into each native gel is displayed.", "section": "FIG", "ner": [ [ 18, 32, "polysaccharide", "chemical" ] ] }, { "sid": 58, "sent": "The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig.\u00a01A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig.\u00a01A; n = 2).", "section": "RESULTS", "ner": [ [ 13, 31, "endo-xyloglucanase", "protein_type" ], [ 39, 44, "XyGUL", "gene" ], [ 46, 51, "BoGH5", "protein" ], [ 80, 94, "polysaccharide", "chemical" ], [ 109, 117, "glucosyl", "chemical" ], [ 139, 165, "xylogluco-oligosaccharides", "chemical" ], [ 167, 172, "XyGOs", "chemical" ], [ 187, 200, "Glc4 backbone", "structure_element" ], [ 206, 241, "variable side-chain galactosylation", "structure_element" ], [ 243, 248, "XyGO1", "chemical" ], [ 312, 350, "controlled digestion and fractionation", "experimental_method" ], [ 354, 383, "size exclusion chromatography", "experimental_method" ], [ 421, 437, "oligosaccharides", "chemical" ], [ 445, 450, "XyGO2", "chemical" ] ] }, { "sid": 59, "sent": "ITC demonstrates that SGBP-A binds to XyG polysaccharide and XyGO2 (based on a Glc8 backbone) with essentially equal affinities, while no binding of XyGO1 (Glc4 backbone) was detectable (Table\u00a01; see Fig.\u00a0S2 and S3 in the supplemental material).", "section": "RESULTS", "ner": [ [ 0, 3, "ITC", "experimental_method" ], [ 22, 28, "SGBP-A", "protein" ], [ 38, 41, "XyG", "chemical" ], [ 42, 56, "polysaccharide", "chemical" ], [ 61, 66, "XyGO2", "chemical" ], [ 79, 92, "Glc8 backbone", "structure_element" ], [ 117, 127, "affinities", "evidence" ], [ 149, 154, "XyGO1", "chemical" ], [ 156, 169, "Glc4 backbone", "structure_element" ] ] }, { "sid": 60, "sent": "Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2.", "section": "RESULTS", "ner": [ [ 11, 17, "SGBP-B", "protein" ], [ 23, 31, "bound to", "protein_state" ], [ 32, 35, "XyG", "chemical" ], [ 40, 45, "XyGO2", "chemical" ], [ 71, 81, "affinities", "evidence" ], [ 107, 109, "Ka", "evidence" ], [ 158, 164, "SGBP-A", "protein" ], [ 186, 192, "SGBP-A", "protein" ], [ 194, 200, "SGBP-B", "protein" ], [ 206, 214, "bound to", "protein_state" ], [ 215, 220, "XyGO1", "chemical" ], [ 230, 238, "affinity", "evidence" ], [ 248, 270, "minimal repeating unit", "structure_element" ], [ 288, 290, "Ka", "evidence" ], [ 340, 343, "XyG", "chemical" ], [ 348, 353, "XyGO2", "chemical" ] ] }, { "sid": 61, "sent": "Together, these data clearly suggest that polysaccharide binding of both SGBPs is fulfilled by a dimer of the minimal repeat, corresponding to XyGO2 (cf.", "section": "RESULTS", "ner": [ [ 42, 56, "polysaccharide", "chemical" ], [ 73, 78, "SGBPs", "protein_type" ], [ 97, 102, "dimer", "oligomeric_state" ], [ 110, 124, "minimal repeat", "structure_element" ], [ 143, 148, "XyGO2", "chemical" ] ] }, { "sid": 62, "sent": "The observation by affinity PAGE that these proteins specifically recognize XyG is further substantiated by their lack of binding for the undecorated oligosaccharide cellotetraose (Table\u00a01; see Fig.\u00a0S3).", "section": "RESULTS", "ner": [ [ 19, 32, "affinity PAGE", "experimental_method" ], [ 76, 79, "XyG", "chemical" ], [ 150, 165, "oligosaccharide", "chemical" ], [ 166, 179, "cellotetraose", "chemical" ] ] }, { "sid": 63, "sent": "Furthermore, SGBP-A binds cellohexaose with ~770-fold weaker affinity than XyG, while SGBP-B displays no detectable binding to this linear hexasaccharide.", "section": "RESULTS", "ner": [ [ 13, 19, "SGBP-A", "protein" ], [ 26, 38, "cellohexaose", "chemical" ], [ 61, 69, "affinity", "evidence" ], [ 75, 78, "XyG", "chemical" ], [ 86, 92, "SGBP-B", "protein" ], [ 139, 153, "hexasaccharide", "chemical" ] ] }, { "sid": 64, "sent": "To provide molecular-level insight into how the XyGUL SGBPs equip B.\u00a0ovatus to specifically harvest XyG from the gut environment, we performed X-ray crystallography analysis of both SGBP-A and SGPB-B in oligosaccharide-complex forms.", "section": "RESULTS", "ner": [ [ 48, 53, "XyGUL", "gene" ], [ 54, 59, "SGBPs", "protein_type" ], [ 66, 75, "B.\u00a0ovatus", "species" ], [ 100, 103, "XyG", "chemical" ], [ 143, 164, "X-ray crystallography", "experimental_method" ], [ 182, 188, "SGBP-A", "protein" ], [ 193, 199, "SGPB-B", "protein" ], [ 203, 232, "oligosaccharide-complex forms", "complex_assembly" ] ] }, { "sid": 65, "sent": "Summary of thermodynamic parameters for wild-type SGBP-A and SGBP-B obtained by isothermal titration calorimetry at 25\u00b0Ca", "section": "TABLE", "ner": [ [ 40, 49, "wild-type", "protein_state" ], [ 50, 56, "SGBP-A", "protein" ], [ 61, 67, "SGBP-B", "protein" ], [ 80, 112, "isothermal titration calorimetry", "experimental_method" ] ] }, { "sid": 66, "sent": "Carbohydrate\tKa (M\u22121)\t\u0394G (kcal\u00a0\u22c5\u00a0mol\u22121)\t\u0394H (kcal\u00a0\u22c5\u00a0mol\u22121)\tT\u0394S (kcal\u00a0\u22c5\u00a0mol\u22121)\t \tSGBP-A\tSGBP-B\tSGBP-A\tSGBP-B\tSGBP-A\tSGBP-B\tSGBP-A\tSGBP-B\t \tXyGb\t(4.4 \u00b1 0.1) \u00d7 105\t(5.7 \u00b1 0.2) \u00d7 104\t\u22127.7\t\u22126.5\t\u221214 \u00b1 3\t\u221214 \u00b1 2\t\u22126.5\t\u22127.6\t \tXyGO2c\t3.0 \u00d7 105\t2.0 \u00d7 104\t\u22127.5\t\u22125.9\t\u221217.2\t\u221217.6\t\u22129.7\t\u221211.7\t \tXyGO1\tNBd\t(2.4 \u00b1 0.1) \u00d7 103\tNB\t\u22124.6\tNB\t\u22124.4 \u00b1 0.2\tNB\t0.2\t \tCellohexaose\t568.0 \u00b1 291.0\tNB\t\u22123.8\tNB\t\u221216 \u00b1 8\tNB\t\u221212.7\tNB\t \tCellotetraose\tNB\tNB\tNB\tNB\tNB\tNB\tNB\tNB\t \t", "section": "TABLE", "ner": [ [ 22, 24, "\u0394G", "evidence" ] ] }, { "sid": 67, "sent": "SGBP-A is a SusD homolog with an extensive glycan-binding platform.", "section": "RESULTS", "ner": [ [ 0, 6, "SGBP-A", "protein" ], [ 12, 16, "SusD", "protein" ], [ 43, 66, "glycan-binding platform", "site" ] ] }, { "sid": 68, "sent": "As anticipated by sequence similarity, the high-resolution tertiary structure of apo-SGBP-A (1.36\u00a0\u00c5, Rwork = 14.7%, Rfree = 17.4%, residues 28 to 546) (Table\u00a02) displays the canonical \u201cSusD-like\u201d protein fold dominated by four tetratrico-peptide repeat (TPR) motifs that cradle the rest of the structure (Fig.\u00a04A).", "section": "RESULTS", "ner": [ [ 68, 77, "structure", "evidence" ], [ 81, 84, "apo", "protein_state" ], [ 85, 91, "SGBP-A", "protein" ], [ 101, 106, "Rwork", "evidence" ], [ 116, 121, "Rfree", "evidence" ], [ 140, 149, "28 to 546", "residue_range" ], [ 184, 208, "\u201cSusD-like\u201d protein fold", "structure_element" ], [ 227, 252, "tetratrico-peptide repeat", "structure_element" ], [ 254, 257, "TPR", "structure_element" ], [ 294, 303, "structure", "evidence" ] ] }, { "sid": 69, "sent": "Specifically, SGBP-A overlays B.\u00a0thetaiotaomicron SusD (BtSusD) with a root mean square deviation (RMSD) value of 2.2\u00a0\u00c5 for 363 C\u03b1 pairs, which is notable given the 26% amino acid identity (40% similarity) between these homologs (Fig.\u00a04C).", "section": "RESULTS", "ner": [ [ 14, 20, "SGBP-A", "protein" ], [ 21, 29, "overlays", "experimental_method" ], [ 30, 49, "B.\u00a0thetaiotaomicron", "species" ], [ 50, 54, "SusD", "protein" ], [ 56, 62, "BtSusD", "protein" ], [ 71, 97, "root mean square deviation", "evidence" ], [ 99, 103, "RMSD", "evidence" ] ] }, { "sid": 70, "sent": "Cocrystallization of SGBP-A with XyGO2 generated a substrate complex structure (2.3\u00a0\u00c5, Rwork = 21.8%, Rfree = 24.8%, residues 36 to 546) (Fig.\u00a04A and B; Table\u00a02) that revealed the distinct binding-site architecture of the XyG binding protein.", "section": "RESULTS", "ner": [ [ 0, 17, "Cocrystallization", "experimental_method" ], [ 21, 27, "SGBP-A", "protein" ], [ 33, 38, "XyGO2", "chemical" ], [ 51, 68, "substrate complex", "complex_assembly" ], [ 69, 78, "structure", "evidence" ], [ 87, 92, "Rwork", "evidence" ], [ 102, 107, "Rfree", "evidence" ], [ 126, 135, "36 to 546", "residue_range" ], [ 189, 201, "binding-site", "site" ], [ 222, 241, "XyG binding protein", "protein_type" ] ] }, { "sid": 71, "sent": "The SGBP-A:XyGO2 complex superimposes closely with the apo structure (RMSD of 0.6\u00a0\u00c5) and demonstrates that no major conformational change occurs upon substrate binding; small deviations in the orientation of several surface loops are likely the result of differential crystal packing.", "section": "RESULTS", "ner": [ [ 4, 16, "SGBP-A:XyGO2", "complex_assembly" ], [ 25, 37, "superimposes", "experimental_method" ], [ 55, 58, "apo", "protein_state" ], [ 59, 68, "structure", "evidence" ], [ 70, 74, "RMSD", "evidence" ] ] }, { "sid": 72, "sent": "It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-\u00c5-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-\u00c5-long, site within SusD that complements the helical conformation of amylose (Fig.\u00a04C and D).", "section": "RESULTS", "ner": [ [ 61, 80, "ligand-binding site", "site" ], [ 84, 93, "conserved", "protein_state" ], [ 102, 108, "SGBP-A", "protein" ], [ 113, 117, "SusD", "protein" ], [ 127, 133, "SGBP-A", "protein" ], [ 157, 174, "aromatic platform", "site" ], [ 211, 214, "XyG", "chemical" ], [ 252, 255, "XyG", "chemical" ], [ 310, 314, "site", "site" ], [ 322, 326, "SusD", "protein" ], [ 372, 379, "amylose", "chemical" ] ] }, { "sid": 73, "sent": "Molecular structure of SGBP-A (Bacova_02651). (A) Overlay of SGBP-A from the apo (rainbow) and XyGO2 (gray) structures.", "section": "FIG", "ner": [ [ 10, 19, "structure", "evidence" ], [ 23, 29, "SGBP-A", "protein" ], [ 31, 43, "Bacova_02651", "gene" ], [ 50, 57, "Overlay", "experimental_method" ], [ 61, 67, "SGBP-A", "protein" ], [ 77, 80, "apo", "protein_state" ], [ 95, 100, "XyGO2", "chemical" ], [ 108, 118, "structures", "evidence" ] ] }, { "sid": 74, "sent": "The apo structure is color ramped from blue to red.", "section": "FIG", "ner": [ [ 4, 7, "apo", "protein_state" ], [ 8, 17, "structure", "evidence" ] ] }, { "sid": 75, "sent": "An omit map (2\u03c3) for XyGO2 (orange and red sticks) is displayed.", "section": "FIG", "ner": [ [ 3, 11, "omit map", "evidence" ], [ 21, 26, "XyGO2", "chemical" ] ] }, { "sid": 76, "sent": "(B) Close-up view of the omit map as in panel A, rotated 90\u00b0 clockwise.", "section": "FIG", "ner": [ [ 25, 33, "omit map", "evidence" ] ] }, { "sid": 77, "sent": "(C) Overlay of the C\u03b1 backbones of SGBP-A (black) with XyGO2 (orange and red spheres) and BtSusD (blue) with maltoheptaose (pink and red spheres), highlighting the conservation of the glycan-binding site location.", "section": "FIG", "ner": [ [ 4, 11, "Overlay", "experimental_method" ], [ 35, 41, "SGBP-A", "protein" ], [ 55, 60, "XyGO2", "chemical" ], [ 90, 96, "BtSusD", "protein" ], [ 109, 122, "maltoheptaose", "chemical" ], [ 184, 203, "glycan-binding site", "site" ] ] }, { "sid": 78, "sent": "(D) Close-up of the SGBP-A (black and orange) and SusD (blue and pink) glycan-binding sites.", "section": "FIG", "ner": [ [ 20, 26, "SGBP-A", "protein" ], [ 50, 54, "SusD", "protein" ], [ 71, 91, "glycan-binding sites", "site" ] ] }, { "sid": 79, "sent": "The approximate length of each glycan-binding site is displayed, colored to match the protein structures. (E) Stereo view of the xyloglucan-binding site of SGBP-A, displaying all residues within 4 \u00c5 of the ligand.", "section": "FIG", "ner": [ [ 31, 50, "glycan-binding site", "site" ], [ 86, 104, "protein structures", "evidence" ], [ 129, 152, "xyloglucan-binding site", "site" ], [ 156, 162, "SGBP-A", "protein" ] ] }, { "sid": 80, "sent": "The backbone glucose residues are numbered from the nonreducing end; xylose residues are labeled X1 and X2.", "section": "FIG", "ner": [ [ 13, 20, "glucose", "chemical" ], [ 69, 75, "xylose", "chemical" ], [ 97, 99, "X1", "residue_name_number" ], [ 104, 106, "X2", "residue_name_number" ] ] }, { "sid": 81, "sent": "Potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms.", "section": "FIG", "ner": [ [ 10, 39, "hydrogen-bonding interactions", "bond_interaction" ] ] }, { "sid": 82, "sent": "Seven of the eight backbone glucosyl residues of XyGO2 could be convincingly modeled in the ligand electron density, and only two \u03b1(1\u21926)-linked xylosyl residues were observed (Fig.\u00a04B; cf.", "section": "RESULTS", "ner": [ [ 28, 36, "glucosyl", "chemical" ], [ 49, 54, "XyGO2", "chemical" ], [ 92, 115, "ligand electron density", "evidence" ], [ 130, 151, "\u03b1(1\u21926)-linked xylosyl", "chemical" ] ] }, { "sid": 83, "sent": "Indeed, the electron density for the ligand suggests some disorder, which may arise from multiple oligosaccharide orientations along the binding site.", "section": "RESULTS", "ner": [ [ 12, 28, "electron density", "evidence" ], [ 98, 113, "oligosaccharide", "chemical" ], [ 137, 149, "binding site", "site" ] ] }, { "sid": 84, "sent": "Three aromatic residues\u2014W82, W283, W306\u2014comprise the flat platform that stacks along the naturally twisted \u03b2-glucan backbone (Fig.\u00a04E).", "section": "RESULTS", "ner": [ [ 24, 27, "W82", "residue_name_number" ], [ 29, 33, "W283", "residue_name_number" ], [ 35, 39, "W306", "residue_name_number" ], [ 53, 66, "flat platform", "site" ], [ 72, 78, "stacks", "bond_interaction" ], [ 107, 115, "\u03b2-glucan", "chemical" ] ] }, { "sid": 85, "sent": "The functional importance of this platform is underscored by the observation that the W82A W283A W306A mutant of SGBP-A, designated SGBP-A*, is completely devoid of XyG affinity (Table\u00a03; see Fig.\u00a0S4 in the supplemental material).", "section": "RESULTS", "ner": [ [ 34, 42, "platform", "site" ], [ 86, 90, "W82A", "mutant" ], [ 91, 96, "W283A", "mutant" ], [ 97, 102, "W306A", "mutant" ], [ 103, 109, "mutant", "protein_state" ], [ 113, 119, "SGBP-A", "protein" ], [ 132, 139, "SGBP-A*", "mutant" ], [ 144, 177, "completely devoid of XyG affinity", "protein_state" ] ] }, { "sid": 86, "sent": "Dissection of the individual contribution of these residues reveals that the W82A mutant displays a significant 4.9-fold decrease in the Ka value for XyG, while the W306A substitution completely abolishes XyG binding.", "section": "RESULTS", "ner": [ [ 77, 81, "W82A", "mutant" ], [ 82, 88, "mutant", "protein_state" ], [ 137, 139, "Ka", "evidence" ], [ 150, 153, "XyG", "chemical" ], [ 165, 170, "W306A", "mutant" ], [ 171, 183, "substitution", "experimental_method" ], [ 195, 216, "abolishes XyG binding", "protein_state" ] ] }, { "sid": 87, "sent": "Contrasting with the clear importance of these hydrophobic interactions, there are remarkably few hydrogen-bonding interactions with the ligand, which are provided by R65, N83, and S308, which are proximal to Glc5 and Glc3.", "section": "RESULTS", "ner": [ [ 47, 71, "hydrophobic interactions", "bond_interaction" ], [ 98, 127, "hydrogen-bonding interactions", "bond_interaction" ], [ 137, 143, "ligand", "chemical" ], [ 167, 170, "R65", "residue_name_number" ], [ 172, 175, "N83", "residue_name_number" ], [ 181, 185, "S308", "residue_name_number" ], [ 209, 213, "Glc5", "residue_name_number" ], [ 218, 222, "Glc3", "residue_name_number" ] ] }, { "sid": 88, "sent": "Most surprising in light of the saccharide-binding data, however, was a lack of extensive recognition of the XyG side chains; only Y84 appeared to provide a hydrophobic interface for a xylosyl residue (Xyl1).", "section": "RESULTS", "ner": [ [ 32, 55, "saccharide-binding data", "evidence" ], [ 109, 112, "XyG", "chemical" ], [ 131, 134, "Y84", "residue_name_number" ], [ 157, 178, "hydrophobic interface", "site" ], [ 185, 192, "xylosyl", "chemical" ], [ 202, 206, "Xyl1", "residue_name_number" ] ] }, { "sid": 89, "sent": "Summary of thermodynamic parameters for site-directed mutants of SGBP-A and SGBP-B obtained by ITC with XyG at 25\u00b0Ca", "section": "TABLE", "ner": [ [ 65, 71, "SGBP-A", "protein" ], [ 76, 82, "SGBP-B", "protein" ], [ 95, 98, "ITC", "experimental_method" ], [ 104, 107, "XyG", "chemical" ] ] }, { "sid": 90, "sent": "Protein name\tKa\t\u0394G (kcal\u00a0\u22c5\u00a0mol\u22121)\t\u0394H (kcal\u00a0\u22c5\u00a0mol\u22121)\tT\u0394S (kcal\u00a0\u22c5\u00a0mol\u22121)\t \tFold changeb\tM\u22121\t \tSGBP-A(W82A W283A W306A)\tND\tNB\tNB\tNB\tNB\t \tSGBP-A(W82A)c\t4.9\t9.1 \u00d7 104\t\u22126.8\t\u22126.3\t0.5\t \tSGBP-A(W306)\tND\tNB\tNB\tNB\tNB\t \tSGBP-B(230\u2013489)\t0.7\t(8.6 \u00b1 0.20) \u00d7 104\t\u22126.7\t\u221214.9 \u00b1 0.1\t\u22128.2\t \tSGBP-B(Y363A)\t19.7\t(2.9 \u00b1 0.10) \u00d7 103\t\u22124.7\t\u221218.1 \u00b1 0.1\t\u221213.3\t \tSGBP-B(W364A)\tND\tWeak\tWeak\tWeak\tWeak\t \tSGBP-B(F414A)\t3.2\t(1.80 \u00b1 0.03) \u00d7 104\t\u22125.8\t\u221211.4 \u00b1 0.1\t\u22125.6\t \t", "section": "TABLE", "ner": [ [ 13, 15, "Ka", "evidence" ], [ 16, 18, "\u0394G", "evidence" ], [ 34, 36, "\u0394H", "evidence" ], [ 52, 55, "T\u0394S", "evidence" ], [ 92, 98, "SGBP-A", "protein" ], [ 99, 103, "W82A", "mutant" ], [ 104, 109, "W283A", "mutant" ], [ 110, 115, "W306A", "mutant" ], [ 134, 140, "SGBP-A", "protein" ], [ 141, 145, "W82A", "mutant" ], [ 178, 184, "SGBP-A", "protein" ], [ 185, 189, "W306", "residue_name_number" ], [ 208, 214, "SGBP-B", "protein" ], [ 215, 222, "230\u2013489", "residue_range" ], [ 271, 277, "SGBP-B", "protein" ], [ 278, 283, "Y363A", "mutant" ], [ 334, 340, "SGBP-B", "protein" ], [ 341, 346, "W364A", "mutant" ], [ 373, 379, "SGBP-B", "protein" ], [ 380, 385, "F414A", "mutant" ] ] }, { "sid": 91, "sent": "Binding thermodynamics are based on the concentration of the binding unit, XyGO2.", "section": "TABLE", "ner": [ [ 75, 80, "XyGO2", "chemical" ] ] }, { "sid": 92, "sent": "Weak binding represents a Ka of <500 M\u22121.", "section": "TABLE", "ner": [ [ 26, 28, "Ka", "evidence" ] ] }, { "sid": 93, "sent": "Ka fold change = Ka of wild-type protein/Ka of mutant protein for xyloglucan binding.", "section": "TABLE", "ner": [ [ 0, 2, "Ka", "evidence" ], [ 17, 19, "Ka", "evidence" ], [ 23, 32, "wild-type", "protein_state" ], [ 41, 43, "Ka", "evidence" ], [ 66, 76, "xyloglucan", "chemical" ] ] }, { "sid": 94, "sent": "SGBP-B has a multimodular structure with a single, C-terminal glycan-binding domain.", "section": "RESULTS", "ner": [ [ 0, 6, "SGBP-B", "protein" ], [ 62, 83, "glycan-binding domain", "structure_element" ] ] }, { "sid": 95, "sent": "The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37\u00a0\u00c5, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table\u00a02) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig.\u00a05A).", "section": "RESULTS", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 25, 36, "full-length", "protein_state" ], [ 37, 43, "SGBP-B", "protein" ], [ 44, 59, "in complex with", "protein_state" ], [ 60, 65, "XyGO2", "chemical" ], [ 75, 80, "Rwork", "evidence" ], [ 90, 95, "Rfree", "evidence" ], [ 114, 123, "34 to 489", "residue_range" ], [ 156, 165, "structure", "evidence" ], [ 184, 223, "tandem immunoglobulin (Ig)-like domains", "structure_element" ], [ 233, 234, "A", "structure_element" ], [ 236, 237, "B", "structure_element" ], [ 243, 244, "C", "structure_element" ], [ 284, 309, "xyloglucan-binding domain", "structure_element" ], [ 318, 319, "D", "structure_element" ] ] }, { "sid": 96, "sent": "Domains A, B, and C display similar \u03b2-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2\u00a0\u00c5, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively).", "section": "RESULTS", "ner": [ [ 8, 9, "A", "structure_element" ], [ 11, 12, "B", "structure_element" ], [ 18, 19, "C", "structure_element" ], [ 36, 52, "\u03b2-sandwich folds", "structure_element" ], [ 62, 63, "B", "structure_element" ], [ 74, 84, "134 to 230", "residue_range" ], [ 90, 91, "C", "structure_element" ], [ 102, 112, "231 to 313", "residue_range" ], [ 121, 133, "superimposed", "experimental_method" ], [ 146, 147, "A", "structure_element" ], [ 158, 167, "34 to 133", "residue_range" ], [ 174, 179, "RMSDs", "evidence" ] ] }, { "sid": 97, "sent": "These domains also display similarity to the C-terminal \u03b2-sandwich domains of many GH13 enzymes, including the cyclodextrin glucanotransferase of Geobacillus stearothermophilus (Fig.\u00a05B).", "section": "RESULTS", "ner": [ [ 0, 13, "These domains", "structure_element" ], [ 56, 74, "\u03b2-sandwich domains", "structure_element" ], [ 83, 95, "GH13 enzymes", "protein_type" ], [ 111, 142, "cyclodextrin glucanotransferase", "protein_type" ], [ 146, 176, "Geobacillus stearothermophilus", "species" ] ] }, { "sid": 98, "sent": "Such domains are not typically involved in carbohydrate binding.", "section": "RESULTS", "ner": [ [ 0, 12, "Such domains", "structure_element" ], [ 43, 55, "carbohydrate", "chemical" ] ] }, { "sid": 99, "sent": "Indeed, visual inspection of the SGBP-B structure, as well as individual production of the A and B domains and affinity PAGE analysis (see Fig.\u00a0S5 in the supplemental material), indicates that these domains do not contribute to XyG capture.", "section": "RESULTS", "ner": [ [ 8, 25, "visual inspection", "experimental_method" ], [ 33, 39, "SGBP-B", "protein" ], [ 40, 49, "structure", "evidence" ], [ 91, 92, "A", "structure_element" ], [ 97, 98, "B", "structure_element" ], [ 111, 124, "affinity PAGE", "experimental_method" ], [ 228, 231, "XyG", "chemical" ] ] }, { "sid": 100, "sent": "On the other hand, production of the fused domains C and D in tandem (SGBP-B residues 230 to 489) retains complete binding of xyloglucan in vitro, with the observed slight increase in affinity likely arising from a reduced potential for steric hindrance of the smaller protein construct during polysaccharide interactions (Table\u00a03).", "section": "RESULTS", "ner": [ [ 19, 29, "production", "experimental_method" ], [ 37, 58, "fused domains C and D", "mutant" ], [ 70, 76, "SGBP-B", "protein" ], [ 86, 96, "230 to 489", "residue_range" ], [ 126, 136, "xyloglucan", "chemical" ], [ 294, 308, "polysaccharide", "chemical" ] ] }, { "sid": 101, "sent": "While neither the full-length protein nor domain D displays structural homology to known XyG-binding proteins, the topology of SGBP-B resembles the xylan-binding protein Bacova_04391 (PDB 3ORJ) encoded within a xylan-targeting PUL of B.\u00a0ovatus (Fig.\u00a05C).", "section": "RESULTS", "ner": [ [ 18, 29, "full-length", "protein_state" ], [ 49, 50, "D", "structure_element" ], [ 89, 109, "XyG-binding proteins", "protein_type" ], [ 127, 133, "SGBP-B", "protein" ], [ 148, 169, "xylan-binding protein", "protein_type" ], [ 170, 182, "Bacova_04391", "protein" ], [ 211, 216, "xylan", "chemical" ], [ 227, 230, "PUL", "gene" ], [ 234, 243, "B.\u00a0ovatus", "species" ] ] }, { "sid": 102, "sent": "The structure-based alignment of these proteins reveals 17% sequence identity, with a core RMSD of 3.6\u00a0\u00c5 for 253 aligned residues.", "section": "RESULTS", "ner": [ [ 4, 29, "structure-based alignment", "experimental_method" ], [ 91, 95, "RMSD", "evidence" ] ] }, { "sid": 103, "sent": "While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below).", "section": "RESULTS", "ner": [ [ 51, 63, "Bacova_04391", "protein" ], [ 79, 91, "binding site", "site" ], [ 116, 120, "W241", "residue_name_number" ], [ 125, 129, "Y404", "residue_name_number" ], [ 157, 174, "XyGO binding site", "site" ], [ 178, 184, "SGBP-B", "protein" ], [ 199, 231, "opposing, clamp-like arrangement", "protein_state" ], [ 235, 249, "these residues", "structure_element" ], [ 253, 265, "Bacova_04391", "protein" ], [ 295, 321, "planar surface arrangement", "site" ], [ 329, 337, "residues", "structure_element" ], [ 357, 360, "XyG", "chemical" ], [ 364, 370, "SGBP-B", "protein" ] ] }, { "sid": 104, "sent": "Multimodular structure of SGBP-B (Bacova_02650). (A) Full-length structure of SGBP-B, color coded by domain as indicated.", "section": "FIG", "ner": [ [ 26, 32, "SGBP-B", "protein" ], [ 34, 46, "Bacova_02650", "gene" ], [ 53, 64, "Full-length", "protein_state" ], [ 65, 74, "structure", "evidence" ], [ 78, 84, "SGBP-B", "protein" ] ] }, { "sid": 105, "sent": "Prolines between domains are indicated as spheres.", "section": "FIG", "ner": [ [ 0, 8, "Prolines", "residue_name" ] ] }, { "sid": 106, "sent": "An omit map (2\u03c3) for XyGO2 is displayed to highlight the location of the glycan-binding site.", "section": "FIG", "ner": [ [ 3, 11, "omit map", "evidence" ], [ 21, 26, "XyGO2", "chemical" ], [ 73, 92, "glycan-binding site", "site" ] ] }, { "sid": 107, "sent": "(B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) C\u03b1 overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink).", "section": "FIG", "ner": [ [ 15, 21, "SGBP-B", "protein" ], [ 30, 31, "A", "structure_element" ], [ 33, 34, "B", "structure_element" ], [ 40, 41, "C", "structure_element" ], [ 85, 99, "Ig-like domain", "structure_element" ], [ 107, 128, "G. stearothermophilus", "species" ], [ 129, 160, "cyclodextrin glucanotransferase", "protein_type" ], [ 181, 191, "375 to 493", "residue_range" ], [ 211, 218, "overlay", "experimental_method" ], [ 222, 228, "SGBP-B", "protein" ], [ 240, 252, "Bacova_04391", "protein" ] ] }, { "sid": 108, "sent": "(D) Close-up omit map for the XyGO2 ligand, contoured at 2\u03c3. (E) Stereo view of the xyloglucan-binding site of SGBP-B, displaying all residues within 4 \u00c5 of the ligand.", "section": "FIG", "ner": [ [ 13, 21, "omit map", "evidence" ], [ 30, 35, "XyGO2", "chemical" ], [ 84, 107, "xyloglucan-binding site", "site" ], [ 111, 117, "SGBP-B", "protein" ] ] }, { "sid": 109, "sent": "The backbone glucose residues are numbered from the nonreducing end, xylose residues are shown as X1, X2, and X3, potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms.", "section": "FIG", "ner": [ [ 13, 20, "glucose", "chemical" ], [ 69, 75, "xylose", "chemical" ], [ 98, 100, "X1", "residue_name_number" ], [ 102, 104, "X2", "residue_name_number" ], [ 110, 112, "X3", "residue_name_number" ], [ 124, 153, "hydrogen-bonding interactions", "bond_interaction" ] ] }, { "sid": 110, "sent": "Inspection of the tertiary structure indicates that domains C and D are effectively inseparable, with a contact interface of 396\u00a0\u00c52.", "section": "RESULTS", "ner": [ [ 27, 36, "structure", "evidence" ], [ 60, 61, "C", "structure_element" ], [ 66, 67, "D", "structure_element" ] ] }, { "sid": 111, "sent": "Domains A, B, and C do not pack against each other.", "section": "RESULTS", "ner": [ [ 8, 9, "A", "structure_element" ], [ 11, 12, "B", "structure_element" ], [ 18, 19, "C", "structure_element" ] ] }, { "sid": 112, "sent": "Moreover, the five-residue linkers between these first three domains all feature a proline as the middle residue, suggesting significant conformational rigidity (Fig.\u00a05A).", "section": "RESULTS", "ner": [ [ 14, 34, "five-residue linkers", "structure_element" ], [ 83, 90, "proline", "residue_name" ], [ 98, 112, "middle residue", "structure_element" ] ] }, { "sid": 113, "sent": "Despite the lack of sequence and structural conservation, a similarly positioned proline joins the Ig-like domains of the xylan-binding Bacova_04391 and the starch-binding proteins SusE and SusF. We speculate that this is a biologically important adaptation that serves to project the glycan binding site of these proteins far from the membrane surface.", "section": "RESULTS", "ner": [ [ 81, 88, "proline", "residue_name" ], [ 99, 114, "Ig-like domains", "structure_element" ], [ 136, 148, "Bacova_04391", "protein" ], [ 157, 180, "starch-binding proteins", "protein_type" ], [ 181, 185, "SusE", "protein" ], [ 190, 194, "SusF", "protein" ], [ 285, 304, "glycan binding site", "site" ] ] }, { "sid": 114, "sent": "Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first \u03b2-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element.", "section": "RESULTS", "ner": [ [ 16, 22, "SGBP-B", "protein" ], [ 123, 143, "eight-residue linker", "structure_element" ], [ 169, 178, "lipidated", "protein_state" ], [ 179, 182, "Cys", "residue_name" ], [ 184, 187, "C28", "residue_name_number" ], [ 197, 211, "first \u03b2-strand", "structure_element" ], [ 222, 223, "A", "structure_element" ], [ 231, 254, "outer membrane proteins", "protein_type" ], [ 268, 284, "Sus-like systems", "complex_assembly" ], [ 303, 339, "10- to 20-amino-acid flexible linker", "structure_element" ], [ 352, 361, "lipidated", "protein_state" ], [ 362, 365, "Cys", "residue_name" ] ] }, { "sid": 115, "sent": "Analogously, the outer membrane-anchored endo-xyloglucanase BoGH5 of the XyGUL contains a 100-amino-acid, all-\u03b2-strand, N-terminal module and flexible linker that imparts conformational flexibility and distances the catalytic module from the cell surface.", "section": "RESULTS", "ner": [ [ 17, 40, "outer membrane-anchored", "protein_state" ], [ 41, 59, "endo-xyloglucanase", "protein_type" ], [ 60, 65, "BoGH5", "protein" ], [ 73, 78, "XyGUL", "gene" ], [ 90, 118, "100-amino-acid, all-\u03b2-strand", "structure_element" ], [ 120, 137, "N-terminal module", "structure_element" ], [ 142, 157, "flexible linker", "structure_element" ], [ 216, 232, "catalytic module", "structure_element" ] ] }, { "sid": 116, "sent": "XyG binds to domain D of SGBP-B at the concave interface of the top \u03b2-sheet, with binding mediated by loops connecting the \u03b2-strands.", "section": "RESULTS", "ner": [ [ 0, 3, "XyG", "chemical" ], [ 4, 12, "binds to", "protein_state" ], [ 20, 21, "D", "structure_element" ], [ 25, 31, "SGBP-B", "protein" ], [ 39, 56, "concave interface", "site" ], [ 68, 75, "\u03b2-sheet", "structure_element" ], [ 102, 107, "loops", "structure_element" ], [ 123, 132, "\u03b2-strands", "structure_element" ] ] }, { "sid": 117, "sent": "Six glucosyl residues, comprising the main chain, and three branching xylosyl residues of XyGO2 can be modeled in the density (Fig.\u00a05D; cf.", "section": "RESULTS", "ner": [ [ 4, 12, "glucosyl", "chemical" ], [ 70, 77, "xylosyl", "chemical" ], [ 90, 95, "XyGO2", "chemical" ], [ 118, 125, "density", "evidence" ] ] }, { "sid": 118, "sent": "The backbone is flat, with less of the \u201ctwisted-ribbon\u201d geometry observed in some cello- and xylogluco-oligosaccharides.", "section": "RESULTS", "ner": [ [ 82, 119, "cello- and xylogluco-oligosaccharides", "chemical" ] ] }, { "sid": 119, "sent": "The aromatic platform created by W330, W364, and Y363 spans four glucosyl residues, compared to the longer platform of SGBP-A, which supports six glucosyl residues (Fig.\u00a05E).", "section": "RESULTS", "ner": [ [ 4, 21, "aromatic platform", "site" ], [ 33, 37, "W330", "residue_name_number" ], [ 39, 43, "W364", "residue_name_number" ], [ 49, 53, "Y363", "residue_name_number" ], [ 65, 73, "glucosyl", "chemical" ], [ 100, 106, "longer", "protein_state" ], [ 107, 115, "platform", "site" ], [ 119, 125, "SGBP-A", "protein" ], [ 146, 154, "glucosyl", "chemical" ] ] }, { "sid": 120, "sent": "The Y363A site-directed mutant of SGBP-B displays a 20-fold decrease in the Ka for XyG, while the W364A mutant lacks XyG binding (Table\u00a03; see Fig.\u00a0S6 in the supplemental material).", "section": "RESULTS", "ner": [ [ 4, 9, "Y363A", "mutant" ], [ 10, 30, "site-directed mutant", "experimental_method" ], [ 34, 40, "SGBP-B", "protein" ], [ 76, 78, "Ka", "evidence" ], [ 83, 86, "XyG", "chemical" ], [ 98, 103, "W364A", "mutant" ], [ 104, 110, "mutant", "protein_state" ], [ 111, 128, "lacks XyG binding", "protein_state" ] ] }, { "sid": 121, "sent": "There are no additional contacts between the protein and the \u03b2-glucan backbone and surprisingly few interactions with the side-chain xylosyl residues, despite that fact that ITC data demonstrate that SGBP-B does not measurably bind the cellohexaose (Table\u00a01).", "section": "RESULTS", "ner": [ [ 61, 69, "\u03b2-glucan", "chemical" ], [ 133, 140, "xylosyl", "chemical" ], [ 174, 177, "ITC", "experimental_method" ], [ 200, 206, "SGBP-B", "protein" ], [ 236, 248, "cellohexaose", "chemical" ] ] }, { "sid": 122, "sent": "F414 stacks with the xylosyl residue of Glc3, while Q407 is positioned for hydrogen bonding with the O4 of xylosyl residue Xyl1.", "section": "RESULTS", "ner": [ [ 0, 4, "F414", "residue_name_number" ], [ 5, 11, "stacks", "bond_interaction" ], [ 21, 28, "xylosyl", "chemical" ], [ 40, 44, "Glc3", "residue_name_number" ], [ 52, 56, "Q407", "residue_name_number" ], [ 75, 91, "hydrogen bonding", "bond_interaction" ], [ 107, 114, "xylosyl", "chemical" ], [ 123, 127, "Xyl1", "residue_name_number" ] ] }, { "sid": 123, "sent": "Surprisingly, an F414A mutant of SGBP-B displays only a mild 3-fold decrease in the Ka value for XyG, again suggesting that glycan recognition is primarily mediated via contact with the \u03b2-glucan backbone (Table\u00a03; see Fig.\u00a0S6).", "section": "RESULTS", "ner": [ [ 17, 22, "F414A", "mutant" ], [ 23, 29, "mutant", "protein_state" ], [ 33, 39, "SGBP-B", "protein" ], [ 84, 86, "Ka", "evidence" ], [ 97, 100, "XyG", "chemical" ], [ 124, 130, "glycan", "chemical" ] ] }, { "sid": 124, "sent": "Additional residues surrounding the binding site, including Y369 and E412, may contribute to the recognition of more highly decorated XyG, but precisely how this is mediated is presently unclear.", "section": "RESULTS", "ner": [ [ 11, 19, "residues", "structure_element" ], [ 36, 48, "binding site", "site" ], [ 60, 64, "Y369", "residue_name_number" ], [ 69, 73, "E412", "residue_name_number" ], [ 134, 137, "XyG", "chemical" ] ] }, { "sid": 125, "sent": "Hoping to achieve a higher-resolution view of the SGBP-B\u2013xyloglucan interaction, we solved the crystal structure of the fused CD domains in complex with XyGO2 (1.57\u00a0\u00c5, Rwork = 15.6%, Rfree = 17.1%, residues 230 to 489) (Table\u00a02).", "section": "RESULTS", "ner": [ [ 50, 56, "SGBP-B", "protein" ], [ 57, 67, "xyloglucan", "chemical" ], [ 84, 90, "solved", "experimental_method" ], [ 95, 112, "crystal structure", "evidence" ], [ 120, 136, "fused CD domains", "mutant" ], [ 137, 152, "in complex with", "protein_state" ], [ 153, 158, "XyGO2", "chemical" ], [ 168, 173, "Rwork", "evidence" ], [ 183, 188, "Rfree", "evidence" ], [ 207, 217, "230 to 489", "residue_range" ] ] }, { "sid": 126, "sent": "The CD domains of the truncated and full-length proteins superimpose with a 0.4-\u00c5 RMSD of the C\u03b1 backbone, with no differences in the position of any of the glycan-binding residues (see Fig.\u00a0S7A in the supplemental material).", "section": "RESULTS", "ner": [ [ 4, 14, "CD domains", "structure_element" ], [ 22, 31, "truncated", "protein_state" ], [ 36, 47, "full-length", "protein_state" ], [ 57, 68, "superimpose", "experimental_method" ], [ 82, 86, "RMSD", "evidence" ], [ 157, 180, "glycan-binding residues", "site" ] ] }, { "sid": 127, "sent": "While density is observed for XyGO2, the ligand could not be unambiguously modeled into this density to achieve a reasonable fit between the X-ray data and the known stereochemistry of the sugar (see Fig.\u00a0S7B and C).", "section": "RESULTS", "ner": [ [ 6, 13, "density", "evidence" ], [ 30, 35, "XyGO2", "chemical" ], [ 93, 100, "density", "evidence" ], [ 141, 151, "X-ray data", "evidence" ] ] }, { "sid": 128, "sent": "While this may occur for a number of reasons in crystal structures, it is likely that the poor ligand density even at higher resolution is due to movement or multiple orientations of the sugar averaged throughout the lattice.", "section": "RESULTS", "ner": [ [ 48, 66, "crystal structures", "evidence" ], [ 187, 192, "sugar", "chemical" ] ] }, { "sid": 129, "sent": "SGBP-A and SGBP-B have distinct, coordinated functions in vivo.", "section": "RESULTS", "ner": [ [ 0, 6, "SGBP-A", "protein" ], [ 11, 17, "SGBP-B", "protein" ] ] }, { "sid": 130, "sent": "The similarity of the glycan specificity of SGBP-A and SGBP-B presents an intriguing conundrum regarding their individual roles in XyG utilization by B.\u00a0ovatus.", "section": "RESULTS", "ner": [ [ 22, 28, "glycan", "chemical" ], [ 44, 50, "SGBP-A", "protein" ], [ 55, 61, "SGBP-B", "protein" ], [ 131, 134, "XyG", "chemical" ], [ 150, 159, "B.\u00a0ovatus", "species" ] ] }, { "sid": 131, "sent": "To disentangle the functions of SGBP-A and SGBP-B in XyG recognition and uptake, we created individual in-frame deletion and complementation mutant strains of B.\u00a0ovatus.", "section": "RESULTS", "ner": [ [ 32, 38, "SGBP-A", "protein" ], [ 43, 49, "SGBP-B", "protein" ], [ 53, 56, "XyG", "chemical" ], [ 103, 147, "in-frame deletion and complementation mutant", "experimental_method" ], [ 159, 168, "B.\u00a0ovatus", "species" ] ] }, { "sid": 132, "sent": "In these growth experiments, overnight cultures of strains grown on minimal medium plus glucose were back-diluted 1:100-fold into minimal medium containing 5\u00a0mg/ml of the reported carbohydrate.", "section": "RESULTS", "ner": [ [ 9, 27, "growth experiments", "experimental_method" ], [ 88, 95, "glucose", "chemical" ], [ 180, 192, "carbohydrate", "chemical" ] ] }, { "sid": 133, "sent": "Growth on glucose displayed the shortest lag time for each strain, and so lag times were normalized for each carbohydrate by subtracting the lag time of that strain in glucose (Fig.\u00a06; see Fig.\u00a0S8 in the supplemental material).", "section": "RESULTS", "ner": [ [ 10, 17, "glucose", "chemical" ], [ 41, 49, "lag time", "evidence" ], [ 74, 83, "lag times", "evidence" ], [ 109, 121, "carbohydrate", "chemical" ], [ 141, 149, "lag time", "evidence" ], [ 168, 175, "glucose", "chemical" ] ] }, { "sid": 134, "sent": "A strain in which the entire XyGUL is deleted displays a lag of 24.5\u00a0h during growth on glucose compared to the isogenic parental wild-type (WT) \u0394tdk strain, for which exponential growth lags for 19.8\u00a0h (see Fig.\u00a0S8D).", "section": "RESULTS", "ner": [ [ 29, 34, "XyGUL", "gene" ], [ 38, 45, "deleted", "experimental_method" ], [ 57, 60, "lag", "evidence" ], [ 88, 95, "glucose", "chemical" ], [ 130, 139, "wild-type", "protein_state" ], [ 141, 143, "WT", "protein_state" ], [ 145, 149, "\u0394tdk", "mutant" ], [ 187, 191, "lags", "evidence" ] ] }, { "sid": 135, "sent": "It is unknown whether this is because cultures were not normalized by the starting optical density (OD) or viable cells or reflects a minor defect for glucose utilization.", "section": "RESULTS", "ner": [ [ 151, 158, "glucose", "chemical" ] ] }, { "sid": 136, "sent": "The former seems more likely as the growth rates are nearly identical for these strains on glucose and xylose.", "section": "RESULTS", "ner": [ [ 91, 98, "glucose", "chemical" ], [ 103, 109, "xylose", "chemical" ] ] }, { "sid": 137, "sent": "The \u0394XyGUL and WT \u0394tdk strains display normalized lag times on xylose within experimental error, and curiously some of the mutant and complemented strains display a nominally shorter lag time on xylose than the WT \u0394tdk strain.", "section": "RESULTS", "ner": [ [ 4, 10, "\u0394XyGUL", "mutant" ], [ 15, 17, "WT", "protein_state" ], [ 18, 22, "\u0394tdk", "mutant" ], [ 50, 59, "lag times", "evidence" ], [ 63, 69, "xylose", "chemical" ], [ 183, 191, "lag time", "evidence" ], [ 195, 201, "xylose", "chemical" ], [ 211, 213, "WT", "protein_state" ], [ 214, 218, "\u0394tdk", "mutant" ] ] }, { "sid": 138, "sent": "Complementation of the \u0394SGBP-A strain (\u0394SGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT \u0394tdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig.\u00a0S8C and F).", "section": "RESULTS", "ner": [ [ 0, 15, "Complementation", "experimental_method" ], [ 23, 30, "\u0394SGBP-A", "mutant" ], [ 39, 46, "\u0394SGBP-A", "mutant" ], [ 48, 54, "SGBP-A", "protein" ], [ 75, 84, "wild-type", "protein_state" ], [ 94, 104, "xyloglucan", "chemical" ], [ 109, 114, "XyGO1", "chemical" ], [ 187, 189, "WT", "protein_state" ], [ 190, 194, "\u0394tdk", "mutant" ], [ 205, 210, "XyGO2", "chemical" ], [ 250, 256, "SGBP-B", "protein" ] ] }, { "sid": 139, "sent": "The reason for this observation on XyGO2 is unclear, as the \u0394SGBP-B mutant does not have a significantly different growth rate from the WT on XyGO2.", "section": "RESULTS", "ner": [ [ 35, 40, "XyGO2", "chemical" ], [ 60, 67, "\u0394SGBP-B", "mutant" ], [ 68, 74, "mutant", "protein_state" ], [ 136, 138, "WT", "protein_state" ], [ 142, 147, "XyGO2", "chemical" ] ] }, { "sid": 140, "sent": "Growth of select XyGUL mutants on xyloglucan and oligosaccharides.", "section": "FIG", "ner": [ [ 17, 22, "XyGUL", "gene" ], [ 34, 44, "xyloglucan", "chemical" ], [ 49, 65, "oligosaccharides", "chemical" ] ] }, { "sid": 141, "sent": "B. ovatus mutants were created in a thymidine kinase deletion (\u0394tdk) mutant as described previously.", "section": "FIG", "ner": [ [ 0, 9, "B. ovatus", "species" ], [ 36, 61, "thymidine kinase deletion", "mutant" ], [ 63, 67, "\u0394tdk", "mutant" ] ] }, { "sid": 142, "sent": "SGBP-A* denotes the Bacova_02651 (W82A W283A W306A) allele, and the GH9 gene is Bacova_02649.", "section": "FIG", "ner": [ [ 0, 7, "SGBP-A*", "mutant" ], [ 20, 32, "Bacova_02651", "gene" ], [ 34, 38, "W82A", "mutant" ], [ 39, 44, "W283A", "mutant" ], [ 45, 50, "W306A", "mutant" ], [ 68, 71, "GH9", "protein" ], [ 80, 92, "Bacova_02649", "gene" ] ] }, { "sid": 143, "sent": "Growth was measured over time in minimal medium containing (A) XyG, (B) XyGO2, (C) XyGO1, (D) glucose, and (E) xylose.", "section": "FIG", "ner": [ [ 63, 66, "XyG", "chemical" ], [ 72, 77, "XyGO2", "chemical" ], [ 83, 88, "XyGO1", "chemical" ], [ 94, 101, "glucose", "chemical" ], [ 111, 117, "xylose", "chemical" ] ] }, { "sid": 144, "sent": "In panel F, the growth rate of each strain on the five carbon sources is displayed, and in panel G, the normalized lag time of each culture, relative to its growth on glucose, is displayed.", "section": "FIG", "ner": [ [ 115, 123, "lag time", "evidence" ], [ 167, 174, "glucose", "chemical" ] ] }, { "sid": 145, "sent": "Solid bars indicate conditions that are not statistically significant from the WT \u0394tdk cultures grown on the indicated carbohydrate, while open bars indicate a P value of <0.005 compared to the WT \u0394tdk strain.", "section": "FIG", "ner": [ [ 79, 81, "WT", "protein_state" ], [ 82, 86, "\u0394tdk", "mutant" ], [ 119, 131, "carbohydrate", "chemical" ], [ 194, 196, "WT", "protein_state" ], [ 197, 201, "\u0394tdk", "mutant" ] ] }, { "sid": 146, "sent": "Conditions denoted by the same letter (b, c, or d) are not statistically significant from each other but are significantly different from the condition labeled \u201ca.\u201d Complementation of \u0394SGBP-A and \u0394SBGP-B was performed by allelic exchange of the wild-type genes back into the genome for expression via the native promoter: these growth curves, quantified rates and lag times are displayed in Fig.\u00a0S8 in the supplemental material.", "section": "FIG", "ner": [ [ 184, 191, "\u0394SGBP-A", "mutant" ], [ 196, 203, "\u0394SBGP-B", "mutant" ], [ 245, 254, "wild-type", "protein_state" ], [ 364, 373, "lag times", "evidence" ] ] }, { "sid": 147, "sent": "The \u0394SGBP-A (\u0394Bacova_02651) strain (cf.", "section": "RESULTS", "ner": [ [ 4, 11, "\u0394SGBP-A", "mutant" ], [ 13, 26, "\u0394Bacova_02651", "mutant" ] ] }, { "sid": 148, "sent": "Fig.\u00a01B) was completely incapable of growth on XyG, XyGO1, and XyGO2, indicating that SGBP-A is essential for XyG utilization (Fig.\u00a06).", "section": "RESULTS", "ner": [ [ 47, 50, "XyG", "chemical" ], [ 52, 57, "XyGO1", "chemical" ], [ 63, 68, "XyGO2", "chemical" ], [ 86, 92, "SGBP-A", "protein" ], [ 110, 113, "XyG", "chemical" ] ] }, { "sid": 149, "sent": "This result mirrors our previous data for the canonical Sus of B.\u00a0thetaiotaomicron, which revealed that a homologous \u0394susD mutant is unable to grow on starch or malto-oligosaccharides, despite normal cell surface expression of all other PUL-encoded proteins.", "section": "RESULTS", "ner": [ [ 56, 59, "Sus", "complex_assembly" ], [ 63, 82, "B.\u00a0thetaiotaomicron", "species" ], [ 117, 122, "\u0394susD", "mutant" ], [ 123, 129, "mutant", "protein_state" ], [ 151, 157, "starch", "chemical" ], [ 161, 183, "malto-oligosaccharides", "chemical" ], [ 237, 240, "PUL", "gene" ] ] }, { "sid": 150, "sent": "More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of \u0394susD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B.\u00a0thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition.", "section": "RESULTS", "ner": [ [ 98, 102, "SusD", "protein" ], [ 104, 119, "complementation", "experimental_method" ], [ 123, 128, "\u0394susD", "mutant" ], [ 134, 139, "SusD*", "mutant" ], [ 143, 170, "triple site-directed mutant", "protein_state" ], [ 172, 176, "W96A", "mutant" ], [ 177, 182, "W320A", "mutant" ], [ 183, 188, "Y296A", "mutant" ], [ 195, 217, "ablates glycan binding", "protein_state" ], [ 228, 247, "B.\u00a0thetaiotaomicron", "species" ], [ 258, 280, "malto-oligosaccharides", "chemical" ], [ 285, 291, "starch", "chemical" ], [ 297, 300, "sus", "gene" ], [ 329, 336, "maltose", "chemical" ] ] }, { "sid": 151, "sent": "Similarly, the function of SGBP-A extends beyond glycan binding.", "section": "RESULTS", "ner": [ [ 27, 33, "SGBP-A", "protein" ], [ 49, 55, "glycan", "chemical" ] ] }, { "sid": 152, "sent": "Complementation of \u0394SGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig.\u00a06; \u0394SGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT.", "section": "RESULTS", "ner": [ [ 0, 15, "Complementation", "experimental_method" ], [ 19, 26, "\u0394SGBP-A", "mutant" ], [ 36, 43, "SGBP-A*", "mutant" ], [ 45, 49, "W82A", "mutant" ], [ 50, 55, "W283A", "mutant" ], [ 56, 61, "W306A", "mutant" ], [ 83, 91, "not bind", "protein_state" ], [ 92, 95, "XyG", "chemical" ], [ 116, 119, "XyG", "chemical" ], [ 124, 129, "XyGOs", "chemical" ], [ 139, 146, "\u0394SGBP-A", "mutant" ], [ 148, 155, "SGBP-A*", "mutant" ], [ 202, 204, "WT", "protein_state" ] ] }, { "sid": 153, "sent": "In previous studies, we observed that carbohydrate binding by SusD enhanced the sensitivity of the cells to limiting concentrations of malto-oligosaccharides by several orders of magnitude, such that the addition of 0.5\u00a0g/liter maltose was required to restore growth of the \u0394susD::SusD* strain on starch, which nonetheless occurred following an extended lag phase.", "section": "RESULTS", "ner": [ [ 38, 50, "carbohydrate", "chemical" ], [ 62, 66, "SusD", "protein" ], [ 228, 235, "maltose", "chemical" ], [ 274, 279, "\u0394susD", "mutant" ], [ 281, 286, "SusD*", "mutant" ], [ 297, 303, "starch", "chemical" ], [ 354, 363, "lag phase", "evidence" ] ] }, { "sid": 154, "sent": "In contrast, the \u0394SGBP-A::SGBP-A* strain does not display an extended lag time on any of the xyloglucan substrates compared to the WT (Fig.\u00a06).", "section": "RESULTS", "ner": [ [ 17, 24, "\u0394SGBP-A", "mutant" ], [ 26, 33, "SGBP-A*", "mutant" ], [ 70, 78, "lag time", "evidence" ], [ 93, 103, "xyloglucan", "chemical" ], [ 131, 133, "WT", "protein_state" ] ] }, { "sid": 155, "sent": "The specific glycan signal that upregulates BoXyGUL is currently unknown.", "section": "RESULTS", "ner": [ [ 13, 19, "glycan", "chemical" ], [ 44, 51, "BoXyGUL", "gene" ] ] }, { "sid": 156, "sent": "From our present data, we cannot eliminate the possibility that the glycan binding by SGBP-A enhances transcriptional activation of the XyGUL.", "section": "RESULTS", "ner": [ [ 68, 74, "glycan", "chemical" ], [ 86, 92, "SGBP-A", "protein" ], [ 136, 141, "XyGUL", "gene" ] ] }, { "sid": 157, "sent": "However, the modest rate defect displayed by the SGBP-A::SGBP-A* strain suggests that recognition of XyG and product import is somewhat less efficient in these cells.", "section": "RESULTS", "ner": [ [ 49, 55, "SGBP-A", "protein" ], [ 57, 64, "SGBP-A*", "mutant" ], [ 101, 104, "XyG", "chemical" ] ] }, { "sid": 158, "sent": "Intriguingly, the \u0394SGBP-B strain (\u0394Bacova_02650) (cf.", "section": "RESULTS", "ner": [ [ 18, 25, "\u0394SGBP-B", "mutant" ], [ 34, 47, "\u0394Bacova_02650", "mutant" ] ] }, { "sid": 159, "sent": "Fig.\u00a01B) exhibited a minor growth defect on both XyG and XyGO2, with rates 84.6% and 93.9% that of the WT \u0394tdk strain.", "section": "RESULTS", "ner": [ [ 49, 52, "XyG", "chemical" ], [ 57, 62, "XyGO2", "chemical" ], [ 103, 105, "WT", "protein_state" ], [ 106, 110, "\u0394tdk", "mutant" ] ] }, { "sid": 160, "sent": "However, growth of the \u0394SGBP-B strain on XyGO1 was 54.2% the rate of the parental strain, despite the fact that SGBP-B binds this substrate ca.", "section": "RESULTS", "ner": [ [ 23, 30, "\u0394SGBP-B", "mutant" ], [ 41, 46, "XyGO1", "chemical" ], [ 112, 118, "SGBP-B", "protein" ] ] }, { "sid": 161, "sent": "10-fold more weakly than XyGO2 and XyG (Fig.\u00a06; Table\u00a01).", "section": "RESULTS", "ner": [ [ 25, 30, "XyGO2", "chemical" ], [ 35, 38, "XyG", "chemical" ] ] }, { "sid": 162, "sent": "As such, the data suggest that SGBP-A can compensate for the loss of function of SGBP-B on longer oligo- and polysaccharides, while SGBP-B may adapt the cell to recognize smaller oligosaccharides efficiently.", "section": "RESULTS", "ner": [ [ 31, 37, "SGBP-A", "protein" ], [ 81, 87, "SGBP-B", "protein" ], [ 98, 124, "oligo- and polysaccharides", "chemical" ], [ 132, 138, "SGBP-B", "protein" ], [ 179, 195, "oligosaccharides", "chemical" ] ] }, { "sid": 163, "sent": "Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (\u0394SGBP-A::SGBP-A*/\u0394SGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1.", "section": "RESULTS", "ner": [ [ 10, 23, "double mutant", "protein_state" ], [ 41, 49, "crippled", "protein_state" ], [ 50, 56, "SGBP-A", "protein" ], [ 63, 74, "deletion of", "experimental_method" ], [ 75, 81, "SGBP-B", "protein" ], [ 83, 90, "\u0394SGBP-A", "mutant" ], [ 92, 99, "SGBP-A*", "mutant" ], [ 100, 107, "\u0394SGBP-B", "mutant" ], [ 131, 139, "lag time", "evidence" ], [ 148, 151, "XyG", "chemical" ], [ 156, 161, "XyGO2", "chemical" ], [ 174, 179, "XyGO1", "chemical" ] ] }, { "sid": 164, "sent": "Taken together, the data indicate that SGBP-A and SGBP-B functionally complement each other in the capture of XyG polysaccharide, while SGBP-B may allow B.\u00a0ovatus to scavenge smaller XyGOs liberated by other gut commensals.", "section": "RESULTS", "ner": [ [ 39, 45, "SGBP-A", "protein" ], [ 50, 56, "SGBP-B", "protein" ], [ 110, 113, "XyG", "chemical" ], [ 114, 128, "polysaccharide", "chemical" ], [ 136, 142, "SGBP-B", "protein" ], [ 153, 162, "B.\u00a0ovatus", "species" ], [ 183, 188, "XyGOs", "chemical" ] ] }, { "sid": 165, "sent": "This additional role of SGBP-B is especially notable in the context of studies on BtSusE and BtSusF (positioned similarly in the archetypal Sus locus) (Fig.\u00a01B), for which growth defects on starch or malto-oligosaccharides have never been observed.", "section": "RESULTS", "ner": [ [ 24, 30, "SGBP-B", "protein" ], [ 82, 88, "BtSusE", "protein" ], [ 93, 99, "BtSusF", "protein" ], [ 140, 149, "Sus locus", "gene" ], [ 190, 196, "starch", "chemical" ], [ 200, 222, "malto-oligosaccharides", "chemical" ] ] }, { "sid": 166, "sent": "Beyond SGBP-A and SGBP-B, we speculated that the catalytically feeble endo-xyloglucanase GH9, which is expendable for growth in the presence of GH5, might also play a role in glycan binding to the cell surface.", "section": "RESULTS", "ner": [ [ 7, 13, "SGBP-A", "protein" ], [ 18, 24, "SGBP-B", "protein" ], [ 49, 69, "catalytically feeble", "protein_state" ], [ 70, 88, "endo-xyloglucanase", "protein_type" ], [ 89, 92, "GH9", "protein" ], [ 144, 147, "GH5", "protein" ], [ 175, 181, "glycan", "chemical" ] ] }, { "sid": 167, "sent": "However, combined deletion of the genes encoding GH9 (encoded by Bacova_02649) and SGBP-B does not exacerbate the growth defect on XyGO1 (Fig.\u00a06; \u0394SGBP-B/\u0394GH9).", "section": "RESULTS", "ner": [ [ 9, 48, "combined deletion of the genes encoding", "experimental_method" ], [ 49, 52, "GH9", "protein" ], [ 65, 77, "Bacova_02649", "gene" ], [ 83, 89, "SGBP-B", "protein" ], [ 131, 136, "XyGO1", "chemical" ], [ 146, 153, "\u0394SGBP-B", "mutant" ], [ 154, 158, "\u0394GH9", "mutant" ] ] }, { "sid": 168, "sent": "The necessity of SGBP-B is elevated in the SGBP-A* strain, as the \u0394SGBP-A::SGBP-A*/ \u0394SGBP-B mutant displays an extended lag during growth on XyG and xylogluco-oligosaccharides, while growth rate differences are more subtle.", "section": "RESULTS", "ner": [ [ 17, 23, "SGBP-B", "protein" ], [ 43, 50, "SGBP-A*", "mutant" ], [ 66, 73, "\u0394SGBP-A", "mutant" ], [ 75, 82, "SGBP-A*", "mutant" ], [ 84, 91, "\u0394SGBP-B", "mutant" ], [ 92, 98, "mutant", "protein_state" ], [ 120, 123, "lag", "evidence" ], [ 141, 144, "XyG", "chemical" ], [ 149, 175, "xylogluco-oligosaccharides", "chemical" ] ] }, { "sid": 169, "sent": "The precise reason for this lag is unclear, but recapitulating our findings on the role of SusD in malto-oligosaccharide sensing in B.\u00a0thetaiotaomicron, this extended lag may be due to inefficient import and thus sensing of xyloglucan in the environment in the absence of glycan binding by essential SGBPs.", "section": "RESULTS", "ner": [ [ 28, 31, "lag", "evidence" ], [ 91, 95, "SusD", "protein" ], [ 99, 120, "malto-oligosaccharide", "chemical" ], [ 132, 151, "B.\u00a0thetaiotaomicron", "species" ], [ 167, 170, "lag", "evidence" ], [ 224, 234, "xyloglucan", "chemical" ], [ 272, 278, "glycan", "chemical" ], [ 300, 305, "SGBPs", "protein_type" ] ] }, { "sid": 170, "sent": "Our previous work demonstrates that B.\u00a0ovatus cells grown in minimal medium plus glucose express low levels of the XyGUL transcript.", "section": "RESULTS", "ner": [ [ 36, 45, "B.\u00a0ovatus", "species" ], [ 81, 88, "glucose", "chemical" ], [ 115, 120, "XyGUL", "gene" ] ] }, { "sid": 171, "sent": "Thus, in our experiments, we presume that each strain, initially grown in glucose, expresses low levels of the XyGUL transcript and thus low levels of the XyGUL-encoded surface proteins, including the vanguard GH5.", "section": "RESULTS", "ner": [ [ 74, 81, "glucose", "chemical" ], [ 111, 116, "XyGUL", "gene" ], [ 155, 160, "XyGUL", "gene" ], [ 210, 213, "GH5", "protein" ] ] }, { "sid": 172, "sent": "Presumably without glycan binding by the SGBPs, the GH5 protein cannot efficiently process xyloglucan, and/or the lack of SGBP function prevents efficient capture and import of the processed oligosaccharides.", "section": "RESULTS", "ner": [ [ 19, 25, "glycan", "chemical" ], [ 41, 46, "SGBPs", "protein_type" ], [ 52, 55, "GH5", "protein" ], [ 91, 101, "xyloglucan", "chemical" ], [ 122, 126, "SGBP", "protein_type" ], [ 191, 207, "oligosaccharides", "chemical" ] ] }, { "sid": 173, "sent": "It may then be that only after a sufficient amount of glycan is processed and imported by the cell is XyGUL upregulated and exponential growth on the glycan can begin.", "section": "RESULTS", "ner": [ [ 54, 60, "glycan", "chemical" ], [ 102, 107, "XyGUL", "gene" ], [ 150, 156, "glycan", "chemical" ] ] }, { "sid": 174, "sent": "We hypothesize that during exponential growth the essential role of SGBP-A extends beyond glycan recognition, perhaps due to a critical interaction with the TBDT.", "section": "RESULTS", "ner": [ [ 68, 74, "SGBP-A", "protein" ], [ 90, 96, "glycan", "chemical" ], [ 157, 161, "TBDT", "protein_type" ] ] }, { "sid": 175, "sent": "In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a \u0394susD mutant cannot grow on starch, but a \u0394susD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied.", "section": "RESULTS", "ner": [ [ 7, 12, "BtSus", "gene" ], [ 14, 18, "SusD", "protein" ], [ 27, 31, "TBDT", "protein_type" ], [ 32, 36, "SusC", "protein" ], [ 103, 109, "glycan", "chemical" ], [ 150, 155, "\u0394susD", "mutant" ], [ 156, 162, "mutant", "protein_state" ], [ 178, 184, "starch", "chemical" ], [ 192, 197, "\u0394susD", "mutant" ], [ 199, 204, "SusD*", "mutant" ], [ 238, 263, "transcriptional activator", "protein_type" ], [ 271, 281, "sus operon", "gene" ] ] }, { "sid": 176, "sent": "Likewise, such cognate interactions between homologous protein pairs such as SGBP-A and its TBDT may underlie our observation that a \u0394SGBP-A mutant cannot grow on xyloglucan.", "section": "RESULTS", "ner": [ [ 77, 83, "SGBP-A", "protein" ], [ 92, 96, "TBDT", "protein_type" ], [ 133, 140, "\u0394SGBP-A", "mutant" ], [ 141, 147, "mutant", "protein_state" ], [ 163, 173, "xyloglucan", "chemical" ] ] }, { "sid": 177, "sent": "However, unlike the Sus, in which elimination of SusE and SusF does not affect growth on starch, SGBP-B appears to have a dedicated role in growth on small xylogluco-oligosaccharides.", "section": "RESULTS", "ner": [ [ 20, 23, "Sus", "complex_assembly" ], [ 34, 48, "elimination of", "experimental_method" ], [ 49, 53, "SusE", "protein" ], [ 58, 62, "SusF", "protein" ], [ 89, 95, "starch", "chemical" ], [ 97, 103, "SGBP-B", "protein" ], [ 156, 182, "xylogluco-oligosaccharides", "chemical" ] ] }, { "sid": 178, "sent": "The ability of gut-adapted microorganisms to thrive in the gastrointestinal tract is critically dependent upon their ability to efficiently recognize, cleave, and import glycans.", "section": "RESULTS", "ner": [ [ 27, 41, "microorganisms", "taxonomy_domain" ], [ 170, 177, "glycans", "chemical" ] ] }, { "sid": 179, "sent": "The human gut, in particular, is a densely packed ecosystem with hundreds of species, in which there is potential for both competition and synergy in the utilization of different substrates.", "section": "RESULTS", "ner": [ [ 4, 9, "human", "species" ] ] }, { "sid": 180, "sent": "Recent work has elucidated that Bacteroidetes cross-feed during growth on many glycans; the glycoside hydrolases expressed by one species liberate oligosaccharides for consumption by other members of the community.", "section": "RESULTS", "ner": [ [ 32, 45, "Bacteroidetes", "taxonomy_domain" ], [ 79, 86, "glycans", "chemical" ], [ 92, 112, "glycoside hydrolases", "protein_type" ], [ 147, 163, "oligosaccharides", "chemical" ] ] }, { "sid": 181, "sent": "Thus, understanding glycan capture at the cell surface is fundamental to explaining, and eventually predicting, how the carbohydrate content of the diet shapes the gut community structure as well as its causative health effects.", "section": "RESULTS", "ner": [ [ 20, 26, "glycan", "chemical" ] ] }, { "sid": 182, "sent": "Here, we demonstrate that the surface glycan binding proteins encoded within the BoXyGUL play unique and essential roles in the acquisition of the ubiquitous and abundant vegetable polysaccharide xyloglucan.", "section": "RESULTS", "ner": [ [ 30, 61, "surface glycan binding proteins", "protein_type" ], [ 81, 88, "BoXyGUL", "gene" ], [ 171, 180, "vegetable", "taxonomy_domain" ], [ 181, 195, "polysaccharide", "chemical" ], [ 196, 206, "xyloglucan", "chemical" ] ] }, { "sid": 183, "sent": "Yet, a number of questions remain regarding the molecular interplay of SGBPs with their cotranscribed cohort of glycoside hydrolases and TonB-dependent transporters.", "section": "RESULTS", "ner": [ [ 71, 76, "SGBPs", "protein_type" ], [ 112, 132, "glycoside hydrolases", "protein_type" ], [ 137, 164, "TonB-dependent transporters", "protein_type" ] ] }, { "sid": 184, "sent": "A particularly understudied aspect of glycan utilization is the mechanism of import via TBDTs (SusC homologs) (Fig.\u00a01), which are ubiquitous and defining components of all PUL.", "section": "RESULTS", "ner": [ [ 38, 44, "glycan", "chemical" ], [ 88, 93, "TBDTs", "protein_type" ], [ 95, 99, "SusC", "protein" ], [ 172, 175, "PUL", "gene" ] ] }, { "sid": 185, "sent": "PUL-encoded TBDTs in Bacteroidetes are larger than the well-characterized iron-targeting TBDTs from many Proteobacteria and are further distinguished as the only known glycan-importing TBDTs coexpressed with an SGBP.", "section": "RESULTS", "ner": [ [ 0, 3, "PUL", "gene" ], [ 12, 17, "TBDTs", "protein_type" ], [ 21, 34, "Bacteroidetes", "taxonomy_domain" ], [ 74, 94, "iron-targeting TBDTs", "protein_type" ], [ 105, 119, "Proteobacteria", "taxonomy_domain" ], [ 168, 190, "glycan-importing TBDTs", "protein_type" ], [ 211, 215, "SGBP", "protein_type" ] ] }, { "sid": 186, "sent": "A direct interaction between the BtSusC TBDT and the SusD SGBP has been previously demonstrated, as has an interaction between the homologous components encoded by an N-glycan-scavenging PUL of Capnocytophaga canimorsus.", "section": "RESULTS", "ner": [ [ 33, 39, "BtSusC", "protein" ], [ 40, 44, "TBDT", "protein_type" ], [ 53, 57, "SusD", "protein" ], [ 58, 62, "SGBP", "protein_type" ], [ 169, 175, "glycan", "chemical" ], [ 187, 190, "PUL", "gene" ], [ 194, 219, "Capnocytophaga canimorsus", "species" ] ] }, { "sid": 187, "sent": "Our observation here that the physical presence of the SusD homolog SGBP-A, independent of XyG-binding ability, is both necessary and sufficient for XyG utilization further supports a model of glycan import whereby the SusC-like TBDTs and the SusD-like SGBPs must be intimately associated to support glycan uptake (Fig.\u00a01C).", "section": "RESULTS", "ner": [ [ 55, 59, "SusD", "protein" ], [ 68, 74, "SGBP-A", "protein" ], [ 91, 94, "XyG", "chemical" ], [ 149, 152, "XyG", "chemical" ], [ 193, 199, "glycan", "chemical" ], [ 219, 234, "SusC-like TBDTs", "protein_type" ], [ 243, 258, "SusD-like SGBPs", "protein_type" ], [ 300, 306, "glycan", "chemical" ] ] }, { "sid": 188, "sent": "It is yet presently unclear whether this interaction is static or dynamic and to what extent the association of cognate TBDT/SGBPs is dependent upon the structure of the carbohydrate to be imported.", "section": "RESULTS", "ner": [ [ 120, 124, "TBDT", "protein_type" ], [ 125, 130, "SGBPs", "protein_type" ], [ 170, 182, "carbohydrate", "chemical" ] ] }, { "sid": 189, "sent": "On the other hand, there is clear evidence for independent TBDTs in Bacteroidetes that do not require SGBP association for activity.", "section": "RESULTS", "ner": [ [ 59, 64, "TBDTs", "protein_type" ], [ 68, 81, "Bacteroidetes", "taxonomy_domain" ], [ 102, 106, "SGBP", "protein_type" ] ] }, { "sid": 190, "sent": "For example, it was recently demonstrated that expression of nanO, which encodes a SusC-like TBDT as part of a sialic-acid-targeting PUL from B.\u00a0fragilis, restored growth on this monosaccharide in a mutant strain of E.\u00a0coli.", "section": "RESULTS", "ner": [ [ 61, 65, "nanO", "gene" ], [ 83, 97, "SusC-like TBDT", "protein_type" ], [ 133, 136, "PUL", "gene" ], [ 142, 153, "B.\u00a0fragilis", "species" ], [ 179, 193, "monosaccharide", "chemical" ], [ 216, 223, "E.\u00a0coli", "species" ] ] }, { "sid": 191, "sent": "In this instance, coexpression of the susD-like gene nanU was not required, nor did the expression of the nanU gene enhance growth kinetics.", "section": "RESULTS", "ner": [ [ 38, 42, "susD", "gene" ], [ 53, 57, "nanU", "gene" ], [ 106, 110, "nanU", "gene" ] ] }, { "sid": 192, "sent": "Similarly, the deletion of BT1762 encoding a fructan-targeting SusD-like protein in B.\u00a0thetaiotaomicron did not result in a dramatic loss of growth on fructans.", "section": "RESULTS", "ner": [ [ 27, 33, "BT1762", "gene" ], [ 45, 80, "fructan-targeting SusD-like protein", "protein_type" ], [ 84, 103, "B.\u00a0thetaiotaomicron", "species" ], [ 151, 159, "fructans", "chemical" ] ] }, { "sid": 193, "sent": "Thus, the strict dependence on a SusD-like SGBP for glycan uptake in the Bacteroidetes may be variable and substrate dependent.", "section": "RESULTS", "ner": [ [ 33, 47, "SusD-like SGBP", "protein_type" ], [ 52, 58, "glycan", "chemical" ], [ 73, 86, "Bacteroidetes", "taxonomy_domain" ] ] }, { "sid": 194, "sent": "Furthermore, considering the broader distribution of TBDTs in PUL lacking SGBPs (sometimes known as carbohydrate utilization containing TBDT [CUT] loci; see reference and reviewed in reference) across bacterial phyla, it appears that the intimate biophysical association of these substrate-transport and -binding proteins is the result of specific evolution within the Bacteroidetes.", "section": "RESULTS", "ner": [ [ 53, 58, "TBDTs", "protein_type" ], [ 62, 65, "PUL", "gene" ], [ 74, 79, "SGBPs", "protein_type" ], [ 100, 151, "carbohydrate utilization containing TBDT [CUT] loci", "gene" ], [ 201, 210, "bacterial", "taxonomy_domain" ], [ 369, 382, "Bacteroidetes", "taxonomy_domain" ] ] }, { "sid": 195, "sent": "Equally intriguing is the observation that while SusD-like proteins such as SGBP-A share moderate primary and high tertiary structural conservation, the genes for the SGBPs encoded immediately downstream (Fig.\u00a01B [sometimes referred to as \u201csusE positioned\u201d]) encode glycan-binding lipoproteins with little or no sequence or structural conservation, even among syntenic PUL that target the same polysaccharide.", "section": "RESULTS", "ner": [ [ 49, 67, "SusD-like proteins", "protein_type" ], [ 76, 82, "SGBP-A", "protein" ], [ 167, 172, "SGBPs", "protein_type" ], [ 266, 293, "glycan-binding lipoproteins", "protein_type" ], [ 369, 372, "PUL", "gene" ], [ 394, 408, "polysaccharide", "chemical" ] ] }, { "sid": 196, "sent": "Such is the case for XyGUL from related Bacteroides species, which may encode either one or two of these predicted SGBPs, and these proteins vary considerably in length.", "section": "RESULTS", "ner": [ [ 21, 26, "XyGUL", "gene" ], [ 40, 51, "Bacteroides", "taxonomy_domain" ], [ 115, 120, "SGBPs", "protein_type" ] ] }, { "sid": 197, "sent": "The extremely low similarity of these SGBPs is striking in light of the moderate sequence conservation observed among homologous GHs in syntenic PUL.", "section": "RESULTS", "ner": [ [ 38, 43, "SGBPs", "protein_type" ], [ 129, 132, "GHs", "protein_type" ], [ 145, 148, "PUL", "gene" ] ] }, { "sid": 198, "sent": "This, together with the observation that these SGBPs, as exemplified by BtSusE and BtSusF and the XyGUL SGBP-B of the present study, are expendable for polysaccharide growth, implies a high degree of evolutionary flexibility to enhance glycan capture at the cell surface.", "section": "RESULTS", "ner": [ [ 47, 52, "SGBPs", "protein_type" ], [ 72, 78, "BtSusE", "protein" ], [ 83, 89, "BtSusF", "protein" ], [ 98, 103, "XyGUL", "gene" ], [ 104, 110, "SGBP-B", "protein" ], [ 152, 166, "polysaccharide", "chemical" ], [ 236, 242, "glycan", "chemical" ] ] }, { "sid": 199, "sent": "Because the intestinal ecosystem is a dense consortium of bacteria that must compete for their nutrients, these multimodular SGBPs may reflect ongoing evolutionary experiments to enhance glycan uptake efficiency.", "section": "RESULTS", "ner": [ [ 58, 66, "bacteria", "taxonomy_domain" ], [ 125, 130, "SGBPs", "protein_type" ], [ 187, 193, "glycan", "chemical" ] ] }, { "sid": 200, "sent": "Whether organisms that express longer SGBPs, extending further above the cell surface toward the extracellular environment, are better equipped to compete for available carbohydrates is presently unknown.", "section": "RESULTS", "ner": [ [ 38, 43, "SGBPs", "protein_type" ], [ 169, 182, "carbohydrates", "chemical" ] ] }, { "sid": 201, "sent": "However, the natural diversity of these proteins represents a rich source for the discovery of unique carbohydrate-binding motifs to both inform gut microbiology and generate new, specific carbohydrate analytical reagents.", "section": "RESULTS", "ner": [ [ 102, 129, "carbohydrate-binding motifs", "structure_element" ], [ 189, 201, "carbohydrate", "chemical" ] ] }, { "sid": 202, "sent": "In conclusion, the present study further illuminates the essential role that surface-glycan binding proteins play in facilitating the catabolism of complex dietary carbohydrates by Bacteroidetes.", "section": "RESULTS", "ner": [ [ 77, 108, "surface-glycan binding proteins", "protein_type" ], [ 164, 177, "carbohydrates", "chemical" ], [ 181, 194, "Bacteroidetes", "taxonomy_domain" ] ] }, { "sid": 203, "sent": "The ability of our resident gut bacteria to recognize polysaccharides is the first committed step of glycan consumption by these organisms, a critical process that influences the community structure and thus the metabolic output (i.e., short-chain fatty acid and metabolite profile) of these organisms.", "section": "RESULTS", "ner": [ [ 32, 40, "bacteria", "taxonomy_domain" ], [ 54, 69, "polysaccharides", "chemical" ], [ 101, 107, "glycan", "chemical" ] ] }, { "sid": 204, "sent": "A molecular understanding of glycan uptake by human gut bacteria is therefore central to the development of strategies to improve human health through manipulation of the microbiota.", "section": "RESULTS", "ner": [ [ 29, 35, "glycan", "chemical" ], [ 46, 51, "human", "species" ], [ 56, 64, "bacteria", "taxonomy_domain" ], [ 130, 135, "human", "species" ], [ 171, 181, "microbiota", "taxonomy_domain" ] ] } ] }, "PMC4869123": { "annotations": [ { "sid": 0, "sent": "Inhibiting complex IL-17A and IL-17RA interactions with a linear peptide", "section": "TITLE", "ner": [ [ 19, 25, "IL-17A", "protein" ], [ 30, 37, "IL-17RA", "protein" ], [ 65, 72, "peptide", "chemical" ] ] }, { "sid": 1, "sent": "IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases.", "section": "ABSTRACT", "ner": [ [ 0, 6, "IL-17A", "protein" ], [ 29, 37, "cytokine", "protein_type" ] ] }, { "sid": 2, "sent": "Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking.", "section": "ABSTRACT", "ner": [ [ 11, 21, "antibodies", "protein_type" ], [ 33, 39, "IL-17A", "protein" ] ] }, { "sid": 3, "sent": "A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions.", "section": "ABSTRACT", "ner": [ [ 2, 41, "high affinity IL-17A peptide antagonist", "chemical" ], [ 43, 46, "HAP", "chemical" ], [ 51, 62, "15 residues", "residue_range" ], [ 86, 109, "phage-display screening", "experimental_method" ], [ 122, 157, "saturation mutagenesis optimization", "experimental_method" ], [ 162, 186, "amino acid substitutions", "experimental_method" ] ] }, { "sid": 4, "sent": "HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA.", "section": "ABSTRACT", "ner": [ [ 0, 3, "HAP", "chemical" ], [ 26, 32, "IL-17A", "protein" ], [ 69, 77, "cytokine", "protein_type" ], [ 87, 95, "receptor", "protein_type" ], [ 97, 104, "IL-17RA", "protein" ] ] }, { "sid": 5, "sent": "Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines.", "section": "ABSTRACT", "ner": [ [ 18, 23, "human", "species" ], [ 31, 34, "HAP", "chemical" ], [ 83, 92, "cytokines", "protein_type" ] ] }, { "sid": 6, "sent": "Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically.", "section": "ABSTRACT", "ner": [ [ 0, 25, "Crystal structure studies", "experimental_method" ], [ 44, 47, "HAP", "chemical" ], [ 70, 76, "IL-17A", "protein" ], [ 77, 82, "dimer", "oligomeric_state" ] ] }, { "sid": 7, "sent": "The N-terminal portions of HAP form a \u03b2-strand that inserts between two IL-17A monomers while the C-terminal section forms an \u03b1 helix that directly blocks IL-17RA from binding to the same region of IL-17A.", "section": "ABSTRACT", "ner": [ [ 27, 30, "HAP", "chemical" ], [ 38, 46, "\u03b2-strand", "structure_element" ], [ 72, 78, "IL-17A", "protein" ], [ 79, 87, "monomers", "oligomeric_state" ], [ 126, 133, "\u03b1 helix", "structure_element" ], [ 155, 162, "IL-17RA", "protein" ], [ 198, 204, "IL-17A", "protein" ] ] }, { "sid": 8, "sent": "The family of IL-17 cytokines and receptors consists of six polypeptides, IL-17A-F, and five receptors, IL-17RA-E. IL-17A is secreted from activated Th17 cells, and several innate immune T cell types including macrophages, neutrophils, natural killer cells, and dendritic cells.", "section": "INTRO", "ner": [ [ 14, 29, "IL-17 cytokines", "protein_type" ], [ 74, 82, "IL-17A-F", "protein" ], [ 104, 113, "IL-17RA-E", "protein" ], [ 115, 121, "IL-17A", "protein" ] ] }, { "sid": 9, "sent": "IL-17A signals through a specific cell surface receptor complex which consists of IL-17RA and IL-17RC.", "section": "INTRO", "ner": [ [ 0, 6, "IL-17A", "protein" ], [ 47, 55, "receptor", "protein_type" ], [ 82, 89, "IL-17RA", "protein" ], [ 94, 101, "IL-17RC", "protein" ] ] }, { "sid": 10, "sent": "IL-17A\u2019s downstream signaling leads to increased production of inflammatory cytokines such as IL-6, IL-8, CCL-20 and CXCL1 by various mechanisms including stimulation of transcription and stabilization of mRNA.", "section": "INTRO", "ner": [ [ 0, 6, "IL-17A", "protein" ], [ 76, 85, "cytokines", "protein_type" ], [ 94, 98, "IL-6", "protein_type" ], [ 100, 104, "IL-8", "protein_type" ], [ 106, 112, "CCL-20", "protein_type" ], [ 117, 122, "CXCL1", "protein_type" ], [ 205, 209, "mRNA", "chemical" ] ] }, { "sid": 11, "sent": "Although various cell types have been reported to express IL-17RA, the highest responses to IL-17A come from epithelial cells, endothelial cells, keratinocytes and fibroblasts.", "section": "INTRO", "ner": [ [ 58, 65, "IL-17RA", "protein" ], [ 92, 98, "IL-17A", "protein" ] ] }, { "sid": 12, "sent": "IL-17A and its signaling is important in host defense against certain fungal and bacterial infections as demonstrated by patients with autoantibodies against IL-17A and IL-17F, or with inborn errors of IL-17 immunity.", "section": "INTRO", "ner": [ [ 0, 6, "IL-17A", "protein" ], [ 158, 164, "IL-17A", "protein" ], [ 169, 175, "IL-17F", "protein" ], [ 202, 207, "IL-17", "protein_type" ] ] }, { "sid": 13, "sent": "In addition to its physiological role, IL-17A is a key pathogenic factor in inflammatory and autoimmune diseases.", "section": "INTRO", "ner": [ [ 39, 45, "IL-17A", "protein" ] ] }, { "sid": 14, "sent": "In phase II and III clinical trials, neutralizing monoclonal antibodies against IL-17A (secukinumab and ixekizumab) or its receptor IL-17RA (brodalumab) are highly efficacious in treating moderate to severe plaque psoriasis and psoriatic arthritis.", "section": "INTRO", "ner": [ [ 61, 71, "antibodies", "protein_type" ], [ 80, 86, "IL-17A", "protein" ], [ 88, 99, "secukinumab", "chemical" ], [ 104, 114, "ixekizumab", "chemical" ], [ 123, 131, "receptor", "protein_type" ], [ 132, 139, "IL-17RA", "protein" ], [ 141, 151, "brodalumab", "chemical" ] ] }, { "sid": 15, "sent": "Secukinumab has been approved recently as a new psoriasis drug by the US Food and Drug Administration (Cosentyx\u2122).", "section": "INTRO", "ner": [ [ 0, 11, "Secukinumab", "chemical" ], [ 103, 112, "Cosentyx\u2122", "chemical" ] ] }, { "sid": 16, "sent": "In addition to psoriasis and psoriatic arthritis, IL-17A blockade has also shown preclinical and clinical efficacies in ankylosing spondylitis and rheumatoid arthritis.", "section": "INTRO", "ner": [ [ 50, 56, "IL-17A", "protein" ] ] }, { "sid": 17, "sent": "Among IL-17 cytokines, IL-17A and IL-17F share the highest homology.", "section": "INTRO", "ner": [ [ 6, 21, "IL-17 cytokines", "protein_type" ], [ 23, 29, "IL-17A", "protein" ], [ 34, 40, "IL-17F", "protein" ] ] }, { "sid": 18, "sent": "These polypeptides form covalent homodimers, and IL-17A and IL-17F also form an IL-17A/IL-17F hetereodimer.", "section": "INTRO", "ner": [ [ 24, 32, "covalent", "protein_state" ], [ 33, 43, "homodimers", "oligomeric_state" ], [ 49, 55, "IL-17A", "protein" ], [ 60, 66, "IL-17F", "protein" ], [ 80, 93, "IL-17A/IL-17F", "complex_assembly" ], [ 94, 106, "hetereodimer", "oligomeric_state" ] ] }, { "sid": 19, "sent": "Structures are known for apo IL-17F and its complex with IL-17RA, for apo IL-17A, its complex with an antibody Fab, and its complex with IL-17RA.", "section": "INTRO", "ner": [ [ 0, 10, "Structures", "evidence" ], [ 25, 28, "apo", "protein_state" ], [ 29, 35, "IL-17F", "protein" ], [ 44, 56, "complex with", "protein_state" ], [ 57, 64, "IL-17RA", "protein" ], [ 70, 73, "apo", "protein_state" ], [ 74, 80, "IL-17A", "protein" ], [ 86, 98, "complex with", "protein_state" ], [ 102, 110, "antibody", "protein_type" ], [ 111, 114, "Fab", "structure_element" ], [ 124, 136, "complex with", "protein_state" ], [ 137, 144, "IL-17RA", "protein" ] ] }, { "sid": 20, "sent": "In these structures, both IL-17A and IL-17F adopt a cysteine-knot fold with two intramolecular disulfides and two interchain disulfide bonds that covalently link two monomers.", "section": "INTRO", "ner": [ [ 9, 19, "structures", "evidence" ], [ 26, 32, "IL-17A", "protein" ], [ 37, 43, "IL-17F", "protein" ], [ 52, 65, "cysteine-knot", "structure_element" ], [ 95, 105, "disulfides", "ptm" ], [ 125, 140, "disulfide bonds", "ptm" ], [ 166, 174, "monomers", "oligomeric_state" ] ] }, { "sid": 21, "sent": "There has been active research in identifying orally available chemical entities that would functionally antagonize IL-17A-mediated signaling.", "section": "INTRO", "ner": [ [ 116, 122, "IL-17A", "protein" ] ] }, { "sid": 22, "sent": "Developing small molecules targeting protein-protein interactions is difficult with particular challenges associated with the large, shallow IL-17A/IL-17RA interfaces.", "section": "INTRO", "ner": [ [ 141, 166, "IL-17A/IL-17RA interfaces", "site" ] ] }, { "sid": 23, "sent": "Since IL-17RA is a shared receptor for at least IL-17A, IL-17F, IL-17A/IL-17F and IL-17E, we chose to seek IL-17A-specific inhibitors that may have more defined pharmacological responses than IL-17RA inhibitors.", "section": "INTRO", "ner": [ [ 6, 13, "IL-17RA", "protein" ], [ 26, 34, "receptor", "protein_type" ], [ 48, 54, "IL-17A", "protein" ], [ 56, 62, "IL-17F", "protein" ], [ 64, 77, "IL-17A/IL-17F", "complex_assembly" ], [ 82, 88, "IL-17E", "protein" ], [ 107, 113, "IL-17A", "protein" ], [ 192, 199, "IL-17RA", "protein" ] ] }, { "sid": 24, "sent": "Our efforts resulted in discovery of a high affinity IL-17A peptide antagonist (HAP), which we attempted to increase the functional production and pharmacokinetics after fusing HAP to antibodies for evaluation as a bispecific therapeutic in animal studies.", "section": "INTRO", "ner": [ [ 39, 78, "high affinity IL-17A peptide antagonist", "chemical" ], [ 80, 83, "HAP", "chemical" ], [ 170, 176, "fusing", "experimental_method" ], [ 177, 180, "HAP", "chemical" ], [ 184, 194, "antibodies", "protein_type" ] ] }, { "sid": 25, "sent": "Unfortunately, this past work revealed stability issues of the uncapped HAP in cell culture Here, we provide the details of the discovery and optimization that led to HAP and report the complex structure of IL-17A with HAP, which provides structure based rationalization of peptide optimization and structure activity relationship (SAR).", "section": "INTRO", "ner": [ [ 63, 71, "uncapped", "protein_state" ], [ 72, 75, "HAP", "chemical" ], [ 167, 170, "HAP", "chemical" ], [ 186, 203, "complex structure", "evidence" ], [ 207, 213, "IL-17A", "protein" ], [ 219, 222, "HAP", "chemical" ], [ 274, 294, "peptide optimization", "experimental_method" ], [ 299, 330, "structure activity relationship", "experimental_method" ], [ 332, 335, "SAR", "experimental_method" ] ] }, { "sid": 26, "sent": "Identification of IL-17A peptide inhibitors", "section": "RESULTS", "ner": [ [ 18, 24, "IL-17A", "protein" ] ] }, { "sid": 27, "sent": "Peptides specifically binding to human IL-17A were identified from phage panning using cyclic and linear peptide libraries (Supplementary Figure S1).", "section": "RESULTS", "ner": [ [ 33, 38, "human", "species" ], [ 39, 45, "IL-17A", "protein" ], [ 67, 80, "phage panning", "experimental_method" ], [ 87, 122, "cyclic and linear peptide libraries", "experimental_method" ] ] }, { "sid": 28, "sent": "Positive phage pools were then sub-cloned into a maltose-binding protein (MBP) fusion system.", "section": "RESULTS", "ner": [ [ 0, 20, "Positive phage pools", "experimental_method" ], [ 31, 41, "sub-cloned", "experimental_method" ], [ 49, 92, "maltose-binding protein (MBP) fusion system", "experimental_method" ] ] }, { "sid": 29, "sent": "Single clones were isolated and sub-cultured in growth medium, and culture supernatants were used in an enzyme-linked immunosorbent assay (ELISA) to identify specific IL-17A-binding clones.", "section": "RESULTS", "ner": [ [ 104, 137, "enzyme-linked immunosorbent assay", "experimental_method" ], [ 139, 144, "ELISA", "experimental_method" ], [ 167, 173, "IL-17A", "protein" ] ] }, { "sid": 30, "sent": "The positive binding supernatants were tested for the ability to block biotinylated IL-17A signaling through IL-17RA in an IL-17A/IL-17RA competition ELISA assay where unlabeled IL-17A was used as positive control to inhibit biotinylated IL-17A binding.", "section": "RESULTS", "ner": [ [ 71, 83, "biotinylated", "protein_state" ], [ 84, 90, "IL-17A", "protein" ], [ 109, 116, "IL-17RA", "protein" ], [ 123, 137, "IL-17A/IL-17RA", "complex_assembly" ], [ 138, 161, "competition ELISA assay", "experimental_method" ], [ 178, 184, "IL-17A", "protein" ], [ 225, 237, "biotinylated", "protein_state" ], [ 238, 244, "IL-17A", "protein" ] ] }, { "sid": 31, "sent": "Approximately 10% of the clones that specifically bound to IL-17A also prevented the cytokine from binding to IL-17RA.", "section": "RESULTS", "ner": [ [ 59, 65, "IL-17A", "protein" ], [ 85, 93, "cytokine", "protein_type" ], [ 110, 117, "IL-17RA", "protein" ] ] }, { "sid": 32, "sent": "Sequences identified from phage clones were chemically synthesized (Supplementary Table 1) and tested for inhibition of IL-17A binding to IL-17RA (Table 1).", "section": "RESULTS", "ner": [ [ 26, 38, "phage clones", "experimental_method" ], [ 44, 66, "chemically synthesized", "experimental_method" ], [ 120, 126, "IL-17A", "protein" ], [ 138, 145, "IL-17RA", "protein" ] ] }, { "sid": 33, "sent": "A 15-mer linear peptide 1 was shown to block IL-17A/IL-17RA binding with an IC50 of 80\u2009nM in the competition ELISA assay (Table 1).", "section": "RESULTS", "ner": [ [ 16, 25, "peptide 1", "chemical" ], [ 45, 59, "IL-17A/IL-17RA", "complex_assembly" ], [ 76, 80, "IC50", "evidence" ], [ 97, 120, "competition ELISA assay", "experimental_method" ] ] }, { "sid": 34, "sent": "This peptide was then tested in a cell-based functional assay wherein production of GRO-\u03b1 in BJ human fibroblast cells was measured as a function of IL-17A stimulation using 1\u2009ng/ml IL-17A.", "section": "RESULTS", "ner": [ [ 34, 61, "cell-based functional assay", "experimental_method" ], [ 84, 89, "GRO-\u03b1", "protein" ], [ 96, 101, "human", "species" ], [ 149, 155, "IL-17A", "protein" ], [ 182, 188, "IL-17A", "protein" ] ] }, { "sid": 35, "sent": "Peptide 1 was found to be active in this functional assay with an IC50 of 370\u2009nM.", "section": "RESULTS", "ner": [ [ 0, 9, "Peptide 1", "chemical" ], [ 41, 57, "functional assay", "experimental_method" ], [ 66, 70, "IC50", "evidence" ] ] }, { "sid": 36, "sent": "Optimization of IL-17A peptide inhibitors", "section": "RESULTS", "ner": [ [ 16, 22, "IL-17A", "protein" ] ] }, { "sid": 37, "sent": "A SAR campaign was undertaken to improve the potency of peptide 1.", "section": "RESULTS", "ner": [ [ 2, 5, "SAR", "experimental_method" ], [ 56, 65, "peptide 1", "chemical" ] ] }, { "sid": 38, "sent": "An alanine scan of peptide 2, an analogue of 1 with a lysine to arginine substitution at position 14, was initiated.", "section": "RESULTS", "ner": [ [ 3, 15, "alanine scan", "experimental_method" ], [ 19, 28, "peptide 2", "chemical" ], [ 45, 46, "1", "chemical" ], [ 54, 60, "lysine", "residue_name" ], [ 64, 72, "arginine", "residue_name" ], [ 73, 85, "substitution", "experimental_method" ], [ 98, 100, "14", "residue_number" ] ] }, { "sid": 39, "sent": "When alanine was already present (positions 7 and 15), substitution was made with lysine (Table 1, peptides 3\u201317).", "section": "RESULTS", "ner": [ [ 5, 12, "alanine", "residue_name" ], [ 44, 45, "7", "residue_number" ], [ 50, 52, "15", "residue_number" ], [ 55, 67, "substitution", "experimental_method" ], [ 82, 88, "lysine", "residue_name" ], [ 99, 112, "peptides 3\u201317", "chemical" ] ] }, { "sid": 40, "sent": "Positions 1, 2, 4, 5, 7, 14 and 15 were shown to be amenable to substitution without significant loss (less than 3-fold) of binding affinity as measured by the IL-17A/IL-17RA competition ELISA.", "section": "RESULTS", "ner": [ [ 10, 11, "1", "residue_number" ], [ 13, 14, "2", "residue_number" ], [ 16, 17, "4", "residue_number" ], [ 19, 20, "5", "residue_number" ], [ 22, 23, "7", "residue_number" ], [ 25, 27, "14", "residue_number" ], [ 32, 34, "15", "residue_number" ], [ 124, 140, "binding affinity", "evidence" ], [ 160, 174, "IL-17A/IL-17RA", "complex_assembly" ], [ 175, 192, "competition ELISA", "experimental_method" ] ] }, { "sid": 41, "sent": "In particular, at position 5 (13), substitution of methionine with alanine resulted in a seven fold improvement in potency (80\u2009nM versus 11\u2009nM respectively).", "section": "RESULTS", "ner": [ [ 27, 28, "5", "residue_number" ], [ 30, 32, "13", "chemical" ], [ 35, 47, "substitution", "experimental_method" ], [ 51, 61, "methionine", "residue_name" ], [ 67, 74, "alanine", "residue_name" ] ] }, { "sid": 42, "sent": "In order to rapidly evaluate the effects of substitution of natural amino acids at tolerant positions identified by the alanine scan, the lead sequence was subjected to site-specific saturation mutagenesis using MBP.", "section": "RESULTS", "ner": [ [ 44, 56, "substitution", "experimental_method" ], [ 120, 132, "alanine scan", "experimental_method" ], [ 169, 205, "site-specific saturation mutagenesis", "experimental_method" ], [ 212, 215, "MBP", "experimental_method" ] ] }, { "sid": 43, "sent": "Each of the seven positions identified by the alanine scan was individually modified while keeping the rest of the sequence constant.", "section": "RESULTS", "ner": [ [ 46, 58, "alanine scan", "experimental_method" ] ] }, { "sid": 44, "sent": "Modifications at positions 2 and 14 were shown to display improvement in binding affinity (data not shown).", "section": "RESULTS", "ner": [ [ 27, 28, "2", "residue_number" ], [ 33, 35, "14", "residue_number" ], [ 73, 89, "binding affinity", "evidence" ] ] }, { "sid": 45, "sent": "Peptides with beneficial point mutations at positions 2, 5, and 14 were synthesized and evaluated in the competition ELISA (Table 1).", "section": "RESULTS", "ner": [ [ 25, 40, "point mutations", "experimental_method" ], [ 54, 55, "2", "residue_number" ], [ 57, 58, "5", "residue_number" ], [ 64, 66, "14", "residue_number" ], [ 72, 83, "synthesized", "experimental_method" ], [ 105, 122, "competition ELISA", "experimental_method" ] ] }, { "sid": 46, "sent": "Two of the changes, V2H (18) or V2T (21) displayed improved binding in the competition ELISA.", "section": "RESULTS", "ner": [ [ 20, 23, "V2H", "mutant" ], [ 25, 27, "18", "chemical" ], [ 32, 35, "V2T", "mutant" ], [ 37, 39, "21", "chemical" ], [ 75, 92, "competition ELISA", "experimental_method" ] ] }, { "sid": 47, "sent": "Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine.", "section": "RESULTS", "ner": [ [ 10, 21, "replacement", "experimental_method" ], [ 25, 35, "methionine", "residue_name" ], [ 48, 49, "5", "residue_number" ], [ 55, 62, "alanine", "residue_name" ], [ 118, 128, "isoleucine", "residue_name" ], [ 130, 132, "24", "chemical" ], [ 135, 142, "leucine", "residue_name" ], [ 144, 146, "25", "chemical" ], [ 152, 158, "valine", "residue_name" ], [ 160, 162, "26", "chemical" ], [ 256, 262, "valine", "residue_name" ], [ 267, 277, "isoleucine", "residue_name" ], [ 278, 290, "replacements", "experimental_method" ], [ 310, 317, "alanine", "residue_name" ] ] }, { "sid": 48, "sent": "Introduction of a methionine (27) or a carboxamide (28 and 29) at position 14 was shown to improve the binding affinity of the lead peptide.", "section": "RESULTS", "ner": [ [ 0, 12, "Introduction", "experimental_method" ], [ 18, 28, "methionine", "residue_name" ], [ 30, 32, "27", "chemical" ], [ 39, 50, "carboxamide", "chemical" ], [ 52, 54, "28", "chemical" ], [ 59, 61, "29", "chemical" ], [ 75, 77, "14", "residue_number" ], [ 103, 119, "binding affinity", "evidence" ] ] }, { "sid": 49, "sent": "In general, there was good agreement between the respective binding affinities of the synthesized peptides and their MBP fusion counterparts, except for substitution of valine at position 2 to a tryptophan (22), which resulted in a fivefold loss of affinity, for the free peptide when compared with the MBP fusion.", "section": "RESULTS", "ner": [ [ 60, 78, "binding affinities", "evidence" ], [ 117, 127, "MBP fusion", "experimental_method" ], [ 153, 165, "substitution", "experimental_method" ], [ 169, 175, "valine", "residue_name" ], [ 188, 189, "2", "residue_number" ], [ 195, 205, "tryptophan", "residue_name" ], [ 207, 209, "22", "chemical" ], [ 249, 257, "affinity", "evidence" ], [ 303, 313, "MBP fusion", "experimental_method" ] ] }, { "sid": 50, "sent": "Combining the key amino-acid residues identified by SAR into a single peptide sequence resulted in peptide 30, named high affinity peptide (HAP), that was found to inhibit IL-17A signaling in a BJ human fibroblast cell assay with an IC50 of 17\u2009nM, a more than 20-fold improvement over the phage peptide 1 (Table 2 and Supplementary Figure S2).", "section": "RESULTS", "ner": [ [ 52, 55, "SAR", "experimental_method" ], [ 99, 109, "peptide 30", "chemical" ], [ 117, 138, "high affinity peptide", "chemical" ], [ 140, 143, "HAP", "chemical" ], [ 172, 178, "IL-17A", "protein" ], [ 197, 202, "human", "species" ], [ 233, 237, "IC50", "evidence" ], [ 289, 294, "phage", "experimental_method" ], [ 295, 304, "peptide 1", "chemical" ] ] }, { "sid": 51, "sent": "We also examined the effect of removing the acetyl group at the N-terminus of HAP (which is present in all the peptides made, see Supplementary Material).", "section": "RESULTS", "ner": [ [ 78, 81, "HAP", "chemical" ] ] }, { "sid": 52, "sent": "The un-capped peptide (31) had an IC50 of 420\u2009nM in the cell-based assay.", "section": "RESULTS", "ner": [ [ 4, 13, "un-capped", "protein_state" ], [ 14, 26, "peptide (31)", "chemical" ], [ 34, 38, "IC50", "evidence" ], [ 56, 72, "cell-based assay", "experimental_method" ] ] }, { "sid": 53, "sent": "The loss of cellular activity of 31 was most likely due to the degradation of the N-terminus of 31, since peptide 31 was shown to be able to bind to IL-17A with similar affinity as HAP itself.", "section": "RESULTS", "ner": [ [ 33, 35, "31", "chemical" ], [ 96, 98, "31", "chemical" ], [ 114, 116, "31", "chemical" ], [ 149, 155, "IL-17A", "protein" ], [ 181, 184, "HAP", "chemical" ] ] }, { "sid": 54, "sent": "Furthermore, our previous work had reported that in antibody fusions the uncapped peptide was degraded under cell assay conditions with removal of the first 1-3 residues to inactive products with the same N-terminal sequences as peptides 32\u201334.", "section": "RESULTS", "ner": [ [ 52, 68, "antibody fusions", "experimental_method" ], [ 73, 81, "uncapped", "protein_state" ], [ 82, 89, "peptide", "chemical" ], [ 136, 146, "removal of", "experimental_method" ], [ 151, 169, "first 1-3 residues", "residue_range" ], [ 229, 243, "peptides 32\u201334", "chemical" ] ] }, { "sid": 55, "sent": "In this work, 32\u201334 are capped by protective acetyl group and reflect the same inactivity as reported.", "section": "RESULTS", "ner": [ [ 14, 19, "32\u201334", "chemical" ], [ 24, 30, "capped", "protein_state" ] ] }, { "sid": 56, "sent": "C-terminal truncations showed a more gradual reduction in activity (35\u201337; Table 2).", "section": "RESULTS", "ner": [ [ 11, 22, "truncations", "experimental_method" ], [ 68, 73, "35\u201337", "chemical" ] ] }, { "sid": 57, "sent": "After deletion of three amino acids from the C-terminal end (37), the peptide is no longer active.", "section": "RESULTS", "ner": [ [ 6, 17, "deletion of", "experimental_method" ], [ 18, 35, "three amino acids", "residue_range" ], [ 61, 63, "37", "chemical" ] ] }, { "sid": 58, "sent": "Dimerization of HAP can further increase its potency", "section": "RESULTS", "ner": [ [ 16, 19, "HAP", "chemical" ] ] }, { "sid": 59, "sent": "We reasoned that since the IL-17A protein is almost exclusively present in a dimeric form, dimerizing the IL-17A binding peptides could result in an improvement in binding affinity and inhibitory activity.", "section": "RESULTS", "ner": [ [ 27, 33, "IL-17A", "protein" ], [ 77, 84, "dimeric", "oligomeric_state" ], [ 91, 101, "dimerizing", "oligomeric_state" ], [ 106, 112, "IL-17A", "protein" ], [ 164, 180, "binding affinity", "evidence" ] ] }, { "sid": 60, "sent": "Homodimers of HAP were made through attachment of polyethylene glycol (PEG) spacers of different lengths at amino acids 4, 7 and 14, as these positions were identified in the alanine scan analysis as not contributing significantly to the activity, and at each N-terminus (Supplementary Table S2).", "section": "RESULTS", "ner": [ [ 0, 10, "Homodimers", "oligomeric_state" ], [ 14, 17, "HAP", "chemical" ], [ 50, 69, "polyethylene glycol", "chemical" ], [ 71, 74, "PEG", "chemical" ], [ 120, 121, "4", "residue_number" ], [ 123, 124, "7", "residue_number" ], [ 129, 131, "14", "residue_number" ], [ 175, 196, "alanine scan analysis", "experimental_method" ] ] }, { "sid": 61, "sent": "Due to the high reactivity of the pentafluoroester (PFP) group used as the activating group in the PEG, the histidine at position 2 and the lysine at position 15 were replaced with threonine and dimethyllysine respectively to prevent formation of side products, which resulted in peptide 38 that was comparable in activity with HAP.", "section": "RESULTS", "ner": [ [ 34, 50, "pentafluoroester", "chemical" ], [ 52, 55, "PFP", "chemical" ], [ 99, 102, "PEG", "chemical" ], [ 108, 117, "histidine", "residue_name" ], [ 130, 131, "2", "residue_number" ], [ 140, 146, "lysine", "residue_name" ], [ 159, 161, "15", "residue_number" ], [ 181, 190, "threonine", "residue_name" ], [ 195, 209, "dimethyllysine", "residue_name" ], [ 280, 290, "peptide 38", "chemical" ], [ 328, 331, "HAP", "chemical" ] ] }, { "sid": 62, "sent": "This exercise revealed that several dimeric peptides with the longer PEG21 spacer were significantly more potent than the monomer peptide in the cell-based assay (Supplementary Table S2).", "section": "RESULTS", "ner": [ [ 36, 43, "dimeric", "oligomeric_state" ], [ 44, 52, "peptides", "chemical" ], [ 69, 74, "PEG21", "chemical" ], [ 122, 129, "monomer", "oligomeric_state" ], [ 145, 161, "cell-based assay", "experimental_method" ] ] }, { "sid": 63, "sent": "Peptide 45, dimerized via attachment of a PEG21 spacer at position 14 (Supplementary Scheme S1 and Figure S3), was the most potent with cellular IC50 of 0.1\u2009nM. This significant improvement in antagonism was not seen in the peptide monomer functionalized with a PEG21 group at position 14 as peptide 48 had an IC50 of 21\u2009nM (Supplementary Scheme S2).", "section": "RESULTS", "ner": [ [ 0, 10, "Peptide 45", "chemical" ], [ 12, 21, "dimerized", "oligomeric_state" ], [ 42, 47, "PEG21", "chemical" ], [ 67, 69, "14", "residue_number" ], [ 145, 149, "IC50", "evidence" ], [ 232, 239, "monomer", "oligomeric_state" ], [ 262, 267, "PEG21", "chemical" ], [ 286, 288, "14", "residue_number" ], [ 292, 302, "peptide 48", "chemical" ], [ 310, 314, "IC50", "evidence" ] ] }, { "sid": 64, "sent": "The species cross-reactivity of the dimeric peptide 45 and HAP were assessed in a murine functional cell assay using 15\u2009ng/ml murine IL-17A.", "section": "RESULTS", "ner": [ [ 36, 43, "dimeric", "oligomeric_state" ], [ 44, 54, "peptide 45", "chemical" ], [ 59, 62, "HAP", "chemical" ], [ 82, 110, "murine functional cell assay", "experimental_method" ], [ 126, 132, "murine", "taxonomy_domain" ], [ 133, 139, "IL-17A", "protein" ] ] }, { "sid": 65, "sent": "Peptide 45 blocked the receptor binding of murine IL-17A although with potency two orders of magnitude weaker than that observed against human IL-17A (IC50\u2009=\u200941\u2009nM vs IC50\u2009=\u20090.1\u2009nM, respectively).", "section": "RESULTS", "ner": [ [ 0, 10, "Peptide 45", "chemical" ], [ 23, 31, "receptor", "protein_type" ], [ 43, 49, "murine", "taxonomy_domain" ], [ 50, 56, "IL-17A", "protein" ], [ 137, 142, "human", "species" ], [ 143, 149, "IL-17A", "protein" ], [ 151, 155, "IC50", "evidence" ], [ 167, 171, "IC50", "evidence" ] ] }, { "sid": 66, "sent": "The monomer HAP was much weaker (IC50 >1\u2009\u03bcM) in inhibiting murine IL-17A signaling (Supplementary Figure S4).", "section": "RESULTS", "ner": [ [ 4, 11, "monomer", "oligomeric_state" ], [ 12, 15, "HAP", "chemical" ], [ 33, 37, "IC50", "evidence" ], [ 59, 65, "murine", "taxonomy_domain" ], [ 66, 72, "IL-17A", "protein" ] ] }, { "sid": 67, "sent": "Although the dimeric peptide 45 is much more potent than HAP in the cell-based assay, in subsequent studies we decided to focus our efforts solely on characterizations of the monomeric peptide HAP in hopes to identify smaller peptide inhibitors containing the best minimal functional group.", "section": "RESULTS", "ner": [ [ 13, 20, "dimeric", "oligomeric_state" ], [ 21, 31, "peptide 45", "chemical" ], [ 57, 60, "HAP", "chemical" ], [ 68, 84, "cell-based assay", "experimental_method" ], [ 175, 184, "monomeric", "oligomeric_state" ], [ 193, 196, "HAP", "chemical" ] ] }, { "sid": 68, "sent": "Orthogonal assays to confirm HAP antagonism", "section": "RESULTS", "ner": [ [ 29, 32, "HAP", "chemical" ] ] }, { "sid": 69, "sent": "To further characterize the interaction of HAP with IL-17A, we set out to determine its in vitro binding affinity, specificity and kinetic profile using Surface Plasmon Resonance (SPR) methods (Fig. 1A).", "section": "RESULTS", "ner": [ [ 43, 46, "HAP", "chemical" ], [ 52, 58, "IL-17A", "protein" ], [ 97, 113, "binding affinity", "evidence" ], [ 131, 146, "kinetic profile", "evidence" ], [ 153, 178, "Surface Plasmon Resonance", "experimental_method" ], [ 180, 183, "SPR", "experimental_method" ] ] }, { "sid": 70, "sent": "HAP binds to immobilized human IL-17A homodimer tightly (Table 3).", "section": "RESULTS", "ner": [ [ 0, 3, "HAP", "chemical" ], [ 25, 30, "human", "species" ], [ 31, 37, "IL-17A", "protein" ], [ 38, 47, "homodimer", "oligomeric_state" ] ] }, { "sid": 71, "sent": "It has slightly weaker affinity for human IL-17A/F heterodimer and >10 fold weaker affinity for mouse IL-17A (Table 3).", "section": "RESULTS", "ner": [ [ 23, 31, "affinity", "evidence" ], [ 36, 41, "human", "species" ], [ 42, 50, "IL-17A/F", "complex_assembly" ], [ 51, 62, "heterodimer", "oligomeric_state" ], [ 83, 91, "affinity", "evidence" ], [ 96, 101, "mouse", "taxonomy_domain" ], [ 102, 108, "IL-17A", "protein" ] ] }, { "sid": 72, "sent": "HAP does not show significant binding to immobilized human IL-17F homodimer or IL-17RA at concentrations up to 100\u2009nM.", "section": "RESULTS", "ner": [ [ 0, 3, "HAP", "chemical" ], [ 53, 58, "human", "species" ], [ 59, 65, "IL-17F", "protein" ], [ 66, 75, "homodimer", "oligomeric_state" ], [ 79, 86, "IL-17RA", "protein" ] ] }, { "sid": 73, "sent": "Additionally, we investigated the antagonism of the human IL-17A/IL-17RA interaction by HAP using orthogonal methods including SPR and F\u00f6rster resonance energy transfer (FRET) competition assays (Fig. 1B,C).", "section": "RESULTS", "ner": [ [ 52, 57, "human", "species" ], [ 58, 72, "IL-17A/IL-17RA", "complex_assembly" ], [ 88, 91, "HAP", "chemical" ], [ 127, 130, "SPR", "experimental_method" ], [ 135, 194, "F\u00f6rster resonance energy transfer (FRET) competition assays", "experimental_method" ] ] }, { "sid": 74, "sent": "In both assays, incubation of IL-17A with HAP effectively blocks the binding of IL-17A to immobilized IL-17RA with similar sub-nM IC50 (Table 3).", "section": "RESULTS", "ner": [ [ 30, 36, "IL-17A", "protein" ], [ 42, 45, "HAP", "chemical" ], [ 80, 86, "IL-17A", "protein" ], [ 90, 101, "immobilized", "protein_state" ], [ 102, 109, "IL-17RA", "protein" ], [ 130, 134, "IC50", "evidence" ] ] }, { "sid": 75, "sent": "HAP blocks IL-17A signaling in a human primary cell assay", "section": "RESULTS", "ner": [ [ 0, 3, "HAP", "chemical" ], [ 11, 17, "IL-17A", "protein" ], [ 33, 38, "human", "species" ] ] }, { "sid": 76, "sent": "While either IL-17A or TNF-\u03b1 alone can stimulate the release of multiple inflammatory cytokines, when acting together they can synergistically enhance each other\u2019s effects (Supplementary Figure S5).", "section": "RESULTS", "ner": [ [ 13, 19, "IL-17A", "protein" ], [ 23, 28, "TNF-\u03b1", "protein" ], [ 86, 95, "cytokines", "protein_type" ] ] }, { "sid": 77, "sent": "These integrative responses to IL-17A and TNF-\u03b1 in human keratinocytes have been reported to account for key inflammatory pathogenic circuits in psoriasis.", "section": "RESULTS", "ner": [ [ 31, 37, "IL-17A", "protein" ], [ 42, 47, "TNF-\u03b1", "protein" ], [ 51, 56, "human", "species" ] ] }, { "sid": 78, "sent": "Thus, we chose to study HAP\u2019s efficacy in blocking the production of IL-8, IL-6 and CCL-20 by primary human keratinocytes stimulated by IL-17A in the presence of TNF-\u03b1, an assay which may be more disease-relevant.", "section": "RESULTS", "ner": [ [ 24, 27, "HAP", "chemical" ], [ 69, 73, "IL-8", "protein_type" ], [ 75, 79, "IL-6", "protein_type" ], [ 84, 90, "CCL-20", "protein_type" ], [ 102, 107, "human", "species" ], [ 136, 142, "IL-17A", "protein" ], [ 162, 167, "TNF-\u03b1", "protein" ] ] }, { "sid": 79, "sent": "HAP inhibits the production of all three cytokines in a dose-dependent fashion (Fig. 1D).", "section": "RESULTS", "ner": [ [ 0, 3, "HAP", "chemical" ], [ 41, 50, "cytokines", "protein_type" ] ] }, { "sid": 80, "sent": "Significantly, the baseline levels of IL-8, IL-6 and CCL-20 stimulated by TNF-\u03b1 alone are not inhibited by HAP, further indicating the selectivity of HAP (Fig. 1D).", "section": "RESULTS", "ner": [ [ 38, 42, "IL-8", "protein_type" ], [ 44, 48, "IL-6", "protein_type" ], [ 53, 59, "CCL-20", "protein_type" ], [ 74, 79, "TNF-\u03b1", "protein" ], [ 107, 110, "HAP", "chemical" ], [ 150, 153, "HAP", "chemical" ] ] }, { "sid": 81, "sent": "Such pharmacological selectivity may be important to suppress inflammatory pathogenic circuits in psoriasis, while sparing the anti-infectious immune responses produced by TNF-\u03b1.", "section": "RESULTS", "ner": [ [ 172, 177, "TNF-\u03b1", "protein" ] ] }, { "sid": 82, "sent": "The relatively high IC50 values in this assay (Table 3) are probably due to the high IL-17A concentration (100\u2009ng/ml) needed for detection of IL-6.", "section": "RESULTS", "ner": [ [ 20, 24, "IC50", "evidence" ], [ 85, 91, "IL-17A", "protein" ], [ 142, 146, "IL-6", "protein_type" ] ] }, { "sid": 83, "sent": "As a reference, a commercial anti-IL-17A antibody (R&D Systems) inhibits the production of IL-8 with an IC50 of 13(\u00b16) nM (N\u2009=\u20093).", "section": "RESULTS", "ner": [ [ 34, 40, "IL-17A", "protein" ], [ 41, 49, "antibody", "protein_type" ], [ 91, 95, "IL-8", "protein_type" ], [ 104, 108, "IC50", "evidence" ] ] }, { "sid": 84, "sent": "Indeed, the IC50 was 14(\u00b19) nM (N\u2009=\u200912) for HAP inhibition of IL-8 production when only 5\u2009ng/ml IL-17A was used in this assay.", "section": "RESULTS", "ner": [ [ 12, 16, "IC50", "evidence" ], [ 44, 47, "HAP", "chemical" ], [ 62, 66, "IL-8", "protein_type" ], [ 96, 102, "IL-17A", "protein" ] ] }, { "sid": 85, "sent": "In patients, the concentration of IL-17A in psoriatic lesions is reported to be 0.01\u2009ng/ml, well below the EC50 (5\u201310ng/ml) of IL-17A induced IL-8 production in vitro.", "section": "RESULTS", "ner": [ [ 34, 40, "IL-17A", "protein" ], [ 127, 133, "IL-17A", "protein" ], [ 142, 146, "IL-8", "protein_type" ] ] }, { "sid": 86, "sent": "Similar to keratinocytes assay results, while HAP inhibits IL-17A stimulated IL-6 production by BJ human fibroblast potently (IC50 of 17\u2009nM), it does not inhibit TNF-\u03b1 stimulated IL-6 production at concentrations up to 10\u2009\u03bcM (Supplementary Figure S2).", "section": "RESULTS", "ner": [ [ 11, 30, "keratinocytes assay", "experimental_method" ], [ 46, 49, "HAP", "chemical" ], [ 59, 65, "IL-17A", "protein" ], [ 77, 81, "IL-6", "protein_type" ], [ 99, 104, "human", "species" ], [ 126, 130, "IC50", "evidence" ], [ 162, 167, "TNF-\u03b1", "protein" ], [ 179, 183, "IL-6", "protein_type" ] ] }, { "sid": 87, "sent": "Crystallization and structure determination", "section": "RESULTS", "ner": [ [ 0, 43, "Crystallization and structure determination", "experimental_method" ] ] }, { "sid": 88, "sent": "Extensive crystallization trials, either by co-crystallization or by soaking HAP into preformed apo IL-17A crystals, failed to lead to an IL-17A/HAP complex crystals.", "section": "RESULTS", "ner": [ [ 10, 32, "crystallization trials", "experimental_method" ], [ 44, 62, "co-crystallization", "experimental_method" ], [ 69, 76, "soaking", "experimental_method" ], [ 77, 80, "HAP", "chemical" ], [ 96, 99, "apo", "protein_state" ], [ 100, 106, "IL-17A", "protein" ], [ 107, 115, "crystals", "evidence" ], [ 138, 148, "IL-17A/HAP", "complex_assembly" ], [ 157, 165, "crystals", "evidence" ] ] }, { "sid": 89, "sent": "We theorized that HAP binding induced large conformational changes in IL-17A that led to the difficulty of getting an IL-17A/HAP binary complex crystal.", "section": "RESULTS", "ner": [ [ 18, 21, "HAP", "chemical" ], [ 70, 76, "IL-17A", "protein" ], [ 118, 128, "IL-17A/HAP", "complex_assembly" ], [ 144, 151, "crystal", "evidence" ] ] }, { "sid": 90, "sent": "It is known that an antibody antigen-binding fragment (Fab) can be used as crystallization chaperones in crystallizing difficult targets.", "section": "RESULTS", "ner": [ [ 20, 28, "antibody", "protein_type" ], [ 29, 53, "antigen-binding fragment", "structure_element" ], [ 55, 58, "Fab", "structure_element" ] ] }, { "sid": 91, "sent": "We hypothesized that HAP may target the N-terminal of IL-17A which is known to be more flexible than its C-terminal and conformational changes needed for HAP binding may be more likely there.", "section": "RESULTS", "ner": [ [ 21, 24, "HAP", "chemical" ], [ 54, 60, "IL-17A", "protein" ], [ 154, 157, "HAP", "chemical" ] ] }, { "sid": 92, "sent": "We designed an antibody Fab known to target the C-terminal half of IL-17A based on a published IL-17A/Fab complex crystal structure, and produced it in HEK293 cells.", "section": "RESULTS", "ner": [ [ 15, 23, "antibody", "protein_type" ], [ 24, 27, "Fab", "structure_element" ], [ 48, 63, "C-terminal half", "structure_element" ], [ 67, 73, "IL-17A", "protein" ], [ 95, 105, "IL-17A/Fab", "complex_assembly" ], [ 114, 131, "crystal structure", "evidence" ] ] }, { "sid": 93, "sent": "In an SPR assay HAP and this Fab were able to co-bind IL-17A without large changes in their binding affinities and kinetics, confirming our hypothesis (Supplementary Figure S6).", "section": "RESULTS", "ner": [ [ 6, 15, "SPR assay", "experimental_method" ], [ 16, 19, "HAP", "chemical" ], [ 29, 32, "Fab", "structure_element" ], [ 54, 60, "IL-17A", "protein" ], [ 92, 110, "binding affinities", "evidence" ], [ 115, 123, "kinetics", "evidence" ] ] }, { "sid": 94, "sent": "Furthermore, since it binds to an area far away from that of HAP (see below), this Fab should have minimum effects on HAP binding conformation.", "section": "RESULTS", "ner": [ [ 61, 64, "HAP", "chemical" ], [ 83, 86, "Fab", "structure_element" ], [ 118, 121, "HAP", "chemical" ] ] }, { "sid": 95, "sent": "Crystals of Fab/IL-17A/HAP ternary complex were obtained readily in crystallization screens.", "section": "RESULTS", "ner": [ [ 0, 8, "Crystals", "evidence" ], [ 12, 26, "Fab/IL-17A/HAP", "complex_assembly" ], [ 68, 91, "crystallization screens", "experimental_method" ] ] }, { "sid": 96, "sent": "Crystallization of IL-17A and its binding partners was accomplished using two forms of IL-17A.", "section": "RESULTS", "ner": [ [ 0, 15, "Crystallization", "experimental_method" ], [ 19, 25, "IL-17A", "protein" ], [ 87, 93, "IL-17A", "protein" ] ] }, { "sid": 97, "sent": "These were, respectively, a presumably more homogeneous form of IL-17A that lacked the disordered N-terminal peptide and a full-length form of the cytokine with a full complement of disulfide bonds.", "section": "RESULTS", "ner": [ [ 64, 70, "IL-17A", "protein" ], [ 76, 82, "lacked", "protein_state" ], [ 87, 97, "disordered", "protein_state" ], [ 98, 116, "N-terminal peptide", "structure_element" ], [ 123, 134, "full-length", "protein_state" ], [ 147, 155, "cytokine", "protein_type" ], [ 182, 197, "disulfide bonds", "ptm" ] ] }, { "sid": 98, "sent": "Crystals of the Fab/truncated IL-17A/HAP complex diffracted to 2.2\u2009\u00c5, and the Fab/full length IL-17A/HAP complex diffracted to 3.0\u2009\u00c5 (Supplementary Table S3).", "section": "RESULTS", "ner": [ [ 0, 8, "Crystals", "evidence" ], [ 16, 40, "Fab/truncated IL-17A/HAP", "complex_assembly" ], [ 78, 104, "Fab/full length IL-17A/HAP", "complex_assembly" ] ] }, { "sid": 99, "sent": "Both structures were solved by molecular replacement.", "section": "RESULTS", "ner": [ [ 5, 15, "structures", "evidence" ], [ 31, 52, "molecular replacement", "experimental_method" ] ] }, { "sid": 100, "sent": "Both complexes crystallized in the space group of P321, with half the complex (1 Fab/1 IL-17A monomer/1 HAP) in the asymmetric unit.", "section": "RESULTS", "ner": [ [ 15, 27, "crystallized", "experimental_method" ], [ 81, 84, "Fab", "structure_element" ], [ 87, 93, "IL-17A", "protein" ], [ 94, 101, "monomer", "oligomeric_state" ], [ 104, 107, "HAP", "chemical" ] ] }, { "sid": 101, "sent": "The intact complex can be generated by applying crystallographic 2-fold symmetry.", "section": "RESULTS", "ner": [ [ 4, 10, "intact", "protein_state" ] ] }, { "sid": 102, "sent": "Electron densities for HAP residues Ile1-Asn14 were readily interpretable with the exception of Lys15, which is disordered.", "section": "RESULTS", "ner": [ [ 0, 18, "Electron densities", "evidence" ], [ 23, 26, "HAP", "chemical" ], [ 36, 46, "Ile1-Asn14", "residue_range" ], [ 96, 101, "Lys15", "residue_name_number" ], [ 112, 122, "disordered", "protein_state" ] ] }, { "sid": 103, "sent": "When considering the protein, the complex structure containing the full length IL-17A is identical to that of the truncated IL-17A, with the exception of Cys106 (Ser106 in the truncated IL-17A), which is disordered.", "section": "RESULTS", "ner": [ [ 34, 51, "complex structure", "evidence" ], [ 67, 78, "full length", "protein_state" ], [ 79, 85, "IL-17A", "protein" ], [ 114, 123, "truncated", "protein_state" ], [ 124, 130, "IL-17A", "protein" ], [ 154, 160, "Cys106", "residue_name_number" ], [ 162, 168, "Ser106", "residue_name_number" ], [ 176, 185, "truncated", "protein_state" ], [ 186, 192, "IL-17A", "protein" ], [ 204, 214, "disordered", "protein_state" ] ] }, { "sid": 104, "sent": "Cys106 is covalently linked to Cys10 that resides in the disordered N-terminal peptide in the full length IL-17A.", "section": "RESULTS", "ner": [ [ 0, 6, "Cys106", "residue_name_number" ], [ 31, 36, "Cys10", "residue_name_number" ], [ 57, 67, "disordered", "protein_state" ], [ 68, 86, "N-terminal peptide", "structure_element" ], [ 94, 105, "full length", "protein_state" ], [ 106, 112, "IL-17A", "protein" ] ] }, { "sid": 105, "sent": "Overall structure of Fab/IL-17A/HAP complex", "section": "RESULTS", "ner": [ [ 8, 17, "structure", "evidence" ], [ 21, 35, "Fab/IL-17A/HAP", "complex_assembly" ] ] }, { "sid": 106, "sent": "In a similar manner to the published structure of Fab/IL-17A complex, two Fab molecules bind symmetrically to the C-terminal of the cytokine dimer, interacting with epitopes from both monomers (Fig. 2A).", "section": "RESULTS", "ner": [ [ 37, 46, "structure", "evidence" ], [ 50, 60, "Fab/IL-17A", "complex_assembly" ], [ 74, 77, "Fab", "structure_element" ], [ 132, 140, "cytokine", "protein_type" ], [ 141, 146, "dimer", "oligomeric_state" ], [ 184, 192, "monomers", "oligomeric_state" ] ] }, { "sid": 107, "sent": "Two copies of HAP bind to the N-terminal of the cytokine dimer, also symmetrically, and each HAP molecule also interacts with both IL-17A monomers (Fig. 2).", "section": "RESULTS", "ner": [ [ 14, 17, "HAP", "chemical" ], [ 48, 56, "cytokine", "protein_type" ], [ 57, 62, "dimer", "oligomeric_state" ], [ 93, 96, "HAP", "chemical" ], [ 131, 137, "IL-17A", "protein" ], [ 138, 146, "monomers", "oligomeric_state" ] ] }, { "sid": 108, "sent": "Based on disclosed epitopes of Secukinumab and Ixekizumab, HAP binds to IL-17A at an area that is also different from those of those two antibodies.", "section": "RESULTS", "ner": [ [ 31, 42, "Secukinumab", "chemical" ], [ 47, 57, "Ixekizumab", "chemical" ], [ 59, 62, "HAP", "chemical" ], [ 72, 78, "IL-17A", "protein" ], [ 137, 147, "antibodies", "protein_type" ] ] }, { "sid": 109, "sent": "The N-terminal 5 residues of HAP, 1IHVTI, form an amphipathic \u03b2-strand that inserts between \u03b2-strand 4 of one IL-17A monomer and \u03b2-strand 0 (the first ordered peptide of IL-17A) of the second monomer.", "section": "RESULTS", "ner": [ [ 15, 25, "5 residues", "residue_range" ], [ 29, 32, "HAP", "chemical" ], [ 34, 40, "1IHVTI", "chemical" ], [ 50, 61, "amphipathic", "protein_state" ], [ 62, 70, "\u03b2-strand", "structure_element" ], [ 92, 102, "\u03b2-strand 4", "structure_element" ], [ 110, 116, "IL-17A", "protein" ], [ 117, 124, "monomer", "oligomeric_state" ], [ 129, 139, "\u03b2-strand 0", "structure_element" ], [ 170, 176, "IL-17A", "protein" ], [ 192, 199, "monomer", "oligomeric_state" ] ] }, { "sid": 110, "sent": "This \u03b2-strand is parallel to both strands 0 and 4 (Fig. 3B).", "section": "RESULTS", "ner": [ [ 5, 13, "\u03b2-strand", "structure_element" ], [ 34, 49, "strands 0 and 4", "structure_element" ] ] }, { "sid": 111, "sent": "Strands 0 of two IL-17A monomer are antiparallel, as appeared in other IL-17A structures.", "section": "RESULTS", "ner": [ [ 0, 9, "Strands 0", "structure_element" ], [ 17, 23, "IL-17A", "protein" ], [ 24, 31, "monomer", "oligomeric_state" ], [ 71, 77, "IL-17A", "protein" ], [ 78, 88, "structures", "evidence" ] ] }, { "sid": 112, "sent": "The C-terminal 8 residues of the HAP that are ordered in the structure, 7ADLWDWIN, form an amphipathic \u03b1-helix interacting with the second IL-17A monomer.", "section": "RESULTS", "ner": [ [ 15, 25, "8 residues", "residue_range" ], [ 33, 36, "HAP", "chemical" ], [ 61, 70, "structure", "evidence" ], [ 72, 81, "7ADLWDWIN", "chemical" ], [ 91, 102, "amphipathic", "protein_state" ], [ 103, 110, "\u03b1-helix", "structure_element" ], [ 139, 145, "IL-17A", "protein" ], [ 146, 153, "monomer", "oligomeric_state" ] ] }, { "sid": 113, "sent": "Pro6 of HAP makes a transition between the N-terminal \u03b2-strand and the C-terminal \u03b1-helix of HAP.", "section": "RESULTS", "ner": [ [ 0, 4, "Pro6", "residue_name_number" ], [ 8, 11, "HAP", "chemical" ], [ 54, 62, "\u03b2-strand", "structure_element" ], [ 82, 89, "\u03b1-helix", "structure_element" ], [ 93, 96, "HAP", "chemical" ] ] }, { "sid": 114, "sent": "As a comparison, an IL-17A/IL-17RA complex structure (PDB code 4HSA) is also shown with IL-17A in the same orientation (Fig. 2C).", "section": "RESULTS", "ner": [ [ 20, 34, "IL-17A/IL-17RA", "complex_assembly" ], [ 35, 52, "complex structure", "evidence" ], [ 88, 94, "IL-17A", "protein" ] ] }, { "sid": 115, "sent": "Inhibition mechanism of IL-17A signaling by HAP", "section": "RESULTS", "ner": [ [ 24, 30, "IL-17A", "protein" ], [ 44, 47, "HAP", "chemical" ] ] }, { "sid": 116, "sent": "IL-17RA binds IL-17A at three regions on the IL-17A homodimer.", "section": "RESULTS", "ner": [ [ 0, 7, "IL-17RA", "protein" ], [ 14, 20, "IL-17A", "protein" ], [ 45, 51, "IL-17A", "protein" ], [ 52, 61, "homodimer", "oligomeric_state" ] ] }, { "sid": 117, "sent": "HAP binds IL-17A at region I. Region I is formed by residues at the ends of \u03b2 strands 0 and 4, and from loops 1\u20132 and 3\u20134 of IL-17A (Fig. 2).", "section": "RESULTS", "ner": [ [ 0, 3, "HAP", "chemical" ], [ 10, 16, "IL-17A", "protein" ], [ 20, 28, "region I", "structure_element" ], [ 30, 38, "Region I", "structure_element" ], [ 76, 93, "\u03b2 strands 0 and 4", "structure_element" ], [ 104, 113, "loops 1\u20132", "structure_element" ], [ 118, 121, "3\u20134", "structure_element" ], [ 125, 131, "IL-17A", "protein" ] ] }, { "sid": 118, "sent": "Conformational changes in region I induced by HAP binding alone may allosterically affect IL-17RA binding, but more importantly, the \u03b1-helix of HAP directly competes with IL-17RA for binding to IL-17A (Fig. 3).", "section": "RESULTS", "ner": [ [ 26, 34, "region I", "structure_element" ], [ 46, 49, "HAP", "chemical" ], [ 90, 97, "IL-17RA", "protein" ], [ 133, 140, "\u03b1-helix", "structure_element" ], [ 144, 147, "HAP", "chemical" ], [ 171, 178, "IL-17RA", "protein" ], [ 194, 200, "IL-17A", "protein" ] ] }, { "sid": 119, "sent": "The most significant interactions between the \u03b1 helix of HAP and IL-17A involve Trp12 of HAP, which binds in a hydrophobic pocket in IL-17A formed by the side chains of Phe110, Tyr62, Pro59 and the hydrophobic portion of the Arg101 side chain (Fig. 3A).", "section": "RESULTS", "ner": [ [ 46, 53, "\u03b1 helix", "structure_element" ], [ 57, 60, "HAP", "chemical" ], [ 65, 71, "IL-17A", "protein" ], [ 80, 85, "Trp12", "residue_name_number" ], [ 89, 92, "HAP", "chemical" ], [ 111, 129, "hydrophobic pocket", "site" ], [ 133, 139, "IL-17A", "protein" ], [ 169, 175, "Phe110", "residue_name_number" ], [ 177, 182, "Tyr62", "residue_name_number" ], [ 184, 189, "Pro59", "residue_name_number" ], [ 225, 231, "Arg101", "residue_name_number" ] ] }, { "sid": 120, "sent": "The Trp12 side chain of HAP donates a hydrogen bond to the main chain oxygen of Pro69 of IL-17A.", "section": "RESULTS", "ner": [ [ 4, 9, "Trp12", "residue_name_number" ], [ 24, 27, "HAP", "chemical" ], [ 38, 51, "hydrogen bond", "bond_interaction" ], [ 80, 85, "Pro69", "residue_name_number" ], [ 89, 95, "IL-17A", "protein" ] ] }, { "sid": 121, "sent": "The positively charged Arg101 side chain of the IL-17A engages in a charge-helix dipole interaction with the main chain oxygen of Trp12.", "section": "RESULTS", "ner": [ [ 23, 29, "Arg101", "residue_name_number" ], [ 48, 54, "IL-17A", "protein" ], [ 68, 99, "charge-helix dipole interaction", "bond_interaction" ], [ 130, 135, "Trp12", "residue_name_number" ] ] }, { "sid": 122, "sent": "Additionally, Leu9 and Ile13 of the HAP have hydrophobic interactions with IL-17A, and the Asp8 side chain has hydrogen bond and ion pair interactions with Tyr62 and Lys114 of IL-17A, respectively.", "section": "RESULTS", "ner": [ [ 14, 18, "Leu9", "residue_name_number" ], [ 23, 28, "Ile13", "residue_name_number" ], [ 36, 39, "HAP", "chemical" ], [ 45, 69, "hydrophobic interactions", "bond_interaction" ], [ 75, 81, "IL-17A", "protein" ], [ 91, 95, "Asp8", "residue_name_number" ], [ 111, 124, "hydrogen bond", "bond_interaction" ], [ 129, 150, "ion pair interactions", "bond_interaction" ], [ 156, 161, "Tyr62", "residue_name_number" ], [ 166, 172, "Lys114", "residue_name_number" ], [ 176, 182, "IL-17A", "protein" ] ] }, { "sid": 123, "sent": "In region I, an IL-17RA peptide interacts with IL-17A in a very similar fashion to the \u03b1-helix of HAP.", "section": "RESULTS", "ner": [ [ 3, 11, "region I", "structure_element" ], [ 16, 23, "IL-17RA", "protein" ], [ 47, 53, "IL-17A", "protein" ], [ 87, 94, "\u03b1-helix", "structure_element" ], [ 98, 101, "HAP", "chemical" ] ] }, { "sid": 124, "sent": "The IL-17RA peptide has sequences of 27LDDSWI, and part of the peptide is also \u03b1-helical (Fig. 3B).", "section": "RESULTS", "ner": [ [ 4, 11, "IL-17RA", "protein" ], [ 37, 45, "27LDDSWI", "chemical" ], [ 79, 88, "\u03b1-helical", "structure_element" ] ] }, { "sid": 125, "sent": "Leu7, Trp31 and Ile32 of IL-17RA interact very similarly with the same residues of IL-17A as Leu9, Trp12 and Ile13 of HAP (Fig. 3B).", "section": "RESULTS", "ner": [ [ 0, 4, "Leu7", "residue_name_number" ], [ 6, 11, "Trp31", "residue_name_number" ], [ 16, 21, "Ile32", "residue_name_number" ], [ 25, 32, "IL-17RA", "protein" ], [ 83, 89, "IL-17A", "protein" ], [ 93, 97, "Leu9", "residue_name_number" ], [ 99, 104, "Trp12", "residue_name_number" ], [ 109, 114, "Ile13", "residue_name_number" ], [ 118, 121, "HAP", "chemical" ] ] }, { "sid": 126, "sent": "In this sense, the \u03b1-helix of HAP with a sequence of 9LWDWI is a good mimetic of the 27LDDSWI peptide of IL-17RA.", "section": "RESULTS", "ner": [ [ 19, 26, "\u03b1-helix", "structure_element" ], [ 30, 33, "HAP", "chemical" ], [ 53, 59, "9LWDWI", "chemical" ], [ 85, 93, "27LDDSWI", "chemical" ], [ 105, 112, "IL-17RA", "protein" ] ] }, { "sid": 127, "sent": "The \u03b2-strand of HAP has no equivalent in IL-17RA.", "section": "RESULTS", "ner": [ [ 4, 12, "\u03b2-strand", "structure_element" ], [ 16, 19, "HAP", "chemical" ], [ 41, 48, "IL-17RA", "protein" ] ] }, { "sid": 128, "sent": "However, it mimics the \u03b2-strand 0 of IL-17A.", "section": "RESULTS", "ner": [ [ 23, 33, "\u03b2-strand 0", "structure_element" ], [ 37, 43, "IL-17A", "protein" ] ] }, { "sid": 129, "sent": "The amphipathic \u03b2-strand of HAP orients the hydrophilic side chains of His2 and Thr4 outwards, and the hydrophobic side chains of Ile1, Val3 and Ile5 inward (Fig. 3A).", "section": "RESULTS", "ner": [ [ 4, 15, "amphipathic", "protein_state" ], [ 16, 24, "\u03b2-strand", "structure_element" ], [ 28, 31, "HAP", "chemical" ], [ 71, 75, "His2", "residue_name_number" ], [ 80, 84, "Thr4", "residue_name_number" ], [ 130, 134, "Ile1", "residue_name_number" ], [ 136, 140, "Val3", "residue_name_number" ], [ 145, 149, "Ile5", "residue_name_number" ] ] }, { "sid": 130, "sent": "\u03b2-strand 0 in IL-17A is also amphipathic with the sequence of 21TVMVNLNI.", "section": "RESULTS", "ner": [ [ 0, 10, "\u03b2-strand 0", "structure_element" ], [ 14, 20, "IL-17A", "protein" ], [ 29, 40, "amphipathic", "protein_state" ], [ 62, 72, "21TVMVNLNI", "chemical" ] ] }, { "sid": 131, "sent": "In all IL-17A structures obtained to date, \u03b2-strand 0 orients the hydrophilic side chains of Thr21, Asn25 and Asn27 outward, and the hydrophobic side chains of Val22, Val24, Leu26 and Ile28 inward.", "section": "RESULTS", "ner": [ [ 7, 13, "IL-17A", "protein" ], [ 14, 24, "structures", "evidence" ], [ 43, 53, "\u03b2-strand 0", "structure_element" ], [ 93, 98, "Thr21", "residue_name_number" ], [ 100, 105, "Asn25", "residue_name_number" ], [ 110, 115, "Asn27", "residue_name_number" ], [ 160, 165, "Val22", "residue_name_number" ], [ 167, 172, "Val24", "residue_name_number" ], [ 174, 179, "Leu26", "residue_name_number" ], [ 184, 189, "Ile28", "residue_name_number" ] ] }, { "sid": 132, "sent": "The binding pocket occupied by either Trp12 of HAP or Trp31 of IL-17RA is not formed in the apo IL-17A structure (Fig. 3C).", "section": "RESULTS", "ner": [ [ 4, 18, "binding pocket", "site" ], [ 38, 43, "Trp12", "residue_name_number" ], [ 47, 50, "HAP", "chemical" ], [ 54, 59, "Trp31", "residue_name_number" ], [ 63, 70, "IL-17RA", "protein" ], [ 92, 95, "apo", "protein_state" ], [ 96, 102, "IL-17A", "protein" ], [ 103, 112, "structure", "evidence" ] ] }, { "sid": 133, "sent": "Conformational changes of IL-17A are needed for both HAP and IL-17RA to bind to that region.", "section": "RESULTS", "ner": [ [ 26, 32, "IL-17A", "protein" ], [ 53, 56, "HAP", "chemical" ], [ 61, 68, "IL-17RA", "protein" ] ] }, { "sid": 134, "sent": "Particularly for HAP, \u03b2-strands 0 have to shift out of the hydrophobic cleft formed by the main body of the IL-17A by as much as 10\u2009\u00c5 between C\u03b1 atoms (Fig. 3C).", "section": "RESULTS", "ner": [ [ 17, 20, "HAP", "chemical" ], [ 22, 33, "\u03b2-strands 0", "structure_element" ], [ 59, 76, "hydrophobic cleft", "site" ], [ 91, 100, "main body", "structure_element" ], [ 108, 114, "IL-17A", "protein" ] ] }, { "sid": 135, "sent": "Disruptions of the apo IL-17A structure by HAP binding are apparently compensated for by formation of the new interactions that involve almost the entire HAP molecule (Fig. 3B).", "section": "RESULTS", "ner": [ [ 19, 22, "apo", "protein_state" ], [ 23, 29, "IL-17A", "protein" ], [ 30, 39, "structure", "evidence" ], [ 43, 46, "HAP", "chemical" ], [ 154, 157, "HAP", "chemical" ] ] }, { "sid": 136, "sent": "Structure basis for the observed SAR of peptides", "section": "RESULTS", "ner": [ [ 33, 36, "SAR", "experimental_method" ] ] }, { "sid": 137, "sent": "The IL-17A/HAP complex structure obtained is very consistent with the observed SAR of our identified peptide inhibitors, explaining well how the evolution of the initial phage peptide 1 to HAP and 45 improved its potency (Supplementary Figure S7).", "section": "RESULTS", "ner": [ [ 4, 14, "IL-17A/HAP", "complex_assembly" ], [ 15, 32, "complex structure", "evidence" ], [ 79, 82, "SAR", "experimental_method" ], [ 170, 175, "phage", "experimental_method" ], [ 176, 185, "peptide 1", "chemical" ], [ 189, 192, "HAP", "chemical" ], [ 197, 199, "45", "chemical" ] ] }, { "sid": 138, "sent": "The important interactions involving Trp12 of HAP explain the >90 times drop in potency of the W12A variant (6 vs 1, Table 1).", "section": "RESULTS", "ner": [ [ 37, 42, "Trp12", "residue_name_number" ], [ 46, 49, "HAP", "chemical" ], [ 95, 99, "W12A", "mutant" ] ] }, { "sid": 139, "sent": "The amphipathic nature of the HAP \u03b2-strand explains the preference of the hydrophilic residues at the 2 and 4 positions of peptides (14, 18, 19, 21 and 23 vs 1 and 22, Table 1).", "section": "RESULTS", "ner": [ [ 4, 15, "amphipathic", "protein_state" ], [ 30, 33, "HAP", "chemical" ], [ 34, 42, "\u03b2-strand", "structure_element" ], [ 102, 103, "2", "residue_number" ], [ 108, 109, "4", "residue_number" ], [ 133, 135, "14", "chemical" ], [ 137, 139, "18", "chemical" ], [ 141, 143, "19", "chemical" ], [ 145, 147, "21", "chemical" ], [ 152, 154, "23", "chemical" ], [ 158, 159, "1", "chemical" ], [ 164, 166, "22", "chemical" ] ] }, { "sid": 140, "sent": "All N-terminal residues of HAP are part of the \u03b2-sheet with \u03b2-stands 0 and 4 of IL-17A, which explains why removal of the first 1\u20133 residues completely abolishes the ability of HAP to block IL-17A cell signaling (31,32 and 33, Table 2).", "section": "RESULTS", "ner": [ [ 27, 30, "HAP", "chemical" ], [ 47, 54, "\u03b2-sheet", "structure_element" ], [ 60, 76, "\u03b2-stands 0 and 4", "structure_element" ], [ 80, 86, "IL-17A", "protein" ], [ 107, 117, "removal of", "experimental_method" ], [ 122, 140, "first 1\u20133 residues", "residue_range" ], [ 177, 180, "HAP", "chemical" ], [ 190, 196, "IL-17A", "protein" ], [ 213, 215, "31", "chemical" ], [ 216, 218, "32", "chemical" ], [ 223, 225, "33", "chemical" ] ] }, { "sid": 141, "sent": "The C-terminal Asn14 and Lys15 of HAP are not directly involved in interactions with IL-17A, and this is reflected in the gradual reduction in activity caused by C-terminal truncations (35 and 36, Table 2).", "section": "RESULTS", "ner": [ [ 15, 20, "Asn14", "residue_name_number" ], [ 25, 30, "Lys15", "residue_name_number" ], [ 34, 37, "HAP", "chemical" ], [ 85, 91, "IL-17A", "protein" ], [ 173, 184, "truncations", "experimental_method" ], [ 186, 188, "35", "chemical" ], [ 193, 195, "36", "chemical" ] ] }, { "sid": 142, "sent": "Each peptide monomer in 45 may not necessarily be more potent than HAP, but two monomer peptides within the same molecule that can simultaneously bind to IL-17A can greatly improve its potency due to avidity effects.", "section": "RESULTS", "ner": [ [ 13, 20, "monomer", "oligomeric_state" ], [ 24, 26, "45", "chemical" ], [ 67, 70, "HAP", "chemical" ], [ 80, 87, "monomer", "oligomeric_state" ], [ 154, 160, "IL-17A", "protein" ] ] }, { "sid": 143, "sent": "HAP targets region I of IL-17A, an area that has the least sequence conservation in IL-17 cytokines.", "section": "RESULTS", "ner": [ [ 0, 3, "HAP", "chemical" ], [ 12, 20, "region I", "structure_element" ], [ 24, 30, "IL-17A", "protein" ], [ 84, 99, "IL-17 cytokines", "protein_type" ] ] }, { "sid": 144, "sent": "This lack of sequence conservation in the HAP binding site explains the observed specificity of HAP binding to human IL-17A.", "section": "RESULTS", "ner": [ [ 42, 58, "HAP binding site", "site" ], [ 96, 99, "HAP", "chemical" ], [ 111, 116, "human", "species" ], [ 117, 123, "IL-17A", "protein" ] ] }, { "sid": 145, "sent": "For example, inspection of the published IL-17F crystal structure (PDB code 1JPY) revealed a pocket of IL-17F similar to that of IL-17A for W12 of HAP binding, but it is occupied by a Phe-Phe motif at the N-terminal peptide of IL-17F.", "section": "RESULTS", "ner": [ [ 41, 47, "IL-17F", "protein" ], [ 48, 65, "crystal structure", "evidence" ], [ 93, 99, "pocket", "site" ], [ 103, 109, "IL-17F", "protein" ], [ 129, 135, "IL-17A", "protein" ], [ 140, 143, "W12", "residue_name_number" ], [ 147, 150, "HAP", "chemical" ], [ 184, 197, "Phe-Phe motif", "structure_element" ], [ 227, 233, "IL-17F", "protein" ] ] }, { "sid": 146, "sent": "This Phe-Phe motif is missing in IL-17A.", "section": "RESULTS", "ner": [ [ 5, 18, "Phe-Phe motif", "structure_element" ], [ 22, 29, "missing", "protein_state" ], [ 33, 39, "IL-17A", "protein" ] ] }, { "sid": 147, "sent": "Sequence alignments between human and mouse IL-17A indicated that among IL-17A residues that interacting with HAP, majority differences occur in strand 0 of IL-17A which interacts with the N-terminal \u03b2-strand of HAP.", "section": "RESULTS", "ner": [ [ 0, 19, "Sequence alignments", "experimental_method" ], [ 28, 33, "human", "species" ], [ 38, 43, "mouse", "taxonomy_domain" ], [ 44, 50, "IL-17A", "protein" ], [ 72, 78, "IL-17A", "protein" ], [ 110, 113, "HAP", "chemical" ], [ 145, 153, "strand 0", "structure_element" ], [ 157, 163, "IL-17A", "protein" ], [ 200, 208, "\u03b2-strand", "structure_element" ], [ 212, 215, "HAP", "chemical" ] ] }, { "sid": 148, "sent": "In human IL-17A the sequences are 21TVMVNLNI, and in mouse they are 21NVKVNLKV.", "section": "RESULTS", "ner": [ [ 3, 8, "human", "species" ], [ 9, 15, "IL-17A", "protein" ], [ 34, 44, "21TVMVNLNI", "chemical" ], [ 53, 58, "mouse", "taxonomy_domain" ], [ 68, 78, "21NVKVNLKV", "chemical" ] ] }, { "sid": 149, "sent": "Using a combination of phage display and SAR we have discovered novel peptides that are IL-17A antagonists.", "section": "DISCUSS", "ner": [ [ 23, 36, "phage display", "experimental_method" ], [ 41, 44, "SAR", "experimental_method" ], [ 88, 94, "IL-17A", "protein" ] ] }, { "sid": 150, "sent": "One of those peptides, HAP, also shows activity in inhibiting the production of multiple inflammatory cytokines by primary human keratinocytes stimulated by IL-17A and TNF-\u03b1, a disease relevant-model.", "section": "DISCUSS", "ner": [ [ 23, 26, "HAP", "chemical" ], [ 102, 111, "cytokines", "protein_type" ], [ 123, 128, "human", "species" ], [ 157, 163, "IL-17A", "protein" ], [ 168, 173, "TNF-\u03b1", "protein" ] ] }, { "sid": 151, "sent": "We have also determined the complex structure of IL-17A/HAP, which provides the structural basis for HAP\u2019s antagonism to IL-17A signaling.", "section": "DISCUSS", "ner": [ [ 13, 23, "determined", "experimental_method" ], [ 28, 45, "complex structure", "evidence" ], [ 49, 59, "IL-17A/HAP", "complex_assembly" ], [ 101, 104, "HAP", "chemical" ], [ 121, 127, "IL-17A", "protein" ] ] }, { "sid": 152, "sent": "During IL-17A signaling, IL-17A binds to one copy of IL-17RA and one copy of IL-17RC.", "section": "DISCUSS", "ner": [ [ 7, 13, "IL-17A", "protein" ], [ 25, 31, "IL-17A", "protein" ], [ 53, 60, "IL-17RA", "protein" ], [ 77, 84, "IL-17RC", "protein" ] ] }, { "sid": 153, "sent": "Since apo IL-17A is a homodimer with 2 fold symmetry, IL-17RA potentially can bind to either face of the IL-17A dimer.", "section": "DISCUSS", "ner": [ [ 6, 9, "apo", "protein_state" ], [ 10, 16, "IL-17A", "protein" ], [ 22, 31, "homodimer", "oligomeric_state" ], [ 54, 61, "IL-17RA", "protein" ], [ 105, 111, "IL-17A", "protein" ], [ 112, 117, "dimer", "oligomeric_state" ] ] }, { "sid": 154, "sent": "With two HAP molecules covering both faces of the IL-17A dimer, HAP can block IL-17RA approaching from either face.", "section": "DISCUSS", "ner": [ [ 9, 12, "HAP", "chemical" ], [ 50, 56, "IL-17A", "protein" ], [ 57, 62, "dimer", "oligomeric_state" ], [ 64, 67, "HAP", "chemical" ], [ 78, 85, "IL-17RA", "protein" ] ] }, { "sid": 155, "sent": "To form the 1:2 complex observed in crystal structure, it is important that there is no strong negative cooperativity in the binding of two HAP molecules.", "section": "DISCUSS", "ner": [ [ 36, 53, "crystal structure", "evidence" ], [ 140, 143, "HAP", "chemical" ] ] }, { "sid": 156, "sent": "In fact, in native electrospray ionization mass spectrometry analysis only 1:2 IL-17A/HAP complex was observed even when IL-17A was in excess (Supplementary Figure S8), indicating a positive binding cooperativity that favors inhibition of IL-17RA binding by HAP.", "section": "DISCUSS", "ner": [ [ 12, 60, "native electrospray ionization mass spectrometry", "experimental_method" ], [ 79, 89, "IL-17A/HAP", "complex_assembly" ], [ 121, 127, "IL-17A", "protein" ], [ 239, 246, "IL-17RA", "protein" ], [ 258, 261, "HAP", "chemical" ] ] }, { "sid": 157, "sent": "HAP, with only 15 residues, can achieve almost the same binding affinity as the much larger IL-17RA molecule, indicating a more efficient way of binding to IL-17A.", "section": "DISCUSS", "ner": [ [ 0, 3, "HAP", "chemical" ], [ 15, 26, "15 residues", "residue_range" ], [ 56, 72, "binding affinity", "evidence" ], [ 92, 99, "IL-17RA", "protein" ], [ 156, 162, "IL-17A", "protein" ] ] }, { "sid": 158, "sent": "The interaction of IL-17A with IL-17RA has an extensive interface, covering ~2,200\u2009\u00c52 surface area of IL-17A.", "section": "DISCUSS", "ner": [ [ 19, 25, "IL-17A", "protein" ], [ 31, 38, "IL-17RA", "protein" ], [ 56, 65, "interface", "site" ], [ 102, 108, "IL-17A", "protein" ] ] }, { "sid": 159, "sent": "Due to the discontinuous nature of the IL-17A/IL-17RA binding interface, it is classified as having tertiary structural epitopes on both binding partners, and is therefore hard to target using small molecules.", "section": "DISCUSS", "ner": [ [ 39, 71, "IL-17A/IL-17RA binding interface", "site" ] ] }, { "sid": 160, "sent": "Our studies of HAP demonstrated an uncommon mode of action for a peptide in inhibiting such a difficult protein-protein interaction target, and suggest further possible improvements in its binding potency.", "section": "DISCUSS", "ner": [ [ 15, 18, "HAP", "chemical" ] ] }, { "sid": 161, "sent": "One way of further improving HAP\u2019s potency is by dimerization.", "section": "DISCUSS", "ner": [ [ 29, 32, "HAP", "chemical" ] ] }, { "sid": 162, "sent": "Homo-dimerization of HAP (45) achieved sub-nanomolar potency against human IL-17A in cell assay.", "section": "DISCUSS", "ner": [ [ 21, 24, "HAP", "chemical" ], [ 26, 28, "45", "chemical" ], [ 69, 74, "human", "species" ], [ 75, 81, "IL-17A", "protein" ] ] }, { "sid": 163, "sent": "In the crystal structure, the distance between the carbonyl of Asn14 of one HAP molecule and the N-terminus of the second is only 15.7\u2009\u00c5, suggesting the potential for more potent dimeric peptides to be designed by using linkers of different lengths at different positions.", "section": "DISCUSS", "ner": [ [ 7, 24, "crystal structure", "evidence" ], [ 63, 68, "Asn14", "residue_name_number" ], [ 76, 79, "HAP", "chemical" ], [ 179, 186, "dimeric", "oligomeric_state" ], [ 187, 195, "peptides", "chemical" ] ] }, { "sid": 164, "sent": "Another direction of improving HAP is by reducing its size.", "section": "DISCUSS", "ner": [ [ 31, 34, "HAP", "chemical" ] ] }, { "sid": 165, "sent": "As demonstrated by the crystal structure, binding of the \u03b1-helix of HAP should be sufficient for preventing IL-17RA binding to IL-17A.", "section": "DISCUSS", "ner": [ [ 23, 40, "crystal structure", "evidence" ], [ 57, 64, "\u03b1-helix", "structure_element" ], [ 68, 71, "HAP", "chemical" ], [ 108, 115, "IL-17RA", "protein" ], [ 127, 133, "IL-17A", "protein" ] ] }, { "sid": 166, "sent": "Theoretically, it is possible to design chemicals such as stapled \u03b1-helical peptides to block \u03b1-helix-mediated IL-17A/IL-17RA interactions.", "section": "DISCUSS", "ner": [ [ 94, 101, "\u03b1-helix", "structure_element" ], [ 111, 125, "IL-17A/IL-17RA", "complex_assembly" ] ] }, { "sid": 167, "sent": "In summary, these peptide-based anti-IL-17A modalities could be further developed as alternative therapeutic options to the reported monoclonal antibodies.", "section": "DISCUSS", "ner": [ [ 37, 43, "IL-17A", "protein" ], [ 144, 154, "antibodies", "protein_type" ] ] }, { "sid": 168, "sent": "We are also very interested in finding non-peptidic small molecule IL-17A antagonists, and HAP can be used as an excellent tool peptide.", "section": "DISCUSS", "ner": [ [ 67, 73, "IL-17A", "protein" ], [ 91, 94, "HAP", "chemical" ] ] }, { "sid": 169, "sent": "The strategy utilized in generating the complex structures of HAP may also be useful for enabling structure based design of some known small molecule IL-17A antagonists.", "section": "DISCUSS", "ner": [ [ 48, 58, "structures", "evidence" ], [ 62, 65, "HAP", "chemical" ] ] }, { "sid": 170, "sent": "Binding of HAP to IL-17A and inhibition of IL-17A/IL-17RA are measured by SPR, FRET and cell-based assays.", "section": "FIG", "ner": [ [ 11, 14, "HAP", "chemical" ], [ 18, 24, "IL-17A", "protein" ], [ 43, 57, "IL-17A/IL-17RA", "complex_assembly" ], [ 74, 77, "SPR", "experimental_method" ], [ 79, 83, "FRET", "experimental_method" ], [ 88, 105, "cell-based assays", "experimental_method" ] ] }, { "sid": 171, "sent": "(A) Typical SPR sensorgrams (black) of HAP at indicated concentrations binding to biotinylated human IL-17A immobilized on a streptavidin chip surface, fitted with single site binding model curves (red).", "section": "FIG", "ner": [ [ 12, 15, "SPR", "experimental_method" ], [ 16, 27, "sensorgrams", "evidence" ], [ 39, 42, "HAP", "chemical" ], [ 82, 94, "biotinylated", "protein_state" ], [ 95, 100, "human", "species" ], [ 101, 107, "IL-17A", "protein" ], [ 164, 196, "single site binding model curves", "evidence" ] ] }, { "sid": 172, "sent": "Kinetic parameters (ka, kd) were obtained by a global fit using three concentrations in triplicate.", "section": "FIG", "ner": [ [ 20, 22, "ka", "evidence" ], [ 24, 26, "kd", "evidence" ] ] }, { "sid": 173, "sent": "KD determined by the standard equation, KD\u2009=\u2009kd/ka. (B) HAP inhibits SPR signaling of IL-17A binding to immobilized IL-17RA.", "section": "FIG", "ner": [ [ 0, 2, "KD", "evidence" ], [ 40, 42, "KD", "evidence" ], [ 45, 47, "kd", "evidence" ], [ 48, 50, "ka", "evidence" ], [ 56, 59, "HAP", "chemical" ], [ 69, 72, "SPR", "experimental_method" ], [ 86, 92, "IL-17A", "protein" ], [ 104, 115, "immobilized", "protein_state" ], [ 116, 123, "IL-17RA", "protein" ] ] }, { "sid": 174, "sent": "Data are mean and error bars of +/\u2212 standard deviation of three measurements. (C) Inhibition of IL-17A and IL-17RA binding by HAP measured by FRET assay.", "section": "FIG", "ner": [ [ 96, 102, "IL-17A", "protein" ], [ 107, 114, "IL-17RA", "protein" ], [ 126, 129, "HAP", "chemical" ], [ 142, 152, "FRET assay", "experimental_method" ] ] }, { "sid": 175, "sent": "Data are mean and error bars of +/\u2212 standard deviation from 299 experiments, each performed in duplicate. (D) Example of HAP selective inhibition of the production of IL-8 (triangles), IL-6 (squares) and CCL-20 (circles) by primary human keratinocyte cells synergistically stimulated by 100\u2009ng/ml IL-17A and 10\u2009ng/ml TNF-\u03b1.", "section": "FIG", "ner": [ [ 121, 124, "HAP", "chemical" ], [ 167, 171, "IL-8", "protein_type" ], [ 185, 189, "IL-6", "protein_type" ], [ 204, 210, "CCL-20", "protein_type" ], [ 232, 237, "human", "species" ], [ 297, 303, "IL-17A", "protein" ], [ 317, 322, "TNF-\u03b1", "protein" ] ] }, { "sid": 176, "sent": "HAP does not inhibit the baseline production of IL-6, IL-8 and CCL-20 stimulated by 10\u2009ng/ml TNF-\u03b1 alone (gray lines and symbols).", "section": "FIG", "ner": [ [ 0, 3, "HAP", "chemical" ], [ 48, 52, "IL-6", "protein_type" ], [ 54, 58, "IL-8", "protein_type" ], [ 63, 69, "CCL-20", "protein_type" ], [ 93, 98, "TNF-\u03b1", "protein" ] ] }, { "sid": 177, "sent": "Overall structure of the Fab/IL-17A/HAP complex in ribbon presentation.", "section": "FIG", "ner": [ [ 8, 17, "structure", "evidence" ], [ 25, 39, "Fab/IL-17A/HAP", "complex_assembly" ] ] }, { "sid": 178, "sent": "Two HAP molecules are colored blue and red, and IL-17A monomers are colored ice blue and pink, respectively.", "section": "FIG", "ner": [ [ 4, 7, "HAP", "chemical" ], [ 48, 54, "IL-17A", "protein" ], [ 55, 63, "monomers", "oligomeric_state" ] ] }, { "sid": 179, "sent": "(A) Overview of the distinct binding sites of Fab and HAP to IL-17A.", "section": "FIG", "ner": [ [ 29, 42, "binding sites", "site" ], [ 46, 49, "Fab", "structure_element" ], [ 54, 57, "HAP", "chemical" ], [ 61, 67, "IL-17A", "protein" ] ] }, { "sid": 180, "sent": "(B) Close-in view of the IL-17A/HAP structure.", "section": "FIG", "ner": [ [ 25, 35, "IL-17A/HAP", "complex_assembly" ], [ 36, 45, "structure", "evidence" ] ] }, { "sid": 181, "sent": "IL-17A \u03b2-strands are labelled.", "section": "FIG", "ner": [ [ 0, 6, "IL-17A", "protein" ], [ 7, 16, "\u03b2-strands", "structure_element" ] ] }, { "sid": 182, "sent": "Each of the two bound HAP interacts with both monomers of the IL-17A dimer.", "section": "FIG", "ner": [ [ 16, 21, "bound", "protein_state" ], [ 22, 25, "HAP", "chemical" ], [ 46, 54, "monomers", "oligomeric_state" ], [ 62, 68, "IL-17A", "protein" ], [ 69, 74, "dimer", "oligomeric_state" ] ] }, { "sid": 183, "sent": "(C) As a comparison, the IL-17A/IL-17RA complex was shown with IL-17A in the same orientation.", "section": "FIG", "ner": [ [ 25, 39, "IL-17A/IL-17RA", "complex_assembly" ], [ 63, 69, "IL-17A", "protein" ] ] }, { "sid": 184, "sent": "Three distinct areas IL-17A/IL-17RA interface are labeled.", "section": "FIG", "ner": [ [ 21, 45, "IL-17A/IL-17RA interface", "site" ] ] }, { "sid": 185, "sent": "Mechanism of the inhibition of the IL-17A/IL-17RA interaction by HAP.", "section": "FIG", "ner": [ [ 35, 49, "IL-17A/IL-17RA", "complex_assembly" ], [ 65, 68, "HAP", "chemical" ] ] }, { "sid": 186, "sent": "(A) HAP binds at region I of IL-17A.", "section": "FIG", "ner": [ [ 4, 7, "HAP", "chemical" ], [ 17, 25, "region I", "structure_element" ], [ 29, 35, "IL-17A", "protein" ] ] }, { "sid": 187, "sent": "IL-17A dimer is in surface presentation (\u03b2-strands 0 shown as ribbons for clarity).", "section": "FIG", "ner": [ [ 0, 6, "IL-17A", "protein" ], [ 7, 12, "dimer", "oligomeric_state" ], [ 41, 52, "\u03b2-strands 0", "structure_element" ] ] }, { "sid": 188, "sent": "Polar interactions are shown in dashes.", "section": "FIG", "ner": [ [ 0, 18, "Polar interactions", "bond_interaction" ] ] }, { "sid": 189, "sent": "HAP residues as well as key IL-17A residues are labeled.", "section": "FIG", "ner": [ [ 0, 3, "HAP", "chemical" ], [ 28, 34, "IL-17A", "protein" ] ] }, { "sid": 190, "sent": "For clarity, a few HAP residues are also shown in stick model with carbon atoms colored green, oxygen in red and nitrogen in blue.", "section": "FIG", "ner": [ [ 19, 22, "HAP", "chemical" ] ] }, { "sid": 191, "sent": "(B) I-17RA (ribbon in gold) peptide Leu27-Ile32 binds to the same area as the HAP \u03b1-helix.", "section": "FIG", "ner": [ [ 4, 10, "I-17RA", "protein" ], [ 36, 47, "Leu27-Ile32", "residue_range" ], [ 78, 81, "HAP", "chemical" ], [ 82, 89, "\u03b1-helix", "structure_element" ] ] }, { "sid": 192, "sent": "Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and \u03b2-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the \u03b2-stand and \u03b1-helix of the HAP.", "section": "FIG", "ner": [ [ 0, 5, "Trp31", "residue_name_number" ], [ 9, 16, "IL-17RA", "protein" ], [ 35, 41, "pocket", "site" ], [ 45, 51, "IL-17A", "protein" ], [ 55, 60, "Trp12", "residue_name_number" ], [ 64, 67, "HAP", "chemical" ], [ 91, 98, "overlay", "experimental_method" ], [ 108, 111, "HAP", "chemical" ], [ 125, 136, "\u03b2-strands 0", "structure_element" ], [ 151, 161, "IL-17A/HAP", "complex_assembly" ], [ 177, 180, "apo", "protein_state" ], [ 181, 187, "IL-17A", "protein" ], [ 188, 197, "structure", "evidence" ], [ 225, 233, "region I", "structure_element" ], [ 237, 243, "IL-17A", "protein" ], [ 279, 286, "\u03b2-stand", "structure_element" ], [ 291, 298, "\u03b1-helix", "structure_element" ], [ 306, 309, "HAP", "chemical" ] ] }, { "sid": 193, "sent": "Notice that the Trp binding pocket for W12 of HAP or W31 of IL-17RA is missing in the apo structure.", "section": "FIG", "ner": [ [ 16, 34, "Trp binding pocket", "site" ], [ 39, 42, "W12", "residue_name_number" ], [ 46, 49, "HAP", "chemical" ], [ 53, 56, "W31", "residue_name_number" ], [ 60, 67, "IL-17RA", "protein" ], [ 86, 89, "apo", "protein_state" ], [ 90, 99, "structure", "evidence" ] ] }, { "sid": 194, "sent": "ELISA competition activity of peptide analogues of 1.", "section": "TABLE", "ner": [ [ 0, 26, "ELISA competition activity", "experimental_method" ] ] } ] }, "PMC4841544": { "annotations": [ { "sid": 0, "sent": "Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA", "section": "TITLE", "ner": [ [ 50, 54, "NadR", "protein" ], [ 60, 85, "Transcriptional Repressor", "protein_type" ], [ 89, 102, "Meningococcal", "taxonomy_domain" ], [ 120, 124, "NadA", "protein" ] ] }, { "sid": 1, "sent": " Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells.", "section": "ABSTRACT", "ner": [ [ 1, 20, "Neisseria adhesin A", "protein" ], [ 22, 26, "NadA", "protein" ], [ 46, 59, "meningococcal", "taxonomy_domain" ], [ 115, 120, "human", "species" ] ] }, { "sid": 2, "sent": "NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus.", "section": "ABSTRACT", "ner": [ [ 0, 4, "NadA", "protein" ], [ 112, 137, "serogroup B meningococcus", "taxonomy_domain" ] ] }, { "sid": 3, "sent": "The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response.", "section": "ABSTRACT", "ner": [ [ 14, 18, "NadA", "protein" ], [ 26, 35, "bacterial", "taxonomy_domain" ], [ 173, 178, "human", "species" ], [ 231, 240, "bacterial", "taxonomy_domain" ] ] }, { "sid": 4, "sent": "It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo.", "section": "ABSTRACT", "ner": [ [ 70, 74, "nadA", "gene" ], [ 136, 161, "transcriptional regulator", "protein_type" ], [ 162, 166, "NadR", "protein" ], [ 168, 197, "Neisseria adhesin A Regulator", "protein" ] ] }, { "sid": 5, "sent": "NadR binds the nadA promoter and represses gene transcription.", "section": "INTRO", "ner": [ [ 0, 4, "NadR", "protein" ], [ 15, 19, "nadA", "gene" ], [ 0, 4, "NadR", "protein" ], [ 15, 19, "nadA", "gene" ] ] }, { "sid": 6, "sent": "In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced.", "section": "ABSTRACT", "ner": [ [ 7, 18, "presence of", "protein_state" ], [ 19, 41, "4-hydroxyphenylacetate", "chemical" ], [ 43, 48, "4-HPA", "chemical" ], [ 75, 80, "human", "species" ], [ 135, 144, "bacterial", "taxonomy_domain" ], [ 171, 175, "NadR", "protein" ], [ 183, 187, "nadA", "gene" ], [ 215, 219, "nadA", "gene" ] ] }, { "sid": 7, "sent": "NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation.", "section": "ABSTRACT", "ner": [ [ 0, 4, "NadR", "protein" ], [ 61, 74, "meningococcal", "taxonomy_domain" ] ] }, { "sid": 8, "sent": "To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies.", "section": "ABSTRACT", "ner": [ [ 40, 44, "NadR", "protein" ], [ 57, 62, "4-HPA", "chemical" ], [ 76, 124, "structural, biochemical, and mutagenesis studies", "experimental_method" ] ] }, { "sid": 9, "sent": "In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of \u2018conformational selection\u2019 by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria.", "section": "ABSTRACT", "ner": [ [ 23, 41, "crystal structures", "evidence" ], [ 45, 56, "ligand-free", "protein_state" ], [ 61, 73, "ligand-bound", "protein_state" ], [ 74, 78, "NadR", "protein" ], [ 172, 177, "4-HPA", "chemical" ], [ 199, 206, "dimeric", "oligomeric_state" ], [ 207, 211, "NadR", "protein" ], [ 329, 349, "hydroxyphenylacetate", "chemical" ], [ 369, 378, "3Cl,4-HPA", "chemical" ], [ 442, 449, "leucine", "residue_name" ], [ 489, 498, "conserved", "protein_state" ], [ 507, 511, "MarR", "protein_type" ], [ 553, 557, "His7", "residue_name_number" ], [ 559, 563, "Ser9", "residue_name_number" ], [ 565, 570, "Asn11", "residue_name_number" ], [ 575, 580, "Phe25", "residue_name_number" ], [ 613, 618, "4-HPA", "chemical" ], [ 697, 705, "bacteria", "taxonomy_domain" ] ] }, { "sid": 10, "sent": "Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis.", "section": "ABSTRACT", "ner": [ [ 120, 124, "nadA", "gene" ], [ 153, 157, "NadR", "protein" ], [ 259, 272, "meningococcal", "taxonomy_domain" ] ] }, { "sid": 11, "sent": "Serogroup B meningococcus (MenB) causes fatal sepsis and invasive meningococcal disease, particularly in young children and adolescents, as highlighted by recent MenB outbreaks in universities of the United States and Canada.", "section": "ABSTRACT", "ner": [ [ 0, 25, "Serogroup B meningococcus", "taxonomy_domain" ], [ 27, 31, "MenB", "species" ], [ 66, 79, "meningococcal", "taxonomy_domain" ], [ 162, 166, "MenB", "species" ] ] }, { "sid": 12, "sent": "The Bexsero vaccine protects against MenB and has recently been approved in > 35 countries worldwide.", "section": "ABSTRACT", "ner": [ [ 37, 41, "MenB", "species" ] ] }, { "sid": 13, "sent": "Neisseria adhesin A (NadA) present on the meningococcal surface can mediate binding to human cells and is one of the three MenB vaccine protein antigens.", "section": "ABSTRACT", "ner": [ [ 0, 19, "Neisseria adhesin A", "protein" ], [ 21, 25, "NadA", "protein" ], [ 42, 55, "meningococcal", "taxonomy_domain" ], [ 87, 92, "human", "species" ], [ 123, 127, "MenB", "species" ] ] }, { "sid": 14, "sent": "The amount of NadA exposed on the meningococcal surface also influences the antibody-mediated serum bactericidal response measured in vitro.", "section": "ABSTRACT", "ner": [ [ 14, 18, "NadA", "protein" ], [ 34, 47, "meningococcal", "taxonomy_domain" ] ] }, { "sid": 15, "sent": "A deep understanding of nadA expression is therefore important, otherwise the contribution of NadA to vaccine-induced protection against meningococcal meningitis may be underestimated.", "section": "ABSTRACT", "ner": [ [ 24, 28, "nadA", "gene" ], [ 94, 98, "NadA", "protein" ], [ 137, 150, "meningococcal", "taxonomy_domain" ] ] }, { "sid": 16, "sent": "The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. Here, we present functional, biochemical and high-resolution structural data on NadR. Our studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by \u2018conformational selection\u2019 of inactive forms.", "section": "ABSTRACT", "ner": [ [ 33, 37, "NadA", "protein" ], [ 58, 101, "ligand-responsive transcriptional repressor", "protein_type" ], [ 102, 106, "NadR", "protein" ], [ 125, 184, "functional, biochemical and high-resolution structural data", "evidence" ], [ 188, 192, "NadR", "protein" ], [ 273, 293, "hydroxyphenylacetate", "chemical" ], [ 377, 381, "NadR", "protein" ], [ 416, 424, "inactive", "protein_state" ] ] }, { "sid": 17, "sent": "These findings shed light on the regulation of NadR, a key MarR-family virulence factor of this important human pathogen.", "section": "ABSTRACT", "ner": [ [ 47, 51, "NadR", "protein" ], [ 59, 63, "MarR", "protein_type" ], [ 106, 111, "human", "species" ] ] }, { "sid": 18, "sent": "The \u2018Reverse Vaccinology\u2019 approach was pioneered to identify antigens for a protein-based vaccine against serogroup B Neisseria meningitidis (MenB), a human pathogen causing potentially-fatal sepsis and invasive meningococcal disease.", "section": "INTRO", "ner": [ [ 5, 24, "Reverse Vaccinology", "experimental_method" ], [ 106, 140, "serogroup B Neisseria meningitidis", "species" ], [ 142, 146, "MenB", "species" ], [ 151, 156, "human", "species" ], [ 212, 225, "meningococcal", "taxonomy_domain" ] ] }, { "sid": 19, "sent": "Indeed, Reverse Vaccinology identified Neisseria adhesin A (NadA), a surface-exposed protein involved in epithelial cell invasion and found in ~30% of clinical isolates.", "section": "INTRO", "ner": [ [ 8, 27, "Reverse Vaccinology", "experimental_method" ], [ 39, 58, "Neisseria adhesin A", "protein" ], [ 60, 64, "NadA", "protein" ] ] }, { "sid": 20, "sent": "Recently, we reported the crystal structure of NadA, providing insights into its biological and immunological functions.", "section": "INTRO", "ner": [ [ 26, 43, "crystal structure", "evidence" ], [ 47, 51, "NadA", "protein" ] ] }, { "sid": 21, "sent": "Recombinant NadA elicits a strong bactericidal immune response and is therefore included in the Bexsero vaccine that protects against MenB and which was recently approved in over 35 countries worldwide.", "section": "INTRO", "ner": [ [ 12, 16, "NadA", "protein" ], [ 134, 138, "MenB", "species" ] ] }, { "sid": 22, "sent": "Previous studies revealed that nadA expression levels are mainly regulated by the Neisseria adhesin A Regulator (NadR).", "section": "INTRO", "ner": [ [ 31, 35, "nadA", "gene" ], [ 82, 111, "Neisseria adhesin A Regulator", "protein" ], [ 113, 117, "NadR", "protein" ] ] }, { "sid": 23, "sent": "Although additional factors influence nadA expression, we focused on its regulation by NadR, the major mediator of NadA phase variable expression.", "section": "INTRO", "ner": [ [ 38, 42, "nadA", "gene" ], [ 87, 91, "NadR", "protein" ], [ 115, 119, "NadA", "protein" ] ] }, { "sid": 24, "sent": "Studies of NadR also have broader implications, since a genome-wide analysis of MenB wild-type and nadR knock-out strains revealed that NadR influences the regulation of > 30 genes, including maf genes, from the multiple adhesin family.", "section": "INTRO", "ner": [ [ 11, 15, "NadR", "protein" ], [ 80, 84, "MenB", "species" ], [ 85, 94, "wild-type", "protein_state" ], [ 99, 103, "nadR", "gene" ], [ 104, 113, "knock-out", "protein_state" ], [ 136, 140, "NadR", "protein" ], [ 221, 228, "adhesin", "protein_type" ] ] }, { "sid": 25, "sent": "These genes encode a wide variety of proteins connected to many biological processes contributing to bacterial survival, adaptation in the host niche, colonization and invasion.", "section": "INTRO", "ner": [ [ 101, 110, "bacterial", "taxonomy_domain" ] ] }, { "sid": 26, "sent": "NadR belongs to the MarR (Multiple Antibiotic Resistance Regulator) family, a group of ligand-responsive transcriptional regulators ubiquitous in bacteria and archaea.", "section": "INTRO", "ner": [ [ 0, 4, "NadR", "protein" ], [ 20, 24, "MarR", "protein_type" ], [ 26, 66, "Multiple Antibiotic Resistance Regulator", "protein_type" ], [ 87, 131, "ligand-responsive transcriptional regulators", "protein_type" ], [ 146, 154, "bacteria", "taxonomy_domain" ], [ 159, 166, "archaea", "taxonomy_domain" ] ] }, { "sid": 27, "sent": "MarR family proteins can promote bacterial survival in the presence of antibiotics, toxic chemicals, organic solvents or reactive oxygen species and can regulate virulence factor expression.", "section": "INTRO", "ner": [ [ 0, 4, "MarR", "protein_type" ], [ 33, 42, "bacterial", "taxonomy_domain" ] ] }, { "sid": 28, "sent": "MarR homologues can act either as transcriptional repressors or as activators.", "section": "INTRO", "ner": [ [ 0, 4, "MarR", "protein_type" ] ] }, { "sid": 29, "sent": "Although > 50 MarR family structures are known, a molecular understanding of their ligand-dependent regulatory mechanisms is still limited, often hampered by lack of identification of their ligands and/or DNA targets.", "section": "INTRO", "ner": [ [ 14, 18, "MarR", "protein_type" ], [ 26, 36, "structures", "evidence" ] ] }, { "sid": 30, "sent": "A potentially interesting exception comes from the ligand-free and salicylate-bound forms of the Methanobacterium thermoautotrophicum protein MTH313 which revealed that two salicylate molecules bind to one MTH313 dimer and induce large conformational changes, apparently sufficient to prevent DNA binding.", "section": "INTRO", "ner": [ [ 51, 62, "ligand-free", "protein_state" ], [ 67, 83, "salicylate-bound", "protein_state" ], [ 97, 133, "Methanobacterium thermoautotrophicum", "species" ], [ 142, 148, "MTH313", "protein" ], [ 173, 183, "salicylate", "chemical" ], [ 206, 212, "MTH313", "protein" ], [ 213, 218, "dimer", "oligomeric_state" ] ] }, { "sid": 31, "sent": "However, the homologous archeal Sulfolobus tokodaii protein ST1710 presented essentially the same structure in ligand-free and salicylate-bound forms, apparently contrasting the mechanism proposed for MTH313.", "section": "INTRO", "ner": [ [ 24, 31, "archeal", "taxonomy_domain" ], [ 32, 51, "Sulfolobus tokodaii", "species" ], [ 60, 66, "ST1710", "protein" ], [ 98, 107, "structure", "evidence" ], [ 111, 122, "ligand-free", "protein_state" ], [ 127, 143, "salicylate-bound", "protein_state" ], [ 201, 207, "MTH313", "protein" ] ] }, { "sid": 32, "sent": "Despite these apparent differences, MTH313 and ST1710 bind salicylate in approximately the same site, between their dimerization and DNA-binding domains.", "section": "INTRO", "ner": [ [ 36, 42, "MTH313", "protein" ], [ 47, 53, "ST1710", "protein" ], [ 59, 69, "salicylate", "chemical" ], [ 116, 152, "dimerization and DNA-binding domains", "structure_element" ] ] }, { "sid": 33, "sent": "However, it is unknown whether salicylate is a relevant in vivo ligand of either of these two proteins, which share ~20% sequence identity with NadR, rendering unclear the interpretation of these findings in relation to the regulatory mechanisms of NadR or other MarR family proteins.", "section": "INTRO", "ner": [ [ 31, 41, "salicylate", "chemical" ], [ 144, 148, "NadR", "protein" ], [ 249, 253, "NadR", "protein" ], [ 263, 267, "MarR", "protein_type" ] ] }, { "sid": 34, "sent": "NadR binds nadA on three different operators (OpI, OpII and OpIII).", "section": "INTRO", "ner": [ [ 0, 4, "NadR", "protein" ], [ 11, 15, "nadA", "gene" ] ] }, { "sid": 35, "sent": "The DNA-binding activity of NadR is attenuated in vitro upon addition of various hydroxyphenylacetate (HPA) derivatives, including 4-HPA.", "section": "INTRO", "ner": [ [ 28, 32, "NadR", "protein" ], [ 81, 101, "hydroxyphenylacetate", "chemical" ], [ 103, 106, "HPA", "chemical" ], [ 131, 136, "4-HPA", "chemical" ] ] }, { "sid": 36, "sent": "4-HPA is a small molecule derived from mammalian aromatic amino acid catabolism and is released in human saliva, where it has been detected at micromolar concentration.", "section": "INTRO", "ner": [ [ 0, 5, "4-HPA", "chemical" ], [ 39, 48, "mammalian", "taxonomy_domain" ], [ 99, 104, "human", "species" ] ] }, { "sid": 37, "sent": "In the presence of 4-HPA, NadR is unable to bind the nadA promoter and nadA gene expression is induced.", "section": "INTRO", "ner": [ [ 19, 24, "4-HPA", "chemical" ], [ 26, 30, "NadR", "protein" ], [ 53, 57, "nadA", "gene" ], [ 71, 75, "nadA", "gene" ] ] }, { "sid": 38, "sent": "In vivo, the presence of 4-HPA in the host niche of N. meningitidis serves as an inducer of NadA production, thereby promoting bacterial adhesion to host cells.", "section": "INTRO", "ner": [ [ 25, 30, "4-HPA", "chemical" ], [ 52, 67, "N. meningitidis", "species" ], [ 92, 96, "NadA", "protein" ], [ 127, 136, "bacterial", "taxonomy_domain" ] ] }, { "sid": 39, "sent": "Further, we recently reported that 3Cl,4-HPA, produced during inflammation, is another inducer of nadA expression.", "section": "INTRO", "ner": [ [ 35, 44, "3Cl,4-HPA", "chemical" ], [ 98, 102, "nadA", "gene" ] ] }, { "sid": 40, "sent": "Extending our previous studies based on hydrogen-deuterium exchange mass spectrometry (HDX-MS), here we sought to reveal the molecular mechanisms and effects of NadR/HPA interactions via X-ray crystallography, NMR spectroscopy and complementary biochemical and in vivo mutagenesis studies.", "section": "INTRO", "ner": [ [ 40, 85, "hydrogen-deuterium exchange mass spectrometry", "experimental_method" ], [ 87, 93, "HDX-MS", "experimental_method" ], [ 161, 165, "NadR", "protein" ], [ 166, 169, "HPA", "chemical" ], [ 187, 208, "X-ray crystallography", "experimental_method" ], [ 210, 226, "NMR spectroscopy", "experimental_method" ], [ 245, 288, "biochemical and in vivo mutagenesis studies", "experimental_method" ] ] }, { "sid": 41, "sent": "We obtained detailed new insights into ligand specificity, how the ligand allosterically influences the DNA-binding ability of NadR, and the regulation of nadA expression, thus also providing a deeper structural understanding of the ligand-responsive MarR super-family.", "section": "INTRO", "ner": [ [ 127, 131, "NadR", "protein" ], [ 155, 159, "nadA", "gene" ], [ 251, 255, "MarR", "protein_type" ] ] }, { "sid": 42, "sent": "Moreover, these findings are important because the activity of NadR impacts the potential coverage provided by anti-NadA antibodies elicited by the Bexsero vaccine and influences host-bacteria interactions that contribute to meningococcal pathogenesis.", "section": "INTRO", "ner": [ [ 63, 67, "NadR", "protein" ], [ 116, 120, "NadA", "protein" ], [ 184, 192, "bacteria", "taxonomy_domain" ], [ 225, 238, "meningococcal", "taxonomy_domain" ] ] }, { "sid": 43, "sent": "NadR is dimeric and is stabilized by specific hydroxyphenylacetate ligands", "section": "RESULTS", "ner": [ [ 0, 4, "NadR", "protein" ], [ 8, 15, "dimeric", "oligomeric_state" ], [ 46, 66, "hydroxyphenylacetate", "chemical" ] ] }, { "sid": 44, "sent": "Recombinant NadR was produced in E. coli using an expression construct prepared from N. meningitidis serogroup B strain MC58.", "section": "RESULTS", "ner": [ [ 12, 16, "NadR", "protein" ], [ 33, 40, "E. coli", "species" ], [ 50, 70, "expression construct", "experimental_method" ], [ 85, 124, "N. meningitidis serogroup B strain MC58", "species" ] ] }, { "sid": 45, "sent": "Standard chromatographic techniques were used to obtain a highly purified sample of NadR (see Materials and Methods).", "section": "RESULTS", "ner": [ [ 84, 88, "NadR", "protein" ] ] }, { "sid": 46, "sent": "In analytical size-exclusion high-performance liquid chromatography (SE-HPLC) experiments coupled with multi-angle laser light scattering (MALLS), NadR presented a single species with an absolute molecular mass of 35 kDa (S1 Fig).", "section": "RESULTS", "ner": [ [ 3, 67, "analytical size-exclusion high-performance liquid chromatography", "experimental_method" ], [ 69, 76, "SE-HPLC", "experimental_method" ], [ 103, 137, "multi-angle laser light scattering", "experimental_method" ], [ 139, 144, "MALLS", "experimental_method" ], [ 147, 151, "NadR", "protein" ] ] }, { "sid": 47, "sent": "These data showed that NadR was dimeric in solution, since the theoretical molecular mass of the NadR dimer is 33.73 kDa; and, there was no change in oligomeric state on addition of 4-HPA.", "section": "RESULTS", "ner": [ [ 23, 27, "NadR", "protein" ], [ 32, 39, "dimeric", "oligomeric_state" ], [ 97, 101, "NadR", "protein" ], [ 102, 107, "dimer", "oligomeric_state" ], [ 182, 187, "4-HPA", "chemical" ] ] }, { "sid": 48, "sent": "The thermal stability of NadR was examined using differential scanning calorimetry (DSC).", "section": "RESULTS", "ner": [ [ 25, 29, "NadR", "protein" ], [ 49, 82, "differential scanning calorimetry", "experimental_method" ], [ 84, 87, "DSC", "experimental_method" ] ] }, { "sid": 49, "sent": "Since ligand-binding often increases protein stability, we also investigated the effect of various HPAs (Fig 1A) on the melting temperature (Tm) of NadR. As a control of specificity, we also tested salicylate, a known ligand of some MarR proteins previously reported to increase the Tm of ST1710 and MTH313.", "section": "RESULTS", "ner": [ [ 99, 103, "HPAs", "chemical" ], [ 120, 139, "melting temperature", "evidence" ], [ 141, 143, "Tm", "evidence" ], [ 148, 152, "NadR", "protein" ], [ 198, 208, "salicylate", "chemical" ], [ 233, 237, "MarR", "protein_type" ], [ 283, 285, "Tm", "evidence" ], [ 289, 295, "ST1710", "protein" ], [ 300, 306, "MTH313", "protein" ] ] }, { "sid": 50, "sent": "The Tm of NadR was 67.4 \u00b1 0.1\u00b0C in the absence of ligand, and was unaffected by salicylate.", "section": "RESULTS", "ner": [ [ 4, 6, "Tm", "evidence" ], [ 10, 14, "NadR", "protein" ], [ 39, 56, "absence of ligand", "protein_state" ], [ 80, 90, "salicylate", "chemical" ] ] }, { "sid": 51, "sent": "However, an increased thermal stability was induced by 4-HPA and, to a lesser extent, by 3-HPA.", "section": "RESULTS", "ner": [ [ 55, 60, "4-HPA", "chemical" ], [ 89, 94, "3-HPA", "chemical" ] ] }, { "sid": 52, "sent": "Interestingly, NadR displayed the greatest Tm increase upon addition of 3Cl,4-HPA (Table 1 and Fig 1B).", "section": "RESULTS", "ner": [ [ 15, 19, "NadR", "protein" ], [ 43, 45, "Tm", "evidence" ], [ 72, 81, "3Cl,4-HPA", "chemical" ] ] }, { "sid": 53, "sent": "Stability of NadR is increased by small molecule ligands.", "section": "FIG", "ner": [ [ 13, 17, "NadR", "protein" ] ] }, { "sid": 54, "sent": " (A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink).", "section": "FIG", "ner": [ [ 29, 34, "3-HPA", "chemical" ], [ 47, 52, "4-HPA", "chemical" ], [ 65, 74, "3Cl,4-HPA", "chemical" ], [ 90, 104, "salicylic acid", "chemical" ], [ 121, 124, "DSC", "experimental_method" ], [ 125, 133, "profiles", "evidence" ], [ 155, 158, "apo", "protein_state" ], [ 159, 163, "NadR", "protein" ], [ 174, 189, "NadR+salicylate", "complex_assembly" ], [ 197, 207, "NadR+3-HPA", "complex_assembly" ], [ 217, 227, "NadR+4-HPA", "complex_assembly" ], [ 236, 250, "NadR+3Cl,4-HPA", "complex_assembly" ] ] }, { "sid": 55, "sent": "All DSC profiles are representative of triplicate experiments.", "section": "FIG", "ner": [ [ 4, 7, "DSC", "experimental_method" ], [ 8, 16, "profiles", "evidence" ] ] }, { "sid": 56, "sent": "Melting-point (Tm) and its ligand-induced increase (\u0394Tm) derived from DSC thermostability experiments.", "section": "TABLE", "ner": [ [ 0, 13, "Melting-point", "evidence" ], [ 15, 17, "Tm", "evidence" ], [ 52, 55, "\u0394Tm", "evidence" ], [ 70, 73, "DSC", "experimental_method" ], [ 74, 101, "thermostability experiments", "experimental_method" ] ] }, { "sid": 57, "sent": "Dissociation constants (KD) of the NadR/ligand interactions from SPR steady-state binding experiments.", "section": "TABLE", "ner": [ [ 0, 22, "Dissociation constants", "evidence" ], [ 24, 26, "KD", "evidence" ], [ 35, 39, "NadR", "protein" ], [ 65, 101, "SPR steady-state binding experiments", "experimental_method" ] ] }, { "sid": 58, "sent": "Ligand\tTm (\u00b0C)\t\u0394Tm (\u00b0C)\tKD (mM)\t \tNo ligand\t67.4 \u00b1 0.1\tn.a.\tn.a.", "section": "TABLE", "ner": [ [ 7, 9, "Tm", "evidence" ], [ 15, 18, "\u0394Tm", "evidence" ], [ 24, 26, "KD", "evidence" ] ] }, { "sid": 59, "sent": "\t \t3-HPA\t70.0 \u00b1 0.1\t2.7\t2.7 \u00b1 0.1\t \t4-HPA\t70.7 \u00b1 0.1\t3.3\t1.5 \u00b1 0.1\t \t3Cl,4-HPA\t71.3 \u00b1 0.2\t3.9\t1.1 \u00b1 0.1\t \t", "section": "TABLE", "ner": [ [ 2, 7, "3-HPA", "chemical" ], [ 35, 40, "4-HPA", "chemical" ], [ 68, 77, "3Cl,4-HPA", "chemical" ] ] }, { "sid": 60, "sent": "NadR displays distinct binding affinities for hydroxyphenylacetate ligands", "section": "RESULTS", "ner": [ [ 0, 4, "NadR", "protein" ], [ 23, 41, "binding affinities", "evidence" ], [ 46, 66, "hydroxyphenylacetate", "chemical" ] ] }, { "sid": 61, "sent": "To further investigate the binding of HPAs to NadR, we used surface plasmon resonance (SPR).", "section": "RESULTS", "ner": [ [ 38, 42, "HPAs", "chemical" ], [ 46, 50, "NadR", "protein" ], [ 60, 85, "surface plasmon resonance", "experimental_method" ], [ 87, 90, "SPR", "experimental_method" ] ] }, { "sid": 62, "sent": "The SPR sensorgrams revealed very fast association and dissociation events, typical of small molecule ligands, thus prohibiting a detailed study of binding kinetics.", "section": "RESULTS", "ner": [ [ 4, 7, "SPR", "experimental_method" ], [ 8, 19, "sensorgrams", "evidence" ] ] }, { "sid": 63, "sent": "However, steady-state SPR analyses of the NadR-HPA interactions allowed determination of the equilibrium dissociation constants (KD) (Table 1 and S2 Fig).", "section": "RESULTS", "ner": [ [ 9, 25, "steady-state SPR", "experimental_method" ], [ 42, 50, "NadR-HPA", "complex_assembly" ], [ 93, 127, "equilibrium dissociation constants", "evidence" ], [ 129, 131, "KD", "evidence" ] ] }, { "sid": 64, "sent": "The interactions of 4-HPA and 3Cl,4-HPA with NadR exhibited KD values of 1.5 mM and 1.1 mM, respectively.", "section": "RESULTS", "ner": [ [ 20, 25, "4-HPA", "chemical" ], [ 30, 39, "3Cl,4-HPA", "chemical" ], [ 45, 49, "NadR", "protein" ], [ 60, 62, "KD", "evidence" ] ] }, { "sid": 65, "sent": "3-HPA showed a weaker interaction, with a KD of 2.7 mM, while salicylate showed only a very weak response that did not reach saturation, indicating a non-specific interaction with NadR. A ranking of these KD values showed that 3Cl,4-HPA was the tightest binder, and thus matched the ranking of ligand-induced Tm increases observed in the DSC experiments.", "section": "RESULTS", "ner": [ [ 0, 5, "3-HPA", "chemical" ], [ 42, 44, "KD", "evidence" ], [ 62, 72, "salicylate", "chemical" ], [ 180, 184, "NadR", "protein" ], [ 205, 207, "KD", "evidence" ], [ 227, 236, "3Cl,4-HPA", "chemical" ], [ 309, 311, "Tm", "evidence" ], [ 338, 341, "DSC", "experimental_method" ] ] }, { "sid": 66, "sent": "Although these KD values indicate rather weak interactions, they are similar to the values reported previously for the MarR/salicylate interaction (KD ~1 mM) and the MTH313/salicylate interaction (KD 2\u20133 mM), and approximately 20-fold tighter than the ST1710/salicylate interaction (KD ~20 mM).", "section": "RESULTS", "ner": [ [ 15, 17, "KD", "evidence" ], [ 119, 123, "MarR", "protein_type" ], [ 124, 134, "salicylate", "chemical" ], [ 166, 172, "MTH313", "protein" ], [ 173, 183, "salicylate", "chemical" ], [ 252, 258, "ST1710", "protein" ], [ 259, 269, "salicylate", "chemical" ] ] }, { "sid": 67, "sent": "Crystal structures of holo-NadR and apo-NadR", "section": "RESULTS", "ner": [ [ 0, 18, "Crystal structures", "evidence" ], [ 22, 26, "holo", "protein_state" ], [ 27, 31, "NadR", "protein" ], [ 36, 39, "apo", "protein_state" ], [ 40, 44, "NadR", "protein" ] ] }, { "sid": 68, "sent": "To fully characterize the NadR/HPA interactions, we sought to determine crystal structures of NadR in ligand-bound (holo) and ligand-free (apo) forms.", "section": "RESULTS", "ner": [ [ 26, 30, "NadR", "protein" ], [ 31, 34, "HPA", "chemical" ], [ 72, 90, "crystal structures", "evidence" ], [ 94, 98, "NadR", "protein" ], [ 102, 114, "ligand-bound", "protein_state" ], [ 116, 120, "holo", "protein_state" ], [ 126, 137, "ligand-free", "protein_state" ], [ 139, 142, "apo", "protein_state" ] ] }, { "sid": 69, "sent": "First, we crystallized NadR (a selenomethionine-labelled derivative) in the presence of a 200-fold molar excess of 4-HPA.", "section": "RESULTS", "ner": [ [ 10, 22, "crystallized", "experimental_method" ], [ 23, 27, "NadR", "protein" ], [ 31, 67, "selenomethionine-labelled derivative", "experimental_method" ], [ 115, 120, "4-HPA", "chemical" ] ] }, { "sid": 70, "sent": "The structure of the NadR/4-HPA complex was determined at 2.3 \u00c5 resolution using a combination of the single-wavelength anomalous dispersion (SAD) and molecular replacement (MR) methods, and was refined to R work/R free values of 20.9/26.0% (Table 2).", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 21, 31, "NadR/4-HPA", "complex_assembly" ], [ 102, 140, "single-wavelength anomalous dispersion", "experimental_method" ], [ 142, 145, "SAD", "experimental_method" ], [ 151, 172, "molecular replacement", "experimental_method" ], [ 174, 176, "MR", "experimental_method" ], [ 206, 219, "R work/R free", "evidence" ] ] }, { "sid": 71, "sent": "Despite numerous attempts, we were unable to obtain high-quality crystals of NadR complexed with 3Cl,4-HPA, 3,4-HPA, 3-HPA or DNA targets.", "section": "RESULTS", "ner": [ [ 65, 73, "crystals", "evidence" ], [ 77, 81, "NadR", "protein" ], [ 82, 96, "complexed with", "protein_state" ], [ 97, 106, "3Cl,4-HPA", "chemical" ], [ 108, 115, "3,4-HPA", "chemical" ], [ 117, 122, "3-HPA", "chemical" ] ] }, { "sid": 72, "sent": "However, it was eventually possible to crystallize apo-NadR, and the structure was determined at 2.7 \u00c5 resolution by MR methods using the NadR/4-HPA complex as the search model.", "section": "RESULTS", "ner": [ [ 39, 50, "crystallize", "experimental_method" ], [ 51, 54, "apo", "protein_state" ], [ 55, 59, "NadR", "protein" ], [ 69, 78, "structure", "evidence" ], [ 117, 119, "MR", "experimental_method" ], [ 138, 148, "NadR/4-HPA", "complex_assembly" ] ] }, { "sid": 73, "sent": "The apo-NadR structure was refined to R work/R free values of 19.1/26.8% (Table 2).", "section": "RESULTS", "ner": [ [ 4, 7, "apo", "protein_state" ], [ 8, 12, "NadR", "protein" ], [ 13, 22, "structure", "evidence" ], [ 38, 51, "R work/R free", "evidence" ] ] }, { "sid": 74, "sent": "Data collection and refinement statistics for NadR structures.", "section": "TABLE", "ner": [ [ 46, 50, "NadR", "protein" ], [ 51, 61, "structures", "evidence" ] ] }, { "sid": 75, "sent": "The asymmetric unit of the NadR/4-HPA crystals (holo-NadR) contained one NadR homodimer, while the apo-NadR crystals contained two homodimers.", "section": "RESULTS", "ner": [ [ 27, 37, "NadR/4-HPA", "complex_assembly" ], [ 38, 46, "crystals", "evidence" ], [ 48, 52, "holo", "protein_state" ], [ 53, 57, "NadR", "protein" ], [ 73, 77, "NadR", "protein" ], [ 78, 87, "homodimer", "oligomeric_state" ], [ 99, 102, "apo", "protein_state" ], [ 103, 107, "NadR", "protein" ], [ 108, 116, "crystals", "evidence" ], [ 131, 141, "homodimers", "oligomeric_state" ] ] }, { "sid": 76, "sent": "In the apo-NadR crystals, the two homodimers were related by a rotation of ~90\u00b0; the observed association of the two dimers was presumably merely an effect of crystal packing, since the interface between the two homodimers is small (< 550 \u00c52 of buried surface area), and is not predicted to be physiologically relevant by the PISA software.", "section": "RESULTS", "ner": [ [ 7, 10, "apo", "protein_state" ], [ 11, 15, "NadR", "protein" ], [ 16, 24, "crystals", "evidence" ], [ 34, 44, "homodimers", "oligomeric_state" ], [ 117, 123, "dimers", "oligomeric_state" ], [ 186, 195, "interface", "site" ], [ 212, 222, "homodimers", "oligomeric_state" ] ] }, { "sid": 77, "sent": "Moreover, our SE-HPLC/MALLS analyses (see above) revealed that in solution NadR is dimeric, and previous studies using native mass spectrometry (MS) revealed dimers, not tetramers.", "section": "RESULTS", "ner": [ [ 14, 27, "SE-HPLC/MALLS", "experimental_method" ], [ 75, 79, "NadR", "protein" ], [ 83, 90, "dimeric", "oligomeric_state" ], [ 119, 143, "native mass spectrometry", "experimental_method" ], [ 145, 147, "MS", "experimental_method" ], [ 158, 164, "dimers", "oligomeric_state" ], [ 170, 179, "tetramers", "oligomeric_state" ] ] }, { "sid": 78, "sent": "The NadR homodimer bound to 4-HPA has a dimerization interface mostly involving the top of its \u2018triangular\u2019 form, while the two DNA-binding domains are located at the base (Fig 2A).", "section": "RESULTS", "ner": [ [ 4, 8, "NadR", "protein" ], [ 9, 18, "homodimer", "oligomeric_state" ], [ 19, 27, "bound to", "protein_state" ], [ 28, 33, "4-HPA", "chemical" ], [ 40, 62, "dimerization interface", "site" ], [ 96, 106, "triangular", "protein_state" ], [ 128, 147, "DNA-binding domains", "structure_element" ] ] }, { "sid": 79, "sent": "High-quality electron density maps allowed clear identification of the bound ligand, 4-HPA (Fig 2B).", "section": "RESULTS", "ner": [ [ 13, 34, "electron density maps", "evidence" ], [ 71, 76, "bound", "protein_state" ], [ 85, 90, "4-HPA", "chemical" ] ] }, { "sid": 80, "sent": "The overall structure of NadR shows dimensions of ~50 \u00d7 65 \u00d7 50 \u00c5 and a large homodimer interface that buries a total surface area of ~ 4800 \u00c52.", "section": "RESULTS", "ner": [ [ 12, 21, "structure", "evidence" ], [ 25, 29, "NadR", "protein" ], [ 78, 97, "homodimer interface", "site" ] ] }, { "sid": 81, "sent": "Each NadR monomer consists of six \u03b1-helices and two short \u03b2-strands, with helices \u03b11, \u03b15, and \u03b16 forming the dimer interface.", "section": "RESULTS", "ner": [ [ 5, 9, "NadR", "protein" ], [ 10, 17, "monomer", "oligomeric_state" ], [ 34, 43, "\u03b1-helices", "structure_element" ], [ 52, 67, "short \u03b2-strands", "structure_element" ], [ 74, 81, "helices", "structure_element" ], [ 82, 84, "\u03b11", "structure_element" ], [ 86, 88, "\u03b15", "structure_element" ], [ 94, 96, "\u03b16", "structure_element" ], [ 109, 124, "dimer interface", "site" ] ] }, { "sid": 82, "sent": "Helices \u03b13 and \u03b14 form a helix-turn-helix motif, followed by the \u201cwing motif\u201d comprised of two short antiparallel \u03b2-strands (\u03b21-\u03b22) linked by a relatively long and flexible loop.", "section": "RESULTS", "ner": [ [ 0, 7, "Helices", "structure_element" ], [ 8, 10, "\u03b13", "structure_element" ], [ 15, 17, "\u03b14", "structure_element" ], [ 25, 47, "helix-turn-helix motif", "structure_element" ], [ 66, 76, "wing motif", "structure_element" ], [ 95, 123, "short antiparallel \u03b2-strands", "structure_element" ], [ 125, 130, "\u03b21-\u03b22", "structure_element" ], [ 173, 177, "loop", "structure_element" ] ] }, { "sid": 83, "sent": "Interestingly, in the \u03b14-\u03b22 region, the stretch of residues from R64-R91 presents seven positively-charged side chains, all available for potential interactions with DNA.", "section": "RESULTS", "ner": [ [ 22, 34, "\u03b14-\u03b22 region", "structure_element" ], [ 65, 72, "R64-R91", "residue_range" ], [ 166, 169, "DNA", "chemical" ] ] }, { "sid": 84, "sent": "Together, these structural elements constitute the winged helix-turn-helix (wHTH) DNA-binding domain and, together with the dimeric organization, are the hallmarks of MarR family structures.", "section": "RESULTS", "ner": [ [ 51, 74, "winged helix-turn-helix", "structure_element" ], [ 76, 80, "wHTH", "structure_element" ], [ 82, 100, "DNA-binding domain", "structure_element" ], [ 124, 131, "dimeric", "oligomeric_state" ], [ 167, 171, "MarR", "protein_type" ], [ 179, 189, "structures", "evidence" ] ] }, { "sid": 85, "sent": "The crystal structure of NadR in complex with 4-HPA.", "section": "FIG", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 25, 29, "NadR", "protein" ], [ 30, 45, "in complex with", "protein_state" ], [ 46, 51, "4-HPA", "chemical" ] ] }, { "sid": 86, "sent": " (A) The holo-NadR homodimer is depicted in green and blue for chains A and B respectively, while yellow sticks depict the 4-HPA ligand (labelled).", "section": "FIG", "ner": [ [ 9, 13, "holo", "protein_state" ], [ 14, 18, "NadR", "protein" ], [ 19, 28, "homodimer", "oligomeric_state" ], [ 63, 77, "chains A and B", "structure_element" ], [ 123, 128, "4-HPA", "chemical" ] ] }, { "sid": 87, "sent": "For simplicity, secondary structure elements are labelled for chain B only.", "section": "FIG", "ner": [ [ 62, 69, "chain B", "structure_element" ] ] }, { "sid": 88, "sent": "Red dashes show hypothetical positions of chain B residues 88\u201390 that were not modeled due to lack of electron density.", "section": "FIG", "ner": [ [ 42, 49, "chain B", "structure_element" ], [ 59, 64, "88\u201390", "residue_range" ], [ 102, 118, "electron density", "evidence" ] ] }, { "sid": 89, "sent": "(B) A zoom into the pocket occupied by 4-HPA shows that the ligand contacts both chains A and B; blue mesh shows electron density around 4-HPA calculated from a composite omit map (omitting 4-HPA), using phenix.", "section": "FIG", "ner": [ [ 20, 26, "pocket", "site" ], [ 39, 44, "4-HPA", "chemical" ], [ 81, 95, "chains A and B", "structure_element" ], [ 113, 129, "electron density", "evidence" ], [ 137, 142, "4-HPA", "chemical" ], [ 161, 179, "composite omit map", "evidence" ], [ 190, 195, "4-HPA", "chemical" ], [ 204, 210, "phenix", "experimental_method" ] ] }, { "sid": 90, "sent": "The map is contoured at 1\u03c3 and the figure was prepared with a density mesh carve factor of 1.7, using Pymol (www.pymol.org).", "section": "FIG", "ner": [ [ 4, 7, "map", "evidence" ], [ 62, 74, "density mesh", "evidence" ] ] }, { "sid": 91, "sent": "A single conserved leucine residue (L130) is crucial for dimerization", "section": "RESULTS", "ner": [ [ 9, 18, "conserved", "protein_state" ], [ 19, 26, "leucine", "residue_name" ], [ 36, 40, "L130", "residue_name_number" ] ] }, { "sid": 92, "sent": "The NadR dimer interface is formed by at least 32 residues, which establish numerous inter-chain salt bridges or hydrogen bonds, and many hydrophobic packing interactions (Fig 3A and 3B).", "section": "RESULTS", "ner": [ [ 4, 8, "NadR", "protein" ], [ 9, 24, "dimer interface", "site" ], [ 97, 109, "salt bridges", "bond_interaction" ], [ 113, 127, "hydrogen bonds", "bond_interaction" ], [ 138, 170, "hydrophobic packing interactions", "bond_interaction" ] ] }, { "sid": 93, "sent": "To determine which residues were most important for dimerization, we studied the interface in silico and identified several residues as potential mediators of key stabilizing interactions.", "section": "RESULTS", "ner": [ [ 81, 90, "interface", "site" ] ] }, { "sid": 94, "sent": "Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface.", "section": "RESULTS", "ner": [ [ 6, 31, "site-directed mutagenesis", "experimental_method" ], [ 50, 56, "mutant", "protein_state" ], [ 57, 61, "NadR", "protein" ], [ 105, 108, "H7A", "mutant" ], [ 110, 113, "S9A", "mutant" ], [ 115, 119, "N11A", "mutant" ], [ 121, 126, "D112A", "mutant" ], [ 128, 133, "R114A", "mutant" ], [ 135, 140, "Y115A", "mutant" ], [ 142, 147, "K126A", "mutant" ], [ 149, 154, "L130K", "mutant" ], [ 159, 164, "L133K", "mutant" ], [ 200, 215, "dimer interface", "site" ] ] }, { "sid": 95, "sent": "Each mutant NadR protein was purified, and then its oligomeric state was examined by analytical SE-HPLC.", "section": "RESULTS", "ner": [ [ 5, 11, "mutant", "protein_state" ], [ 12, 16, "NadR", "protein" ], [ 85, 103, "analytical SE-HPLC", "experimental_method" ] ] }, { "sid": 96, "sent": "Almost all the mutants showed the same elution profile as the wild-type (WT) NadR protein.", "section": "RESULTS", "ner": [ [ 62, 71, "wild-type", "protein_state" ], [ 73, 75, "WT", "protein_state" ], [ 77, 81, "NadR", "protein" ] ] }, { "sid": 97, "sent": "Only the L130K mutation induced a notable change in the oligomeric state of NadR (Fig 3C).", "section": "RESULTS", "ner": [ [ 9, 14, "L130K", "mutant" ], [ 76, 80, "NadR", "protein" ] ] }, { "sid": 98, "sent": "Further, in SE-MALLS analyses, the L130K mutant displayed two distinct species in solution, approximately 80% being monomeric (a 19 kDa species), and only 20% retaining the typical native dimeric state (a 35 kDa species) (Fig 3D), demonstrating that Leu130 is crucial for stable dimerization.", "section": "RESULTS", "ner": [ [ 12, 20, "SE-MALLS", "experimental_method" ], [ 35, 40, "L130K", "mutant" ], [ 41, 47, "mutant", "protein_state" ], [ 116, 125, "monomeric", "oligomeric_state" ], [ 188, 195, "dimeric", "oligomeric_state" ], [ 250, 256, "Leu130", "residue_name_number" ] ] }, { "sid": 99, "sent": "It is notable that L130 is usually present as Leu, or an alternative bulky hydrophobic amino acid (e.g. Phe, Val), in many MarR family proteins, suggesting a conserved role in stabilizing the dimer interface.", "section": "RESULTS", "ner": [ [ 19, 23, "L130", "residue_name_number" ], [ 46, 49, "Leu", "residue_name" ], [ 104, 107, "Phe", "residue_name" ], [ 109, 112, "Val", "residue_name" ], [ 123, 127, "MarR", "protein_type" ], [ 158, 167, "conserved", "protein_state" ], [ 192, 207, "dimer interface", "site" ] ] }, { "sid": 100, "sent": "In contrast, most of the other residues identified in the NadR dimer interface were poorly conserved in the MarR family.", "section": "RESULTS", "ner": [ [ 58, 62, "NadR", "protein" ], [ 63, 78, "dimer interface", "site" ], [ 84, 100, "poorly conserved", "protein_state" ], [ 108, 112, "MarR", "protein_type" ] ] }, { "sid": 101, "sent": "Analysis of the NadR dimer interface.", "section": "FIG", "ner": [ [ 16, 20, "NadR", "protein" ], [ 21, 36, "dimer interface", "site" ] ] }, { "sid": 102, "sent": " (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137.", "section": "FIG", "ner": [ [ 28, 35, "chain A", "structure_element" ], [ 145, 157, "salt bridges", "bond_interaction" ], [ 161, 175, "hydrogen bonds", "bond_interaction" ], [ 186, 188, "Q4", "residue_name_number" ], [ 190, 192, "S5", "residue_name_number" ], [ 194, 196, "K6", "residue_name_number" ], [ 198, 200, "H7", "residue_name_number" ], [ 202, 204, "S9", "residue_name_number" ], [ 206, 209, "I10", "residue_name_number" ], [ 211, 214, "N11", "residue_name_number" ], [ 216, 219, "I15", "residue_name_number" ], [ 221, 224, "Q16", "residue_name_number" ], [ 226, 229, "R18", "residue_name_number" ], [ 231, 234, "D36", "residue_name_number" ], [ 236, 239, "R43", "residue_name_number" ], [ 241, 244, "A46", "residue_name_number" ], [ 246, 249, "Q59", "residue_name_number" ], [ 251, 254, "C61", "residue_name_number" ], [ 256, 260, "Y104", "residue_name_number" ], [ 262, 266, "D112", "residue_name_number" ], [ 268, 272, "R114", "residue_name_number" ], [ 274, 278, "Y115", "residue_name_number" ], [ 280, 284, "D116", "residue_name_number" ], [ 286, 290, "E119", "residue_name_number" ], [ 292, 296, "K126", "residue_name_number" ], [ 298, 302, "E136", "residue_name_number" ], [ 304, 308, "E141", "residue_name_number" ], [ 310, 314, "N145", "residue_name_number" ], [ 324, 356, "hydrophobic packing interactions", "bond_interaction" ], [ 367, 370, "I10", "residue_name_number" ], [ 372, 375, "I12", "residue_name_number" ], [ 377, 380, "L14", "residue_name_number" ], [ 382, 385, "I15", "residue_name_number" ], [ 387, 390, "R18", "residue_name_number" ], [ 392, 396, "Y115", "residue_name_number" ], [ 398, 402, "I118", "residue_name_number" ], [ 404, 408, "L130", "residue_name_number" ], [ 410, 414, "L133", "residue_name_number" ], [ 416, 420, "L134", "residue_name_number" ], [ 425, 429, "L137", "residue_name_number" ] ] }, { "sid": 103, "sent": "Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix \u03b16 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains).", "section": "FIG", "ner": [ [ 0, 7, "Chain B", "structure_element" ], [ 70, 95, "site-directed mutagenesis", "experimental_method" ], [ 97, 101, "E136", "residue_name_number" ], [ 115, 126, "salt bridge", "bond_interaction" ], [ 132, 136, "K126", "residue_name_number" ], [ 178, 183, "K126A", "mutant" ], [ 226, 243, "ionic interaction", "bond_interaction" ], [ 249, 251, "H7", "residue_name_number" ], [ 277, 284, "monomer", "oligomeric_state" ], [ 285, 286, "A", "structure_element" ], [ 294, 310, "electron density", "evidence" ], [ 327, 334, "monomer", "oligomeric_state" ], [ 335, 336, "B", "structure_element" ], [ 374, 379, "helix", "structure_element" ], [ 380, 382, "\u03b16", "structure_element" ], [ 403, 407, "L130", "residue_name_number" ], [ 408, 415, "chain B", "structure_element" ], [ 448, 480, "hydrophobic packing interactions", "bond_interaction" ], [ 486, 490, "L130", "residue_name_number" ], [ 492, 496, "L133", "residue_name_number" ], [ 498, 502, "L134", "residue_name_number" ], [ 507, 511, "L137", "residue_name_number" ], [ 515, 522, "chain A", "structure_element" ] ] }, { "sid": 104, "sent": "(C) SE-HPLC analyses of all mutant forms of NadR are compared with the wild-type (WT) protein.", "section": "FIG", "ner": [ [ 4, 11, "SE-HPLC", "experimental_method" ], [ 28, 34, "mutant", "protein_state" ], [ 44, 48, "NadR", "protein" ], [ 71, 80, "wild-type", "protein_state" ], [ 82, 84, "WT", "protein_state" ] ] }, { "sid": 105, "sent": "The WT and most of the mutants show a single elution peak with an absorbance maximum at 17.5 min.", "section": "FIG", "ner": [ [ 4, 6, "WT", "protein_state" ] ] }, { "sid": 106, "sent": "Only the mutation L130K has a noteworthy effect on the oligomeric state, inducing a second peak with a longer retention time and a second peak maximum at 18.6 min.", "section": "FIG", "ner": [ [ 18, 23, "L130K", "mutant" ] ] }, { "sid": 107, "sent": "To a much lesser extent, the L133K mutation also appears to induce a \u2018shoulder\u2019 to the main peak, suggesting very weak ability to disrupt the dimer. (D) SE-HPLC/MALLS analyses of the L130K mutant, shows 20% dimer and 80% monomer.", "section": "FIG", "ner": [ [ 29, 34, "L133K", "mutant" ], [ 142, 147, "dimer", "oligomeric_state" ], [ 153, 166, "SE-HPLC/MALLS", "experimental_method" ], [ 183, 188, "L130K", "mutant" ], [ 189, 195, "mutant", "protein_state" ], [ 207, 212, "dimer", "oligomeric_state" ], [ 221, 228, "monomer", "oligomeric_state" ] ] }, { "sid": 108, "sent": "The holo-NadR structure presents only one occupied ligand-binding pocket", "section": "RESULTS", "ner": [ [ 4, 8, "holo", "protein_state" ], [ 9, 13, "NadR", "protein" ], [ 14, 23, "structure", "evidence" ], [ 51, 72, "ligand-binding pocket", "site" ] ] }, { "sid": 109, "sent": "The NadR/4-HPA structure revealed the ligand-binding site nestled between the dimerization and DNA-binding domains (Fig 2).", "section": "RESULTS", "ner": [ [ 4, 14, "NadR/4-HPA", "complex_assembly" ], [ 15, 24, "structure", "evidence" ], [ 38, 57, "ligand-binding site", "site" ], [ 78, 114, "dimerization and DNA-binding domains", "structure_element" ] ] }, { "sid": 110, "sent": "The ligand showed a different position and orientation compared to salicylate complexed with MTH313 and ST1710 (see Discussion).", "section": "RESULTS", "ner": [ [ 67, 77, "salicylate", "chemical" ], [ 78, 92, "complexed with", "protein_state" ], [ 93, 99, "MTH313", "protein" ], [ 104, 110, "ST1710", "protein" ] ] }, { "sid": 111, "sent": "The binding pocket was almost entirely filled by 4-HPA and one water molecule, although there also remained a small tunnel 2-4\u00c5 in diameter and 5-6\u00c5 long leading from the pocket (proximal to the 4-hydroxyl position) to the protein surface.", "section": "RESULTS", "ner": [ [ 4, 18, "binding pocket", "site" ], [ 49, 54, "4-HPA", "chemical" ], [ 63, 68, "water", "chemical" ], [ 116, 122, "tunnel", "site" ], [ 171, 177, "pocket", "site" ] ] }, { "sid": 112, "sent": "The tunnel was lined with rather hydrophobic amino acids, and did not contain water molecules.", "section": "RESULTS", "ner": [ [ 4, 10, "tunnel", "site" ], [ 78, 83, "water", "chemical" ] ] }, { "sid": 113, "sent": "Unexpectedly, only one monomer of the holo-NadR homodimer contained 4-HPA in the binding pocket, whereas the corresponding pocket of the other monomer was unoccupied by ligand, despite the large excess of 4-HPA used in the crystallization conditions.", "section": "RESULTS", "ner": [ [ 23, 30, "monomer", "oligomeric_state" ], [ 38, 42, "holo", "protein_state" ], [ 43, 47, "NadR", "protein" ], [ 48, 57, "homodimer", "oligomeric_state" ], [ 68, 73, "4-HPA", "chemical" ], [ 81, 95, "binding pocket", "site" ], [ 123, 129, "pocket", "site" ], [ 143, 150, "monomer", "oligomeric_state" ], [ 205, 210, "4-HPA", "chemical" ] ] }, { "sid": 114, "sent": "Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A).", "section": "RESULTS", "ner": [ [ 18, 52, "protein-ligand interaction network", "site" ], [ 76, 80, "NadR", "protein" ], [ 148, 166, "hydrogen (H)-bonds", "bond_interaction" ], [ 171, 189, "ionic interactions", "bond_interaction" ], [ 237, 242, "4-HPA", "chemical" ], [ 247, 251, "Ser9", "residue_name_number" ], [ 255, 262, "chain A", "structure_element" ], [ 264, 269, "SerA9", "residue_name_number" ], [ 276, 283, "chain B", "structure_element" ], [ 293, 299, "TrpB39", "residue_name_number" ], [ 301, 307, "ArgB43", "residue_name_number" ], [ 312, 319, "TyrB115", "residue_name_number" ] ] }, { "sid": 115, "sent": "At the other \u2018end\u2019 of the ligand, the 4-hydroxyl group was proximal to AspB36, with which it may establish an H-bond (see bond distances in Table 3).", "section": "RESULTS", "ner": [ [ 71, 77, "AspB36", "residue_name_number" ], [ 110, 116, "H-bond", "bond_interaction" ] ] }, { "sid": 116, "sent": "The water molecule observed in the pocket was bound by the carboxylate group and the side chains of SerA9 and AsnA11.", "section": "RESULTS", "ner": [ [ 4, 9, "water", "chemical" ], [ 100, 105, "SerA9", "residue_name_number" ], [ 110, 116, "AsnA11", "residue_name_number" ] ] }, { "sid": 117, "sent": "Atomic details of NadR/HPA interactions.", "section": "FIG", "ner": [ [ 18, 22, "NadR", "protein" ], [ 23, 26, "HPA", "chemical" ] ] }, { "sid": 118, "sent": " A) A stereo-view zoom into the binding pocket showing side chain sticks for all interactions between NadR and 4-HPA.", "section": "FIG", "ner": [ [ 32, 46, "binding pocket", "site" ], [ 102, 106, "NadR", "protein" ], [ 111, 116, "4-HPA", "chemical" ] ] }, { "sid": 119, "sent": "Green and blue ribbons depict NadR chains A and B, respectively.", "section": "FIG", "ner": [ [ 30, 34, "NadR", "protein" ], [ 35, 49, "chains A and B", "structure_element" ] ] }, { "sid": 120, "sent": "4-HPA is shown in yellow sticks, with oxygen atoms in red.", "section": "FIG", "ner": [ [ 0, 5, "4-HPA", "chemical" ] ] }, { "sid": 121, "sent": "A water molecule is shown by the red sphere.", "section": "FIG", "ner": [ [ 2, 7, "water", "chemical" ] ] }, { "sid": 122, "sent": "H-bonds up to 3.6\u00c5 are shown as dashed lines.", "section": "FIG", "ner": [ [ 0, 7, "H-bonds", "bond_interaction" ] ] }, { "sid": 123, "sent": "The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually).", "section": "FIG", "ner": [ [ 34, 41, "H-bonds", "bond_interaction" ], [ 45, 64, "non-bonded contacts", "bond_interaction" ], [ 70, 75, "4-HPA", "chemical" ], [ 91, 96, "SerA9", "residue_name_number" ], [ 98, 104, "AsnA11", "residue_name_number" ], [ 106, 112, "LeuB21", "residue_name_number" ], [ 114, 120, "MetB22", "residue_name_number" ], [ 122, 128, "PheB25", "residue_name_number" ], [ 130, 136, "LeuB29", "residue_name_number" ], [ 138, 144, "AspB36", "residue_name_number" ], [ 146, 152, "TrpB39", "residue_name_number" ], [ 154, 160, "ArgB43", "residue_name_number" ], [ 162, 169, "ValB111", "residue_name_number" ], [ 174, 181, "TyrB115", "residue_name_number" ], [ 218, 224, "PDBsum", "experimental_method" ] ] }, { "sid": 124, "sent": "Residues AsnA11 and ArgB18 likely make indirect yet local contributions to ligand binding, mainly by stabilizing the position of AspB36.", "section": "FIG", "ner": [ [ 9, 15, "AsnA11", "residue_name_number" ], [ 20, 26, "ArgB18", "residue_name_number" ], [ 129, 135, "AspB36", "residue_name_number" ] ] }, { "sid": 125, "sent": "Side chains mediating hydrophobic interactions are shown in orange. (B) A model was prepared to visualize putative interactions of 3Cl,4-HPA (pink) with NadR, revealing the potential for additional contacts (dashed lines) of the chloro moiety (green stick) with LeuB29 and AspB36.", "section": "FIG", "ner": [ [ 22, 46, "hydrophobic interactions", "bond_interaction" ], [ 131, 140, "3Cl,4-HPA", "chemical" ], [ 153, 157, "NadR", "protein" ], [ 262, 268, "LeuB29", "residue_name_number" ], [ 273, 279, "AspB36", "residue_name_number" ] ] }, { "sid": 126, "sent": "List of 4-HPA atoms bound to NadR via ionic interactions and/or H-bonds.", "section": "TABLE", "ner": [ [ 8, 13, "4-HPA", "chemical" ], [ 29, 33, "NadR", "protein" ], [ 38, 56, "ionic interactions", "bond_interaction" ], [ 64, 71, "H-bonds", "bond_interaction" ] ] }, { "sid": 127, "sent": "4-HPA atom\tNadR residue/atom\tDistance (\u00c5)\t \tO2\tTrpB39/NE1\t2.83\t \tO2\tArgB43/NH1\t2.76\t \tO1\tArgB43/NH1\t3.84\t \tO1\tSerA9/OG\t2.75\t \tO1\tTyrB115/OH\t2.50\t \tO2\tWater (*Ser9/Asn11)\t2.88\t \tOH\tAspB36/OD1/OD2\t3.6/3.7\t \t", "section": "TABLE", "ner": [ [ 0, 5, "4-HPA", "chemical" ], [ 11, 15, "NadR", "protein" ], [ 47, 53, "TrpB39", "residue_name_number" ], [ 68, 74, "ArgB43", "residue_name_number" ], [ 89, 95, "ArgB43", "residue_name_number" ], [ 110, 115, "SerA9", "residue_name_number" ], [ 129, 136, "TyrB115", "residue_name_number" ], [ 150, 155, "Water", "chemical" ], [ 158, 162, "Ser9", "residue_name_number" ], [ 163, 168, "Asn11", "residue_name_number" ], [ 180, 186, "AspB36", "residue_name_number" ] ] }, { "sid": 128, "sent": "* Bond distance between the ligand carboxylate group and the water molecule, which in turn makes H-bond to the SerA9 and AsnA11 side chains.", "section": "TABLE", "ner": [ [ 61, 66, "water", "chemical" ], [ 97, 103, "H-bond", "bond_interaction" ], [ 111, 116, "SerA9", "residue_name_number" ], [ 121, 127, "AsnA11", "residue_name_number" ] ] }, { "sid": 129, "sent": "In addition to the H-bonds involving the carboxylate and hydroxyl groups of 4-HPA, binding of the phenyl moiety appeared to be stabilized by several van der Waals\u2019 contacts, particularly those involving the hydrophobic side chain atoms of LeuB21, MetB22, PheB25, LeuB29 and ValB111 (Fig 4A).", "section": "RESULTS", "ner": [ [ 19, 26, "H-bonds", "bond_interaction" ], [ 76, 81, "4-HPA", "chemical" ], [ 149, 172, "van der Waals\u2019 contacts", "bond_interaction" ], [ 239, 245, "LeuB21", "residue_name_number" ], [ 247, 253, "MetB22", "residue_name_number" ], [ 255, 261, "PheB25", "residue_name_number" ], [ 263, 269, "LeuB29", "residue_name_number" ], [ 274, 281, "ValB111", "residue_name_number" ] ] }, { "sid": 130, "sent": "Notably, the phenyl ring of PheB25 was positioned parallel to the phenyl ring of 4-HPA, potentially forming \u03c0-\u03c0 parallel-displaced stacking interactions.", "section": "RESULTS", "ner": [ [ 28, 34, "PheB25", "residue_name_number" ], [ 81, 86, "4-HPA", "chemical" ], [ 108, 152, "\u03c0-\u03c0 parallel-displaced stacking interactions", "bond_interaction" ] ] }, { "sid": 131, "sent": "Consequently, residues in the 4-HPA binding pocket are mostly contributed by NadR chain B, and effectively created a polar \u2018floor\u2019 and a hydrophobic \u2018ceiling\u2019, which house the ligand.", "section": "RESULTS", "ner": [ [ 30, 50, "4-HPA binding pocket", "site" ], [ 77, 81, "NadR", "protein" ], [ 82, 89, "chain B", "structure_element" ] ] }, { "sid": 132, "sent": "Collectively, this mixed network of polar and hydrophobic interactions endows NadR with a strong recognition pattern for HPAs, with additional medium-range interactions potentially established with the hydroxyl group at the 4-position.", "section": "RESULTS", "ner": [ [ 36, 70, "polar and hydrophobic interactions", "bond_interaction" ], [ 78, 82, "NadR", "protein" ], [ 121, 125, "HPAs", "chemical" ] ] }, { "sid": 133, "sent": "Structure-activity relationships: molecular basis of enhanced stabilization by 3Cl,4-HPA", "section": "RESULTS", "ner": [ [ 79, 88, "3Cl,4-HPA", "chemical" ] ] }, { "sid": 134, "sent": "We modelled the binding of other HPAs by in silico superposition onto 4-HPA in the holo-NadR structure, and thereby obtained molecular explanations for the binding specificities of diverse ligands.", "section": "RESULTS", "ner": [ [ 3, 11, "modelled", "experimental_method" ], [ 33, 37, "HPAs", "chemical" ], [ 41, 64, "in silico superposition", "experimental_method" ], [ 70, 75, "4-HPA", "chemical" ], [ 83, 87, "holo", "protein_state" ], [ 88, 92, "NadR", "protein" ], [ 93, 102, "structure", "evidence" ] ] }, { "sid": 135, "sent": "For example, similar to 4-HPA, the binding of 3Cl,4-HPA could involve multiple bonds towards the carboxylate group of the ligand and some to the 4-hydroxyl group.", "section": "RESULTS", "ner": [ [ 24, 29, "4-HPA", "chemical" ], [ 46, 55, "3Cl,4-HPA", "chemical" ] ] }, { "sid": 136, "sent": "Additionally, the side chains of LeuB29 and AspB36 would be only 2.6\u20133.5 \u00c5 from the chlorine atom, thus providing van der Waals\u2019 interactions or H-bonds to generate the additional binding affinity observed for 3Cl,4-HPA (Fig 4B).", "section": "RESULTS", "ner": [ [ 33, 39, "LeuB29", "residue_name_number" ], [ 44, 50, "AspB36", "residue_name_number" ], [ 114, 141, "van der Waals\u2019 interactions", "bond_interaction" ], [ 145, 152, "H-bonds", "bond_interaction" ], [ 180, 196, "binding affinity", "evidence" ], [ 210, 219, "3Cl,4-HPA", "chemical" ] ] }, { "sid": 137, "sent": "The presence of a single hydroxyl group at position 2, as in 2-HPA, rather than at position 4, would eliminate the possibility of favorable interactions with AspB36, resulting in the lack of NadR regulation by 2-HPA described previously.", "section": "RESULTS", "ner": [ [ 61, 66, "2-HPA", "chemical" ], [ 158, 164, "AspB36", "residue_name_number" ], [ 191, 195, "NadR", "protein" ], [ 210, 215, "2-HPA", "chemical" ] ] }, { "sid": 138, "sent": "Finally, salicylate is presumably unable to specifically bind NadR due to the 2-hydroxyl substitution and the shorter aliphatic chain connecting its carboxylate group (Fig 1A): the compound simply seems too small to simultaneously establish the network of beneficial bonds observed in the NadR/HPA interactions.", "section": "RESULTS", "ner": [ [ 9, 19, "salicylate", "chemical" ], [ 62, 66, "NadR", "protein" ], [ 289, 293, "NadR", "protein" ], [ 294, 297, "HPA", "chemical" ] ] }, { "sid": 139, "sent": "Analysis of the pockets reveals the molecular basis for asymmetric binding and stoichiometry", "section": "RESULTS", "ner": [ [ 16, 23, "pockets", "site" ] ] }, { "sid": 140, "sent": "However, studies based on tryptophan fluorescence were confounded by the fluorescence of the HPA ligands, and isothermal titration calorimetry (ITC) was unfeasible due to the need for very high concentrations of NadR in the ITC chamber (due to the relatively low affinity), which exceeded the solubility limits of the protein.", "section": "RESULTS", "ner": [ [ 26, 49, "tryptophan fluorescence", "experimental_method" ], [ 93, 96, "HPA", "chemical" ], [ 110, 142, "isothermal titration calorimetry", "experimental_method" ], [ 144, 147, "ITC", "experimental_method" ], [ 212, 216, "NadR", "protein" ], [ 224, 227, "ITC", "experimental_method" ] ] }, { "sid": 141, "sent": "However, it was possible to calculate the binding stoichiometry of the NadR-HPA interactions using an SPR-based approach.", "section": "RESULTS", "ner": [ [ 42, 63, "binding stoichiometry", "evidence" ], [ 71, 79, "NadR-HPA", "complex_assembly" ], [ 102, 105, "SPR", "experimental_method" ] ] }, { "sid": 142, "sent": "In SPR, the signal measured is proportional to the total molecular mass proximal to the sensor surface; consequently, if the molecular weights of the interactors are known, then the stoichiometry of the resulting complex can be determined.", "section": "RESULTS", "ner": [ [ 3, 6, "SPR", "experimental_method" ] ] }, { "sid": 143, "sent": "This approach relies on the assumption that the captured protein (\u2018the ligand\u2019, according to SPR conventions) is 100% active and freely-accessible to potential interactors (\u2018the analytes\u2019).", "section": "RESULTS", "ner": [ [ 93, 96, "SPR", "experimental_method" ] ] }, { "sid": 144, "sent": "Firstly, NadR is expected to be covalently immobilized on the sensor chip as a dimer in random orientations, since it is a stable dimer in solution and has sixteen lysines well-distributed around its surface, all able to act as potential sites for amine coupling to the chip, and none of which are close to the ligand-binding pocket.", "section": "RESULTS", "ner": [ [ 9, 13, "NadR", "protein" ], [ 79, 84, "dimer", "oligomeric_state" ], [ 123, 129, "stable", "protein_state" ], [ 130, 135, "dimer", "oligomeric_state" ], [ 164, 171, "lysines", "residue_name" ], [ 311, 332, "ligand-binding pocket", "site" ] ] }, { "sid": 145, "sent": "Secondly, the HPA analytes are all very small (MW 150\u2013170, Fig 1A) and therefore are expected to be able to diffuse readily into all potential binding sites, irrespective of the random orientations of the immobilized NadR dimers on the chip.", "section": "RESULTS", "ner": [ [ 14, 17, "HPA", "chemical" ], [ 143, 156, "binding sites", "site" ], [ 217, 221, "NadR", "protein" ], [ 222, 228, "dimers", "oligomeric_state" ] ] }, { "sid": 146, "sent": "The stoichiometry of the NadR-HPA interactions was determined using Eq 1 (see Materials and Methods), and revealed stoichiometries of 1.13 for 4-HPA, 1.02 for 3-HPA, and 1.21 for 3Cl,4-HPA, strongly suggesting that one NadR dimer bound to 1 HPA analyte molecule.", "section": "RESULTS", "ner": [ [ 25, 33, "NadR-HPA", "complex_assembly" ], [ 115, 130, "stoichiometries", "evidence" ], [ 143, 148, "4-HPA", "chemical" ], [ 159, 164, "3-HPA", "chemical" ], [ 179, 188, "3Cl,4-HPA", "chemical" ], [ 219, 223, "NadR", "protein" ], [ 224, 229, "dimer", "oligomeric_state" ], [ 230, 238, "bound to", "protein_state" ], [ 241, 244, "HPA", "chemical" ] ] }, { "sid": 147, "sent": "The crystallographic data, supported by the SPR studies of binding stoichiometry, revealed the lack of a second 4-HPA molecule in the homodimer, suggesting negative co-operativity, a phenomenon previously described for the MTH313/salicylate interaction and for other MarR family proteins.", "section": "RESULTS", "ner": [ [ 4, 25, "crystallographic data", "evidence" ], [ 44, 47, "SPR", "experimental_method" ], [ 59, 80, "binding stoichiometry", "evidence" ], [ 112, 117, "4-HPA", "chemical" ], [ 134, 143, "homodimer", "oligomeric_state" ], [ 223, 229, "MTH313", "protein" ], [ 230, 240, "salicylate", "chemical" ], [ 267, 271, "MarR", "protein_type" ] ] }, { "sid": 148, "sent": "To explore the molecular basis of asymmetry in holo-NadR, we superposed its ligand-free monomer (chain A) onto the ligand-occupied monomer (chain B).", "section": "RESULTS", "ner": [ [ 47, 51, "holo", "protein_state" ], [ 52, 56, "NadR", "protein" ], [ 61, 71, "superposed", "experimental_method" ], [ 76, 87, "ligand-free", "protein_state" ], [ 88, 95, "monomer", "oligomeric_state" ], [ 97, 104, "chain A", "structure_element" ], [ 115, 130, "ligand-occupied", "protein_state" ], [ 131, 138, "monomer", "oligomeric_state" ], [ 140, 147, "chain B", "structure_element" ] ] }, { "sid": 149, "sent": "Overall, the superposition revealed a high degree of structural similarity (C\u03b1 root mean square deviation (rmsd) of 1.5\u00c5), though on closer inspection a rotational difference of ~9 degrees along the long axis of helix \u03b16 was observed, suggesting that 4-HPA induced a slight conformational change (Fig 5A).", "section": "RESULTS", "ner": [ [ 13, 26, "superposition", "experimental_method" ], [ 79, 105, "root mean square deviation", "evidence" ], [ 107, 111, "rmsd", "evidence" ], [ 212, 217, "helix", "structure_element" ], [ 218, 220, "\u03b16", "structure_element" ], [ 251, 256, "4-HPA", "chemical" ] ] }, { "sid": 150, "sent": "However, since residues of helix \u03b16 were not directly involved in ligand binding, an explanation for the lack of 4-HPA in monomer A did not emerge by analyzing only these backbone atom positions, suggesting that a more complex series of allosteric events may occur.", "section": "RESULTS", "ner": [ [ 27, 32, "helix", "structure_element" ], [ 33, 35, "\u03b16", "structure_element" ], [ 113, 118, "4-HPA", "chemical" ], [ 122, 129, "monomer", "oligomeric_state" ], [ 130, 131, "A", "structure_element" ] ] }, { "sid": 151, "sent": "Indeed, we noted interesting differences in the side chains of Met22, Phe25 and Arg43, which in monomer B are used to contact the ligand while in monomer A they partially occupied the pocket and collectively reduced its volume significantly.", "section": "RESULTS", "ner": [ [ 63, 68, "Met22", "residue_name_number" ], [ 70, 75, "Phe25", "residue_name_number" ], [ 80, 85, "Arg43", "residue_name_number" ], [ 96, 103, "monomer", "oligomeric_state" ], [ 104, 105, "B", "structure_element" ], [ 146, 153, "monomer", "oligomeric_state" ], [ 154, 155, "A", "structure_element" ], [ 184, 190, "pocket", "site" ] ] }, { "sid": 152, "sent": "Specifically, upon analysis with the CASTp software, the pocket in chain B containing the 4-HPA exhibited a total volume of approximately 370 \u00c53, while the pocket in chain A was occupied by these three side chains that adopted \u2018inward\u2019 positions and thereby divided the space into a few much smaller pockets, each with volume < 50 \u00c53, evidently rendering chain A unfavorable for ligand binding.", "section": "RESULTS", "ner": [ [ 37, 42, "CASTp", "experimental_method" ], [ 57, 63, "pocket", "site" ], [ 67, 74, "chain B", "structure_element" ], [ 90, 95, "4-HPA", "chemical" ], [ 156, 162, "pocket", "site" ], [ 166, 173, "chain A", "structure_element" ], [ 228, 234, "inward", "protein_state" ], [ 355, 362, "chain A", "structure_element" ] ] }, { "sid": 153, "sent": "Most notably, atomic clashes between the ligand and the side chains of MetA22, PheA25 and ArgA43 would occur if 4-HPA were present in the monomer A pocket (Fig 5B).", "section": "RESULTS", "ner": [ [ 71, 77, "MetA22", "residue_name_number" ], [ 79, 85, "PheA25", "residue_name_number" ], [ 90, 96, "ArgA43", "residue_name_number" ], [ 112, 117, "4-HPA", "chemical" ], [ 138, 145, "monomer", "oligomeric_state" ], [ 146, 147, "A", "structure_element" ], [ 148, 154, "pocket", "site" ] ] }, { "sid": 154, "sent": "Subsequently, analyses of the pockets in apo-NadR revealed that in the absence of ligand the long Arg43 side chain was always in the open \u2018outward\u2019 position compatible with binding to the 4-HPA carboxylate group.", "section": "RESULTS", "ner": [ [ 30, 37, "pockets", "site" ], [ 41, 44, "apo", "protein_state" ], [ 45, 49, "NadR", "protein" ], [ 71, 88, "absence of ligand", "protein_state" ], [ 98, 103, "Arg43", "residue_name_number" ], [ 139, 146, "outward", "protein_state" ], [ 188, 193, "4-HPA", "chemical" ] ] }, { "sid": 155, "sent": "In contrast, the apo-form Met22 and Phe25 residues were still encroaching the spaces of the 4-hydroxyl group and the phenyl ring of the ligand, respectively (Fig 5C).", "section": "RESULTS", "ner": [ [ 17, 20, "apo", "protein_state" ], [ 26, 31, "Met22", "residue_name_number" ], [ 36, 41, "Phe25", "residue_name_number" ] ] }, { "sid": 156, "sent": "The \u2018outward\u2019 position of Arg43 generated an open apo-form pocket with volume approximately 380\u00c53.", "section": "RESULTS", "ner": [ [ 5, 12, "outward", "protein_state" ], [ 26, 31, "Arg43", "residue_name_number" ], [ 45, 49, "open", "protein_state" ], [ 50, 53, "apo", "protein_state" ], [ 59, 65, "pocket", "site" ] ] }, { "sid": 157, "sent": "Taken together, these observations suggest that Arg43 is a major determinant of ligand binding, and that its \u2018inward\u2019 position inhibits the binding of 4-HPA to the empty pocket of holo-NadR.", "section": "RESULTS", "ner": [ [ 48, 53, "Arg43", "residue_name_number" ], [ 110, 116, "inward", "protein_state" ], [ 151, 156, "4-HPA", "chemical" ], [ 170, 176, "pocket", "site" ], [ 180, 184, "holo", "protein_state" ], [ 185, 189, "NadR", "protein" ] ] }, { "sid": 158, "sent": "Structural differences of NadR in ligand-bound or free forms.", "section": "FIG", "ner": [ [ 26, 30, "NadR", "protein" ], [ 34, 46, "ligand-bound", "protein_state" ], [ 50, 54, "free", "protein_state" ] ] }, { "sid": 159, "sent": " (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix \u03b16. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt \u2018inward\u2019 positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these \u2018inward\u2019 conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively.", "section": "FIG", "ner": [ [ 5, 12, "Aligned", "experimental_method" ], [ 13, 21, "monomers", "oligomeric_state" ], [ 25, 29, "holo", "protein_state" ], [ 30, 34, "NadR", "protein" ], [ 36, 43, "chain A", "structure_element" ], [ 52, 59, "chain B", "structure_element" ], [ 117, 122, "helix", "structure_element" ], [ 123, 125, "\u03b16", "structure_element" ], [ 131, 141, "Comparison", "experimental_method" ], [ 153, 168, "binding pockets", "site" ], [ 172, 176, "holo", "protein_state" ], [ 177, 181, "NadR", "protein" ], [ 200, 211, "ligand-free", "protein_state" ], [ 212, 219, "monomer", "oligomeric_state" ], [ 220, 221, "A", "structure_element" ], [ 239, 244, "Met22", "residue_name_number" ], [ 246, 251, "Phe25", "residue_name_number" ], [ 256, 261, "Arg43", "residue_name_number" ], [ 269, 275, "inward", "protein_state" ], [ 327, 342, "ligand-occupied", "protein_state" ], [ 343, 349, "pocket", "site" ], [ 374, 380, "inward", "protein_state" ], [ 430, 435, "4-HPA", "chemical" ] ] }, { "sid": 160, "sent": "In these crystals, the ArgA43 side chain showed two alternate conformations, modelled with 50% occupancy in each state, as indicated by the two \u2018mirrored\u2019 arrows.", "section": "FIG", "ner": [ [ 9, 17, "crystals", "evidence" ], [ 23, 29, "ArgA43", "residue_name_number" ] ] }, { "sid": 161, "sent": "The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the \u2018outward\u2019 conformation.", "section": "FIG", "ner": [ [ 67, 72, "4-HPA", "chemical" ], [ 115, 121, "pocket", "site" ], [ 127, 131, "holo", "protein_state" ], [ 132, 136, "NadR", "protein" ], [ 174, 181, "pockets", "site" ], [ 185, 188, "apo", "protein_state" ], [ 189, 193, "NadR", "protein" ], [ 229, 239, "absence of", "protein_state" ], [ 240, 245, "4-HPA", "chemical" ], [ 250, 255, "Arg43", "residue_name_number" ], [ 294, 301, "outward", "protein_state" ] ] }, { "sid": 162, "sent": "Finally, we applied 15N heteronuclear solution NMR spectroscopy to examine the interaction of 4-HPA with apo NadR. We collected NMR spectra on NadR in the presence and absence of 4-HPA (see Materials and Methods).", "section": "RESULTS", "ner": [ [ 20, 63, "15N heteronuclear solution NMR spectroscopy", "experimental_method" ], [ 94, 99, "4-HPA", "chemical" ], [ 105, 108, "apo", "protein_state" ], [ 109, 113, "NadR", "protein" ], [ 128, 131, "NMR", "experimental_method" ], [ 132, 139, "spectra", "evidence" ], [ 143, 147, "NadR", "protein" ], [ 148, 163, "in the presence", "protein_state" ], [ 168, 178, "absence of", "protein_state" ], [ 179, 184, "4-HPA", "chemical" ] ] }, { "sid": 163, "sent": "The 1H-15N TROSY-HSQC spectrum of apo-NadR, acquired at 25\u00b0C, displayed approximately 140 distinct peaks (Fig 6A), most of which correspond to backbone amide N-H groups.", "section": "RESULTS", "ner": [ [ 4, 21, "1H-15N TROSY-HSQC", "experimental_method" ], [ 22, 30, "spectrum", "evidence" ], [ 34, 37, "apo", "protein_state" ], [ 38, 42, "NadR", "protein" ] ] }, { "sid": 164, "sent": "The broad spectral dispersion and the number of peaks observed, which is close to the number of expected backbone amide N-H groups for this polypeptide, confirmed that apo-NadR is well-folded under these conditions and exhibits one conformation appreciable on the NMR timescale, i.e. in the NMR experiments at 25\u00b0C, two or more distinct conformations of apo-NadR monomers were not readily apparent.", "section": "RESULTS", "ner": [ [ 168, 171, "apo", "protein_state" ], [ 172, 176, "NadR", "protein" ], [ 180, 191, "well-folded", "protein_state" ], [ 264, 267, "NMR", "experimental_method" ], [ 291, 294, "NMR", "experimental_method" ], [ 354, 357, "apo", "protein_state" ], [ 358, 362, "NadR", "protein" ], [ 363, 371, "monomers", "oligomeric_state" ] ] }, { "sid": 165, "sent": "Upon the addition of 4-HPA, over 45 peaks showed chemical shift perturbations, i.e. changed position in the spectrum or disappeared, while the remaining peaks remained unchanged.", "section": "RESULTS", "ner": [ [ 21, 26, "4-HPA", "chemical" ] ] }, { "sid": 166, "sent": "This observation showed that 4-HPA was able to bind NadR and induce notable changes in specific regions of the protein.", "section": "RESULTS", "ner": [ [ 29, 34, "4-HPA", "chemical" ], [ 52, 56, "NadR", "protein" ] ] }, { "sid": 167, "sent": "NMR spectra of NadR in the presence and absence of 4-HPA.", "section": "FIG", "ner": [ [ 0, 3, "NMR", "experimental_method" ], [ 4, 11, "spectra", "evidence" ], [ 15, 19, "NadR", "protein" ], [ 20, 35, "in the presence", "protein_state" ], [ 40, 50, "absence of", "protein_state" ], [ 51, 56, "4-HPA", "chemical" ] ] }, { "sid": 168, "sent": " (A) Superposition of two 1H-15N TROSY-HSQC spectra recorded at 25\u00b0C on apo-NadR (cyan) and on NadR in the presence of 4-HPA (red).", "section": "FIG", "ner": [ [ 5, 18, "Superposition", "experimental_method" ], [ 26, 43, "1H-15N TROSY-HSQC", "experimental_method" ], [ 44, 51, "spectra", "evidence" ], [ 72, 75, "apo", "protein_state" ], [ 76, 80, "NadR", "protein" ], [ 95, 99, "NadR", "protein" ], [ 107, 118, "presence of", "protein_state" ], [ 119, 124, "4-HPA", "chemical" ] ] }, { "sid": 169, "sent": "(B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25\u00b0C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10\u00b0C of apo-NadR (blue) and NadR/4-HPA (green).", "section": "FIG", "ner": [ [ 6, 13, "Overlay", "experimental_method" ], [ 41, 58, "1H-15N TROSY-HSQC", "experimental_method" ], [ 59, 66, "spectra", "evidence" ], [ 87, 90, "apo", "protein_state" ], [ 91, 95, "NadR", "protein" ], [ 107, 117, "NadR/4-HPA", "complex_assembly" ], [ 124, 136, "superimposed", "experimental_method" ], [ 146, 153, "spectra", "evidence" ], [ 174, 177, "apo", "protein_state" ], [ 178, 182, "NadR", "protein" ], [ 194, 204, "NadR/4-HPA", "complex_assembly" ] ] }, { "sid": 170, "sent": "The spectra acquired at 10\u00b0C are excluded from panel A for simplicity.", "section": "FIG", "ner": [ [ 4, 11, "spectra", "evidence" ] ] }, { "sid": 171, "sent": "However, in the presence of 4-HPA, the 1H-15N TROSY-HSQC spectrum of NadR displayed approximately 140 peaks, as for apo-NadR, i.e. two distinct stable conformations (that might have potentially revealed the molecular asymmetry observed crystallographically) were not notable.", "section": "RESULTS", "ner": [ [ 16, 27, "presence of", "protein_state" ], [ 28, 33, "4-HPA", "chemical" ], [ 39, 56, "1H-15N TROSY-HSQC", "experimental_method" ], [ 57, 65, "spectrum", "evidence" ], [ 69, 73, "NadR", "protein" ], [ 116, 119, "apo", "protein_state" ], [ 120, 124, "NadR", "protein" ], [ 236, 256, "crystallographically", "experimental_method" ] ] }, { "sid": 172, "sent": "Considering the small size, fast diffusion and relatively low binding affinity of 4-HPA, it would not be surprising if the ligand associates and dissociates rapidly on the NMR time scale, resulting in only one set of peaks whose chemical shifts represent the average environment of the bound and unbound states.", "section": "RESULTS", "ner": [ [ 62, 78, "binding affinity", "evidence" ], [ 82, 87, "4-HPA", "chemical" ], [ 172, 175, "NMR", "experimental_method" ], [ 286, 291, "bound", "protein_state" ], [ 296, 303, "unbound", "protein_state" ] ] }, { "sid": 173, "sent": "Interestingly, by cooling the samples to 10\u00b0C, we observed that a number of those peaks strongly affected by 4-HPA (and therefore likely to be in the ligand-binding site) demonstrated evidence of peak splitting, i.e. a tendency to become two distinct peaks rather than one single peak (Fig 6B and 6C).", "section": "RESULTS", "ner": [ [ 109, 114, "4-HPA", "chemical" ], [ 150, 169, "ligand-binding site", "site" ] ] }, { "sid": 174, "sent": "These doubled peaks may therefore reveal that the cooler temperature partially trapped the existence in solution of two distinct states, in presence or absence of 4-HPA, with minor conformational differences occurring at least in proximity to the binding pocket.", "section": "RESULTS", "ner": [ [ 140, 148, "presence", "protein_state" ], [ 152, 162, "absence of", "protein_state" ], [ 163, 168, "4-HPA", "chemical" ], [ 247, 261, "binding pocket", "site" ] ] }, { "sid": 175, "sent": "Although more comprehensive NMR experiments and full chemical shift assignment of the spectra would be required to precisely define this multi-state behavior, the NMR data clearly demonstrate that NadR exhibits conformational flexibility which is modulated by 4-HPA in solution.", "section": "RESULTS", "ner": [ [ 28, 31, "NMR", "experimental_method" ], [ 86, 93, "spectra", "evidence" ], [ 163, 166, "NMR", "experimental_method" ], [ 197, 201, "NadR", "protein" ], [ 260, 265, "4-HPA", "chemical" ] ] }, { "sid": 176, "sent": "Apo-NadR structures reveal intrinsic conformational flexibility", "section": "RESULTS", "ner": [ [ 0, 3, "Apo", "protein_state" ], [ 4, 8, "NadR", "protein" ], [ 9, 19, "structures", "evidence" ] ] }, { "sid": 177, "sent": "The apo-NadR crystal structure contained two homodimers in the asymmetric unit (chains A+B and chains C+D).", "section": "RESULTS", "ner": [ [ 4, 7, "apo", "protein_state" ], [ 8, 12, "NadR", "protein" ], [ 13, 30, "crystal structure", "evidence" ], [ 45, 55, "homodimers", "oligomeric_state" ], [ 80, 90, "chains A+B", "structure_element" ], [ 95, 105, "chains C+D", "structure_element" ] ] }, { "sid": 178, "sent": "Upon overall structural superposition, these dimers revealed a few minor differences in the \u03b16 helix (a major component of the dimer interface) and the helices \u03b14-\u03b15 (the DNA binding region), and an rmsd of 1.55\u00c5 (Fig 7A).", "section": "RESULTS", "ner": [ [ 13, 37, "structural superposition", "experimental_method" ], [ 45, 51, "dimers", "oligomeric_state" ], [ 92, 100, "\u03b16 helix", "structure_element" ], [ 127, 142, "dimer interface", "site" ], [ 152, 159, "helices", "structure_element" ], [ 160, 165, "\u03b14-\u03b15", "structure_element" ], [ 171, 189, "DNA binding region", "site" ], [ 199, 203, "rmsd", "evidence" ] ] }, { "sid": 179, "sent": "Similarly, the entire holo-homodimer could be closely superposed onto each of the apo-homodimers, showing rmsd values of 1.29\u00c5 and 1.31\u00c5, and with more notable differences in the \u03b16 helix positions (Fig 7B).", "section": "RESULTS", "ner": [ [ 22, 26, "holo", "protein_state" ], [ 27, 36, "homodimer", "oligomeric_state" ], [ 46, 64, "closely superposed", "experimental_method" ], [ 82, 85, "apo", "protein_state" ], [ 86, 96, "homodimers", "oligomeric_state" ], [ 106, 110, "rmsd", "evidence" ], [ 179, 187, "\u03b16 helix", "structure_element" ] ] }, { "sid": 180, "sent": "The slightly larger rmsd between the two apo-homodimers, rather than between apo- and holo-homodimers, further indicate that apo-NadR possesses a notable degree of intrinsic conformational flexibility.", "section": "RESULTS", "ner": [ [ 20, 24, "rmsd", "evidence" ], [ 41, 44, "apo", "protein_state" ], [ 45, 55, "homodimers", "oligomeric_state" ], [ 77, 80, "apo", "protein_state" ], [ 86, 90, "holo", "protein_state" ], [ 91, 101, "homodimers", "oligomeric_state" ], [ 125, 128, "apo", "protein_state" ], [ 129, 133, "NadR", "protein" ] ] }, { "sid": 181, "sent": "Overall apo- and holo-NadR structures are similar.", "section": "FIG", "ner": [ [ 8, 11, "apo", "protein_state" ], [ 17, 21, "holo", "protein_state" ], [ 22, 26, "NadR", "protein" ], [ 27, 37, "structures", "evidence" ] ] }, { "sid": 182, "sent": " (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers.", "section": "FIG", "ner": [ [ 5, 23, "Pairwise alignment", "experimental_method" ], [ 44, 47, "apo", "protein_state" ], [ 48, 52, "NadR", "protein" ], [ 53, 63, "homodimers", "oligomeric_state" ], [ 65, 67, "AB", "structure_element" ], [ 72, 74, "CD", "structure_element" ], [ 91, 94, "apo", "protein_state" ], [ 95, 99, "NadR", "protein" ], [ 100, 108, "crystals", "evidence" ], [ 114, 123, "Alignment", "experimental_method" ], [ 131, 135, "holo", "protein_state" ], [ 136, 140, "NadR", "protein" ], [ 141, 150, "homodimer", "oligomeric_state" ], [ 184, 187, "apo", "protein_state" ], [ 188, 192, "NadR", "protein" ], [ 193, 203, "homodimers", "oligomeric_state" ] ] }, { "sid": 183, "sent": "Here, larger differences are observed in the \u03b16 helices (top).", "section": "FIG", "ner": [ [ 45, 55, "\u03b16 helices", "structure_element" ] ] }, { "sid": 184, "sent": "4-HPA stabilizes concerted conformational changes in NadR that prevent DNA-binding", "section": "RESULTS", "ner": [ [ 0, 5, "4-HPA", "chemical" ], [ 53, 57, "NadR", "protein" ] ] }, { "sid": 185, "sent": "To further investigate the conformational rearrangements of NadR, we performed local structural alignments using only a subset of residues in the DNA-binding helix (\u03b14).", "section": "RESULTS", "ner": [ [ 60, 64, "NadR", "protein" ], [ 79, 106, "local structural alignments", "experimental_method" ], [ 146, 163, "DNA-binding helix", "structure_element" ], [ 165, 167, "\u03b14", "structure_element" ] ] }, { "sid": 186, "sent": "By selecting and aligning residues Arg64-Ala77 of one \u03b14 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure.", "section": "RESULTS", "ner": [ [ 3, 12, "selecting", "experimental_method" ], [ 17, 25, "aligning", "experimental_method" ], [ 35, 46, "Arg64-Ala77", "residue_range" ], [ 54, 62, "\u03b14 helix", "structure_element" ], [ 67, 72, "dimer", "oligomeric_state" ], [ 74, 87, "superposition", "experimental_method" ], [ 95, 99, "holo", "protein_state" ], [ 100, 109, "homodimer", "oligomeric_state" ], [ 123, 126, "apo", "protein_state" ], [ 127, 137, "homodimers", "oligomeric_state" ], [ 166, 173, "monomer", "oligomeric_state" ], [ 196, 205, "structure", "evidence" ] ] }, { "sid": 187, "sent": "While one monomer from each structure was closely superimposable (Fig 8A, left side), the second monomer displayed quite large differences (Fig 8A, right side).", "section": "RESULTS", "ner": [ [ 10, 17, "monomer", "oligomeric_state" ], [ 28, 37, "structure", "evidence" ], [ 97, 104, "monomer", "oligomeric_state" ] ] }, { "sid": 188, "sent": "Most notably, the position of the DNA-binding helix \u03b14 shifted by as much as 6 \u00c5 (Fig 8B).", "section": "RESULTS", "ner": [ [ 34, 37, "DNA", "chemical" ], [ 46, 51, "helix", "structure_element" ], [ 52, 54, "\u03b14", "structure_element" ] ] }, { "sid": 189, "sent": "Accordingly, helix \u03b14 was also found to be one of the most dynamic regions in previous HDX-MS analyses of apo-NadR in solution.", "section": "RESULTS", "ner": [ [ 13, 18, "helix", "structure_element" ], [ 19, 21, "\u03b14", "structure_element" ], [ 87, 93, "HDX-MS", "experimental_method" ], [ 106, 109, "apo", "protein_state" ], [ 110, 114, "NadR", "protein" ] ] }, { "sid": 190, "sent": "Structural comparisons of NadR and modelling of interactions with DNA.", "section": "FIG", "ner": [ [ 0, 22, "Structural comparisons", "experimental_method" ], [ 26, 30, "NadR", "protein" ], [ 66, 69, "DNA", "chemical" ] ] }, { "sid": 191, "sent": " (A) The holo-homodimer structure is shown as green and blue cartoons, for chain A and B, respectively, while the two homodimers of apo-NadR are both cyan and pale blue for chains A/C and B/D, respectively.", "section": "FIG", "ner": [ [ 9, 13, "holo", "protein_state" ], [ 14, 23, "homodimer", "oligomeric_state" ], [ 24, 33, "structure", "evidence" ], [ 75, 88, "chain A and B", "structure_element" ], [ 118, 128, "homodimers", "oligomeric_state" ], [ 132, 135, "apo", "protein_state" ], [ 136, 140, "NadR", "protein" ], [ 180, 183, "A/C", "structure_element" ], [ 188, 191, "B/D", "structure_element" ] ] }, { "sid": 192, "sent": "The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix \u03b14 (left).", "section": "FIG", "ner": [ [ 10, 20, "homodimers", "oligomeric_state" ], [ 29, 31, "AB", "structure_element" ], [ 32, 36, "holo", "protein_state" ], [ 38, 40, "AB", "structure_element" ], [ 41, 44, "apo", "protein_state" ], [ 50, 52, "CD", "structure_element" ], [ 53, 56, "apo", "protein_state" ], [ 63, 71, "overlaid", "experimental_method" ], [ 75, 95, "structural alignment", "experimental_method" ], [ 139, 146, "R64-A77", "residue_range" ], [ 196, 197, "A", "structure_element" ], [ 198, 202, "holo", "protein_state" ], [ 204, 205, "A", "structure_element" ], [ 206, 209, "apo", "protein_state" ], [ 215, 216, "C", "structure_element" ], [ 217, 220, "apo", "protein_state" ], [ 235, 240, "helix", "structure_element" ], [ 241, 243, "\u03b14", "structure_element" ] ] }, { "sid": 193, "sent": "The \u03b14 helices aligned closely, C\u03b1 rmsd 0.2\u00c5 for 14 residues.", "section": "FIG", "ner": [ [ 4, 14, "\u03b14 helices", "structure_element" ], [ 35, 39, "rmsd", "evidence" ] ] }, { "sid": 194, "sent": "(B) The relative positions of the \u03b14 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue.", "section": "FIG", "ner": [ [ 34, 44, "\u03b14 helices", "structure_element" ], [ 52, 63, "4-HPA-bound", "protein_state" ], [ 64, 68, "holo", "protein_state" ], [ 69, 78, "homodimer", "oligomeric_state" ], [ 79, 86, "chain B", "structure_element" ], [ 102, 105, "apo", "protein_state" ], [ 106, 116, "homodimers", "oligomeric_state" ], [ 117, 119, "AB", "structure_element" ], [ 124, 126, "CD", "structure_element" ], [ 136, 150, "chains B and D", "structure_element" ] ] }, { "sid": 195, "sent": "Dashes indicate the Ala77 C\u03b1 atoms, in the most highly shifted region of the \u2018non-fixed\u2019 \u03b14 helix.", "section": "FIG", "ner": [ [ 20, 25, "Ala77", "residue_name_number" ], [ 89, 97, "\u03b14 helix", "structure_element" ] ] }, { "sid": 196, "sent": "(C) The double-stranded DNA molecule (grey cartoon) from the OhrR-ohrA complex is shown after superposition with NadR, to highlight the expected positions of the NadR \u03b14 helices in the DNA major grooves.", "section": "FIG", "ner": [ [ 24, 27, "DNA", "chemical" ], [ 61, 70, "OhrR-ohrA", "complex_assembly" ], [ 94, 107, "superposition", "experimental_method" ], [ 113, 117, "NadR", "protein" ], [ 162, 166, "NadR", "protein" ], [ 167, 177, "\u03b14 helices", "structure_element" ], [ 185, 188, "DNA", "chemical" ] ] }, { "sid": 197, "sent": "For clarity, only the \u03b14 helices are shown in panels (B) and (C). (D) Upon comparison with the experimentally-determined OhrR:ohrA structure (grey), the \u03b14 helix of holo-NadR (blue) is shifted ~8\u00c5 out of the major groove.", "section": "FIG", "ner": [ [ 22, 32, "\u03b14 helices", "structure_element" ], [ 121, 130, "OhrR:ohrA", "complex_assembly" ], [ 131, 140, "structure", "evidence" ], [ 153, 161, "\u03b14 helix", "structure_element" ], [ 165, 169, "holo", "protein_state" ], [ 170, 174, "NadR", "protein" ] ] }, { "sid": 198, "sent": "However, structural comparisons revealed that the shift of holo-NadR helix \u03b14 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the \u03b16 helices (S3 Fig).", "section": "RESULTS", "ner": [ [ 9, 31, "structural comparisons", "experimental_method" ], [ 59, 63, "holo", "protein_state" ], [ 64, 68, "NadR", "protein" ], [ 69, 74, "helix", "structure_element" ], [ 75, 77, "\u03b14", "structure_element" ], [ 93, 104, "presence of", "protein_state" ], [ 105, 110, "4-HPA", "chemical" ], [ 158, 162, "holo", "protein_state" ], [ 163, 178, "dimer interface", "site" ], [ 249, 252, "apo", "protein_state" ], [ 253, 269, "dimer interfaces", "site" ], [ 311, 321, "\u03b16 helices", "structure_element" ] ] }, { "sid": 199, "sent": "In summary, compared to ligand-stabilized holo-NadR, apo-NadR displayed an intrinsic flexibility focused in the DNA-binding region.", "section": "RESULTS", "ner": [ [ 24, 41, "ligand-stabilized", "protein_state" ], [ 42, 46, "holo", "protein_state" ], [ 47, 51, "NadR", "protein" ], [ 53, 56, "apo", "protein_state" ], [ 57, 61, "NadR", "protein" ], [ 112, 130, "DNA-binding region", "site" ] ] }, { "sid": 200, "sent": "This was also evident in the greater disorder (i.e. less well-defined electron density) in the \u03b21-\u03b22 loops of the apo dimers (density for 16 residues per dimer was missing) compared to the holo dimer (density for only 3 residues was missing).", "section": "RESULTS", "ner": [ [ 70, 86, "electron density", "evidence" ], [ 95, 106, "\u03b21-\u03b22 loops", "structure_element" ], [ 114, 117, "apo", "protein_state" ], [ 118, 124, "dimers", "oligomeric_state" ], [ 126, 133, "density", "evidence" ], [ 154, 159, "dimer", "oligomeric_state" ], [ 189, 193, "holo", "protein_state" ], [ 194, 199, "dimer", "oligomeric_state" ], [ 201, 208, "density", "evidence" ] ] }, { "sid": 201, "sent": "In holo-NadR, the distance separating the two DNA-binding \u03b14 helices was 32 \u00c5, while in apo-NadR it was 29 \u00c5 for homodimer AB, and 34 \u00c5 for homodimer CD (Fig 8C).", "section": "RESULTS", "ner": [ [ 3, 7, "holo", "protein_state" ], [ 8, 12, "NadR", "protein" ], [ 58, 68, "\u03b14 helices", "structure_element" ], [ 88, 91, "apo", "protein_state" ], [ 92, 96, "NadR", "protein" ], [ 113, 122, "homodimer", "oligomeric_state" ], [ 123, 125, "AB", "structure_element" ], [ 140, 149, "homodimer", "oligomeric_state" ], [ 150, 152, "CD", "structure_element" ] ] }, { "sid": 202, "sent": "Thus, the apo-homodimer AB presented the DNA-binding helices in a conformation similar to that observed in the protein:DNA complex of OhrR:ohrA from Bacillus subtilis (Fig 8C).", "section": "RESULTS", "ner": [ [ 10, 13, "apo", "protein_state" ], [ 14, 23, "homodimer", "oligomeric_state" ], [ 24, 26, "AB", "structure_element" ], [ 41, 60, "DNA-binding helices", "structure_element" ], [ 134, 143, "OhrR:ohrA", "complex_assembly" ], [ 149, 166, "Bacillus subtilis", "species" ] ] }, { "sid": 203, "sent": "Interestingly, OhrR contacts ohrA across 22 base pairs (bp), and similarly the main NadR target sites identified in the nadA promoter (the operators Op I and Op II) both span 22 bp.", "section": "RESULTS", "ner": [ [ 15, 19, "OhrR", "protein" ], [ 29, 33, "ohrA", "gene" ], [ 84, 88, "NadR", "protein" ], [ 89, 101, "target sites", "site" ], [ 120, 124, "nadA", "gene" ] ] }, { "sid": 204, "sent": "Pairwise superpositions showed that the NadR apo-homodimer AB was the most similar to OhrR (rmsd 2.6 \u00c5), while the holo-homodimer was the most divergent (rmsd 3.3 \u00c5) (Fig 8C).", "section": "RESULTS", "ner": [ [ 0, 23, "Pairwise superpositions", "experimental_method" ], [ 40, 44, "NadR", "protein" ], [ 45, 48, "apo", "protein_state" ], [ 49, 58, "homodimer", "oligomeric_state" ], [ 59, 61, "AB", "structure_element" ], [ 86, 90, "OhrR", "protein" ], [ 92, 96, "rmsd", "evidence" ], [ 115, 119, "holo", "protein_state" ], [ 120, 129, "homodimer", "oligomeric_state" ], [ 154, 158, "rmsd", "evidence" ] ] }, { "sid": 205, "sent": "Assuming the same DNA-binding mechanism is used by OhrR and NadR, the apo-homodimer AB seems ideally pre-configured for DNA binding, while 4-HPA appeared to stabilize holo-NadR in a conformation poorly suited for DNA binding.", "section": "RESULTS", "ner": [ [ 18, 21, "DNA", "chemical" ], [ 51, 55, "OhrR", "protein" ], [ 60, 64, "NadR", "protein" ], [ 70, 73, "apo", "protein_state" ], [ 74, 83, "homodimer", "oligomeric_state" ], [ 84, 86, "AB", "structure_element" ], [ 120, 123, "DNA", "chemical" ], [ 139, 144, "4-HPA", "chemical" ], [ 167, 171, "holo", "protein_state" ], [ 172, 176, "NadR", "protein" ], [ 213, 216, "DNA", "chemical" ] ] }, { "sid": 206, "sent": "Specifically, in addition to the different inter-helical translational distances, the \u03b14 helices in the holo-NadR homodimer were also reoriented, resulting in movement of \u03b14 out of the major groove, by up to 8\u00c5, and presumably preventing efficient DNA binding in the presence of 4-HPA (Fig 8D).", "section": "RESULTS", "ner": [ [ 43, 80, "inter-helical translational distances", "evidence" ], [ 86, 96, "\u03b14 helices", "structure_element" ], [ 104, 108, "holo", "protein_state" ], [ 109, 113, "NadR", "protein" ], [ 114, 123, "homodimer", "oligomeric_state" ], [ 171, 173, "\u03b14", "structure_element" ], [ 248, 251, "DNA", "chemical" ], [ 279, 284, "4-HPA", "chemical" ] ] }, { "sid": 207, "sent": "When aligned with OhrR, the apo-homodimer CD presented yet another different intermediate conformation (rmsd 2.9\u00c5), apparently not ideally pre-configured for DNA binding, but which in solution can presumably readily adopt the AB conformation due to the intrinsic flexibility described above.", "section": "RESULTS", "ner": [ [ 5, 12, "aligned", "experimental_method" ], [ 18, 22, "OhrR", "protein" ], [ 28, 31, "apo", "protein_state" ], [ 32, 41, "homodimer", "oligomeric_state" ], [ 42, 44, "CD", "structure_element" ], [ 104, 108, "rmsd", "evidence" ], [ 158, 161, "DNA", "chemical" ], [ 226, 228, "AB", "structure_element" ] ] }, { "sid": 208, "sent": "NadR residues His7, Ser9, Asn11 and Phe25 are essential for regulation of NadA expression in vivo", "section": "RESULTS", "ner": [ [ 0, 4, "NadR", "protein" ], [ 14, 18, "His7", "residue_name_number" ], [ 20, 24, "Ser9", "residue_name_number" ], [ 26, 31, "Asn11", "residue_name_number" ], [ 36, 41, "Phe25", "residue_name_number" ], [ 74, 78, "NadA", "protein" ] ] }, { "sid": 209, "sent": "While previous studies had correctly suggested the involvement of several NadR residues in ligand binding, the crystal structures presented here revealed additional residues with previously unknown roles in dimerization and/or binding to 4-HPA.", "section": "RESULTS", "ner": [ [ 74, 78, "NadR", "protein" ], [ 111, 129, "crystal structures", "evidence" ], [ 238, 243, "4-HPA", "chemical" ] ] }, { "sid": 210, "sent": "To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-\u03941843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants.", "section": "RESULTS", "ner": [ [ 99, 103, "NadR", "protein" ], [ 113, 116, "H7A", "mutant" ], [ 118, 121, "S9A", "mutant" ], [ 123, 127, "N11A", "mutant" ], [ 132, 136, "F25A", "mutant" ], [ 189, 199, "MC58-\u03941843", "mutant" ], [ 200, 204, "nadR", "gene" ], [ 210, 216, "mutant", "protein_state" ], [ 258, 267, "wild-type", "protein_state" ], [ 268, 272, "nadR", "gene" ], [ 283, 287, "nadR", "gene" ], [ 288, 295, "mutants", "protein_state" ] ] }, { "sid": 211, "sent": "NadA protein abundance levels were assessed by Western blotting to evaluate the ability of the NadR mutants to repress the nadA promoter, in the presence or absence of 4-HPA.", "section": "RESULTS", "ner": [ [ 0, 4, "NadA", "protein" ], [ 47, 63, "Western blotting", "experimental_method" ], [ 95, 99, "NadR", "protein" ], [ 100, 107, "mutants", "protein_state" ], [ 123, 127, "nadA", "gene" ], [ 168, 173, "4-HPA", "chemical" ] ] }, { "sid": 212, "sent": "The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9).", "section": "RESULTS", "ner": [ [ 4, 8, "nadR", "gene" ], [ 9, 12, "H7A", "mutant" ], [ 14, 17, "S9A", "mutant" ], [ 22, 26, "F25A", "mutant" ], [ 75, 79, "nadA", "gene" ], [ 129, 133, "nadA", "gene" ], [ 160, 164, "NadR", "protein" ], [ 165, 167, "WT", "protein_state" ], [ 214, 219, "4-HPA", "chemical" ], [ 248, 257, "wild-type", "protein_state" ], [ 258, 262, "nadR", "gene" ], [ 294, 298, "NadA", "protein" ], [ 323, 328, "4-HPA", "chemical" ] ] }, { "sid": 213, "sent": "Interestingly, and on the contrary, the nadR N11A complemented strain showed hypo-repression (i.e. exhibited high expression of nadA both in absence and presence of 4-HPA).", "section": "RESULTS", "ner": [ [ 40, 44, "nadR", "gene" ], [ 45, 49, "N11A", "mutant" ], [ 128, 132, "nadA", "gene" ], [ 165, 170, "4-HPA", "chemical" ] ] }, { "sid": 214, "sent": "This mutagenesis data revealed that NadR residues His7, Ser9, Asn11 and Phe25 play key roles in the ligand-mediated regulation of NadR; they are each involved in the controlled de-repression of the nadA promoter and synthesis of NadA in response to 4-HPA in vivo.", "section": "RESULTS", "ner": [ [ 5, 16, "mutagenesis", "experimental_method" ], [ 36, 40, "NadR", "protein" ], [ 50, 54, "His7", "residue_name_number" ], [ 56, 60, "Ser9", "residue_name_number" ], [ 62, 67, "Asn11", "residue_name_number" ], [ 72, 77, "Phe25", "residue_name_number" ], [ 130, 134, "NadR", "protein" ], [ 198, 202, "nadA", "gene" ], [ 229, 233, "NadA", "protein" ], [ 249, 254, "4-HPA", "chemical" ] ] }, { "sid": 215, "sent": "Structure-based point mutations shed light on ligand-induced regulation of NadR.", "section": "FIG", "ner": [ [ 0, 31, "Structure-based point mutations", "experimental_method" ], [ 75, 79, "NadR", "protein" ] ] }, { "sid": 216, "sent": "Western blot analyses of wild-type (WT) strain (lanes 1\u20132) or isogenic nadR knockout strains (\u0394NadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3\u201312) or not complemented (lanes 13\u201314), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression.", "section": "FIG", "ner": [ [ 0, 12, "Western blot", "experimental_method" ], [ 25, 34, "wild-type", "protein_state" ], [ 36, 38, "WT", "protein_state" ], [ 71, 75, "nadR", "gene" ], [ 94, 99, "\u0394NadR", "mutant" ], [ 139, 143, "NadR", "protein" ], [ 144, 146, "WT", "protein_state" ], [ 150, 156, "mutant", "protein_state" ], [ 279, 284, "4-HPA", "chemical" ], [ 294, 298, "NadA", "protein" ], [ 303, 307, "NadR", "protein" ] ] }, { "sid": 217, "sent": "Complementation of \u0394NadR with WT NadR enables induction of nadA expression by 4-HPA.", "section": "FIG", "ner": [ [ 19, 24, "\u0394NadR", "mutant" ], [ 30, 32, "WT", "protein_state" ], [ 33, 37, "NadR", "protein" ], [ 59, 63, "nadA", "gene" ], [ 78, 83, "4-HPA", "chemical" ] ] }, { "sid": 218, "sent": "The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control).", "section": "FIG", "ner": [ [ 4, 7, "H7A", "mutant" ], [ 9, 12, "S9A", "mutant" ], [ 17, 21, "F25A", "mutant" ], [ 50, 54, "nadA", "gene" ], [ 102, 104, "WT", "protein_state" ], [ 105, 109, "NadR", "protein" ], [ 115, 119, "N11A", "mutant" ], [ 120, 126, "mutant", "protein_state" ], [ 156, 160, "nadA", "gene" ], [ 205, 210, "4-HPA", "chemical" ], [ 249, 262, "meningococcal", "taxonomy_domain" ], [ 263, 287, "factor H binding protein", "protein" ], [ 289, 293, "fHbp", "protein" ] ] }, { "sid": 219, "sent": "NadA is a surface-exposed meningococcal protein contributing to pathogenesis, and is one of three main antigens present in the vaccine Bexsero.", "section": "DISCUSS", "ner": [ [ 0, 4, "NadA", "protein" ], [ 26, 39, "meningococcal", "taxonomy_domain" ] ] }, { "sid": 220, "sent": "A detailed understanding of the in vitro repression of nadA expression by the transcriptional regulator NadR is important, both because it is a relevant disease-related model of how small-molecule ligands can regulate MarR family proteins and thereby impact bacterial virulence, and because nadA expression levels are linked to the prediction of vaccine coverage.", "section": "DISCUSS", "ner": [ [ 55, 59, "nadA", "gene" ], [ 78, 103, "transcriptional regulator", "protein_type" ], [ 104, 108, "NadR", "protein" ], [ 218, 222, "MarR", "protein_type" ], [ 258, 267, "bacterial", "taxonomy_domain" ], [ 291, 295, "nadA", "gene" ] ] }, { "sid": 221, "sent": "The repressive activity of NadR can be relieved by hydroxyphenylacetate (HPA) ligands, and HDX-MS studies previously indicated that 4-HPA stabilizes dimeric NadR in a configuration incompatible with DNA binding.", "section": "DISCUSS", "ner": [ [ 27, 31, "NadR", "protein" ], [ 51, 71, "hydroxyphenylacetate", "chemical" ], [ 73, 76, "HPA", "chemical" ], [ 91, 97, "HDX-MS", "experimental_method" ], [ 132, 137, "4-HPA", "chemical" ], [ 149, 156, "dimeric", "oligomeric_state" ], [ 157, 161, "NadR", "protein" ] ] }, { "sid": 222, "sent": "Despite these and other studies, the molecular mechanisms by which ligands regulate MarR family proteins are relatively poorly understood and likely differ depending on the specific ligand.", "section": "DISCUSS", "ner": [ [ 84, 88, "MarR", "protein_type" ] ] }, { "sid": 223, "sent": "Given the importance of NadR-mediated regulation of NadA levels in the contexts of meningococcal pathogenesis, we sought to characterize NadR, and its interaction with ligands, at atomic resolution.", "section": "DISCUSS", "ner": [ [ 24, 28, "NadR", "protein" ], [ 52, 56, "NadA", "protein" ], [ 83, 96, "meningococcal", "taxonomy_domain" ], [ 137, 141, "NadR", "protein" ] ] }, { "sid": 224, "sent": "Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric.", "section": "DISCUSS", "ner": [ [ 27, 31, "NadR", "protein" ], [ 35, 42, "dimeric", "oligomeric_state" ], [ 92, 99, "dimeric", "oligomeric_state" ], [ 113, 124, "presence of", "protein_state" ], [ 125, 130, "4-HPA", "chemical" ], [ 163, 172, "monomeric", "oligomeric_state" ], [ 207, 212, "4-HPA", "chemical" ], [ 223, 227, "NadR", "protein" ], [ 295, 299, "NadR", "protein" ], [ 316, 319, "SEC", "experimental_method" ], [ 324, 341, "mass spectrometry", "experimental_method" ], [ 352, 376, "crystallographic studies", "experimental_method" ], [ 398, 402, "MarR", "protein_type" ], [ 418, 425, "dimeric", "oligomeric_state" ] ] }, { "sid": 225, "sent": "We also used structure-guided site-directed mutagenesis to identify an important conserved residue, Leu130, which stabilizes the NadR dimer interface, knowledge of which may also inform future studies to explore the regulatory mechanisms of other MarR family proteins.", "section": "DISCUSS", "ner": [ [ 13, 55, "structure-guided site-directed mutagenesis", "experimental_method" ], [ 81, 90, "conserved", "protein_state" ], [ 100, 106, "Leu130", "residue_name_number" ], [ 129, 133, "NadR", "protein" ], [ 134, 149, "dimer interface", "site" ], [ 247, 251, "MarR", "protein_type" ] ] }, { "sid": 226, "sent": "Secondly, we assessed the thermal stability and unfolding of NadR in the presence or absence of ligands.", "section": "DISCUSS", "ner": [ [ 13, 43, "assessed the thermal stability", "experimental_method" ], [ 61, 65, "NadR", "protein" ], [ 66, 81, "in the presence", "protein_state" ], [ 85, 95, "absence of", "protein_state" ] ] }, { "sid": 227, "sent": "All DSC profiles showed a single peak, suggesting that a single unfolding event simultaneously disrupted the dimer and the monomer.", "section": "DISCUSS", "ner": [ [ 4, 7, "DSC", "experimental_method" ], [ 8, 16, "profiles", "evidence" ], [ 109, 114, "dimer", "oligomeric_state" ], [ 123, 130, "monomer", "oligomeric_state" ] ] }, { "sid": 228, "sent": "HPA ligands specifically increased the stability of NadR. The largest effects were induced by the naturally-occurring compounds 4-HPA and 3Cl,4-HPA, which, in SPR assays, were found to bind NadR with KD values of 1.5 mM and 1.1 mM, respectively.", "section": "DISCUSS", "ner": [ [ 52, 56, "NadR", "protein" ], [ 128, 133, "4-HPA", "chemical" ], [ 138, 147, "3Cl,4-HPA", "chemical" ], [ 159, 169, "SPR assays", "experimental_method" ], [ 190, 194, "NadR", "protein" ], [ 200, 202, "KD", "evidence" ] ] }, { "sid": 229, "sent": "Although these NadR/HPA interactions appeared rather weak, their distinct affinities and specificities matched their in vitro effects and their biological relevance appears similar to previous proposals that certain small molecules, including some antibiotics, in the millimolar concentration range may be broad inhibitors of MarR family proteins.", "section": "DISCUSS", "ner": [ [ 15, 19, "NadR", "protein" ], [ 20, 23, "HPA", "chemical" ], [ 326, 330, "MarR", "protein_type" ] ] }, { "sid": 230, "sent": "Indeed, 4-HPA is found in human saliva and 3Cl,4-HPA is produced during inflammatory processes, suggesting that these natural ligands are encountered by N. meningitidis in the mucosa of the oropharynx during infections.", "section": "DISCUSS", "ner": [ [ 8, 13, "4-HPA", "chemical" ], [ 26, 31, "human", "species" ], [ 43, 52, "3Cl,4-HPA", "chemical" ], [ 153, 168, "N. meningitidis", "species" ] ] }, { "sid": 231, "sent": "It is also possible that NadR responds to currently unidentified HPA analogues.", "section": "DISCUSS", "ner": [ [ 25, 29, "NadR", "protein" ], [ 65, 68, "HPA", "chemical" ] ] }, { "sid": 232, "sent": "Indeed, in the NadR/4-HPA complex there was a water molecule close to the carboxylate group and also a small unfilled tunnel ~5\u00c5 long, both factors suggesting that alternative larger ligands could occupy the pocket.", "section": "DISCUSS", "ner": [ [ 15, 25, "NadR/4-HPA", "complex_assembly" ], [ 46, 51, "water", "chemical" ], [ 118, 124, "tunnel", "site" ] ] }, { "sid": 233, "sent": "The ability to respond to various ligands might enable NadR in vivo to orchestrate multiple response mechanisms and modulate expression of genes other than nadA. Ultimately, confirmation of the relevance of each ligand will require a deeper understanding of the available concentration in vivo in the host niche during bacterial colonization and inflammation.", "section": "DISCUSS", "ner": [ [ 55, 59, "NadR", "protein" ], [ 156, 160, "nadA", "gene" ], [ 319, 328, "bacterial", "taxonomy_domain" ] ] }, { "sid": 234, "sent": "Here, we determined the first crystal structures of apo-NadR and holo-NadR. These experimentally-determined structures enabled a new detailed characterization of the ligand-binding pocket.", "section": "DISCUSS", "ner": [ [ 30, 48, "crystal structures", "evidence" ], [ 52, 55, "apo", "protein_state" ], [ 56, 60, "NadR", "protein" ], [ 65, 69, "holo", "protein_state" ], [ 70, 74, "NadR", "protein" ], [ 108, 118, "structures", "evidence" ], [ 166, 187, "ligand-binding pocket", "site" ] ] }, { "sid": 235, "sent": "In holo-NadR, 4-HPA interacted directly with at least 11 polar and hydrophobic residues.", "section": "DISCUSS", "ner": [ [ 3, 7, "holo", "protein_state" ], [ 8, 12, "NadR", "protein" ], [ 14, 19, "4-HPA", "chemical" ] ] }, { "sid": 236, "sent": "Several, but not all, of these interactions were predicted previously by homology modelling combined with ligand docking in silico.", "section": "DISCUSS", "ner": [ [ 73, 91, "homology modelling", "experimental_method" ], [ 106, 120, "ligand docking", "experimental_method" ] ] }, { "sid": 237, "sent": "Subsequently, we established the functional importance of His7, Ser9, Asn11 and Phe25 in the in vitro response of meningococcus to 4-HPA, via site-directed mutagenesis.", "section": "DISCUSS", "ner": [ [ 58, 62, "His7", "residue_name_number" ], [ 64, 68, "Ser9", "residue_name_number" ], [ 70, 75, "Asn11", "residue_name_number" ], [ 80, 85, "Phe25", "residue_name_number" ], [ 114, 127, "meningococcus", "taxonomy_domain" ], [ 131, 136, "4-HPA", "chemical" ], [ 142, 167, "site-directed mutagenesis", "experimental_method" ] ] }, { "sid": 238, "sent": "More unexpectedly, the crystal structure revealed that only one molecule of 4-HPA was bound per NadR dimer.", "section": "DISCUSS", "ner": [ [ 23, 40, "crystal structure", "evidence" ], [ 76, 81, "4-HPA", "chemical" ], [ 86, 91, "bound", "protein_state" ], [ 96, 100, "NadR", "protein" ], [ 101, 106, "dimer", "oligomeric_state" ] ] }, { "sid": 239, "sent": "We confirmed this stoichiometry in solution using SPR methods.", "section": "DISCUSS", "ner": [ [ 50, 53, "SPR", "experimental_method" ] ] }, { "sid": 240, "sent": "We also used heteronuclear NMR spectroscopy to detect substantial conformational changes of NadR occurring in solution upon addition of 4-HPA.", "section": "DISCUSS", "ner": [ [ 13, 43, "heteronuclear NMR spectroscopy", "experimental_method" ], [ 92, 96, "NadR", "protein" ], [ 136, 141, "4-HPA", "chemical" ] ] }, { "sid": 241, "sent": "Moreover, NMR spectra at 10\u00b0C suggested the existence of two distinct conformations of NadR in the vicinity of the ligand-binding pocket.", "section": "DISCUSS", "ner": [ [ 10, 13, "NMR", "experimental_method" ], [ 14, 21, "spectra", "evidence" ], [ 87, 91, "NadR", "protein" ], [ 115, 136, "ligand-binding pocket", "site" ] ] }, { "sid": 242, "sent": "More powerfully, our unique crystallographic observation of this \u2018occupied vs unoccupied site\u2019 asymmetry in the NadR/4-HPA interaction is, to our knowledge, the first example reported for a MarR family protein.", "section": "DISCUSS", "ner": [ [ 28, 56, "crystallographic observation", "evidence" ], [ 66, 74, "occupied", "protein_state" ], [ 78, 88, "unoccupied", "protein_state" ], [ 112, 122, "NadR/4-HPA", "complex_assembly" ], [ 190, 194, "MarR", "protein_type" ] ] }, { "sid": 243, "sent": "Structural analyses suggested that \u2018inward\u2019 side chain positions of Met22, Phe25 and especially Arg43 precluded binding of a second ligand molecule.", "section": "DISCUSS", "ner": [ [ 0, 19, "Structural analyses", "experimental_method" ], [ 36, 42, "inward", "protein_state" ], [ 68, 73, "Met22", "residue_name_number" ], [ 75, 80, "Phe25", "residue_name_number" ], [ 96, 101, "Arg43", "residue_name_number" ] ] }, { "sid": 244, "sent": "Such a mechanism indicates negative cooperativity, which may enhance the ligand-responsiveness of NadR.", "section": "DISCUSS", "ner": [ [ 98, 102, "NadR", "protein" ] ] }, { "sid": 245, "sent": "Comparisons of the NadR/4-HPA complex with available MarR family/salicylate complexes revealed that 4-HPA has a previously unobserved binding mode.", "section": "DISCUSS", "ner": [ [ 19, 29, "NadR/4-HPA", "complex_assembly" ], [ 53, 57, "MarR", "protein_type" ], [ 65, 75, "salicylate", "chemical" ], [ 100, 105, "4-HPA", "chemical" ] ] }, { "sid": 246, "sent": "Briefly, in the M. thermoautotrophicum MTH313 dimer, one molecule of salicylate binds in the pocket of each monomer, though with two rather different positions and orientations, only one of which (site-1) is thought to be biologically relevant (Fig 10A).", "section": "DISCUSS", "ner": [ [ 16, 38, "M. thermoautotrophicum", "species" ], [ 39, 45, "MTH313", "protein" ], [ 46, 51, "dimer", "oligomeric_state" ], [ 69, 79, "salicylate", "chemical" ], [ 93, 99, "pocket", "site" ], [ 108, 115, "monomer", "oligomeric_state" ], [ 197, 203, "site-1", "site" ] ] }, { "sid": 247, "sent": "In the S. tokodaii protein ST1710, salicylate binds to the same position in each monomer of the dimer, in a site equivalent to the putative biologically relevant site of MTH313 (Fig 10B).", "section": "DISCUSS", "ner": [ [ 7, 18, "S. tokodaii", "species" ], [ 27, 33, "ST1710", "protein" ], [ 35, 45, "salicylate", "chemical" ], [ 81, 88, "monomer", "oligomeric_state" ], [ 96, 101, "dimer", "oligomeric_state" ], [ 170, 176, "MTH313", "protein" ] ] }, { "sid": 248, "sent": "Unlike other MarR family proteins which revealed multiple ligand binding interactions, we observed only 1 molecule of 4-HPA bound to NadR, suggesting a more specific and less promiscuous interaction.", "section": "DISCUSS", "ner": [ [ 13, 17, "MarR", "protein_type" ], [ 118, 123, "4-HPA", "chemical" ], [ 124, 132, "bound to", "protein_state" ], [ 133, 137, "NadR", "protein" ] ] }, { "sid": 249, "sent": "In NadR, the single molecule of 4-HPA binds in a position distinctly different from the salicylate binding site: translated by > 10 \u00c5 and with a 180\u00b0 inverted orientation (Fig 10C).", "section": "DISCUSS", "ner": [ [ 3, 7, "NadR", "protein" ], [ 32, 37, "4-HPA", "chemical" ], [ 88, 111, "salicylate binding site", "site" ] ] }, { "sid": 250, "sent": "NadR shows a ligand binding site distinct from other MarR homologues.", "section": "FIG", "ner": [ [ 0, 4, "NadR", "protein" ], [ 13, 32, "ligand binding site", "site" ], [ 53, 57, "MarR", "protein_type" ] ] }, { "sid": 251, "sent": " (A) A structural alignment of MTH313 chains A and B shows that salicylate is bound in distinct locations in each monomer; site-1 (thought to be the biologically relevant site) and site-2 differ by ~7\u00c5 (indicated by black dotted line) and also by ligand orientation.", "section": "FIG", "ner": [ [ 7, 27, "structural alignment", "experimental_method" ], [ 31, 37, "MTH313", "protein" ], [ 38, 52, "chains A and B", "structure_element" ], [ 64, 74, "salicylate", "chemical" ], [ 78, 83, "bound", "protein_state" ], [ 114, 121, "monomer", "oligomeric_state" ], [ 123, 129, "site-1", "site" ], [ 181, 187, "site-2", "site" ] ] }, { "sid": 252, "sent": "(B) A structural alignment of MTH313 chain A and ST1710 (pink) (C\u03b1 rmsd 2.3\u00c5), shows that they bind salicylate in equivalent sites (differing by only ~3\u00c5) and with the same orientation.", "section": "FIG", "ner": [ [ 6, 26, "structural alignment", "experimental_method" ], [ 30, 36, "MTH313", "protein" ], [ 37, 44, "chain A", "structure_element" ], [ 49, 55, "ST1710", "protein" ], [ 67, 71, "rmsd", "evidence" ], [ 100, 110, "salicylate", "chemical" ] ] }, { "sid": 253, "sent": "(C) Addition of holo-NadR (chain B, blue) to the alignment reveals that bound 4-HPA differs in position by > 10 \u00c5 compared to salicylate, and adopts a novel orientation.", "section": "FIG", "ner": [ [ 16, 20, "holo", "protein_state" ], [ 21, 25, "NadR", "protein" ], [ 27, 34, "chain B", "structure_element" ], [ 49, 58, "alignment", "experimental_method" ], [ 72, 77, "bound", "protein_state" ], [ 78, 83, "4-HPA", "chemical" ], [ 126, 136, "salicylate", "chemical" ] ] }, { "sid": 254, "sent": "Interestingly, a crystal structure was previously reported for a functionally-uncharacterized meningococcal homologue of NadR, termed NMB1585, which shares 16% sequence identity with NadR. The two structures can be closely aligned (rmsd 2.3 \u00c5), but NMB1585 appears unsuited for binding HPAs, since its corresponding \u2018pocket\u2019 region is occupied by several bulky hydrophobic side chains.", "section": "DISCUSS", "ner": [ [ 17, 34, "crystal structure", "evidence" ], [ 94, 107, "meningococcal", "taxonomy_domain" ], [ 121, 125, "NadR", "protein" ], [ 134, 141, "NMB1585", "protein" ], [ 183, 187, "NadR", "protein" ], [ 197, 207, "structures", "evidence" ], [ 232, 236, "rmsd", "evidence" ], [ 249, 256, "NMB1585", "protein" ], [ 286, 290, "HPAs", "chemical" ], [ 316, 323, "\u2018pocket", "site" ] ] }, { "sid": 255, "sent": "It can be speculated that MarR family members have evolved separately to engage distinct signaling molecules, thus enabling bacteria to use the overall conserved MarR scaffold to adapt and respond to diverse changing environmental stimuli experienced in their natural niches.", "section": "DISCUSS", "ner": [ [ 26, 30, "MarR", "protein_type" ], [ 124, 132, "bacteria", "taxonomy_domain" ], [ 162, 166, "MarR", "protein_type" ] ] }, { "sid": 256, "sent": "Alternatively, it is possible that other MarR homologues (e.g. NMB1585) may have no extant functional binding pocket and thus may have lost the ability to respond to a ligand, acting instead as constitutive DNA-binding regulatory proteins.", "section": "DISCUSS", "ner": [ [ 41, 45, "MarR", "protein_type" ], [ 63, 70, "NMB1585", "protein" ], [ 102, 116, "binding pocket", "site" ], [ 207, 210, "DNA", "chemical" ] ] }, { "sid": 257, "sent": "The apo-NadR crystal structures revealed two dimers with slightly different conformations, most divergent in the DNA-binding domain.", "section": "DISCUSS", "ner": [ [ 4, 7, "apo", "protein_state" ], [ 8, 12, "NadR", "protein" ], [ 13, 31, "crystal structures", "evidence" ], [ 45, 51, "dimers", "oligomeric_state" ], [ 113, 131, "DNA-binding domain", "structure_element" ] ] }, { "sid": 258, "sent": "It is not unusual for a crystal structure to reveal multiple copies of the same protein in very slightly different conformations, which are likely representative of the lowest-energy conformations sampled by the dynamic ensemble of molecular states occurring in solution, and which likely have only small energetic differences, as described previously for MexR (a MarR protein) or more recently for the solute-binding protein FhuD2.", "section": "DISCUSS", "ner": [ [ 24, 41, "crystal structure", "evidence" ], [ 356, 360, "MexR", "protein" ], [ 364, 368, "MarR", "protein_type" ], [ 403, 425, "solute-binding protein", "protein_type" ], [ 426, 431, "FhuD2", "protein" ] ] }, { "sid": 259, "sent": "Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3\u00c5), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA.", "section": "DISCUSS", "ner": [ [ 13, 17, "holo", "protein_state" ], [ 18, 22, "NadR", "protein" ], [ 23, 32, "structure", "evidence" ], [ 73, 76, "apo", "protein_state" ], [ 77, 81, "NadR", "protein" ], [ 82, 92, "structures", "evidence" ], [ 94, 98, "rmsd", "evidence" ], [ 193, 197, "NadR", "protein" ], [ 231, 236, "4-HPA", "chemical" ], [ 357, 363, "active", "protein_state" ], [ 368, 376, "inactive", "protein_state" ], [ 430, 434, "NadR", "protein" ], [ 457, 460, "DNA", "chemical" ] ] }, { "sid": 260, "sent": "Comparisons of the apo- and holo-NadR structures revealed that the largest differences occurred in the DNA-binding helix \u03b14.", "section": "DISCUSS", "ner": [ [ 19, 22, "apo", "protein_state" ], [ 28, 32, "holo", "protein_state" ], [ 33, 37, "NadR", "protein" ], [ 38, 48, "structures", "evidence" ], [ 103, 106, "DNA", "chemical" ], [ 115, 120, "helix", "structure_element" ], [ 121, 123, "\u03b14", "structure_element" ] ] }, { "sid": 261, "sent": "The shift of helix \u03b14 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices \u03b11, \u03b15, and \u03b16, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex.", "section": "DISCUSS", "ner": [ [ 13, 18, "helix", "structure_element" ], [ 19, 21, "\u03b14", "structure_element" ], [ 25, 29, "holo", "protein_state" ], [ 30, 34, "NadR", "protein" ], [ 81, 96, "dimer interface", "site" ], [ 108, 115, "helices", "structure_element" ], [ 116, 118, "\u03b11", "structure_element" ], [ 120, 122, "\u03b15", "structure_element" ], [ 128, 130, "\u03b16", "structure_element" ], [ 141, 145, "holo", "protein_state" ], [ 178, 181, "DNA", "chemical" ], [ 219, 228, "OhrR:ohrA", "complex_assembly" ] ] }, { "sid": 262, "sent": "While some flexibility of helix \u03b14 was also observed in the two apo-structures, concomitant changes in the dimer interfaces were not observed, possibly due to the absence of ligand.", "section": "DISCUSS", "ner": [ [ 26, 31, "helix", "structure_element" ], [ 32, 34, "\u03b14", "structure_element" ], [ 64, 67, "apo", "protein_state" ], [ 68, 78, "structures", "evidence" ], [ 107, 123, "dimer interfaces", "site" ], [ 163, 180, "absence of ligand", "protein_state" ] ] }, { "sid": 263, "sent": "One of the two conformations of apo-NadR appeared ideally suited for DNA-binding.", "section": "DISCUSS", "ner": [ [ 32, 35, "apo", "protein_state" ], [ 36, 40, "NadR", "protein" ], [ 69, 72, "DNA", "chemical" ] ] }, { "sid": 264, "sent": "Overall, these analyses suggest that the apo-NadR dimer has a pre-existing equilibrium that samples a variety of conformations, some of which are compatible with DNA binding.", "section": "DISCUSS", "ner": [ [ 41, 44, "apo", "protein_state" ], [ 45, 49, "NadR", "protein" ], [ 50, 55, "dimer", "oligomeric_state" ], [ 162, 165, "DNA", "chemical" ] ] }, { "sid": 265, "sent": "The noted flexibility may also explain how NadR can adapt to bind various DNA target sequences with slightly different structural features.", "section": "DISCUSS", "ner": [ [ 43, 47, "NadR", "protein" ], [ 74, 77, "DNA", "chemical" ] ] }, { "sid": 266, "sent": "Subsequently, upon ligand binding, holo-NadR adopts a structure less suited for DNA-binding and this conformation is selected and stabilized by a network of protein-ligand interactions and concomitant rearrangements at the NadR holo dimer interface.", "section": "DISCUSS", "ner": [ [ 35, 39, "holo", "protein_state" ], [ 40, 44, "NadR", "protein" ], [ 80, 83, "DNA", "chemical" ], [ 223, 227, "NadR", "protein" ], [ 228, 232, "holo", "protein_state" ], [ 233, 248, "dimer interface", "site" ] ] }, { "sid": 267, "sent": "In an alternative and less extensive manner, the binding of two salicylate molecules to the M. thermoautotrophicum protein MTH313 appeared to induce large changes in the wHTH domain, which was associated with reduced DNA-binding activity.", "section": "DISCUSS", "ner": [ [ 64, 74, "salicylate", "chemical" ], [ 92, 114, "M. thermoautotrophicum", "species" ], [ 123, 129, "MTH313", "protein" ], [ 170, 181, "wHTH domain", "structure_element" ] ] }, { "sid": 268, "sent": "Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family\u2013i.e. NadR represses these genes in the presence but not absence of 4-HPA.", "section": "DISCUSS", "ner": [ [ 31, 49, "crystal structures", "evidence" ], [ 57, 77, "transcription factor", "protein_type" ], [ 79, 83, "NadR", "protein" ], [ 119, 132, "meningococcal", "taxonomy_domain" ], [ 187, 191, "NadA", "protein" ], [ 202, 221, "structural analyses", "experimental_method" ], [ 295, 299, "NadR", "protein" ], [ 336, 349, "meningococcal", "taxonomy_domain" ], [ 369, 373, "NadR", "protein" ], [ 385, 389, "nadA", "gene" ], [ 405, 409, "NadR", "protein" ], [ 493, 497, "mafA", "gene" ], [ 534, 538, "NadR", "protein" ], [ 596, 601, "4-HPA", "chemical" ] ] }, { "sid": 269, "sent": "The latter may influence the surface abundance or secretion of maf proteins, an emerging class of highly conserved meningococcal putative adhesins and toxins with many important roles.", "section": "DISCUSS", "ner": [ [ 98, 114, "highly conserved", "protein_state" ], [ 115, 128, "meningococcal", "taxonomy_domain" ] ] }, { "sid": 270, "sent": "Further work is required to investigate how the two different promoter types influence the ligand-responsiveness of NadR during bacterial infection and may provide insights into the regulatory mechanisms occurring during these host-pathogen interactions.", "section": "DISCUSS", "ner": [ [ 116, 120, "NadR", "protein" ], [ 128, 137, "bacterial", "taxonomy_domain" ] ] }, { "sid": 271, "sent": "Ultimately, knowledge of the ligand-dependent activity of NadR will continue to deepen our understanding of nadA expression levels, which influence meningococcal pathogenesis.", "section": "DISCUSS", "ner": [ [ 58, 62, "NadR", "protein" ], [ 108, 112, "nadA", "gene" ], [ 148, 161, "meningococcal", "taxonomy_domain" ] ] }, { "sid": 272, "sent": "Structure of an OhrR-ohrA operator complex reveals the DNA binding mechanism of the MarR family", "section": "REF", "ner": [ [ 21, 25, "ohrA", "gene" ] ] }, { "sid": 273, "sent": "The structure of NMB1585, a MarR-family regulator from Neisseria meningitidis", "section": "REF", "ner": [ [ 17, 24, "NMB1585", "protein" ] ] } ] }, "PMC4888278": { "annotations": [ { "sid": 0, "sent": "Structural determinant for inducing RORgamma specific inverse agonism triggered by a synthetic benzoxazinone ligand", "section": "TITLE", "ner": [ [ 36, 44, "RORgamma", "protein" ], [ 95, 108, "benzoxazinone", "chemical" ] ] }, { "sid": 1, "sent": "The nuclear hormone receptor ROR\u03b3 regulates transcriptional genes involved in the production of the pro-inflammatory interleukin IL-17 which has been linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease.", "section": "ABSTRACT", "ner": [ [ 4, 28, "nuclear hormone receptor", "protein_type" ], [ 29, 33, "ROR\u03b3", "protein" ], [ 117, 128, "interleukin", "protein_type" ], [ 129, 134, "IL-17", "protein_type" ] ] }, { "sid": 2, "sent": "This transcriptional activity of ROR\u03b3 is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of ROR\u03b3 and a conserved LXXLL helix motif on coactivator proteins.", "section": "ABSTRACT", "ner": [ [ 33, 37, "ROR\u03b3", "protein" ], [ 103, 136, "activation function 2 (AF2) helix", "structure_element" ], [ 144, 165, "ligand binding domain", "structure_element" ], [ 169, 173, "ROR\u03b3", "protein" ], [ 180, 189, "conserved", "protein_state" ], [ 190, 207, "LXXLL helix motif", "structure_element" ] ] }, { "sid": 3, "sent": "Our goal was to develop a ROR\u03b3 specific inverse agonist that would help down regulate pro-inflammatory gene transcription by disrupting the protein protein interaction with coactivator proteins as a therapeutic agent.", "section": "ABSTRACT", "ner": [ [ 26, 30, "ROR\u03b3", "protein" ], [ 40, 55, "inverse agonist", "protein_state" ] ] }, { "sid": 4, "sent": "We identified a novel series of synthetic benzoxazinone ligands having an agonist (BIO592) and inverse agonist (BIO399) mode of action in a FRET based assay.", "section": "ABSTRACT", "ner": [ [ 42, 55, "benzoxazinone", "chemical" ], [ 74, 81, "agonist", "protein_state" ], [ 83, 89, "BIO592", "chemical" ], [ 95, 110, "inverse agonist", "protein_state" ], [ 112, 118, "BIO399", "chemical" ], [ 140, 156, "FRET based assay", "experimental_method" ] ] }, { "sid": 5, "sent": "We show that the AF2 helix of ROR\u03b3 is proteolytically sensitive when inverse agonist BIO399 binds.", "section": "ABSTRACT", "ner": [ [ 17, 26, "AF2 helix", "structure_element" ], [ 30, 34, "ROR\u03b3", "protein" ], [ 38, 63, "proteolytically sensitive", "protein_state" ], [ 69, 84, "inverse agonist", "protein_state" ], [ 85, 91, "BIO399", "chemical" ] ] }, { "sid": 6, "sent": "Using x-ray crystallography we show how small modifications on the benzoxazinone agonist BIO592 trigger inverse agonism of ROR\u03b3.", "section": "ABSTRACT", "ner": [ [ 6, 27, "x-ray crystallography", "experimental_method" ], [ 67, 80, "benzoxazinone", "chemical" ], [ 81, 88, "agonist", "protein_state" ], [ 89, 95, "BIO592", "chemical" ], [ 123, 127, "ROR\u03b3", "protein" ] ] }, { "sid": 7, "sent": "Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for ROR\u03b3 over ROR sub-family members \u03b1 and \u03b2.", "section": "ABSTRACT", "ner": [ [ 9, 31, "in vivo reporter assay", "experimental_method" ], [ 50, 65, "inverse agonist", "protein_state" ], [ 66, 72, "BIO399", "chemical" ], [ 99, 103, "ROR\u03b3", "protein" ], [ 109, 112, "ROR", "protein_type" ], [ 132, 133, "\u03b1", "protein" ], [ 138, 139, "\u03b2", "protein" ] ] }, { "sid": 8, "sent": "The synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of ROR\u03b3.", "section": "ABSTRACT", "ner": [ [ 14, 27, "benzoxazinone", "chemical" ], [ 54, 64, "FRET assay", "experimental_method" ], [ 73, 80, "agonist", "protein_state" ], [ 82, 88, "BIO592", "chemical" ], [ 93, 108, "inverse agonist", "protein_state" ], [ 110, 116, "BIO399", "chemical" ], [ 161, 168, "agonist", "protein_state" ], [ 185, 189, "ROR\u03b3", "protein" ] ] }, { "sid": 9, "sent": "The proteolytic sensitivity of the AF2 helix of ROR\u03b3 demonstrates that it destabilizes upon BIO399 inverse agonist binding perturbing the coactivator protein binding site.", "section": "ABSTRACT", "ner": [ [ 35, 44, "AF2 helix", "structure_element" ], [ 48, 52, "ROR\u03b3", "protein" ], [ 92, 98, "BIO399", "chemical" ], [ 99, 114, "inverse agonist", "protein_state" ], [ 138, 170, "coactivator protein binding site", "site" ] ] }, { "sid": 10, "sent": "Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on ROR\u03b3 as the trigger for ROR\u03b3 specific inverse agonism.", "section": "ABSTRACT", "ner": [ [ 4, 28, "structural investigation", "experimental_method" ], [ 36, 42, "BIO592", "chemical" ], [ 43, 50, "agonist", "protein_state" ], [ 55, 61, "BIO399", "chemical" ], [ 62, 77, "inverse agonist", "protein_state" ], [ 78, 88, "structures", "evidence" ], [ 108, 114, "Met358", "residue_name_number" ], [ 118, 122, "ROR\u03b3", "protein" ], [ 142, 146, "ROR\u03b3", "protein" ] ] }, { "sid": 11, "sent": "Retinoid-related orphan receptor gamma (ROR\u03b3) is a transcription factor belonging to a sub-family of nuclear receptors that includes two closely related members ROR\u03b1 and ROR\u03b2.", "section": "INTRO", "ner": [ [ 0, 38, "Retinoid-related orphan receptor gamma", "protein" ], [ 40, 44, "ROR\u03b3", "protein" ], [ 51, 71, "transcription factor", "protein_type" ], [ 101, 118, "nuclear receptors", "protein_type" ], [ 161, 165, "ROR\u03b1", "protein" ], [ 170, 174, "ROR\u03b2", "protein" ] ] }, { "sid": 12, "sent": "Even though a high degree of sequence similarity exists between the RORs, their functional roles in regulation for physiological processes involved in development and immunity are distinct.", "section": "INTRO", "ner": [ [ 68, 72, "RORs", "protein_type" ] ] }, { "sid": 13, "sent": "During development, ROR\u03b3 regulates the transcriptional genes involved in the functioning of multiple pro-inflammatory lymphocyte lineages including T helper cells (TH17cells) which are necessary for IL-17 production.", "section": "INTRO", "ner": [ [ 20, 24, "ROR\u03b3", "protein" ], [ 199, 204, "IL-17", "protein_type" ] ] }, { "sid": 14, "sent": "IL-17 is a pro-inflammatory interleukin linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease; making its transcriptional regulation through ROR\u03b3 an attractive therapeutic target.", "section": "INTRO", "ner": [ [ 0, 5, "IL-17", "protein_type" ], [ 28, 39, "interleukin", "protein_type" ], [ 197, 201, "ROR\u03b3", "protein" ] ] }, { "sid": 15, "sent": "ROR\u03b3 consists of an N-terminal DNA binding domain (DBD) connected to a C-terminal ligand binding domain (LBD) via a flexible hinge region.", "section": "INTRO", "ner": [ [ 0, 4, "ROR\u03b3", "protein" ], [ 31, 49, "DNA binding domain", "structure_element" ], [ 51, 54, "DBD", "structure_element" ], [ 82, 103, "ligand binding domain", "structure_element" ], [ 105, 108, "LBD", "structure_element" ], [ 125, 137, "hinge region", "structure_element" ] ] }, { "sid": 16, "sent": "The DBD is composed of two zinc fingers that allow it to interact with specifically encoded regions on the DNA called the nuclear receptor response elements.", "section": "INTRO", "ner": [ [ 4, 7, "DBD", "structure_element" ], [ 27, 39, "zinc fingers", "structure_element" ], [ 122, 156, "nuclear receptor response elements", "structure_element" ] ] }, { "sid": 17, "sent": "The LBD consists of a coactivator protein binding pocket and a hydrophobic ligand binding site (LBS) which are responsible for regulating transcription.", "section": "INTRO", "ner": [ [ 4, 7, "LBD", "structure_element" ], [ 22, 56, "coactivator protein binding pocket", "site" ], [ 63, 94, "hydrophobic ligand binding site", "site" ], [ 96, 99, "LBS", "site" ] ] }, { "sid": 18, "sent": "The coactivator binding pocket of ROR\u03b3 recognizes a conserved helix motif LXXLL (where X can be any amino acid) on transcriptional coactivator complexes and recruits it to activate transcription.", "section": "INTRO", "ner": [ [ 4, 30, "coactivator binding pocket", "site" ], [ 34, 38, "ROR\u03b3", "protein" ], [ 52, 61, "conserved", "protein_state" ], [ 62, 79, "helix motif LXXLL", "structure_element" ] ] }, { "sid": 19, "sent": "Like other nuclear hormone receptors, ROR\u03b3\u2019s helix12 which makes up the C-termini of the LBD is an essential part of the coactivator binding pocket and is commonly referred to as the activation function helix 2 (AF2).", "section": "INTRO", "ner": [ [ 11, 36, "nuclear hormone receptors", "protein_type" ], [ 38, 42, "ROR\u03b3", "protein" ], [ 45, 52, "helix12", "structure_element" ], [ 89, 92, "LBD", "structure_element" ], [ 121, 147, "coactivator binding pocket", "site" ], [ 183, 210, "activation function helix 2", "structure_element" ], [ 212, 215, "AF2", "structure_element" ] ] }, { "sid": 20, "sent": "In ROR\u03b3, the conformation of the AF2 helix required to form the coactivator binding pocket is mediated by a salt bridge between His479 and Tyr502 in addition to \u03c0- \u03c0 interactions between Tyr502 and Phe506.", "section": "INTRO", "ner": [ [ 3, 7, "ROR\u03b3", "protein" ], [ 33, 42, "AF2 helix", "structure_element" ], [ 64, 90, "coactivator binding pocket", "site" ], [ 108, 119, "salt bridge", "bond_interaction" ], [ 128, 134, "His479", "residue_name_number" ], [ 139, 145, "Tyr502", "residue_name_number" ], [ 161, 178, "\u03c0- \u03c0 interactions", "bond_interaction" ], [ 187, 193, "Tyr502", "residue_name_number" ], [ 198, 204, "Phe506", "residue_name_number" ] ] }, { "sid": 21, "sent": "The conformation of the AF2 helix can be modulated through targeted ligands which bind the LBS and increase the binding of the coactivator protein (agonists) or disrupt binding (inverse agonists) thereby enhancing or inhibiting transcription.", "section": "INTRO", "ner": [ [ 24, 33, "AF2 helix", "structure_element" ], [ 91, 94, "LBS", "site" ] ] }, { "sid": 22, "sent": "Since ROR\u03b3 has been demonstrated to play an important role in pro-inflammatory gene expression patterns implicated in several major autoimmune diseases, our aim was to develop ROR\u03b3 inverse agonists that would help down regulate pro-inflammatory gene transcription.", "section": "INTRO", "ner": [ [ 6, 10, "ROR\u03b3", "protein" ], [ 176, 180, "ROR\u03b3", "protein" ] ] }, { "sid": 23, "sent": "FRET results for agonist BIO592 (a) and Inverse Agonist BIO399 (b)", "section": "FIG", "ner": [ [ 0, 12, "FRET results", "evidence" ], [ 17, 24, "agonist", "protein_state" ], [ 25, 31, "BIO592", "chemical" ], [ 40, 55, "Inverse Agonist", "protein_state" ], [ 56, 62, "BIO399", "chemical" ] ] }, { "sid": 24, "sent": "Here we present the identification of two synthetic benzoxazinone ROR\u03b3 ligands, a weak agonist BIO592 (Fig.\u00a01a) and an inverse agonist BIO399 (Fig.\u00a01b) which were identified using a Fluorescence Resonance Energy transfer (FRET) based assay that monitored coactivator peptide recruitment.", "section": "INTRO", "ner": [ [ 52, 65, "benzoxazinone", "chemical" ], [ 66, 70, "ROR\u03b3", "protein" ], [ 87, 94, "agonist", "protein_state" ], [ 95, 101, "BIO592", "chemical" ], [ 119, 134, "inverse agonist", "protein_state" ], [ 135, 141, "BIO399", "chemical" ], [ 182, 239, "Fluorescence Resonance Energy transfer (FRET) based assay", "experimental_method" ] ] }, { "sid": 25, "sent": "Using partial proteolysis in combination with mass spectrometry analysis we demonstrate that the AF2 helix of ROR\u03b3 destabilizes upon BIO399 (inverse agonist) binding.", "section": "INTRO", "ner": [ [ 6, 25, "partial proteolysis", "experimental_method" ], [ 46, 63, "mass spectrometry", "experimental_method" ], [ 97, 106, "AF2 helix", "structure_element" ], [ 110, 114, "ROR\u03b3", "protein" ], [ 133, 139, "BIO399", "chemical" ], [ 141, 156, "inverse agonist", "protein_state" ] ] }, { "sid": 26, "sent": "Finally, comparing binding modes of our benzoxazinone ROR\u03b3 crystal structures to other ROR structures, we hypothesize a new mode of action for achieving inverse agonism and selectivity.", "section": "INTRO", "ner": [ [ 19, 32, "binding modes", "evidence" ], [ 40, 53, "benzoxazinone", "chemical" ], [ 54, 58, "ROR\u03b3", "protein" ], [ 59, 77, "crystal structures", "evidence" ], [ 87, 90, "ROR", "protein_type" ], [ 91, 101, "structures", "evidence" ] ] }, { "sid": 27, "sent": "Using a FRET based assay we discovered agonist BIO592 (Fig.\u00a01a) which increased the coactivator peptide TRAP220 recruitment to ROR\u03b3 (EC50 0f 58nM and Emax of 130\u00a0%) and a potent inverse agonist BIO399 (Fig.\u00a01b) which inhibited coactivator recruitment (IC50: 4.7nM).", "section": "RESULTS", "ner": [ [ 8, 24, "FRET based assay", "experimental_method" ], [ 39, 46, "agonist", "protein_state" ], [ 47, 53, "BIO592", "chemical" ], [ 104, 111, "TRAP220", "chemical" ], [ 127, 131, "ROR\u03b3", "protein" ], [ 133, 137, "EC50", "evidence" ], [ 150, 154, "Emax", "evidence" ], [ 178, 193, "inverse agonist", "protein_state" ], [ 194, 200, "BIO399", "chemical" ], [ 252, 256, "IC50", "evidence" ] ] }, { "sid": 28, "sent": "Interestingly, the structural difference between the agonist BIO592 and inverse agonist BIO399 was minor; with the 2,3-dihydrobenzo[1,4]oxazepin-4-one ring system of BIO399 being 3 atoms larger than the benzo[1,4]oxazine-3-one ring system of BIO592.", "section": "RESULTS", "ner": [ [ 53, 60, "agonist", "protein_state" ], [ 61, 67, "BIO592", "chemical" ], [ 72, 87, "inverse agonist", "protein_state" ], [ 88, 94, "BIO399", "chemical" ], [ 115, 150, "2,3-dihydrobenzo[1,4]oxazepin-4-one", "chemical" ], [ 166, 172, "BIO399", "chemical" ], [ 203, 226, "benzo[1,4]oxazine-3-one", "chemical" ], [ 242, 248, "BIO592", "chemical" ] ] }, { "sid": 29, "sent": "In order to understand how small changes in the core ring system leads to inverse agonism, we wanted to structurally determine the binding mode of both BIO592 and BIO399 in the LBS of ROR\u03b3 using x-ray crystallography.", "section": "RESULTS", "ner": [ [ 152, 158, "BIO592", "chemical" ], [ 163, 169, "BIO399", "chemical" ], [ 177, 180, "LBS", "site" ], [ 184, 188, "ROR\u03b3", "protein" ], [ 195, 216, "x-ray crystallography", "experimental_method" ] ] }, { "sid": 30, "sent": "Structure of the ROR\u03b3518-BIO592-EBI96 ternary complex is in a transcriptionally active conformation", "section": "RESULTS", "ner": [ [ 0, 9, "Structure", "evidence" ], [ 17, 37, "ROR\u03b3518-BIO592-EBI96", "complex_assembly" ], [ 80, 86, "active", "protein_state" ] ] }, { "sid": 31, "sent": " a The ternary structure of ROR\u03b3518 BIO592 and EBI96.", "section": "FIG", "ner": [ [ 7, 24, "ternary structure", "evidence" ], [ 28, 35, "ROR\u03b3518", "protein" ], [ 36, 42, "BIO592", "chemical" ], [ 47, 52, "EBI96", "chemical" ] ] }, { "sid": 32, "sent": "b ROR\u03b3 AF2 helix in the agonist conformation.", "section": "FIG", "ner": [ [ 2, 6, "ROR\u03b3", "protein" ], [ 7, 16, "AF2 helix", "structure_element" ], [ 24, 31, "agonist", "protein_state" ] ] }, { "sid": 33, "sent": "c EBI96 coactivator peptide bound in the coactivator pocket of ROR\u03b3", "section": "FIG", "ner": [ [ 2, 7, "EBI96", "chemical" ], [ 28, 36, "bound in", "protein_state" ], [ 41, 59, "coactivator pocket", "site" ], [ 63, 67, "ROR\u03b3", "protein" ] ] }, { "sid": 34, "sent": "ROR\u03b3518 bound to agonist BIO592 was crystallized with a truncated form of the coactivator peptide EBI96 to a resolution of 2.6\u00a0\u00c5 (Fig.\u00a02a).", "section": "RESULTS", "ner": [ [ 0, 7, "ROR\u03b3518", "protein" ], [ 8, 16, "bound to", "protein_state" ], [ 17, 24, "agonist", "protein_state" ], [ 25, 31, "BIO592", "chemical" ], [ 36, 48, "crystallized", "experimental_method" ], [ 56, 65, "truncated", "protein_state" ], [ 98, 103, "EBI96", "chemical" ] ] }, { "sid": 35, "sent": "The structure of the ternary complex had features similar to other ROR agonist coactivator structures in a transcriptionally active canonical three layer helix fold with the AF2 helix in the agonist conformation.", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 67, 70, "ROR", "protein_type" ], [ 71, 78, "agonist", "protein_state" ], [ 91, 101, "structures", "evidence" ], [ 107, 131, "transcriptionally active", "protein_state" ], [ 132, 164, "canonical three layer helix fold", "protein_state" ], [ 174, 183, "AF2 helix", "structure_element" ], [ 191, 198, "agonist", "protein_state" ] ] }, { "sid": 36, "sent": "The agonist conformation is stabilized by a hydrogen bond between His479 and Tyr502, in addition to \u03c0-\u03c0 interactions between His479, Tyr502 and Phe506 (Fig.\u00a02b).", "section": "RESULTS", "ner": [ [ 4, 11, "agonist", "protein_state" ], [ 44, 57, "hydrogen bond", "bond_interaction" ], [ 66, 72, "His479", "residue_name_number" ], [ 77, 83, "Tyr502", "residue_name_number" ], [ 100, 116, "\u03c0-\u03c0 interactions", "bond_interaction" ], [ 125, 131, "His479", "residue_name_number" ], [ 133, 139, "Tyr502", "residue_name_number" ], [ 144, 150, "Phe506", "residue_name_number" ] ] }, { "sid": 37, "sent": "The hydrogen bond between His479 and Tyr502 has been reported to be critical for ROR\u03b3 agonist activity.", "section": "RESULTS", "ner": [ [ 4, 17, "hydrogen bond", "bond_interaction" ], [ 26, 32, "His479", "residue_name_number" ], [ 37, 43, "Tyr502", "residue_name_number" ], [ 81, 85, "ROR\u03b3", "protein" ], [ 86, 93, "agonist", "protein_state" ] ] }, { "sid": 38, "sent": "Disrupting this interaction through mutagenesis reduced transcriptional activity of ROR\u03b3.", "section": "RESULTS", "ner": [ [ 36, 47, "mutagenesis", "experimental_method" ], [ 84, 88, "ROR\u03b3", "protein" ] ] }, { "sid": 39, "sent": "This reduced transcriptional activity has been attributed to the inability of the AF2 helix to complete the formation of the coactivator binding pocket necessary for coactivator proteins to bind.", "section": "RESULTS", "ner": [ [ 82, 91, "AF2 helix", "structure_element" ], [ 125, 151, "coactivator binding pocket", "site" ] ] }, { "sid": 40, "sent": "Electron density for the coactivator peptide EBI96 was observed for residues EFPYLLSLLG which formed a \u03b1-helix stabilized through hydrophobic interactions with the coactivator binding pocket on ROR\u03b3 (Fig.\u00a02c).", "section": "RESULTS", "ner": [ [ 0, 16, "Electron density", "evidence" ], [ 45, 50, "EBI96", "chemical" ], [ 77, 87, "EFPYLLSLLG", "structure_element" ], [ 103, 110, "\u03b1-helix", "structure_element" ], [ 130, 154, "hydrophobic interactions", "bond_interaction" ], [ 164, 190, "coactivator binding pocket", "site" ], [ 194, 198, "ROR\u03b3", "protein" ] ] }, { "sid": 41, "sent": "This interaction is further stabilized through a conserved charged clamp wherein the backbone amide of Tyr7 and carbonyl of Leu11 of EBI96 form hydrogen bonds with Glu504 (helix12) and Lys336 (helix3) of ROR\u03b3.", "section": "RESULTS", "ner": [ [ 49, 58, "conserved", "protein_state" ], [ 59, 72, "charged clamp", "structure_element" ], [ 103, 107, "Tyr7", "residue_name_number" ], [ 124, 129, "Leu11", "residue_name_number" ], [ 133, 138, "EBI96", "chemical" ], [ 144, 158, "hydrogen bonds", "bond_interaction" ], [ 164, 170, "Glu504", "residue_name_number" ], [ 172, 179, "helix12", "structure_element" ], [ 185, 191, "Lys336", "residue_name_number" ], [ 193, 199, "helix3", "structure_element" ], [ 204, 208, "ROR\u03b3", "protein" ] ] }, { "sid": 42, "sent": "Formation of this charged clamp is essential for ROR\u03b3\u2019s function for playing a role in transcriptional activation and this has been corroborated through mutagenic studies in this region.", "section": "RESULTS", "ner": [ [ 18, 31, "charged clamp", "structure_element" ], [ 49, 53, "ROR\u03b3", "protein" ], [ 153, 170, "mutagenic studies", "experimental_method" ] ] }, { "sid": 43, "sent": "BIO592 binds in a collapsed conformation stabilizing the agonist conformation of ROR\u03b3", "section": "RESULTS", "ner": [ [ 0, 6, "BIO592", "chemical" ], [ 18, 27, "collapsed", "protein_state" ], [ 57, 64, "agonist", "protein_state" ], [ 81, 85, "ROR\u03b3", "protein" ] ] }, { "sid": 44, "sent": " a Collapsed binding mode of agonist BIO592 in the hydrophobic LBS of ROR\u03b3.", "section": "FIG", "ner": [ [ 29, 36, "agonist", "protein_state" ], [ 37, 43, "BIO592", "chemical" ], [ 63, 66, "LBS", "site" ], [ 70, 74, "ROR\u03b3", "protein" ] ] }, { "sid": 45, "sent": "b Benzoxazinone ring system of agonist BIO592 packing against His479 of ROR\u03b3 stabilizing agonist conformation of the AF2 helix", "section": "FIG", "ner": [ [ 2, 15, "Benzoxazinone", "chemical" ], [ 31, 38, "agonist", "protein_state" ], [ 39, 45, "BIO592", "chemical" ], [ 62, 68, "His479", "residue_name_number" ], [ 72, 76, "ROR\u03b3", "protein" ], [ 89, 96, "agonist", "protein_state" ], [ 117, 126, "AF2 helix", "structure_element" ] ] }, { "sid": 46, "sent": "BIO592 bound in a collapsed conformational state in the LBS of ROR\u03b3 with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig.\u00a03a, Additional file 3).", "section": "RESULTS", "ner": [ [ 0, 6, "BIO592", "chemical" ], [ 7, 15, "bound in", "protein_state" ], [ 18, 27, "collapsed", "protein_state" ], [ 56, 59, "LBS", "site" ], [ 63, 67, "ROR\u03b3", "protein" ], [ 77, 83, "xylene", "chemical" ], [ 121, 127, "pocket", "site" ], [ 135, 159, "hydrophobic interactions", "bond_interaction" ], [ 165, 171, "Val376", "residue_name_number" ], [ 173, 179, "Phe378", "residue_name_number" ], [ 181, 187, "Phe388", "residue_name_number" ], [ 192, 198, "Phe401", "residue_name_number" ], [ 209, 228, "ethyl-benzoxazinone", "chemical" ], [ 249, 273, "hydrophobic interactions", "bond_interaction" ], [ 279, 285, "Trp317", "residue_name_number" ], [ 287, 293, "Leu324", "residue_name_number" ], [ 295, 301, "Met358", "residue_name_number" ], [ 303, 309, "Leu391", "residue_name_number" ], [ 311, 318, "Ile 400", "residue_name_number" ], [ 323, 329, "His479", "residue_name_number" ] ] }, { "sid": 47, "sent": "The sulfonyl group faces the entrance of the pocket, while the CF3 makes a hydrophobic contact with Ala327.", "section": "RESULTS", "ner": [ [ 4, 12, "sulfonyl", "chemical" ], [ 45, 51, "pocket", "site" ], [ 75, 94, "hydrophobic contact", "bond_interaction" ], [ 100, 106, "Ala327", "residue_name_number" ] ] }, { "sid": 48, "sent": "Hydrophobic interaction between the ethyl group of the benzoxazinone and His479 reinforce the His479 sidechain position for making the hydrogen bond with Tyr502 thereby stabilizing the agonist conformation (Fig.\u00a03b).", "section": "RESULTS", "ner": [ [ 0, 23, "Hydrophobic interaction", "bond_interaction" ], [ 55, 68, "benzoxazinone", "chemical" ], [ 73, 79, "His479", "residue_name_number" ], [ 94, 100, "His479", "residue_name_number" ], [ 135, 148, "hydrogen bond", "bond_interaction" ], [ 154, 160, "Tyr502", "residue_name_number" ], [ 185, 192, "agonist", "protein_state" ] ] }, { "sid": 49, "sent": "ROR\u03b3 AF2 helix is sensitive to proteolysis in the presence of Inverse Agonist BIO399", "section": "RESULTS", "ner": [ [ 0, 4, "ROR\u03b3", "protein" ], [ 5, 14, "AF2 helix", "structure_element" ], [ 50, 61, "presence of", "protein_state" ], [ 62, 77, "Inverse Agonist", "protein_state" ], [ 78, 84, "BIO399", "chemical" ] ] }, { "sid": 50, "sent": "Next, we attempted co-crystallization with the inverse agonist BIO399.", "section": "RESULTS", "ner": [ [ 19, 37, "co-crystallization", "experimental_method" ], [ 47, 62, "inverse agonist", "protein_state" ], [ 63, 69, "BIO399", "chemical" ] ] }, { "sid": 51, "sent": "However, extensive crystallization efforts with BIO399 and ROR\u03b3518 or other AF2 intact constructs did not produce crystals.", "section": "RESULTS", "ner": [ [ 19, 34, "crystallization", "experimental_method" ], [ 48, 54, "BIO399", "chemical" ], [ 59, 66, "ROR\u03b3518", "protein" ], [ 76, 79, "AF2", "structure_element" ], [ 80, 86, "intact", "protein_state" ], [ 114, 122, "crystals", "evidence" ] ] }, { "sid": 52, "sent": "We hypothesized that the ROR\u03b3518 coactivator peptide interaction in the FRET assay was disrupted upon BIO399 binding and that a conformational rearrangement of the AF2 helix could have occurred, hindering crystallization.", "section": "RESULTS", "ner": [ [ 25, 32, "ROR\u03b3518", "protein" ], [ 72, 82, "FRET assay", "experimental_method" ], [ 102, 108, "BIO399", "chemical" ], [ 164, 173, "AF2 helix", "structure_element" ], [ 205, 220, "crystallization", "experimental_method" ] ] }, { "sid": 53, "sent": "Specific proteolytic positions on ROR\u03b3518 when treated with Actinase E alone (Green) or in the presence of BIO399 (Red) and shared proteolytic sites (Yellow)", "section": "FIG", "ner": [ [ 34, 41, "ROR\u03b3518", "protein" ], [ 47, 59, "treated with", "experimental_method" ], [ 60, 70, "Actinase E", "protein" ], [ 95, 106, "presence of", "protein_state" ], [ 107, 113, "BIO399", "chemical" ], [ 131, 148, "proteolytic sites", "site" ] ] }, { "sid": 54, "sent": "The unfolding of the AF2 helix has been observed for other nuclear hormone receptors when bound to an inverse agonist or antagonist.", "section": "RESULTS", "ner": [ [ 21, 30, "AF2 helix", "structure_element" ], [ 59, 84, "nuclear hormone receptors", "protein_type" ], [ 90, 98, "bound to", "protein_state" ], [ 102, 117, "inverse agonist", "protein_state" ] ] }, { "sid": 55, "sent": "We used partial proteolysis in combination with mass spectrometry to determine if BIO399 was causing the AF2 helix to unfold.", "section": "RESULTS", "ner": [ [ 8, 27, "partial proteolysis", "experimental_method" ], [ 48, 65, "mass spectrometry", "experimental_method" ], [ 82, 88, "BIO399", "chemical" ], [ 105, 114, "AF2 helix", "structure_element" ] ] }, { "sid": 56, "sent": "Results of the Actinase E proteolysis experiments on ROR\u03b3518, the ternary complex of ROR\u03b3518 with agonist BIO592 and coactivator EBI96, or in the presence of inverse agonist BIO399 supported our hypothesis.", "section": "RESULTS", "ner": [ [ 15, 37, "Actinase E proteolysis", "experimental_method" ], [ 53, 60, "ROR\u03b3518", "protein" ], [ 85, 92, "ROR\u03b3518", "protein" ], [ 98, 105, "agonist", "protein_state" ], [ 106, 112, "BIO592", "chemical" ], [ 129, 134, "EBI96", "chemical" ], [ 146, 157, "presence of", "protein_state" ], [ 158, 173, "inverse agonist", "protein_state" ], [ 174, 180, "BIO399", "chemical" ] ] }, { "sid": 57, "sent": "Analysis of the fragmentation pattern showed minimal proteolytic removal of the AF2 helix by Actinase E on ROR\u03b3518 alone (ending at 504 to 506) and the ternary complex remained primarily intact (ending at 515/518) (Additional file 4).", "section": "RESULTS", "ner": [ [ 16, 37, "fragmentation pattern", "evidence" ], [ 80, 89, "AF2 helix", "structure_element" ], [ 93, 103, "Actinase E", "protein" ], [ 107, 114, "ROR\u03b3518", "protein" ], [ 132, 142, "504 to 506", "residue_range" ], [ 152, 167, "ternary complex", "protein_state" ], [ 205, 208, "515", "residue_number" ], [ 209, 212, "518", "residue_number" ] ] }, { "sid": 58, "sent": "However, in the presence of inverse agonist BIO399, the proteolytic pattern showed significantly less protection, albeit the products were more heterogeneous (majority ending at 494/495), indicating the destabilization of the AF2 helix compared to either the APO or ternary agonist complex (Fig.\u00a04, Additional file 5).", "section": "RESULTS", "ner": [ [ 16, 27, "presence of", "protein_state" ], [ 28, 43, "inverse agonist", "protein_state" ], [ 44, 50, "BIO399", "chemical" ], [ 56, 75, "proteolytic pattern", "evidence" ], [ 178, 181, "494", "residue_number" ], [ 182, 185, "495", "residue_number" ], [ 226, 235, "AF2 helix", "structure_element" ], [ 259, 262, "APO", "protein_state" ], [ 266, 289, "ternary agonist complex", "protein_state" ] ] }, { "sid": 59, "sent": "Several rounds of cocrystallization attempts with ROR\u03b3518 or other ROR\u03b3 AF2 helix containing constructs complexed with BIO399 had not produced crystals.", "section": "RESULTS", "ner": [ [ 18, 35, "cocrystallization", "experimental_method" ], [ 50, 57, "ROR\u03b3518", "protein" ], [ 67, 71, "ROR\u03b3", "protein" ], [ 72, 81, "AF2 helix", "structure_element" ], [ 104, 118, "complexed with", "protein_state" ], [ 119, 125, "BIO399", "chemical" ], [ 143, 151, "crystals", "evidence" ] ] }, { "sid": 60, "sent": "We attributed the inability to form crystals to the unfolding of the AF2 helix induced by BIO399.", "section": "RESULTS", "ner": [ [ 36, 44, "crystals", "evidence" ], [ 69, 78, "AF2 helix", "structure_element" ], [ 90, 96, "BIO399", "chemical" ] ] }, { "sid": 61, "sent": "We reasoned that if we could remove the unfolded AF2 helix using proteolysis we could produce a binary complex more amenable to crystallization.", "section": "RESULTS", "ner": [ [ 40, 48, "unfolded", "protein_state" ], [ 49, 58, "AF2 helix", "structure_element" ], [ 65, 76, "proteolysis", "experimental_method" ], [ 128, 143, "crystallization", "experimental_method" ] ] }, { "sid": 62, "sent": "AF2 truncated ROR\u03b3 BIO399 complex is more amenable to crystallization", "section": "RESULTS", "ner": [ [ 0, 13, "AF2 truncated", "protein_state" ], [ 14, 25, "ROR\u03b3 BIO399", "complex_assembly" ], [ 54, 69, "crystallization", "experimental_method" ] ] }, { "sid": 63, "sent": " a The binary structure of AF2-truncated ROR\u03b3 and BIO399.", "section": "FIG", "ner": [ [ 14, 23, "structure", "evidence" ], [ 27, 40, "AF2-truncated", "protein_state" ], [ 41, 45, "ROR\u03b3", "protein" ], [ 50, 56, "BIO399", "chemical" ] ] }, { "sid": 64, "sent": "b The superposition of inverse agonist BIO399 (Cyan) and agonist BIO592 (Green).", "section": "FIG", "ner": [ [ 6, 19, "superposition", "experimental_method" ], [ 23, 38, "inverse agonist", "protein_state" ], [ 39, 45, "BIO399", "chemical" ], [ 57, 64, "agonist", "protein_state" ], [ 65, 71, "BIO592", "chemical" ] ] }, { "sid": 65, "sent": "c Movement of Met358 and His479 in the BIO399 (Cyan) and BIO592 (Green) structures", "section": "FIG", "ner": [ [ 14, 20, "Met358", "residue_name_number" ], [ 25, 31, "His479", "residue_name_number" ], [ 39, 45, "BIO399", "chemical" ], [ 57, 63, "BIO592", "chemical" ], [ 72, 82, "structures", "evidence" ] ] }, { "sid": 66, "sent": "The Actinase E treated ROR\u03b3518 BIO399 ternary complex (aeROR\u03b3493/4) co-crystallized readily in several PEG based conditions.", "section": "RESULTS", "ner": [ [ 4, 14, "Actinase E", "protein" ], [ 23, 37, "ROR\u03b3518 BIO399", "complex_assembly" ], [ 55, 66, "aeROR\u03b3493/4", "complex_assembly" ], [ 68, 83, "co-crystallized", "experimental_method" ] ] }, { "sid": 67, "sent": "The structure of aeROR\u03b3493/4 BIO399 complex was solved to 2.3\u00a0\u00c5 and adopted a similar core fold to the BIO592 agonist crystal structure (Fig.\u00a05a, Additional file 3).", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 17, 35, "aeROR\u03b3493/4 BIO399", "complex_assembly" ], [ 48, 54, "solved", "experimental_method" ], [ 103, 109, "BIO592", "chemical" ], [ 110, 117, "agonist", "protein_state" ], [ 118, 135, "crystal structure", "evidence" ] ] }, { "sid": 68, "sent": "The aeROR\u03b3493/4 BIO399 structure diverged at the c-terminal end of Helix 11 from the ROR\u03b3518 BIO592 EBI96 structure, where helix 11 unwinds into a random coil after residue L475.", "section": "RESULTS", "ner": [ [ 4, 22, "aeROR\u03b3493/4 BIO399", "complex_assembly" ], [ 23, 32, "structure", "evidence" ], [ 67, 75, "Helix 11", "structure_element" ], [ 85, 105, "ROR\u03b3518 BIO592 EBI96", "complex_assembly" ], [ 106, 115, "structure", "evidence" ], [ 123, 131, "helix 11", "structure_element" ], [ 173, 177, "L475", "residue_name_number" ] ] }, { "sid": 69, "sent": "Inverse agonist BIO399 uses Met358 as a trigger for inverse agonism", "section": "RESULTS", "ner": [ [ 0, 15, "Inverse agonist", "protein_state" ], [ 16, 22, "BIO399", "chemical" ], [ 28, 34, "Met358", "residue_name_number" ] ] }, { "sid": 70, "sent": "BIO399 binds to the ligand binding site of ROR\u03b3 adopting a collapsed conformation as seen with BIO592 where the two compounds superimpose with an RMSD of 0.72\u00a0\u00c5 (Fig.\u00a05b).", "section": "RESULTS", "ner": [ [ 0, 6, "BIO399", "chemical" ], [ 20, 39, "ligand binding site", "site" ], [ 43, 47, "ROR\u03b3", "protein" ], [ 59, 68, "collapsed", "protein_state" ], [ 95, 101, "BIO592", "chemical" ], [ 126, 137, "superimpose", "experimental_method" ], [ 146, 150, "RMSD", "evidence" ] ] }, { "sid": 71, "sent": "The majority of the side chains within 4\u00a0\u00c5 of BIO399 and BIO592 adopt similar rotomer conformations with the exceptions of Met358 and His479 (Fig.\u00a05c).", "section": "RESULTS", "ner": [ [ 46, 52, "BIO399", "chemical" ], [ 57, 63, "BIO592", "chemical" ], [ 123, 129, "Met358", "residue_name_number" ], [ 134, 140, "His479", "residue_name_number" ] ] }, { "sid": 72, "sent": "The difference density map showed clear positive density for Met358 in an alternate rotomer conformation compared to the one observed in the molecular replacement model or the other agonist containing models (Additional file 6).", "section": "RESULTS", "ner": [ [ 4, 26, "difference density map", "evidence" ], [ 40, 56, "positive density", "evidence" ], [ 61, 67, "Met358", "residue_name_number" ], [ 141, 168, "molecular replacement model", "experimental_method" ], [ 182, 189, "agonist", "protein_state" ] ] }, { "sid": 73, "sent": "We tried to refine Met358 in the same conformation as the molecular replacement model or the other agonist containing models, but the results clearly indicated that this was not possible, thus confirming the new rotamer conformation for the Met358 sidechain in the inverse agonist bound structure.", "section": "RESULTS", "ner": [ [ 19, 25, "Met358", "residue_name_number" ], [ 58, 85, "molecular replacement model", "experimental_method" ], [ 99, 106, "agonist", "protein_state" ], [ 241, 247, "Met358", "residue_name_number" ], [ 265, 286, "inverse agonist bound", "protein_state" ], [ 287, 296, "structure", "evidence" ] ] }, { "sid": 74, "sent": "The change in rotomer conformation of Met358 between the agonist and inverse agonist structures is attributed to the gem-dimethyl group on the larger 7 membered benzoxazinone ring system of BIO399.", "section": "RESULTS", "ner": [ [ 38, 44, "Met358", "residue_name_number" ], [ 57, 64, "agonist", "protein_state" ], [ 69, 84, "inverse agonist", "protein_state" ], [ 85, 95, "structures", "evidence" ], [ 161, 174, "benzoxazinone", "chemical" ], [ 190, 196, "BIO399", "chemical" ] ] }, { "sid": 75, "sent": "The comparison of the two structures shows that the agonist conformation observed in the BIO592 structure would be perturbed by BIO399 pushing Met358 into Phe506 of the AF2 helix indicating that Met358 is a trigger for inducing inverse agonism in ROR\u03b3 (Fig.\u00a05c).", "section": "RESULTS", "ner": [ [ 4, 14, "comparison", "experimental_method" ], [ 26, 36, "structures", "evidence" ], [ 52, 59, "agonist", "protein_state" ], [ 89, 95, "BIO592", "chemical" ], [ 96, 105, "structure", "evidence" ], [ 128, 134, "BIO399", "chemical" ], [ 143, 149, "Met358", "residue_name_number" ], [ 155, 161, "Phe506", "residue_name_number" ], [ 169, 178, "AF2 helix", "structure_element" ], [ 195, 201, "Met358", "residue_name_number" ], [ 247, 251, "ROR\u03b3", "protein" ] ] }, { "sid": 76, "sent": "BIO399 and Inverse agonist T0901317 bind in a collapsed conformation distinct from other ROR\u03b3 Inverse Agonists Cocrystal structures", "section": "RESULTS", "ner": [ [ 0, 6, "BIO399", "chemical" ], [ 11, 26, "Inverse agonist", "protein_state" ], [ 27, 35, "T0901317", "chemical" ], [ 46, 55, "collapsed", "protein_state" ], [ 89, 93, "ROR\u03b3", "protein" ], [ 111, 131, "Cocrystal structures", "evidence" ] ] }, { "sid": 77, "sent": " a Overlay of ROR\u03b3 structures bound to BIO596 (Green), BIO399 (Cyan) and T0901317 (Pink).", "section": "FIG", "ner": [ [ 3, 10, "Overlay", "experimental_method" ], [ 14, 18, "ROR\u03b3", "protein" ], [ 19, 29, "structures", "evidence" ], [ 30, 38, "bound to", "protein_state" ], [ 39, 45, "BIO596", "chemical" ], [ 55, 61, "BIO399", "chemical" ], [ 73, 81, "T0901317", "chemical" ] ] }, { "sid": 78, "sent": "b Overlay of M358 in ROR\u03b3 structure BIO596 (Green), BIO399 (Cyan), Digoxin (Yellow), Compound 2 (Grey), Compound 48 (Salmon) and Compound 4j (Orange)", "section": "FIG", "ner": [ [ 2, 9, "Overlay", "experimental_method" ], [ 13, 17, "M358", "residue_name_number" ], [ 21, 25, "ROR\u03b3", "protein" ], [ 26, 35, "structure", "evidence" ], [ 36, 42, "BIO596", "chemical" ], [ 52, 58, "BIO399", "chemical" ], [ 67, 74, "Digoxin", "chemical" ] ] }, { "sid": 79, "sent": "The co-crystal structure of ROR\u03b3 with T0901317 (PDB code: 4NB6), an inverse agonist of ROR\u03b3 (IC50 of 54nM in an SRC1 displacement FRET assay and an IC50 of 59nM in our FRET assay (Additional file 7)) shows that it adopts a collapsed conformation similar to the structure of BIO399 described here.", "section": "RESULTS", "ner": [ [ 4, 24, "co-crystal structure", "evidence" ], [ 28, 32, "ROR\u03b3", "protein" ], [ 38, 46, "T0901317", "chemical" ], [ 68, 83, "inverse agonist", "protein_state" ], [ 87, 91, "ROR\u03b3", "protein" ], [ 93, 97, "IC50", "evidence" ], [ 112, 140, "SRC1 displacement FRET assay", "experimental_method" ], [ 148, 152, "IC50", "evidence" ], [ 168, 178, "FRET assay", "experimental_method" ], [ 223, 232, "collapsed", "protein_state" ], [ 261, 270, "structure", "evidence" ], [ 274, 280, "BIO399", "chemical" ] ] }, { "sid": 80, "sent": "The two compounds superimpose with an RMSD of 0.81\u00a0\u00c5 (Fig.\u00a06a).", "section": "RESULTS", "ner": [ [ 18, 29, "superimpose", "experimental_method" ], [ 38, 42, "RMSD", "evidence" ] ] }, { "sid": 81, "sent": "The CF3 group on the hexafluoropropanol group of T0901317 was reported to fit the electron density in two conformations one of which pushes Met358 into the vicinity of Phe506 in the ROR\u03b3 BIO592 agonist structure.", "section": "RESULTS", "ner": [ [ 21, 39, "hexafluoropropanol", "chemical" ], [ 49, 57, "T0901317", "chemical" ], [ 82, 98, "electron density", "evidence" ], [ 140, 146, "Met358", "residue_name_number" ], [ 168, 174, "Phe506", "residue_name_number" ], [ 182, 186, "ROR\u03b3", "protein" ], [ 187, 193, "BIO592", "chemical" ], [ 194, 201, "agonist", "protein_state" ], [ 202, 211, "structure", "evidence" ] ] }, { "sid": 82, "sent": "We hypothesize that since the Met358 sidechain conformation in the T0901317 ROR\u03b3 structure is not in the BIO399 conformation, this difference could account for the 10-fold reduction in the inverse agonism for T0901317 compared to BIO399 in the FRET assay.", "section": "RESULTS", "ner": [ [ 30, 36, "Met358", "residue_name_number" ], [ 67, 75, "T0901317", "chemical" ], [ 76, 80, "ROR\u03b3", "protein" ], [ 81, 90, "structure", "evidence" ], [ 105, 111, "BIO399", "chemical" ], [ 209, 217, "T0901317", "chemical" ], [ 230, 236, "BIO399", "chemical" ], [ 244, 254, "FRET assay", "experimental_method" ] ] }, { "sid": 83, "sent": "Co-crystal structures of ROR\u03b3 have been generated with several potent inverse agonists adopting a linear conformation distinct from the collapsed conformations seen for BIO399 and T090131718.", "section": "RESULTS", "ner": [ [ 0, 21, "Co-crystal structures", "evidence" ], [ 25, 29, "ROR\u03b3", "protein" ], [ 98, 104, "linear", "protein_state" ], [ 136, 145, "collapsed", "protein_state" ], [ 169, 175, "BIO399", "chemical" ], [ 180, 190, "T090131718", "chemical" ] ] }, { "sid": 84, "sent": "The inverse agonist activity for these compounds has been attributed to orientating Trp317 to clash with Tyr502 or a direct inverse agonist hydrogen bonding event with His479, both of which would perturb the agonist conformation of ROR\u03b3.", "section": "RESULTS", "ner": [ [ 4, 19, "inverse agonist", "protein_state" ], [ 84, 90, "Trp317", "residue_name_number" ], [ 105, 111, "Tyr502", "residue_name_number" ], [ 124, 139, "inverse agonist", "protein_state" ], [ 140, 156, "hydrogen bonding", "bond_interaction" ], [ 168, 174, "His479", "residue_name_number" ], [ 208, 215, "agonist", "protein_state" ], [ 232, 236, "ROR\u03b3", "protein" ] ] }, { "sid": 85, "sent": "BIO399 neither orients the sidechain of Trp317 toward Tyr502 nor forms a hydrogen bond with His479 suggesting its mode of action is distinct from linear inverse agonists (Additional file 8).", "section": "RESULTS", "ner": [ [ 0, 6, "BIO399", "chemical" ], [ 40, 46, "Trp317", "residue_name_number" ], [ 54, 60, "Tyr502", "residue_name_number" ], [ 73, 86, "hydrogen bond", "bond_interaction" ], [ 92, 98, "His479", "residue_name_number" ] ] }, { "sid": 86, "sent": "In the linear inverse agonist crystal structures the side chain of Met358 resides in a similar position as the rotomer observed in ROR\u03b3 agonist structures with BIO592 described here or as observed in the hydroxycholesterol derivatives and therefore would not trigger inverse agonism with these ligands (Fig.\u00a06b).", "section": "RESULTS", "ner": [ [ 14, 29, "inverse agonist", "protein_state" ], [ 30, 48, "crystal structures", "evidence" ], [ 67, 73, "Met358", "residue_name_number" ], [ 131, 135, "ROR\u03b3", "protein" ], [ 136, 143, "agonist", "protein_state" ], [ 144, 154, "structures", "evidence" ], [ 160, 166, "BIO592", "chemical" ], [ 204, 222, "hydroxycholesterol", "chemical" ] ] }, { "sid": 87, "sent": "BIO399 shows selectivity for ROR\u03b3 over ROR\u03b1 and ROR\u03b2 in a GAL4 Cellular Reporter Assay", "section": "RESULTS", "ner": [ [ 0, 6, "BIO399", "chemical" ], [ 29, 33, "ROR\u03b3", "protein" ], [ 39, 43, "ROR\u03b1", "protein" ], [ 48, 52, "ROR\u03b2", "protein" ], [ 58, 86, "GAL4 Cellular Reporter Assay", "experimental_method" ] ] }, { "sid": 88, "sent": "GAL4 cell assay selectivity profile for BIO399 toward ROR\u03b1 and ROR\u03b2 in GAL4", "section": "TABLE", "ner": [ [ 0, 15, "GAL4 cell assay", "experimental_method" ], [ 40, 46, "BIO399", "chemical" ], [ 54, 58, "ROR\u03b1", "protein" ], [ 63, 67, "ROR\u03b2", "protein" ], [ 71, 75, "GAL4", "protein" ] ] }, { "sid": 89, "sent": " a Overlay of ROR\u03b1 (yellow), \u03b2 (pink) and \u03b3 (cyan) showing side chain differences at Met358 inverse agonism trigger position and (b) around the benzoxazinone ring system of BIO399", "section": "FIG", "ner": [ [ 3, 10, "Overlay", "experimental_method" ], [ 14, 18, "ROR\u03b1", "protein" ], [ 29, 30, "\u03b2", "protein" ], [ 42, 43, "\u03b3", "protein" ], [ 85, 91, "Met358", "residue_name_number" ], [ 144, 157, "benzoxazinone", "chemical" ], [ 173, 179, "BIO399", "chemical" ] ] }, { "sid": 90, "sent": "In order to assess the in vivo selectivity profile of BIO399 a cellular reporter assay was implemented where the ligand binding domains of ROR \u03b1, \u03b2 and \u03b3 were fused to the DNA binding domain of the transcriptional factor GAL4.", "section": "RESULTS", "ner": [ [ 54, 60, "BIO399", "chemical" ], [ 63, 86, "cellular reporter assay", "experimental_method" ], [ 113, 135, "ligand binding domains", "structure_element" ], [ 139, 142, "ROR", "protein_type" ], [ 143, 144, "\u03b1", "protein" ], [ 146, 147, "\u03b2", "protein" ], [ 152, 153, "\u03b3", "protein" ], [ 159, 167, "fused to", "experimental_method" ], [ 172, 190, "DNA binding domain", "structure_element" ], [ 198, 220, "transcriptional factor", "protein_type" ], [ 221, 225, "GAL4", "protein" ] ] }, { "sid": 91, "sent": "The ROR-GAL4 fusion proteins were expressed in cells with the luciferase reporter gene under the control of a GAL4 promoter.", "section": "RESULTS", "ner": [ [ 4, 7, "ROR", "protein_type" ], [ 8, 12, "GAL4", "protein" ], [ 110, 114, "GAL4", "protein" ] ] }, { "sid": 92, "sent": "BIO399 inhibited the luciferase activity when added to the cells expressing the ROR\u03b3-GAL4 fusion with an in vivo IC50 of 42.5nM while showing >235 and 28 fold selectivity over cells expressing GAL4 fused to the LBD of ROR \u03b1 or \u03b2, respectively (Table\u00a01).", "section": "RESULTS", "ner": [ [ 0, 6, "BIO399", "chemical" ], [ 80, 84, "ROR\u03b3", "protein" ], [ 85, 89, "GAL4", "protein" ], [ 113, 117, "IC50", "evidence" ], [ 193, 197, "GAL4", "protein" ], [ 211, 214, "LBD", "structure_element" ], [ 218, 221, "ROR", "protein_type" ], [ 222, 223, "\u03b1", "protein" ], [ 227, 228, "\u03b2", "protein" ] ] }, { "sid": 93, "sent": "The LBS of RORs share a high degree of similarity.", "section": "RESULTS", "ner": [ [ 4, 7, "LBS", "site" ], [ 11, 15, "RORs", "protein_type" ] ] }, { "sid": 94, "sent": "However, the inverse agonism trigger of BIO399, residue Met358, is a leucine in both ROR\u03b1 and \u03b2.", "section": "RESULTS", "ner": [ [ 40, 46, "BIO399", "chemical" ], [ 56, 62, "Met358", "residue_name_number" ], [ 69, 76, "leucine", "residue_name" ], [ 85, 89, "ROR\u03b1", "protein" ], [ 94, 95, "\u03b2", "protein" ] ] }, { "sid": 95, "sent": "This selectivity profile for BIO399 is attributed to the shorter leucine side chain in ROR\u03b1 and \u03b2 which would not reach the phenylalanine on the AF2 helix further underscoring the role of Met358 as a trigger for ROR\u03b3 specific inverse agonism (Fig.\u00a07a).", "section": "RESULTS", "ner": [ [ 29, 35, "BIO399", "chemical" ], [ 65, 72, "leucine", "residue_name" ], [ 87, 91, "ROR\u03b1", "protein" ], [ 96, 97, "\u03b2", "protein" ], [ 124, 137, "phenylalanine", "residue_name" ], [ 145, 154, "AF2 helix", "structure_element" ], [ 188, 194, "Met358", "residue_name_number" ], [ 212, 216, "ROR\u03b3", "protein" ] ] }, { "sid": 96, "sent": "Furthermore, ROR\u03b1 contains two phenylalanine residues in its LBS whereas ROR\u03b2 and \u03b3 have a leucine in the same position (Fig.\u00a06b).", "section": "RESULTS", "ner": [ [ 13, 17, "ROR\u03b1", "protein" ], [ 31, 44, "phenylalanine", "residue_name" ], [ 61, 64, "LBS", "site" ], [ 73, 77, "ROR\u03b2", "protein" ], [ 82, 83, "\u03b3", "protein" ], [ 91, 98, "leucine", "residue_name" ] ] }, { "sid": 97, "sent": "We hypothesize that the two phenylalanine residues in the LBS of ROR\u03b1 occlude the dihydrobenzoxazepinone ring system of BIO399 from binding it and responsible for the increase in selectivity for ROR\u03b1 over \u03b2.", "section": "RESULTS", "ner": [ [ 28, 41, "phenylalanine", "residue_name" ], [ 58, 61, "LBS", "site" ], [ 65, 69, "ROR\u03b1", "protein" ], [ 82, 104, "dihydrobenzoxazepinone", "chemical" ], [ 120, 126, "BIO399", "chemical" ], [ 195, 199, "ROR\u03b1", "protein" ], [ 205, 206, "\u03b2", "protein" ] ] }, { "sid": 98, "sent": "We have identified a novel series of synthetic benzoxazinone ligands which modulate the transcriptional activity of ROR\u03b3 in a FRET based assay.", "section": "CONCL", "ner": [ [ 47, 60, "benzoxazinone", "chemical" ], [ 116, 120, "ROR\u03b3", "protein" ], [ 126, 142, "FRET based assay", "experimental_method" ] ] }, { "sid": 99, "sent": "Using partial proteolysis we show a conformational change which destabilizes the AF2 helix of ROR\u03b3 when the inverse agonist BIO399 binds.", "section": "CONCL", "ner": [ [ 6, 25, "partial proteolysis", "experimental_method" ], [ 81, 90, "AF2 helix", "structure_element" ], [ 94, 98, "ROR\u03b3", "protein" ], [ 108, 123, "inverse agonist", "protein_state" ], [ 124, 130, "BIO399", "chemical" ] ] }, { "sid": 100, "sent": "The two ROR\u03b3 co-crystal structures reported here show how a small change to the core ring system can modulate the mode of action from agonist (BIO592) to inverse agonism (BIO399).", "section": "CONCL", "ner": [ [ 8, 12, "ROR\u03b3", "protein" ], [ 13, 34, "co-crystal structures", "evidence" ], [ 134, 141, "agonist", "protein_state" ], [ 143, 149, "BIO592", "chemical" ], [ 171, 177, "BIO399", "chemical" ] ] }, { "sid": 101, "sent": "Finally, we are reporting a newly identified trigger for achieving ROR\u03b3 specific inverse agonism in an in vivo setting through Met358 which perturbs the agonist conformation of the AF2 helix and prevents coactivator protein binding.", "section": "CONCL", "ner": [ [ 67, 71, "ROR\u03b3", "protein" ], [ 127, 133, "Met358", "residue_name_number" ], [ 153, 160, "agonist", "protein_state" ], [ 181, 190, "AF2 helix", "structure_element" ] ] } ] }, "PMC4784909": { "annotations": [ { "sid": 0, "sent": "The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle", "section": "TITLE", "ner": [ [ 24, 34, "Coenzyme A", "chemical" ], [ 50, 59, "Bacterial", "taxonomy_domain" ] ] }, { "sid": 1, "sent": "Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla.", "section": "ABSTRACT", "ner": [ [ 0, 9, "Bacterial", "taxonomy_domain" ], [ 10, 27, "Microcompartments", "complex_assembly" ], [ 29, 33, "BMCs", "complex_assembly" ] ] }, { "sid": 2, "sent": "The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate.", "section": "ABSTRACT", "ner": [ [ 16, 25, "catabolic", "protein_state" ], [ 26, 30, "BMCs", "complex_assembly" ], [ 32, 45, "metabolosomes", "complex_assembly" ], [ 141, 149, "aldehyde", "chemical" ] ] }, { "sid": 3, "sent": "The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source.", "section": "ABSTRACT", "ner": [ [ 16, 35, "phosphotransacylase", "protein_type" ], [ 37, 41, "PTAC", "protein_type" ], [ 52, 62, "Coenzyme A", "chemical" ], [ 80, 94, "acyl phosphate", "chemical" ] ] }, { "sid": 4, "sent": "The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta).", "section": "ABSTRACT", "ner": [ [ 4, 8, "PTAC", "protein_type" ], [ 39, 52, "metabolosomes", "complex_assembly" ], [ 54, 58, "PduL", "protein_type" ], [ 92, 96, "PTAC", "protein_type" ], [ 114, 135, "fermentative bacteria", "taxonomy_domain" ], [ 137, 140, "Pta", "protein_type" ] ] }, { "sid": 5, "sent": "Here, we report two high-resolution PduL crystal structures with bound substrates.", "section": "ABSTRACT", "ner": [ [ 36, 40, "PduL", "protein_type" ], [ 41, 59, "crystal structures", "evidence" ], [ 60, 81, "with bound substrates", "protein_state" ] ] }, { "sid": 6, "sent": "The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC.", "section": "ABSTRACT", "ner": [ [ 4, 8, "PduL", "protein_type" ], [ 9, 13, "fold", "structure_element" ], [ 30, 34, "that", "structure_element" ], [ 38, 41, "Pta", "protein_type" ], [ 57, 76, "dimetal active site", "site" ], [ 137, 149, "housekeeping", "protein_state" ], [ 150, 154, "PTAC", "protein_type" ] ] }, { "sid": 7, "sent": "Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution.", "section": "ABSTRACT", "ner": [ [ 13, 17, "PduL", "protein_type" ], [ 22, 25, "Pta", "protein_type" ] ] }, { "sid": 8, "sent": "The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen.", "section": "ABSTRACT", "ner": [ [ 4, 8, "PduL", "protein_type" ], [ 9, 18, "structure", "evidence" ], [ 139, 151, "metabolosome", "complex_assembly" ] ] }, { "sid": 9, "sent": "This study describes the structure of a novel phosphotransacylase enzyme that facilitates the recycling of the essential cofactor acetyl-CoA within a bacterial organelle and discusses the properties of the enzyme's active site and how it is packaged into the organelle.", "section": "ABSTRACT", "ner": [ [ 25, 34, "structure", "evidence" ], [ 46, 65, "phosphotransacylase", "protein_type" ], [ 130, 140, "acetyl-CoA", "chemical" ], [ 150, 159, "bacterial", "taxonomy_domain" ], [ 215, 226, "active site", "site" ] ] }, { "sid": 10, "sent": "In metabolism, molecules with \u201chigh-energy\u201d bonds (e.g., ATP and Acetyl~CoA) are critical for both catabolic and anabolic processes.", "section": "ABSTRACT", "ner": [ [ 57, 60, "ATP", "chemical" ], [ 65, 75, "Acetyl~CoA", "chemical" ] ] }, { "sid": 11, "sent": "The phosphotransacylase (Pta) enzyme catalyzes the conversion between acyl-CoA and acyl-phosphate.", "section": "ABSTRACT", "ner": [ [ 4, 23, "phosphotransacylase", "protein_type" ], [ 25, 28, "Pta", "protein_type" ], [ 70, 78, "acyl-CoA", "chemical" ], [ 83, 97, "acyl-phosphate", "chemical" ] ] }, { "sid": 12, "sent": "This reaction directly links an acyl-CoA with ATP generation via substrate-level phosphorylation, producing short-chain fatty acids (e.g., acetate), and also provides a path for short-chain fatty acids to enter central metabolism.", "section": "ABSTRACT", "ner": [ [ 32, 40, "acyl-CoA", "chemical" ], [ 46, 49, "ATP", "chemical" ], [ 108, 131, "short-chain fatty acids", "chemical" ], [ 139, 146, "acetate", "chemical" ], [ 178, 201, "short-chain fatty acids", "chemical" ] ] }, { "sid": 13, "sent": "Due to this key function, Pta is conserved across the bacterial kingdom.", "section": "ABSTRACT", "ner": [ [ 33, 42, "conserved", "protein_state" ], [ 54, 71, "bacterial kingdom", "taxonomy_domain" ] ] }, { "sid": 14, "sent": "Recently, a new type of phosphotransacylase was described that shares no evolutionary relation to Pta.", "section": "ABSTRACT", "ner": [ [ 24, 43, "phosphotransacylase", "protein_type" ], [ 98, 101, "Pta", "protein_type" ] ] }, { "sid": 15, "sent": "This enzyme, PduL, is exclusively associated with organelles called bacterial microcompartments, which are used to catabolize various compounds.", "section": "ABSTRACT", "ner": [ [ 13, 17, "PduL", "protein_type" ], [ 22, 33, "exclusively", "protein_state" ], [ 68, 77, "bacterial", "taxonomy_domain" ], [ 78, 95, "microcompartments", "complex_assembly" ] ] }, { "sid": 16, "sent": "Not only does PduL facilitate substrate level phosphorylation, but it also is critical for cofactor recycling within, and product efflux from, the organelle.", "section": "ABSTRACT", "ner": [ [ 14, 18, "PduL", "protein_type" ] ] }, { "sid": 17, "sent": "We solved the structure of this convergent phosphotransacylase and show that it is completely structurally different from Pta, including its active site architecture.", "section": "ABSTRACT", "ner": [ [ 3, 9, "solved", "experimental_method" ], [ 14, 23, "structure", "evidence" ], [ 32, 42, "convergent", "protein_state" ], [ 43, 62, "phosphotransacylase", "protein_type" ], [ 122, 125, "Pta", "protein_type" ], [ 141, 152, "active site", "site" ] ] }, { "sid": 18, "sent": "Bacterial Microcompartments (BMCs) are organelles that encapsulate enzymes for sequential biochemical reactions within a protein shell.", "section": "INTRO", "ner": [ [ 0, 9, "Bacterial", "taxonomy_domain" ], [ 10, 27, "Microcompartments", "complex_assembly" ], [ 29, 33, "BMCs", "complex_assembly" ], [ 129, 134, "shell", "structure_element" ] ] }, { "sid": 19, "sent": "The shell is typically composed of three types of protein subunits, which form either hexagonal (BMC-H and BMC-T) or pentagonal (BMC-P) tiles that assemble into a polyhedral shell.", "section": "INTRO", "ner": [ [ 4, 9, "shell", "structure_element" ], [ 86, 95, "hexagonal", "protein_state" ], [ 97, 102, "BMC-H", "complex_assembly" ], [ 107, 112, "BMC-T", "complex_assembly" ], [ 117, 127, "pentagonal", "protein_state" ], [ 129, 134, "BMC-P", "complex_assembly" ], [ 163, 173, "polyhedral", "protein_state" ], [ 174, 179, "shell", "structure_element" ] ] }, { "sid": 20, "sent": "The facets of the shell are composed primarily of hexamers that are typically perforated by pores lined with highly conserved, polar residues that presumably function as the conduits for metabolites into and out of the shell.", "section": "INTRO", "ner": [ [ 18, 23, "shell", "structure_element" ], [ 50, 58, "hexamers", "oligomeric_state" ], [ 92, 97, "pores", "site" ], [ 109, 125, "highly conserved", "protein_state" ], [ 127, 132, "polar", "protein_state" ], [ 133, 141, "residues", "structure_element" ], [ 219, 224, "shell", "structure_element" ] ] }, { "sid": 21, "sent": "The vitamin B12-dependent propanediol-utilizing (PDU) BMC was one of the first functionally characterized catabolic BMCs; subsequently, other types have been implicated in the degradation of ethanolamine, choline, fucose, rhamnose, and ethanol, all of which produce different aldehyde intermediates (Table 1).", "section": "INTRO", "ner": [ [ 4, 57, "vitamin B12-dependent propanediol-utilizing (PDU) BMC", "complex_assembly" ], [ 106, 115, "catabolic", "protein_state" ], [ 116, 120, "BMCs", "complex_assembly" ], [ 191, 203, "ethanolamine", "chemical" ], [ 205, 212, "choline", "chemical" ], [ 214, 220, "fucose", "chemical" ], [ 222, 230, "rhamnose", "chemical" ], [ 236, 243, "ethanol", "chemical" ], [ 276, 284, "aldehyde", "chemical" ] ] }, { "sid": 22, "sent": "More recently, bioinformatic studies have demonstrated the widespread distribution of BMCs among diverse bacterial phyla and grouped them into 23 different functional types.", "section": "INTRO", "ner": [ [ 15, 36, "bioinformatic studies", "experimental_method" ], [ 86, 90, "BMCs", "complex_assembly" ], [ 105, 120, "bacterial phyla", "taxonomy_domain" ] ] }, { "sid": 23, "sent": "The reactions carried out in the majority of catabolic BMCs (also known as metabolosomes) fit a generalized biochemical paradigm for the oxidation of aldehydes (Fig 1).", "section": "INTRO", "ner": [ [ 45, 54, "catabolic", "protein_state" ], [ 55, 59, "BMCs", "complex_assembly" ], [ 75, 88, "metabolosomes", "complex_assembly" ], [ 150, 159, "aldehydes", "chemical" ] ] }, { "sid": 24, "sent": "This involves a BMC-encapsulated signature enzyme that generates a toxic and/or volatile aldehyde that the BMC shell sequesters from the cytosol.", "section": "INTRO", "ner": [ [ 16, 19, "BMC", "complex_assembly" ], [ 89, 97, "aldehyde", "chemical" ], [ 107, 110, "BMC", "complex_assembly" ], [ 111, 116, "shell", "structure_element" ] ] }, { "sid": 25, "sent": "The aldehyde is subsequently converted into an acyl-CoA by aldehyde dehydrogenase, which uses NAD+ and CoA as cofactors.", "section": "INTRO", "ner": [ [ 4, 12, "aldehyde", "chemical" ], [ 47, 55, "acyl-CoA", "chemical" ], [ 59, 81, "aldehyde dehydrogenase", "protein_type" ], [ 94, 98, "NAD+", "chemical" ], [ 103, 106, "CoA", "chemical" ] ] }, { "sid": 26, "sent": "These two cofactors are relatively large, and their diffusion across the protein shell is thought to be restricted, necessitating their regeneration within the BMC lumen.", "section": "INTRO", "ner": [ [ 73, 86, "protein shell", "structure_element" ], [ 160, 163, "BMC", "complex_assembly" ] ] }, { "sid": 27, "sent": "NAD+ is recycled via alcohol dehydrogenase, and CoA is recycled via phosphotransacetylase (PTAC) (Fig 1).", "section": "INTRO", "ner": [ [ 0, 4, "NAD+", "chemical" ], [ 21, 42, "alcohol dehydrogenase", "protein_type" ], [ 48, 51, "CoA", "chemical" ], [ 68, 89, "phosphotransacetylase", "protein_type" ], [ 91, 95, "PTAC", "protein_type" ] ] }, { "sid": 28, "sent": "The final product of the BMC, an acyl-phosphate, can then be used to generate ATP via acyl kinase, or revert back to acyl-CoA by Pta for biosynthesis.", "section": "INTRO", "ner": [ [ 25, 28, "BMC", "complex_assembly" ], [ 33, 47, "acyl-phosphate", "chemical" ], [ 78, 81, "ATP", "chemical" ], [ 86, 97, "acyl kinase", "protein_type" ], [ 117, 125, "acyl-CoA", "chemical" ], [ 129, 132, "Pta", "protein_type" ] ] }, { "sid": 29, "sent": "Collectively, the aldehyde and alcohol dehydrogenases, as well as the PTAC, constitute the common metabolosome core.", "section": "INTRO", "ner": [ [ 18, 53, "aldehyde and alcohol dehydrogenases", "protein_type" ], [ 70, 74, "PTAC", "protein_type" ], [ 98, 110, "metabolosome", "complex_assembly" ] ] }, { "sid": 30, "sent": "General biochemical model of aldehyde-degrading BMCs (metabolosomes) illustrating the common metabolosome core enzymes and reactions.", "section": "FIG", "ner": [ [ 29, 47, "aldehyde-degrading", "protein_state" ], [ 48, 52, "BMCs", "complex_assembly" ], [ 54, 67, "metabolosomes", "complex_assembly" ], [ 93, 105, "metabolosome", "complex_assembly" ] ] }, { "sid": 31, "sent": "Substrates and cofactors involving the PTAC reaction are shown in red; other substrates and enzymes are shown in black, and other cofactors are shown in gray.", "section": "FIG", "ner": [ [ 39, 43, "PTAC", "protein_type" ] ] }, { "sid": 32, "sent": "Characterized and predicted catabolic BMC (metabolosome) types that represent the aldehyde-degrading paradigm (for definition of types see Kerfeld and Erbilgin).", "section": "TABLE", "ner": [ [ 28, 37, "catabolic", "protein_state" ], [ 38, 41, "BMC", "complex_assembly" ], [ 43, 55, "metabolosome", "complex_assembly" ], [ 82, 90, "aldehyde", "chemical" ] ] }, { "sid": 33, "sent": "Name\tPTAC Type\tSequestered Aldehyde\t \tPDU*\tPduL\tpropionaldehyde\t \tEUT1\tPTA_PTB\tacetaldehyde\t \tEUT2\tPduL\tacetaldehyde\t \tETU\tNone\tacetaldehyde\t \tGRM1/CUT\tPduL\tacetaldehyde\t \tGRM2\tPduL\tacetaldehyde\t \tGRM3*,4\tPduL\tpropionaldehyde\t \tGRM5/GRP\tPduL\tpropionaldehyde\t \tPVM*\tPduL\tlactaldehyde\t \tRMM1,2\tNone\tunknown\t \tSPU\tPduL\tunknown\t \t", "section": "TABLE", "ner": [ [ 5, 9, "PTAC", "protein_type" ], [ 27, 35, "Aldehyde", "chemical" ], [ 38, 41, "PDU", "complex_assembly" ], [ 43, 47, "PduL", "protein_type" ], [ 48, 63, "propionaldehyde", "chemical" ], [ 66, 70, "EUT1", "complex_assembly" ], [ 71, 78, "PTA_PTB", "protein_type" ], [ 79, 91, "acetaldehyde", "chemical" ], [ 94, 98, "EUT2", "complex_assembly" ], [ 99, 103, "PduL", "protein_type" ], [ 104, 116, "acetaldehyde", "chemical" ], [ 119, 122, "ETU", "complex_assembly" ], [ 128, 140, "acetaldehyde", "chemical" ], [ 143, 151, "GRM1/CUT", "complex_assembly" ], [ 152, 156, "PduL", "protein_type" ], [ 157, 169, "acetaldehyde", "chemical" ], [ 172, 176, "GRM2", "complex_assembly" ], [ 177, 181, "PduL", "protein_type" ], [ 182, 194, "acetaldehyde", "chemical" ], [ 197, 204, "GRM3*,4", "complex_assembly" ], [ 205, 209, "PduL", "protein_type" ], [ 210, 225, "propionaldehyde", "chemical" ], [ 228, 236, "GRM5/GRP", "complex_assembly" ], [ 237, 241, "PduL", "protein_type" ], [ 242, 257, "propionaldehyde", "chemical" ], [ 260, 263, "PVM", "complex_assembly" ], [ 265, 269, "PduL", "protein_type" ], [ 270, 282, "lactaldehyde", "chemical" ], [ 285, 291, "RMM1,2", "complex_assembly" ], [ 307, 310, "SPU", "complex_assembly" ], [ 311, 315, "PduL", "protein_type" ] ] }, { "sid": 34, "sent": "* PduL from these functional types of metabolosomes were purified in this study.", "section": "TABLE", "ner": [ [ 2, 6, "PduL", "protein_type" ], [ 38, 51, "metabolosomes", "complex_assembly" ] ] }, { "sid": 35, "sent": "The activities of core enzymes are not confined to BMC-associated functions: aldehyde and alcohol dehydrogenases are utilized in diverse metabolic reactions, and PTAC catalyzes a key biochemical reaction in the process of obtaining energy during fermentation.", "section": "INTRO", "ner": [ [ 51, 54, "BMC", "complex_assembly" ], [ 77, 112, "aldehyde and alcohol dehydrogenases", "protein_type" ], [ 162, 166, "PTAC", "protein_type" ] ] }, { "sid": 36, "sent": "The concerted functioning of a PTAC and an acetate kinase (Ack) is crucial for ATP generation in the fermentation of pyruvate to acetate (see Reactions 1 and 2).", "section": "INTRO", "ner": [ [ 31, 35, "PTAC", "protein_type" ], [ 43, 57, "acetate kinase", "protein_type" ], [ 59, 62, "Ack", "protein_type" ], [ 79, 82, "ATP", "chemical" ], [ 117, 125, "pyruvate", "chemical" ], [ 129, 136, "acetate", "chemical" ] ] }, { "sid": 37, "sent": "Both enzymes are, however, not restricted to fermentative organisms.", "section": "INTRO", "ner": [ [ 45, 67, "fermentative organisms", "taxonomy_domain" ] ] }, { "sid": 38, "sent": "They can also work in the reverse direction to activate acetate to the CoA-thioester.", "section": "INTRO", "ner": [ [ 56, 63, "acetate", "chemical" ], [ 71, 84, "CoA-thioester", "chemical" ] ] }, { "sid": 39, "sent": "This occurs, for example, during acetoclastic methanogenesis in the archaeal Methanosarcina species.", "section": "INTRO", "ner": [ [ 68, 76, "archaeal", "taxonomy_domain" ], [ 77, 99, "Methanosarcina species", "taxonomy_domain" ] ] }, { "sid": 40, "sent": " Reaction 1: acetyl-S-CoA + Pi \u2190\u2192 acetyl phosphate + CoA-SH (PTAC)", "section": "INTRO", "ner": [ [ 13, 25, "acetyl-S-CoA", "chemical" ], [ 28, 30, "Pi", "chemical" ], [ 34, 50, "acetyl phosphate", "chemical" ], [ 53, 59, "CoA-SH", "chemical" ], [ 61, 65, "PTAC", "protein_type" ] ] }, { "sid": 41, "sent": " Reaction 2: acetyl phosphate + ADP \u2190\u2192 acetate + ATP (Ack)", "section": "INTRO", "ner": [ [ 13, 29, "acetyl phosphate", "chemical" ], [ 32, 35, "ADP", "chemical" ], [ 39, 46, "acetate", "chemical" ], [ 49, 52, "ATP", "chemical" ], [ 54, 57, "Ack", "protein_type" ] ] }, { "sid": 42, "sent": "The canonical PTAC, Pta, is an ancient enzyme found in some eukaryotes and archaea, and widespread among the bacteria; 90% of the bacterial genomes in the Integrated Microbial Genomes database contain a gene encoding the PTA_PTB phosphotransacylase (Pfam domain PF01515).", "section": "INTRO", "ner": [ [ 14, 18, "PTAC", "protein_type" ], [ 20, 23, "Pta", "protein_type" ], [ 60, 70, "eukaryotes", "taxonomy_domain" ], [ 75, 82, "archaea", "taxonomy_domain" ], [ 109, 117, "bacteria", "taxonomy_domain" ], [ 130, 139, "bacterial", "taxonomy_domain" ], [ 221, 248, "PTA_PTB phosphotransacylase", "protein_type" ], [ 262, 269, "PF01515", "structure_element" ] ] }, { "sid": 43, "sent": "Pta has been extensively characterized due to its key role in fermentation.", "section": "INTRO", "ner": [ [ 0, 3, "Pta", "protein_type" ] ] }, { "sid": 44, "sent": "More recently, a second type of PTAC without any sequence homology to Pta was identified.", "section": "INTRO", "ner": [ [ 32, 36, "PTAC", "protein_type" ], [ 70, 73, "Pta", "protein_type" ] ] }, { "sid": 45, "sent": "This protein, PduL (Pfam domain PF06130), was shown to catalyze the conversion of propionyl-CoA to propionyl-phosphate and is associated with a BMC involved in propanediol utilization, the PDU BMC.", "section": "INTRO", "ner": [ [ 14, 18, "PduL", "protein_type" ], [ 32, 39, "PF06130", "structure_element" ], [ 82, 95, "propionyl-CoA", "chemical" ], [ 99, 118, "propionyl-phosphate", "chemical" ], [ 144, 147, "BMC", "complex_assembly" ], [ 189, 196, "PDU BMC", "complex_assembly" ] ] }, { "sid": 46, "sent": "Both pduL and pta genes can be found in genetic loci of functionally distinct BMCs, although the PduL type is much more prevalent, being found in all but one type of metabolosome locus: EUT1 (Table 1).", "section": "INTRO", "ner": [ [ 5, 9, "pduL", "gene" ], [ 14, 17, "pta", "gene" ], [ 78, 82, "BMCs", "complex_assembly" ], [ 97, 101, "PduL", "protein_type" ], [ 166, 184, "metabolosome locus", "gene" ], [ 186, 190, "EUT1", "gene" ] ] }, { "sid": 47, "sent": "Furthermore, in the Integrated Microbial Genomes Database, 91% of genomes that encode PF06130 also encode genes for shell proteins.", "section": "INTRO", "ner": [ [ 86, 93, "PF06130", "structure_element" ] ] }, { "sid": 48, "sent": "As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives.", "section": "INTRO", "ner": [ [ 70, 88, "aldehyde-oxidizing", "protein_state" ], [ 89, 102, "metabolosomes", "complex_assembly" ], [ 104, 108, "PduL", "protein_type" ], [ 197, 200, "Pta", "protein_type" ], [ 246, 256, "acetyl-CoA", "chemical" ], [ 258, 262, "PduL", "protein_type" ], [ 272, 279, "PDU BMC", "complex_assembly" ], [ 283, 302, "Salmonella enterica", "species" ], [ 310, 323, "propionyl-CoA", "chemical" ], [ 329, 339, "acetyl-CoA", "chemical" ], [ 363, 367, "PduL", "protein_type" ], [ 402, 406, "BMCs", "complex_assembly" ], [ 450, 453, "CoA", "chemical" ] ] }, { "sid": 49, "sent": "Another distinctive feature of BMC-associated PduL homologs is an N-terminal encapsulation peptide (EP) that is thought to \u201ctarget\u201d proteins for encapsulation by the BMC shell.", "section": "INTRO", "ner": [ [ 31, 45, "BMC-associated", "protein_state" ], [ 46, 50, "PduL", "protein_type" ], [ 77, 98, "encapsulation peptide", "structure_element" ], [ 100, 102, "EP", "structure_element" ], [ 166, 169, "BMC", "complex_assembly" ], [ 170, 175, "shell", "structure_element" ] ] }, { "sid": 50, "sent": "EPs are frequently found on BMC-associated proteins and have been shown to interact with shell proteins.", "section": "INTRO", "ner": [ [ 0, 3, "EPs", "structure_element" ], [ 28, 51, "BMC-associated proteins", "protein_type" ] ] }, { "sid": 51, "sent": "EPs have also been observed to cause proteins to aggregate, and this has recently been suggested to be functionally relevant as an initial step in metabolosome assembly, in which a multifunctional protein core is formed, around which the shell assembles.", "section": "INTRO", "ner": [ [ 0, 3, "EPs", "structure_element" ], [ 147, 159, "metabolosome", "complex_assembly" ], [ 238, 243, "shell", "structure_element" ] ] }, { "sid": 52, "sent": "Of the three common metabolosome core enzymes, crystal structures are available for both the alcohol and aldehyde dehydrogenases.", "section": "INTRO", "ner": [ [ 20, 32, "metabolosome", "complex_assembly" ], [ 47, 65, "crystal structures", "evidence" ], [ 93, 128, "alcohol and aldehyde dehydrogenases", "protein_type" ] ] }, { "sid": 53, "sent": "In contrast, the structure of PduL, the PTAC found in the vast majority of catabolic BMCs, has not been determined.", "section": "INTRO", "ner": [ [ 17, 26, "structure", "evidence" ], [ 30, 34, "PduL", "protein_type" ], [ 40, 44, "PTAC", "protein_type" ], [ 75, 84, "catabolic", "protein_state" ], [ 85, 89, "BMCs", "complex_assembly" ] ] }, { "sid": 54, "sent": "This is a major gap in our understanding of metabolosome-encapsulated biochemistry and cofactor recycling.", "section": "INTRO", "ner": [ [ 44, 56, "metabolosome", "complex_assembly" ] ] }, { "sid": 55, "sent": "Moreover, it will be useful for guiding efforts to engineer novel BMC cores for biotechnological applications.", "section": "INTRO", "ner": [ [ 66, 69, "BMC", "complex_assembly" ] ] }, { "sid": 56, "sent": "The primary structure of PduL homologs is subdivided into two PF06130 domains, each roughly 80 residues in length.", "section": "INTRO", "ner": [ [ 25, 29, "PduL", "protein_type" ], [ 62, 69, "PF06130", "structure_element" ], [ 92, 113, "80 residues in length", "residue_range" ] ] }, { "sid": 57, "sent": "No available protein structures contain the PF06130 domain, and homology searches using the primary structure of PduL do not return any significant results that would allow prediction of the structure.", "section": "INTRO", "ner": [ [ 44, 51, "PF06130", "structure_element" ], [ 64, 81, "homology searches", "experimental_method" ], [ 113, 117, "PduL", "protein_type" ], [ 191, 200, "structure", "evidence" ] ] }, { "sid": 58, "sent": "Moreover, the evident novelty of PduL makes its structure interesting in the context of convergent evolution of PTAC function; to-date, only the Pta active site and catalytic mechanism is known.", "section": "INTRO", "ner": [ [ 33, 37, "PduL", "protein_type" ], [ 48, 57, "structure", "evidence" ], [ 112, 116, "PTAC", "protein_type" ], [ 145, 148, "Pta", "protein_type" ], [ 149, 160, "active site", "site" ] ] }, { "sid": 59, "sent": "Here we report high-resolution crystal structures of a PduL-type PTAC in both CoA- and phosphate-bound forms, completing our understanding of the structural basis of catalysis by the metabolosome common core enzymes.", "section": "INTRO", "ner": [ [ 31, 49, "crystal structures", "evidence" ], [ 55, 69, "PduL-type PTAC", "protein_type" ], [ 78, 82, "CoA-", "protein_state" ], [ 87, 102, "phosphate-bound", "protein_state" ], [ 183, 195, "metabolosome", "complex_assembly" ] ] }, { "sid": 60, "sent": "We propose a catalytic mechanism analogous but yet distinct from the ubiquitous Pta enzyme, highlighting the functional convergence of two enzymes with completely different structures and metal requirements.", "section": "INTRO", "ner": [ [ 80, 83, "Pta", "protein_type" ] ] }, { "sid": 61, "sent": "We also investigate the quaternary structures of three different PduL homologs and situate our findings in the context of organelle biogenesis in functionally diverse BMCs.", "section": "INTRO", "ner": [ [ 65, 69, "PduL", "protein_type" ], [ 167, 171, "BMCs", "complex_assembly" ] ] }, { "sid": 62, "sent": "Structure Determination of PduL", "section": "RESULTS", "ner": [ [ 0, 23, "Structure Determination", "experimental_method" ], [ 27, 31, "PduL", "protein_type" ] ] }, { "sid": 63, "sent": "We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL).", "section": "RESULTS", "ner": [ [ 3, 34, "cloned, expressed, and purified", "experimental_method" ], [ 51, 55, "PduL", "protein_type" ], [ 92, 96, "BMCs", "complex_assembly" ], [ 130, 139, "pdu locus", "gene" ], [ 143, 170, "S. enterica Typhimurium LT2", "species" ], [ 172, 177, "sPduL", "protein" ], [ 212, 221, "pvm locus", "gene" ], [ 225, 249, "Planctomyces limnophilus", "species" ], [ 251, 256, "pPduL", "protein" ], [ 272, 282, "grm3 locus", "gene" ], [ 286, 319, "Rhodopseudomonas palustris BisB18", "species" ], [ 321, 326, "rPduL", "protein" ] ] }, { "sid": 64, "sent": "While purifying full-length sPduL, we observed a tendency to aggregation as described previously, with a large fraction of the expressed protein found in the insoluble fraction in a white, cake-like pellet.", "section": "RESULTS", "ner": [ [ 16, 27, "full-length", "protein_state" ], [ 28, 33, "sPduL", "protein" ] ] }, { "sid": 65, "sent": "Remarkably, after removing the N-terminal putative EP (27 amino acids), most of the sPduL\u0394EP protein was in the soluble fraction upon cell lysis.", "section": "RESULTS", "ner": [ [ 18, 26, "removing", "experimental_method" ], [ 51, 53, "EP", "structure_element" ], [ 55, 69, "27 amino acids", "residue_range" ], [ 84, 92, "sPduL\u0394EP", "mutant" ] ] }, { "sid": 66, "sent": "Similar differences in solubility were observed for pPduL and rPduL when comparing EP-truncated forms to the full-length protein, but none were quite as dramatic as for sPduL. We confirmed that all homologs were active (S1a and S1b Fig).", "section": "RESULTS", "ner": [ [ 52, 57, "pPduL", "protein" ], [ 62, 67, "rPduL", "protein" ], [ 83, 95, "EP-truncated", "protein_state" ], [ 109, 120, "full-length", "protein_state" ], [ 169, 174, "sPduL", "protein" ], [ 212, 218, "active", "protein_state" ] ] }, { "sid": 67, "sent": "Among these, we were only able to obtain diffraction-quality crystals of rPduL after removing the N-terminal putative EP (33 amino acids, also see Fig 2a) (rPduL\u0394EP).", "section": "RESULTS", "ner": [ [ 41, 69, "diffraction-quality crystals", "evidence" ], [ 73, 78, "rPduL", "protein" ], [ 85, 93, "removing", "experimental_method" ], [ 118, 120, "EP", "structure_element" ], [ 122, 136, "33 amino acids", "residue_range" ], [ 156, 164, "rPduL\u0394EP", "mutant" ] ] }, { "sid": 68, "sent": "Truncated rPduL\u0394EP had comparable enzymatic activity to the full-length enzyme (S1a Fig).", "section": "RESULTS", "ner": [ [ 0, 9, "Truncated", "protein_state" ], [ 10, 18, "rPduL\u0394EP", "mutant" ], [ 60, 71, "full-length", "protein_state" ] ] }, { "sid": 69, "sent": "Structural overview of R. palustris PduL from the grm3 locus.", "section": "FIG", "ner": [ [ 23, 35, "R. palustris", "species" ], [ 36, 40, "PduL", "protein_type" ], [ 50, 60, "grm3 locus", "gene" ] ] }, { "sid": 70, "sent": "(a) Primary and secondary structure of rPduL (tubes represent \u03b1-helices, arrows \u03b2-sheets and dashed line residues disordered in the structure.", "section": "FIG", "ner": [ [ 39, 44, "rPduL", "protein" ], [ 62, 71, "\u03b1-helices", "structure_element" ], [ 80, 88, "\u03b2-sheets", "structure_element" ], [ 132, 141, "structure", "evidence" ] ] }, { "sid": 71, "sent": "The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, \u03b10); the truncated rPduL\u0394EP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red).", "section": "FIG", "ner": [ [ 4, 24, "first 33 amino acids", "residue_range" ], [ 95, 97, "EP", "structure_element" ], [ 98, 109, "alpha helix", "structure_element" ], [ 111, 113, "\u03b10", "structure_element" ], [ 120, 129, "truncated", "protein_state" ], [ 130, 138, "rPduL\u0394EP", "mutant" ], [ 148, 160, "crystallized", "experimental_method" ], [ 173, 174, "M", "residue_name" ], [ 175, 176, "G", "residue_name" ], [ 177, 178, "V", "residue_name" ], [ 225, 233, "domain 1", "structure_element" ], [ 234, 241, "D36-N46", "residue_range" ], [ 242, 251, "Q155-C224", "residue_range" ], [ 259, 273, "loop insertion", "structure_element" ], [ 274, 281, "G61-E81", "residue_range" ], [ 289, 297, "domain 2", "structure_element" ], [ 298, 305, "R47-F60", "residue_range" ], [ 306, 314, "E82-A154", "residue_range" ] ] }, { "sid": 72, "sent": "Metal coordination residues are highlighted in light blue and CoA contacting residues in magenta, residues contacting the CoA of the other chain are also outlined.", "section": "FIG", "ner": [ [ 0, 27, "Metal coordination residues", "site" ], [ 62, 85, "CoA contacting residues", "site" ], [ 122, 125, "CoA", "chemical" ] ] }, { "sid": 73, "sent": "(b) Cartoon representation of the structure colored by domains and including secondary structure numbering.", "section": "FIG", "ner": [ [ 34, 43, "structure", "evidence" ], [ 87, 96, "structure", "evidence" ] ] }, { "sid": 74, "sent": "Coenzyme A is shown in magenta sticks and Zinc (grey) as spheres.", "section": "FIG", "ner": [ [ 0, 10, "Coenzyme A", "chemical" ], [ 42, 46, "Zinc", "chemical" ] ] }, { "sid": 75, "sent": "We collected a native dataset from rPduL\u0394EP crystals diffracting to a resolution of 1.54 \u00c5 (Table 2).", "section": "RESULTS", "ner": [ [ 3, 29, "collected a native dataset", "experimental_method" ], [ 35, 43, "rPduL\u0394EP", "mutant" ], [ 44, 52, "crystals", "evidence" ] ] }, { "sid": 76, "sent": "Using a mercury-derivative crystal form diffracting to 1.99 \u00c5 (Table 2), we obtained high quality electron density for model building and used the initial model to refine against the native data to Rwork/Rfree values of 18.9/22.1%.", "section": "RESULTS", "ner": [ [ 8, 34, "mercury-derivative crystal", "experimental_method" ], [ 98, 114, "electron density", "evidence" ], [ 198, 203, "Rwork", "evidence" ], [ 204, 209, "Rfree", "evidence" ] ] }, { "sid": 77, "sent": "There are two PduL molecules in the asymmetric unit of the P212121 unit cell.", "section": "RESULTS", "ner": [ [ 14, 18, "PduL", "protein_type" ] ] }, { "sid": 78, "sent": "We were able to fit all of the primary structure of PduL\u0394EP into the electron density with the exception of three amino acids at the N-terminus and two amino acids at the C-terminus (Fig 2a); the model is of excellent quality (Table 2).", "section": "RESULTS", "ner": [ [ 52, 59, "PduL\u0394EP", "mutant" ], [ 69, 85, "electron density", "evidence" ] ] }, { "sid": 79, "sent": "A CoA cofactor as well as two metal ions are clearly resolved in the density (for omit maps of CoA see S2 Fig).", "section": "RESULTS", "ner": [ [ 2, 5, "CoA", "chemical" ], [ 69, 76, "density", "evidence" ], [ 82, 91, "omit maps", "evidence" ], [ 95, 98, "CoA", "chemical" ] ] }, { "sid": 80, "sent": "Structurally, PduL consists of two domains (Fig 2, blue/red), each a beta-barrel that is capped on both ends by short \u03b1-helices.", "section": "RESULTS", "ner": [ [ 14, 18, "PduL", "protein_type" ], [ 35, 42, "domains", "structure_element" ], [ 69, 80, "beta-barrel", "structure_element" ], [ 118, 127, "\u03b1-helices", "structure_element" ] ] }, { "sid": 81, "sent": "\u03b2-Barrel 1 consists of the N-terminal \u03b2 strand and \u03b2 strands from the C-terminal half of the polypeptide chain (\u03b21, \u03b210-\u03b214; residues 37\u201346 and 155\u2013224).", "section": "RESULTS", "ner": [ [ 0, 10, "\u03b2-Barrel 1", "structure_element" ], [ 38, 46, "\u03b2 strand", "structure_element" ], [ 51, 60, "\u03b2 strands", "structure_element" ], [ 70, 85, "C-terminal half", "structure_element" ], [ 112, 114, "\u03b21", "structure_element" ], [ 116, 123, "\u03b210-\u03b214", "structure_element" ], [ 134, 139, "37\u201346", "residue_range" ], [ 144, 151, "155\u2013224", "residue_range" ] ] }, { "sid": 82, "sent": "\u03b2-Barrel 2 consists mainly of the central segment of primary structure (\u03b22, \u03b25\u2013\u03b29; residues 47\u201360 and 82\u2013154) (Fig 2, red), but is interrupted by a short two-strand beta sheet (\u03b23-\u03b24, residues 61\u201381).", "section": "RESULTS", "ner": [ [ 0, 10, "\u03b2-Barrel 2", "structure_element" ], [ 72, 74, "\u03b22", "structure_element" ], [ 76, 81, "\u03b25\u2013\u03b29", "structure_element" ], [ 92, 97, "47\u201360", "residue_range" ], [ 102, 108, "82\u2013154", "residue_range" ], [ 148, 175, "short two-strand beta sheet", "structure_element" ], [ 177, 182, "\u03b23-\u03b24", "structure_element" ], [ 193, 198, "61\u201381", "residue_range" ] ] }, { "sid": 83, "sent": "This \u03b2-sheet is involved in contacts between the two domains and forms a lid over the active site.", "section": "RESULTS", "ner": [ [ 5, 12, "\u03b2-sheet", "structure_element" ], [ 86, 97, "active site", "site" ] ] }, { "sid": 84, "sent": "Residues in this region (Gln42, Pro43, Gly44), covering the active site, are strongly conserved (Fig 3).", "section": "RESULTS", "ner": [ [ 25, 30, "Gln42", "residue_name_number" ], [ 32, 37, "Pro43", "residue_name_number" ], [ 39, 44, "Gly44", "residue_name_number" ], [ 60, 71, "active site", "site" ], [ 77, 95, "strongly conserved", "protein_state" ] ] }, { "sid": 85, "sent": "This structural arrangement is completely different from the functionally related Pta, which is composed of two domains, each consisting of a central flat beta sheet with alpha-helices on the top and bottom.", "section": "RESULTS", "ner": [ [ 82, 85, "Pta", "protein_type" ], [ 112, 119, "domains", "structure_element" ], [ 155, 165, "beta sheet", "structure_element" ], [ 171, 184, "alpha-helices", "structure_element" ] ] }, { "sid": 86, "sent": "Primary structure conservation of the PduL protein family.", "section": "FIG", "ner": [ [ 38, 42, "PduL", "protein_type" ] ] }, { "sid": 87, "sent": "Sequence logo calculated from the multiple sequence alignment of PduL homologs (see Materials and Methods), but not including putative EP sequences.", "section": "FIG", "ner": [ [ 34, 61, "multiple sequence alignment", "experimental_method" ], [ 65, 69, "PduL", "protein_type" ], [ 112, 125, "not including", "protein_state" ], [ 135, 137, "EP", "structure_element" ] ] }, { "sid": 88, "sent": "Residues 100% conserved across all PduL homologs in our dataset are noted with an asterisk, and residues conserved in over 90% of sequences are noted with a colon.", "section": "FIG", "ner": [ [ 35, 39, "PduL", "protein_type" ] ] }, { "sid": 89, "sent": "The sequences aligning to the PF06130 domain (determined by BLAST) are highlighted in red and blue.", "section": "FIG", "ner": [ [ 30, 37, "PF06130", "structure_element" ] ] }, { "sid": 90, "sent": "The position numbers shown correspond to the residue numbering of rPduL; note that some positions in the logo represent gaps in the rPduL sequence.", "section": "FIG", "ner": [ [ 66, 71, "rPduL", "protein" ], [ 132, 137, "rPduL", "protein" ] ] }, { "sid": 91, "sent": "There are two PduL molecules in the asymmetric unit forming a butterfly-shaped dimer (Fig 4c).", "section": "RESULTS", "ner": [ [ 14, 18, "PduL", "protein_type" ], [ 62, 78, "butterfly-shaped", "protein_state" ], [ 79, 84, "dimer", "oligomeric_state" ] ] }, { "sid": 92, "sent": "Consistent with this, results from size exclusion chromatography of rPduL\u0394EP suggest that it is a dimer in solution (Fig 5e).", "section": "RESULTS", "ner": [ [ 35, 64, "size exclusion chromatography", "experimental_method" ], [ 68, 76, "rPduL\u0394EP", "mutant" ], [ 98, 103, "dimer", "oligomeric_state" ] ] }, { "sid": 93, "sent": "The interface between the two chains buries 882 \u00c52 per monomer and is mainly formed by \u03b1-helices 2 and 4 and parts of \u03b2-sheets 12 and 14, as well as a \u03c0\u2013\u03c0 stacking of the adenine moiety of CoA with Phe116 of the adjacent chain (Fig 4c).", "section": "RESULTS", "ner": [ [ 4, 13, "interface", "site" ], [ 55, 62, "monomer", "oligomeric_state" ], [ 87, 104, "\u03b1-helices 2 and 4", "structure_element" ], [ 118, 136, "\u03b2-sheets 12 and 14", "structure_element" ], [ 171, 178, "adenine", "chemical" ], [ 189, 192, "CoA", "chemical" ], [ 198, 204, "Phe116", "residue_name_number" ] ] }, { "sid": 94, "sent": "The folds of the two chains in the asymmetric unit are very similar, superimposing with a rmsd of 0.16 \u00c5 over 2,306 aligned atom pairs.", "section": "RESULTS", "ner": [ [ 69, 82, "superimposing", "experimental_method" ], [ 90, 94, "rmsd", "evidence" ] ] }, { "sid": 95, "sent": "The peripheral helices and the short antiparallel \u03b23\u20134 sheet mediate most of the crystal contacts.", "section": "RESULTS", "ner": [ [ 15, 22, "helices", "structure_element" ], [ 31, 60, "short antiparallel \u03b23\u20134 sheet", "structure_element" ] ] }, { "sid": 96, "sent": "Details of active site, dimeric assembly, and sequence conservation of PduL.", "section": "FIG", "ner": [ [ 11, 22, "active site", "site" ], [ 24, 31, "dimeric", "oligomeric_state" ], [ 71, 75, "PduL", "protein_type" ] ] }, { "sid": 97, "sent": "(a,b) Proposed active site of PduL with relevant residues shown as sticks in atom coloring (nitrogen blue, oxygen red, sulfur yellow), zinc as grey colored spheres and coordinating ordered water molecules in red.", "section": "FIG", "ner": [ [ 15, 26, "active site", "site" ], [ 30, 34, "PduL", "protein_type" ], [ 92, 100, "nitrogen", "chemical" ], [ 107, 113, "oxygen", "chemical" ], [ 119, 125, "sulfur", "chemical" ], [ 135, 139, "zinc", "chemical" ], [ 189, 194, "water", "chemical" ] ] }, { "sid": 98, "sent": "Distances between atom centers are indicated in \u00c5. (a) Coenzyme A containing, (b) phosphate-bound structure.", "section": "FIG", "ner": [ [ 55, 65, "Coenzyme A", "chemical" ], [ 82, 97, "phosphate-bound", "protein_state" ], [ 98, 107, "structure", "evidence" ] ] }, { "sid": 99, "sent": "(c) View of the dimer in the asymmetric unit from the side, domains 1 and 2 colored as in Fig 2 and the two chains differentiated by blue/red versus slate/firebrick.", "section": "FIG", "ner": [ [ 16, 21, "dimer", "oligomeric_state" ], [ 60, 75, "domains 1 and 2", "structure_element" ] ] }, { "sid": 100, "sent": "The asterisk and double arrow marks the location of the \u03c0\u2013\u03c0 interaction between F116 and the CoA base of the other dimer chain.", "section": "FIG", "ner": [ [ 56, 71, "\u03c0\u2013\u03c0 interaction", "bond_interaction" ], [ 80, 84, "F116", "residue_name_number" ], [ 93, 96, "CoA", "chemical" ], [ 115, 120, "dimer", "oligomeric_state" ] ] }, { "sid": 101, "sent": "(d) Surface representation of the structure with indicated conservation (red: high, white: intermediate, yellow: low).", "section": "FIG", "ner": [ [ 34, 43, "structure", "evidence" ] ] }, { "sid": 102, "sent": "Size exclusion chromatography of PduL homologs.", "section": "FIG", "ner": [ [ 0, 29, "Size exclusion chromatography", "experimental_method" ], [ 33, 37, "PduL", "protein_type" ] ] }, { "sid": 103, "sent": "(a)\u2013(c): Chromatograms of sPduL (a), rPduL (b), and pPduL (c) with (orange) or without (blue) the predicted EP, post-nickel affinity purification, applied over a preparative size exclusion column (see Materials and Methods).", "section": "FIG", "ner": [ [ 9, 22, "Chromatograms", "evidence" ], [ 26, 31, "sPduL", "protein" ], [ 37, 42, "rPduL", "protein" ], [ 52, 57, "pPduL", "protein" ], [ 108, 110, "EP", "structure_element" ], [ 117, 145, "nickel affinity purification", "experimental_method" ] ] }, { "sid": 104, "sent": "(d)\u2013(f): Chromatograms of sPduL (d), rPduL (e), and pPduL (f) post-preparative size exclusion chromatography with different size fractions separated, applied over an analytical size exclusion column (see Materials and Methods).", "section": "FIG", "ner": [ [ 9, 22, "Chromatograms", "evidence" ], [ 26, 31, "sPduL", "protein" ], [ 37, 42, "rPduL", "protein" ], [ 52, 57, "pPduL", "protein" ], [ 79, 108, "size exclusion chromatography", "experimental_method" ] ] }, { "sid": 105, "sent": "All chromatograms are cropped to show only the linear range of separation based on standard runs, shown in black squares with a dashed linear trend line.", "section": "FIG", "ner": [ [ 4, 17, "chromatograms", "evidence" ] ] }, { "sid": 106, "sent": "Active Site Properties", "section": "RESULTS", "ner": [ [ 0, 11, "Active Site", "site" ] ] }, { "sid": 107, "sent": "CoA and the metal ions bind between the two domains, presumably in the active site (Figs 2b and 4a).", "section": "RESULTS", "ner": [ [ 0, 3, "CoA", "chemical" ], [ 71, 82, "active site", "site" ] ] }, { "sid": 108, "sent": "To identify the bound metals, we performed an X-ray fluorescence scan on the crystals at various wavelengths (corresponding to the K-edges of Mn, Fe, Co, Ni, Cu, and Zn).", "section": "RESULTS", "ner": [ [ 46, 69, "X-ray fluorescence scan", "experimental_method" ], [ 77, 85, "crystals", "evidence" ], [ 142, 144, "Mn", "chemical" ], [ 146, 148, "Fe", "chemical" ], [ 150, 152, "Co", "chemical" ], [ 154, 156, "Ni", "chemical" ], [ 158, 160, "Cu", "chemical" ], [ 166, 168, "Zn", "chemical" ] ] }, { "sid": 109, "sent": "There was a large signal at the zinc edge, and we tested for the presence of zinc by collecting full data sets before and after the Zn K-edge (1.2861 and 1.2822 \u00c5, respectively).", "section": "RESULTS", "ner": [ [ 77, 81, "zinc", "chemical" ], [ 85, 162, "collecting full data sets before and after the Zn K-edge (1.2861 and 1.2822 \u00c5", "experimental_method" ] ] }, { "sid": 110, "sent": "The large differences between the anomalous signals confirm the presence of zinc at both metal sites (S3 Fig).", "section": "RESULTS", "ner": [ [ 76, 80, "zinc", "chemical" ] ] }, { "sid": 111, "sent": "The first zinc ion (Zn1) is in a tetrahedral coordination state with His48, His50, Glu109, and the CoA sulfur (Fig 4a).", "section": "RESULTS", "ner": [ [ 10, 14, "zinc", "chemical" ], [ 20, 23, "Zn1", "chemical" ], [ 69, 74, "His48", "residue_name_number" ], [ 76, 81, "His50", "residue_name_number" ], [ 83, 89, "Glu109", "residue_name_number" ], [ 99, 102, "CoA", "chemical" ], [ 103, 109, "sulfur", "chemical" ] ] }, { "sid": 112, "sent": "The second (Zn2) is in octahedral coordination by three conserved histidine residues (His157, His159 and His204) as well as three water molecules (Fig 4a).", "section": "RESULTS", "ner": [ [ 12, 15, "Zn2", "chemical" ], [ 56, 65, "conserved", "protein_state" ], [ 66, 75, "histidine", "residue_name" ], [ 86, 92, "His157", "residue_name_number" ], [ 94, 100, "His159", "residue_name_number" ], [ 105, 111, "His204", "residue_name_number" ], [ 130, 135, "water", "chemical" ] ] }, { "sid": 113, "sent": "The nitrogen atom coordinating the zinc is the N\u03b5 in each histidine residue, as is typical for this interaction.", "section": "RESULTS", "ner": [ [ 35, 39, "zinc", "chemical" ], [ 58, 67, "histidine", "residue_name" ] ] }, { "sid": 114, "sent": "When the crystals were soaked in a sodium phosphate solution for 2 d prior to data collection, the CoA dissociates, and density for a phosphate molecule is visible at the active site (Table 2, Fig 4b).", "section": "RESULTS", "ner": [ [ 9, 29, "crystals were soaked", "experimental_method" ], [ 35, 51, "sodium phosphate", "chemical" ], [ 99, 102, "CoA", "chemical" ], [ 120, 127, "density", "evidence" ], [ 134, 143, "phosphate", "chemical" ], [ 171, 182, "active site", "site" ] ] }, { "sid": 115, "sent": "The phosphate-bound structure aligns well with the CoA-bound structure (0.43 \u00c5 rmsd over 2,361 atoms for the monomer, 0.83 \u00c5 over 5,259 aligned atoms for the dimer).", "section": "RESULTS", "ner": [ [ 4, 19, "phosphate-bound", "protein_state" ], [ 20, 29, "structure", "evidence" ], [ 30, 36, "aligns", "experimental_method" ], [ 51, 60, "CoA-bound", "protein_state" ], [ 61, 70, "structure", "evidence" ], [ 79, 83, "rmsd", "evidence" ], [ 109, 116, "monomer", "oligomeric_state" ], [ 158, 163, "dimer", "oligomeric_state" ] ] }, { "sid": 116, "sent": "The phosphate contacts both zinc atoms (Fig 4b) and replaces the coordination by CoA at Zn1; the coordination for Zn2 changes from octahedral with three bound waters to tetrahedral with a phosphate ion as one of the ligands (Fig 4b).", "section": "RESULTS", "ner": [ [ 4, 13, "phosphate", "chemical" ], [ 28, 32, "zinc", "chemical" ], [ 81, 84, "CoA", "chemical" ], [ 88, 91, "Zn1", "chemical" ], [ 114, 117, "Zn2", "chemical" ], [ 159, 165, "waters", "chemical" ], [ 188, 197, "phosphate", "chemical" ] ] }, { "sid": 117, "sent": "Conserved Arg103 seems to be involved in maintaining the phosphate in that position.", "section": "RESULTS", "ner": [ [ 0, 9, "Conserved", "protein_state" ], [ 10, 16, "Arg103", "residue_name_number" ], [ 57, 66, "phosphate", "chemical" ] ] }, { "sid": 118, "sent": "The two zinc atoms are slightly closer together in the phosphate-bound form (5.8 \u00c5 vs 6.3 \u00c5), possibly due to the bridging effect of the phosphate.", "section": "RESULTS", "ner": [ [ 8, 12, "zinc", "chemical" ], [ 55, 70, "phosphate-bound", "protein_state" ], [ 137, 146, "phosphate", "chemical" ] ] }, { "sid": 119, "sent": "An additional phosphate molecule is bound at a crystal contact interface, perhaps accounting for the 14 \u00c5 shorter c-axis in the phosphate-bound crystal form (Table 2).", "section": "RESULTS", "ner": [ [ 14, 23, "phosphate", "chemical" ], [ 128, 143, "phosphate-bound", "protein_state" ] ] }, { "sid": 120, "sent": "Oligomeric States of PduL Orthologs Are Influenced by the EP", "section": "RESULTS", "ner": [ [ 21, 25, "PduL", "protein_type" ], [ 58, 60, "EP", "structure_element" ] ] }, { "sid": 121, "sent": "Interestingly, some of the residues important for dimerization of rPduL, particularly Phe116, are poorly conserved across PduL homologs associated with functionally diverse BMCs (Figs 4c and 3), suggesting that they may have alternative oligomeric states.", "section": "RESULTS", "ner": [ [ 66, 71, "rPduL", "protein" ], [ 86, 92, "Phe116", "residue_name_number" ], [ 98, 114, "poorly conserved", "protein_state" ], [ 122, 126, "PduL", "protein_type" ], [ 173, 177, "BMCs", "complex_assembly" ] ] }, { "sid": 122, "sent": "We tested this hypothesis by performing size exclusion chromatography on both full-length and truncated variants (lacking the EP, \u0394EP) of sPduL, rPduL, and pPduL. These three homologs are found in functionally distinct BMCs (Table 1).", "section": "RESULTS", "ner": [ [ 40, 69, "size exclusion chromatography", "experimental_method" ], [ 78, 89, "full-length", "protein_state" ], [ 114, 121, "lacking", "protein_state" ], [ 126, 128, "EP", "structure_element" ], [ 130, 133, "\u0394EP", "mutant" ], [ 138, 143, "sPduL", "protein" ], [ 145, 150, "rPduL", "protein" ], [ 156, 161, "pPduL", "protein" ], [ 219, 223, "BMCs", "complex_assembly" ] ] }, { "sid": 123, "sent": "It has been proposed that the catabolic BMCs may assemble in a core-first manner, with the luminal enzymes (signature enzyme, aldehyde, and alcohol dehydrogenases and the BMC PTAC) forming an initial bolus, or prometabolosome, around which a shell assembles.", "section": "RESULTS", "ner": [ [ 30, 39, "catabolic", "protein_state" ], [ 40, 44, "BMCs", "complex_assembly" ], [ 126, 162, "aldehyde, and alcohol dehydrogenases", "protein_type" ], [ 171, 174, "BMC", "complex_assembly" ], [ 175, 179, "PTAC", "protein_type" ], [ 242, 247, "shell", "structure_element" ] ] }, { "sid": 124, "sent": "Given the diversity of signature enzymes (Table 1), it is plausible that PduL orthologs may adopt different oligomeric states that reflect the differences in the proteins being packaged with them in the organelle lumen.", "section": "RESULTS", "ner": [ [ 73, 77, "PduL", "protein_type" ] ] }, { "sid": 125, "sent": "We found that not only did the different orthologs appear to assemble into different oligomeric states, but that quaternary structure was dependent on whether or not the EP was present.", "section": "RESULTS", "ner": [ [ 170, 172, "EP", "structure_element" ] ] }, { "sid": 126, "sent": "Full-length sPduL was unstable in solution\u2014precipitating over time\u2014and eluted throughout the entire volume of a size exclusion column, indicating it was nonspecifically aggregating.", "section": "RESULTS", "ner": [ [ 0, 11, "Full-length", "protein_state" ], [ 12, 17, "sPduL", "protein" ] ] }, { "sid": 127, "sent": "However, when the putative EP (residues 1\u201327) was removed (sPduL \u0394EP), the truncated protein was stable and eluted as a single peak (Fig 5a) consistent with the size of a monomer (Fig 5d, blue curve).", "section": "RESULTS", "ner": [ [ 27, 29, "EP", "structure_element" ], [ 40, 44, "1\u201327", "residue_range" ], [ 50, 57, "removed", "experimental_method" ], [ 59, 68, "sPduL \u0394EP", "mutant" ], [ 75, 84, "truncated", "protein_state" ], [ 171, 178, "monomer", "oligomeric_state" ] ] }, { "sid": 128, "sent": "In contrast, both full-length rPduL and pPduL appeared to exist in two distinct oligomeric states (Fig 5b and 5c respectively, orange curves), one form of the approximate size of a dimer and the second, a higher molecular weight oligomer (~150 kDa).", "section": "RESULTS", "ner": [ [ 18, 29, "full-length", "protein_state" ], [ 30, 35, "rPduL", "protein" ], [ 40, 45, "pPduL", "protein" ], [ 181, 186, "dimer", "oligomeric_state" ], [ 229, 237, "oligomer", "oligomeric_state" ] ] }, { "sid": 129, "sent": "Upon deletion of the putative EP (residues 1\u201347 for rPduL, and 1\u201320 for pPduL), there was a distinct change in the elution profiles (Fig 5b and 5c respectively, blue curves).", "section": "RESULTS", "ner": [ [ 5, 13, "deletion", "experimental_method" ], [ 30, 32, "EP", "structure_element" ], [ 43, 47, "1\u201347", "residue_range" ], [ 52, 57, "rPduL", "protein" ], [ 63, 67, "1\u201320", "residue_range" ], [ 72, 77, "pPduL", "protein" ] ] }, { "sid": 130, "sent": "pPduL\u0394EP eluted as two smaller forms, possibly corresponding to a trimer and a monomer.", "section": "RESULTS", "ner": [ [ 0, 8, "pPduL\u0394EP", "mutant" ], [ 66, 72, "trimer", "oligomeric_state" ], [ 79, 86, "monomer", "oligomeric_state" ] ] }, { "sid": 131, "sent": "In contrast, rPduL\u0394EP eluted as one smaller oligomer, possibly a dimer.", "section": "RESULTS", "ner": [ [ 13, 21, "rPduL\u0394EP", "mutant" ], [ 65, 70, "dimer", "oligomeric_state" ] ] }, { "sid": 132, "sent": "We also analyzed purified rPduL and rPduL\u0394EP by size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) for a complementary approach to assessing oligomeric state.", "section": "RESULTS", "ner": [ [ 26, 31, "rPduL", "protein" ], [ 36, 44, "rPduL\u0394EP", "mutant" ], [ 48, 77, "size exclusion chromatography", "experimental_method" ], [ 91, 118, "multiangle light scattering", "experimental_method" ], [ 120, 128, "SEC-MALS", "experimental_method" ] ] }, { "sid": 133, "sent": "SEC-MALS analysis of rPdul\u0394EP is consistent with a dimer (as observed in the crystal structure) with a weighted average (Mw) and number average (Mn) of the molar mass of 58.4 kDa +/\u2212 11.2% and 58.8 kDa +/\u2212 10.9%, respectively (S4a Fig).", "section": "RESULTS", "ner": [ [ 0, 8, "SEC-MALS", "experimental_method" ], [ 21, 29, "rPdul\u0394EP", "mutant" ], [ 51, 56, "dimer", "oligomeric_state" ], [ 77, 94, "crystal structure", "evidence" ], [ 103, 166, "weighted average (Mw) and number average (Mn) of the molar mass", "evidence" ] ] }, { "sid": 134, "sent": "rPduL full length runs as Mw = 140.3 kDa +/\u2212 1.2% and Mn = 140.5 kDa +/\u2212 1.2%.", "section": "RESULTS", "ner": [ [ 0, 5, "rPduL", "protein" ], [ 6, 17, "full length", "protein_state" ], [ 26, 28, "Mw", "evidence" ], [ 54, 56, "Mn", "evidence" ] ] }, { "sid": 135, "sent": "This corresponds to an oligomeric state of six subunits (calculated molecular weight of 144 kDa).", "section": "RESULTS", "ner": [ [ 43, 55, "six subunits", "oligomeric_state" ], [ 68, 84, "molecular weight", "evidence" ] ] }, { "sid": 136, "sent": "Collectively, these data strongly suggest that the N-terminal EP of PduL plays a role in defining the quaternary structure of the protein.", "section": "RESULTS", "ner": [ [ 62, 64, "EP", "structure_element" ], [ 68, 72, "PduL", "protein_type" ] ] }, { "sid": 137, "sent": "The BMC shell not only sequesters specific enzymes but also their cofactors, thereby establishing a private cofactor pool dedicated to the encapsulated reactions.", "section": "DISCUSS", "ner": [ [ 4, 7, "BMC", "complex_assembly" ], [ 8, 13, "shell", "structure_element" ] ] }, { "sid": 138, "sent": "In catabolic BMCs, CoA and NAD+ must be continually recycled within the organelle (Fig 1).", "section": "DISCUSS", "ner": [ [ 3, 12, "catabolic", "protein_state" ], [ 13, 17, "BMCs", "complex_assembly" ], [ 19, 22, "CoA", "chemical" ], [ 27, 31, "NAD+", "chemical" ] ] }, { "sid": 139, "sent": "Homologs of the predominant cofactor utilizer (aldehyde dehydrogenase) and NAD+ regenerator (alcohol dehydrogenase) have been structurally characterized, but until now structural information was lacking for PduL, which recycles CoA in the organelle lumen.", "section": "DISCUSS", "ner": [ [ 47, 69, "aldehyde dehydrogenase", "protein_type" ], [ 75, 79, "NAD+", "chemical" ], [ 93, 114, "alcohol dehydrogenase", "protein_type" ], [ 207, 211, "PduL", "protein_type" ], [ 228, 231, "CoA", "chemical" ] ] }, { "sid": 140, "sent": "Curiously, while the housekeeping Pta could provide this function, and indeed does so in the case of one type of ethanolamine-utilizing (EUT) BMC, the evolutionarily unrelated PduL fulfills this function for the majority of metabolosomes using a novel structure and active site for convergent evolution of function.", "section": "DISCUSS", "ner": [ [ 21, 33, "housekeeping", "protein_state" ], [ 34, 37, "Pta", "protein_type" ], [ 113, 145, "ethanolamine-utilizing (EUT) BMC", "complex_assembly" ], [ 176, 180, "PduL", "protein_type" ], [ 224, 237, "metabolosomes", "complex_assembly" ], [ 252, 261, "structure", "evidence" ], [ 266, 277, "active site", "site" ] ] }, { "sid": 141, "sent": "The Tertiary Structure of PduL Is Formed by Discontinuous Segments of Primary Structure", "section": "DISCUSS", "ner": [ [ 26, 30, "PduL", "protein_type" ] ] }, { "sid": 142, "sent": "The structure of PduL consists of two \u03b2-barrel domains capped by short alpha helical segments (Fig 2b).", "section": "DISCUSS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 17, 21, "PduL", "protein_type" ], [ 34, 54, "two \u03b2-barrel domains", "structure_element" ], [ 65, 93, "short alpha helical segments", "structure_element" ] ] }, { "sid": 143, "sent": "The two domains are structurally very similar (superimposing with a rmsd of 1.34 \u00c5 (over 123 out of 320/348 aligned backbone atoms, S5a Fig).", "section": "DISCUSS", "ner": [ [ 47, 60, "superimposing", "experimental_method" ], [ 68, 72, "rmsd", "evidence" ] ] }, { "sid": 144, "sent": "However, the amino acid sequences of the two domains are only 16% identical (mainly the RHxH motif, \u03b22 and \u03b210), and 34% similar.", "section": "DISCUSS", "ner": [ [ 88, 98, "RHxH motif", "structure_element" ], [ 100, 102, "\u03b22", "structure_element" ], [ 107, 110, "\u03b210", "structure_element" ] ] }, { "sid": 145, "sent": "Our structure reveals that the two assigned PF06130 domains (Fig 3) do not form structurally discrete units; this reduces the apparent sequence conservation at the level of primary structure.", "section": "DISCUSS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 44, 51, "PF06130", "structure_element" ] ] }, { "sid": 146, "sent": "One strand of the domain 1 beta barrel (shown in blue in Fig 2) is contributed by the N-terminus, while the rest of the domain is formed by the residues from the C-terminal half of the protein.", "section": "DISCUSS", "ner": [ [ 4, 10, "strand", "structure_element" ], [ 18, 26, "domain 1", "structure_element" ], [ 27, 38, "beta barrel", "structure_element" ], [ 162, 177, "C-terminal half", "structure_element" ], [ 185, 192, "protein", "protein_type" ] ] }, { "sid": 147, "sent": "When aligned by structure, the \u03b21 strand of the first domain (Fig 2a and 2b, blue) corresponds to the final strand of the second domain (\u03b29), effectively making the domains continuous if the first strand was transplanted to the C-terminus.", "section": "DISCUSS", "ner": [ [ 5, 12, "aligned", "experimental_method" ], [ 16, 25, "structure", "evidence" ], [ 31, 40, "\u03b21 strand", "structure_element" ], [ 48, 60, "first domain", "structure_element" ], [ 102, 114, "final strand", "structure_element" ], [ 122, 135, "second domain", "structure_element" ], [ 137, 139, "\u03b29", "structure_element" ] ] }, { "sid": 148, "sent": "Refined domain assignment based on our structure should be able to predict domains of PF06130 homologs much more accurately.", "section": "DISCUSS", "ner": [ [ 39, 48, "structure", "evidence" ], [ 86, 93, "PF06130", "structure_element" ] ] }, { "sid": 149, "sent": "The closest structural homolog of the PduL barrel domain is a subdomain of a multienzyme complex, the alpha subunit of ethylbenzene dehydrogenase (S5b Fig, rmsd of 2.26 \u00c5 over 226 aligned atoms consisting of one beta barrel and one capping helix).", "section": "DISCUSS", "ner": [ [ 38, 42, "PduL", "protein_type" ], [ 43, 56, "barrel domain", "structure_element" ], [ 102, 115, "alpha subunit", "structure_element" ], [ 119, 145, "ethylbenzene dehydrogenase", "protein_type" ], [ 156, 160, "rmsd", "evidence" ], [ 212, 223, "beta barrel", "structure_element" ], [ 232, 245, "capping helix", "structure_element" ] ] }, { "sid": 150, "sent": "In contrast to PduL, there is only one barrel present in ethylbenzene dehydrogenase, and there is no comparable active site arrangement.", "section": "DISCUSS", "ner": [ [ 15, 19, "PduL", "protein_type" ], [ 39, 45, "barrel", "structure_element" ], [ 57, 83, "ethylbenzene dehydrogenase", "protein_type" ], [ 112, 123, "active site", "site" ] ] }, { "sid": 151, "sent": "The PduL signature primary structure, two PF06130 domains, occurs in some multidomain proteins, most of them annotated as Acks, suggesting that PduL may also replace Pta in variants of the phosphotransacetylase-Ack pathway.", "section": "DISCUSS", "ner": [ [ 4, 8, "PduL", "protein_type" ], [ 42, 49, "PF06130", "structure_element" ], [ 122, 126, "Acks", "protein_type" ], [ 144, 148, "PduL", "protein_type" ], [ 166, 169, "Pta", "protein_type" ], [ 189, 210, "phosphotransacetylase", "protein_type" ], [ 211, 214, "Ack", "protein_type" ] ] }, { "sid": 152, "sent": "These PduL homologs lack EPs, and their fusion to Ack may have evolved as a way to facilitate substrate channeling between the two enzymes.", "section": "DISCUSS", "ner": [ [ 6, 10, "PduL", "protein_type" ], [ 20, 24, "lack", "protein_state" ], [ 25, 28, "EPs", "structure_element" ], [ 34, 39, "their", "protein_type" ], [ 50, 53, "Ack", "protein_type" ] ] }, { "sid": 153, "sent": "Implications for Metabolosome Core Assembly", "section": "DISCUSS", "ner": [ [ 17, 29, "Metabolosome", "complex_assembly" ] ] }, { "sid": 154, "sent": "For BMC-encapsulated proteins to properly function together, they must be targeted to the lumen and assemble into an organization that facilitates substrate/product channeling among the different catalytic sites of the signature and core enzymes.", "section": "DISCUSS", "ner": [ [ 4, 7, "BMC", "complex_assembly" ], [ 196, 211, "catalytic sites", "site" ] ] }, { "sid": 155, "sent": "The N-terminal extension on PduL homologs may serve both of these functions.", "section": "DISCUSS", "ner": [ [ 4, 24, "N-terminal extension", "structure_element" ], [ 28, 32, "PduL", "protein_type" ] ] }, { "sid": 156, "sent": "The extension shares many features with previously characterized EPs: it is present only in homologs associated with BMC loci, and it is predicted to form an amphipathic \u03b1-helix.", "section": "DISCUSS", "ner": [ [ 0, 13, "The extension", "structure_element" ], [ 65, 68, "EPs", "structure_element" ], [ 117, 125, "BMC loci", "gene" ], [ 158, 169, "amphipathic", "protein_state" ], [ 170, 177, "\u03b1-helix", "structure_element" ] ] }, { "sid": 157, "sent": "Moreover, its removal affects the oligomeric state of the protein.", "section": "DISCUSS", "ner": [ [ 14, 21, "removal", "experimental_method" ] ] }, { "sid": 158, "sent": "EP-mediated oligomerization has been observed for the signature and core BMC enzymes; for example, full-length propanediol dehydratase and ethanolamine ammonia-lyase (signature enzymes for PDU and EUT BMCs) subunits are also insoluble, but become soluble upon removal of the predicted EP.", "section": "DISCUSS", "ner": [ [ 0, 2, "EP", "structure_element" ], [ 73, 76, "BMC", "complex_assembly" ], [ 99, 110, "full-length", "protein_state" ], [ 111, 134, "propanediol dehydratase", "protein_type" ], [ 139, 165, "ethanolamine ammonia-lyase", "protein_type" ], [ 189, 192, "PDU", "complex_assembly" ], [ 197, 205, "EUT BMCs", "complex_assembly" ], [ 285, 287, "EP", "structure_element" ] ] }, { "sid": 159, "sent": "sPduL has also previously been reported to localize to inclusion bodies when overexpressed; we show here that this is dependent on the presence of the EP.", "section": "DISCUSS", "ner": [ [ 0, 5, "sPduL", "protein" ], [ 77, 90, "overexpressed", "experimental_method" ], [ 151, 153, "EP", "structure_element" ] ] }, { "sid": 160, "sent": "This propensity of the EP to cause proteins to form complexes (Fig 5) might not be a coincidence, but could be a necessary step in the assembly of BMCs.", "section": "DISCUSS", "ner": [ [ 23, 25, "EP", "structure_element" ], [ 147, 151, "BMCs", "complex_assembly" ] ] }, { "sid": 161, "sent": "Structured aggregation of the core enzymes has been proposed to be the initial step in metabolosome assembly and is known to be the first step of \u03b2-carboxysome biogenesis, where the core enzyme Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO) is aggregated by the CcmM protein.", "section": "DISCUSS", "ner": [ [ 87, 99, "metabolosome", "complex_assembly" ], [ 194, 237, "Ribulose Bisphosphate Carboxylase/Oxygenase", "protein_type" ], [ 239, 246, "RuBisCO", "protein_type" ], [ 269, 273, "CcmM", "protein_type" ] ] }, { "sid": 162, "sent": "Likewise, CsoS2, a protein in the \u03b1-carboxysome core, also aggregates when purified and is proposed to facilitate the nucleation and encapsulation of RuBisCO molecules in the lumen of the organelle.", "section": "DISCUSS", "ner": [ [ 10, 15, "CsoS2", "protein_type" ], [ 34, 47, "\u03b1-carboxysome", "complex_assembly" ], [ 150, 157, "RuBisCO", "protein_type" ] ] }, { "sid": 163, "sent": "This role for EPs in BMC assembly is in addition to their interaction with shell proteins.", "section": "DISCUSS", "ner": [ [ 14, 17, "EPs", "structure_element" ], [ 21, 24, "BMC", "complex_assembly" ] ] }, { "sid": 164, "sent": "Moreover, the PduL crystal structures offer a clue as to how required cofactors enter the BMC lumen during assembly.", "section": "DISCUSS", "ner": [ [ 14, 18, "PduL", "protein_type" ], [ 19, 37, "crystal structures", "evidence" ], [ 90, 93, "BMC", "complex_assembly" ] ] }, { "sid": 165, "sent": "Free CoA and NAD+/H could potentially be bound to the enzymes as the core assembles and is encapsulated.", "section": "DISCUSS", "ner": [ [ 5, 8, "CoA", "chemical" ], [ 13, 17, "NAD+", "chemical" ], [ 18, 19, "H", "chemical" ] ] }, { "sid": 166, "sent": "Our PduL crystals contained CoA that was captured from the Escherichia coli cytosol, indicating that the \u201cground state\u201d of PduL is in the CoA-bound form; this could provide an elegantly simple means of guaranteeing a 1:1 ratio of CoA:PduL within the metabolosome lumen.", "section": "DISCUSS", "ner": [ [ 4, 8, "PduL", "protein_type" ], [ 9, 17, "crystals", "evidence" ], [ 28, 31, "CoA", "chemical" ], [ 59, 75, "Escherichia coli", "species" ], [ 123, 127, "PduL", "protein_type" ], [ 138, 147, "CoA-bound", "protein_state" ], [ 230, 238, "CoA:PduL", "complex_assembly" ], [ 250, 262, "metabolosome", "complex_assembly" ] ] }, { "sid": 167, "sent": "Active Site Identification and Structural Insights into Catalysis", "section": "DISCUSS", "ner": [ [ 0, 11, "Active Site", "site" ] ] }, { "sid": 168, "sent": "The active site of PduL is formed at the interface of the two structural domains (Fig 2b).", "section": "DISCUSS", "ner": [ [ 4, 15, "active site", "site" ], [ 19, 23, "PduL", "protein_type" ], [ 41, 50, "interface", "site" ], [ 73, 80, "domains", "structure_element" ] ] }, { "sid": 169, "sent": "As expected, the amino acid sequence conservation is highest in the region around the proposed active site (Fig 4d); highly conserved residues are also involved in CoA binding (Figs 2a and 3, residues Ser45, Lys70, Arg97, Leu99, His204, Asn211).", "section": "DISCUSS", "ner": [ [ 95, 106, "active site", "site" ], [ 117, 133, "highly conserved", "protein_state" ], [ 164, 167, "CoA", "chemical" ], [ 201, 206, "Ser45", "residue_name_number" ], [ 208, 213, "Lys70", "residue_name_number" ], [ 215, 220, "Arg97", "residue_name_number" ], [ 222, 227, "Leu99", "residue_name_number" ], [ 229, 235, "His204", "residue_name_number" ], [ 237, 243, "Asn211", "residue_name_number" ] ] }, { "sid": 170, "sent": "All of the metal-coordinating residues (Fig 2a) are absolutely conserved, implicating them in catalysis or the correct spatial orientation of the substrates.", "section": "DISCUSS", "ner": [ [ 11, 38, "metal-coordinating residues", "site" ], [ 52, 72, "absolutely conserved", "protein_state" ] ] }, { "sid": 171, "sent": "Arg103, which contacts the phosphate (Fig 4b), is present in all PduL homologs.", "section": "DISCUSS", "ner": [ [ 0, 6, "Arg103", "residue_name_number" ], [ 27, 36, "phosphate", "chemical" ], [ 65, 69, "PduL", "protein_type" ] ] }, { "sid": 172, "sent": "The close resemblance between the structures binding CoA and phosphate likely indicates that no large changes in protein conformation are involved in catalysis, and that our crystal structures are representative of the active form.", "section": "DISCUSS", "ner": [ [ 53, 56, "CoA", "chemical" ], [ 61, 70, "phosphate", "chemical" ], [ 174, 192, "crystal structures", "evidence" ], [ 219, 225, "active", "protein_state" ] ] }, { "sid": 173, "sent": "The native substrate for the forward reaction of rPduL and pPduL, propionyl-CoA, most likely binds to the enzyme in the same way at the observed nucleotide and pantothenic acid moiety, but the propionyl group in the CoA-thioester might point in a different direction.", "section": "DISCUSS", "ner": [ [ 49, 54, "rPduL", "protein" ], [ 59, 64, "pPduL", "protein" ], [ 66, 79, "propionyl-CoA", "chemical" ], [ 145, 155, "nucleotide", "chemical" ], [ 160, 176, "pantothenic acid", "chemical" ], [ 216, 229, "CoA-thioester", "chemical" ] ] }, { "sid": 174, "sent": "There is a pocket nearby the active site between the well-conserved residues Ser45 and Ala154, which could accommodate the propionyl group (S6 Fig).", "section": "DISCUSS", "ner": [ [ 11, 17, "pocket", "site" ], [ 29, 40, "active site", "site" ], [ 53, 67, "well-conserved", "protein_state" ], [ 77, 82, "Ser45", "residue_name_number" ], [ 87, 93, "Ala154", "residue_name_number" ] ] }, { "sid": 175, "sent": "A homology model of sPduL indicates that the residues making up this pocket and the surrounding active site region are identical to that of rPduL, which is not surprising, because these two homologs presumably have the same propionyl-CoA substrate.", "section": "DISCUSS", "ner": [ [ 2, 16, "homology model", "experimental_method" ], [ 20, 25, "sPduL", "protein" ], [ 69, 75, "pocket", "site" ], [ 96, 107, "active site", "site" ], [ 140, 145, "rPduL", "protein" ], [ 224, 237, "propionyl-CoA", "chemical" ] ] }, { "sid": 176, "sent": "The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus.", "section": "DISCUSS", "ner": [ [ 4, 18, "homology model", "experimental_method" ], [ 22, 27, "pPduL", "protein" ], [ 70, 76, "pocket", "site" ], [ 127, 138, "active site", "site" ], [ 140, 145, "Gln77", "residue_name_number" ], [ 149, 154, "rPduL", "protein" ], [ 172, 180, "tyrosine", "residue_name" ], [ 182, 187, "Tyr77", "residue_name_number" ], [ 192, 197, "pPduL", "protein" ], [ 230, 235, "pPduL", "protein" ], [ 263, 273, "lactyl-CoA", "chemical" ], [ 331, 344, "propionyl-CoA", "chemical" ], [ 365, 373, "aromatic", "protein_state" ], [ 374, 381, "residue", "structure_element" ], [ 444, 448, "PduL", "protein_type" ], [ 465, 474, "pvm locus", "gene" ] ] }, { "sid": 177, "sent": "Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms.", "section": "DISCUSS", "ner": [ [ 27, 32, "PduLs", "protein_type" ], [ 44, 52, "pvm loci", "gene" ], [ 54, 59, "Gln77", "residue_name_number" ], [ 87, 90, "Tyr", "residue_name" ], [ 94, 97, "Phe", "residue_name" ], [ 125, 128, "Gln", "residue_name" ], [ 132, 135, "Glu", "residue_name" ], [ 139, 144, "PduLs", "protein_type" ], [ 158, 161, "BMC", "complex_assembly" ], [ 181, 188, "acetyl-", "chemical" ], [ 192, 205, "propionyl-CoA", "chemical" ], [ 209, 219, "comparison", "experimental_method" ], [ 227, 231, "PduL", "protein_type" ], [ 232, 243, "active site", "site" ], [ 282, 285, "Pta", "protein_type" ] ] }, { "sid": 178, "sent": "The catalytic mechanism of Pta involves the abstraction of a thiol hydrogen by an aspartate residue, resulting in the nucleophilic attack of thiolate upon the carbonyl carbon of acetyl-phosphate, oriented by an arginine and stabilized by a serine \u2014there are no metals involved.", "section": "DISCUSS", "ner": [ [ 27, 30, "Pta", "protein_type" ], [ 82, 91, "aspartate", "residue_name" ], [ 178, 194, "acetyl-phosphate", "chemical" ], [ 211, 219, "arginine", "residue_name" ], [ 240, 246, "serine", "residue_name" ] ] }, { "sid": 179, "sent": "In contrast, in the rPduL structure, there are no conserved aspartate residues in or around the active site, and the only well-conserved glutamate residue in the active site is involved in coordinating one of the metal ions.", "section": "DISCUSS", "ner": [ [ 20, 25, "rPduL", "protein" ], [ 26, 35, "structure", "evidence" ], [ 60, 69, "aspartate", "residue_name" ], [ 96, 107, "active site", "site" ], [ 122, 136, "well-conserved", "protein_state" ], [ 137, 146, "glutamate", "residue_name" ], [ 162, 173, "active site", "site" ], [ 189, 201, "coordinating", "bond_interaction" ] ] }, { "sid": 180, "sent": "These observations strongly suggest that an acidic residue is not directly involved in catalysis by PduL. Instead, the dimetal active site of PduL may create a nucleophile from one of the hydroxyl groups on free phosphate to attack the carbonyl carbon of the thioester bond of an acyl-CoA. In the reverse direction, the metal ion(s) could stabilize the thiolate anion that would attack the carbonyl carbon of an acyl-phosphate; a similar mechanism has been described for phosphatases where hydroxyl groups or hydroxide ions can act as a base when coordinated by a dimetal active site.", "section": "DISCUSS", "ner": [ [ 44, 50, "acidic", "protein_state" ], [ 51, 58, "residue", "structure_element" ], [ 100, 104, "PduL", "protein_type" ], [ 119, 138, "dimetal active site", "site" ], [ 142, 146, "PduL", "protein_type" ], [ 212, 221, "phosphate", "chemical" ], [ 280, 288, "acyl-CoA", "chemical" ], [ 412, 426, "acyl-phosphate", "chemical" ], [ 471, 483, "phosphatases", "protein_type" ], [ 564, 583, "dimetal active site", "site" ] ] }, { "sid": 181, "sent": "Our structures provide the foundation for studies to elucidate the details of the catalytic mechanism of PduL. Conserved residues in the active site that may contribute to substrate binding and/or transition state stabilization include Ser127, Arg103, Arg194, Gln107, Gln74, and Gln/Glu77.", "section": "DISCUSS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 105, 109, "PduL", "protein_type" ], [ 111, 120, "Conserved", "protein_state" ], [ 137, 148, "active site", "site" ], [ 236, 242, "Ser127", "residue_name_number" ], [ 244, 250, "Arg103", "residue_name_number" ], [ 252, 258, "Arg194", "residue_name_number" ], [ 260, 266, "Gln107", "residue_name_number" ], [ 268, 273, "Gln74", "residue_name_number" ], [ 279, 282, "Gln", "residue_name_number" ], [ 283, 288, "Glu77", "residue_name_number" ] ] }, { "sid": 182, "sent": "In the phosphate-bound crystal structure, Ser127 and Arg103 appear to position the phosphate (Fig 4b).", "section": "DISCUSS", "ner": [ [ 7, 22, "phosphate-bound", "protein_state" ], [ 23, 40, "crystal structure", "evidence" ], [ 42, 48, "Ser127", "residue_name_number" ], [ 53, 59, "Arg103", "residue_name_number" ], [ 83, 92, "phosphate", "chemical" ] ] }, { "sid": 183, "sent": "Alternatively, Arg103 might act as a base to render the phosphate more nucleophilic.", "section": "DISCUSS", "ner": [ [ 15, 21, "Arg103", "residue_name_number" ], [ 56, 65, "phosphate", "chemical" ] ] }, { "sid": 184, "sent": "The functional groups of Gln74, Gln/Glu77, and Arg194 are directed away from the active site in both CoA and phosphate-bound crystal structures and do not appear to be involved in hydrogen bonding with these substrates, although they could be important for positioning an acyl-phosphate.", "section": "DISCUSS", "ner": [ [ 25, 30, "Gln74", "residue_name_number" ], [ 32, 35, "Gln", "residue_name_number" ], [ 36, 41, "Glu77", "residue_name_number" ], [ 47, 53, "Arg194", "residue_name_number" ], [ 81, 92, "active site", "site" ], [ 101, 104, "CoA", "protein_state" ], [ 109, 124, "phosphate-bound", "protein_state" ], [ 125, 143, "crystal structures", "evidence" ], [ 180, 196, "hydrogen bonding", "bond_interaction" ], [ 272, 286, "acyl-phosphate", "chemical" ] ] }, { "sid": 185, "sent": "The free CoA-bound form is presumably poised for attack upon an acyl-phosphate, indicating that the enzyme initially binds CoA as opposed to acyl-phosphate.", "section": "DISCUSS", "ner": [ [ 9, 18, "CoA-bound", "protein_state" ], [ 64, 78, "acyl-phosphate", "chemical" ], [ 123, 126, "CoA", "chemical" ], [ 141, 155, "acyl-phosphate", "chemical" ] ] }, { "sid": 186, "sent": "This hypothesis is strengthened by the fact that the CoA-bound crystals were obtained without added CoA, indicating that the protein bound CoA from the E. coli expression strain and retained it throughout purification and crystallization.", "section": "DISCUSS", "ner": [ [ 53, 62, "CoA-bound", "protein_state" ], [ 63, 71, "crystals", "evidence" ], [ 100, 103, "CoA", "chemical" ], [ 133, 138, "bound", "protein_state" ], [ 139, 142, "CoA", "chemical" ], [ 152, 159, "E. coli", "species" ] ] }, { "sid": 187, "sent": "The phosphate-bound structure indicates that in the opposite reaction direction phosphate is bound first, and then an acyl-CoA enters.", "section": "DISCUSS", "ner": [ [ 4, 19, "phosphate-bound", "protein_state" ], [ 20, 29, "structure", "evidence" ], [ 80, 89, "phosphate", "chemical" ], [ 118, 126, "acyl-CoA", "chemical" ] ] }, { "sid": 188, "sent": "The two high-resolution crystal structures presented here will serve as the foundation for mechanistic studies on this noncanonical PTAC enzyme to determine how the dimetal active site functions to catalyze both forward and reverse reactions.", "section": "DISCUSS", "ner": [ [ 24, 42, "crystal structures", "evidence" ], [ 132, 136, "PTAC", "protein_type" ], [ 165, 184, "dimetal active site", "site" ] ] }, { "sid": 189, "sent": "Functional, but Not Structural, Convergence of PduL and Pta", "section": "DISCUSS", "ner": [ [ 47, 51, "PduL", "protein_type" ], [ 56, 59, "Pta", "protein_type" ] ] }, { "sid": 190, "sent": "PduL and Pta are mechanistically and structurally distinct enzymes that catalyze the same reaction, a prime example of evolutionary convergence upon a function.", "section": "DISCUSS", "ner": [ [ 0, 4, "PduL", "protein_type" ], [ 9, 12, "Pta", "protein_type" ] ] }, { "sid": 191, "sent": "There are several examples of such functional convergence of enzymes, although typically the enzymes have independently evolved similar, or even identical active sites; for example, the carbonic anhydrase family.", "section": "DISCUSS", "ner": [ [ 155, 167, "active sites", "site" ], [ 186, 204, "carbonic anhydrase", "protein_type" ] ] }, { "sid": 192, "sent": "However, apparently less frequent is functional convergence that is supported by distinctly different active sites and accordingly catalytic mechanism, as revealed by comparison of the structures of Pta and PduL. One well-studied example of this is the \u03b2-lactamase family of enzymes, in which the active site of Class A and Class C enzymes involve serine-based catalysis, but Class B enzymes are metalloproteins.", "section": "DISCUSS", "ner": [ [ 102, 114, "active sites", "site" ], [ 199, 202, "Pta", "protein_type" ], [ 207, 211, "PduL", "protein_type" ], [ 253, 264, "\u03b2-lactamase", "protein_type" ], [ 297, 308, "active site", "site" ], [ 396, 411, "metalloproteins", "protein_type" ] ] }, { "sid": 193, "sent": "This is not surprising, as \u03b2-lactamases are not so widespread among bacteria and therefore would be expected to have evolved independently several times as a defense mechanism against \u03b2-lactam antibiotics.", "section": "DISCUSS", "ner": [ [ 27, 39, "\u03b2-lactamases", "protein_type" ], [ 68, 76, "bacteria", "taxonomy_domain" ] ] }, { "sid": 194, "sent": "However, nearly all bacteria encode Pta, and it is not immediately clear why the Pta/PduL functional convergence should have evolved: it would seem to be evolutionarily more resourceful for the Pta-encoding gene to be duplicated and repurposed for BMCs, as is apparently the case in one type of BMC\u2014EUT1 (Table 1).", "section": "DISCUSS", "ner": [ [ 20, 28, "bacteria", "taxonomy_domain" ], [ 36, 39, "Pta", "protein_type" ], [ 81, 84, "Pta", "protein_type" ], [ 85, 89, "PduL", "protein_type" ], [ 194, 211, "Pta-encoding gene", "gene" ], [ 248, 252, "BMCs", "complex_assembly" ], [ 295, 303, "BMC\u2014EUT1", "complex_assembly" ] ] }, { "sid": 195, "sent": "There could be some intrinsic biochemical difference between the two enzymes that renders PduL a more attractive candidate for encapsulation in a BMC\u2014for example, PduL might be more amenable to tight packaging, or is better suited for the chemical microenvironment formed within the lumen of the BMC, which can be quite different from the cytosol.", "section": "DISCUSS", "ner": [ [ 90, 94, "PduL", "protein_type" ], [ 146, 149, "BMC", "complex_assembly" ], [ 163, 167, "PduL", "protein_type" ], [ 296, 299, "BMC", "complex_assembly" ] ] }, { "sid": 196, "sent": "Further biochemical comparison between the two PTACs will likely yield exciting results that could answer this evolutionary question.", "section": "DISCUSS", "ner": [ [ 47, 52, "PTACs", "protein_type" ] ] }, { "sid": 197, "sent": "BMCs are now known to be widespread among the bacteria and are involved in critical segments of both autotrophic and heterotrophic biochemical pathways that confer to the host organism a competitive (metabolic) advantage in select niches.", "section": "DISCUSS", "ner": [ [ 0, 4, "BMCs", "complex_assembly" ], [ 46, 54, "bacteria", "taxonomy_domain" ] ] }, { "sid": 198, "sent": "As one of the three common metabolosome core enzymes, the structure of PduL provides a key missing piece to our structural picture of the shared core biochemistry (Fig 1) of functionally diverse catabolic BMCs.", "section": "DISCUSS", "ner": [ [ 27, 39, "metabolosome", "complex_assembly" ], [ 58, 67, "structure", "evidence" ], [ 71, 75, "PduL", "protein_type" ], [ 195, 204, "catabolic", "protein_state" ], [ 205, 209, "BMCs", "complex_assembly" ] ] }, { "sid": 199, "sent": "We have observed the oligomeric state differences of PduL to correlate with the presence of an EP, providing new insight into the function of this sequence extension in BMC assembly.", "section": "DISCUSS", "ner": [ [ 53, 57, "PduL", "protein_type" ], [ 95, 97, "EP", "structure_element" ], [ 169, 172, "BMC", "complex_assembly" ] ] }, { "sid": 200, "sent": "Moreover, our results suggest a means for Coenzyme A incorporation during metabolosome biogenesis.", "section": "DISCUSS", "ner": [ [ 42, 52, "Coenzyme A", "chemical" ], [ 74, 86, "metabolosome", "complex_assembly" ] ] }, { "sid": 201, "sent": "A detailed understanding of the underlying principles governing the assembly and internal structural organization of BMCs is a requisite for synthetic biologists to design custom nanoreactors that use BMC architectures as a template.", "section": "DISCUSS", "ner": [ [ 117, 121, "BMCs", "complex_assembly" ], [ 201, 204, "BMC", "complex_assembly" ] ] }, { "sid": 202, "sent": "Furthermore, given the growing number of metabolosomes implicated in pathogenesis, the PduL structure will be useful in the development of therapeutics.", "section": "DISCUSS", "ner": [ [ 41, 54, "metabolosomes", "complex_assembly" ], [ 87, 91, "PduL", "protein_type" ], [ 92, 101, "structure", "evidence" ] ] }, { "sid": 203, "sent": "The fact that PduL is confined almost exclusively to metabolosomes can be used to develop an inhibitor that blocks only PduL and not Pta as a way to selectively disrupt BMC-based metabolism, while not affecting most commensal organisms that require PTAC activity.", "section": "DISCUSS", "ner": [ [ 14, 18, "PduL", "protein_type" ], [ 53, 66, "metabolosomes", "complex_assembly" ], [ 120, 124, "PduL", "protein_type" ], [ 133, 136, "Pta", "protein_type" ], [ 169, 172, "BMC", "complex_assembly" ], [ 249, 253, "PTAC", "protein_type" ] ] } ] }, "PMC4795551": { "annotations": [ { "sid": 0, "sent": "Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily", "section": "TITLE", "ner": [ [ 17, 34, "Crystal Structure", "evidence" ], [ 38, 54, "Ectoine Synthase", "protein_type" ], [ 58, 74, "Metal-Containing", "protein_state" ], [ 89, 106, "Cupin Superfamily", "protein_type" ] ] }, { "sid": 1, "sent": "Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth.", "section": "ABSTRACT", "ner": [ [ 0, 7, "Ectoine", "chemical" ], [ 84, 92, "Bacteria", "taxonomy_domain" ], [ 103, 110, "Archaea", "taxonomy_domain" ] ] }, { "sid": 2, "sent": "Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction.", "section": "ABSTRACT", "ner": [ [ 0, 16, "Ectoine synthase", "protein_type" ], [ 18, 22, "EctC", "protein_type" ], [ 51, 58, "ectoine", "chemical" ], [ 117, 157, "N-gamma-acetyl-L-2,4-diaminobutyric acid", "chemical" ], [ 168, 173, "water", "chemical" ] ] }, { "sid": 3, "sent": "However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking.", "section": "ABSTRACT", "ner": [ [ 13, 30, "crystal structure", "evidence" ], [ 34, 50, "ectoine synthase", "protein_type" ] ] }, { "sid": 4, "sent": "Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form.", "section": "ABSTRACT", "ner": [ [ 10, 26, "ectoine synthase", "protein_type" ], [ 49, 65, "marine bacterium", "taxonomy_domain" ], [ 66, 89, "Sphingopyxis alaskensis", "species" ], [ 91, 93, "Sa", "species" ], [ 163, 167, "EctC", "protein" ], [ 199, 216, "crystal structure", "evidence" ], [ 224, 227, "apo", "protein_state" ] ] }, { "sid": 5, "sent": "Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily.", "section": "ABSTRACT", "ner": [ [ 0, 19, "Structural analysis", "experimental_method" ], [ 36, 38, "Sa", "species" ], [ 39, 43, "EctC", "protein" ], [ 71, 88, "cupin superfamily", "protein_type" ] ] }, { "sid": 6, "sent": "EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure.", "section": "ABSTRACT", "ner": [ [ 0, 4, "EctC", "protein" ], [ 13, 18, "dimer", "oligomeric_state" ], [ 26, 38, "head-to-tail", "protein_state" ], [ 80, 97, "crystal structure", "evidence" ] ] }, { "sid": 7, "sent": "The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer.", "section": "ABSTRACT", "ner": [ [ 4, 13, "interface", "site" ], [ 21, 26, "dimer", "oligomeric_state" ], [ 81, 105, "hydrophobic interactions", "bond_interaction" ], [ 122, 133, "beta-sheets", "structure_element" ], [ 146, 153, "monomer", "oligomeric_state" ] ] }, { "sid": 8, "sent": "We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme.", "section": "ABSTRACT", "ner": [ [ 32, 48, "ectoine synthase", "protein_type" ], [ 83, 88, "metal", "chemical" ], [ 100, 146, "metal depletion and reconstitution experiments", "experimental_method" ], [ 160, 164, "EctC", "protein" ], [ 180, 194, "iron-dependent", "protein_state" ] ] }, { "sid": 9, "sent": "We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency.", "section": "ABSTRACT", "ner": [ [ 14, 18, "EctC", "protein" ], [ 71, 111, "N-gamma-acetyl-L-2,4-diaminobutyric acid", "chemical" ], [ 117, 124, "ectoine", "chemical" ], [ 199, 239, "N-alpha-acetyl-L-2,4-diaminobutyric acid", "chemical" ], [ 292, 312, "catalytic efficiency", "evidence" ] ] }, { "sid": 10, "sent": "Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants.", "section": "ABSTRACT", "ner": [ [ 0, 42, "Structure-guided site-directed mutagenesis", "experimental_method" ], [ 94, 125, "evolutionarily highly conserved", "protein_state" ], [ 145, 164, "EctC protein family", "protein_type" ], [ 206, 223, "iron-binding site", "site" ], [ 296, 300, "EctC", "protein" ] ] }, { "sid": 11, "sent": "An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.", "section": "ABSTRACT", "ner": [ [ 37, 41, "iron", "chemical" ], [ 130, 141, "active site", "site" ], [ 176, 180, "EctC", "protein" ], [ 181, 193, "cupin barrel", "structure_element" ] ] }, { "sid": 12, "sent": "Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] and its derivative 5-hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] are such compatible solutes.", "section": "INTRO", "ner": [ [ 0, 7, "Ectoine", "chemical" ], [ 9, 68, "(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid", "chemical" ], [ 89, 105, "5-hydroxyectoine", "chemical" ], [ 107, 180, "(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid", "chemical" ] ] }, { "sid": 13, "sent": "Both marine and terrestrial microorganisms produce them widely in response to osmotic or temperature stress.", "section": "INTRO", "ner": [ [ 5, 42, "marine and terrestrial microorganisms", "taxonomy_domain" ] ] }, { "sid": 14, "sent": "Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-\u00df-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).", "section": "INTRO", "ner": [ [ 13, 20, "ectoine", "chemical" ], [ 61, 87, "L-aspartate-\u00df-semialdehyde", "chemical" ], [ 146, 180, "L-2,4-diaminobutyrate transaminase", "protein_type" ], [ 182, 186, "EctB", "protein_type" ], [ 202, 239, "2,4-diaminobutyrate acetyltransferase", "protein_type" ], [ 241, 245, "EctA", "protein_type" ], [ 266, 282, "ectoine synthase", "protein_type" ], [ 284, 288, "EctC", "protein_type" ] ] }, { "sid": 15, "sent": "The ectoine derivative 5-hydroxyectoine, a highly effective stress protectant in its own right, is synthesized by a substantial subgroup of the ectoine producers.", "section": "INTRO", "ner": [ [ 4, 11, "ectoine", "chemical" ], [ 23, 39, "5-hydroxyectoine", "chemical" ], [ 144, 151, "ectoine", "chemical" ] ] }, { "sid": 16, "sent": "This stereospecific chemical modification of ectoine (Fig 1) is catalyzed by the ectoine hydroxylase (EctD) (EC 1.14.11), a member of the non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily.", "section": "INTRO", "ner": [ [ 45, 52, "ectoine", "chemical" ], [ 81, 100, "ectoine hydroxylase", "protein_type" ], [ 102, 106, "EctD", "protein_type" ], [ 138, 219, "non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily", "protein_type" ] ] }, { "sid": 17, "sent": "The remarkable function preserving effects of ectoines for macromolecules and cells, frequently also addressed as chemical chaperones, led to a substantial interest in exploiting these compounds for biotechnological purposes and medical applications.", "section": "INTRO", "ner": [ [ 46, 54, "ectoines", "chemical" ] ] }, { "sid": 18, "sent": "Biosynthetic routes for ectoine and 5-hydroxyectoine.", "section": "FIG", "ner": [ [ 24, 31, "ectoine", "chemical" ], [ 36, 52, "5-hydroxyectoine", "chemical" ] ] }, { "sid": 19, "sent": "Scheme of the ectoine and 5-hydroxyectoine biosynthetic pathway.", "section": "FIG", "ner": [ [ 14, 21, "ectoine", "chemical" ], [ 26, 42, "5-hydroxyectoine", "chemical" ] ] }, { "sid": 20, "sent": "Here we focus on ectoine synthase (EctC), the key enzyme of the ectoine biosynthetic route (Fig 1).", "section": "INTRO", "ner": [ [ 17, 33, "ectoine synthase", "protein_type" ], [ 35, 39, "EctC", "protein" ], [ 64, 71, "ectoine", "chemical" ] ] }, { "sid": 21, "sent": "Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.", "section": "INTRO", "ner": [ [ 12, 29, "characterizations", "experimental_method" ], [ 33, 50, "ectoine synthases", "protein_type" ], [ 60, 73, "extremophiles", "taxonomy_domain" ], [ 74, 92, "Halomonas elongata", "species" ], [ 94, 123, "Methylomicrobium alcaliphilum", "species" ], [ 129, 149, "Acidiphilium cryptum", "species" ], [ 164, 183, "nitrifying archaeon", "taxonomy_domain" ], [ 184, 208, "Nitrosopumilus maritimus", "species" ] ] }, { "sid": 22, "sent": "Each of these enzymes catalyzes as their main activity the cyclization of N-\u03b3-acetyl-L-2,4-diaminobutyric acid (N-\u03b3-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1).", "section": "INTRO", "ner": [ [ 74, 110, "N-\u03b3-acetyl-L-2,4-diaminobutyric acid", "chemical" ], [ 112, 121, "N-\u03b3-ADABA", "chemical" ], [ 152, 189, "2,4-diaminobutyrate acetyltransferase", "protein_type" ], [ 191, 195, "EctA", "protein_type" ], [ 201, 208, "ectoine", "chemical" ], [ 243, 248, "water", "chemical" ] ] }, { "sid": 23, "sent": "In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes.", "section": "INTRO", "ner": [ [ 19, 23, "EctC", "protein" ], [ 85, 129, "5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate", "chemical" ], [ 131, 135, "ADPC", "chemical" ], [ 176, 185, "glutamine", "chemical" ], [ 250, 257, "ectoine", "chemical" ], [ 272, 279, "ectoine", "chemical" ] ] }, { "sid": 24, "sent": "Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa).", "section": "INTRO", "ner": [ [ 84, 100, "ectoine synthase", "protein_type" ], [ 135, 144, "structure", "evidence" ], [ 248, 275, "biochemically characterized", "experimental_method" ], [ 280, 296, "ectoine synthase", "protein_type" ], [ 319, 335, "marine bacterium", "taxonomy_domain" ], [ 336, 359, "Sphingopyxis alaskensis", "species" ], [ 361, 363, "Sa", "species" ] ] }, { "sid": 25, "sent": "We demonstrate here for the first time that the ectoine synthase is a metal-dependent enzyme, with iron as the most likely physiologically relevant co-factor.", "section": "INTRO", "ner": [ [ 48, 64, "ectoine synthase", "protein_type" ], [ 70, 75, "metal", "chemical" ], [ 99, 103, "iron", "chemical" ] ] }, { "sid": 26, "sent": "The EctC protein forms a dimer in solution and our structural analysis identifies it as a member of the cupin superfamily.", "section": "INTRO", "ner": [ [ 4, 8, "EctC", "protein" ], [ 25, 30, "dimer", "oligomeric_state" ], [ 51, 70, "structural analysis", "experimental_method" ], [ 104, 121, "cupin superfamily", "protein_type" ] ] }, { "sid": 27, "sent": "The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 \u00c5 and 2.0 \u00c5, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.", "section": "INTRO", "ner": [ [ 8, 26, "crystal structures", "evidence" ], [ 56, 58, "Sa", "species" ], [ 59, 63, "EctC", "protein" ], [ 157, 182, "site-directed mutagenesis", "experimental_method" ], [ 205, 236, "evolutionarily highly conserved", "protein_state" ], [ 266, 278, "EctC protein", "protein_type" ], [ 337, 351, "catalytic core", "site" ], [ 359, 375, "ectoine synthase", "protein_type" ] ] }, { "sid": 28, "sent": "Overproduction, purification and oligomeric state of the ectoine synthase in solution", "section": "RESULTS", "ner": [ [ 0, 14, "Overproduction", "experimental_method" ], [ 16, 28, "purification", "experimental_method" ], [ 57, 73, "ectoine synthase", "protein_type" ] ] }, { "sid": 29, "sent": "We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.", "section": "RESULTS", "ner": [ [ 15, 49, "biochemical and structural studies", "experimental_method" ], [ 57, 73, "ectoine synthase", "protein_type" ], [ 79, 92, "S. alaskensis", "species" ], [ 95, 97, "Sa", "species" ], [ 98, 102, "EctC", "protein" ], [ 120, 147, "marine ultra-microbacterium", "taxonomy_domain" ], [ 192, 209, "crystal structure", "evidence" ], [ 217, 236, "ectoine hydroxylase", "protein_type" ], [ 238, 242, "EctD", "protein_type" ], [ 244, 259, "in complex with", "protein_state" ] ] }, { "sid": 30, "sent": "We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.", "section": "RESULTS", "ner": [ [ 46, 59, "S. alaskensis", "species" ], [ 60, 64, "ectC", "gene" ], [ 73, 80, "E. coli", "species" ], [ 149, 178, "Strep-tag II affinity peptide", "experimental_method" ], [ 209, 211, "Sa", "species" ], [ 212, 216, "EctC", "protein" ], [ 217, 229, "Strep-Tag-II", "experimental_method" ], [ 241, 264, "affinity chromatography", "experimental_method" ] ] }, { "sid": 31, "sent": "The (Sa)EctC protein was overproduced and isolated with good yields (30\u201340 mg L-1 of culture) and purity (S2a Fig).", "section": "RESULTS", "ner": [ [ 5, 7, "Sa", "species" ], [ 8, 12, "EctC", "protein" ] ] }, { "sid": 32, "sent": "Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution.", "section": "RESULTS", "ner": [ [ 13, 42, "size-exclusion chromatography", "experimental_method" ], [ 44, 47, "SEC", "experimental_method" ], [ 73, 75, "Sa", "species" ], [ 76, 80, "EctC", "protein" ], [ 162, 168, "dimers", "oligomeric_state" ] ] }, { "sid": 33, "sent": "High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 \u00b1 2.3 kDa (S2b Fig).", "section": "RESULTS", "ner": [ [ 0, 38, "High performance liquid chromatography", "experimental_method" ], [ 52, 90, "multi-angle light-scattering detection", "experimental_method" ], [ 92, 101, "HPLC-MALS", "experimental_method" ], [ 161, 163, "Sa", "species" ], [ 164, 168, "EctC", "protein" ] ] }, { "sid": 34, "sent": "This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa).", "section": "RESULTS", "ner": [ [ 89, 91, "Sa", "species" ], [ 92, 96, "EctC", "protein" ], [ 97, 102, "dimer", "oligomeric_state" ], [ 126, 133, "monomer", "oligomeric_state" ], [ 149, 178, "Strep-tag II affinity peptide", "experimental_method" ] ] }, { "sid": 35, "sent": "Such a quaternary assembly as dimer has also been reported for the EctC proteins from H. elongata and N. maritimus.", "section": "RESULTS", "ner": [ [ 30, 35, "dimer", "oligomeric_state" ], [ 67, 80, "EctC proteins", "protein_type" ], [ 86, 97, "H. elongata", "species" ], [ 102, 114, "N. maritimus", "species" ] ] }, { "sid": 36, "sent": "Biochemical properties of the ectoine synthase", "section": "RESULTS", "ner": [ [ 30, 46, "ectoine synthase", "protein_type" ] ] }, { "sid": 37, "sent": "The EctA-produced substrate of the ectoine synthase, N-\u03b3-acetyl-L-2,4-diaminobutyric acid (N-\u03b3-ADABA) (Fig 1), is commercially not available.", "section": "RESULTS", "ner": [ [ 4, 8, "EctA", "protein" ], [ 35, 51, "ectoine synthase", "protein_type" ], [ 53, 89, "N-\u03b3-acetyl-L-2,4-diaminobutyric acid", "chemical" ], [ 91, 100, "N-\u03b3-ADABA", "chemical" ] ] }, { "sid": 38, "sent": "We used alkaline hydrolysis of ectoine and subsequent chromatography on silica gel columns to obtain N-\u03b3-ADABA in chemically highly purified form (S1a Fig).", "section": "RESULTS", "ner": [ [ 31, 38, "ectoine", "chemical" ], [ 101, 110, "N-\u03b3-ADABA", "chemical" ] ] }, { "sid": 39, "sent": "This procedure also yielded the isomer of N-\u03b3-ADABA, N-\u03b1-acetyl-L-2,4-diaminobutyric acid (N-\u03b1-ADABA) (S1b Fig).", "section": "RESULTS", "ner": [ [ 42, 51, "N-\u03b3-ADABA", "chemical" ], [ 53, 89, "N-\u03b1-acetyl-L-2,4-diaminobutyric acid", "chemical" ], [ 91, 100, "N-\u03b1-ADABA", "chemical" ] ] }, { "sid": 40, "sent": "N-\u03b1-ADABA has so far not been considered as a substrate for EctC, but microorganisms that use ectoine as a nutrient produce it as an intermediate during catabolism.", "section": "RESULTS", "ner": [ [ 0, 9, "N-\u03b1-ADABA", "chemical" ], [ 60, 64, "EctC", "protein" ], [ 70, 84, "microorganisms", "taxonomy_domain" ], [ 94, 101, "ectoine", "chemical" ] ] }, { "sid": 41, "sent": "Using N-\u03b3-ADABA as the substrate, we initially evaluated a set of biochemical parameters of the recombinant (Sa)EctC protein.", "section": "RESULTS", "ner": [ [ 6, 15, "N-\u03b3-ADABA", "chemical" ], [ 109, 111, "Sa", "species" ], [ 112, 116, "EctC", "protein" ] ] }, { "sid": 42, "sent": "S. alaskensis, from which the studied ectoine synthase was originally derived, is a microorganism that is well-adapted to a life in permanently cold ocean waters.", "section": "RESULTS", "ner": [ [ 0, 13, "S. alaskensis", "species" ], [ 38, 54, "ectoine synthase", "protein_type" ], [ 84, 97, "microorganism", "taxonomy_domain" ] ] }, { "sid": 43, "sent": "Consistent with the physicochemical attributes of this habitat, the (Sa)EctC protein was already enzymatically active at 5\u00b0C, had a temperature optimum of 15\u00b0C and was able to function over a broad range of temperatures (S3a Fig).", "section": "RESULTS", "ner": [ [ 69, 71, "Sa", "species" ], [ 72, 76, "EctC", "protein" ], [ 97, 117, "enzymatically active", "protein_state" ] ] }, { "sid": 44, "sent": "It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).", "section": "RESULTS", "ner": [ [ 16, 24, "alkaline", "protein_state" ], [ 77, 94, "ectoine synthases", "protein_type" ], [ 104, 117, "halo-tolerant", "protein_state" ], [ 118, 129, "H. elongata", "species" ], [ 162, 173, "alkaliphile", "taxonomy_domain" ], [ 174, 189, "M. alcaliphilum", "species" ], [ 219, 229, "acidophile", "taxonomy_domain" ], [ 230, 250, "Acidiphilium cryptum", "species" ], [ 291, 295, "EctC", "protein" ], [ 309, 321, "N. maritimus", "species" ], [ 328, 338, "neutral pH", "protein_state" ] ] }, { "sid": 45, "sent": "The salinity of the assay buffer had a significant influence on the maximal enzyme activity of the (Sa)EctC protein.", "section": "RESULTS", "ner": [ [ 100, 102, "Sa", "species" ], [ 103, 107, "EctC", "protein" ] ] }, { "sid": 46, "sent": "An increase in either the NaCl or the KCl concentration led to an approximately 5-fold enhancement of the ectoine synthase activity.", "section": "RESULTS", "ner": [ [ 26, 30, "NaCl", "chemical" ], [ 38, 41, "KCl", "chemical" ], [ 106, 122, "ectoine synthase", "protein_type" ] ] }, { "sid": 47, "sent": "The maximum enzyme activity of (Sa)EctC occurred around 250 mM NaCl or KCl, respectively.", "section": "RESULTS", "ner": [ [ 32, 34, "Sa", "species" ], [ 35, 39, "EctC", "protein" ], [ 63, 67, "NaCl", "chemical" ], [ 71, 74, "KCl", "chemical" ] ] }, { "sid": 48, "sent": "(Sa)EctC is a highly salt-tolerant enzyme since it exhibited substantial enzyme activity even at NaCl and KCl concentrations of 1 M in the assay buffer (S3c and S3d Fig).", "section": "RESULTS", "ner": [ [ 1, 3, "Sa", "species" ], [ 4, 8, "EctC", "protein" ], [ 97, 101, "NaCl", "chemical" ], [ 106, 109, "KCl", "chemical" ] ] }, { "sid": 49, "sent": "The stimulation of EctC enzyme activity by salts has previously also been observed for other ectoine synthases.", "section": "RESULTS", "ner": [ [ 19, 23, "EctC", "protein" ], [ 93, 110, "ectoine synthases", "protein_type" ] ] }, { "sid": 50, "sent": "The ectoine synthase is a metal-containing protein", "section": "RESULTS", "ner": [ [ 4, 20, "ectoine synthase", "protein_type" ], [ 26, 50, "metal-containing protein", "protein_type" ] ] }, { "sid": 51, "sent": "Considerations based on bioinformatics suggests that EctC belongs to the cupin superfamily.", "section": "RESULTS", "ner": [ [ 53, 57, "EctC", "protein" ], [ 73, 90, "cupin superfamily", "protein_type" ] ] }, { "sid": 52, "sent": "Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel.", "section": "RESULTS", "ner": [ [ 87, 91, "iron", "chemical" ], [ 93, 99, "copper", "chemical" ], [ 101, 105, "zinc", "chemical" ], [ 107, 116, "manganese", "chemical" ], [ 118, 124, "cobalt", "chemical" ], [ 129, 135, "nickel", "chemical" ] ] }, { "sid": 53, "sent": "Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal).", "section": "RESULTS", "ner": [ [ 0, 6, "Cupins", "protein_type" ], [ 19, 28, "conserved", "protein_state" ], [ 37, 57, "G(X)5HXH(X)3,4E(X)6G", "structure_element" ], [ 62, 80, "G(X)5PXG(X)2H(X)3N", "structure_element" ], [ 153, 158, "metal", "chemical" ] ] }, { "sid": 54, "sent": "Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family.", "section": "RESULTS", "ner": [ [ 25, 62, "alignment of the amino acid sequences", "experimental_method" ], [ 70, 88, "EctC-type proteins", "protein_type" ], [ 117, 136, "metal-binding motif", "structure_element" ], [ 143, 162, "cupin-type proteins", "protein_type" ], [ 166, 179, "not conserved", "protein_state" ], [ 210, 241, "ectoine synthase protein family", "protein_type" ] ] }, { "sid": 55, "sent": "An abbreviated alignment of the amino acid sequence of EctC-type proteins is shown in Fig 2.", "section": "RESULTS", "ner": [ [ 15, 51, "alignment of the amino acid sequence", "experimental_method" ], [ 55, 73, "EctC-type proteins", "protein_type" ] ] }, { "sid": 56, "sent": "Abbreviated alignment of EctC-type proteins.", "section": "FIG", "ner": [ [ 12, 21, "alignment", "experimental_method" ], [ 25, 43, "EctC-type proteins", "protein_type" ] ] }, { "sid": 57, "sent": "The amino acid sequences of 20 selected EctC-type proteins are compared.", "section": "FIG", "ner": [ [ 40, 58, "EctC-type proteins", "protein_type" ] ] }, { "sid": 58, "sent": "Strictly conserved amino acid residues are shown in yellow.", "section": "FIG", "ner": [ [ 0, 18, "Strictly conserved", "protein_state" ] ] }, { "sid": 59, "sent": "Dots shown above the (Sa)EctC protein sequence indicate residues likely to be involved in iron-binding (red), ligand-binding (green) and stabilization of the loop-architecture (blue).", "section": "FIG", "ner": [ [ 22, 24, "Sa", "species" ], [ 25, 29, "EctC", "protein" ], [ 90, 94, "iron", "chemical" ] ] }, { "sid": 60, "sent": "The conserved residue Tyr-52 with so-far undefined functions is indicated by a green dot circled in red.", "section": "FIG", "ner": [ [ 4, 13, "conserved", "protein_state" ], [ 22, 28, "Tyr-52", "residue_name_number" ] ] }, { "sid": 61, "sent": "Secondary structural elements (\u03b1-helices and \u03b2-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins.", "section": "FIG", "ner": [ [ 31, 40, "\u03b1-helices", "structure_element" ], [ 45, 53, "\u03b2-sheets", "structure_element" ], [ 69, 71, "Sa", "species" ], [ 72, 76, "EctC", "protein" ], [ 77, 94, "crystal structure", "evidence" ], [ 142, 160, "EctC-type proteins", "protein_type" ] ] }, { "sid": 62, "sent": "Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS).", "section": "RESULTS", "ner": [ [ 40, 59, "metal-binding motif", "structure_element" ], [ 140, 145, "metal", "chemical" ], [ 178, 180, "Sa", "species" ], [ 181, 185, "EctC", "protein" ], [ 197, 239, "inductive-coupled plasma mass spectrometry", "experimental_method" ], [ 241, 247, "ICP-MS", "experimental_method" ] ] }, { "sid": 63, "sent": "For this analysis we used recombinant (Sa)EctC preparations from three independent protein overproduction and purification experiments.", "section": "RESULTS", "ner": [ [ 39, 41, "Sa", "species" ], [ 42, 46, "EctC", "protein" ] ] }, { "sid": 64, "sent": "The ICP-MS analyses yielded an iron content of 0.66 \u00b1 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein).", "section": "RESULTS", "ner": [ [ 4, 10, "ICP-MS", "experimental_method" ], [ 31, 35, "iron", "chemical" ], [ 63, 67, "iron", "chemical" ], [ 101, 103, "Sa", "species" ], [ 104, 108, "EctC", "protein" ], [ 163, 167, "zinc", "chemical" ], [ 178, 182, "zinc", "chemical" ] ] }, { "sid": 65, "sent": "All other assayed metals (copper and nickel) were only present in trace amounts (0.01 mol metal per mol of protein, respectively).", "section": "RESULTS", "ner": [ [ 26, 32, "copper", "chemical" ], [ 37, 43, "nickel", "chemical" ], [ 90, 95, "metal", "chemical" ] ] }, { "sid": 66, "sent": "The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 \u00b1 0.05 mol per mol of (Sa)EctC protein.", "section": "RESULTS", "ner": [ [ 16, 20, "iron", "chemical" ], [ 31, 33, "Sa", "species" ], [ 34, 38, "EctC", "protein" ], [ 87, 106, "colorimetric method", "experimental_method" ], [ 127, 131, "iron", "chemical" ], [ 178, 182, "iron", "chemical" ], [ 222, 224, "Sa", "species" ], [ 225, 229, "EctC", "protein" ] ] }, { "sid": 67, "sent": "Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein.", "section": "RESULTS", "ner": [ [ 12, 18, "ICP-MS", "experimental_method" ], [ 27, 46, "colorimetric method", "experimental_method" ], [ 99, 115, "ectoine synthase", "protein_type" ], [ 121, 134, "S. alaskensis", "species" ], [ 141, 145, "iron", "chemical" ] ] }, { "sid": 68, "sent": "We note in this context, that the values obtained for the iron content of the (Sa)EctC proteins varied by approximately 10 to 20% between the two methods.", "section": "RESULTS", "ner": [ [ 58, 62, "iron", "chemical" ], [ 79, 81, "Sa", "species" ], [ 82, 86, "EctC", "protein" ] ] }, { "sid": 69, "sent": "The reason for this difference is not known, but indicates that the well established colorimetric assay probably overestimates the iron content of (Sa)EctC protein preparations to a certain degree.", "section": "RESULTS", "ner": [ [ 85, 103, "colorimetric assay", "experimental_method" ], [ 131, 135, "iron", "chemical" ], [ 148, 150, "Sa", "species" ], [ 151, 155, "EctC", "protein" ] ] }, { "sid": 70, "sent": "A metal cofactor is important for the catalytic activity of EctC", "section": "RESULTS", "ner": [ [ 2, 7, "metal", "chemical" ], [ 60, 64, "EctC", "protein" ] ] }, { "sid": 71, "sent": "The iron detected in the (Sa)EctC protein preparations could serve a structural role, or most likely, could be critical for enzyme catalysis as is the case for many members of the cupin superfamily.", "section": "RESULTS", "ner": [ [ 4, 8, "iron", "chemical" ], [ 26, 28, "Sa", "species" ], [ 29, 33, "EctC", "protein" ], [ 180, 197, "cupin superfamily", "protein_type" ] ] }, { "sid": 72, "sent": "To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.", "section": "RESULTS", "ner": [ [ 31, 40, "incubated", "experimental_method" ], [ 46, 48, "Sa", "species" ], [ 49, 53, "EctC", "protein" ], [ 61, 91, "with increasing concentrations", "experimental_method" ], [ 99, 104, "metal", "chemical" ], [ 114, 147, "ethylene-diamine-tetraacetic-acid", "chemical" ], [ 149, 153, "EDTA", "chemical" ], [ 180, 196, "ectoine synthase", "protein_type" ] ] }, { "sid": 73, "sent": "The addition of very low concentrations of EDTA (0.05 mM) to the EctC enzyme already led to a noticeable inhibition of the ectoine synthase activity and the presence of 1 mM EDTA completely inhibited the enzyme (Fig 3a).", "section": "RESULTS", "ner": [ [ 43, 47, "EDTA", "chemical" ], [ 65, 69, "EctC", "protein" ], [ 123, 139, "ectoine synthase", "protein_type" ], [ 174, 178, "EDTA", "chemical" ] ] }, { "sid": 74, "sent": "Dependency of the ectoine synthase activity on metals.", "section": "FIG", "ner": [ [ 18, 34, "ectoine synthase", "protein_type" ] ] }, { "sid": 75, "sent": "(a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (Sa)EctC protein.", "section": "FIG", "ner": [ [ 18, 22, "iron", "chemical" ], [ 32, 36, "EDTA", "chemical" ], [ 77, 79, "Sa", "species" ], [ 80, 84, "EctC", "protein" ] ] }, { "sid": 76, "sent": "Metal depletion and reconstitution experiments with (b) stoichiometric and (c) excess amounts of metals.", "section": "FIG", "ner": [ [ 0, 46, "Metal depletion and reconstitution experiments", "experimental_method" ] ] }, { "sid": 77, "sent": "The (Sa)EctC protein was present at a concentration of 10 \u03bcM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added.", "section": "FIG", "ner": [ [ 5, 7, "Sa", "species" ], [ 8, 12, "EctC", "protein" ], [ 139, 155, "ectoine synthase", "protein_type" ], [ 156, 169, "enzyme assays", "experimental_method" ], [ 184, 189, "FeCl2", "chemical" ] ] }, { "sid": 78, "sent": "We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 \u03bcM) of various metals to the (Sa)EctC enzyme.", "section": "RESULTS", "ner": [ [ 21, 32, "inactivated", "protein_state" ], [ 65, 69, "EDTA", "chemical" ], [ 73, 81, "dialysis", "experimental_method" ], [ 150, 152, "Sa", "species" ], [ 153, 157, "EctC", "protein" ] ] }, { "sid": 79, "sent": "The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively.", "section": "RESULTS", "ner": [ [ 16, 21, "FeCl2", "chemical" ], [ 29, 41, "enzyme assay", "experimental_method" ], [ 105, 110, "ZnCl2", "chemical" ], [ 114, 119, "CoCl2", "chemical" ], [ 129, 131, "Sa", "species" ], [ 132, 136, "EctC", "protein" ] ] }, { "sid": 80, "sent": "All other tested metals, including Fe3+, were unable to restore activity (Fig 3b).", "section": "RESULTS", "ner": [ [ 35, 39, "Fe3+", "chemical" ] ] }, { "sid": 81, "sent": "When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c).", "section": "RESULTS", "ner": [ [ 52, 64, "enzyme assay", "experimental_method" ], [ 89, 93, "Fe2+", "chemical" ], [ 233, 235, "Sa", "species" ], [ 236, 240, "EctC", "protein" ], [ 300, 304, "EDTA", "chemical" ] ] }, { "sid": 82, "sent": "However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c).", "section": "RESULTS", "ner": [ [ 64, 68, "zinc", "chemical" ], [ 70, 76, "cobalt", "chemical" ], [ 78, 84, "nickel", "chemical" ], [ 86, 92, "copper", "chemical" ], [ 98, 107, "manganese", "chemical" ], [ 143, 160, "cupin superfamily", "protein_type" ], [ 191, 207, "ectoine synthase", "protein_type" ] ] }, { "sid": 83, "sent": "This is in line with literature data showing that cupin-type enzymes are often promiscuous with respect to the use of the catalytically important metal.", "section": "RESULTS", "ner": [ [ 50, 68, "cupin-type enzymes", "protein_type" ], [ 146, 151, "metal", "chemical" ] ] }, { "sid": 84, "sent": "Kinetic parameters of EctC for N-\u03b3-ADABA and N-\u03b1-ADABA", "section": "RESULTS", "ner": [ [ 22, 26, "EctC", "protein" ], [ 31, 40, "N-\u03b3-ADABA", "chemical" ], [ 45, 54, "N-\u03b1-ADABA", "chemical" ] ] }, { "sid": 85, "sent": "Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-\u03b3-ADABA and the isomer N-\u03b1-ADABA.", "section": "RESULTS", "ner": [ [ 66, 80, "activity assay", "experimental_method" ], [ 89, 105, "ectoine synthase", "protein_type" ], [ 109, 122, "S. alaskensis", "species" ], [ 181, 183, "Sa", "species" ], [ 184, 188, "EctC", "protein" ], [ 227, 236, "N-\u03b3-ADABA", "chemical" ], [ 252, 261, "N-\u03b1-ADABA", "chemical" ] ] }, { "sid": 86, "sent": "The EctC-catalyzed ring-closure of N-\u03b3-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 \u00b1 0.5 mM, a vmax of 25.0 \u00b1 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig).", "section": "RESULTS", "ner": [ [ 4, 8, "EctC", "protein" ], [ 35, 44, "N-\u03b3-ADABA", "chemical" ], [ 53, 60, "ectoine", "chemical" ], [ 71, 96, "Michaelis-Menten-kinetics", "experimental_method" ], [ 114, 116, "Km", "evidence" ], [ 136, 140, "vmax", "evidence" ], [ 166, 170, "kcat", "evidence" ] ] }, { "sid": 87, "sent": "Given the chemical relatedness of N-\u03b1-ADABA to the natural substrate (N-\u03b3-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-\u03b1-ADABA to produce ectoine.", "section": "RESULTS", "ner": [ [ 34, 43, "N-\u03b1-ADABA", "chemical" ], [ 70, 79, "N-\u03b3-ADABA", "chemical" ], [ 88, 104, "ectoine synthase", "protein_type" ], [ 145, 147, "Sa", "species" ], [ 148, 152, "EctC", "protein" ], [ 168, 177, "N-\u03b1-ADABA", "chemical" ], [ 189, 196, "ectoine", "chemical" ] ] }, { "sid": 88, "sent": "(Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 \u00b1 2.9 mM, a vmax of 24.6 \u00b1 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig).", "section": "RESULTS", "ner": [ [ 1, 3, "Sa", "species" ], [ 4, 8, "EctC", "protein" ], [ 38, 63, "Michaelis-Menten-kinetics", "experimental_method" ], [ 87, 89, "Km", "evidence" ], [ 110, 114, "vmax", "evidence" ], [ 140, 144, "kcat", "evidence" ] ] }, { "sid": 89, "sent": "Hence, N-\u03b1-ADABA is a newly recognized substrate for ectoine synthase.", "section": "RESULTS", "ner": [ [ 7, 16, "N-\u03b1-ADABA", "chemical" ], [ 53, 69, "ectoine synthase", "protein_type" ] ] }, { "sid": 90, "sent": "However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-\u03b3-ADABA.", "section": "RESULTS", "ner": [ [ 18, 26, "affinity", "evidence" ], [ 28, 30, "Km", "evidence" ], [ 40, 42, "Sa", "species" ], [ 43, 47, "EctC", "protein" ], [ 64, 84, "catalytic efficiency", "evidence" ], [ 86, 93, "kcat/Km", "evidence" ], [ 136, 145, "N-\u03b3-ADABA", "chemical" ] ] }, { "sid": 91, "sent": "The Km dropped fife-fold from 4.9 \u00b1 0.5 mM to 25.4 \u00b1 2.9 mM, and the catalytic efficiency was reduced from 1.47 mM-1 s-1 to 0.02 mM-1 s-1, a 73-fold decrease.", "section": "RESULTS", "ner": [ [ 4, 6, "Km", "evidence" ], [ 69, 89, "catalytic efficiency", "evidence" ] ] }, { "sid": 92, "sent": "Both N-\u03b3-ADABA and N-\u03b1-ADABA are concomitantly formed during the enzymatic hydrolysis of the ectoine ring during catabolism.", "section": "RESULTS", "ner": [ [ 5, 14, "N-\u03b3-ADABA", "chemical" ], [ 19, 28, "N-\u03b1-ADABA", "chemical" ], [ 93, 100, "ectoine", "chemical" ] ] }, { "sid": 93, "sent": "Our finding that N-\u03b1-ADABA is a substrate for ectoine synthase has bearings for an understanding of the physiology of those microorganisms that can both synthesize and catabolize ectoine.", "section": "RESULTS", "ner": [ [ 17, 26, "N-\u03b1-ADABA", "chemical" ], [ 46, 62, "ectoine synthase", "protein_type" ], [ 124, 138, "microorganisms", "taxonomy_domain" ], [ 179, 186, "ectoine", "chemical" ] ] }, { "sid": 94, "sent": "However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-\u03b3-ADABA and N-\u03b1-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig).", "section": "RESULTS", "ner": [ [ 24, 38, "microorganisms", "taxonomy_domain" ], [ 102, 110, "affinity", "evidence" ], [ 114, 130, "ectoine synthase", "protein_type" ], [ 135, 144, "N-\u03b3-ADABA", "chemical" ], [ 149, 158, "N-\u03b1-ADABA", "chemical" ], [ 168, 188, "catalytic efficiency", "evidence" ] ] }, { "sid": 95, "sent": "Crystallization of the (Sa)EctC protein", "section": "RESULTS", "ner": [ [ 0, 15, "Crystallization", "experimental_method" ], [ 24, 26, "Sa", "species" ], [ 27, 31, "EctC", "protein" ] ] }, { "sid": 96, "sent": "Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein.", "section": "RESULTS", "ner": [ [ 9, 26, "crystal structure", "evidence" ], [ 30, 46, "ectoine synthase", "protein_type" ], [ 80, 91, "crystallize", "experimental_method" ], [ 97, 99, "Sa", "species" ], [ 100, 104, "EctC", "protein" ] ] }, { "sid": 97, "sent": "Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-\u03b3-ADABA or its reaction product ectoine were not successful.", "section": "RESULTS", "ner": [ [ 19, 27, "crystals", "evidence" ], [ 32, 34, "Sa", "species" ], [ 35, 39, "EctC", "protein" ], [ 40, 50, "in complex", "protein_state" ], [ 77, 86, "N-\u03b3-ADABA", "chemical" ], [ 111, 118, "ectoine", "chemical" ] ] }, { "sid": 98, "sent": "However, two crystal forms of the (Sa)EctC protein in the absence of the substrate were obtained.", "section": "RESULTS", "ner": [ [ 13, 26, "crystal forms", "evidence" ], [ 35, 37, "Sa", "species" ], [ 38, 42, "EctC", "protein" ], [ 58, 68, "absence of", "protein_state" ] ] }, { "sid": 99, "sent": "Attempts to solve the crystal structure of the (Sa)EctC protein by molecular replacement has previously failed.", "section": "RESULTS", "ner": [ [ 22, 39, "crystal structure", "evidence" ], [ 48, 50, "Sa", "species" ], [ 51, 55, "EctC", "protein" ], [ 67, 88, "molecular replacement", "experimental_method" ] ] }, { "sid": 100, "sent": "However, we were able to obtain crystals of form B that were derivatized with mercury and these diffracted up to 2.8 \u00c5 (S1 Table).", "section": "RESULTS", "ner": [ [ 32, 40, "crystals", "evidence" ], [ 78, 85, "mercury", "chemical" ] ] }, { "sid": 101, "sent": "This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 \u00c5) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).", "section": "RESULTS", "ner": [ [ 43, 59, "structural model", "evidence" ], [ 68, 70, "Sa", "species" ], [ 71, 75, "EctC", "protein" ], [ 130, 151, "molecular replacement", "experimental_method" ], [ 274, 282, "monomers", "oligomeric_state" ], [ 287, 289, "Sa", "species" ], [ 290, 294, "EctC", "protein" ], [ 319, 336, "crystal structure", "evidence" ], [ 360, 366, "Rcryst", "evidence" ], [ 383, 388, "Rfree", "evidence" ] ] }, { "sid": 102, "sent": "Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 \u00c5) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table).", "section": "RESULTS", "ner": [ [ 11, 18, "monomer", "oligomeric_state" ], [ 27, 36, "structure", "evidence" ], [ 64, 85, "molecular replacement", "experimental_method" ], [ 192, 198, "Rcryst", "evidence" ], [ 215, 220, "Rfree", "evidence" ] ] }, { "sid": 103, "sent": "Overall fold of the (Sa)EctC protein", "section": "RESULTS", "ner": [ [ 21, 23, "Sa", "species" ], [ 24, 28, "EctC", "protein" ] ] }, { "sid": 104, "sent": "The two EctC structures that we determined revealed that the ectoine synthase belongs to the cupin superfamily with respect to its overall fold (Fig 4a\u20134c).", "section": "RESULTS", "ner": [ [ 8, 12, "EctC", "protein" ], [ 13, 23, "structures", "evidence" ], [ 61, 77, "ectoine synthase", "protein_type" ], [ 93, 110, "cupin superfamily", "protein_type" ] ] }, { "sid": 105, "sent": "However, they represent two different states of the 137 amino acids comprising (Sa)EctC protein (Fig 2).", "section": "RESULTS", "ner": [ [ 52, 67, "137 amino acids", "residue_range" ], [ 80, 82, "Sa", "species" ], [ 83, 87, "EctC", "protein" ] ] }, { "sid": 106, "sent": "First, the 1.2 \u00c5 structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.", "section": "RESULTS", "ner": [ [ 17, 26, "structure", "evidence" ], [ 69, 71, "Sa", "species" ], [ 72, 76, "EctC", "protein" ], [ 109, 125, "Met-1 to Glu-115", "residue_range" ], [ 137, 142, "lacks", "protein_state" ], [ 143, 157, "22 amino acids", "residue_range" ], [ 165, 181, "carboxy-terminus", "structure_element" ], [ 200, 202, "Sa", "species" ], [ 203, 207, "EctC", "protein" ] ] }, { "sid": 107, "sent": "This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the \u201copen\u201d (Sa)EctC structure (Fig 4b).", "section": "RESULTS", "ner": [ [ 5, 14, "structure", "evidence" ], [ 25, 29, "open", "protein_state" ], [ 79, 92, "cupin barrels", "structure_element" ], [ 139, 143, "open", "protein_state" ], [ 146, 148, "Sa", "species" ], [ 149, 153, "EctC", "protein" ], [ 154, 163, "structure", "evidence" ] ] }, { "sid": 108, "sent": "In this structure no metal co-factor was identified.", "section": "RESULTS", "ner": [ [ 8, 17, "structure", "evidence" ], [ 21, 26, "metal", "chemical" ] ] }, { "sid": 109, "sent": "The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 \u00c5 and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.", "section": "RESULTS", "ner": [ [ 11, 28, "crystal structure", "evidence" ], [ 37, 39, "Sa", "species" ], [ 40, 44, "EctC", "protein" ], [ 57, 63, "solved", "experimental_method" ], [ 165, 173, "protomer", "oligomeric_state" ], [ 174, 175, "A", "structure_element" ], [ 197, 213, "Met-1 to Gly-121", "residue_range" ], [ 227, 233, "closed", "protein_state" ] ] }, { "sid": 110, "sent": "Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2).", "section": "RESULTS", "ner": [ [ 16, 21, "lacks", "protein_state" ], [ 22, 35, "16 amino acid", "residue_range" ], [ 52, 68, "carboxy-terminus", "structure_element" ], [ 86, 101, "137 amino acids", "residue_range" ], [ 114, 116, "Sa", "species" ], [ 117, 121, "EctC", "protein" ] ] }, { "sid": 111, "sent": "We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the \u201csemi-closed\u201d (Sa)EctC structure.", "section": "RESULTS", "ner": [ [ 38, 55, "crystal structure", "evidence" ], [ 79, 91, "fully closed", "protein_state" ], [ 105, 121, "ectoine synthase", "protein_type" ], [ 167, 178, "semi-closed", "protein_state" ], [ 181, 183, "Sa", "species" ], [ 184, 188, "EctC", "protein" ], [ 189, 198, "structure", "evidence" ] ] }, { "sid": 112, "sent": "Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the \u201copen\u201d EctC structure.", "section": "RESULTS", "ner": [ [ 31, 39, "monomers", "oligomeric_state" ], [ 86, 102, "Met-1 to Glu-115", "residue_range" ], [ 144, 148, "open", "protein_state" ], [ 150, 154, "EctC", "protein" ], [ 155, 164, "structure", "evidence" ] ] }, { "sid": 113, "sent": "Overall structure of the \u201copen\u201d and \u201csemi-closed\u201d crystal structures of (Sa)EctC.", "section": "FIG", "ner": [ [ 8, 17, "structure", "evidence" ], [ 26, 30, "open", "protein_state" ], [ 37, 48, "semi-closed", "protein_state" ], [ 50, 68, "crystal structures", "evidence" ], [ 73, 75, "Sa", "species" ], [ 76, 80, "EctC", "protein" ] ] }, { "sid": 114, "sent": "(a) The overall structure of the \u201csemi-closed\u201d (Sa)EctC resolved at 2.0 \u00c5 is depicted in green in a cartoon (upper panel) and surface (lower panel) representation.", "section": "FIG", "ner": [ [ 16, 25, "structure", "evidence" ], [ 34, 45, "semi-closed", "protein_state" ], [ 48, 50, "Sa", "species" ], [ 51, 55, "EctC", "protein" ] ] }, { "sid": 115, "sent": "The \u03b2-strands are numbered \u03b21-\u03b211 and the helices \u03b1-I to \u03b1-II.", "section": "FIG", "ner": [ [ 4, 13, "\u03b2-strands", "structure_element" ], [ 27, 33, "\u03b21-\u03b211", "structure_element" ], [ 42, 49, "helices", "structure_element" ], [ 50, 61, "\u03b1-I to \u03b1-II", "structure_element" ] ] }, { "sid": 116, "sent": "(b) The overall structure of the \u201copen\u201d (Sa)EctC was resolved at 1.2 \u00c5 and is depicted in yellow in a cartoon (upper panel) and surface (lower panel) representation.", "section": "FIG", "ner": [ [ 16, 25, "structure", "evidence" ], [ 34, 38, "open", "protein_state" ], [ 41, 43, "Sa", "species" ], [ 44, 48, "EctC", "protein" ] ] }, { "sid": 117, "sent": "The entrance to the active site of the ectoine synthase is marked.", "section": "FIG", "ner": [ [ 20, 31, "active site", "site" ], [ 39, 55, "ectoine synthase", "protein_type" ] ] }, { "sid": 118, "sent": "(c) Overlay of the \u201csemi-closed\u201d and \u201copen\u201d (Sa)EctC structures.", "section": "FIG", "ner": [ [ 4, 11, "Overlay", "experimental_method" ], [ 20, 31, "semi-closed", "protein_state" ], [ 38, 42, "open", "protein_state" ], [ 45, 47, "Sa", "species" ], [ 48, 52, "EctC", "protein" ], [ 53, 63, "structures", "evidence" ] ] }, { "sid": 119, "sent": "The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the \u201csemi-closed\u201d structure.", "section": "RESULTS", "ner": [ [ 12, 21, "structure", "evidence" ], [ 26, 28, "Sa", "species" ], [ 29, 33, "EctC", "protein" ], [ 64, 72, "crystals", "evidence" ], [ 88, 104, "carboxy-terminus", "structure_element" ], [ 148, 160, "cupin barrel", "structure_element" ], [ 186, 193, "monomer", "oligomeric_state" ], [ 194, 195, "A", "structure_element" ], [ 204, 215, "semi-closed", "protein_state" ], [ 217, 226, "structure", "evidence" ] ] }, { "sid": 120, "sent": "This is reflected by the calculated root mean square deviation (RMSD) of the C\u03b1 atoms that was about 0.56 \u00c5 (over 117 residues) when the four \u201copen\u201d monomers were compared with each other.", "section": "RESULTS", "ner": [ [ 36, 62, "root mean square deviation", "evidence" ], [ 64, 68, "RMSD", "evidence" ], [ 143, 147, "open", "protein_state" ], [ 149, 157, "monomers", "oligomeric_state" ] ] }, { "sid": 121, "sent": "However, the \u201csemi-closed\u201d monomer has a slightly higher RMSD of 1.4 \u00c5 (over 117 residues) when compared with the \u201copen\u201d 2.0 \u00c5 structure.", "section": "RESULTS", "ner": [ [ 14, 25, "semi-closed", "protein_state" ], [ 27, 34, "monomer", "oligomeric_state" ], [ 57, 61, "RMSD", "evidence" ], [ 115, 119, "open", "protein_state" ], [ 127, 136, "structure", "evidence" ] ] }, { "sid": 122, "sent": "Therefore, we describe in the following the overall structure for the \u201csemi-closed\u201d form of the (Sa)EctC protein and subsequently highlight the structural differences between the \u201copen\u201d and \u201csemi-closed\u201d forms in more detail.", "section": "RESULTS", "ner": [ [ 52, 61, "structure", "evidence" ], [ 71, 82, "semi-closed", "protein_state" ], [ 97, 99, "Sa", "species" ], [ 100, 104, "EctC", "protein" ], [ 180, 184, "open", "protein_state" ], [ 191, 202, "semi-closed", "protein_state" ] ] }, { "sid": 123, "sent": "The structure of the \u201csemi-closed\u201d (Sa)EctC protein consists of 11 \u03b2-strands (\u03b21-\u03b211) and two \u03b1-helices (\u03b1-I and \u03b1-II) (Fig 4a).", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 22, 33, "semi-closed", "protein_state" ], [ 36, 38, "Sa", "species" ], [ 39, 43, "EctC", "protein" ], [ 67, 76, "\u03b2-strands", "structure_element" ], [ 78, 84, "\u03b21-\u03b211", "structure_element" ], [ 94, 103, "\u03b1-helices", "structure_element" ], [ 105, 108, "\u03b1-I", "structure_element" ], [ 113, 117, "\u03b1-II", "structure_element" ] ] }, { "sid": 124, "sent": "The \u03b2-strands form two anti-parallel \u03b2-sheets: \u03b22 \u03b23, \u03b24, \u03b211, \u03b26, and \u03b29, and a smaller three-stranded \u03b2-sheet (\u03b27, \u03b28, and \u03b210), respectively.", "section": "RESULTS", "ner": [ [ 4, 13, "\u03b2-strands", "structure_element" ], [ 23, 45, "anti-parallel \u03b2-sheets", "structure_element" ], [ 47, 49, "\u03b22", "structure_element" ], [ 50, 52, "\u03b23", "structure_element" ], [ 54, 56, "\u03b24", "structure_element" ], [ 58, 61, "\u03b211", "structure_element" ], [ 63, 65, "\u03b26", "structure_element" ], [ 71, 73, "\u03b29", "structure_element" ], [ 89, 111, "three-stranded \u03b2-sheet", "structure_element" ], [ 113, 115, "\u03b27", "structure_element" ], [ 117, 119, "\u03b28", "structure_element" ], [ 125, 128, "\u03b210", "structure_element" ] ] }, { "sid": 125, "sent": "These two \u03b2-sheets pack against each other, forming a cup-shaped \u03b2-sandwich with a topology characteristic for the cupin-fold.", "section": "RESULTS", "ner": [ [ 10, 18, "\u03b2-sheets", "structure_element" ], [ 54, 75, "cup-shaped \u03b2-sandwich", "structure_element" ], [ 115, 125, "cupin-fold", "structure_element" ] ] }, { "sid": 126, "sent": "Hence, (Sa)EctC adopts an overall bowl shape in which one side is opened towards the solvent (Fig 4a to 4c).", "section": "RESULTS", "ner": [ [ 8, 10, "Sa", "species" ], [ 11, 15, "EctC", "protein" ] ] }, { "sid": 127, "sent": "In the \u201csemi-closed\u201d structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (\u03b1-II) that closes the active site of the (Sa)EctC protein (Fig 4a).", "section": "RESULTS", "ner": [ [ 8, 19, "semi-closed", "protein_state" ], [ 21, 30, "structure", "evidence" ], [ 41, 62, "carboxy-terminal tail", "structure_element" ], [ 81, 97, "electron density", "evidence" ], [ 114, 125, "small helix", "structure_element" ], [ 127, 131, "\u03b1-II", "structure_element" ], [ 149, 160, "active site", "site" ], [ 169, 171, "Sa", "species" ], [ 172, 176, "EctC", "protein" ] ] }, { "sid": 128, "sent": "The formation of this \u03b1-II helix induces a reorientation and shift of a long unstructured loop (as observed in the \u201copen\u201d structure) connecting \u03b24 and \u03b26, resulting in the formation of the stable \u03b2-strand \u03b25 as observed in the \u201csemi-closed\u201dstate of the (Sa)EctC protein (Fig 4a).", "section": "RESULTS", "ner": [ [ 22, 32, "\u03b1-II helix", "structure_element" ], [ 77, 89, "unstructured", "protein_state" ], [ 90, 94, "loop", "structure_element" ], [ 116, 120, "open", "protein_state" ], [ 122, 131, "structure", "evidence" ], [ 144, 146, "\u03b24", "structure_element" ], [ 151, 153, "\u03b26", "structure_element" ], [ 189, 195, "stable", "protein_state" ], [ 196, 204, "\u03b2-strand", "structure_element" ], [ 205, 207, "\u03b25", "structure_element" ], [ 228, 239, "semi-closed", "protein_state" ], [ 254, 256, "Sa", "species" ], [ 257, 261, "EctC", "protein" ] ] }, { "sid": 129, "sent": "Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily.", "section": "RESULTS", "ner": [ [ 0, 30, "Structural comparison analyses", "experimental_method" ], [ 41, 52, "DALI server", "experimental_method" ], [ 68, 70, "Sa", "species" ], [ 71, 75, "EctC", "protein" ], [ 122, 139, "cupin superfamily", "protein_type" ] ] }, { "sid": 130, "sent": "The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 \u00c5 over 104 C\u03b1-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 \u00c5 over 103 C\u03b1-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 \u00c5 over 102 C\u03b1-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 \u00c5 over 104 C\u03b1-atoms).", "section": "RESULTS", "ner": [ [ 57, 96, "Cupin 2 conserved barrel domain protein", "protein" ], [ 98, 109, "YP_751781.1", "protein" ], [ 116, 140, "Shewanella frigidimarina", "species" ], [ 175, 182, "Z-score", "evidence" ], [ 198, 202, "RMSD", "evidence" ], [ 332, 341, "structure", "evidence" ], [ 369, 395, "manganese-containing cupin", "protein" ], [ 397, 403, "TM1459", "protein" ], [ 410, 429, "Thermotoga maritima", "species" ], [ 464, 471, "Z-score", "evidence" ], [ 487, 491, "RMSD", "evidence" ], [ 524, 531, "cyclase", "protein_type" ], [ 532, 536, "RemF", "protein" ], [ 542, 571, "Streptomyces resistomycificus", "species" ], [ 605, 612, "Z-score", "evidence" ], [ 628, 632, "RMSD", "evidence" ], [ 669, 692, "auxin-binding protein 1", "protein" ], [ 698, 706, "Zea mays", "species" ], [ 742, 749, "Z-score", "evidence" ], [ 765, 769, "RMSD", "evidence" ] ] }, { "sid": 131, "sent": "Our data classify EctC, in addition to the polyketide cyclase RemF, as the second known cupin-related enzyme that catalyze a cyclocondensation reaction.", "section": "RESULTS", "ner": [ [ 18, 22, "EctC", "protein" ], [ 43, 61, "polyketide cyclase", "protein_type" ], [ 62, 66, "RemF", "protein" ], [ 88, 101, "cupin-related", "protein_type" ] ] }, { "sid": 132, "sent": "Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily.", "section": "RESULTS", "ner": [ [ 8, 12, "RemF", "protein" ], [ 21, 46, "aldos-2-ulose dehydratase", "protein_type" ], [ 47, 56, "isomerase", "protein_type" ], [ 62, 78, "ectoine synthase", "protein_type" ], [ 111, 122, "dehydratase", "protein_type" ], [ 134, 151, "cupin superfamily", "protein_type" ] ] }, { "sid": 133, "sent": "Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure", "section": "RESULTS", "ner": [ [ 16, 20, "EctC", "protein" ], [ 21, 36, "dimer interface", "site" ], [ 57, 59, "Sa", "species" ], [ 60, 64, "EctC", "protein" ], [ 65, 82, "crystal structure", "evidence" ] ] }, { "sid": 134, "sent": "Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution.", "section": "RESULTS", "ner": [ [ 9, 12, "SEC", "experimental_method" ], [ 30, 39, "HPLC-MALS", "experimental_method" ], [ 82, 98, "ectoine synthase", "protein_type" ], [ 104, 117, "S. alaskensis", "species" ], [ 123, 128, "dimer", "oligomeric_state" ] ] }, { "sid": 135, "sent": "The crystal structure of this protein reflects this quaternary arrangement.", "section": "RESULTS", "ner": [ [ 4, 21, "crystal structure", "evidence" ] ] }, { "sid": 136, "sent": "In the \u201csemi-closed\u201d crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit.", "section": "RESULTS", "ner": [ [ 8, 19, "semi-closed", "protein_state" ], [ 21, 38, "crystal structure", "evidence" ], [ 41, 43, "Sa", "species" ], [ 44, 48, "EctC", "protein" ], [ 53, 65, "crystallized", "experimental_method" ], [ 71, 76, "dimer", "oligomeric_state" ], [ 80, 86, "dimers", "oligomeric_state" ] ] }, { "sid": 137, "sent": "This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel \u03b2-strands, \u03b2-strand \u03b21 (sequence 1MIVRN5) from monomer A and \u03b2-strand \u03b28 from monomer B (sequence 82GVMYAL87) (Fig 5c).", "section": "RESULTS", "ner": [ [ 5, 10, "dimer", "oligomeric_state" ], [ 46, 54, "monomers", "oligomeric_state" ], [ 69, 81, "head-to-tail", "protein_state" ], [ 152, 174, "antiparallel \u03b2-strands", "structure_element" ], [ 176, 184, "\u03b2-strand", "structure_element" ], [ 185, 187, "\u03b21", "structure_element" ], [ 198, 205, "1MIVRN5", "structure_element" ], [ 212, 219, "monomer", "oligomeric_state" ], [ 220, 221, "A", "structure_element" ], [ 226, 234, "\u03b2-strand", "structure_element" ], [ 235, 237, "\u03b28", "structure_element" ], [ 243, 250, "monomer", "oligomeric_state" ], [ 251, 252, "B", "structure_element" ], [ 263, 273, "82GVMYAL87", "structure_element" ] ] }, { "sid": 138, "sent": "The strong interactions between these \u03b2-strands rely primarily on backbone contacts.", "section": "RESULTS", "ner": [ [ 38, 47, "\u03b2-strands", "structure_element" ] ] }, { "sid": 139, "sent": "In addition to these interactions, some weaker hydrophobic interactions are also observed between the two monomers in some loops connecting the \u03b2-strands.", "section": "RESULTS", "ner": [ [ 47, 71, "hydrophobic interactions", "bond_interaction" ], [ 106, 114, "monomers", "oligomeric_state" ], [ 123, 128, "loops", "structure_element" ], [ 144, 153, "\u03b2-strands", "structure_element" ] ] }, { "sid": 140, "sent": "As calculated with PDBePISA, the surface area buried upon dimer formation is 1462 \u00c52, which is 20.5% of the total accessible surface of a monomer of this protein.", "section": "RESULTS", "ner": [ [ 19, 27, "PDBePISA", "experimental_method" ], [ 58, 63, "dimer", "oligomeric_state" ], [ 138, 145, "monomer", "oligomeric_state" ] ] }, { "sid": 141, "sent": "Both values fall within the range for known functional dimers.", "section": "RESULTS", "ner": [ [ 55, 61, "dimers", "oligomeric_state" ] ] }, { "sid": 142, "sent": "Crystal structure of (Sa)EctC.", "section": "FIG", "ner": [ [ 0, 17, "Crystal structure", "evidence" ], [ 22, 24, "Sa", "species" ], [ 25, 29, "EctC", "protein" ] ] }, { "sid": 143, "sent": "(a) Top-view of the dimer of the (Sa)EctC protein.", "section": "FIG", "ner": [ [ 20, 25, "dimer", "oligomeric_state" ], [ 34, 36, "Sa", "species" ], [ 37, 41, "EctC", "protein" ] ] }, { "sid": 144, "sent": "The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two \u03b2-strands of each monomer.", "section": "FIG", "ner": [ [ 20, 25, "water", "chemical" ], [ 92, 100, "monomers", "oligomeric_state" ], [ 142, 144, "Sa", "species" ], [ 145, 149, "EctC", "protein" ], [ 150, 155, "dimer", "oligomeric_state" ], [ 186, 201, "dimer interface", "site" ], [ 216, 225, "\u03b2-strands", "structure_element" ], [ 234, 241, "monomer", "oligomeric_state" ] ] }, { "sid": 145, "sent": "(c) Close-up representation of the dimer interface mediated by beta-strand \u03b21 and \u03b26.", "section": "FIG", "ner": [ [ 35, 50, "dimer interface", "site" ], [ 63, 74, "beta-strand", "structure_element" ], [ 75, 77, "\u03b21", "structure_element" ], [ 82, 84, "\u03b26", "structure_element" ] ] }, { "sid": 146, "sent": "In the \u201copen\u201d (Sa)EctC structure, one monomer is present in the asymmetric unit.", "section": "RESULTS", "ner": [ [ 8, 12, "open", "protein_state" ], [ 15, 17, "Sa", "species" ], [ 18, 22, "EctC", "protein" ], [ 23, 32, "structure", "evidence" ], [ 38, 45, "monomer", "oligomeric_state" ] ] }, { "sid": 147, "sent": "We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well.", "section": "RESULTS", "ner": [ [ 35, 42, "packing", "experimental_method" ], [ 60, 67, "monomer", "oligomeric_state" ], [ 68, 75, "monomer", "oligomeric_state" ], [ 169, 174, "dimer", "oligomeric_state" ], [ 202, 214, "crystal form", "evidence" ] ] }, { "sid": 148, "sent": "Indeed, a similar dimer configuration to the one described for the \u201csemi-closed\u201d (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two \u03b2-sheets.", "section": "RESULTS", "ner": [ [ 18, 23, "dimer", "oligomeric_state" ], [ 68, 79, "semi-closed", "protein_state" ], [ 82, 84, "Sa", "species" ], [ 85, 89, "EctC", "protein" ], [ 90, 99, "structure", "evidence" ], [ 126, 133, "monomer", "oligomeric_state" ], [ 134, 141, "monomer", "oligomeric_state" ], [ 175, 183, "\u03b2-sheets", "structure_element" ] ] }, { "sid": 149, "sent": "The crystallographic two-fold axis present within the crystal symmetry is located exactly in between the two monomers, resulting in a monomer within the asymmetric unit.", "section": "RESULTS", "ner": [ [ 109, 117, "monomers", "oligomeric_state" ], [ 134, 141, "monomer", "oligomeric_state" ] ] }, { "sid": 150, "sent": "Hence, the same dimer observed in the \u201csemi-closed\u201d structure of (Sa)EctC can also be observed in the \u201copen\u201d structure.", "section": "RESULTS", "ner": [ [ 16, 21, "dimer", "oligomeric_state" ], [ 39, 50, "semi-closed", "protein_state" ], [ 52, 61, "structure", "evidence" ], [ 66, 68, "Sa", "species" ], [ 69, 73, "EctC", "protein" ], [ 103, 107, "open", "protein_state" ], [ 109, 118, "structure", "evidence" ] ] }, { "sid": 151, "sent": "Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5).", "section": "RESULTS", "ner": [ [ 62, 73, "DALI search", "experimental_method" ], [ 105, 109, "EctC", "protein" ], [ 135, 141, "dimers", "oligomeric_state" ], [ 161, 168, "monomer", "oligomeric_state" ], [ 169, 176, "monomer", "oligomeric_state" ], [ 201, 206, "dimer", "oligomeric_state" ] ] }, { "sid": 152, "sent": "Structural rearrangements of the flexible (Sa)EctC carboxy-terminus", "section": "RESULTS", "ner": [ [ 33, 41, "flexible", "protein_state" ], [ 43, 45, "Sa", "species" ], [ 46, 50, "EctC", "protein" ], [ 51, 67, "carboxy-terminus", "structure_element" ] ] }, { "sid": 153, "sent": "The cupin core represents the structural framework of ectoine synthase (Figs 4 and 5).", "section": "RESULTS", "ner": [ [ 54, 70, "ectoine synthase", "protein_type" ] ] }, { "sid": 154, "sent": "The major difference in the two crystal structures of the (Sa)EctC protein reported here is the orientation of the carboxy-terminus.", "section": "RESULTS", "ner": [ [ 32, 50, "crystal structures", "evidence" ], [ 59, 61, "Sa", "species" ], [ 62, 66, "EctC", "protein" ], [ 115, 131, "carboxy-terminus", "structure_element" ] ] }, { "sid": 155, "sent": "Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family.", "section": "RESULTS", "ner": [ [ 32, 55, "carboxy-terminal region", "structure_element" ], [ 63, 78, "137 amino acids", "residue_range" ], [ 91, 93, "Sa", "species" ], [ 94, 98, "EctC", "protein" ], [ 111, 127, "highly conserved", "protein_state" ], [ 147, 155, "extended", "protein_state" ], [ 156, 168, "EctC protein", "protein_type" ] ] }, { "sid": 156, "sent": "At the end of \u03b2-strand \u03b211, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.", "section": "RESULTS", "ner": [ [ 14, 22, "\u03b2-strand", "structure_element" ], [ 23, 26, "\u03b211", "structure_element" ], [ 44, 53, "conserved", "protein_state" ], [ 54, 61, "proline", "residue_name" ], [ 72, 79, "Pro-109", "residue_name_number" ], [ 84, 91, "Pro-110", "residue_name_number" ], [ 163, 165, "Sa", "species" ], [ 166, 170, "EctC", "protein" ] ] }, { "sid": 157, "sent": "In the \u201csemi-closed\u201d (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.", "section": "RESULTS", "ner": [ [ 8, 19, "semi-closed", "protein_state" ], [ 22, 24, "Sa", "species" ], [ 25, 29, "EctC", "protein" ], [ 30, 39, "structure", "evidence" ], [ 53, 69, "electron density", "evidence" ], [ 77, 93, "carboxy-terminus", "structure_element" ], [ 109, 130, "7 amino acid residues", "residue_range" ], [ 152, 159, "Gly-121", "residue_name_number" ] ] }, { "sid": 158, "sent": "These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a).", "section": "RESULTS", "ner": [ [ 41, 52, "small helix", "structure_element" ], [ 70, 74, "open", "protein_state" ], [ 75, 81, "cavity", "site" ], [ 89, 99, "cupin-fold", "structure_element" ], [ 108, 110, "Sa", "species" ], [ 111, 115, "EctC", "protein" ] ] }, { "sid": 159, "sent": "Furthermore, this helix is stabilized via interactions with the loop region between \u03b2-strands \u03b24 and \u03b26, thereby inducing a structural rearrangement.", "section": "RESULTS", "ner": [ [ 18, 23, "helix", "structure_element" ], [ 64, 75, "loop region", "structure_element" ], [ 84, 93, "\u03b2-strands", "structure_element" ], [ 94, 96, "\u03b24", "structure_element" ], [ 101, 103, "\u03b26", "structure_element" ] ] }, { "sid": 160, "sent": "This induces the formation of \u03b2-strand \u03b25, which is not present when the small C-terminal helix is absent as observed in the \u201copen\u201d (Sa)EctC structure.", "section": "RESULTS", "ner": [ [ 30, 38, "\u03b2-strand", "structure_element" ], [ 39, 41, "\u03b25", "structure_element" ], [ 73, 95, "small C-terminal helix", "structure_element" ], [ 99, 105, "absent", "protein_state" ], [ 126, 130, "open", "protein_state" ], [ 133, 135, "Sa", "species" ], [ 136, 140, "EctC", "protein" ], [ 141, 150, "structure", "evidence" ] ] }, { "sid": 161, "sent": "As a result, the newly formed \u03b2-strand \u03b25 is reoriented and moved by 2.4 \u00c5 within the \u201csemi-closed\u201d (Sa)EctC structure (Fig 4a to 4c).", "section": "RESULTS", "ner": [ [ 30, 38, "\u03b2-strand", "structure_element" ], [ 39, 41, "\u03b25", "structure_element" ], [ 87, 98, "semi-closed", "protein_state" ], [ 101, 103, "Sa", "species" ], [ 104, 108, "EctC", "protein" ], [ 109, 118, "structure", "evidence" ] ] }, { "sid": 162, "sent": "It is worth mentioning that \u03b2-strand \u03b25 is located next to His-93, which in all likelihood involved in metal binding (see below).", "section": "RESULTS", "ner": [ [ 28, 36, "\u03b2-strand", "structure_element" ], [ 37, 39, "\u03b25", "structure_element" ], [ 59, 65, "His-93", "residue_name_number" ], [ 103, 108, "metal", "chemical" ] ] }, { "sid": 163, "sent": "The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of \u03b2-strand \u03b25.", "section": "RESULTS", "ner": [ [ 21, 24, "His", "residue_name" ], [ 62, 64, "Sa", "species" ], [ 65, 69, "EctC", "protein" ], [ 70, 80, "structures", "evidence" ], [ 120, 128, "\u03b2-strand", "structure_element" ], [ 129, 131, "\u03b25", "structure_element" ] ] }, { "sid": 164, "sent": "Therefore the sealing of the cupin fold, as described above, seem to have an indirect influence on the architecture of the postulated iron-binding site.", "section": "RESULTS", "ner": [ [ 29, 39, "cupin fold", "structure_element" ], [ 134, 151, "iron-binding site", "site" ] ] }, { "sid": 165, "sent": "The consecutive Pro-109 and Pro-110 residues found at the end of \u03b2-strand \u03b211are highly conserved in EctC-type proteins (Fig 2).", "section": "RESULTS", "ner": [ [ 16, 23, "Pro-109", "residue_name_number" ], [ 28, 35, "Pro-110", "residue_name_number" ], [ 65, 73, "\u03b2-strand", "structure_element" ], [ 74, 77, "\u03b211", "structure_element" ], [ 81, 97, "highly conserved", "protein_state" ], [ 101, 119, "EctC-type proteins", "protein_type" ] ] }, { "sid": 166, "sent": "They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold.", "section": "RESULTS", "ner": [ [ 69, 85, "carboxy-terminus", "structure_element" ], [ 87, 109, "27 amino acid residues", "residue_range" ], [ 115, 117, "Sa", "species" ], [ 118, 122, "EctC", "protein" ], [ 136, 146, "cupin fold", "structure_element" ] ] }, { "sid": 167, "sent": "In the \u201csemi-closed\u201d structure this results in a complete closure of the entry of the cupin barrel (Fig 4a to 4c).", "section": "RESULTS", "ner": [ [ 8, 19, "semi-closed", "protein_state" ], [ 21, 30, "structure", "evidence" ], [ 86, 98, "cupin barrel", "structure_element" ] ] }, { "sid": 168, "sent": "In the \u201copen\u201d (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.", "section": "RESULTS", "ner": [ [ 8, 12, "open", "protein_state" ], [ 15, 17, "Sa", "species" ], [ 18, 22, "EctC", "protein" ], [ 23, 32, "structure", "evidence" ], [ 39, 46, "proline", "residue_name" ], [ 75, 91, "electron density", "evidence" ], [ 124, 131, "Pro-110", "residue_name_number" ], [ 137, 153, "electron density", "evidence" ], [ 213, 229, "carboxy-terminus", "structure_element" ] ] }, { "sid": 169, "sent": "A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the \u201copen\u201d and \u201csemi-closed\u201d (Sa)EctC structures.", "section": "RESULTS", "ner": [ [ 39, 46, "Pro-109", "residue_name_number" ], [ 121, 127, "His-55", "residue_name_number" ], [ 152, 156, "open", "protein_state" ], [ 163, 174, "semi-closed", "protein_state" ], [ 177, 179, "Sa", "species" ], [ 180, 184, "EctC", "protein" ], [ 185, 195, "structures", "evidence" ] ] }, { "sid": 170, "sent": "The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of \u03b2-strand \u03b211.", "section": "RESULTS", "ner": [ [ 4, 11, "Pro-109", "residue_name_number" ], [ 12, 18, "His-55", "residue_name_number" ], [ 43, 49, "stable", "protein_state" ], [ 70, 77, "proline", "residue_name" ], [ 101, 109, "\u03b2-strand", "structure_element" ], [ 110, 113, "\u03b211", "structure_element" ] ] }, { "sid": 171, "sent": "Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein.", "section": "RESULTS", "ner": [ [ 12, 19, "proline", "residue_name" ], [ 49, 72, "carboxy-terminal region", "structure_element" ], [ 81, 83, "Sa", "species" ], [ 84, 88, "EctC", "protein" ], [ 117, 123, "His-55", "residue_name_number" ], [ 129, 136, "Pro-109", "residue_name_number" ] ] }, { "sid": 172, "sent": "In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.", "section": "RESULTS", "ner": [ [ 40, 47, "Pro-109", "residue_name_number" ], [ 52, 58, "His-55", "residue_name_number" ], [ 64, 87, "carboxy-terminal region", "structure_element" ], [ 92, 94, "Sa", "species" ], [ 95, 99, "EctC", "protein" ], [ 142, 149, "Glu-115", "residue_name_number" ], [ 155, 161, "His-55", "residue_name_number" ], [ 204, 215, "small helix", "structure_element" ], [ 223, 239, "carboxy-terminus", "structure_element" ] ] }, { "sid": 173, "sent": "The interaction between Glu-115 and His-55 is only visible in the \u201csemi-closed\u201d structure where the partially extended carboxy-terminus is resolved in the electron density.", "section": "RESULTS", "ner": [ [ 24, 31, "Glu-115", "residue_name_number" ], [ 36, 42, "His-55", "residue_name_number" ], [ 67, 78, "semi-closed", "protein_state" ], [ 80, 89, "structure", "evidence" ], [ 100, 118, "partially extended", "protein_state" ], [ 119, 135, "carboxy-terminus", "structure_element" ], [ 155, 171, "electron density", "evidence" ] ] }, { "sid": 174, "sent": "In the \u201copen\u201d structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b).", "section": "RESULTS", "ner": [ [ 8, 12, "open", "protein_state" ], [ 14, 23, "structure", "evidence" ], [ 32, 34, "Sa", "species" ], [ 35, 39, "EctC", "protein" ], [ 87, 94, "Glu-115", "residue_name_number" ] ] }, { "sid": 175, "sent": "Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the \u201copen\u201d (Sa)EctC structure and consequently results in less well defined electron density in this region.", "section": "RESULTS", "ner": [ [ 105, 121, "carboxy-terminus", "structure_element" ], [ 130, 134, "open", "protein_state" ], [ 137, 139, "Sa", "species" ], [ 140, 144, "EctC", "protein" ], [ 145, 154, "structure", "evidence" ], [ 201, 217, "electron density", "evidence" ] ] }, { "sid": 176, "sent": "Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus.", "section": "FIG", "ner": [ [ 29, 47, "metal-binding site", "site" ], [ 56, 58, "Sa", "species" ], [ 59, 63, "EctC", "protein" ], [ 80, 88, "flexible", "protein_state" ], [ 89, 105, "carboxy-terminus", "structure_element" ] ] }, { "sid": 177, "sent": "(a) The described water molecule (depicted as orange sphere) is bound via interactions with the side chains of Glu-57, Tyr-85, and His-93.", "section": "FIG", "ner": [ [ 18, 23, "water", "chemical" ], [ 111, 117, "Glu-57", "residue_name_number" ], [ 119, 125, "Tyr-85", "residue_name_number" ], [ 131, 137, "His-93", "residue_name_number" ] ] }, { "sid": 178, "sent": "The position occupied by this water molecule represents probably the position of the Fe2+ cofactor in the active side of the ectoine synthase.", "section": "FIG", "ner": [ [ 30, 35, "water", "chemical" ], [ 85, 89, "Fe2+", "chemical" ], [ 106, 117, "active side", "site" ], [ 125, 141, "ectoine synthase", "protein_type" ] ] }, { "sid": 179, "sent": "His-55 interacts with the double proline motif (Pro-109 and Pro-110).", "section": "FIG", "ner": [ [ 0, 6, "His-55", "residue_name_number" ], [ 26, 46, "double proline motif", "structure_element" ], [ 48, 55, "Pro-109", "residue_name_number" ], [ 60, 67, "Pro-110", "residue_name_number" ] ] }, { "sid": 180, "sent": "It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the \u201csemi-closed\u201d (Sa)EctC structure.", "section": "FIG", "ner": [ [ 67, 74, "Glu-115", "residue_name_number" ], [ 101, 109, "flexible", "protein_state" ], [ 110, 126, "carboxy-terminus", "structure_element" ], [ 151, 153, "Sa", "species" ], [ 154, 158, "EctC", "protein" ], [ 183, 194, "semi-closed", "protein_state" ], [ 197, 199, "Sa", "species" ], [ 200, 204, "EctC", "protein" ], [ 205, 214, "structure", "evidence" ] ] }, { "sid": 181, "sent": "(b) An overlay of the \u201copen\u201d (colored in light blue) and the \u201csemi-closed\u201d (colored in green) structure of the (Sa)EctC protein.", "section": "FIG", "ner": [ [ 7, 14, "overlay", "experimental_method" ], [ 23, 27, "open", "protein_state" ], [ 62, 73, "semi-closed", "protein_state" ], [ 94, 103, "structure", "evidence" ], [ 112, 114, "Sa", "species" ], [ 115, 119, "EctC", "protein" ] ] }, { "sid": 182, "sent": "The putative iron binding site of (Sa)EctC", "section": "RESULTS", "ner": [ [ 13, 30, "iron binding site", "site" ], [ 35, 37, "Sa", "species" ], [ 38, 42, "EctC", "protein" ] ] }, { "sid": 183, "sent": "In the \u201csemi-closed\u201d structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel.", "section": "RESULTS", "ner": [ [ 8, 19, "semi-closed", "protein_state" ], [ 21, 30, "structure", "evidence" ], [ 35, 37, "Sa", "species" ], [ 38, 42, "EctC", "protein" ], [ 61, 69, "monomers", "oligomeric_state" ], [ 120, 136, "electron density", "evidence" ], [ 159, 171, "cupin barrel", "structure_element" ] ] }, { "sid": 184, "sent": "Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy.", "section": "RESULTS", "ner": [ [ 7, 9, "Sa", "species" ], [ 10, 14, "EctC", "protein" ], [ 20, 25, "metal", "chemical" ], [ 77, 81, "Fe2+", "chemical" ], [ 86, 90, "Zn2+", "chemical" ], [ 106, 113, "density", "evidence" ], [ 123, 140, "refined occupancy", "experimental_method" ] ] }, { "sid": 185, "sent": "Only the refinement of Fe2+ resulted in a visibly improved electron density, however with a low degree of occupancy.", "section": "RESULTS", "ner": [ [ 23, 27, "Fe2+", "chemical" ], [ 59, 75, "electron density", "evidence" ] ] }, { "sid": 186, "sent": "This possible iron molecule is bound via interactions with Glu-57, Tyr-85 and His-93 (Fig 6a and 6b).", "section": "RESULTS", "ner": [ [ 14, 18, "iron", "chemical" ], [ 59, 65, "Glu-57", "residue_name_number" ], [ 67, 73, "Tyr-85", "residue_name_number" ], [ 78, 84, "His-93", "residue_name_number" ] ] }, { "sid": 187, "sent": "The distance between the side chains of these residues and the (putative) iron co-factor is 3.1 \u00c5 for Glu-57, 2.9 \u00c5 for Tyr-85, and 2.9 \u00c5 for His-93, respectively.", "section": "RESULTS", "ner": [ [ 74, 78, "iron", "chemical" ], [ 102, 108, "Glu-57", "residue_name_number" ], [ 120, 126, "Tyr-85", "residue_name_number" ], [ 142, 148, "His-93", "residue_name_number" ] ] }, { "sid": 188, "sent": "These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure.", "section": "RESULTS", "ner": [ [ 51, 69, "iron binding sites", "site" ], [ 106, 116, "absence of", "protein_state" ], [ 146, 148, "Sa", "species" ], [ 149, 153, "EctC", "protein" ], [ 154, 171, "crystal structure", "evidence" ] ] }, { "sid": 189, "sent": "Since both the refinement and the distance did not clearly identify an iron molecule, we decided to conservatively place a water molecule at this position.", "section": "RESULTS", "ner": [ [ 71, 75, "iron", "chemical" ], [ 123, 128, "water", "chemical" ] ] }, { "sid": 190, "sent": "The position of this water molecule is described in more detail below and is highlighted in Figs 5a and 5b and 6a and 6b as a sphere.", "section": "RESULTS", "ner": [ [ 21, 26, "water", "chemical" ] ] }, { "sid": 191, "sent": "Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2).", "section": "RESULTS", "ner": [ [ 55, 60, "water", "chemical" ], [ 74, 92, "strictly conserved", "protein_state" ], [ 103, 112, "alignment", "experimental_method" ], [ 135, 147, "EctC protein", "protein_type" ], [ 188, 206, "EctC-type proteins", "protein_type" ] ] }, { "sid": 192, "sent": "In the \u201copen\u201d structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the \u201csemi-closed\u201d structure.", "section": "RESULTS", "ner": [ [ 8, 12, "open", "protein_state" ], [ 14, 23, "structure", "evidence" ], [ 32, 34, "Sa", "species" ], [ 35, 39, "EctC", "protein" ], [ 49, 65, "electron density", "evidence" ], [ 99, 103, "iron", "chemical" ], [ 126, 137, "semi-closed", "protein_state" ], [ 139, 148, "structure", "evidence" ] ] }, { "sid": 193, "sent": "However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 \u00c5) of the \u201copen\u201d (Sa)EctC structure.", "section": "RESULTS", "ner": [ [ 14, 30, "electron density", "evidence" ], [ 51, 56, "water", "chemical" ], [ 80, 84, "iron", "chemical" ], [ 94, 99, "water", "chemical" ], [ 190, 194, "open", "protein_state" ], [ 197, 199, "Sa", "species" ], [ 200, 204, "EctC", "protein" ], [ 205, 214, "structure", "evidence" ] ] }, { "sid": 194, "sent": "In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the \u201csemi-closed\u201d structure match nicely with those of the corresponding residues of the \u201ciron-free\u201d \u201copen\u201d structure (Fig 6b).", "section": "RESULTS", "ner": [ [ 5, 20, "superimposition", "experimental_method" ], [ 30, 32, "Sa", "species" ], [ 33, 37, "EctC", "protein" ], [ 38, 56, "crystal structures", "evidence" ], [ 128, 134, "Glu-57", "residue_name_number" ], [ 136, 142, "Tyr-85", "residue_name_number" ], [ 148, 154, "His-93", "residue_name_number" ], [ 178, 182, "iron", "chemical" ], [ 191, 202, "semi-closed", "protein_state" ], [ 204, 213, "structure", "evidence" ], [ 276, 285, "iron-free", "protein_state" ], [ 288, 292, "open", "protein_state" ], [ 294, 303, "structure", "evidence" ] ] }, { "sid": 195, "sent": "Only His-93 is slightly rotated inwards in the \u201csemi-closed\u201d structure, most likely due to formation of \u03b2-strand \u03b25 as described above.", "section": "RESULTS", "ner": [ [ 5, 11, "His-93", "residue_name_number" ], [ 48, 59, "semi-closed", "protein_state" ], [ 61, 70, "structure", "evidence" ], [ 104, 112, "\u03b2-strand", "structure_element" ], [ 113, 115, "\u03b25", "structure_element" ] ] }, { "sid": 196, "sent": "Taken together, this observations indicate, that the architecture of the presumptive iron-binding site is pre-set for the binding of the catalytically important metal by the ectoine synthase.", "section": "RESULTS", "ner": [ [ 85, 102, "iron-binding site", "site" ], [ 161, 166, "metal", "chemical" ], [ 174, 190, "ectoine synthase", "protein_type" ] ] }, { "sid": 197, "sent": "Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of \u03b2-strand \u03b25) in the \u201copen\u201d and \u201csemi-closed\u201d (Sa)EctC structures.", "section": "RESULTS", "ner": [ [ 66, 72, "Tyr-52", "residue_name_number" ], [ 87, 91, "loop", "structure_element" ], [ 109, 117, "\u03b2-strand", "structure_element" ], [ 118, 120, "\u03b25", "structure_element" ], [ 130, 134, "open", "protein_state" ], [ 141, 152, "semi-closed", "protein_state" ], [ 155, 157, "Sa", "species" ], [ 158, 162, "EctC", "protein" ], [ 163, 173, "structures", "evidence" ] ] }, { "sid": 198, "sent": "In the \u201csemi-closed\u201d structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 \u00c5) makes it highly unlikely that Tyr-52 is directly involved in metal binding.", "section": "RESULTS", "ner": [ [ 8, 19, "semi-closed", "protein_state" ], [ 21, 30, "structure", "evidence" ], [ 72, 78, "Tyr-52", "residue_name_number" ], [ 98, 102, "iron", "chemical" ], [ 189, 195, "Tyr-52", "residue_name_number" ], [ 220, 225, "metal", "chemical" ] ] }, { "sid": 199, "sent": "Nevertheless, its substitution by an Ala residue causes a strong decrease in iron-content and enzyme activity of the mutant protein (Table 1).", "section": "RESULTS", "ner": [ [ 18, 30, "substitution", "experimental_method" ], [ 37, 40, "Ala", "residue_name" ], [ 77, 81, "iron", "chemical" ], [ 117, 123, "mutant", "protein_state" ] ] }, { "sid": 200, "sent": "It becomes apparent from an overlay of the \u201copen\u201d and \u201csemi-closed\u201d (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).", "section": "RESULTS", "ner": [ [ 28, 35, "overlay", "experimental_method" ], [ 44, 48, "open", "protein_state" ], [ 55, 66, "semi-closed", "protein_state" ], [ 69, 71, "Sa", "species" ], [ 72, 76, "EctC", "protein" ], [ 77, 95, "crystal structures", "evidence" ], [ 119, 125, "Tyr-52", "residue_name_number" ], [ 176, 180, "iron", "chemical" ], [ 253, 258, "metal", "chemical" ], [ 269, 275, "Glu-57", "residue_name_number" ], [ 277, 283, "Tyr-85", "residue_name_number" ], [ 289, 295, "His-93", "residue_name_number" ] ] }, { "sid": 201, "sent": "Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-\u03b3-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.", "section": "RESULTS", "ner": [ [ 6, 12, "Tyr-52", "residue_name_number" ], [ 16, 34, "strictly conserved", "protein_state" ], [ 41, 50, "alignment", "experimental_method" ], [ 58, 76, "EctC-type proteins", "protein_type" ], [ 160, 176, "ectoine synthase", "protein_type" ], [ 190, 200, "absence of", "protein_state" ], [ 201, 210, "N-\u03b3-ADABA", "chemical" ], [ 219, 221, "Sa", "species" ], [ 222, 226, "EctC", "protein" ], [ 227, 245, "crystal structures", "evidence" ], [ 276, 282, "Tyr-52", "residue_name_number" ] ] }, { "sid": 202, "sent": "To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity.", "section": "RESULTS", "ner": [ [ 32, 49, "iron binding site", "site" ], [ 73, 115, "structure-guided site-directed mutagenesis", "experimental_method" ], [ 144, 146, "Sa", "species" ], [ 147, 151, "EctC", "protein" ], [ 171, 175, "iron", "chemical" ] ] }, { "sid": 203, "sent": "When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).", "section": "RESULTS", "ner": [ [ 27, 33, "Glu-57", "residue_name_number" ], [ 35, 41, "Tyr-85", "residue_name_number" ], [ 43, 49, "His-93", "residue_name_number" ], [ 72, 96, "mono-nuclear iron center", "site" ], [ 105, 107, "Sa", "species" ], [ 108, 112, "EctC", "protein" ], [ 113, 130, "crystal structure", "evidence" ], [ 149, 157, "replaced", "experimental_method" ], [ 164, 167, "Ala", "residue_name" ], [ 213, 217, "iron", "chemical" ], [ 233, 239, "mutant", "protein_state" ] ] }, { "sid": 204, "sent": "For some of the presumptive iron-coordinating residues, additional site-directed mutagenesis experiments were carried out.", "section": "RESULTS", "ner": [ [ 28, 54, "iron-coordinating residues", "site" ], [ 67, 92, "site-directed mutagenesis", "experimental_method" ] ] }, { "sid": 205, "sent": "To verify the importance of the negative charge in the position of Glu-57, we created an Asp variant.", "section": "RESULTS", "ner": [ [ 67, 73, "Glu-57", "residue_name_number" ], [ 89, 92, "Asp", "residue_name" ], [ 93, 100, "variant", "protein_state" ] ] }, { "sid": 206, "sent": "This mutant protein rescued the enzyme activity and iron content of the Ala substitution substantially (Table 1).", "section": "RESULTS", "ner": [ [ 5, 11, "mutant", "protein_state" ], [ 52, 56, "iron", "chemical" ], [ 72, 75, "Ala", "residue_name" ], [ 76, 88, "substitution", "experimental_method" ] ] }, { "sid": 207, "sent": "We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative.", "section": "RESULTS", "ner": [ [ 8, 16, "replaced", "experimental_method" ], [ 17, 23, "Tyr-85", "residue_name_number" ], [ 38, 41, "Phe", "residue_name" ], [ 47, 50, "Trp", "residue_name" ], [ 68, 74, "mutant", "protein_state" ], [ 126, 130, "iron", "chemical" ] ] }, { "sid": 208, "sent": "Collectively, these data suggest that the hydroxyl group of the Tyr-85 side chain is needed for the binding of the iron (Fig 6a).", "section": "RESULTS", "ner": [ [ 64, 70, "Tyr-85", "residue_name_number" ], [ 115, 119, "iron", "chemical" ] ] }, { "sid": 209, "sent": "We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).", "section": "RESULTS", "ner": [ [ 8, 16, "replaced", "experimental_method" ], [ 33, 53, "iron-binding residue", "site" ], [ 54, 60, "His-93", "residue_name_number" ], [ 67, 70, "Asn", "residue_name" ], [ 92, 94, "Sa", "species" ], [ 95, 99, "EctC", "protein" ], [ 161, 165, "iron", "chemical" ], [ 210, 219, "wild-type", "protein_state" ] ] }, { "sid": 210, "sent": "Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance.", "section": "RESULTS", "ner": [ [ 68, 94, "iron-coordinating residues", "site" ], [ 96, 102, "Glu-57", "residue_name_number" ], [ 104, 110, "Tyr-85", "residue_name_number" ], [ 112, 118, "His-93", "residue_name_number" ], [ 149, 153, "Fe2+", "chemical" ], [ 170, 172, "Sa", "species" ], [ 173, 177, "EctC", "protein" ] ] }, { "sid": 211, "sent": "A chemically undefined ligand in the (Sa)EctC structure provides clues for the binding of the N-\u03b3-ADABA substrate", "section": "RESULTS", "ner": [ [ 38, 40, "Sa", "species" ], [ 41, 45, "EctC", "protein" ], [ 46, 55, "structure", "evidence" ], [ 94, 103, "N-\u03b3-ADABA", "chemical" ] ] }, { "sid": 212, "sent": "Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-\u03b3-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).", "section": "RESULTS", "ner": [ [ 47, 65, "co-crystallization", "experimental_method" ], [ 69, 88, "soaking experiments", "experimental_method" ], [ 120, 122, "Sa", "species" ], [ 123, 127, "EctC", "protein" ], [ 128, 146, "crystal structures", "evidence" ], [ 183, 192, "N-\u03b3-ADABA", "chemical" ], [ 197, 204, "ectoine", "chemical" ], [ 230, 246, "ectoine synthase", "protein_type" ] ] }, { "sid": 213, "sent": "However, in the \u201csemi-closed\u201d (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.", "section": "RESULTS", "ner": [ [ 17, 28, "semi-closed", "protein_state" ], [ 31, 33, "Sa", "species" ], [ 34, 38, "EctC", "protein" ], [ 39, 48, "structure", "evidence" ], [ 59, 80, "carboxy-terminal loop", "structure_element" ], [ 119, 135, "electron density", "evidence" ], [ 174, 185, "active site", "site" ], [ 227, 254, "crystallographic refinement", "experimental_method" ] ] }, { "sid": 214, "sent": "This is in contrast to the high-resolution \u201copen\u201d structure of the (Sa)EctC protein where no additional electron density was observed after refinement.", "section": "RESULTS", "ner": [ [ 44, 48, "open", "protein_state" ], [ 50, 59, "structure", "evidence" ], [ 68, 70, "Sa", "species" ], [ 71, 75, "EctC", "protein" ], [ 104, 120, "electron density", "evidence" ] ] }, { "sid": 215, "sent": "We tried to fit all compounds used in the buffers during purification and crystallization into the observed electron density, but none matched.", "section": "RESULTS", "ner": [ [ 57, 69, "purification", "experimental_method" ], [ 74, 89, "crystallization", "experimental_method" ], [ 108, 124, "electron density", "evidence" ] ] }, { "sid": 216, "sent": "This observation indicates that the chemically undefined ligand was either trapped by the (Sa)EctC protein during its heterologous production in E. coli or during crystallization.", "section": "RESULTS", "ner": [ [ 91, 93, "Sa", "species" ], [ 94, 98, "EctC", "protein" ], [ 145, 152, "E. coli", "species" ], [ 163, 178, "crystallization", "experimental_method" ] ] }, { "sid": 217, "sent": "Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments.", "section": "RESULTS", "ner": [ [ 14, 17, "PEG", "chemical" ], [ 76, 83, "density", "evidence" ], [ 121, 124, "PEG", "chemical" ], [ 159, 162, "PEG", "chemical" ], [ 207, 210, "PEG", "chemical" ] ] }, { "sid": 218, "sent": "Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density.", "section": "RESULTS", "ner": [ [ 38, 62, "electron density feature", "evidence" ], [ 125, 127, "Sa", "species" ], [ 128, 132, "EctC", "protein" ], [ 146, 161, "hexane-1,6-diol", "chemical" ], [ 218, 234, "electron density", "evidence" ] ] }, { "sid": 219, "sent": "However, to the best of our knowledge, hexane-1,6-diol is not part of the E. coli metabolome.", "section": "RESULTS", "ner": [ [ 39, 54, "hexane-1,6-diol", "chemical" ], [ 74, 81, "E. coli", "species" ] ] }, { "sid": 220, "sent": "Despite these notable limitations, we considered the serendipitously trapped compound as a mock ligand that might provide useful insights into the spatial positioning of the true EctC substrate and those residues that coordinate it within the ectoine synthase active site.", "section": "RESULTS", "ner": [ [ 179, 183, "EctC", "protein" ], [ 243, 259, "ectoine synthase", "protein_type" ], [ 260, 271, "active site", "site" ] ] }, { "sid": 221, "sent": "We note that both N-\u03b3-ADABA and hexane-1,6-diol are both C6-compounds and display similar length (Fig 7a).", "section": "RESULTS", "ner": [ [ 18, 27, "N-\u03b3-ADABA", "chemical" ], [ 32, 47, "hexane-1,6-diol", "chemical" ] ] }, { "sid": 222, "sent": "A chemically undefined ligand is captured in the active site of the \u201csemi-closed\u201d (Sa)EctC crystal structure.", "section": "FIG", "ner": [ [ 49, 60, "active site", "site" ], [ 69, 80, "semi-closed", "protein_state" ], [ 83, 85, "Sa", "species" ], [ 86, 90, "EctC", "protein" ], [ 91, 108, "crystal structure", "evidence" ] ] }, { "sid": 223, "sent": "(a) The observed electron density in the active site of the \u201csemi-closed\u201d structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-\u03b3-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.", "section": "FIG", "ner": [ [ 17, 33, "electron density", "evidence" ], [ 41, 52, "active site", "site" ], [ 61, 72, "semi-closed", "protein_state" ], [ 74, 83, "structure", "evidence" ], [ 88, 90, "Sa", "species" ], [ 91, 95, "EctC", "protein" ], [ 112, 127, "hexane-1,6-diol", "chemical" ], [ 159, 175, "electron density", "evidence" ], [ 183, 192, "N-\u03b3-ADABA", "chemical" ], [ 210, 226, "ectoine synthase", "protein_type" ] ] }, { "sid": 224, "sent": "(b) The presumable binding site of the iron co-factor and of the modeled hexane-1,6-diol molecule is depicted.", "section": "FIG", "ner": [ [ 19, 31, "binding site", "site" ], [ 39, 43, "iron", "chemical" ], [ 73, 88, "hexane-1,6-diol", "chemical" ] ] }, { "sid": 225, "sent": "The amino acid side chains involved in iron-ligand binding are colored in blue and those involved in the binding of the chemically undefined ligand are colored in green using a ball and stick representation.", "section": "FIG", "ner": [ [ 39, 43, "iron", "chemical" ] ] }, { "sid": 226, "sent": "The flexible carboxy-terminal loop of (Sa)EctC is highlighted in orange.", "section": "FIG", "ner": [ [ 4, 12, "flexible", "protein_state" ], [ 13, 34, "carboxy-terminal loop", "structure_element" ], [ 39, 41, "Sa", "species" ], [ 42, 46, "EctC", "protein" ] ] }, { "sid": 227, "sent": "The electron density was calculated as an omit map and contoured at 1.0 \u03c3.", "section": "FIG", "ner": [ [ 4, 20, "electron density", "evidence" ], [ 42, 50, "omit map", "evidence" ] ] }, { "sid": 228, "sent": "We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%.", "section": "RESULTS", "ner": [ [ 3, 10, "refined", "experimental_method" ], [ 16, 18, "Sa", "species" ], [ 19, 23, "EctC", "protein" ], [ 24, 33, "structure", "evidence" ], [ 116, 135, "R- and Rfree-factor", "evidence" ] ] }, { "sid": 229, "sent": "We also calculated an omit map and the electron density reappeared (Fig 7b).", "section": "RESULTS", "ner": [ [ 22, 30, "omit map", "evidence" ], [ 39, 55, "electron density", "evidence" ] ] }, { "sid": 230, "sent": "When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of \u03b2-sheet \u03b23, Thr-40 located in \u03b2-sheet \u03b24, and Cys-105 and Phe-107, which are both part of \u03b2-sheet \u03b211.", "section": "RESULTS", "ner": [ [ 61, 63, "Sa", "species" ], [ 64, 68, "EctC", "protein" ], [ 98, 103, "bound", "protein_state" ], [ 126, 132, "Trp-21", "residue_name_number" ], [ 137, 143, "Ser-23", "residue_name_number" ], [ 147, 154, "\u03b2-sheet", "structure_element" ], [ 155, 157, "\u03b23", "structure_element" ], [ 159, 165, "Thr-40", "residue_name_number" ], [ 177, 184, "\u03b2-sheet", "structure_element" ], [ 185, 187, "\u03b24", "structure_element" ], [ 193, 200, "Cys-105", "residue_name_number" ], [ 205, 212, "Phe-107", "residue_name_number" ], [ 237, 244, "\u03b2-sheet", "structure_element" ], [ 245, 248, "\u03b211", "structure_element" ] ] }, { "sid": 231, "sent": "Remarkably, all of these residues are highly conserved throughout the extended EctC protein family (Fig 2).", "section": "RESULTS", "ner": [ [ 38, 54, "highly conserved", "protein_state" ], [ 79, 91, "EctC protein", "protein_type" ] ] }, { "sid": 232, "sent": "Structure-guided site-directed mutagenesis of the catalytic core of the ectoine synthase", "section": "RESULTS", "ner": [ [ 0, 42, "Structure-guided site-directed mutagenesis", "experimental_method" ], [ 50, 64, "catalytic core", "site" ], [ 72, 88, "ectoine synthase", "protein_type" ] ] }, { "sid": 233, "sent": "In a previous alignment of the amino acid sequences of 440 EctC-type proteins, 13 amino acids were identified as strictly conserved residues.", "section": "RESULTS", "ner": [ [ 14, 51, "alignment of the amino acid sequences", "experimental_method" ], [ 59, 77, "EctC-type proteins", "protein_type" ], [ 113, 131, "strictly conserved", "protein_state" ] ] }, { "sid": 234, "sent": "These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).", "section": "RESULTS", "ner": [ [ 32, 38, "Thr-40", "residue_name_number" ], [ 40, 46, "Tyr-52", "residue_name_number" ], [ 48, 54, "His-55", "residue_name_number" ], [ 56, 62, "Glu-57", "residue_name_number" ], [ 64, 70, "Gly-64", "residue_name_number" ], [ 72, 78, "Tyr-85", "residue_name_number" ], [ 80, 86, "Leu-87", "residue_name_number" ], [ 88, 94, "His-93", "residue_name_number" ], [ 96, 103, "Phe-107", "residue_name_number" ], [ 105, 112, "Pro-109", "residue_name_number" ], [ 114, 121, "Gly-113", "residue_name_number" ], [ 123, 130, "Glu-115", "residue_name_number" ], [ 136, 143, "His-117", "residue_name_number" ], [ 152, 154, "Sa", "species" ], [ 155, 159, "EctC", "protein" ] ] }, { "sid": 235, "sent": "Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of \u03b2-strands and \u03b1-helices.", "section": "RESULTS", "ner": [ [ 20, 26, "Gly-64", "residue_name_number" ], [ 28, 35, "Pro-109", "residue_name_number" ], [ 41, 48, "Gly-113", "residue_name_number" ], [ 148, 157, "\u03b2-strands", "structure_element" ], [ 162, 171, "\u03b1-helices", "structure_element" ] ] }, { "sid": 236, "sent": "We considered the remaining ten residues as important either for ligand binding, for catalysis, or for the structurally correct orientation of the flexible carboxy-terminus of the (Sa)EctC protein.", "section": "RESULTS", "ner": [ [ 147, 155, "flexible", "protein_state" ], [ 156, 172, "carboxy-terminus", "structure_element" ], [ 181, 183, "Sa", "species" ], [ 184, 188, "EctC", "protein" ] ] }, { "sid": 237, "sent": "As described above, the side chains of Glu-57, Tyr-85, and His-93 are probably involved in iron binding (Table 1 and Fig 6a).", "section": "RESULTS", "ner": [ [ 39, 45, "Glu-57", "residue_name_number" ], [ 47, 53, "Tyr-85", "residue_name_number" ], [ 59, 65, "His-93", "residue_name_number" ], [ 91, 95, "iron", "chemical" ] ] }, { "sid": 238, "sent": "In view of the (Sa)EctC structure with the serendipitously trapped compound (Fig 7b), we probed the functional importance of the seven residues that contact this ligand by structure-guided site-directed mutagenesis (Table 1).", "section": "RESULTS", "ner": [ [ 16, 18, "Sa", "species" ], [ 19, 23, "EctC", "protein" ], [ 24, 33, "structure", "evidence" ], [ 172, 214, "structure-guided site-directed mutagenesis", "experimental_method" ] ] }, { "sid": 239, "sent": "Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations.", "section": "RESULTS", "ner": [ [ 14, 20, "mutant", "protein_state" ], [ 22, 24, "Sa", "species" ], [ 25, 29, "EctC", "protein" ], [ 59, 66, "E. coli", "species" ], [ 83, 106, "affinity chromatography", "experimental_method" ] ] }, { "sid": 240, "sent": "We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 \u03bcM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-\u03b3-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.", "section": "RESULTS", "ner": [ [ 36, 38, "Sa", "species" ], [ 39, 43, "EctC", "protein" ], [ 58, 88, "single time-point enzyme assay", "experimental_method" ], [ 125, 134, "wild-type", "protein_state" ], [ 136, 138, "Sa", "species" ], [ 139, 143, "EctC", "protein" ], [ 199, 208, "N-\u03b3-ADABA", "chemical" ], [ 230, 237, "ectoine", "chemical" ] ] }, { "sid": 241, "sent": "In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1).", "section": "RESULTS", "ner": [ [ 31, 35, "iron", "chemical" ], [ 59, 65, "mutant", "protein_state" ], [ 67, 69, "Sa", "species" ], [ 70, 74, "EctC", "protein" ], [ 88, 106, "colorimetric assay", "experimental_method" ] ] }, { "sid": 242, "sent": "The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the \u201csemi-closed\u201d (Sa)EctC structure (Fig 7b).", "section": "RESULTS", "ner": [ [ 23, 47, "evolutionarily conserved", "protein_state" ], [ 48, 54, "Trp-21", "residue_name_number" ], [ 56, 62, "Ser-23", "residue_name_number" ], [ 64, 70, "Thr-40", "residue_name_number" ], [ 72, 79, "Cys-105", "residue_name_number" ], [ 85, 92, "Phe-107", "residue_name_number" ], [ 186, 197, "semi-closed", "protein_state" ], [ 200, 202, "Sa", "species" ], [ 203, 207, "EctC", "protein" ], [ 208, 217, "structure", "evidence" ] ] }, { "sid": 243, "sent": "We replaced each of these residues with an Ala residue and found that none of them had an influence on the iron content of the mutant proteins.", "section": "RESULTS", "ner": [ [ 3, 11, "replaced", "experimental_method" ], [ 43, 46, "Ala", "residue_name" ], [ 107, 111, "iron", "chemical" ], [ 127, 133, "mutant", "protein_state" ] ] }, { "sid": 244, "sent": "Thr-40 is positioned on \u03b2-strand \u03b25 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b).", "section": "RESULTS", "ner": [ [ 0, 6, "Thr-40", "residue_name_number" ], [ 24, 32, "\u03b2-strand", "structure_element" ], [ 33, 35, "\u03b25", "structure_element" ], [ 87, 99, "cupin barrel", "structure_element" ], [ 115, 117, "Sa", "species" ], [ 118, 122, "EctC", "protein" ] ] }, { "sid": 245, "sent": "We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.", "section": "RESULTS", "ner": [ [ 8, 16, "replaced", "experimental_method" ], [ 17, 24, "Phe-107", "residue_name_number" ], [ 40, 43, "Tyr", "residue_name" ], [ 50, 53, "Trp", "residue_name" ], [ 67, 78, "Phe-107/Tyr", "mutant" ], [ 79, 91, "substitution", "experimental_method" ], [ 107, 116, "wild-type", "protein_state" ], [ 158, 162, "iron", "chemical" ], [ 180, 191, "Phe-107/Trp", "mutant" ], [ 192, 204, "substitution", "experimental_method" ], [ 248, 252, "iron", "chemical" ], [ 277, 286, "wild-type", "protein_state" ] ] }, { "sid": 246, "sent": "The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding.", "section": "RESULTS", "ner": [ [ 24, 30, "mutant", "protein_state" ], [ 90, 93, "107", "residue_number" ], [ 98, 100, "Sa", "species" ], [ 101, 105, "EctC", "protein" ], [ 134, 146, "substitution", "experimental_method" ], [ 260, 264, "iron", "chemical" ] ] }, { "sid": 247, "sent": "Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.", "section": "RESULTS", "ner": [ [ 0, 11, "Replacement", "experimental_method" ], [ 24, 27, "Cys", "residue_name" ], [ 40, 42, "Sa", "species" ], [ 43, 47, "EctC", "protein" ], [ 49, 56, "Cys-105", "residue_name_number" ], [ 70, 73, "Ser", "residue_name" ], [ 130, 143, "EctC proteins", "protein_type" ], [ 197, 199, "Sa", "species" ], [ 200, 204, "EctC", "protein" ], [ 205, 212, "variant", "protein_state" ], [ 222, 231, "wild-type", "protein_state" ], [ 248, 252, "iron", "chemical" ], [ 284, 293, "wild-type", "protein_state" ] ] }, { "sid": 248, "sent": "However, the Cys-105/Ala variant was practically catalytically inactive while largely maintaining its iron content (Table 1).", "section": "RESULTS", "ner": [ [ 13, 24, "Cys-105/Ala", "mutant" ], [ 25, 32, "variant", "protein_state" ], [ 49, 71, "catalytically inactive", "protein_state" ], [ 102, 106, "iron", "chemical" ] ] }, { "sid": 249, "sent": "Since the side-chains of Cys residues are chemically reactive and often participate in enzyme catalysis, Cys-105 (or Ser-105) might serve such a role for ectoine synthase.", "section": "RESULTS", "ner": [ [ 25, 28, "Cys", "residue_name" ], [ 105, 112, "Cys-105", "residue_name_number" ], [ 117, 124, "Ser-105", "residue_name_number" ], [ 154, 170, "ectoine synthase", "protein_type" ] ] }, { "sid": 250, "sent": "We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1).", "section": "RESULTS", "ner": [ [ 16, 40, "amino acid substitutions", "experimental_method" ], [ 99, 103, "iron", "chemical" ], [ 128, 138, "Tyr-52/Ala", "mutant" ], [ 147, 157, "His-55/Ala", "mutant" ], [ 159, 161, "Sa", "species" ], [ 162, 166, "EctC", "protein" ] ] }, { "sid": 251, "sent": "Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1).", "section": "RESULTS", "ner": [ [ 14, 16, "Sa", "species" ], [ 17, 21, "EctC", "protein" ], [ 22, 40, "crystal structures", "evidence" ], [ 110, 121, "replacement", "experimental_method" ], [ 125, 131, "Tyr-52", "residue_name_number" ], [ 135, 138, "Ala", "residue_name" ], [ 167, 171, "iron", "chemical" ] ] }, { "sid": 252, "sent": "This is different for the His-55/Ala substitution.", "section": "RESULTS", "ner": [ [ 26, 36, "His-55/Ala", "mutant" ] ] }, { "sid": 253, "sent": "The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b).", "section": "RESULTS", "ner": [ [ 4, 27, "carboxy-terminal region", "structure_element" ], [ 36, 38, "Sa", "species" ], [ 39, 43, "EctC", "protein" ], [ 98, 105, "Glu-115", "residue_name_number" ], [ 111, 117, "His-55", "residue_name_number" ], [ 125, 131, "His-55", "residue_name_number" ], [ 155, 162, "Pro-110", "residue_name_number" ] ] }, { "sid": 254, "sent": "Each of these residues is evolutionarily highly conserved.", "section": "RESULTS", "ner": [ [ 26, 57, "evolutionarily highly conserved", "protein_state" ] ] }, { "sid": 255, "sent": "The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity.", "section": "RESULTS", "ner": [ [ 15, 27, "substitution", "experimental_method" ], [ 38, 45, "Glu-115", "residue_name_number" ], [ 49, 55, "His-55", "residue_name_number" ], [ 62, 65, "Ala", "residue_name" ], [ 103, 122, "interactive network", "site" ] ] }, { "sid": 256, "sent": "Indeed, the Glu-115/Ala and the His-55/Ala substitutions possessed only 21% and 16% activity of the wild-type protein, respectively (Table 1).", "section": "RESULTS", "ner": [ [ 12, 23, "Glu-115/Ala", "mutant" ], [ 32, 42, "His-55/Ala", "mutant" ], [ 100, 109, "wild-type", "protein_state" ] ] }, { "sid": 257, "sent": "The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1).", "section": "RESULTS", "ner": [ [ 4, 15, "Glu-115/Ala", "mutant" ], [ 16, 22, "mutant", "protein_state" ], [ 33, 42, "wild-type", "protein_state" ], [ 53, 57, "iron", "chemical" ], [ 71, 75, "iron", "chemical" ], [ 91, 101, "His-55/Ala", "mutant" ], [ 138, 147, "wild-type", "protein_state" ] ] }, { "sid": 258, "sent": "We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.", "section": "RESULTS", "ner": [ [ 8, 16, "replaced", "experimental_method" ], [ 17, 24, "Glu-115", "residue_name_number" ], [ 60, 63, "Asp", "residue_name" ], [ 72, 74, "Sa", "species" ], [ 75, 79, "EctC", "protein" ], [ 98, 107, "wild-type", "protein_state" ], [ 118, 122, "iron", "chemical" ], [ 150, 159, "wild-type", "protein_state" ] ] }, { "sid": 259, "sent": "Collectively, these data suggest that the correct positioning of the carboxy-terminus of the (Sa)EctC protein is of structural and functional importance for the activity of the ectoine synthase.", "section": "RESULTS", "ner": [ [ 69, 85, "carboxy-terminus", "structure_element" ], [ 94, 96, "Sa", "species" ], [ 97, 101, "EctC", "protein" ], [ 177, 193, "ectoine synthase", "protein_type" ] ] }, { "sid": 260, "sent": "Residues Leu-87 and Asp-91 are highly conserved in the ectoine synthase protein family.", "section": "RESULTS", "ner": [ [ 9, 15, "Leu-87", "residue_name_number" ], [ 20, 26, "Asp-91", "residue_name_number" ], [ 31, 47, "highly conserved", "protein_state" ], [ 55, 71, "ectoine synthase", "protein_type" ] ] }, { "sid": 261, "sent": "The replacement of Leu-87 by Ala led to a substantial drop in enzyme activity (Table 1).", "section": "RESULTS", "ner": [ [ 4, 15, "replacement", "experimental_method" ], [ 19, 25, "Leu-87", "residue_name_number" ], [ 29, 32, "Ala", "residue_name" ] ] }, { "sid": 262, "sent": "Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1).", "section": "RESULTS", "ner": [ [ 16, 27, "replacement", "experimental_method" ], [ 31, 37, "Asp-91", "residue_name_number" ], [ 41, 44, "Ala", "residue_name" ], [ 49, 52, "Glu", "residue_name" ], [ 67, 69, "Sa", "species" ], [ 70, 74, "EctC", "protein" ] ] }, { "sid": 263, "sent": "We currently cannot comment on possible functional role Asp-91.", "section": "RESULTS", "ner": [ [ 56, 62, "Asp-91", "residue_name_number" ] ] }, { "sid": 264, "sent": "However, Leu-87 is positioned at the end of one of the \u03b2-sheets that form the dimer interface (Fig 5c) and it might therefore possess a structural role.", "section": "RESULTS", "ner": [ [ 9, 15, "Leu-87", "residue_name_number" ], [ 55, 63, "\u03b2-sheets", "structure_element" ], [ 78, 93, "dimer interface", "site" ] ] }, { "sid": 265, "sent": "It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects.", "section": "RESULTS", "ner": [ [ 24, 30, "Tyr-85", "residue_name_number" ], [ 81, 85, "iron", "chemical" ], [ 108, 110, "Sa", "species" ], [ 111, 115, "EctC", "protein" ], [ 116, 127, "active site", "site" ] ] }, { "sid": 266, "sent": "His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1).", "section": "RESULTS", "ner": [ [ 0, 7, "His-117", "residue_name_number" ], [ 13, 31, "strictly conserved", "protein_state" ], [ 48, 60, "substitution", "experimental_method" ], [ 67, 70, "Ala", "residue_name" ], [ 137, 141, "iron", "chemical" ] ] }, { "sid": 267, "sent": "We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase.", "section": "RESULTS", "ner": [ [ 13, 20, "His-117", "residue_name_number" ], [ 81, 83, "Sa", "species" ], [ 84, 88, "EctC", "protein" ], [ 89, 98, "structure", "evidence" ], [ 178, 194, "ectoine synthase", "protein_type" ] ] }, { "sid": 268, "sent": "As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues.", "section": "RESULTS", "ner": [ [ 31, 54, "mutagenesis experiments", "experimental_method" ], [ 64, 75, "substituted", "experimental_method" ], [ 76, 82, "Thr-41", "residue_name_number" ], [ 87, 93, "His-51", "residue_name_number" ], [ 117, 145, "not evolutionarily conserved", "protein_state" ], [ 149, 167, "EctC-type proteins", "protein_type" ], [ 173, 176, "Ala", "residue_name" ] ] }, { "sid": 269, "sent": "Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1).", "section": "RESULTS", "ner": [ [ 6, 8, "Sa", "species" ], [ 9, 13, "EctC", "protein" ], [ 41, 50, "wild-type", "protein_state" ], [ 91, 95, "iron", "chemical" ], [ 125, 134, "wild-type", "protein_state" ] ] }, { "sid": 270, "sent": "This illustrates that not every amino acid substitution in the (Sa)EctC protein leads to an indiscriminate impairment of enzyme function and iron content.", "section": "RESULTS", "ner": [ [ 64, 66, "Sa", "species" ], [ 67, 71, "EctC", "protein" ], [ 141, 145, "iron", "chemical" ] ] }, { "sid": 271, "sent": "The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily.", "section": "DISCUSS", "ner": [ [ 4, 25, "crystallographic data", "evidence" ], [ 57, 73, "ectoine synthase", "protein_type" ], [ 75, 79, "EctC", "protein" ], [ 127, 136, "microbial", "taxonomy_domain" ], [ 175, 182, "ectoine", "chemical" ], [ 207, 224, "cupin superfamily", "protein_type" ] ] }, { "sid": 272, "sent": "The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 \u03b2-strands (\u03b21-\u03b211) and two \u03b1-helices (\u03b1-I and \u03b1-II) closely adheres to the design principles typically found in crystal structures of cupins.", "section": "DISCUSS", "ner": [ [ 40, 42, "Sa", "species" ], [ 43, 47, "EctC", "protein" ], [ 83, 92, "\u03b2-strands", "structure_element" ], [ 94, 100, "\u03b21-\u03b211", "structure_element" ], [ 110, 119, "\u03b1-helices", "structure_element" ], [ 121, 124, "\u03b1-I", "structure_element" ], [ 129, 133, "\u03b1-II", "structure_element" ], [ 195, 213, "crystal structures", "evidence" ], [ 217, 223, "cupins", "protein_type" ] ] }, { "sid": 273, "sent": "In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different.", "section": "DISCUSS", "ner": [ [ 19, 35, "ectoine synthase", "protein_type" ], [ 41, 59, "polyketide cyclase", "protein_type" ], [ 60, 64, "RemF", "protein" ], [ 99, 112, "cupin-related", "protein_type" ], [ 190, 194, "EctC", "protein" ], [ 199, 203, "RemF", "protein" ] ] }, { "sid": 274, "sent": "As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as \u201cRemF-like\u201d proteins.", "section": "DISCUSS", "ner": [ [ 50, 54, "EctC", "protein" ], [ 59, 63, "RemF", "protein" ], [ 162, 180, "EctC-type proteins", "protein_type" ], [ 215, 224, "microbial", "taxonomy_domain" ], [ 246, 255, "RemF-like", "protein_type" ] ] }, { "sid": 275, "sent": "The pro- and eukaryotic members of the cupin superfamily perform a variety of both enzymatic and non-enzymatic functions that are built upon a common structural scaffold.", "section": "DISCUSS", "ner": [ [ 4, 8, "pro-", "taxonomy_domain" ], [ 13, 23, "eukaryotic", "taxonomy_domain" ], [ 39, 56, "cupin superfamily", "protein_type" ] ] }, { "sid": 276, "sent": "Most cupins contain transition state metals that can promote different types of chemical reactions.", "section": "DISCUSS", "ner": [ [ 5, 11, "cupins", "protein_type" ] ] }, { "sid": 277, "sent": "Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites.", "section": "DISCUSS", "ner": [ [ 16, 38, "cupin-related proteins", "protein_type" ], [ 64, 82, "metallo-chaperones", "protein_type" ], [ 88, 93, "bound", "protein_state" ], [ 94, 99, "metal", "chemical" ], [ 138, 150, "active sites", "site" ] ] }, { "sid": 278, "sent": "We report here for the first time that the ectoine synthase is a metal-dependent enzyme.", "section": "DISCUSS", "ner": [ [ 43, 59, "ectoine synthase", "protein_type" ], [ 65, 70, "metal", "chemical" ] ] }, { "sid": 279, "sent": "ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction.", "section": "DISCUSS", "ner": [ [ 0, 6, "ICP-MS", "experimental_method" ], [ 8, 54, "metal-depletion and reconstitution experiments", "experimental_method" ], [ 85, 89, "iron", "chemical" ], [ 124, 129, "metal", "chemical" ], [ 138, 142, "EctC", "protein" ] ] }, { "sid": 280, "sent": "However, as observed with other cupins, EctC is a somewhat promiscuous enzyme as far as the catalytically important metal is concerned when they are provided in large molar excess (Fig 3c).", "section": "DISCUSS", "ner": [ [ 32, 38, "cupins", "protein_type" ], [ 40, 44, "EctC", "protein" ], [ 116, 121, "metal", "chemical" ] ] }, { "sid": 281, "sent": "Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1).", "section": "DISCUSS", "ner": [ [ 114, 119, "metal", "chemical" ], [ 132, 134, "Sa", "species" ], [ 135, 139, "EctC", "protein" ], [ 145, 187, "structure-guided site-directed mutagenesis", "experimental_method" ], [ 226, 247, "iron-binding residues", "site" ] ] }, { "sid": 282, "sent": "The architecture of the metal center of ectoine synthase seems to be subjected to considerable evolutionary constraints.", "section": "DISCUSS", "ner": [ [ 24, 36, "metal center", "site" ], [ 40, 56, "ectoine synthase", "protein_type" ] ] }, { "sid": 283, "sent": "The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2).", "section": "DISCUSS", "ner": [ [ 20, 26, "Glu-57", "residue_name_number" ], [ 28, 34, "Tyr-85", "residue_name_number" ], [ 36, 42, "His-93", "residue_name_number" ], [ 88, 106, "strictly conserved", "protein_state" ], [ 132, 150, "EctC-type proteins", "protein_type" ], [ 171, 180, "bacterial", "taxonomy_domain" ], [ 191, 199, "archaeal", "taxonomy_domain" ] ] }, { "sid": 284, "sent": "We also show here for the first time that, in addition to its natural substrate N-\u03b3-ADABA, EctC also converts the isomer N-\u03b1-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig).", "section": "DISCUSS", "ner": [ [ 80, 89, "N-\u03b3-ADABA", "chemical" ], [ 91, 95, "EctC", "protein" ], [ 121, 130, "N-\u03b1-ADABA", "chemical" ], [ 136, 143, "ectoine", "chemical" ], [ 175, 195, "catalytic efficiency", "evidence" ] ] }, { "sid": 285, "sent": "Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules.", "section": "DISCUSS", "ner": [ [ 11, 22, "active site", "site" ], [ 26, 42, "ectoine synthase", "protein_type" ], [ 147, 151, "EctC", "protein" ], [ 207, 211, "ADPC", "chemical" ], [ 251, 260, "glutamine", "chemical" ] ] }, { "sid": 286, "sent": "Our finding that N-\u03b1-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-\u03b1-ADABA is produced as an intermediate in the catabolic route.", "section": "DISCUSS", "ner": [ [ 17, 26, "N-\u03b1-ADABA", "chemical" ], [ 53, 69, "ectoine synthase", "protein_type" ], [ 123, 137, "microorganisms", "taxonomy_domain" ], [ 178, 185, "ectoine", "chemical" ], [ 263, 272, "N-\u03b1-ADABA", "chemical" ] ] }, { "sid": 287, "sent": "Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a).", "section": "DISCUSS", "ner": [ [ 60, 62, "C6", "chemical" ], [ 97, 99, "Sa", "species" ], [ 100, 104, "EctC", "protein" ], [ 105, 114, "structure", "evidence" ], [ 204, 220, "ectoine synthase", "protein_type" ] ] }, { "sid": 288, "sent": "We assumed that its location and mode of binding gives, in all likelihood, clues as to the position of the true substrate N-\u03b3-ADABA within the EctC active site.", "section": "DISCUSS", "ner": [ [ 122, 131, "N-\u03b3-ADABA", "chemical" ], [ 143, 147, "EctC", "protein" ], [ 148, 159, "active site", "site" ] ] }, { "sid": 289, "sent": "Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1).", "section": "DISCUSS", "ner": [ [ 8, 33, "site-directed mutagenesis", "experimental_method" ], [ 112, 114, "Sa", "species" ], [ 115, 119, "EctC", "protein" ], [ 177, 186, "wild-type", "protein_state" ], [ 197, 201, "iron", "chemical" ] ] }, { "sid": 290, "sent": "This set of data and the fact that the targeted residues are strongly conserved among EctC-type proteins (Fig 2) is consistent with their potential role in N-\u03b3-ADABA binding or enzyme catalysis.", "section": "DISCUSS", "ner": [ [ 61, 79, "strongly conserved", "protein_state" ], [ 86, 104, "EctC-type proteins", "protein_type" ], [ 156, 165, "N-\u03b3-ADABA", "chemical" ] ] }, { "sid": 291, "sent": "We therefore surmise that our crystallographic data and the site-directed mutagenesis study reported here provide a structural and functional view into the architecture of the EctC active site (Fig 7b).", "section": "DISCUSS", "ner": [ [ 30, 51, "crystallographic data", "evidence" ], [ 60, 91, "site-directed mutagenesis study", "experimental_method" ], [ 176, 180, "EctC", "protein" ], [ 181, 192, "active site", "site" ] ] }, { "sid": 292, "sent": "The ectoine synthase from the cold-adapted marine bacterium S. alaskensis can be considered as a psychrophilic enzyme (S3a Fig), types of proteins with a considerable structural flexibility.", "section": "DISCUSS", "ner": [ [ 4, 20, "ectoine synthase", "protein_type" ], [ 43, 59, "marine bacterium", "taxonomy_domain" ], [ 60, 73, "S. alaskensis", "species" ] ] }, { "sid": 293, "sent": "This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-\u03b3-ADABA or ectoine.", "section": "DISCUSS", "ner": [ [ 64, 82, "crystal structures", "evidence" ], [ 90, 101, "full-length", "protein_state" ], [ 103, 105, "Sa", "species" ], [ 106, 110, "EctC", "protein" ], [ 119, 134, "in complex with", "protein_state" ], [ 142, 151, "N-\u03b3-ADABA", "chemical" ], [ 155, 162, "ectoine", "chemical" ] ] }, { "sid": 294, "sent": "Because microbial ectoine producers can colonize ecological niches with rather different physicochemical attributes, it seems promising to exploit this considerable biodiversity to identify EctC proteins with enhanced protein stability.", "section": "DISCUSS", "ner": [ [ 8, 17, "microbial", "taxonomy_domain" ], [ 18, 25, "ectoine", "chemical" ], [ 190, 203, "EctC proteins", "protein_type" ] ] }, { "sid": 295, "sent": "It is hoped that these can be further employed to obtain EctC crystal structures with either the substrate or the reaction product.", "section": "DISCUSS", "ner": [ [ 57, 61, "EctC", "protein" ], [ 62, 80, "crystal structures", "evidence" ] ] }, { "sid": 296, "sent": "Together with our finding that ectoine synthase is metal dependent, these crystal structures should allow a more detailed understanding of the chemistry underlying the EctC-catalyzed cyclocondensation reaction.", "section": "DISCUSS", "ner": [ [ 31, 47, "ectoine synthase", "protein_type" ], [ 51, 66, "metal dependent", "protein_state" ], [ 74, 92, "crystal structures", "evidence" ], [ 168, 172, "EctC", "protein" ] ] } ] }, "PMC4772114": { "annotations": [ { "sid": 0, "sent": "Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions", "section": "TITLE", "ner": [ [ 61, 70, "Regnase-1", "protein" ] ] }, { "sid": 1, "sent": "Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses.", "section": "ABSTRACT", "ner": [ [ 0, 9, "Regnase-1", "protein" ], [ 16, 21, "RNase", "protein_type" ], [ 44, 49, "mRNAs", "chemical" ], [ 80, 84, "IL-6", "protein_type" ], [ 89, 97, "IL-12p40", "protein_type" ] ] }, { "sid": 2, "sent": "Here, we report the structures of four domains of Regnase-1 from Mus musculus\u2014the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD).", "section": "ABSTRACT", "ner": [ [ 20, 30, "structures", "evidence" ], [ 50, 59, "Regnase-1", "protein" ], [ 65, 77, "Mus musculus", "species" ], [ 82, 99, "N-terminal domain", "structure_element" ], [ 101, 104, "NTD", "structure_element" ], [ 107, 127, "PilT N-terminus like", "structure_element" ], [ 129, 132, "PIN", "structure_element" ], [ 142, 153, "zinc finger", "structure_element" ], [ 155, 157, "ZF", "structure_element" ], [ 170, 187, "C-terminal domain", "structure_element" ], [ 189, 192, "CTD", "structure_element" ] ] }, { "sid": 3, "sent": "The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity.", "section": "ABSTRACT", "ner": [ [ 4, 7, "PIN", "structure_element" ], [ 27, 32, "RNase", "protein_type" ], [ 33, 49, "catalytic center", "site" ] ] }, { "sid": 4, "sent": "We found that the NTD associates with the PIN domain and significantly enhances its RNase activity.", "section": "ABSTRACT", "ner": [ [ 18, 21, "NTD", "structure_element" ], [ 42, 45, "PIN", "structure_element" ], [ 84, 89, "RNase", "protein_type" ] ] }, { "sid": 5, "sent": "The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site.", "section": "ABSTRACT", "ner": [ [ 4, 7, "PIN", "structure_element" ], [ 23, 35, "head-to-tail", "protein_state" ], [ 36, 44, "oligomer", "oligomeric_state" ], [ 53, 68, "dimer interface", "site" ], [ 87, 103, "NTD binding site", "site" ] ] }, { "sid": 6, "sent": "Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro.", "section": "ABSTRACT", "ner": [ [ 15, 24, "mutations", "experimental_method" ], [ 34, 37, "PIN", "structure_element" ], [ 61, 66, "RNase", "protein_type" ], [ 118, 121, "NTD", "structure_element" ], [ 146, 151, "RNase", "protein_type" ] ] }, { "sid": 7, "sent": "These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions.", "section": "ABSTRACT", "ner": [ [ 27, 36, "Regnase-1", "protein" ], [ 37, 42, "RNase", "protein_type" ], [ 98, 101, "NTD", "structure_element" ], [ 102, 105, "PIN", "structure_element" ], [ 127, 130, "PIN", "structure_element" ], [ 131, 134, "PIN", "structure_element" ] ] }, { "sid": 8, "sent": "The initial sensing of infection is mediated by a set of pattern-recognition receptors (PRRs) such Toll-like receptors (TLRs) and the intracellular signaling cascades triggered by TLRs evoke transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells.", "section": "INTRO", "ner": [ [ 57, 86, "pattern-recognition receptors", "protein_type" ], [ 88, 92, "PRRs", "protein_type" ], [ 99, 118, "Toll-like receptors", "protein_type" ], [ 120, 124, "TLRs", "protein_type" ], [ 180, 184, "TLRs", "protein_type" ] ] }, { "sid": 9, "sent": "Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1\u03b2, IL-2, and IL-12p40.", "section": "INTRO", "ner": [ [ 0, 9, "Regnase-1", "protein" ], [ 25, 32, "Zc3h12a", "protein" ], [ 37, 43, "MCPIP1", "protein" ], [ 51, 56, "RNase", "protein_type" ], [ 97, 116, "lipopolysaccharides", "chemical" ], [ 191, 196, "mRNAs", "chemical" ], [ 239, 245, "(IL)-6", "protein_type" ], [ 247, 252, "IL-1\u03b2", "protein_type" ], [ 254, 258, "IL-2", "protein_type" ], [ 264, 272, "IL-12p40", "protein_type" ] ] }, { "sid": 10, "sent": "Regnase-1 accelerates target mRNA degradation via their 3\u2032-terminal untranslated region (3\u2032UTR), and also degrades its own mRNA.", "section": "INTRO", "ner": [ [ 0, 9, "Regnase-1", "protein" ], [ 29, 33, "mRNA", "chemical" ], [ 56, 87, "3\u2032-terminal untranslated region", "structure_element" ], [ 89, 94, "3\u2032UTR", "structure_element" ], [ 123, 127, "mRNA", "chemical" ] ] }, { "sid": 11, "sent": "Regnase-1 is a member of Regnase family and is composed of a PilT N-terminus like (PIN) domain followed by a CCCH-type zinc\u2013finger (ZF) domain, which are conserved among Regnase family members.", "section": "INTRO", "ner": [ [ 0, 9, "Regnase-1", "protein" ], [ 25, 39, "Regnase family", "protein_type" ], [ 61, 81, "PilT N-terminus like", "structure_element" ], [ 83, 86, "PIN", "structure_element" ], [ 109, 130, "CCCH-type zinc\u2013finger", "structure_element" ], [ 132, 134, "ZF", "structure_element" ], [ 154, 163, "conserved", "protein_state" ], [ 170, 192, "Regnase family members", "protein_type" ] ] }, { "sid": 12, "sent": "Recently, the crystal structure of the Regnase-1 PIN domain derived from Homo sapiens was reported.", "section": "INTRO", "ner": [ [ 14, 31, "crystal structure", "evidence" ], [ 39, 48, "Regnase-1", "protein" ], [ 49, 52, "PIN", "structure_element" ], [ 73, 85, "Homo sapiens", "species" ] ] }, { "sid": 13, "sent": "The structure combined with functional analyses revealed that four catalytically important Asp residues form the catalytic center and stabilize Mg2+ binding that is crucial for RNase activity.", "section": "INTRO", "ner": [ [ 4, 13, "structure", "evidence" ], [ 91, 94, "Asp", "residue_name" ], [ 113, 129, "catalytic center", "site" ], [ 144, 148, "Mg2+", "chemical" ], [ 177, 182, "RNase", "protein_type" ] ] }, { "sid": 14, "sent": "Several CCCH-type ZF motifs in RNA-binding proteins have been reported to directly bind RNA.", "section": "INTRO", "ner": [ [ 8, 27, "CCCH-type ZF motifs", "structure_element" ], [ 31, 51, "RNA-binding proteins", "protein_type" ], [ 88, 91, "RNA", "chemical" ] ] }, { "sid": 15, "sent": "In addition, Regnase-1 has been predicted to possess other domains in the N- and C- terminal regions.", "section": "INTRO", "ner": [ [ 13, 22, "Regnase-1", "protein" ], [ 74, 100, "N- and C- terminal regions", "structure_element" ] ] }, { "sid": 16, "sent": "However, the structure and function of the ZF domain, N-terminal domain (NTD) and C-terminal domain (CTD) of Regnase-1 have not been solved.", "section": "INTRO", "ner": [ [ 13, 22, "structure", "evidence" ], [ 43, 45, "ZF", "structure_element" ], [ 54, 71, "N-terminal domain", "structure_element" ], [ 73, 76, "NTD", "structure_element" ], [ 82, 99, "C-terminal domain", "structure_element" ], [ 101, 104, "CTD", "structure_element" ], [ 109, 118, "Regnase-1", "protein" ] ] }, { "sid": 17, "sent": "Here, we performed structural and functional analyses of individual domains of Regnase-1 derived from Mus musculus in order to understand the catalytic activity in vitro.", "section": "INTRO", "ner": [ [ 19, 53, "structural and functional analyses", "experimental_method" ], [ 79, 88, "Regnase-1", "protein" ], [ 102, 114, "Mus musculus", "species" ] ] }, { "sid": 18, "sent": "Our data revealed that the catalytic activity of Regnase-1 is regulated through both intra and intermolecular domain interactions in vitro.", "section": "INTRO", "ner": [ [ 49, 58, "Regnase-1", "protein" ] ] }, { "sid": 19, "sent": "The NTD plays a crucial role in efficient cleavage of target mRNA, through intramolecular NTD-PIN interactions.", "section": "INTRO", "ner": [ [ 4, 7, "NTD", "structure_element" ], [ 61, 65, "mRNA", "chemical" ], [ 90, 93, "NTD", "structure_element" ], [ 94, 97, "PIN", "structure_element" ] ] }, { "sid": 20, "sent": "Moreover, Regnase-1 functions as a dimer through intermolecular PIN-PIN interactions during cleavage of target mRNA.", "section": "INTRO", "ner": [ [ 10, 19, "Regnase-1", "protein" ], [ 35, 40, "dimer", "oligomeric_state" ], [ 64, 67, "PIN", "structure_element" ], [ 68, 71, "PIN", "structure_element" ], [ 111, 115, "mRNA", "chemical" ] ] }, { "sid": 21, "sent": "Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer.", "section": "INTRO", "ner": [ [ 26, 35, "Regnase-1", "protein" ], [ 55, 59, "mRNA", "chemical" ], [ 66, 79, "NTD-activated", "protein_state" ], [ 80, 90, "functional", "protein_state" ], [ 91, 94, "PIN", "structure_element" ], [ 95, 100, "dimer", "oligomeric_state" ], [ 112, 114, "ZF", "structure_element" ], [ 125, 128, "RNA", "chemical" ], [ 161, 164, "PIN", "structure_element" ], [ 165, 170, "dimer", "oligomeric_state" ] ] }, { "sid": 22, "sent": "Domain structures of Regnase-1", "section": "RESULTS", "ner": [ [ 7, 17, "structures", "evidence" ], [ 21, 30, "Regnase-1", "protein" ] ] }, { "sid": 23, "sent": "We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a\u2013e).", "section": "RESULTS", "ner": [ [ 12, 21, "Rengase-1", "protein" ], [ 35, 47, "Mus musculus", "species" ], [ 52, 58, "solved", "experimental_method" ], [ 63, 73, "structures", "evidence" ], [ 95, 98, "NTD", "structure_element" ], [ 100, 103, "PIN", "structure_element" ], [ 105, 107, "ZF", "structure_element" ], [ 113, 116, "CTD", "structure_element" ], [ 133, 154, "X-ray crystallography", "experimental_method" ], [ 158, 161, "NMR", "experimental_method" ] ] }, { "sid": 24, "sent": "X-ray crystallography was attempted for the fragment containing both the PIN and ZF domains, however, electron density was observed only for the PIN domain (Fig. 1c), consistent with a previous report on Regnase-1 derived from Homo sapiens.", "section": "RESULTS", "ner": [ [ 0, 21, "X-ray crystallography", "experimental_method" ], [ 73, 76, "PIN", "structure_element" ], [ 81, 83, "ZF", "structure_element" ], [ 102, 118, "electron density", "evidence" ], [ 145, 148, "PIN", "structure_element" ], [ 204, 213, "Regnase-1", "protein" ], [ 227, 239, "Homo sapiens", "species" ] ] }, { "sid": 25, "sent": "This suggests that the PIN and ZF domains exist independently without interacting with each other.", "section": "RESULTS", "ner": [ [ 23, 26, "PIN", "structure_element" ], [ 31, 33, "ZF", "structure_element" ] ] }, { "sid": 26, "sent": "The domain structures of NTD, ZF, and CTD were determined by NMR (Fig. 1b,d,e).", "section": "RESULTS", "ner": [ [ 11, 21, "structures", "evidence" ], [ 25, 28, "NTD", "structure_element" ], [ 30, 32, "ZF", "structure_element" ], [ 38, 41, "CTD", "structure_element" ], [ 61, 64, "NMR", "experimental_method" ] ] }, { "sid": 27, "sent": "The NTD and CTD are both composed of three \u03b1 helices, and structurally resemble ubiquitin conjugating enzyme E2 K (PDB ID: 3K9O) and ubiquitin associated protein 1 (PDB ID: 4AE4), respectively, according to the Dali server.", "section": "RESULTS", "ner": [ [ 4, 7, "NTD", "structure_element" ], [ 12, 15, "CTD", "structure_element" ], [ 43, 52, "\u03b1 helices", "structure_element" ], [ 80, 113, "ubiquitin conjugating enzyme E2 K", "protein" ], [ 133, 163, "ubiquitin associated protein 1", "protein" ], [ 211, 222, "Dali server", "experimental_method" ] ] }, { "sid": 28, "sent": "Contribution of each domain of Regnase-1 to the mRNA binding activity", "section": "RESULTS", "ner": [ [ 31, 40, "Regnase-1", "protein" ], [ 48, 52, "mRNA", "chemical" ] ] }, { "sid": 29, "sent": "Although the PIN domain is responsible for the catalytic activity of Regnase-1, the roles of the other domains are largely unknown.", "section": "RESULTS", "ner": [ [ 13, 16, "PIN", "structure_element" ], [ 69, 78, "Regnase-1", "protein" ] ] }, { "sid": 30, "sent": "First, we evaluated a role of the NTD and ZF domains for mRNA binding by an in vitro gel shift assay (Fig. 1f).", "section": "RESULTS", "ner": [ [ 34, 37, "NTD", "structure_element" ], [ 42, 44, "ZF", "structure_element" ], [ 57, 61, "mRNA", "chemical" ], [ 76, 100, "in vitro gel shift assay", "experimental_method" ] ] }, { "sid": 31, "sent": "Fluorescently 5\u2032-labeled RNA corresponding to nucleotides 82\u2013106 of the IL-6 mRNA 3\u2032UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1\u2014hereafter referred to as the DDNN mutant\u2014were utilized.", "section": "RESULTS", "ner": [ [ 0, 24, "Fluorescently 5\u2032-labeled", "protein_state" ], [ 25, 28, "RNA", "chemical" ], [ 72, 76, "IL-6", "protein_type" ], [ 77, 81, "mRNA", "chemical" ], [ 82, 87, "3\u2032UTR", "structure_element" ], [ 110, 118, "inactive", "protein_state" ], [ 119, 125, "mutant", "protein_state" ], [ 127, 132, "D226N", "mutant" ], [ 137, 142, "D244N", "mutant" ], [ 147, 156, "Regnase-1", "protein" ], [ 186, 190, "DDNN", "mutant" ], [ 191, 197, "mutant", "protein_state" ] ] }, { "sid": 32, "sent": "Upon addition of a larger amount of Regnase-1, the fluorescence of free RNA decreased, indicating that Regnase-1 bound to the RNA.", "section": "RESULTS", "ner": [ [ 36, 45, "Regnase-1", "protein" ], [ 51, 63, "fluorescence", "evidence" ], [ 67, 71, "free", "protein_state" ], [ 72, 75, "RNA", "chemical" ], [ 103, 112, "Regnase-1", "protein" ], [ 113, 121, "bound to", "protein_state" ], [ 126, 129, "RNA", "chemical" ] ] }, { "sid": 33, "sent": "Based on the decrease in the free RNA fluorescence band, we evaluated the contribution of each domain of Regnase-1 to RNA binding.", "section": "RESULTS", "ner": [ [ 34, 37, "RNA", "chemical" ], [ 105, 114, "Regnase-1", "protein" ], [ 118, 121, "RNA", "chemical" ] ] }, { "sid": 34, "sent": "While the RNA binding ability was not significantly changed in the presence of NTD, it increased in the presence of the ZF domain (Fig. 1f,g and Supplementary Fig. 1).", "section": "RESULTS", "ner": [ [ 10, 13, "RNA", "chemical" ], [ 67, 78, "presence of", "protein_state" ], [ 79, 82, "NTD", "structure_element" ], [ 104, 115, "presence of", "protein_state" ], [ 120, 122, "ZF", "structure_element" ] ] }, { "sid": 35, "sent": "Direct binding of the ZF domain and RNA were confirmed by NMR spectral changes.", "section": "RESULTS", "ner": [ [ 22, 24, "ZF", "structure_element" ], [ 36, 39, "RNA", "chemical" ], [ 58, 61, "NMR", "experimental_method" ], [ 62, 78, "spectral changes", "evidence" ] ] }, { "sid": 36, "sent": "The fitting of the titration curve of Y314 resulted in an apparent dissociation constant (Kd) of 10\u2009\u00b1\u20091.1\u2009\u03bcM (Supplementary Fig. 2).", "section": "RESULTS", "ner": [ [ 19, 34, "titration curve", "evidence" ], [ 38, 42, "Y314", "residue_name_number" ], [ 67, 88, "dissociation constant", "evidence" ], [ 90, 92, "Kd", "evidence" ] ] }, { "sid": 37, "sent": "These results indicate that not only the PIN but also the ZF domain contribute to RNA binding, while the NTD is not likely to be involved in direct interaction with RNA.", "section": "RESULTS", "ner": [ [ 41, 44, "PIN", "structure_element" ], [ 58, 60, "ZF", "structure_element" ], [ 82, 85, "RNA", "chemical" ], [ 105, 108, "NTD", "structure_element" ], [ 165, 168, "RNA", "chemical" ] ] }, { "sid": 38, "sent": "Contribution of each domain of Regnase-1 to RNase activity", "section": "RESULTS", "ner": [ [ 31, 40, "Regnase-1", "protein" ], [ 44, 49, "RNase", "protein_type" ] ] }, { "sid": 39, "sent": "In order to characterize the role of each domain in the RNase activity of Regnase-1, we performed an in vitro cleavage assay using fluorescently 5\u2032-labeled RNA corresponding to nucleotides 82\u2013106 of the IL-6 mRNA 3\u2032UTR (Fig. 1g).", "section": "RESULTS", "ner": [ [ 56, 61, "RNase", "protein_type" ], [ 74, 83, "Regnase-1", "protein" ], [ 101, 124, "in vitro cleavage assay", "experimental_method" ], [ 131, 155, "fluorescently 5\u2032-labeled", "protein_state" ], [ 156, 159, "RNA", "chemical" ], [ 203, 207, "IL-6", "protein_type" ], [ 208, 212, "mRNA", "chemical" ], [ 213, 218, "3\u2032UTR", "structure_element" ] ] }, { "sid": 40, "sent": "Regnase-1 constructs consisting of NTD-PIN-ZF completely cleaved the target mRNA and generated the cleaved products.", "section": "RESULTS", "ner": [ [ 0, 9, "Regnase-1", "protein" ], [ 35, 45, "NTD-PIN-ZF", "mutant" ], [ 76, 80, "mRNA", "chemical" ] ] }, { "sid": 41, "sent": "The apparent half-life (T1/2) of the RNase activity was about 20\u2009minutes.", "section": "RESULTS", "ner": [ [ 37, 42, "RNase", "protein_type" ] ] }, { "sid": 42, "sent": "Regnase-1 lacking the ZF domain generated a smaller but appreciable amount of cleaved product (T1/2\u2009~\u200970\u2009minutes), while those lacking the NTD did not generate cleaved products (T1/2\u2009>\u200990\u2009minutes).", "section": "RESULTS", "ner": [ [ 0, 9, "Regnase-1", "protein" ], [ 10, 17, "lacking", "protein_state" ], [ 22, 24, "ZF", "structure_element" ], [ 127, 134, "lacking", "protein_state" ], [ 139, 142, "NTD", "structure_element" ] ] }, { "sid": 43, "sent": "It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain.", "section": "RESULTS", "ner": [ [ 24, 40, "NTD-PIN(DDNN)-ZF", "mutant" ], [ 62, 65, "NTD", "structure_element" ], [ 70, 75, "lacks", "protein_state" ], [ 80, 98, "catalytic residues", "site" ], [ 102, 105, "PIN", "structure_element" ], [ 127, 132, "RNase", "protein_type" ], [ 199, 204, "RNase", "protein_type" ], [ 205, 221, "catalytic center", "site" ], [ 240, 243, "PIN", "structure_element" ] ] }, { "sid": 44, "sent": "Taken together with the results in the previous section, we conclude that the NTD is crucial for the RNase activity of Regnase-1 in vitro, although it does not contribute to the direct mRNA binding.", "section": "RESULTS", "ner": [ [ 78, 81, "NTD", "structure_element" ], [ 101, 106, "RNase", "protein_type" ], [ 119, 128, "Regnase-1", "protein" ], [ 185, 189, "mRNA", "chemical" ] ] }, { "sid": 45, "sent": "Dimer formation of the PIN domains", "section": "RESULTS", "ner": [ [ 0, 5, "Dimer", "oligomeric_state" ], [ 23, 26, "PIN", "structure_element" ] ] }, { "sid": 46, "sent": "During purification by gel filtration, the PIN domain exhibited extremely asymmetric elution peaks in a concentration dependent manner (Fig. 2a).", "section": "RESULTS", "ner": [ [ 7, 19, "purification", "experimental_method" ], [ 23, 37, "gel filtration", "experimental_method" ], [ 43, 46, "PIN", "structure_element" ] ] }, { "sid": 47, "sent": "By comparison with the elution volume of standard marker proteins, the PIN domain was assumed to be in equilibrium between a monomer and a dimer in solution at concentrations in the 20\u2013200\u2009\u03bcM range.", "section": "RESULTS", "ner": [ [ 3, 65, "comparison with the elution volume of standard marker proteins", "experimental_method" ], [ 71, 74, "PIN", "structure_element" ], [ 125, 132, "monomer", "oligomeric_state" ], [ 139, 144, "dimer", "oligomeric_state" ] ] }, { "sid": 48, "sent": "The crystal structure of the PIN domain has been determined in three distinct crystal forms with a space group of P3121 (form I in this study and PDB ID 3V33), P3221 (form II in this study), and P41 (PDB ID 3V32 and 3V34), respectively.", "section": "RESULTS", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 29, 32, "PIN", "structure_element" ], [ 78, 91, "crystal forms", "evidence" ] ] }, { "sid": 49, "sent": "We found that the PIN domain formed a head-to-tail oligomer that was commonly observed in all three crystal forms in spite of the different crystallization conditions (Supplementary Fig. 3).", "section": "RESULTS", "ner": [ [ 18, 21, "PIN", "structure_element" ], [ 38, 50, "head-to-tail", "protein_state" ], [ 51, 59, "oligomer", "oligomeric_state" ], [ 100, 113, "crystal forms", "evidence" ] ] }, { "sid": 50, "sent": "Mutation of Arg215, whose side chain faces to the opposite side of the oligomeric surface, to Glu preserved the monomer/dimer equilibrium, similar to the wild type.", "section": "RESULTS", "ner": [ [ 0, 8, "Mutation", "experimental_method" ], [ 12, 18, "Arg215", "residue_name_number" ], [ 71, 89, "oligomeric surface", "site" ], [ 94, 97, "Glu", "residue_name" ], [ 112, 119, "monomer", "oligomeric_state" ], [ 120, 125, "dimer", "oligomeric_state" ], [ 154, 163, "wild type", "protein_state" ] ] }, { "sid": 51, "sent": "On the other hand, single mutations of side chains involved in the PIN\u2013PIN oligomeric interaction resulted in monomer formation, judging from gel filtration (Fig. 2a,b).", "section": "RESULTS", "ner": [ [ 19, 35, "single mutations", "experimental_method" ], [ 67, 70, "PIN", "structure_element" ], [ 71, 74, "PIN", "structure_element" ], [ 110, 117, "monomer", "oligomeric_state" ], [ 142, 156, "gel filtration", "experimental_method" ] ] }, { "sid": 52, "sent": "Wild type and monomeric PIN mutants (P212A and D278R) were also analyzed by NMR.", "section": "RESULTS", "ner": [ [ 0, 9, "Wild type", "protein_state" ], [ 14, 23, "monomeric", "oligomeric_state" ], [ 24, 27, "PIN", "structure_element" ], [ 28, 35, "mutants", "protein_state" ], [ 37, 42, "P212A", "mutant" ], [ 47, 52, "D278R", "mutant" ], [ 76, 79, "NMR", "experimental_method" ] ] }, { "sid": 53, "sent": "The spectra indicate that the dimer interface of the wild type PIN domain were significantly broadened compared to the monomeric mutants (Supplementary Fig. 4).", "section": "RESULTS", "ner": [ [ 4, 11, "spectra", "evidence" ], [ 30, 45, "dimer interface", "site" ], [ 53, 62, "wild type", "protein_state" ], [ 63, 66, "PIN", "structure_element" ], [ 119, 128, "monomeric", "oligomeric_state" ], [ 129, 136, "mutants", "protein_state" ] ] }, { "sid": 54, "sent": "These results indicate that the PIN domain forms a head-to-tail oligomer in solution similar to the crystal structure.", "section": "RESULTS", "ner": [ [ 32, 35, "PIN", "structure_element" ], [ 51, 63, "head-to-tail", "protein_state" ], [ 64, 72, "oligomer", "oligomeric_state" ], [ 100, 117, "crystal structure", "evidence" ] ] }, { "sid": 55, "sent": "Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c).", "section": "RESULTS", "ner": [ [ 19, 28, "monomeric", "oligomeric_state" ], [ 29, 32, "PIN", "structure_element" ], [ 33, 40, "mutants", "protein_state" ], [ 41, 46, "P212A", "mutant" ], [ 48, 53, "R214A", "mutant" ], [ 59, 64, "D278R", "mutant" ], [ 84, 89, "RNase", "protein_type" ], [ 103, 107, "IL-6", "protein_type" ], [ 108, 112, "mRNA", "chemical" ] ] }, { "sid": 56, "sent": "The side chains of these residues point away from the catalytic center on the same molecule (Fig. 2b).", "section": "RESULTS", "ner": [ [ 54, 70, "catalytic center", "site" ] ] }, { "sid": 57, "sent": "Therefore, we concluded that head-to-tail PIN dimerization, together with the NTD, are required for Regnase-1 RNase activity in vitro.", "section": "RESULTS", "ner": [ [ 29, 41, "head-to-tail", "protein_state" ], [ 42, 45, "PIN", "structure_element" ], [ 78, 81, "NTD", "structure_element" ], [ 100, 109, "Regnase-1", "protein" ], [ 110, 115, "RNase", "protein_type" ] ] }, { "sid": 58, "sent": "Domain-domain interaction between the NTD and the PIN domain", "section": "RESULTS", "ner": [ [ 38, 41, "NTD", "structure_element" ], [ 50, 53, "PIN", "structure_element" ] ] }, { "sid": 59, "sent": "While the NTD does not contribute to RNA binding (Fig. 1f,g, and Supplementary Fig. 1), it increases the RNase activity of Regnase-1 (Fig. 1h).", "section": "RESULTS", "ner": [ [ 10, 13, "NTD", "structure_element" ], [ 37, 40, "RNA", "chemical" ], [ 105, 110, "RNase", "protein_type" ], [ 123, 132, "Regnase-1", "protein" ] ] }, { "sid": 60, "sent": "In order to gain insight into the molecular mechanism of the NTD-mediated enhancement of Regnase-1 RNase activity, we further investigated the domain-domain interaction between the NTD and the PIN domain using NMR.", "section": "RESULTS", "ner": [ [ 61, 64, "NTD", "structure_element" ], [ 89, 98, "Regnase-1", "protein" ], [ 99, 104, "RNase", "protein_type" ], [ 181, 184, "NTD", "structure_element" ], [ 193, 196, "PIN", "structure_element" ], [ 210, 213, "NMR", "experimental_method" ] ] }, { "sid": 61, "sent": "We used the catalytically inactive monomeric PIN mutant possessing both the DDNN and D278R mutations to avoid dimer formation of the PIN domain.", "section": "RESULTS", "ner": [ [ 12, 34, "catalytically inactive", "protein_state" ], [ 35, 44, "monomeric", "oligomeric_state" ], [ 45, 48, "PIN", "structure_element" ], [ 49, 55, "mutant", "protein_state" ], [ 76, 80, "DDNN", "mutant" ], [ 85, 90, "D278R", "mutant" ], [ 110, 115, "dimer", "oligomeric_state" ], [ 133, 136, "PIN", "structure_element" ] ] }, { "sid": 62, "sent": "The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a).", "section": "RESULTS", "ner": [ [ 4, 7, "NMR", "experimental_method" ], [ 25, 28, "PIN", "structure_element" ], [ 46, 50, "V177", "residue_name_number" ], [ 52, 61, "F210-T211", "residue_range" ], [ 63, 67, "R214", "residue_name_number" ], [ 69, 78, "F228-L232", "residue_range" ], [ 84, 93, "F234-S236", "residue_range" ], [ 145, 156, "addition of", "experimental_method" ], [ 161, 164, "NTD", "structure_element" ] ] }, { "sid": 63, "sent": "Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5).", "section": "RESULTS", "ner": [ [ 15, 26, "addition of", "experimental_method" ], [ 31, 34, "PIN", "structure_element" ], [ 43, 46, "NMR", "experimental_method" ], [ 68, 71, "R56", "residue_name_number" ], [ 73, 80, "L58-G59", "residue_range" ], [ 86, 93, "V86-H88", "residue_range" ], [ 101, 104, "NTD", "structure_element" ], [ 157, 160, "D53", "residue_name_number" ], [ 162, 165, "F55", "residue_name_number" ], [ 167, 170, "K57", "residue_name_number" ], [ 172, 179, "Y60-S61", "residue_range" ], [ 181, 184, "V68", "residue_name_number" ], [ 186, 193, "T80-G83", "residue_range" ], [ 195, 198, "L85", "residue_name_number" ], [ 204, 207, "G89", "residue_name_number" ], [ 215, 218, "NTD", "structure_element" ], [ 258, 261, "N79", "residue_name_number" ] ] }, { "sid": 64, "sent": "These results clearly indicate a direct interaction between the PIN domain and the NTD.", "section": "RESULTS", "ner": [ [ 64, 67, "PIN", "structure_element" ], [ 83, 86, "NTD", "structure_element" ] ] }, { "sid": 65, "sent": "Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110\u2009\u00b1\u20095.8\u2009\u03bcM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein.", "section": "RESULTS", "ner": [ [ 13, 28, "titration curve", "evidence" ], [ 37, 59, "chemical shift changes", "evidence" ], [ 63, 66, "L58", "residue_name_number" ], [ 81, 83, "Kd", "evidence" ], [ 105, 108, "NTD", "structure_element" ], [ 113, 116, "PIN", "structure_element" ], [ 181, 184, "NTD", "structure_element" ], [ 189, 192, "PIN", "structure_element" ], [ 219, 225, "linker", "structure_element" ], [ 238, 254, "binding affinity", "evidence" ], [ 286, 292, "native", "protein_state" ] ] }, { "sid": 66, "sent": "Mapping the residues with chemical shift changes reveals the putative PIN/NTD interface, which includes a helix that harbors catalytic residues D225 and D226 on the PIN domain (Fig. 3a).", "section": "RESULTS", "ner": [ [ 70, 87, "PIN/NTD interface", "site" ], [ 106, 111, "helix", "structure_element" ], [ 144, 148, "D225", "residue_name_number" ], [ 153, 157, "D226", "residue_name_number" ], [ 165, 168, "PIN", "structure_element" ] ] }, { "sid": 67, "sent": "Interestingly, the putative binding site for the NTD overlaps with the PIN-PIN dimer interface, implying that NTD binding can \u201cterminate\u201d PIN-PIN oligomerization (Fig. 2b).", "section": "RESULTS", "ner": [ [ 28, 40, "binding site", "site" ], [ 49, 52, "NTD", "structure_element" ], [ 71, 94, "PIN-PIN dimer interface", "site" ], [ 110, 113, "NTD", "structure_element" ], [ 138, 141, "PIN", "structure_element" ], [ 142, 145, "PIN", "structure_element" ] ] }, { "sid": 68, "sent": "An in silico docking of the NTD and PIN domains using chemical shift restraints provided a model consistent with the NMR experiments (Fig. 3c).", "section": "RESULTS", "ner": [ [ 3, 20, "in silico docking", "experimental_method" ], [ 28, 31, "NTD", "structure_element" ], [ 36, 39, "PIN", "structure_element" ], [ 54, 79, "chemical shift restraints", "evidence" ], [ 117, 120, "NMR", "experimental_method" ] ] }, { "sid": 69, "sent": "Residues critical for Regnase-1 RNase activity", "section": "RESULTS", "ner": [ [ 22, 31, "Regnase-1", "protein" ], [ 32, 37, "RNase", "protein_type" ] ] }, { "sid": 70, "sent": "To gain insight into the residues critical for Regnase-1 RNase activity, each basic or aromatic residue located around the catalytic site of the PIN oligomer was mutated to alanine, and the oligomerization and RNase activity were investigated (Fig. 4).", "section": "RESULTS", "ner": [ [ 47, 56, "Regnase-1", "protein" ], [ 57, 62, "RNase", "protein_type" ], [ 123, 137, "catalytic site", "site" ], [ 145, 148, "PIN", "structure_element" ], [ 149, 157, "oligomer", "oligomeric_state" ], [ 162, 172, "mutated to", "experimental_method" ], [ 173, 180, "alanine", "residue_name" ], [ 210, 215, "RNase", "protein_type" ] ] }, { "sid": 71, "sent": "From the gel filtration assays, all mutants except R214A formed dimers, suggesting that any lack of RNase activity in the mutants, except R214A, was directly due to mutational effects of the specific residues and not to abrogation of dimer formation.", "section": "RESULTS", "ner": [ [ 9, 30, "gel filtration assays", "experimental_method" ], [ 36, 43, "mutants", "protein_state" ], [ 51, 56, "R214A", "mutant" ], [ 64, 70, "dimers", "oligomeric_state" ], [ 100, 105, "RNase", "protein_type" ], [ 122, 129, "mutants", "protein_state" ], [ 138, 143, "R214A", "mutant" ], [ 234, 239, "dimer", "oligomeric_state" ] ] }, { "sid": 72, "sent": "The W182A, R183A, and R214A mutants markedly lost cleavage activity for IL-6 mRNA as well as for Regnase-1 mRNA.", "section": "RESULTS", "ner": [ [ 4, 9, "W182A", "mutant" ], [ 11, 16, "R183A", "mutant" ], [ 22, 27, "R214A", "mutant" ], [ 28, 35, "mutants", "protein_state" ], [ 72, 76, "IL-6", "protein_type" ], [ 77, 81, "mRNA", "chemical" ], [ 97, 106, "Regnase-1", "protein" ], [ 107, 111, "mRNA", "chemical" ] ] }, { "sid": 73, "sent": "The K184A, R215A, and R220A mutants moderately but significantly decreased the cleavage activity for both target mRNAs.", "section": "RESULTS", "ner": [ [ 4, 9, "K184A", "mutant" ], [ 11, 16, "R215A", "mutant" ], [ 22, 27, "R220A", "mutant" ], [ 28, 35, "mutants", "protein_state" ], [ 113, 118, "mRNAs", "chemical" ] ] }, { "sid": 74, "sent": "The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA.", "section": "RESULTS", "ner": [ [ 18, 22, "K219", "residue_name_number" ], [ 27, 31, "R247", "residue_name_number" ], [ 59, 63, "IL-6", "protein_type" ], [ 68, 77, "Regnase-1", "protein" ], [ 78, 82, "mRNA", "chemical" ], [ 89, 93, "K219", "residue_name_number" ], [ 98, 102, "R247", "residue_name_number" ], [ 142, 146, "IL-6", "protein_type" ], [ 147, 151, "mRNA", "chemical" ], [ 161, 170, "Regnase-1", "protein" ], [ 171, 175, "mRNA", "chemical" ] ] }, { "sid": 75, "sent": "The other mutated residues\u2014K152, R158, R188, R200, K204, K206, K257, and R258\u2014were not critical for RNase activity.", "section": "RESULTS", "ner": [ [ 27, 31, "K152", "residue_name_number" ], [ 33, 37, "R158", "residue_name_number" ], [ 39, 43, "R188", "residue_name_number" ], [ 45, 49, "R200", "residue_name_number" ], [ 51, 55, "K204", "residue_name_number" ], [ 57, 61, "K206", "residue_name_number" ], [ 63, 67, "K257", "residue_name_number" ], [ 73, 77, "R258", "residue_name_number" ], [ 100, 105, "RNase", "protein_type" ] ] }, { "sid": 76, "sent": "The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the \u201csecondary\u201d chain\u2019s residue K184 is positioned near the \u201cprimary\u201d chain\u2019s catalytic site (Fig. 4).", "section": "RESULTS", "ner": [ [ 27, 31, "W182", "residue_name_number" ], [ 36, 40, "R183", "residue_name_number" ], [ 83, 92, "monomeric", "oligomeric_state" ], [ 93, 96, "PIN", "structure_element" ], [ 97, 106, "structure", "evidence" ], [ 139, 144, "RNase", "protein_type" ], [ 145, 159, "catalytic site", "site" ], [ 196, 200, "K184", "residue_name_number" ], [ 229, 240, "active site", "site" ], [ 296, 305, "structure", "evidence" ], [ 348, 352, "K184", "residue_name_number" ], [ 377, 385, "primary\u201d", "protein_state" ], [ 394, 408, "catalytic site", "site" ] ] }, { "sid": 77, "sent": "In contrast, R214 is important for oligomerization of the PIN domain and the \u201csecondary\u201d chain\u2019s residue R214 is also positioned near the \u201cprimary\u201d chain\u2019s active site within the dimer interface.", "section": "RESULTS", "ner": [ [ 13, 17, "R214", "residue_name_number" ], [ 58, 61, "PIN", "structure_element" ], [ 105, 109, "R214", "residue_name_number" ], [ 139, 146, "primary", "protein_state" ], [ 156, 167, "active site", "site" ], [ 179, 194, "dimer interface", "site" ] ] }, { "sid": 78, "sent": "It should be noted that the putative-RNA binding residues K184 and R214 are unique to Regnase-1 among PIN domains.", "section": "RESULTS", "ner": [ [ 28, 57, "putative-RNA binding residues", "site" ], [ 58, 62, "K184", "residue_name_number" ], [ 67, 71, "R214", "residue_name_number" ], [ 86, 95, "Regnase-1", "protein" ], [ 102, 105, "PIN", "structure_element" ] ] }, { "sid": 79, "sent": "Molecular mechanism of target mRNA cleavage by the PIN dimer", "section": "RESULTS", "ner": [ [ 30, 34, "mRNA", "chemical" ], [ 51, 54, "PIN", "structure_element" ], [ 55, 60, "dimer", "oligomeric_state" ] ] }, { "sid": 80, "sent": "Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain.", "section": "RESULTS", "ner": [ [ 4, 26, "mutational experiments", "experimental_method" ], [ 55, 60, "dimer", "oligomeric_state" ], [ 100, 109, "secondary", "protein_state" ], [ 110, 113, "PIN", "structure_element" ], [ 136, 145, "Regnase-1", "protein" ], [ 153, 173, "RNA binding residues", "site" ], [ 183, 194, "active site", "site" ], [ 202, 209, "primary", "protein_state" ], [ 210, 213, "PIN", "structure_element" ] ] }, { "sid": 81, "sent": "If this model is correct, then we reasoned that a catalytically inactive PIN and a PIN lacking the putative RNA-binding residues ought to be inactive in isolation but become active when mixed together.", "section": "RESULTS", "ner": [ [ 50, 72, "catalytically inactive", "protein_state" ], [ 73, 76, "PIN", "structure_element" ], [ 83, 86, "PIN", "structure_element" ], [ 87, 94, "lacking", "protein_state" ], [ 108, 128, "RNA-binding residues", "site" ], [ 141, 149, "inactive", "protein_state" ], [ 174, 180, "active", "protein_state" ] ] }, { "sid": 82, "sent": "In order to test this hypothesis, we performed in vitro cleavage assays using combinations of Regnase-1 mutants that had no or decreased RNase activities by themselves (Fig. 5).", "section": "RESULTS", "ner": [ [ 47, 71, "in vitro cleavage assays", "experimental_method" ], [ 94, 103, "Regnase-1", "protein" ], [ 104, 111, "mutants", "protein_state" ], [ 137, 142, "RNase", "protein_type" ] ] }, { "sid": 83, "sent": "One group consisted of catalytically active PIN domains with mutation of basic residues found in the previous section to confer decreased RNase activity (Fig. 4).", "section": "RESULTS", "ner": [ [ 23, 43, "catalytically active", "protein_state" ], [ 44, 47, "PIN", "structure_element" ], [ 61, 72, "mutation of", "experimental_method" ], [ 138, 143, "RNase", "protein_type" ] ] }, { "sid": 84, "sent": "These were paired with a DDNN mutant that had no RNase activity by itself.", "section": "RESULTS", "ner": [ [ 25, 29, "DDNN", "mutant" ], [ 30, 36, "mutant", "protein_state" ], [ 49, 54, "RNase", "protein_type" ] ] }, { "sid": 85, "sent": "When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a).", "section": "RESULTS", "ner": [ [ 59, 71, "heterodimers", "oligomeric_state" ], [ 108, 112, "DDNN", "mutant" ], [ 113, 120, "primary", "protein_state" ], [ 121, 124, "PIN", "structure_element" ], [ 145, 151, "mutant", "protein_state" ], [ 152, 161, "secondary", "protein_state" ], [ 162, 165, "PIN", "structure_element" ], [ 196, 201, "RNase", "protein_type" ], [ 253, 259, "mutant", "protein_state" ], [ 260, 267, "primary", "protein_state" ], [ 268, 271, "PIN", "structure_element" ], [ 278, 282, "DDNN", "mutant" ], [ 283, 292, "secondary", "protein_state" ], [ 293, 296, "PIN", "structure_element" ], [ 324, 329, "RNase", "protein_type" ] ] }, { "sid": 86, "sent": "When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b).", "section": "RESULTS", "ner": [ [ 21, 43, "fluorescence intensity", "evidence" ], [ 47, 56, "uncleaved", "protein_state" ], [ 57, 61, "IL-6", "protein_type" ], [ 62, 66, "mRNA", "chemical" ], [ 82, 89, "mutants", "protein_state" ], [ 90, 95, "W182A", "mutant" ], [ 97, 102, "K184A", "mutant" ], [ 104, 109, "R214A", "mutant" ], [ 115, 120, "R220A", "mutant" ], [ 155, 159, "DDNN", "mutant" ], [ 160, 166, "mutant", "protein_state" ] ] }, { "sid": 87, "sent": "Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c).", "section": "RESULTS", "ner": [ [ 35, 57, "fluorescence intensity", "evidence" ], [ 65, 74, "uncleaved", "protein_state" ], [ 75, 84, "Regnase-1", "protein" ], [ 85, 89, "mRNA", "chemical" ], [ 105, 112, "mutants", "protein_state" ], [ 113, 118, "K184A", "mutant" ], [ 123, 128, "R214A", "mutant" ], [ 163, 167, "DDNN", "mutant" ], [ 168, 174, "mutant", "protein_state" ] ] }, { "sid": 88, "sent": "Rescue of K184A and R214A by the DDNN mutant was also confirmed by a significant increase in the cleaved products.", "section": "RESULTS", "ner": [ [ 10, 15, "K184A", "mutant" ], [ 20, 25, "R214A", "mutant" ], [ 33, 37, "DDNN", "mutant" ], [ 38, 44, "mutant", "protein_state" ] ] }, { "sid": 89, "sent": "This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN.", "section": "RESULTS", "ner": [ [ 60, 64, "K184", "residue_name_number" ], [ 69, 73, "R214", "residue_name_number" ], [ 81, 88, "primary", "protein_state" ], [ 89, 92, "PIN", "structure_element" ], [ 126, 142, "catalytic center", "site" ], [ 163, 172, "secondary", "protein_state" ], [ 173, 176, "PIN", "structure_element" ], [ 193, 209, "catalytic center", "site" ], [ 217, 224, "primary", "protein_state" ], [ 225, 228, "PIN", "structure_element" ] ] }, { "sid": 90, "sent": "R214 is an important residue for dimer formation as shown in Fig. 2, therefore, R214A in the secondary PIN cannot dimerize.", "section": "RESULTS", "ner": [ [ 0, 4, "R214", "residue_name_number" ], [ 33, 38, "dimer", "oligomeric_state" ], [ 80, 85, "R214A", "mutant" ], [ 93, 102, "secondary", "protein_state" ], [ 103, 106, "PIN", "structure_element" ] ] }, { "sid": 91, "sent": "According to the proposed model, an R214A PIN domain can only form a dimer when the DDNN PIN acts as the secondary PIN.", "section": "RESULTS", "ner": [ [ 36, 41, "R214A", "mutant" ], [ 42, 45, "PIN", "structure_element" ], [ 69, 74, "dimer", "oligomeric_state" ], [ 84, 88, "DDNN", "mutant" ], [ 89, 92, "PIN", "structure_element" ], [ 105, 114, "secondary", "protein_state" ], [ 115, 118, "PIN", "structure_element" ] ] }, { "sid": 92, "sent": "Taken together, the rescue experiments above support the proposed model in which the head-to-tail dimer is functional in vitro.", "section": "RESULTS", "ner": [ [ 85, 97, "head-to-tail", "protein_state" ], [ 98, 103, "dimer", "oligomeric_state" ] ] }, { "sid": 93, "sent": "We determined the individual domain structures of Regnase-1 by NMR and X-ray crystallography.", "section": "DISCUSS", "ner": [ [ 36, 46, "structures", "evidence" ], [ 50, 59, "Regnase-1", "protein" ], [ 63, 66, "NMR", "experimental_method" ], [ 71, 92, "X-ray crystallography", "experimental_method" ] ] }, { "sid": 94, "sent": "Although the function of the CTD remains elusive, we revealed the functions of the NTD, PIN, and ZF domains.", "section": "DISCUSS", "ner": [ [ 29, 32, "CTD", "structure_element" ], [ 83, 86, "NTD", "structure_element" ], [ 88, 91, "PIN", "structure_element" ], [ 97, 99, "ZF", "structure_element" ] ] }, { "sid": 95, "sent": "A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions.", "section": "DISCUSS", "ner": [ [ 2, 11, "Regnase-1", "protein" ], [ 36, 39, "PIN", "structure_element" ], [ 44, 46, "ZF", "structure_element" ], [ 68, 80, "Mus musculus", "species" ], [ 85, 97, "crystallized", "experimental_method" ], [ 112, 128, "electron density", "evidence" ], [ 136, 138, "ZF", "structure_element" ], [ 175, 177, "ZF", "structure_element" ], [ 188, 201, "highly mobile", "protein_state" ], [ 209, 219, "absence of", "protein_state" ], [ 227, 231, "mRNA", "chemical" ] ] }, { "sid": 96, "sent": "Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10\u2009\u00b1\u20091.1\u2009\u03bcM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel.", "section": "DISCUSS", "ner": [ [ 4, 7, "NMR", "experimental_method" ], [ 52, 54, "ZF", "structure_element" ], [ 65, 69, "IL-6", "protein_type" ], [ 70, 74, "mRNA", "chemical" ], [ 82, 84, "Kd", "evidence" ], [ 117, 141, "in vitro gel shift assay", "experimental_method" ], [ 157, 166, "Regnase-1", "protein" ], [ 182, 184, "ZF", "structure_element" ], [ 208, 212, "mRNA", "chemical" ], [ 238, 241, "RNA", "chemical" ] ] }, { "sid": 97, "sent": "These results indicate that Regnase-1 directly binds to RNA and precipitates under such experimental conditions.", "section": "DISCUSS", "ner": [ [ 28, 37, "Regnase-1", "protein" ], [ 56, 59, "RNA", "chemical" ] ] }, { "sid": 98, "sent": "Due to this limitation, it is difficult to perform further structural analyses of mRNA-Regnase-1 complexes by X-ray crystallography or NMR.", "section": "DISCUSS", "ner": [ [ 59, 78, "structural analyses", "experimental_method" ], [ 82, 96, "mRNA-Regnase-1", "complex_assembly" ], [ 110, 131, "X-ray crystallography", "experimental_method" ], [ 135, 138, "NMR", "experimental_method" ] ] }, { "sid": 99, "sent": "The previously reported crystal structure of the Regnase-1 PIN domain derived from Homo sapiens is nearly identical to the one derived from Mus musculus in this study, with a backbone RMSD of 0.2\u2009\u00c5. The amino acid sequences corresponding to PIN (residues 134\u2013295) are the two non-identical residues are substituted with similar amino acids.", "section": "DISCUSS", "ner": [ [ 24, 41, "crystal structure", "evidence" ], [ 49, 58, "Regnase-1", "protein" ], [ 59, 62, "PIN", "structure_element" ], [ 83, 95, "Homo sapiens", "species" ], [ 140, 152, "Mus musculus", "species" ], [ 184, 188, "RMSD", "evidence" ], [ 241, 244, "PIN", "structure_element" ], [ 255, 262, "134\u2013295", "residue_range" ] ] }, { "sid": 100, "sent": "Both the mouse and human PIN domains form head-to-tail oligomers in three distinct crystal forms.", "section": "DISCUSS", "ner": [ [ 9, 14, "mouse", "taxonomy_domain" ], [ 19, 24, "human", "species" ], [ 25, 28, "PIN", "structure_element" ], [ 42, 54, "head-to-tail", "protein_state" ], [ 55, 64, "oligomers", "oligomeric_state" ], [ 83, 96, "crystal forms", "evidence" ] ] }, { "sid": 101, "sent": "Rao and co-workers previously argued that PIN dimerization is likely to be a crystallographic artifact with no physiological significance, since monomers were dominant in their analytical ultra-centrifugation experiments.", "section": "DISCUSS", "ner": [ [ 42, 45, "PIN", "structure_element" ], [ 145, 153, "monomers", "oligomeric_state" ], [ 177, 208, "analytical ultra-centrifugation", "experimental_method" ] ] }, { "sid": 102, "sent": "In contrast, our gel filtration data, mutational analyses, and NMR spectra all indicate that the PIN domain forms a head-to-tail dimer in solution in a manner similar to the crystal structure.", "section": "DISCUSS", "ner": [ [ 17, 31, "gel filtration", "experimental_method" ], [ 38, 57, "mutational analyses", "experimental_method" ], [ 63, 66, "NMR", "experimental_method" ], [ 67, 74, "spectra", "evidence" ], [ 97, 100, "PIN", "structure_element" ], [ 116, 128, "head-to-tail", "protein_state" ], [ 129, 134, "dimer", "oligomeric_state" ], [ 174, 191, "crystal structure", "evidence" ] ] }, { "sid": 103, "sent": "This inconsistency might be due to difference in the analytical methods and/or protein concentrations used in each experiment, since the oligomer formation of PIN was dependent on the protein concentration in our study.", "section": "DISCUSS", "ner": [ [ 137, 145, "oligomer", "oligomeric_state" ], [ 159, 162, "PIN", "structure_element" ] ] }, { "sid": 104, "sent": "Single mutations to residues involved in the putative oligomeric interaction of PIN monomerized as expected and these mutants lost their RNase activity as well.", "section": "DISCUSS", "ner": [ [ 0, 16, "Single mutations", "experimental_method" ], [ 80, 83, "PIN", "structure_element" ], [ 84, 95, "monomerized", "oligomeric_state" ], [ 118, 125, "mutants", "protein_state" ], [ 137, 142, "RNase", "protein_type" ] ] }, { "sid": 105, "sent": "Since the NMR spectra of monomeric mutants overlaps with those of the oligomeric forms, it is unlikely that the tertiary structure of the monomeric mutants were affected by the mutations. (Supplementary Fig. 4b,c).", "section": "DISCUSS", "ner": [ [ 10, 13, "NMR", "experimental_method" ], [ 14, 21, "spectra", "evidence" ], [ 25, 34, "monomeric", "oligomeric_state" ], [ 35, 42, "mutants", "protein_state" ], [ 138, 147, "monomeric", "oligomeric_state" ], [ 148, 155, "mutants", "protein_state" ] ] }, { "sid": 106, "sent": "Based on these observations, we concluded that PIN-PIN dimer formation is critical for Regnase-1 RNase activity in vitro.", "section": "DISCUSS", "ner": [ [ 47, 50, "PIN", "structure_element" ], [ 51, 54, "PIN", "structure_element" ], [ 55, 60, "dimer", "oligomeric_state" ], [ 87, 96, "Regnase-1", "protein" ], [ 97, 102, "RNase", "protein_type" ] ] }, { "sid": 107, "sent": "Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the \u201cprimary\u201d and \u201csecondary\u201d PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6).", "section": "DISCUSS", "ner": [ [ 11, 28, "crystal structure", "evidence" ], [ 36, 39, "PIN", "structure_element" ], [ 40, 45, "dimer", "oligomeric_state" ], [ 51, 60, "Regnase-1", "protein" ], [ 97, 104, "primary", "protein_state" ], [ 111, 120, "secondary", "protein_state" ], [ 122, 126, "PINs", "structure_element" ], [ 150, 164, "catalytic site", "site" ], [ 180, 183, "PIN", "structure_element" ] ] }, { "sid": 108, "sent": "Moreover, our structure-based mutational analyses showed these two Regnase-1 specific basic regions were essential for target mRNA cleavage in vitro.", "section": "DISCUSS", "ner": [ [ 14, 49, "structure-based mutational analyses", "experimental_method" ], [ 67, 76, "Regnase-1", "protein" ], [ 126, 130, "mRNA", "chemical" ] ] }, { "sid": 109, "sent": "The cleavage assay also showed that the NTD is crucial for efficient mRNA cleavage.", "section": "DISCUSS", "ner": [ [ 4, 18, "cleavage assay", "experimental_method" ], [ 40, 43, "NTD", "structure_element" ], [ 69, 73, "mRNA", "chemical" ] ] }, { "sid": 110, "sent": "Moreover, we found that the NTD associates with the oligomeric surface of the primary PIN, docking to a helix that harbors its catalytic residues (Figs 2b and 3a).", "section": "DISCUSS", "ner": [ [ 28, 31, "NTD", "structure_element" ], [ 52, 70, "oligomeric surface", "site" ], [ 78, 85, "primary", "protein_state" ], [ 86, 89, "PIN", "structure_element" ], [ 104, 109, "helix", "structure_element" ], [ 127, 145, "catalytic residues", "site" ] ] }, { "sid": 111, "sent": "Taken together, this suggests that the NTD and the PIN domain compete for a common binding site.", "section": "DISCUSS", "ner": [ [ 39, 42, "NTD", "structure_element" ], [ 51, 54, "PIN", "structure_element" ], [ 76, 95, "common binding site", "site" ] ] }, { "sid": 112, "sent": "The affinity of the domain-domain interaction between two PIN domains (Kd\u2009=\u2009~10\u22124\u2009M) is similar to that of the NTD-PIN (Kd\u2009=\u2009110\u2009\u00b1\u20095.8\u2009\u03bcM) interactions; however, the covalent connection corresponding to residues 90\u2013133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1.", "section": "DISCUSS", "ner": [ [ 4, 12, "affinity", "evidence" ], [ 58, 61, "PIN", "structure_element" ], [ 71, 73, "Kd", "evidence" ], [ 111, 114, "NTD", "structure_element" ], [ 115, 118, "PIN", "structure_element" ], [ 120, 122, "Kd", "evidence" ], [ 212, 218, "90\u2013133", "residue_range" ], [ 231, 234, "NTD", "structure_element" ], [ 243, 250, "primary", "protein_state" ], [ 251, 254, "PIN", "structure_element" ], [ 329, 340, "full-length", "protein_state" ], [ 341, 350, "Regnase-1", "protein" ] ] }, { "sid": 113, "sent": "While further analyses are necessary to prove this point, our preliminary docking and molecular dynamics simulations indicate that NTD-binding rearranges the catalytic residues of the PIN domain toward an active conformation suitable for binding Mg2+.", "section": "DISCUSS", "ner": [ [ 74, 116, "docking and molecular dynamics simulations", "experimental_method" ], [ 131, 134, "NTD", "structure_element" ], [ 158, 176, "catalytic residues", "site" ], [ 184, 187, "PIN", "structure_element" ], [ 205, 211, "active", "protein_state" ], [ 246, 250, "Mg2+", "chemical" ] ] }, { "sid": 114, "sent": "In this context, it is interesting that, in response to TCR stimulation, Malt1 cleaves Regnase-1 at R111 to control immune responses in vivo.", "section": "DISCUSS", "ner": [ [ 73, 78, "Malt1", "protein" ], [ 87, 96, "Regnase-1", "protein" ], [ 100, 104, "R111", "residue_name_number" ] ] }, { "sid": 115, "sent": "This result is consistent with a model in which the NTD acts as an enhancer, and cleavage of the linker lowers enzymatic activity dramatically.", "section": "DISCUSS", "ner": [ [ 52, 55, "NTD", "structure_element" ], [ 97, 103, "linker", "structure_element" ] ] }, { "sid": 116, "sent": "Based on these structural and functional analyses of Regnase-1 domain-domain interactions, we performed docking simulations of the NTD, PIN dimer, and IL-6 mRNA.", "section": "DISCUSS", "ner": [ [ 15, 49, "structural and functional analyses", "experimental_method" ], [ 53, 62, "Regnase-1", "protein" ], [ 104, 123, "docking simulations", "experimental_method" ], [ 131, 134, "NTD", "structure_element" ], [ 136, 139, "PIN", "structure_element" ], [ 140, 145, "dimer", "oligomeric_state" ], [ 151, 155, "IL-6", "protein_type" ], [ 156, 160, "mRNA", "chemical" ] ] }, { "sid": 117, "sent": "We incorporated information from the cleavage site of IL-6 mRNA in vitro is indicated by denaturing polyacrylamide gel electrophoresis (Supplementary Fig. 7a,b).", "section": "DISCUSS", "ner": [ [ 37, 50, "cleavage site", "site" ], [ 54, 58, "IL-6", "protein_type" ], [ 59, 63, "mRNA", "chemical" ], [ 100, 134, "polyacrylamide gel electrophoresis", "experimental_method" ] ] }, { "sid": 118, "sent": "The docking result revealed multiple RNA binding modes that satisfied the experimental results in vitro (Supplementary Fig. 7c,d), however, it should be noted that, in vivo, there would likely be many other RNA-binding proteins that would protect loop regions from cleavage by Regnase-1.", "section": "DISCUSS", "ner": [ [ 4, 11, "docking", "experimental_method" ], [ 37, 40, "RNA", "chemical" ], [ 207, 227, "RNA-binding proteins", "protein_type" ], [ 247, 251, "loop", "structure_element" ], [ 277, 286, "Regnase-1", "protein" ] ] }, { "sid": 119, "sent": "The overall model of regulation of Regnase-1 RNase activity through domain-domain interactions in vitro is summarized in Fig. 6.", "section": "DISCUSS", "ner": [ [ 35, 44, "Regnase-1", "protein" ], [ 45, 50, "RNase", "protein_type" ] ] }, { "sid": 120, "sent": "In the absence of target mRNA, the PIN domain forms head-to-tail oligomers at high concentration.", "section": "DISCUSS", "ner": [ [ 7, 17, "absence of", "protein_state" ], [ 25, 29, "mRNA", "chemical" ], [ 35, 38, "PIN", "structure_element" ], [ 52, 64, "head-to-tail", "protein_state" ], [ 65, 74, "oligomers", "oligomeric_state" ] ] }, { "sid": 121, "sent": "A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN).", "section": "DISCUSS", "ner": [ [ 2, 14, "fully active", "protein_state" ], [ 15, 31, "catalytic center", "site" ], [ 60, 63, "NTD", "structure_element" ], [ 84, 92, "oligomer", "oligomeric_state" ], [ 108, 111, "PIN", "structure_element" ], [ 141, 153, "head-to-tail", "protein_state" ], [ 154, 162, "oligomer", "oligomeric_state" ], [ 191, 198, "primary", "protein_state" ], [ 199, 202, "PIN", "structure_element" ], [ 217, 227, "functional", "protein_state" ], [ 228, 233, "dimer", "oligomeric_state" ], [ 264, 267, "PIN", "structure_element" ], [ 269, 278, "secondary", "protein_state" ], [ 279, 282, "PIN", "structure_element" ] ] }, { "sid": 122, "sent": "While further investigations on the domain-domain interactions of Regnase-1 in vivo are necessary, these intramolecular and intermolecular domain interactions of Regnase-1 appear to structurally constrain Regnase-1activity, which, in turn, enables tight regulation of immune responses.", "section": "DISCUSS", "ner": [ [ 66, 75, "Regnase-1", "protein" ], [ 162, 171, "Regnase-1", "protein" ], [ 205, 214, "Regnase-1", "protein" ] ] }, { "sid": 123, "sent": "Structural and functional analyses of Regnase-1.", "section": "FIG", "ner": [ [ 0, 34, "Structural and functional analyses", "experimental_method" ], [ 38, 47, "Regnase-1", "protein" ] ] }, { "sid": 124, "sent": "(a) Domain architecture of Regnase-1. (b) Solution structure of the NTD. (c) Crystal structure of the PIN domain.", "section": "FIG", "ner": [ [ 27, 36, "Regnase-1", "protein" ], [ 42, 60, "Solution structure", "evidence" ], [ 68, 71, "NTD", "structure_element" ], [ 77, 94, "Crystal structure", "evidence" ], [ 102, 105, "PIN", "structure_element" ] ] }, { "sid": 125, "sent": "Catalytic Asp residues were shown in sticks.", "section": "FIG", "ner": [ [ 0, 9, "Catalytic", "protein_state" ], [ 10, 13, "Asp", "residue_name" ] ] }, { "sid": 126, "sent": "(d) Solution structure of the ZF domain.", "section": "FIG", "ner": [ [ 4, 22, "Solution structure", "evidence" ], [ 30, 32, "ZF", "structure_element" ] ] }, { "sid": 127, "sent": "Three Cys residues and one His residue responsible for Zn2+-binding were shown in sticks.", "section": "FIG", "ner": [ [ 6, 9, "Cys", "residue_name" ], [ 27, 30, "His", "residue_name" ] ] }, { "sid": 128, "sent": "(e) Solution structure of the CTD.", "section": "FIG", "ner": [ [ 4, 22, "Solution structure", "evidence" ], [ 30, 33, "CTD", "structure_element" ] ] }, { "sid": 129, "sent": "All the structures were colored in rainbow from N-terminus (blue) to C-terminus (red).", "section": "FIG", "ner": [ [ 8, 18, "structures", "evidence" ] ] }, { "sid": 130, "sent": "(f) In vitro gel shift binding assay between Regnase-1 and IL-6 mRNA.", "section": "FIG", "ner": [ [ 4, 36, "In vitro gel shift binding assay", "experimental_method" ], [ 45, 54, "Regnase-1", "protein" ], [ 59, 63, "IL-6", "protein_type" ], [ 64, 68, "mRNA", "chemical" ] ] }, { "sid": 131, "sent": "Fluorescence intensity of the free IL-6 in each sample was indicated as the percentage against that in the absence of Regnase-1.", "section": "FIG", "ner": [ [ 0, 22, "Fluorescence intensity", "evidence" ], [ 30, 34, "free", "protein_state" ], [ 35, 39, "IL-6", "protein_type" ], [ 107, 117, "absence of", "protein_state" ], [ 118, 127, "Regnase-1", "protein" ] ] }, { "sid": 132, "sent": "(g) Binding of Regnase-1 and IL-6 mRNA was plotted.", "section": "FIG", "ner": [ [ 15, 24, "Regnase-1", "protein" ], [ 29, 33, "IL-6", "protein_type" ], [ 34, 38, "mRNA", "chemical" ] ] }, { "sid": 133, "sent": "The percentage of the bound IL-6 was calculated based on the fluorescence intensities of the free IL-6 quantified in (f).", "section": "FIG", "ner": [ [ 28, 32, "IL-6", "protein_type" ], [ 61, 85, "fluorescence intensities", "evidence" ], [ 98, 102, "IL-6", "protein_type" ] ] }, { "sid": 134, "sent": "(h) In vitro cleavage assay of Regnase-1 to IL-6 mRNA.", "section": "FIG", "ner": [ [ 4, 27, "In vitro cleavage assay", "experimental_method" ], [ 31, 40, "Regnase-1", "protein" ], [ 44, 48, "IL-6", "protein_type" ], [ 49, 53, "mRNA", "chemical" ] ] }, { "sid": 135, "sent": "Fluorescence intensity of the uncleaved IL-6 mRNA was indicated as the percentage against that in the absence of Regnase-1.", "section": "FIG", "ner": [ [ 0, 22, "Fluorescence intensity", "evidence" ], [ 30, 39, "uncleaved", "protein_state" ], [ 40, 44, "IL-6", "protein_type" ], [ 45, 49, "mRNA", "chemical" ], [ 102, 112, "absence of", "protein_state" ], [ 113, 122, "Regnase-1", "protein" ] ] }, { "sid": 136, "sent": "Head-to-tail oligomer formation of the PIN domain is crucial for the RNase activity of Regnase-1.", "section": "FIG", "ner": [ [ 0, 12, "Head-to-tail", "protein_state" ], [ 13, 21, "oligomer", "oligomeric_state" ], [ 39, 42, "PIN", "structure_element" ], [ 69, 74, "RNase", "protein_type" ], [ 87, 96, "Regnase-1", "protein" ] ] }, { "sid": 137, "sent": "(a) Gel filtration analyses of the PIN domain.", "section": "FIG", "ner": [ [ 4, 27, "Gel filtration analyses", "experimental_method" ], [ 35, 38, "PIN", "structure_element" ] ] }, { "sid": 138, "sent": "(b) Dimer structure of the PIN domain.", "section": "FIG", "ner": [ [ 4, 9, "Dimer", "oligomeric_state" ], [ 10, 19, "structure", "evidence" ], [ 27, 30, "PIN", "structure_element" ] ] }, { "sid": 139, "sent": "Two PIN molecules in the crystal were colored white and green, respectively.", "section": "FIG", "ner": [ [ 4, 7, "PIN", "structure_element" ], [ 25, 32, "crystal", "evidence" ] ] }, { "sid": 140, "sent": "Catalytic residues and mutated residues were shown in sticks.", "section": "FIG", "ner": [ [ 0, 18, "Catalytic residues", "site" ] ] }, { "sid": 141, "sent": "Residues important for the oligomeric interaction were colored red, while R215 that was dispensable for the oligomeric interaction was colored blue. (c) RNase activity of monomeric mutants for IL-6 mRNA was analyzed.", "section": "FIG", "ner": [ [ 74, 78, "R215", "residue_name_number" ], [ 153, 158, "RNase", "protein_type" ], [ 171, 180, "monomeric", "oligomeric_state" ], [ 181, 188, "mutants", "protein_state" ], [ 193, 197, "IL-6", "protein_type" ], [ 198, 202, "mRNA", "chemical" ] ] }, { "sid": 142, "sent": "Domain-domain interaction between the NTD and the PIN domain.", "section": "FIG", "ner": [ [ 38, 41, "NTD", "structure_element" ], [ 50, 53, "PIN", "structure_element" ] ] }, { "sid": 143, "sent": "(a) NMR analyses of the NTD-binding to the PIN domain.", "section": "FIG", "ner": [ [ 4, 16, "NMR analyses", "experimental_method" ], [ 24, 27, "NTD", "structure_element" ], [ 43, 46, "PIN", "structure_element" ] ] }, { "sid": 144, "sent": "The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red on the surface and ribbon structure of the PIN domain (right).", "section": "FIG", "ner": [ [ 73, 81, "overlaid", "experimental_method" ], [ 82, 89, "spectra", "evidence" ], [ 156, 159, "PIN", "structure_element" ] ] }, { "sid": 145, "sent": "Pro and the residues without analysis were colored black and gray, respectively.", "section": "FIG", "ner": [ [ 0, 3, "Pro", "residue_name" ] ] }, { "sid": 146, "sent": "(b) NMR analyses of the PIN-binding to the NTD.", "section": "FIG", "ner": [ [ 4, 16, "NMR analyses", "experimental_method" ], [ 24, 27, "PIN", "structure_element" ], [ 43, 46, "NTD", "structure_element" ] ] }, { "sid": 147, "sent": "The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red, yellow, or green on the surface and ribbon structure of the NTD.", "section": "FIG", "ner": [ [ 18, 52, "significant chemical shift changes", "evidence" ], [ 73, 81, "overlaid", "experimental_method" ], [ 82, 89, "spectra", "evidence" ], [ 174, 177, "NTD", "structure_element" ] ] }, { "sid": 148, "sent": "S62 was colored gray and excluded from the analysis, due to low signal intensity.", "section": "FIG", "ner": [ [ 0, 3, "S62", "residue_name_number" ] ] }, { "sid": 149, "sent": "(c) Docking model of the NTD and the PIN domain.", "section": "FIG", "ner": [ [ 25, 28, "NTD", "structure_element" ], [ 37, 40, "PIN", "structure_element" ] ] }, { "sid": 150, "sent": "The NTD and the PIN domain are shown in cyan and white, respectively.", "section": "FIG", "ner": [ [ 4, 7, "NTD", "structure_element" ], [ 16, 19, "PIN", "structure_element" ] ] }, { "sid": 151, "sent": "Residues in close proximity (<5\u2009\u00c5) to each other in the docking structure were colored yellow.", "section": "FIG", "ner": [ [ 56, 73, "docking structure", "evidence" ] ] }, { "sid": 152, "sent": "Catalytic residues of the PIN domain are shown in sticks, and the residues that exhibited significant chemical shift changes in (a,b) were labeled.", "section": "FIG", "ner": [ [ 0, 18, "Catalytic residues", "site" ], [ 26, 29, "PIN", "structure_element" ], [ 90, 124, "significant chemical shift changes", "evidence" ] ] }, { "sid": 153, "sent": "Critical residues in the PIN domain for the RNase activity of Regnase-1.", "section": "FIG", "ner": [ [ 25, 28, "PIN", "structure_element" ], [ 44, 49, "RNase", "protein_type" ], [ 62, 71, "Regnase-1", "protein" ] ] }, { "sid": 154, "sent": "(a) In vitro cleavage assay of basic residue mutants for IL-6 mRNA.", "section": "FIG", "ner": [ [ 4, 27, "In vitro cleavage assay", "experimental_method" ], [ 45, 52, "mutants", "protein_state" ], [ 57, 61, "IL-6", "protein_type" ], [ 62, 66, "mRNA", "chemical" ] ] }, { "sid": 155, "sent": "(b) In vitro cleavage assay of basic residue mutants for Regnase-1 mRNA.", "section": "FIG", "ner": [ [ 4, 27, "In vitro cleavage assay", "experimental_method" ], [ 45, 52, "mutants", "protein_state" ], [ 57, 66, "Regnase-1", "protein" ], [ 67, 71, "mRNA", "chemical" ] ] }, { "sid": 156, "sent": "The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer structure.", "section": "FIG", "ner": [ [ 4, 26, "fluorescence intensity", "evidence" ], [ 34, 43, "uncleaved", "protein_state" ], [ 44, 48, "mRNA", "chemical" ], [ 99, 102, "PIN", "structure_element" ], [ 103, 108, "dimer", "oligomeric_state" ], [ 109, 118, "structure", "evidence" ] ] }, { "sid": 157, "sent": "Mutated basic residues were shown in sticks and those with significantly reduced RNase activities were colored red or yellow.", "section": "FIG", "ner": [ [ 81, 86, "RNase", "protein_type" ] ] }, { "sid": 158, "sent": "Heterodimer formation by combination of the Regnase-1 basic residue mutants and the DDNN mutant restored the RNase activity.", "section": "FIG", "ner": [ [ 44, 53, "Regnase-1", "protein" ], [ 68, 75, "mutants", "protein_state" ], [ 84, 88, "DDNN", "mutant" ], [ 89, 95, "mutant", "protein_state" ], [ 109, 114, "RNase", "protein_type" ] ] }, { "sid": 159, "sent": "(a) Cartoon representation of the concept of the experiment. (b) In vitro cleavage assay of Regnase-1 for IL-6 mRNA.", "section": "FIG", "ner": [ [ 65, 88, "In vitro cleavage assay", "experimental_method" ], [ 92, 101, "Regnase-1", "protein" ], [ 106, 110, "IL-6", "protein_type" ], [ 111, 115, "mRNA", "chemical" ] ] }, { "sid": 160, "sent": "(c) In vitro cleavage assay of Regnase-1 for Regnase-1 mRNA.", "section": "FIG", "ner": [ [ 4, 27, "In vitro cleavage assay", "experimental_method" ], [ 31, 40, "Regnase-1", "protein" ], [ 45, 54, "Regnase-1", "protein" ], [ 55, 59, "mRNA", "chemical" ] ] }, { "sid": 161, "sent": "The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer.", "section": "FIG", "ner": [ [ 4, 26, "fluorescence intensity", "evidence" ], [ 34, 43, "uncleaved", "protein_state" ], [ 44, 48, "mRNA", "chemical" ], [ 99, 102, "PIN", "structure_element" ], [ 103, 108, "dimer", "oligomeric_state" ] ] }, { "sid": 162, "sent": "The mutations whose RNase activities were not increased in the presence of DDNN mutant were colored in blue on the primary PIN.", "section": "FIG", "ner": [ [ 20, 25, "RNase", "protein_type" ], [ 63, 74, "presence of", "protein_state" ], [ 75, 79, "DDNN", "mutant" ], [ 80, 86, "mutant", "protein_state" ], [ 123, 126, "PIN", "structure_element" ] ] }, { "sid": 163, "sent": "The mutations whose RNase activities were restored in the presence of DDNN mutant were colored in red or yellow on the primary PIN.", "section": "FIG", "ner": [ [ 20, 25, "RNase", "protein_type" ], [ 58, 69, "presence of", "protein_state" ], [ 70, 74, "DDNN", "mutant" ], [ 75, 81, "mutant", "protein_state" ], [ 127, 130, "PIN", "structure_element" ] ] }, { "sid": 164, "sent": "Schematic representation of regulation of the Regnase-1 catalytic activity through the domain-domain interactions.", "section": "FIG", "ner": [ [ 46, 55, "Regnase-1", "protein" ] ] } ] }, "PMC4850288": { "annotations": [ { "sid": 0, "sent": "Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome*", "section": "TITLE", "ner": [ [ 0, 17, "Crystal Structure", "evidence" ], [ 22, 38, "Activity Studies", "experimental_method" ], [ 46, 49, "C11", "protein_type" ], [ 50, 68, "Cysteine Peptidase", "protein_type" ], [ 74, 96, "Parabacteroides merdae", "species" ], [ 104, 109, "Human", "species" ] ] }, { "sid": 1, "sent": "Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms.", "section": "ABSTRACT", "ner": [ [ 0, 27, "Clan CD cysteine peptidases", "protein_type" ], [ 61, 71, "peptidases", "protein_type" ], [ 85, 94, "mammalian", "taxonomy_domain" ], [ 95, 103, "caspases", "protein_type" ] ] }, { "sid": 2, "sent": "However, for the clostripain family (denoted C11), little is currently known.", "section": "ABSTRACT", "ner": [ [ 17, 35, "clostripain family", "protein_type" ], [ 45, 48, "C11", "protein_type" ] ] }, { "sid": 3, "sent": "Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-\u212b resolution.", "section": "ABSTRACT", "ner": [ [ 28, 45, "crystal structure", "evidence" ], [ 51, 54, "C11", "protein_type" ], [ 72, 77, "human", "species" ], [ 82, 91, "bacterium", "taxonomy_domain" ], [ 93, 115, "Parabacteroides merdae", "species" ], [ 117, 122, "PmC11", "protein" ] ] }, { "sid": 4, "sent": "PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like \u03b1/\u03b2/\u03b1 sandwich and an unusual C-terminal domain.", "section": "ABSTRACT", "ner": [ [ 0, 5, "PmC11", "protein" ], [ 11, 20, "monomeric", "oligomeric_state" ], [ 21, 39, "cysteine peptidase", "protein_type" ], [ 58, 94, "extended caspase-like \u03b1/\u03b2/\u03b1 sandwich", "structure_element" ], [ 110, 127, "C-terminal domain", "structure_element" ] ] }, { "sid": 5, "sent": "It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan.", "section": "ABSTRACT", "ner": [ [ 40, 67, "clan CD cysteine peptidases", "protein_type" ] ] }, { "sid": 6, "sent": "These studies also revealed a well ordered break in the polypeptide chain at Lys147, resulting in a large conformational rearrangement close to the active site.", "section": "ABSTRACT", "ner": [ [ 77, 83, "Lys147", "residue_name_number" ], [ 148, 159, "active site", "site" ] ] }, { "sid": 7, "sent": "Biochemical and kinetic analysis revealed Lys147 to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation.", "section": "ABSTRACT", "ner": [ [ 0, 32, "Biochemical and kinetic analysis", "experimental_method" ], [ 42, 48, "Lys147", "residue_name_number" ], [ 58, 88, "intramolecular processing site", "site" ], [ 98, 106, "cleavage", "ptm" ], [ 123, 138, "full activation", "protein_state" ], [ 146, 152, "enzyme", "protein" ] ] }, { "sid": 8, "sent": "PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca2+ for activity.", "section": "ABSTRACT", "ner": [ [ 0, 5, "PmC11", "protein" ], [ 13, 34, "acidic binding pocket", "site" ], [ 102, 105, "Arg", "residue_name" ], [ 110, 113, "Lys", "residue_name" ], [ 117, 119, "P1", "residue_number" ], [ 141, 145, "Ca2+", "chemical" ] ] }, { "sid": 9, "sent": "Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms.", "section": "ABSTRACT", "ner": [ [ 77, 82, "PmC11", "protein" ], [ 123, 137, "C11 peptidases", "protein_type" ] ] }, { "sid": 10, "sent": "Cysteine peptidases play crucial roles in the virulence of bacterial and other eukaryotic pathogens.", "section": "INTRO", "ner": [ [ 0, 19, "Cysteine peptidases", "protein_type" ], [ 59, 68, "bacterial", "taxonomy_domain" ], [ 79, 89, "eukaryotic", "taxonomy_domain" ] ] }, { "sid": 11, "sent": "In the MEROPS peptidase database, clan CD contains groups (or families) of cysteine peptidases that share some highly conserved structural elements.", "section": "INTRO", "ner": [ [ 34, 41, "clan CD", "protein_type" ], [ 75, 94, "cysteine peptidases", "protein_type" ], [ 111, 127, "highly conserved", "protein_state" ] ] }, { "sid": 12, "sent": "Clan CD families are typically described using the name of their archetypal, or founding, member and also given an identification number preceded by a \u201cC,\u201d to denote cysteine peptidase.", "section": "INTRO", "ner": [ [ 0, 16, "Clan CD families", "protein_type" ], [ 166, 184, "cysteine peptidase", "protein_type" ] ] }, { "sid": 13, "sent": "Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80).", "section": "INTRO", "ner": [ [ 110, 128, "crystal structures", "evidence" ], [ 166, 174, "legumain", "protein" ], [ 176, 179, "C13", "protein_type" ], [ 182, 189, "caspase", "protein" ], [ 191, 195, "C14a", "protein_type" ], [ 198, 209, "paracaspase", "protein" ], [ 211, 217, "C14b(P", "protein_type" ], [ 220, 231, "metacaspase", "protein" ], [ 233, 239, "C14b(M", "protein_type" ], [ 242, 251, "gingipain", "protein" ], [ 253, 256, "C25", "protein_type" ], [ 267, 292, "cysteine peptidase domain", "structure_element" ], [ 294, 297, "CPD", "structure_element" ], [ 318, 321, "C80", "protein_type" ] ] }, { "sid": 14, "sent": "No structural information is available for clostripain (C11), separase (C50), or PrtH-peptidase (C85).", "section": "INTRO", "ner": [ [ 43, 54, "clostripain", "protein" ], [ 56, 59, "C11", "protein_type" ], [ 62, 70, "separase", "protein" ], [ 72, 75, "C50", "protein_type" ], [ 81, 95, "PrtH-peptidase", "protein" ], [ 97, 100, "C85", "protein_type" ] ] }, { "sid": 15, "sent": "Clan CD enzymes have a highly conserved His/Cys catalytic dyad and exhibit strict specificity for the P1 residue of their substrates.", "section": "INTRO", "ner": [ [ 0, 15, "Clan CD enzymes", "protein_type" ], [ 23, 39, "highly conserved", "protein_state" ], [ 40, 62, "His/Cys catalytic dyad", "site" ], [ 102, 104, "P1", "residue_number" ] ] }, { "sid": 16, "sent": "However, despite these similarities, clan CD forms a functionally diverse group of enzymes: the overall structural diversity between (and at times within) the various families provides these peptidases with a wide variety of substrate specificities and activation mechanisms.", "section": "INTRO", "ner": [ [ 37, 44, "clan CD", "protein_type" ], [ 191, 201, "peptidases", "protein_type" ] ] }, { "sid": 17, "sent": "The archetypal and arguably most notable family in the clan is that of the mammalian caspases (C14a), although clan CD members are distributed throughout the entire phylogenetic kingdom and are often required in fundamental biological processes.", "section": "INTRO", "ner": [ [ 75, 84, "mammalian", "taxonomy_domain" ], [ 85, 93, "caspases", "protein_type" ], [ 95, 99, "C14a", "protein_type" ], [ 111, 118, "clan CD", "protein_type" ] ] }, { "sid": 18, "sent": "Interestingly, little is known about the structure or function of the C11 proteins, despite their widespread distribution and its archetypal member, clostripain from Clostridium histolyticum, first reported in the literature in 1938.", "section": "INTRO", "ner": [ [ 70, 73, "C11", "protein_type" ], [ 149, 160, "clostripain", "protein" ], [ 166, 190, "Clostridium histolyticum", "species" ] ] }, { "sid": 19, "sent": "Clostripain has been described as an arginine-specific peptidase with a requirement for Ca2+ and loss of an internal nonapeptide for full activation; lack of structural information on the family appears to have prohibited further investigation.", "section": "INTRO", "ner": [ [ 0, 11, "Clostripain", "protein" ], [ 37, 64, "arginine-specific peptidase", "protein_type" ], [ 88, 92, "Ca2+", "chemical" ], [ 108, 128, "internal nonapeptide", "structure_element" ], [ 133, 148, "full activation", "protein_state" ] ] }, { "sid": 20, "sent": "As part of an ongoing project to characterize commensal bacteria in the microbiome that inhabit the human gut, the structure of C11 peptidase, PmC11, from Parabacteroides merdae was determined using the Joint Center for Structural Genomics (JCSG)4 HTP structural biology pipeline.", "section": "INTRO", "ner": [ [ 56, 64, "bacteria", "taxonomy_domain" ], [ 100, 105, "human", "species" ], [ 115, 124, "structure", "evidence" ], [ 128, 141, "C11 peptidase", "protein_type" ], [ 143, 148, "PmC11", "protein" ], [ 155, 177, "Parabacteroides merdae", "species" ] ] }, { "sid": 21, "sent": "The structure was analyzed, and the enzyme was biochemically characterized to provide the first structure/function correlation for a C11 peptidase.", "section": "INTRO", "ner": [ [ 4, 26, "structure was analyzed", "experimental_method" ], [ 47, 74, "biochemically characterized", "experimental_method" ], [ 133, 146, "C11 peptidase", "protein_type" ] ] }, { "sid": 22, "sent": "Structure of PmC11", "section": "RESULTS", "ner": [ [ 0, 9, "Structure", "evidence" ], [ 13, 18, "PmC11", "protein" ] ] }, { "sid": 23, "sent": "The crystal structure of the catalytically active form of PmC11 revealed an extended caspase-like \u03b1/\u03b2/\u03b1 sandwich architecture comprised of a central nine-stranded \u03b2-sheet, with an unusual C-terminal domain (CTD), starting at Lys250.", "section": "RESULTS", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 29, 49, "catalytically active", "protein_state" ], [ 58, 63, "PmC11", "protein" ], [ 76, 112, "extended caspase-like \u03b1/\u03b2/\u03b1 sandwich", "structure_element" ], [ 149, 170, "nine-stranded \u03b2-sheet", "structure_element" ], [ 188, 205, "C-terminal domain", "structure_element" ], [ 207, 210, "CTD", "structure_element" ], [ 225, 231, "Lys250", "residue_name_number" ] ] }, { "sid": 24, "sent": "A single cleavage was observed in the polypeptide chain at Lys147 (Fig. 1, A and B), where both ends of the cleavage site are fully visible and well ordered in the electron density.", "section": "RESULTS", "ner": [ [ 2, 17, "single cleavage", "ptm" ], [ 59, 65, "Lys147", "residue_name_number" ], [ 108, 121, "cleavage site", "site" ], [ 164, 180, "electron density", "evidence" ] ] }, { "sid": 25, "sent": "The central nine-stranded \u03b2-sheet (\u03b21\u2013\u03b29) of PmC11 consists of six parallel and three anti-parallel \u03b2-strands with 4\u21913\u21932\u21911\u21915\u21916\u21917\u21938\u21939\u2191 topology (Fig. 1A) and the overall structure includes 14 \u03b1-helices with six (\u03b11\u2013\u03b12 and \u03b14\u2013\u03b17) closely surrounding the \u03b2-sheet in an approximately parallel orientation.", "section": "RESULTS", "ner": [ [ 12, 33, "nine-stranded \u03b2-sheet", "structure_element" ], [ 35, 40, "\u03b21\u2013\u03b29", "structure_element" ], [ 45, 50, "PmC11", "protein" ], [ 67, 75, "parallel", "structure_element" ], [ 86, 109, "anti-parallel \u03b2-strands", "structure_element" ], [ 169, 178, "structure", "evidence" ], [ 191, 200, "\u03b1-helices", "structure_element" ], [ 211, 216, "\u03b11\u2013\u03b12", "structure_element" ], [ 221, 226, "\u03b14\u2013\u03b17", "structure_element" ], [ 252, 259, "\u03b2-sheet", "structure_element" ] ] }, { "sid": 26, "sent": "Helices \u03b11, \u03b17, and \u03b16 are located on one side of the \u03b2-sheet with \u03b12, \u03b14, and \u03b15 on the opposite side (Fig. 1A).", "section": "RESULTS", "ner": [ [ 0, 7, "Helices", "structure_element" ], [ 8, 10, "\u03b11", "structure_element" ], [ 12, 14, "\u03b17", "structure_element" ], [ 20, 22, "\u03b16", "structure_element" ], [ 54, 61, "\u03b2-sheet", "structure_element" ], [ 67, 69, "\u03b12", "structure_element" ], [ 71, 73, "\u03b14", "structure_element" ], [ 79, 81, "\u03b15", "structure_element" ] ] }, { "sid": 27, "sent": "Helix \u03b13 sits at the end of the loop following \u03b25 (L5), just preceding the Lys147 cleavage site, with both L5 and \u03b13 pointing away from the central \u03b2-sheet and toward the CTD, which starts with \u03b18.", "section": "RESULTS", "ner": [ [ 0, 5, "Helix", "structure_element" ], [ 6, 8, "\u03b13", "structure_element" ], [ 32, 36, "loop", "structure_element" ], [ 47, 49, "\u03b25", "structure_element" ], [ 51, 53, "L5", "structure_element" ], [ 75, 81, "Lys147", "residue_name_number" ], [ 82, 95, "cleavage site", "site" ], [ 107, 109, "L5", "structure_element" ], [ 114, 116, "\u03b13", "structure_element" ], [ 148, 155, "\u03b2-sheet", "structure_element" ], [ 171, 174, "CTD", "structure_element" ], [ 194, 196, "\u03b18", "structure_element" ] ] }, { "sid": 28, "sent": "The structure also includes two short \u03b2-hairpins (\u03b2A\u2013\u03b2B and \u03b2D\u2013\u03b2E) and a small \u03b2-sheet (\u03b2C\u2013\u03b2F), which is formed from two distinct regions of the sequence (\u03b2C precedes \u03b111, \u03b112 and \u03b29, whereas \u03b2F follows the \u03b2D-\u03b2E hairpin) in the middle of the CTD (Fig. 1B).", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 38, 48, "\u03b2-hairpins", "structure_element" ], [ 50, 55, "\u03b2A\u2013\u03b2B", "structure_element" ], [ 60, 65, "\u03b2D\u2013\u03b2E", "structure_element" ], [ 73, 86, "small \u03b2-sheet", "structure_element" ], [ 88, 93, "\u03b2C\u2013\u03b2F", "structure_element" ], [ 155, 157, "\u03b2C", "structure_element" ], [ 167, 170, "\u03b111", "structure_element" ], [ 172, 175, "\u03b112", "structure_element" ], [ 180, 182, "\u03b29", "structure_element" ], [ 192, 194, "\u03b2F", "structure_element" ], [ 207, 212, "\u03b2D-\u03b2E", "structure_element" ], [ 213, 220, "hairpin", "structure_element" ], [ 243, 246, "CTD", "structure_element" ] ] }, { "sid": 29, "sent": "Crystal structure of a C11 peptidase from P. merdae.", "section": "FIG", "ner": [ [ 0, 17, "Crystal structure", "evidence" ], [ 23, 36, "C11 peptidase", "protein_type" ], [ 42, 51, "P. merdae", "species" ] ] }, { "sid": 30, "sent": " A, primary sequence alignment of PmC11 (Uniprot ID A7A9N3) and clostripain (Uniprot ID P09870) from C. histolyticum with identical residues highlighted in gray shading.", "section": "FIG", "ner": [ [ 4, 30, "primary sequence alignment", "experimental_method" ], [ 34, 39, "PmC11", "protein" ], [ 64, 75, "clostripain", "protein" ], [ 101, 116, "C. histolyticum", "species" ] ] }, { "sid": 31, "sent": "The secondary structure of PmC11 from the crystal structure is mapped onto its sequence with the position of the PmC11 catalytic dyad, autocatalytic cleavage site (Lys147), and S1 binding pocket Asp (Asp177) highlighted by a red star, a red downturned triangle, and a red upturned triangle, respectively.", "section": "FIG", "ner": [ [ 27, 32, "PmC11", "protein" ], [ 42, 59, "crystal structure", "evidence" ], [ 113, 118, "PmC11", "protein" ], [ 119, 133, "catalytic dyad", "site" ], [ 135, 162, "autocatalytic cleavage site", "site" ], [ 164, 170, "Lys147", "residue_name_number" ], [ 177, 194, "S1 binding pocket", "site" ], [ 195, 198, "Asp", "residue_name" ], [ 200, 206, "Asp177", "residue_name_number" ] ] }, { "sid": 32, "sent": "Connecting loops are colored gray, the main \u03b2-sheet is in orange, with other strands in olive, \u03b1-helices are in blue, and the nonapeptide linker of clostripain that is excised upon autocleavage is underlined in red.", "section": "FIG", "ner": [ [ 11, 16, "loops", "structure_element" ], [ 44, 51, "\u03b2-sheet", "structure_element" ], [ 95, 104, "\u03b1-helices", "structure_element" ], [ 126, 144, "nonapeptide linker", "structure_element" ], [ 148, 159, "clostripain", "protein" ], [ 181, 193, "autocleavage", "ptm" ] ] }, { "sid": 33, "sent": "Sequences around the catalytic site of clostripain and PmC11 align well.", "section": "FIG", "ner": [ [ 21, 35, "catalytic site", "site" ], [ 39, 50, "clostripain", "protein" ], [ 55, 60, "PmC11", "protein" ] ] }, { "sid": 34, "sent": "B, topology diagram of PmC11 colored as in A except that additional (non-core) \u03b2-strands are in yellow.", "section": "FIG", "ner": [ [ 23, 28, "PmC11", "protein" ], [ 79, 88, "\u03b2-strands", "structure_element" ] ] }, { "sid": 35, "sent": "Helices found on either side of the central \u03b2-sheet are shown above and below the sheet, respectively.", "section": "FIG", "ner": [ [ 44, 51, "\u03b2-sheet", "structure_element" ], [ 82, 87, "sheet", "structure_element" ] ] }, { "sid": 36, "sent": "The position of the catalytic dyad (H, C) and the processing site (Lys147) are highlighted.", "section": "FIG", "ner": [ [ 20, 34, "catalytic dyad", "site" ], [ 36, 37, "H", "residue_name" ], [ 39, 40, "C", "residue_name" ], [ 50, 65, "processing site", "site" ], [ 67, 73, "Lys147", "residue_name_number" ] ] }, { "sid": 37, "sent": "Helices (1\u201314) and \u03b2-strands (1\u20139 and A-F) are numbered from the N terminus.", "section": "FIG", "ner": [ [ 19, 28, "\u03b2-strands", "structure_element" ] ] }, { "sid": 38, "sent": "The core caspase-fold is highlighted in a box.", "section": "FIG", "ner": [ [ 4, 21, "core caspase-fold", "structure_element" ] ] }, { "sid": 39, "sent": "C, tertiary structure of PmC11.", "section": "FIG", "ner": [ [ 25, 30, "PmC11", "protein" ] ] }, { "sid": 40, "sent": "The N and C termini (N and C) of PmC11 along with the central \u03b2-sheet (1\u20139), helix \u03b15, and helices \u03b18, \u03b111, and \u03b113 from the C-terminal domain, are all labeled.", "section": "FIG", "ner": [ [ 33, 38, "PmC11", "protein" ], [ 62, 69, "\u03b2-sheet", "structure_element" ], [ 77, 82, "helix", "structure_element" ], [ 83, 85, "\u03b15", "structure_element" ], [ 91, 98, "helices", "structure_element" ], [ 99, 101, "\u03b18", "structure_element" ], [ 103, 106, "\u03b111", "structure_element" ], [ 112, 115, "\u03b113", "structure_element" ], [ 125, 142, "C-terminal domain", "structure_element" ] ] }, { "sid": 41, "sent": "Loops are colored gray, the main \u03b2-sheet is in orange, with other \u03b2-strands in yellow, and \u03b1-helices are in blue.", "section": "FIG", "ner": [ [ 33, 40, "\u03b2-sheet", "structure_element" ], [ 66, 75, "\u03b2-strands", "structure_element" ], [ 91, 100, "\u03b1-helices", "structure_element" ] ] }, { "sid": 42, "sent": "The CTD of PmC11 is composed of a tight helical bundle formed from helices \u03b18\u2013\u03b114 and includes strands \u03b2C and \u03b2F, and \u03b2-hairpin \u03b2D\u2013\u03b2E. The CTD sits entirely on one side of the enzyme interacting only with \u03b13, \u03b15, \u03b29, and the loops surrounding \u03b28.", "section": "RESULTS", "ner": [ [ 4, 7, "CTD", "structure_element" ], [ 11, 16, "PmC11", "protein" ], [ 34, 54, "tight helical bundle", "structure_element" ], [ 67, 74, "helices", "structure_element" ], [ 75, 81, "\u03b18\u2013\u03b114", "structure_element" ], [ 95, 102, "strands", "structure_element" ], [ 103, 105, "\u03b2C", "structure_element" ], [ 110, 112, "\u03b2F", "structure_element" ], [ 118, 127, "\u03b2-hairpin", "structure_element" ], [ 128, 133, "\u03b2D\u2013\u03b2E", "structure_element" ], [ 139, 142, "CTD", "structure_element" ], [ 205, 207, "\u03b13", "structure_element" ], [ 209, 211, "\u03b15", "structure_element" ], [ 213, 215, "\u03b29", "structure_element" ], [ 225, 230, "loops", "structure_element" ], [ 243, 245, "\u03b28", "structure_element" ] ] }, { "sid": 43, "sent": "Of the interacting secondary structure elements, \u03b15 is perhaps the most interesting.", "section": "RESULTS", "ner": [ [ 49, 51, "\u03b15", "structure_element" ] ] }, { "sid": 44, "sent": "This helix makes a total of eight hydrogen bonds with the CTD, including one salt bridge (Arg191-Asp255) and is surrounded by the CTD on one side and the main core of the enzyme on the other, acting like a linchpin holding both components together (Fig. 1C).", "section": "RESULTS", "ner": [ [ 0, 10, "This helix", "structure_element" ], [ 34, 48, "hydrogen bonds", "bond_interaction" ], [ 58, 61, "CTD", "structure_element" ], [ 77, 88, "salt bridge", "bond_interaction" ], [ 90, 96, "Arg191", "residue_name_number" ], [ 97, 103, "Asp255", "residue_name_number" ], [ 130, 133, "CTD", "structure_element" ], [ 154, 163, "main core", "structure_element" ] ] }, { "sid": 45, "sent": "PmC11 is, as expected, most structurally similar to other members of clan CD with the top hits in a search of known structures being caspase-7, gingipain-K, and legumain (PBD codes 4hq0, 4tkx, and 4aw9, respectively) (Table 2).", "section": "RESULTS", "ner": [ [ 0, 5, "PmC11", "protein" ], [ 69, 76, "clan CD", "protein_type" ], [ 116, 126, "structures", "evidence" ], [ 133, 142, "caspase-7", "protein" ], [ 144, 155, "gingipain-K", "protein" ], [ 161, 169, "legumain", "protein" ] ] }, { "sid": 46, "sent": "The C-terminal domain is unique to PmC11 within clan CD and structure comparisons for this domain alone does not produce any hits in the PDB (DaliLite, PDBeFold), suggesting a completely novel fold.", "section": "RESULTS", "ner": [ [ 4, 21, "C-terminal domain", "structure_element" ], [ 35, 40, "PmC11", "protein" ], [ 48, 55, "clan CD", "protein_type" ], [ 60, 81, "structure comparisons", "experimental_method" ], [ 86, 103, "this domain alone", "structure_element" ], [ 142, 150, "DaliLite", "experimental_method" ], [ 152, 160, "PDBeFold", "experimental_method" ] ] }, { "sid": 47, "sent": "As the archetypal and arguably most well studied member of clan CD, the caspases were used as the basis to investigate the structure/function relationships in PmC11, with caspase-7 as the representative member.", "section": "RESULTS", "ner": [ [ 59, 66, "clan CD", "protein_type" ], [ 72, 80, "caspases", "protein_type" ], [ 159, 164, "PmC11", "protein" ], [ 171, 180, "caspase-7", "protein" ] ] }, { "sid": 48, "sent": "Six of the central \u03b2-strands in PmC11 (\u03b21\u2013\u03b22 and \u03b25\u2013\u03b28) share the same topology as the six-stranded \u03b2-sheet found in caspases, with strands \u03b23, \u03b24, and \u03b29 located on the outside of this core structure (Fig. 1B, box).", "section": "RESULTS", "ner": [ [ 19, 28, "\u03b2-strands", "structure_element" ], [ 32, 37, "PmC11", "protein" ], [ 39, 44, "\u03b21\u2013\u03b22", "structure_element" ], [ 49, 54, "\u03b25\u2013\u03b28", "structure_element" ], [ 87, 107, "six-stranded \u03b2-sheet", "structure_element" ], [ 117, 125, "caspases", "protein_type" ], [ 132, 139, "strands", "structure_element" ], [ 140, 142, "\u03b23", "structure_element" ], [ 144, 146, "\u03b24", "structure_element" ], [ 152, 154, "\u03b29", "structure_element" ], [ 186, 200, "core structure", "structure_element" ] ] }, { "sid": 49, "sent": "His133 and Cys179 were found at locations structurally homologous to the caspase catalytic dyad, and other clan CD structures, at the C termini of strands \u03b25 and \u03b26, respectively (Figs. 1, A and B, and 2A).", "section": "RESULTS", "ner": [ [ 0, 6, "His133", "residue_name_number" ], [ 11, 17, "Cys179", "residue_name_number" ], [ 73, 80, "caspase", "protein_type" ], [ 81, 95, "catalytic dyad", "site" ], [ 107, 114, "clan CD", "protein_type" ], [ 115, 125, "structures", "evidence" ], [ 147, 154, "strands", "structure_element" ], [ 155, 157, "\u03b25", "structure_element" ], [ 162, 164, "\u03b26", "structure_element" ] ] }, { "sid": 50, "sent": "A multiple sequence alignment of C11 proteins revealed that these residues are highly conserved (data not shown).", "section": "RESULTS", "ner": [ [ 2, 29, "multiple sequence alignment", "experimental_method" ], [ 33, 36, "C11", "protein_type" ], [ 79, 95, "highly conserved", "protein_state" ] ] }, { "sid": 51, "sent": "Summary of PDBeFOLD superposition of structures found to be most similar to PmC11 in the PBD based on DaliLite", "section": "TABLE", "ner": [ [ 11, 33, "PDBeFOLD superposition", "experimental_method" ], [ 76, 81, "PmC11", "protein" ], [ 102, 110, "DaliLite", "experimental_method" ] ] }, { "sid": 52, "sent": "Biochemical and structural characterization of PmC11.", "section": "FIG", "ner": [ [ 0, 43, "Biochemical and structural characterization", "experimental_method" ], [ 47, 52, "PmC11", "protein" ] ] }, { "sid": 53, "sent": " A, ribbon representation of the overall structure of PmC11 illustrating the catalytic site, cleavage site displacement, and potential S1 binding site.", "section": "FIG", "ner": [ [ 54, 59, "PmC11", "protein" ], [ 77, 91, "catalytic site", "site" ], [ 135, 150, "S1 binding site", "site" ] ] }, { "sid": 54, "sent": "The overall structure of PmC11 is shown in gray, looking down into the catalytic site with the catalytic dyad in red.", "section": "FIG", "ner": [ [ 12, 21, "structure", "evidence" ], [ 25, 30, "PmC11", "protein" ], [ 71, 85, "catalytic site", "site" ], [ 95, 109, "catalytic dyad", "site" ] ] }, { "sid": 55, "sent": "The two ends of the autolytic cleavage site (Lys147 and Ala148, green) are displaced by 19.5 \u212b (thin black line) from one another and residues in the potential substrate binding pocket are highlighted in blue.", "section": "FIG", "ner": [ [ 20, 43, "autolytic cleavage site", "site" ], [ 45, 51, "Lys147", "residue_name_number" ], [ 45, 51, "Lys147", "residue_name_number" ], [ 56, 62, "Ala148", "residue_name_number" ], [ 160, 184, "substrate binding pocket", "site" ] ] }, { "sid": 56, "sent": "B, size exclusion chromatography of PmC11.", "section": "FIG", "ner": [ [ 3, 32, "size exclusion chromatography", "experimental_method" ], [ 36, 41, "PmC11", "protein" ] ] }, { "sid": 57, "sent": "PmC11 migrates as a monomer with a molecular mass around 41 kDa calculated from protein standards of known molecular weights.", "section": "FIG", "ner": [ [ 20, 27, "monomer", "oligomeric_state" ] ] }, { "sid": 58, "sent": "Elution fractions across the major peak (1\u20136) were analyzed by SDS-PAGE on a 4\u201312% gel in MES buffer.", "section": "FIG", "ner": [ [ 63, 71, "SDS-PAGE", "experimental_method" ] ] }, { "sid": 59, "sent": "C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro.", "section": "FIG", "ner": [ [ 7, 13, "active", "protein_state" ], [ 22, 27, "PmC11", "protein" ], [ 45, 55, "PmC11C179A", "mutant" ], [ 64, 74, "PmC11K147A", "mutant" ], [ 97, 105, "SDS-PAGE", "experimental_method" ], [ 119, 131, "Western blot", "experimental_method" ], [ 187, 192, "PmC11", "protein" ], [ 193, 206, "autoprocesses", "ptm" ], [ 225, 235, "PmC11C179A", "mutant" ], [ 240, 250, "PmC11K147A", "mutant" ], [ 264, 278, "autoprocessing", "ptm" ] ] }, { "sid": 60, "sent": "D, cysteine peptidase activity of PmC11.", "section": "FIG", "ner": [ [ 34, 39, "PmC11", "protein" ] ] }, { "sid": 61, "sent": "Km and Vmax of PmC11 and K147A mutant were determined by monitoring change in the fluorescence corresponding to AMC release from Bz-R-AMC.", "section": "FIG", "ner": [ [ 7, 11, "Vmax", "evidence" ], [ 15, 20, "PmC11", "protein" ], [ 25, 30, "K147A", "mutant" ], [ 129, 137, "Bz-R-AMC", "chemical" ] ] }, { "sid": 62, "sent": "E, intermolecular processing of PmC11C179A by PmC11.", "section": "FIG", "ner": [ [ 3, 28, "intermolecular processing", "ptm" ], [ 32, 42, "PmC11C179A", "mutant" ], [ 46, 51, "PmC11", "protein" ] ] }, { "sid": 63, "sent": "PmC11C179A (20 \u03bcg) was incubated overnight at 37 \u00b0C with increasing amounts of processed PmC11 and analyzed on a 10% SDS-PAGE gel.", "section": "FIG", "ner": [ [ 89, 94, "PmC11", "protein" ], [ 117, 125, "SDS-PAGE", "experimental_method" ] ] }, { "sid": 64, "sent": "Inactive PmC11C179A was not processed to a major extent by active PmC11 until around a ratio of 1:4 (5 \u03bcg of active PmC11).", "section": "FIG", "ner": [ [ 9, 19, "PmC11C179A", "mutant" ], [ 59, 65, "active", "protein_state" ], [ 66, 71, "PmC11", "protein" ], [ 109, 115, "active", "protein_state" ], [ 116, 121, "PmC11", "protein" ] ] }, { "sid": 65, "sent": "A single lane of 20 \u03bcg of active PmC11 (labeled 20) is shown for comparison.", "section": "FIG", "ner": [ [ 26, 32, "active", "protein_state" ], [ 33, 38, "PmC11", "protein" ] ] }, { "sid": 66, "sent": "F, activity of PmC11 against basic substrates.", "section": "FIG", "ner": [ [ 3, 11, "activity", "evidence" ], [ 15, 20, "PmC11", "protein" ] ] }, { "sid": 67, "sent": "G, electrostatic surface potential of PmC11 shown in a similar orientation, where blue and red denote positively and negatively charged surface potential, respectively, contoured at \u00b15 kT/e.", "section": "FIG", "ner": [ [ 38, 43, "PmC11", "protein" ] ] }, { "sid": 68, "sent": "The position of the catalytic dyad, one potential key substrate binding residue Asp177, and the ends of the cleavage site Lys147 and Ala148 are indicated.", "section": "FIG", "ner": [ [ 20, 34, "catalytic dyad", "site" ], [ 50, 79, "key substrate binding residue", "site" ], [ 80, 86, "Asp177", "residue_name_number" ], [ 108, 121, "cleavage site", "site" ], [ 122, 128, "Lys147", "residue_name_number" ], [ 133, 139, "Ala148", "residue_name_number" ] ] }, { "sid": 69, "sent": "Five of the \u03b1-helices surrounding the \u03b2-sheet of PmC11 (\u03b11, \u03b12, \u03b14, \u03b16, and \u03b17) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80.", "section": "RESULTS", "ner": [ [ 12, 21, "\u03b1-helices", "structure_element" ], [ 38, 45, "\u03b2-sheet", "structure_element" ], [ 49, 54, "PmC11", "protein" ], [ 56, 58, "\u03b11", "structure_element" ], [ 60, 62, "\u03b12", "structure_element" ], [ 64, 66, "\u03b14", "structure_element" ], [ 68, 70, "\u03b16", "structure_element" ], [ 76, 78, "\u03b17", "structure_element" ], [ 123, 145, "structurally conserved", "protein_state" ], [ 146, 153, "helices", "structure_element" ], [ 157, 165, "caspases", "protein_type" ], [ 187, 194, "clan CD", "protein_type" ], [ 214, 217, "C80", "protein_type" ] ] }, { "sid": 70, "sent": "Other than its more extended \u03b2-sheet, PmC11 differs most significantly from other clan CD members at its C terminus, where the CTD contains a further seven \u03b1-helices and four \u03b2-strands after \u03b28.", "section": "RESULTS", "ner": [ [ 20, 36, "extended \u03b2-sheet", "structure_element" ], [ 38, 43, "PmC11", "protein" ], [ 82, 89, "clan CD", "protein_type" ], [ 127, 130, "CTD", "structure_element" ], [ 156, 165, "\u03b1-helices", "structure_element" ], [ 175, 184, "\u03b2-strands", "structure_element" ], [ 191, 193, "\u03b28", "structure_element" ] ] }, { "sid": 71, "sent": "Autoprocessing of PmC11", "section": "RESULTS", "ner": [ [ 0, 14, "Autoprocessing", "ptm" ], [ 18, 23, "PmC11", "protein" ] ] }, { "sid": 72, "sent": "Purification of recombinant PmC11 (molecular mass = 42.6 kDa) revealed partial processing into two cleavage products of 26.4 and 16.2 kDa, related to the observed cleavage at Lys147 in the crystal structure (Fig. 2A).", "section": "RESULTS", "ner": [ [ 0, 12, "Purification", "experimental_method" ], [ 28, 33, "PmC11", "protein" ], [ 163, 171, "cleavage", "ptm" ], [ 175, 181, "Lys147", "residue_name_number" ], [ 189, 206, "crystal structure", "evidence" ] ] }, { "sid": 73, "sent": "Incubation of PmC11 at 37 \u00b0C for 16 h, resulted in a fully processed enzyme that remained as an intact monomer when applied to a size-exclusion column (Fig. 2B).", "section": "RESULTS", "ner": [ [ 0, 10, "Incubation", "experimental_method" ], [ 14, 19, "PmC11", "protein" ], [ 53, 68, "fully processed", "protein_state" ], [ 96, 102, "intact", "protein_state" ], [ 103, 110, "monomer", "oligomeric_state" ] ] }, { "sid": 74, "sent": "The single cleavage site of PmC11 at Lys147 is found immediately after \u03b13, in loop L5 within the central \u03b2-sheet (Figs. 1, A and B, and 2A).", "section": "RESULTS", "ner": [ [ 11, 24, "cleavage site", "site" ], [ 28, 33, "PmC11", "protein" ], [ 37, 43, "Lys147", "residue_name_number" ], [ 71, 73, "\u03b13", "structure_element" ], [ 78, 82, "loop", "structure_element" ], [ 83, 85, "L5", "structure_element" ], [ 105, 112, "\u03b2-sheet", "structure_element" ] ] }, { "sid": 75, "sent": "The two ends of the cleavage site are remarkably well ordered in the crystal structure and displaced from one another by 19.5 \u212b (Fig. 2A).", "section": "RESULTS", "ner": [ [ 20, 33, "cleavage site", "site" ], [ 69, 86, "crystal structure", "evidence" ] ] }, { "sid": 76, "sent": "Moreover, the C-terminal side of the cleavage site resides near the catalytic dyad with Ala148 being 4.5 and 5.7 \u212b from His133 and Cys179, respectively.", "section": "RESULTS", "ner": [ [ 37, 50, "cleavage site", "site" ], [ 68, 82, "catalytic dyad", "site" ], [ 88, 94, "Ala148", "residue_name_number" ], [ 120, 126, "His133", "residue_name_number" ], [ 131, 137, "Cys179", "residue_name_number" ] ] }, { "sid": 77, "sent": "Consequently, it appears feasible that the helix attached to Lys147 (\u03b13) could be responsible for steric autoinhibition of PmC11 when Lys147 is covalently bonded to Ala148.", "section": "RESULTS", "ner": [ [ 43, 48, "helix", "structure_element" ], [ 61, 67, "Lys147", "residue_name_number" ], [ 69, 71, "\u03b13", "structure_element" ], [ 123, 128, "PmC11", "protein" ], [ 134, 140, "Lys147", "residue_name_number" ], [ 165, 171, "Ala148", "residue_name_number" ] ] }, { "sid": 78, "sent": "Thus, the cleavage would be required for full activation of PmC11.", "section": "RESULTS", "ner": [ [ 10, 18, "cleavage", "ptm" ], [ 41, 56, "full activation", "protein_state" ], [ 60, 65, "PmC11", "protein" ] ] }, { "sid": 79, "sent": "To investigate this possibility, two mutant forms of the enzyme were created: PmC11C179A (a catalytically inactive mutant) and PmC11K147A (a cleavage-site mutant).", "section": "RESULTS", "ner": [ [ 78, 88, "PmC11C179A", "mutant" ], [ 92, 121, "catalytically inactive mutant", "protein_state" ], [ 127, 137, "PmC11K147A", "mutant" ], [ 141, 161, "cleavage-site mutant", "protein_state" ] ] }, { "sid": 80, "sent": "Initial SDS-PAGE and Western blot analysis of both mutants revealed no discernible processing occurred as compared with active PmC11 (Fig. 2C).", "section": "RESULTS", "ner": [ [ 8, 16, "SDS-PAGE", "experimental_method" ], [ 21, 33, "Western blot", "experimental_method" ], [ 120, 126, "active", "protein_state" ], [ 127, 132, "PmC11", "protein" ] ] }, { "sid": 81, "sent": "The PmC11K147A mutant enzyme had a markedly different reaction rate (Vmax) compared with WT, where the reaction velocity of PmC11 was 10 times greater than that of PmC11K147A (Fig. 2D).", "section": "RESULTS", "ner": [ [ 4, 14, "PmC11K147A", "mutant" ], [ 15, 21, "mutant", "protein_state" ], [ 54, 67, "reaction rate", "evidence" ], [ 69, 73, "Vmax", "evidence" ], [ 89, 91, "WT", "protein_state" ], [ 103, 120, "reaction velocity", "evidence" ], [ 124, 129, "PmC11", "protein" ], [ 164, 174, "PmC11K147A", "mutant" ] ] }, { "sid": 82, "sent": "Taken together, these data reveal that PmC11 requires processing at Lys147 for optimum activity.", "section": "RESULTS", "ner": [ [ 39, 44, "PmC11", "protein" ], [ 68, 74, "Lys147", "residue_name_number" ] ] }, { "sid": 83, "sent": "To investigate whether processing is a result of intra- or intermolecular cleavage, the PmC11C179A mutant was incubated with increasing concentrations of processed and activated PmC11.", "section": "RESULTS", "ner": [ [ 88, 98, "PmC11C179A", "mutant" ], [ 99, 105, "mutant", "protein_state" ], [ 110, 150, "incubated with increasing concentrations", "experimental_method" ], [ 154, 163, "processed", "protein_state" ], [ 168, 177, "activated", "protein_state" ], [ 178, 183, "PmC11", "protein" ] ] }, { "sid": 84, "sent": "These studies revealed that there was no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11:PmC11C179A) was increased to \u223c1:10 and 1:4, with complete cleavage observed at a ratio of 1:1 (Fig. 2E).", "section": "RESULTS", "ner": [ [ 62, 72, "PmC11C179A", "mutant" ], [ 80, 86, "active", "protein_state" ], [ 94, 115, "at low concentrations", "experimental_method" ], [ 119, 124, "PmC11", "protein" ], [ 187, 193, "active", "protein_state" ], [ 202, 207, "PmC11", "protein" ], [ 208, 218, "PmC11C179A", "mutant" ], [ 224, 250, "increased to \u223c1:10 and 1:4", "experimental_method" ], [ 289, 301, "ratio of 1:1", "experimental_method" ] ] }, { "sid": 85, "sent": "This suggests that cleavage of PmC11C179A was most likely an effect of the increasing concentration of PmC11 and intermolecular cleavage.", "section": "RESULTS", "ner": [ [ 19, 27, "cleavage", "ptm" ], [ 31, 41, "PmC11C179A", "mutant" ], [ 103, 108, "PmC11", "protein" ] ] }, { "sid": 86, "sent": "Collectively, these data suggest that the pro-form of PmC11 is autoinhibited by a section of L5 blocking access to the active site, prior to intramolecular cleavage at Lys147.", "section": "RESULTS", "ner": [ [ 42, 50, "pro-form", "protein_state" ], [ 54, 59, "PmC11", "protein" ], [ 63, 76, "autoinhibited", "protein_state" ], [ 93, 95, "L5", "structure_element" ], [ 119, 130, "active site", "site" ], [ 141, 164, "intramolecular cleavage", "ptm" ], [ 168, 174, "Lys147", "residue_name_number" ] ] }, { "sid": 87, "sent": "This cleavage subsequently allows movement of the region containing Lys147 and the active site to open up for substrate access.", "section": "RESULTS", "ner": [ [ 5, 13, "cleavage", "ptm" ], [ 68, 74, "Lys147", "residue_name_number" ], [ 83, 94, "active site", "site" ], [ 98, 102, "open", "protein_state" ] ] }, { "sid": 88, "sent": "Substrate Specificity of PmC11", "section": "RESULTS", "ner": [ [ 25, 30, "PmC11", "protein" ] ] }, { "sid": 89, "sent": "The autocatalytic cleavage of PmC11 at Lys147 (sequence KLK\u2227A) demonstrates that the enzyme accepts substrates with Lys in the P1 position.", "section": "RESULTS", "ner": [ [ 4, 26, "autocatalytic cleavage", "ptm" ], [ 30, 35, "PmC11", "protein" ], [ 39, 45, "Lys147", "residue_name_number" ], [ 116, 119, "Lys", "residue_name" ], [ 127, 129, "P1", "residue_number" ] ] }, { "sid": 90, "sent": "As expected, PmC11 showed no activity against substrates with Pro or Asp in P1 but was active toward substrates with a basic residue in P1 such as Bz-R-AMC, Z-GGR-AMC, and BOC-VLK-AMC.", "section": "RESULTS", "ner": [ [ 13, 18, "PmC11", "protein" ], [ 62, 65, "Pro", "residue_name" ], [ 69, 72, "Asp", "residue_name" ], [ 76, 78, "P1", "residue_number" ], [ 87, 93, "active", "protein_state" ], [ 136, 138, "P1", "residue_number" ], [ 147, 155, "Bz-R-AMC", "chemical" ], [ 157, 166, "Z-GGR-AMC", "chemical" ], [ 172, 183, "BOC-VLK-AMC", "chemical" ] ] }, { "sid": 91, "sent": "The rate of cleavage was \u223c3-fold greater toward the single Arg substrate Bz-R-AMC than for the other two (Fig. 2F) and, unexpectedly, PmC11 showed no activity toward BOC-K-AMC.", "section": "RESULTS", "ner": [ [ 59, 62, "Arg", "residue_name" ], [ 73, 81, "Bz-R-AMC", "chemical" ], [ 134, 139, "PmC11", "protein" ], [ 166, 175, "BOC-K-AMC", "chemical" ] ] }, { "sid": 92, "sent": "These results confirm that PmC11 accepts substrates containing Arg or Lys in P1 with a possible preference for Arg.", "section": "RESULTS", "ner": [ [ 27, 32, "PmC11", "protein" ], [ 63, 66, "Arg", "residue_name" ], [ 70, 73, "Lys", "residue_name" ], [ 77, 79, "P1", "residue_number" ], [ 111, 114, "Arg", "residue_name" ] ] }, { "sid": 93, "sent": "The catalytic dyad of PmC11 sits near the bottom of an open pocket on the surface of the enzyme at a conserved location in the clan CD family.", "section": "RESULTS", "ner": [ [ 4, 18, "catalytic dyad", "site" ], [ 22, 27, "PmC11", "protein" ], [ 55, 59, "open", "protein_state" ], [ 60, 66, "pocket", "site" ], [ 101, 119, "conserved location", "protein_state" ], [ 132, 141, "CD family", "protein_type" ] ] }, { "sid": 94, "sent": "The PmC11 structure reveals that the catalytic dyad forms part of a large acidic pocket (Fig. 2G), consistent with a binding site for a basic substrate.", "section": "RESULTS", "ner": [ [ 4, 9, "PmC11", "protein" ], [ 10, 19, "structure", "evidence" ], [ 37, 51, "catalytic dyad", "site" ], [ 74, 87, "acidic pocket", "site" ], [ 117, 129, "binding site", "site" ] ] }, { "sid": 95, "sent": "This pocket is lined with the potential functional side chains of Asn50, Asp177, and Thr204 with Gly134, Asp207, and Met205 also contributing to the pocket (Fig. 2A).", "section": "RESULTS", "ner": [ [ 5, 11, "pocket", "site" ], [ 66, 71, "Asn50", "residue_name_number" ], [ 73, 79, "Asp177", "residue_name_number" ], [ 85, 91, "Thr204", "residue_name_number" ], [ 97, 103, "Gly134", "residue_name_number" ], [ 105, 111, "Asp207", "residue_name_number" ], [ 117, 123, "Met205", "residue_name_number" ], [ 149, 155, "pocket", "site" ] ] }, { "sid": 96, "sent": "Interestingly, these residues are in regions that are structurally similar to those involved in the S1 binding pockets of other clan CD members (shown in Ref.).", "section": "RESULTS", "ner": [ [ 54, 74, "structurally similar", "protein_state" ], [ 100, 118, "S1 binding pockets", "site" ], [ 128, 143, "clan CD members", "protein_type" ] ] }, { "sid": 97, "sent": "Because PmC11 recognizes basic substrates, the tetrapeptide inhibitor Z-VRPR-FMK was tested as an enzyme inhibitor and was found to inhibit both the autoprocessing and activity of PmC11 (Fig. 3A).", "section": "RESULTS", "ner": [ [ 8, 13, "PmC11", "protein" ], [ 70, 80, "Z-VRPR-FMK", "chemical" ], [ 132, 139, "inhibit", "protein_state" ], [ 149, 163, "autoprocessing", "ptm" ], [ 180, 185, "PmC11", "protein" ] ] }, { "sid": 98, "sent": "Z-VRPR-FMK was also shown to bind to the enzyme: a size-shift was observed, by SDS-PAGE analysis, in the larger processed product of PmC11 suggesting that the inhibitor bound to the active site (Fig. 3B).", "section": "RESULTS", "ner": [ [ 0, 10, "Z-VRPR-FMK", "chemical" ], [ 51, 61, "size-shift", "evidence" ], [ 79, 87, "SDS-PAGE", "experimental_method" ], [ 133, 138, "PmC11", "protein" ], [ 159, 174, "inhibitor bound", "protein_state" ], [ 182, 193, "active site", "site" ] ] }, { "sid": 99, "sent": "A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.).", "section": "RESULTS", "ner": [ [ 2, 19, "structure overlay", "experimental_method" ], [ 23, 28, "PmC11", "protein" ], [ 38, 57, "MALT1-paracacaspase", "protein" ], [ 59, 66, "MALT1-P", "protein" ], [ 72, 79, "complex", "protein_state" ], [ 85, 95, "Z-VRPR-FMK", "chemical" ], [ 115, 120, "PmC11", "protein" ], [ 121, 125, "dyad", "site" ], [ 169, 175, "active", "protein_state" ], [ 176, 183, "MALT1-P", "protein" ], [ 193, 198, "Asn50", "residue_name_number" ], [ 200, 206, "Asp177", "residue_name_number" ], [ 212, 218, "Asp207", "residue_name_number" ], [ 255, 262, "MALT1-P", "protein" ], [ 263, 289, "inhibitor binding residues", "site" ], [ 291, 297, "Asp365", "residue_name_number" ], [ 299, 305, "Asp462", "residue_name_number" ], [ 311, 317, "Glu500", "residue_name_number" ], [ 333, 341, "VRPR-FMK", "chemical" ], [ 347, 354, "MALT1-P", "protein" ], [ 378, 383, "PmC11", "protein" ], [ 402, 420, "structural overlay", "experimental_method" ] ] }, { "sid": 100, "sent": "Asp177 is located near the catalytic cysteine and is conserved throughout the C11 family, suggesting it is the primary S1 binding site residue.", "section": "RESULTS", "ner": [ [ 0, 6, "Asp177", "residue_name_number" ], [ 27, 36, "catalytic", "protein_state" ], [ 37, 45, "cysteine", "residue_name" ], [ 53, 73, "conserved throughout", "protein_state" ], [ 78, 88, "C11 family", "protein_type" ], [ 119, 142, "S1 binding site residue", "site" ] ] }, { "sid": 101, "sent": "In the structure of PmC11, Asp207 resides on a flexible loop pointing away from the S1 binding pocket (Fig. 3C).", "section": "RESULTS", "ner": [ [ 7, 16, "structure", "evidence" ], [ 20, 25, "PmC11", "protein" ], [ 27, 33, "Asp207", "residue_name_number" ], [ 56, 60, "loop", "structure_element" ], [ 84, 101, "S1 binding pocket", "site" ] ] }, { "sid": 102, "sent": "However, this loop has been shown to be important for substrate binding in clan CD and this residue could easily rotate and be involved in substrate binding in PmC11.", "section": "RESULTS", "ner": [ [ 14, 18, "loop", "structure_element" ], [ 75, 82, "clan CD", "protein_type" ], [ 160, 165, "PmC11", "protein" ] ] }, { "sid": 103, "sent": "Thus, Asn50, Asp177, and Asp207 are most likely responsible for the substrate specificity of PmC11.", "section": "RESULTS", "ner": [ [ 6, 11, "Asn50", "residue_name_number" ], [ 13, 19, "Asp177", "residue_name_number" ], [ 25, 31, "Asp207", "residue_name_number" ], [ 93, 98, "PmC11", "protein" ] ] }, { "sid": 104, "sent": "Asp177 is highly conserved throughout the clan CD C11 peptidases and is thought to be primarily responsible for substrate specificity of the clan CD enzymes, as also illustrated from the proximity of these residues relative to the inhibitor Z-VRPR-FMK when PmC11 is overlaid on the MALT1-P structure (Fig. 3C).", "section": "RESULTS", "ner": [ [ 0, 6, "Asp177", "residue_name_number" ], [ 10, 26, "highly conserved", "protein_state" ], [ 42, 64, "clan CD C11 peptidases", "protein_type" ], [ 141, 156, "clan CD enzymes", "protein_type" ], [ 241, 251, "Z-VRPR-FMK", "chemical" ], [ 257, 262, "PmC11", "protein" ], [ 266, 274, "overlaid", "experimental_method" ], [ 282, 289, "MALT1-P", "protein" ], [ 290, 299, "structure", "evidence" ] ] }, { "sid": 105, "sent": "PmC11 binds and is inhibited by Z-VRPR-FMK and does not require Ca2+ for activity.", "section": "FIG", "ner": [ [ 0, 5, "PmC11", "protein" ], [ 32, 42, "Z-VRPR-FMK", "chemical" ], [ 64, 68, "Ca2+", "chemical" ] ] }, { "sid": 106, "sent": " A, PmC11 activity is inhibited by Z-VRPR-FMK.", "section": "FIG", "ner": [ [ 35, 45, "Z-VRPR-FMK", "chemical" ] ] }, { "sid": 107, "sent": "Cleavage of Bz-R-AMC by PmC11 was measured in a fluorometric activity assay with (+, purple) and without (\u2212, red) Z-VRPR-FMK.", "section": "FIG", "ner": [ [ 12, 20, "Bz-R-AMC", "chemical" ], [ 24, 29, "PmC11", "protein" ], [ 48, 75, "fluorometric activity assay", "experimental_method" ], [ 114, 124, "Z-VRPR-FMK", "chemical" ] ] }, { "sid": 108, "sent": "B, gel-shift assay reveals that Z-VRPR-FMK binds to PmC11.", "section": "FIG", "ner": [ [ 3, 18, "gel-shift assay", "experimental_method" ], [ 32, 42, "Z-VRPR-FMK", "chemical" ], [ 52, 57, "PmC11", "protein" ] ] }, { "sid": 109, "sent": "PmC11 was incubated with (+) or without (\u2212) Z-VRPR-FMK and the samples analyzed on a 10% SDS-PAGE gel.", "section": "FIG", "ner": [ [ 10, 19, "incubated", "experimental_method" ], [ 44, 54, "Z-VRPR-FMK", "chemical" ], [ 89, 97, "SDS-PAGE", "experimental_method" ] ] }, { "sid": 110, "sent": "A size shift can be observed in the larger processed product of PmC11 (26.1 kDa).", "section": "FIG", "ner": [ [ 2, 12, "size shift", "evidence" ], [ 64, 69, "PmC11", "protein" ] ] }, { "sid": 111, "sent": "C, PmC11 with the Z-VRPR-FMK from the MALT1-paracacaspase (MALT1-P) superimposed.", "section": "FIG", "ner": [ [ 3, 8, "PmC11", "protein" ], [ 18, 28, "Z-VRPR-FMK", "chemical" ], [ 38, 57, "MALT1-paracacaspase", "protein" ], [ 59, 66, "MALT1-P", "protein" ], [ 68, 80, "superimposed", "experimental_method" ] ] }, { "sid": 112, "sent": "A three-dimensional structural overlay of Z-VRPR-FMK from the MALT1-P complex onto PmC11.", "section": "FIG", "ner": [ [ 2, 38, "three-dimensional structural overlay", "experimental_method" ], [ 42, 52, "Z-VRPR-FMK", "chemical" ], [ 62, 69, "MALT1-P", "protein" ], [ 83, 88, "PmC11", "protein" ] ] }, { "sid": 113, "sent": "The position and orientation of Z-VRPR-FMK was taken from superposition of the PmC11 and MALTI_P structures and indicates the presumed active site of PmC11.", "section": "FIG", "ner": [ [ 32, 42, "Z-VRPR-FMK", "chemical" ], [ 58, 71, "superposition", "experimental_method" ], [ 79, 84, "PmC11", "protein" ], [ 89, 96, "MALTI_P", "protein" ], [ 97, 107, "structures", "evidence" ], [ 135, 146, "active site", "site" ], [ 150, 155, "PmC11", "protein" ] ] }, { "sid": 114, "sent": "Residues surrounding the inhibitor are labeled and represent potentially important binding site residues, labeled in black and shown in an atomic representation.", "section": "FIG", "ner": [ [ 83, 104, "binding site residues", "site" ] ] }, { "sid": 115, "sent": "C, divalent cations do not increase the activity of PmC11.", "section": "FIG", "ner": [ [ 52, 57, "PmC11", "protein" ] ] }, { "sid": 116, "sent": "The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min).", "section": "FIG", "ner": [ [ 16, 24, "Bz-R-AMC", "chemical" ], [ 28, 33, "PmC11", "protein" ], [ 78, 82, "Ca2+", "chemical" ], [ 84, 88, "Mn2+", "chemical" ], [ 90, 94, "Zn2+", "chemical" ], [ 96, 100, "Co2+", "chemical" ], [ 102, 106, "Cu2+", "chemical" ], [ 108, 112, "Mg2+", "chemical" ], [ 118, 122, "Fe3+", "chemical" ], [ 128, 132, "EGTA", "chemical" ], [ 160, 203, "relative fluorescence measured against time", "experimental_method" ] ] }, { "sid": 117, "sent": "The addition of cations produced no improvement in activity of PmC11 when compared in the presence of EGTA, suggesting that PmC11 does not require metal ions for proteolytic activity.", "section": "FIG", "ner": [ [ 4, 23, "addition of cations", "experimental_method" ], [ 63, 68, "PmC11", "protein" ], [ 102, 106, "EGTA", "chemical" ], [ 124, 129, "PmC11", "protein" ] ] }, { "sid": 118, "sent": "Furthermore, Cu2+, Fe2+, and Zn2+ appear to inhibit PmC11.", "section": "FIG", "ner": [ [ 13, 17, "Cu2+", "chemical" ], [ 19, 23, "Fe2+", "chemical" ], [ 29, 33, "Zn2+", "chemical" ], [ 44, 51, "inhibit", "protein_state" ], [ 52, 57, "PmC11", "protein" ] ] }, { "sid": 119, "sent": "Comparison with Clostripain", "section": "RESULTS", "ner": [ [ 16, 27, "Clostripain", "protein" ] ] }, { "sid": 120, "sent": "Clostripain from C. histolyticum is the founding member of the C11 family of peptidases and contains an additional 149 residues compared with PmC11.", "section": "RESULTS", "ner": [ [ 0, 11, "Clostripain", "protein" ], [ 17, 32, "C. histolyticum", "species" ], [ 63, 73, "C11 family", "protein_type" ], [ 77, 87, "peptidases", "protein_type" ], [ 115, 127, "149 residues", "residue_range" ], [ 142, 147, "PmC11", "protein" ] ] }, { "sid": 121, "sent": "A multiple sequence alignment revealed that most of the secondary structural elements are conserved between the two enzymes, although they are only \u223c23% identical (Fig. 1A).", "section": "RESULTS", "ner": [ [ 2, 29, "multiple sequence alignment", "experimental_method" ], [ 56, 85, "secondary structural elements", "structure_element" ], [ 90, 99, "conserved", "protein_state" ] ] }, { "sid": 122, "sent": "Nevertheless, PmC11 may be a good model for the core structure of clostripain.", "section": "RESULTS", "ner": [ [ 14, 19, "PmC11", "protein" ], [ 66, 77, "clostripain", "protein" ] ] }, { "sid": 123, "sent": "The primary structural alignment also shows that the catalytic dyad in PmC11 is structurally conserved in clostripain (Fig. 1A).", "section": "RESULTS", "ner": [ [ 4, 32, "primary structural alignment", "experimental_method" ], [ 53, 67, "catalytic dyad", "site" ], [ 71, 76, "PmC11", "protein" ], [ 80, 102, "structurally conserved", "protein_state" ], [ 106, 117, "clostripain", "protein" ] ] }, { "sid": 124, "sent": "Unlike PmC11, clostripain has two cleavage sites (Arg181 and Arg190), which results in the removal of a nonapeptide, and is required for full activation of the enzyme (highlighted in Fig. 1A).", "section": "RESULTS", "ner": [ [ 7, 12, "PmC11", "protein" ], [ 14, 25, "clostripain", "protein" ], [ 34, 48, "cleavage sites", "site" ], [ 50, 56, "Arg181", "residue_name_number" ], [ 61, 67, "Arg190", "residue_name_number" ], [ 104, 115, "nonapeptide", "structure_element" ], [ 137, 152, "full activation", "protein_state" ] ] }, { "sid": 125, "sent": "Interestingly, Arg190 was found to align with Lys147 in PmC11.", "section": "RESULTS", "ner": [ [ 15, 21, "Arg190", "residue_name_number" ], [ 46, 52, "Lys147", "residue_name_number" ], [ 56, 61, "PmC11", "protein" ] ] }, { "sid": 126, "sent": "In addition, the predicted primary S1-binding residue in PmC11 Asp177 also overlays with the residue predicted to be the P1 specificity determining residue in clostripain (Asp229, Fig. 1A).", "section": "RESULTS", "ner": [ [ 35, 53, "S1-binding residue", "site" ], [ 57, 62, "PmC11", "protein" ], [ 63, 69, "Asp177", "residue_name_number" ], [ 75, 83, "overlays", "experimental_method" ], [ 121, 155, "P1 specificity determining residue", "site" ], [ 159, 170, "clostripain", "protein" ], [ 172, 178, "Asp229", "residue_name_number" ] ] }, { "sid": 127, "sent": "As studies on clostripain revealed addition of Ca2+ ions are required for full activation, the Ca2+ dependence of PmC11 was examined.", "section": "RESULTS", "ner": [ [ 14, 25, "clostripain", "protein" ], [ 47, 51, "Ca2+", "chemical" ], [ 74, 89, "full activation", "protein_state" ], [ 95, 99, "Ca2+", "chemical" ], [ 114, 119, "PmC11", "protein" ] ] }, { "sid": 128, "sent": "Surprisingly, Ca2+ did not enhance PmC11 activity and, furthermore, other divalent cations, Mg2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+, were not necessary for PmC11 activity (Fig. 3D).", "section": "RESULTS", "ner": [ [ 14, 18, "Ca2+", "chemical" ], [ 35, 40, "PmC11", "protein" ], [ 92, 96, "Mg2+", "chemical" ], [ 98, 102, "Mn2+", "chemical" ], [ 104, 108, "Co2+", "chemical" ], [ 110, 114, "Fe2+", "chemical" ], [ 116, 120, "Zn2+", "chemical" ], [ 126, 130, "Cu2+", "chemical" ], [ 155, 160, "PmC11", "protein" ] ] }, { "sid": 129, "sent": "In support of these findings, EGTA did not inhibit PmC11 suggesting that, unlike clostripain, PmC11 does not require Ca2+ or other divalent cations, for activity.", "section": "RESULTS", "ner": [ [ 30, 34, "EGTA", "chemical" ], [ 51, 56, "PmC11", "protein" ], [ 81, 92, "clostripain", "protein" ], [ 94, 99, "PmC11", "protein" ], [ 117, 121, "Ca2+", "chemical" ] ] }, { "sid": 130, "sent": "The crystal structure of PmC11 now provides three-dimensional information for a member of the clostripain C11 family of cysteine peptidases.", "section": "DISCUSS", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 25, 30, "PmC11", "protein" ], [ 94, 105, "clostripain", "protein" ], [ 106, 116, "C11 family", "protein_type" ], [ 120, 139, "cysteine peptidases", "protein_type" ] ] }, { "sid": 131, "sent": "The enzyme exhibits all of the key structural elements of clan CD members, but is unusual in that it has a nine-stranded central \u03b2-sheet with a novel C-terminal domain.", "section": "DISCUSS", "ner": [ [ 58, 73, "clan CD members", "protein_type" ], [ 129, 136, "\u03b2-sheet", "structure_element" ], [ 150, 167, "C-terminal domain", "structure_element" ] ] }, { "sid": 132, "sent": "The structural similarity of PmC11 with its nearest structural neighbors in the PDB is decidedly low, overlaying better with six-stranded caspase-7 than any of the other larger members of the clan (Table 2).", "section": "DISCUSS", "ner": [ [ 29, 34, "PmC11", "protein" ], [ 138, 147, "caspase-7", "protein" ] ] }, { "sid": 133, "sent": "The substrate specificity of PmC11 is Arg/Lys and the crystal structure revealed an acidic pocket for specific binding of such basic substrates.", "section": "DISCUSS", "ner": [ [ 29, 34, "PmC11", "protein" ], [ 38, 41, "Arg", "residue_name" ], [ 42, 45, "Lys", "residue_name" ], [ 54, 71, "crystal structure", "evidence" ], [ 84, 97, "acidic pocket", "site" ] ] }, { "sid": 134, "sent": "In addition, the structure suggested a mechanism of self-inhibition in both PmC11 and clostripain and an activation mechanism that requires autoprocessing.", "section": "DISCUSS", "ner": [ [ 17, 26, "structure", "evidence" ], [ 76, 81, "PmC11", "protein" ], [ 86, 97, "clostripain", "protein" ], [ 140, 154, "autoprocessing", "ptm" ] ] }, { "sid": 135, "sent": "PmC11 differs from clostripain in that is does not appear to require divalent cations for activation.", "section": "DISCUSS", "ner": [ [ 0, 5, "PmC11", "protein" ], [ 19, 30, "clostripain", "protein" ] ] }, { "sid": 136, "sent": "Several other members of clan CD require processing for full activation including legumain, gingipain-R, MARTX-CPD, and the effector caspases, e.g. caspase-7.", "section": "DISCUSS", "ner": [ [ 25, 32, "clan CD", "protein_type" ], [ 41, 51, "processing", "ptm" ], [ 56, 71, "full activation", "protein_state" ], [ 82, 90, "legumain", "protein" ], [ 92, 103, "gingipain-R", "protein" ], [ 105, 114, "MARTX-CPD", "protein" ], [ 124, 141, "effector caspases", "protein_type" ], [ 148, 157, "caspase-7", "protein" ] ] }, { "sid": 137, "sent": "To date, the effector caspases are the only group of enzymes that require cleavage of a loop within the central \u03b2-sheet.", "section": "DISCUSS", "ner": [ [ 13, 30, "effector caspases", "protein_type" ], [ 74, 82, "cleavage", "ptm" ], [ 88, 92, "loop", "structure_element" ], [ 112, 119, "\u03b2-sheet", "structure_element" ] ] }, { "sid": 138, "sent": "This is also the case in PmC11, although the cleavage loop is structurally different to that found in the caspases and follows the catalytic His (Fig. 1A), as opposed to the Cys in the caspases.", "section": "DISCUSS", "ner": [ [ 25, 30, "PmC11", "protein" ], [ 45, 53, "cleavage", "ptm" ], [ 54, 58, "loop", "structure_element" ], [ 106, 114, "caspases", "protein_type" ], [ 131, 140, "catalytic", "protein_state" ], [ 141, 144, "His", "residue_name" ], [ 174, 177, "Cys", "residue_name" ], [ 185, 193, "caspases", "protein_type" ] ] }, { "sid": 139, "sent": "All other clan CD members requiring cleavage for full activation do so at sites external to their central sheets.", "section": "DISCUSS", "ner": [ [ 10, 25, "clan CD members", "protein_type" ], [ 36, 44, "cleavage", "ptm" ], [ 49, 64, "full activation", "protein_state" ], [ 74, 79, "sites", "site" ], [ 106, 112, "sheets", "structure_element" ] ] }, { "sid": 140, "sent": "The caspases and gingipain-R both undergo intermolecular (trans) cleavage and legumain and MARTX-CPD are reported to perform intramolecular (cis) cleavage.", "section": "DISCUSS", "ner": [ [ 4, 12, "caspases", "protein_type" ], [ 17, 28, "gingipain-R", "protein" ], [ 42, 73, "intermolecular (trans) cleavage", "ptm" ], [ 78, 86, "legumain", "protein" ], [ 91, 100, "MARTX-CPD", "protein" ], [ 125, 154, "intramolecular (cis) cleavage", "ptm" ] ] }, { "sid": 141, "sent": "In addition, several members of clan CD exhibit self-inhibition, whereby regions of the enzyme block access to the active site.", "section": "DISCUSS", "ner": [ [ 32, 39, "clan CD", "protein_type" ], [ 73, 80, "regions", "structure_element" ], [ 115, 126, "active site", "site" ] ] }, { "sid": 142, "sent": "Like PmC11, these structures show preformed catalytic machinery and, for a substrate to gain access, movement and/or cleavage of the blocking region is required.", "section": "DISCUSS", "ner": [ [ 5, 10, "PmC11", "protein" ], [ 117, 125, "cleavage", "ptm" ], [ 133, 148, "blocking region", "structure_element" ] ] }, { "sid": 143, "sent": "The structure of PmC11 gives the first insight into this class of relatively unexplored family of proteins and should allow important catalytic and substrate binding residues to be identified in a variety of orthologues.", "section": "DISCUSS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 17, 22, "PmC11", "protein" ] ] }, { "sid": 144, "sent": "Indeed, insights gained from an analysis of the PmC11 structure revealed the identity of the Trypanosoma brucei PNT1 protein as a C11 cysteine peptidase with an essential role in organelle replication.", "section": "DISCUSS", "ner": [ [ 48, 53, "PmC11", "protein" ], [ 54, 63, "structure", "evidence" ], [ 93, 111, "Trypanosoma brucei", "species" ], [ 112, 116, "PNT1", "protein" ], [ 130, 152, "C11 cysteine peptidase", "protein_type" ] ] }, { "sid": 145, "sent": "The PmC11 structure should provide a good basis for structural modeling and, given the importance of other clan CD enzymes, this work should also advance the exploration of these peptidases and potentially identify new biologically important substrates.", "section": "DISCUSS", "ner": [ [ 4, 9, "PmC11", "protein" ], [ 10, 19, "structure", "evidence" ], [ 52, 71, "structural modeling", "experimental_method" ], [ 107, 122, "clan CD enzymes", "protein_type" ], [ 179, 189, "peptidases", "protein_type" ] ] } ] }, "PMC4872110": { "annotations": [ { "sid": 0, "sent": "Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans", "section": "TITLE", "ner": [ [ 0, 26, "Ribosome biogenesis factor", "protein_type" ], [ 27, 31, "Tsr3", "protein" ], [ 39, 69, "aminocarboxypropyl transferase", "protein_type" ], [ 86, 94, "18S rRNA", "chemical" ], [ 116, 121, "yeast", "taxonomy_domain" ], [ 126, 132, "humans", "species" ] ] }, { "sid": 1, "sent": "The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3\u03a8) located next to the P-site tRNA on the small subunit 18S rRNA.", "section": "ABSTRACT", "ner": [ [ 44, 54, "eukaryotic", "taxonomy_domain" ], [ 55, 59, "rRNA", "chemical" ], [ 67, 76, "conserved", "protein_state" ], [ 77, 90, "hypermodified", "protein_state" ], [ 91, 101, "nucleotide", "chemical" ], [ 102, 147, "N1-methyl-N3-aminocarboxypropyl-pseudouridine", "chemical" ], [ 149, 156, "m1acp3\u03a8", "chemical" ], [ 178, 184, "P-site", "site" ], [ 185, 189, "tRNA", "chemical" ], [ 211, 219, "18S rRNA", "chemical" ] ] }, { "sid": 2, "sent": "While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive.", "section": "ABSTRACT", "ner": [ [ 6, 26, "S-adenosylmethionine", "chemical" ], [ 63, 81, "aminocarboxypropyl", "chemical" ], [ 83, 86, "acp", "chemical" ], [ 143, 146, "acp", "chemical" ] ] }, { "sid": 3, "sent": "Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells.", "section": "ABSTRACT", "ner": [ [ 61, 65, "Tsr3", "protein" ], [ 95, 100, "yeast", "taxonomy_domain" ], [ 105, 110, "human", "species" ] ] }, { "sid": 4, "sent": "In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation.", "section": "ABSTRACT", "ner": [ [ 25, 29, "Tsr3", "protein" ], [ 30, 37, "mutants", "protein_state" ], [ 58, 61, "acp", "chemical" ], [ 110, 122, "20S pre-rRNA", "chemical" ] ] }, { "sid": 5, "sent": "The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode.", "section": "ABSTRACT", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 25, 33, "archaeal", "taxonomy_domain" ], [ 34, 38, "Tsr3", "protein" ], [ 77, 111, "SPOUT-class RNA-methyltransferases", "protein_type" ], [ 127, 143, "SAM binding mode", "site" ] ] }, { "sid": 6, "sent": "This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate.", "section": "ABSTRACT", "ner": [ [ 12, 28, "SAM binding mode", "site" ], [ 42, 46, "Tsr3", "protein" ], [ 61, 64, "acp", "chemical" ], [ 93, 96, "SAM", "chemical" ] ] }, { "sid": 7, "sent": "Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes.", "section": "ABSTRACT", "ner": [ [ 14, 18, "Tsr3", "protein" ], [ 57, 72, "acp transferase", "protein_type" ] ] }, { "sid": 8, "sent": "Eukaryotic ribosome biogenesis is highly complex and requires a large number of non-ribosomal proteins and small non-coding RNAs in addition to ribosomal RNAs (rRNAs) and proteins.", "section": "INTRO", "ner": [ [ 0, 10, "Eukaryotic", "taxonomy_domain" ], [ 107, 128, "small non-coding RNAs", "chemical" ], [ 144, 158, "ribosomal RNAs", "chemical" ], [ 160, 165, "rRNAs", "chemical" ] ] }, { "sid": 9, "sent": "During eukaryotic ribosome biogenesis several dozens of rRNA nucleotides become chemically modified.", "section": "INTRO", "ner": [ [ 7, 17, "eukaryotic", "taxonomy_domain" ], [ 56, 60, "rRNA", "chemical" ], [ 61, 72, "nucleotides", "chemical" ] ] }, { "sid": 10, "sent": "The most abundant rRNA modifications are methylations at the 2\u2032-OH ribose moieties and isomerizations of uridine residues to pseudouridine, catalyzed by small nucleolar ribonucleoprotein particles (snoRNPs).", "section": "INTRO", "ner": [ [ 18, 22, "rRNA", "chemical" ], [ 41, 53, "methylations", "ptm" ], [ 67, 73, "ribose", "chemical" ], [ 105, 112, "uridine", "chemical" ], [ 125, 138, "pseudouridine", "chemical" ], [ 153, 196, "small nucleolar ribonucleoprotein particles", "complex_assembly" ], [ 198, 205, "snoRNPs", "complex_assembly" ] ] }, { "sid": 11, "sent": "In addition, 18S and 25S (yeast)/ 28S (humans) rRNAs contain several base modifications catalyzed by site-specific and snoRNA-independent enzymes.", "section": "INTRO", "ner": [ [ 13, 16, "18S", "chemical" ], [ 21, 24, "25S", "chemical" ], [ 26, 31, "yeast", "taxonomy_domain" ], [ 34, 37, "28S", "chemical" ], [ 39, 45, "humans", "species" ], [ 47, 52, "rRNAs", "chemical" ], [ 119, 125, "snoRNA", "chemical" ] ] }, { "sid": 12, "sent": "In Saccharomyces cerevisiae 18S rRNA contains four base methylations, two acetylations and a single 3-amino-3-carboxypropyl (acp) modification, whereas six base methylations are present in the 25S rRNA.", "section": "INTRO", "ner": [ [ 3, 27, "Saccharomyces cerevisiae", "species" ], [ 28, 36, "18S rRNA", "chemical" ], [ 56, 68, "methylations", "ptm" ], [ 74, 86, "acetylations", "ptm" ], [ 100, 123, "3-amino-3-carboxypropyl", "chemical" ], [ 125, 128, "acp", "chemical" ], [ 161, 173, "methylations", "ptm" ], [ 193, 201, "25S rRNA", "chemical" ] ] }, { "sid": 13, "sent": "While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA.", "section": "INTRO", "ner": [ [ 9, 15, "humans", "species" ], [ 20, 28, "18S rRNA", "chemical" ], [ 52, 68, "highly conserved", "protein_state" ], [ 88, 93, "yeast", "taxonomy_domain" ], [ 126, 132, "ScRrp8", "protein" ], [ 133, 138, "HsNML", "protein" ], [ 140, 146, "ScRcm1", "protein" ], [ 147, 154, "HsNSUN5", "protein" ], [ 159, 165, "ScNop2", "protein" ], [ 166, 173, "HsNSUN1", "protein" ], [ 209, 214, "human", "species" ], [ 215, 223, "28S rRNA", "chemical" ] ] }, { "sid": 14, "sent": "Ribosomal RNA modifications have been suggested to optimize ribosome function, although in most cases this remains to be clearly established.", "section": "INTRO", "ner": [ [ 0, 13, "Ribosomal RNA", "chemical" ] ] }, { "sid": 15, "sent": "They might contribute to increased RNA stability by providing additional hydrogen bonds (pseudouridines), improved base stacking (pseudouridines and base methylations) or an increased resistance against hydrolysis (ribose methylations).", "section": "INTRO", "ner": [ [ 35, 38, "RNA", "chemical" ], [ 73, 87, "hydrogen bonds", "bond_interaction" ], [ 89, 103, "pseudouridines", "chemical" ], [ 115, 128, "base stacking", "bond_interaction" ], [ 130, 144, "pseudouridines", "chemical" ], [ 149, 166, "base methylations", "ptm" ], [ 215, 234, "ribose methylations", "ptm" ] ] }, { "sid": 16, "sent": "Most modified rRNA nucleotides cluster in the vicinity of the decoding or the peptidyl transferase center, suggesting an influence on ribosome functionality and stability.", "section": "INTRO", "ner": [ [ 14, 18, "rRNA", "chemical" ], [ 19, 30, "nucleotides", "chemical" ], [ 62, 70, "decoding", "site" ], [ 78, 105, "peptidyl transferase center", "site" ] ] }, { "sid": 17, "sent": "Defects of rRNA modification enzymes often lead to disturbed ribosome biogenesis or functionally impaired ribosomes, although the lack of individual rRNA modifications often has no or only a slight influence on the cell.", "section": "INTRO", "ner": [ [ 11, 15, "rRNA", "chemical" ], [ 149, 153, "rRNA", "chemical" ] ] }, { "sid": 18, "sent": "The chemically most complex modification is located in the loop capping helix 31 of 18S rRNA (Supplementary Figure S1B).", "section": "INTRO", "ner": [ [ 59, 80, "loop capping helix 31", "structure_element" ], [ 84, 92, "18S rRNA", "chemical" ] ] }, { "sid": 19, "sent": "There a uridine (U1191 in yeast) is modified to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3\u03a8, Figure 1A).", "section": "INTRO", "ner": [ [ 8, 15, "uridine", "residue_name" ], [ 17, 22, "U1191", "residue_name_number" ], [ 26, 31, "yeast", "taxonomy_domain" ], [ 48, 98, "1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine", "chemical" ], [ 100, 107, "m1acp3\u03a8", "chemical" ] ] }, { "sid": 20, "sent": "This base modification was first described in 1968 for hamster cells and is conserved in eukaryotes.", "section": "INTRO", "ner": [ [ 55, 62, "hamster", "taxonomy_domain" ], [ 76, 88, "conserved in", "protein_state" ], [ 89, 99, "eukaryotes", "taxonomy_domain" ] ] }, { "sid": 21, "sent": "This hypermodified nucleotide, which is located at the P-site tRNA, is synthesized in three steps beginning with the snR35 H/ACA snoRNP guided conversion of uridine into pseudouridine.", "section": "INTRO", "ner": [ [ 5, 18, "hypermodified", "protein_state" ], [ 19, 29, "nucleotide", "chemical" ], [ 55, 61, "P-site", "site" ], [ 62, 66, "tRNA", "chemical" ], [ 117, 122, "snR35", "chemical" ], [ 123, 128, "H/ACA", "structure_element" ], [ 129, 135, "snoRNP", "complex_assembly" ], [ 157, 164, "uridine", "chemical" ], [ 170, 183, "pseudouridine", "chemical" ] ] }, { "sid": 22, "sent": "In a second step, the essential SPOUT-class methyltransferase Nep1/Emg1 modifies the pseudouridine to N1-methylpseudouridine.", "section": "INTRO", "ner": [ [ 32, 61, "SPOUT-class methyltransferase", "protein_type" ], [ 62, 66, "Nep1", "protein" ], [ 67, 71, "Emg1", "protein" ], [ 85, 98, "pseudouridine", "chemical" ], [ 102, 124, "N1-methylpseudouridine", "chemical" ] ] }, { "sid": 23, "sent": "Methylation can only occur once pseudouridylation has taken place, as the latter reaction generates the substrate for the former.", "section": "INTRO", "ner": [ [ 0, 11, "Methylation", "ptm" ], [ 32, 49, "pseudouridylation", "ptm" ] ] }, { "sid": 24, "sent": "The final acp modification leading to N1-methyl-N3-aminocarboxypropyl-pseudouridine occurs late during 40S biogenesis in the cytoplasm, while the two former reactions are taking place in the nucleolus and nucleus, and is independent from pseudouridylation or methylation.", "section": "INTRO", "ner": [ [ 10, 13, "acp", "chemical" ], [ 38, 83, "N1-methyl-N3-aminocarboxypropyl-pseudouridine", "chemical" ], [ 103, 106, "40S", "complex_assembly" ], [ 238, 255, "pseudouridylation", "ptm" ] ] }, { "sid": 25, "sent": "Both the methyl and the acp group are derived from S-adenosylmethionine (SAM), but the enzyme responsible for acp modification remained elusive for more than 40 years.", "section": "INTRO", "ner": [ [ 51, 71, "S-adenosylmethionine", "chemical" ], [ 73, 76, "SAM", "chemical" ], [ 110, 113, "acp", "chemical" ] ] }, { "sid": 26, "sent": "Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3\u03a8 is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3.", "section": "FIG", "ner": [ [ 0, 4, "Tsr3", "protein" ], [ 22, 25, "acp", "chemical" ], [ 42, 50, "18S rRNA", "chemical" ], [ 54, 59, "yeast", "taxonomy_domain" ], [ 64, 69, "human", "species" ], [ 75, 88, "Hypermodified", "protein_state" ], [ 89, 99, "nucleotide", "chemical" ], [ 100, 107, "m1acp3\u03a8", "chemical" ], [ 139, 156, "pseudouridylation", "ptm" ], [ 170, 178, "snoRNP35", "complex_assembly" ], [ 180, 194, "N1-methylation", "ptm" ], [ 208, 225, "methyltransferase", "protein_type" ], [ 226, 230, "Nep1", "protein" ], [ 238, 241, "acp", "chemical" ], [ 268, 272, "Tsr3", "protein" ] ] }, { "sid": 27, "sent": "The asterisk indicates the C1-atom labeled in the 14C-incorporation assay.", "section": "FIG", "ner": [ [ 50, 73, "14C-incorporation assay", "experimental_method" ] ] }, { "sid": 28, "sent": "(B) RP-HPLC elution profile of yeast 18S rRNA nucleosides.", "section": "FIG", "ner": [ [ 4, 11, "RP-HPLC", "experimental_method" ], [ 12, 27, "elution profile", "evidence" ], [ 31, 36, "yeast", "taxonomy_domain" ], [ 37, 45, "18S rRNA", "chemical" ], [ 46, 57, "nucleosides", "chemical" ] ] }, { "sid": 29, "sent": "Hypermodified m1acp3\u03a8 elutes at 7.4 min (wild type, left profile) and is missing in \u0394tsr3 (middle profile) and \u0394nep1 \u0394nop6 mutants (right profile).", "section": "FIG", "ner": [ [ 0, 13, "Hypermodified", "protein_state" ], [ 14, 21, "m1acp3\u03a8", "chemical" ], [ 41, 50, "wild type", "protein_state" ], [ 84, 89, "\u0394tsr3", "mutant" ], [ 111, 122, "\u0394nep1 \u0394nop6", "mutant" ] ] }, { "sid": 30, "sent": "(C) 14C-acp labeling of 18S rRNAs.", "section": "FIG", "ner": [ [ 4, 11, "14C-acp", "chemical" ], [ 24, 33, "18S rRNAs", "chemical" ] ] }, { "sid": 31, "sent": "Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the\u00a0U1191A mutant plasmid encoded 18S rRNA (U1191A) and \u0394tsr3 mutants (\u0394tsr3).", "section": "FIG", "ner": [ [ 0, 9, "Wild type", "protein_state" ], [ 11, 13, "WT", "protein_state" ], [ 35, 43, "18S rRNA", "chemical" ], [ 45, 51, "U1191U", "mutant" ], [ 62, 69, "14C-acp", "chemical" ], [ 90, 97, "14C-acp", "chemical" ], [ 123, 129, "U1191A", "mutant" ], [ 130, 136, "mutant", "protein_state" ], [ 153, 161, "18S rRNA", "chemical" ], [ 163, 169, "U1191A", "mutant" ], [ 175, 180, "\u0394tsr3", "mutant" ], [ 190, 195, "\u0394tsr3", "mutant" ] ] }, { "sid": 32, "sent": "Upper lanes show the ethidium bromide staining of the 18S rRNAs for quantification.", "section": "FIG", "ner": [ [ 21, 37, "ethidium bromide", "chemical" ], [ 54, 63, "18S rRNAs", "chemical" ] ] }, { "sid": 33, "sent": "All samples were loaded on the gel with two different amounts of 5 and 10 \u03bcl. (D) Primer extension analysis of acp modification in yeast 18S rRNA (right gel) including a sequencing ladder (left gel).", "section": "FIG", "ner": [ [ 82, 107, "Primer extension analysis", "experimental_method" ], [ 111, 114, "acp", "chemical" ], [ 131, 136, "yeast", "taxonomy_domain" ], [ 137, 145, "18S rRNA", "chemical" ] ] }, { "sid": 34, "sent": "The primer extension stop at nucleotide 1191 is missing exclusively in \u0394tsr3 mutants and \u0394tsr3 \u0394snr35 recombinants.", "section": "FIG", "ner": [ [ 40, 44, "1191", "residue_number" ], [ 71, 76, "\u0394tsr3", "mutant" ], [ 89, 101, "\u0394tsr3 \u0394snr35", "mutant" ] ] }, { "sid": 35, "sent": "(E) Primer extension analysis of human 18S rRNA after siRNA knockdown of HsNEP1/EMG1 (541, 542 and 543) and HsTSR3 (544 and 545) (right gel), including a sequencing ladder (left gel).", "section": "FIG", "ner": [ [ 4, 29, "Primer extension analysis", "experimental_method" ], [ 33, 38, "human", "species" ], [ 39, 47, "18S rRNA", "chemical" ], [ 54, 69, "siRNA knockdown", "experimental_method" ], [ 73, 79, "HsNEP1", "protein" ], [ 80, 84, "EMG1", "protein" ], [ 108, 114, "HsTSR3", "protein" ] ] }, { "sid": 36, "sent": "The primer extension arrest is reduced in HTC116 cells transfected with siRNAs 544 and 545.", "section": "FIG", "ner": [ [ 72, 78, "siRNAs", "chemical" ] ] }, { "sid": 37, "sent": "The efficiency of siRNA mediated HsTSR3 repression correlates with the primer extension signals (see Supplementary Figure S2A).", "section": "FIG", "ner": [ [ 18, 23, "siRNA", "chemical" ], [ 33, 39, "HsTSR3", "protein" ], [ 71, 95, "primer extension signals", "evidence" ] ] }, { "sid": 38, "sent": "Only a few acp transferring enzymes have been characterized until now.", "section": "INTRO", "ner": [ [ 11, 14, "acp", "chemical" ] ] }, { "sid": 39, "sent": "During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom.", "section": "INTRO", "ner": [ [ 27, 37, "wybutosine", "chemical" ], [ 51, 61, "nucleoside", "chemical" ], [ 73, 83, "eukaryotic", "taxonomy_domain" ], [ 88, 96, "archaeal", "taxonomy_domain" ], [ 97, 110, "phenylalanine", "chemical" ], [ 111, 115, "tRNA", "chemical" ], [ 117, 121, "Tyw2", "protein" ], [ 123, 128, "Trm12", "protein" ], [ 132, 137, "yeast", "taxonomy_domain" ], [ 152, 155, "acp", "chemical" ], [ 167, 170, "SAM", "chemical" ] ] }, { "sid": 40, "sent": "Archaeal Tyw2 has a structure very similar to Rossmann-fold (class I) RNA-methyltransferases, but its distinctive SAM-binding mode enables the transfer of the acp group instead of the methyl group of the cofactor.", "section": "INTRO", "ner": [ [ 0, 8, "Archaeal", "taxonomy_domain" ], [ 9, 13, "Tyw2", "protein" ], [ 20, 29, "structure", "evidence" ], [ 46, 92, "Rossmann-fold (class I) RNA-methyltransferases", "protein_type" ], [ 114, 130, "SAM-binding mode", "site" ], [ 159, 162, "acp", "chemical" ] ] }, { "sid": 41, "sent": "Another acp modification has been described in the diphtamide biosynthesis pathway, where an acp group is transferred from SAM to the carbon atom of a histidine residue of eukaryotic translation elongation factor 2 by use of a radical mechanism.", "section": "INTRO", "ner": [ [ 8, 11, "acp", "chemical" ], [ 51, 61, "diphtamide", "chemical" ], [ 93, 96, "acp", "chemical" ], [ 123, 126, "SAM", "chemical" ], [ 151, 160, "histidine", "residue_name" ], [ 172, 182, "eukaryotic", "taxonomy_domain" ], [ 183, 214, "translation elongation factor 2", "protein_type" ] ] }, { "sid": 42, "sent": "In a recent bioinformatic study, the uncharacterized yeast gene YOR006c was predicted to be involved in ribosome biogenesis.", "section": "INTRO", "ner": [ [ 53, 58, "yeast", "taxonomy_domain" ], [ 64, 71, "YOR006c", "gene" ] ] }, { "sid": 43, "sent": "It is highly conserved among eukaryotes and archaea (Supplementary Figure S1A) and its deletion leads to an accumulation of the 20S pre-rRNA precursor of 18S rRNA, suggesting an influence on D-site cleavage during the maturation of the small ribosomal subunit.", "section": "INTRO", "ner": [ [ 6, 22, "highly conserved", "protein_state" ], [ 29, 39, "eukaryotes", "taxonomy_domain" ], [ 44, 51, "archaea", "taxonomy_domain" ], [ 128, 140, "20S pre-rRNA", "chemical" ], [ 154, 162, "18S rRNA", "chemical" ], [ 191, 197, "D-site", "site" ] ] }, { "sid": 44, "sent": "On this basis, YOR006C was renamed \u2018Twenty S rRNA accumulation 3\u2032 (TSR3).", "section": "INTRO", "ner": [ [ 15, 22, "YOR006C", "gene" ], [ 36, 64, "Twenty S rRNA accumulation 3", "protein" ], [ 67, 71, "TSR3", "protein" ] ] }, { "sid": 45, "sent": "However, its function remained unclear although recently a putative nuclease function during 18S rRNA maturation was predicted.", "section": "INTRO", "ner": [ [ 93, 101, "18S rRNA", "chemical" ] ] }, { "sid": 46, "sent": "Here, we identify Tsr3 as the long-sought acp transferase that catalyzes the last step in the biosynthesis of the hypermodified nucleotide m1acp3\u03a8 in yeast and human cells.", "section": "INTRO", "ner": [ [ 18, 22, "Tsr3", "protein" ], [ 42, 57, "acp transferase", "protein_type" ], [ 114, 127, "hypermodified", "protein_state" ], [ 128, 138, "nucleotide", "chemical" ], [ 139, 146, "m1acp3\u03a8", "chemical" ], [ 150, 155, "yeast", "taxonomy_domain" ], [ 160, 165, "human", "species" ] ] }, { "sid": 47, "sent": "Furthermore using catalytically defective mutants of yeast Tsr3 we demonstrated that the acp modification is required for 18S rRNA maturation.", "section": "INTRO", "ner": [ [ 18, 41, "catalytically defective", "protein_state" ], [ 53, 58, "yeast", "taxonomy_domain" ], [ 59, 63, "Tsr3", "protein" ], [ 89, 92, "acp", "chemical" ], [ 122, 130, "18S rRNA", "chemical" ] ] }, { "sid": 48, "sent": "Surprisingly, the crystal structures of archaeal homologs revealed that Tsr3 is structurally similar to the SPOUT-class RNA methyltransferases.", "section": "INTRO", "ner": [ [ 18, 36, "crystal structures", "evidence" ], [ 40, 48, "archaeal", "taxonomy_domain" ], [ 72, 76, "Tsr3", "protein" ], [ 108, 142, "SPOUT-class RNA methyltransferases", "protein_type" ] ] }, { "sid": 49, "sent": "In contrast, the only other structurally characterized acp transferase enzyme Tyw2 belongs to the Rossmann-fold class of methyltransferase proteins.", "section": "INTRO", "ner": [ [ 55, 70, "acp transferase", "protein_type" ], [ 78, 82, "Tyw2", "protein" ], [ 98, 147, "Rossmann-fold class of methyltransferase proteins", "protein_type" ] ] }, { "sid": 50, "sent": "Interestingly, the two structurally very different enzymes use similar strategies in binding the SAM-cofactor in order to ensure that in contrast to methyltransferases the acp and not the methyl group of SAM is transferred to the substrate.", "section": "INTRO", "ner": [ [ 97, 100, "SAM", "chemical" ], [ 149, 167, "methyltransferases", "protein_type" ], [ 172, 175, "acp", "chemical" ], [ 204, 207, "SAM", "chemical" ] ] }, { "sid": 51, "sent": "Tsr3 is the enzyme responsible for 18S rRNA acp modification in yeast and humans", "section": "RESULTS", "ner": [ [ 0, 4, "Tsr3", "protein" ], [ 35, 43, "18S rRNA", "chemical" ], [ 44, 47, "acp", "chemical" ], [ 64, 69, "yeast", "taxonomy_domain" ], [ 74, 80, "humans", "species" ] ] }, { "sid": 52, "sent": "The S. cerevisiae 18S rRNA acp transferase was identified in a systematic genetic screen where numerous deletion mutants from the EUROSCARF strain collection (www.euroscarf.de) were analyzed by HPLC for alterations in 18S rRNA base modifications.", "section": "RESULTS", "ner": [ [ 4, 17, "S. cerevisiae", "species" ], [ 18, 42, "18S rRNA acp transferase", "protein_type" ], [ 194, 198, "HPLC", "experimental_method" ], [ 218, 226, "18S rRNA", "chemical" ] ] }, { "sid": 53, "sent": "For the \u0394tsr3 deletion strain the HPLC elution profile of 18S rRNA nucleosides (Figure 1B) was very similar to that of the pseudouridine-N1 methyltransferase mutant \u0394nep1, where a shoulder at \u223c 7.4 min elution time was missing in the elution profile.", "section": "RESULTS", "ner": [ [ 8, 13, "\u0394tsr3", "mutant" ], [ 34, 54, "HPLC elution profile", "evidence" ], [ 58, 66, "18S rRNA", "chemical" ], [ 67, 78, "nucleosides", "chemical" ], [ 123, 157, "pseudouridine-N1 methyltransferase", "protein_type" ], [ 158, 164, "mutant", "protein_state" ], [ 165, 170, "\u0394nep1", "mutant" ] ] }, { "sid": 54, "sent": "As previously reported this shoulder was identified by ESI-MS as corresponding to m1acp3\u03a8.", "section": "RESULTS", "ner": [ [ 55, 61, "ESI-MS", "experimental_method" ], [ 82, 89, "m1acp3\u03a8", "chemical" ] ] }, { "sid": 55, "sent": "In order to directly analyze the presence of the acp modification of nucleotide 1191 we used an in vivo14C incorporation assay with 1-14C-methionine.", "section": "RESULTS", "ner": [ [ 49, 52, "acp", "chemical" ], [ 69, 79, "nucleotide", "chemical" ], [ 80, 84, "1191", "residue_number" ], [ 96, 126, "in vivo14C incorporation assay", "experimental_method" ], [ 132, 148, "1-14C-methionine", "chemical" ] ] }, { "sid": 56, "sent": "Whereas the acp labeling of 18S rRNA was clearly present in the wild type strain no radioactive labeling could be observed in a \u0394tsr3 strain (Figure 1C).", "section": "RESULTS", "ner": [ [ 12, 15, "acp", "chemical" ], [ 28, 36, "18S rRNA", "chemical" ], [ 64, 73, "wild type", "protein_state" ], [ 128, 133, "\u0394tsr3", "mutant" ] ] }, { "sid": 57, "sent": "No radioactive labeling was detected in the 18S U1191A mutant which served as a control for the specificity of the 14C-aminocarboxypropyl incorporation.", "section": "RESULTS", "ner": [ [ 44, 54, "18S U1191A", "mutant" ], [ 55, 61, "mutant", "protein_state" ], [ 115, 137, "14C-aminocarboxypropyl", "chemical" ] ] }, { "sid": 58, "sent": "As previously shown, only the acp but none of the other modifications at U1191 of yeast 18S rRNA blocks reverse transcriptase activity.", "section": "RESULTS", "ner": [ [ 30, 33, "acp", "chemical" ], [ 73, 78, "U1191", "residue_name_number" ], [ 82, 87, "yeast", "taxonomy_domain" ], [ 88, 96, "18S rRNA", "chemical" ] ] }, { "sid": 59, "sent": "Therefore the presence of the acp modification can be directly assessed by primer extension.", "section": "RESULTS", "ner": [ [ 30, 33, "acp", "chemical" ], [ 75, 91, "primer extension", "experimental_method" ] ] }, { "sid": 60, "sent": "Indeed, in wild-type yeast a strong primer extension stop signal occurred at position 1192.", "section": "RESULTS", "ner": [ [ 11, 20, "wild-type", "protein_state" ], [ 21, 26, "yeast", "taxonomy_domain" ], [ 36, 64, "primer extension stop signal", "evidence" ], [ 86, 90, "1192", "residue_number" ] ] }, { "sid": 61, "sent": "In contrast, in a \u0394tsr3 mutant no primer extension stop signal was present at this position.", "section": "RESULTS", "ner": [ [ 18, 23, "\u0394tsr3", "mutant" ], [ 24, 30, "mutant", "protein_state" ] ] }, { "sid": 62, "sent": "As expected, in a \u0394snr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a \u0394nep1 deletion strain where pseudouridine is not methylated (resulting in acp3\u03a8) a primer extension stop signal of similar intensity as in the wild type was observed.", "section": "RESULTS", "ner": [ [ 18, 24, "\u0394snr35", "mutant" ], [ 25, 33, "deletion", "experimental_method" ], [ 45, 62, "pseudouridylation", "ptm" ], [ 67, 81, "N1-methylation", "ptm" ], [ 96, 101, "acp3U", "chemical" ], [ 119, 124, "\u0394nep1", "mutant" ], [ 147, 160, "pseudouridine", "chemical" ], [ 164, 178, "not methylated", "protein_state" ], [ 193, 198, "acp3\u03a8", "chemical" ], [ 202, 230, "primer extension stop signal", "evidence" ], [ 262, 271, "wild type", "protein_state" ] ] }, { "sid": 63, "sent": "In a \u0394tsr3 \u0394snr35 double deletion strain the 18S rRNA contains an unmodified U and the primer extension stop signal was missing (Figure 1D).", "section": "RESULTS", "ner": [ [ 5, 17, "\u0394tsr3 \u0394snr35", "mutant" ], [ 45, 53, "18S rRNA", "chemical" ], [ 66, 76, "unmodified", "protein_state" ], [ 77, 78, "U", "chemical" ] ] }, { "sid": 64, "sent": "The Tsr3 protein is highly conserved in yeast and humans (50% identity).", "section": "RESULTS", "ner": [ [ 4, 8, "Tsr3", "protein" ], [ 20, 36, "highly conserved", "protein_state" ], [ 40, 45, "yeast", "taxonomy_domain" ], [ 50, 56, "humans", "species" ] ] }, { "sid": 65, "sent": "Human 18S rRNA has also been shown to contain m1acp3\u03a8 in the 18S rRNA at position 1248.", "section": "RESULTS", "ner": [ [ 0, 5, "Human", "species" ], [ 6, 14, "18S rRNA", "chemical" ], [ 46, 53, "m1acp3\u03a8", "ptm" ], [ 61, 69, "18S rRNA", "chemical" ], [ 82, 86, "1248", "residue_number" ] ] }, { "sid": 66, "sent": "After siRNA-mediated depletion of Tsr3 in human colon carcinoma HCT116(+/+) cells the acp primer extension arrest was reduced in comparison to cells transfected with a non-targeting scramble siRNA control (Figure 1E, compare lanes 544 and scramble).", "section": "RESULTS", "ner": [ [ 6, 30, "siRNA-mediated depletion", "experimental_method" ], [ 34, 38, "Tsr3", "protein" ], [ 42, 47, "human", "species" ], [ 86, 113, "acp primer extension arrest", "evidence" ], [ 191, 196, "siRNA", "chemical" ] ] }, { "sid": 67, "sent": "The efficiency of siRNA-mediated depletion was established by RT-qPCR and found to be very high with siRNA 544 (Supplementary Figure S2A, remaining TSR3 mRNA level of 2%).", "section": "RESULTS", "ner": [ [ 18, 23, "siRNA", "chemical" ], [ 62, 69, "RT-qPCR", "experimental_method" ], [ 101, 106, "siRNA", "chemical" ], [ 148, 152, "TSR3", "protein" ] ] }, { "sid": 68, "sent": "By comparison, treating cells with siRNA 545, which only reduced the TSR3 mRNA to 20%, did not markedly reduced the acp signal.", "section": "RESULTS", "ner": [ [ 35, 40, "siRNA", "chemical" ], [ 69, 73, "TSR3", "protein" ], [ 116, 119, "acp", "chemical" ] ] }, { "sid": 69, "sent": "This suggests that low residual levels of HsTsr3 are sufficient to modify the RNA.", "section": "RESULTS", "ner": [ [ 42, 48, "HsTsr3", "protein" ], [ 78, 81, "RNA", "chemical" ] ] }, { "sid": 70, "sent": "Thus, HsTsr3 is also responsible for the acp modification of 18S rRNA nucleotide \u03a81248 in helix 31.", "section": "RESULTS", "ner": [ [ 6, 12, "HsTsr3", "protein" ], [ 41, 44, "acp", "chemical" ], [ 61, 69, "18S rRNA", "chemical" ], [ 70, 80, "nucleotide", "chemical" ], [ 81, 86, "\u03a81248", "ptm" ], [ 90, 98, "helix 31", "structure_element" ] ] }, { "sid": 71, "sent": "Similar to yeast, siRNA-mediated depletion of the \u03a81248 N1-methyltransferase Nep1/Emg1 had no influence on the primer extension arrest (Figure 1E).", "section": "RESULTS", "ner": [ [ 11, 16, "yeast", "taxonomy_domain" ], [ 18, 42, "siRNA-mediated depletion", "experimental_method" ], [ 50, 76, "\u03a81248 N1-methyltransferase", "protein_type" ], [ 77, 81, "Nep1", "protein" ], [ 82, 86, "Emg1", "protein" ], [ 111, 134, "primer extension arrest", "evidence" ] ] }, { "sid": 72, "sent": "Phenotypic characterization of \u0394tsr3 mutants", "section": "RESULTS", "ner": [ [ 31, 36, "\u0394tsr3", "mutant" ] ] }, { "sid": 73, "sent": "Although the acp modification of 18S rRNA is highly conserved in eukaryotes, yeast \u0394tsr3 mutants showed only a minor growth defect.", "section": "RESULTS", "ner": [ [ 13, 16, "acp", "chemical" ], [ 33, 41, "18S rRNA", "chemical" ], [ 45, 61, "highly conserved", "protein_state" ], [ 65, 75, "eukaryotes", "taxonomy_domain" ], [ 77, 82, "yeast", "taxonomy_domain" ], [ 83, 88, "\u0394tsr3", "mutant" ] ] }, { "sid": 74, "sent": "However, the \u0394tsr3 deletion was synthetic sick with a \u0394snr35 deletion preventing pseudouridylation and Nep1-catalyzed methylation of nucleotide 1191 (Figure 2A).", "section": "RESULTS", "ner": [ [ 13, 18, "\u0394tsr3", "mutant" ], [ 54, 60, "\u0394snr35", "mutant" ], [ 81, 98, "pseudouridylation", "ptm" ], [ 103, 107, "Nep1", "protein" ], [ 144, 148, "1191", "residue_number" ] ] }, { "sid": 75, "sent": "Interestingly, no increased growth defect could be observed for \u0394tsr3 \u0394nep1 recombinants containing the nep1 suppressor mutation \u0394nop6 as well as for \u0394tsr3 \u0394snr35 \u0394nep1 recombinants with unmodified U1191 (Supplementary Figure S2D and E).", "section": "RESULTS", "ner": [ [ 64, 75, "\u0394tsr3 \u0394nep1", "mutant" ], [ 104, 108, "nep1", "gene" ], [ 129, 134, "\u0394nop6", "mutant" ], [ 150, 168, "\u0394tsr3 \u0394snr35 \u0394nep1", "mutant" ], [ 187, 197, "unmodified", "protein_state" ], [ 198, 203, "U1191", "residue_name_number" ] ] }, { "sid": 76, "sent": "Phenotypic characterization of yeast TSR3 deletion (\u0394trs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, \u0394tsr3, \u0394snr35 and \u0394tsr3 \u0394snr35 segregants after meiosis and tetrad dissection of \u0394tsr3/TSR3 \u0394snr35/SNR35 heterozygous diploids.", "section": "FIG", "ner": [ [ 31, 36, "yeast", "taxonomy_domain" ], [ 37, 41, "TSR3", "protein" ], [ 52, 57, "\u0394trs3", "mutant" ], [ 63, 68, "human", "species" ], [ 69, 73, "TSR3", "protein" ], [ 85, 91, "siRNAs", "chemical" ], [ 134, 139, "yeast", "taxonomy_domain" ], [ 140, 144, "Tsr3", "protein" ], [ 160, 165, "yeast", "taxonomy_domain" ], [ 166, 175, "wild type", "protein_state" ], [ 177, 182, "\u0394tsr3", "mutant" ], [ 184, 190, "\u0394snr35", "mutant" ], [ 195, 207, "\u0394tsr3 \u0394snr35", "mutant" ], [ 258, 263, "\u0394tsr3", "mutant" ], [ 264, 268, "TSR3", "protein" ], [ 269, 275, "\u0394snr35", "mutant" ], [ 276, 281, "SNR35", "protein" ] ] }, { "sid": 77, "sent": "The \u0394tsr3 deletion is synthetic sick with a \u0394snr35 deletion preventing U1191 pseudouridylation.", "section": "FIG", "ner": [ [ 4, 9, "\u0394tsr3", "mutant" ], [ 44, 50, "\u0394snr35", "mutant" ], [ 71, 76, "U1191", "residue_name_number" ] ] }, { "sid": 78, "sent": "(B) In agar diffusion assays the yeast \u0394tsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with \u0394snr35. (C) Northern blot analysis with\u00a0an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control.", "section": "FIG", "ner": [ [ 7, 28, "agar diffusion assays", "experimental_method" ], [ 33, 38, "yeast", "taxonomy_domain" ], [ 39, 44, "\u0394tsr3", "mutant" ], [ 45, 60, "deletion mutant", "protein_state" ], [ 94, 105, "paromomycin", "chemical" ], [ 110, 122, "hygromycin B", "chemical" ], [ 172, 178, "\u0394snr35", "mutant" ], [ 184, 206, "Northern blot analysis", "experimental_method" ], [ 246, 261, "siRNA depletion", "experimental_method" ], [ 265, 271, "HsTSR3", "protein" ], [ 273, 279, "siRNAs", "chemical" ], [ 309, 314, "siRNA", "chemical" ] ] }, { "sid": 79, "sent": "The accumulation of 18SE and 47S and/or 45S pre-RNAs is enforced upon HsTSR3 depletion.", "section": "FIG", "ner": [ [ 20, 24, "18SE", "chemical" ], [ 29, 32, "47S", "chemical" ], [ 40, 52, "45S pre-RNAs", "chemical" ], [ 70, 76, "HsTSR3", "protein" ] ] }, { "sid": 80, "sent": "Right gel: Ethidium bromide staining showing 18S and 28S rRNAs.", "section": "FIG", "ner": [ [ 45, 48, "18S", "chemical" ], [ 53, 62, "28S rRNAs", "chemical" ] ] }, { "sid": 81, "sent": "(D) Cytoplasmic localization of yeast Tsr3 shown by fluorescence microscopy of GFP-fused Tsr3.", "section": "FIG", "ner": [ [ 32, 37, "yeast", "taxonomy_domain" ], [ 38, 42, "Tsr3", "protein" ], [ 52, 75, "fluorescence microscopy", "experimental_method" ], [ 79, 93, "GFP-fused Tsr3", "mutant" ] ] }, { "sid": 82, "sent": "From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part).", "section": "FIG", "ner": [ [ 20, 54, "differential interference contrast", "experimental_method" ], [ 56, 59, "DIC", "experimental_method" ], [ 84, 92, "GFP-Tsr3", "mutant" ], [ 114, 124, "Nop56-mRFP", "mutant" ], [ 159, 167, "GFP-Tsr3", "mutant" ], [ 168, 178, "Nop56-mRFP", "mutant" ], [ 184, 187, "DIC", "experimental_method" ], [ 193, 208, "Elution profile", "evidence" ], [ 222, 249, "sucrose gradient separation", "experimental_method" ], [ 253, 258, "yeast", "taxonomy_domain" ], [ 259, 277, "ribosomal subunits", "complex_assembly" ], [ 282, 291, "polysomes", "complex_assembly" ], [ 309, 321, "western blot", "experimental_method" ], [ 334, 338, "3xHA", "chemical" ], [ 346, 350, "Tsr3", "protein" ], [ 352, 361, "Tsr3-3xHA", "mutant" ], [ 369, 377, "SDS-PAGE", "experimental_method" ] ] }, { "sid": 83, "sent": "The TSR3 gene was genetically modified at its native locus, resulting in a C-terminal fusion of Tsr3 with a 3xHA epitope expressed by the native promotor in yeast strain CEN.BM258-5B.", "section": "FIG", "ner": [ [ 4, 8, "TSR3", "protein" ], [ 86, 92, "fusion", "protein_state" ], [ 96, 100, "Tsr3", "protein" ], [ 108, 112, "3xHA", "chemical" ], [ 157, 162, "yeast", "taxonomy_domain" ] ] }, { "sid": 84, "sent": "The influence of the acp modification of nucleotide 1191 on ribosome function was analyzed by treating \u0394tsr3 mutants with protein synthesis inhibitors.", "section": "RESULTS", "ner": [ [ 21, 24, "acp", "chemical" ], [ 41, 51, "nucleotide", "chemical" ], [ 52, 56, "1191", "residue_number" ], [ 103, 108, "\u0394tsr3", "mutant" ] ] }, { "sid": 85, "sent": "Similar to a temperature-sensitive nep1 mutant, the \u0394tsr3 deletion caused hypersensitivity to paromomycin and, to a lesser extent, to hygromycin B (Figure 2B), but not to G418 or cycloheximide (data not shown).", "section": "RESULTS", "ner": [ [ 35, 39, "nep1", "gene" ], [ 40, 46, "mutant", "protein_state" ], [ 52, 57, "\u0394tsr3", "mutant" ], [ 94, 105, "paromomycin", "chemical" ], [ 134, 146, "hygromycin B", "chemical" ], [ 171, 175, "G418", "chemical" ], [ 179, 192, "cycloheximide", "chemical" ] ] }, { "sid": 86, "sent": "In accordance with the synthetic sick growth phenotype the paromomycin and hygromycin B hypersensitivity further increased in a \u0394tsr3 \u0394snr35 recombination strain (Figure 2B).", "section": "RESULTS", "ner": [ [ 59, 70, "paromomycin", "chemical" ], [ 75, 87, "hygromycin B", "chemical" ], [ 128, 140, "\u0394tsr3 \u0394snr35", "mutant" ] ] }, { "sid": 87, "sent": "In a yeast \u0394tsr3 strain as well as in the \u0394tsr3 \u0394snr35 recombinant 20S pre-rRNA accumulated significantly and the level of mature 18S rRNA was reduced (Supplementary Figures S2C and S3D), as reported previously.", "section": "RESULTS", "ner": [ [ 5, 10, "yeast", "taxonomy_domain" ], [ 11, 16, "\u0394tsr3", "mutant" ], [ 42, 54, "\u0394tsr3 \u0394snr35", "mutant" ], [ 67, 79, "20S pre-rRNA", "chemical" ], [ 130, 138, "18S rRNA", "chemical" ] ] }, { "sid": 88, "sent": "A minor effect on 20S rRNA accumulation was also observed for \u0394snr35, but - probably due to different strain backgrounds \u2013 to a weaker extent than described earlier.", "section": "RESULTS", "ner": [ [ 18, 26, "20S rRNA", "chemical" ], [ 62, 68, "\u0394snr35", "mutant" ] ] }, { "sid": 89, "sent": "In human cells, the depletion of HsTsr3 in HCT116(+/+) cells caused an accumulation of the human 20S pre-rRNA equivalent 18S-E suggesting an evolutionary conserved role of Tsr3 in the late steps of 18S rRNA processing (Figure 2C and Supplementary Figure S2B).", "section": "RESULTS", "ner": [ [ 3, 8, "human", "species" ], [ 20, 32, "depletion of", "experimental_method" ], [ 33, 39, "HsTsr3", "protein" ], [ 91, 96, "human", "species" ], [ 97, 109, "20S pre-rRNA", "chemical" ], [ 121, 126, "18S-E", "chemical" ], [ 172, 176, "Tsr3", "protein" ], [ 198, 206, "18S rRNA", "chemical" ] ] }, { "sid": 90, "sent": "Surprisingly, early nucleolar processing reactions were also inhibited, and this was observed in both yeast \u0394tsr3 cells (see accumulation of 35S in Supplementary Figure S2C) and Tsr3 depleted human cells (see 47S/45S accumulation in Figure 2C and Northern blot quantification in Supplementary Figure S2B).", "section": "RESULTS", "ner": [ [ 102, 107, "yeast", "taxonomy_domain" ], [ 108, 113, "\u0394tsr3", "mutant" ], [ 141, 144, "35S", "complex_assembly" ], [ 178, 182, "Tsr3", "protein" ], [ 192, 197, "human", "species" ], [ 209, 212, "47S", "complex_assembly" ], [ 213, 216, "45S", "complex_assembly" ], [ 247, 260, "Northern blot", "experimental_method" ] ] }, { "sid": 91, "sent": "Consistent with its role in late 18S rRNA processing, TSR3 deletion leads to a ribosomal subunit imbalance with a reduced 40S to 60S ratio of 0.81 (\u03c3 = 0.024) which was further increased in a \u0394tsr3 \u0394snr35 recombinant to 0.73 (\u03c3 = 0.023) (Supplementary Figure S2F).", "section": "RESULTS", "ner": [ [ 33, 41, "18S rRNA", "chemical" ], [ 54, 58, "TSR3", "protein" ], [ 122, 125, "40S", "complex_assembly" ], [ 129, 132, "60S", "complex_assembly" ], [ 192, 204, "\u0394tsr3 \u0394snr35", "mutant" ] ] }, { "sid": 92, "sent": "In polysome profiles, a reduced level of 80S ribosomes and a strong signal for free 60S subunits was observed in line with the 40S subunit deficiency (Supplementary Figure S2G).", "section": "RESULTS", "ner": [ [ 3, 20, "polysome profiles", "evidence" ], [ 41, 54, "80S ribosomes", "complex_assembly" ], [ 84, 87, "60S", "complex_assembly" ], [ 127, 130, "40S", "complex_assembly" ] ] }, { "sid": 93, "sent": "Cellular localization of Tsr3 in S. cerevisiae", "section": "RESULTS", "ner": [ [ 25, 29, "Tsr3", "protein" ], [ 33, 46, "S. cerevisiae", "species" ] ] }, { "sid": 94, "sent": "Fluorescence microscopy of GFP-tagged Tsr3 localized the fusion protein in the cytoplasm of yeast cells and no co-localization with the nucleolar marker protein Nop56 could be observed (Figure 2D).", "section": "RESULTS", "ner": [ [ 0, 23, "Fluorescence microscopy", "experimental_method" ], [ 27, 37, "GFP-tagged", "protein_state" ], [ 38, 42, "Tsr3", "protein" ], [ 92, 97, "yeast", "taxonomy_domain" ], [ 161, 166, "Nop56", "protein" ] ] }, { "sid": 95, "sent": "This agrees with previous biochemical data suggesting that the acp modification of 18S rRNA occurs late during 40S subunit biogenesis in the cytoplasm, and makes an additional nuclear localization as reported in a previous large-scale analysis unlikely.", "section": "RESULTS", "ner": [ [ 63, 66, "acp", "chemical" ], [ 83, 91, "18S rRNA", "chemical" ], [ 111, 114, "40S", "complex_assembly" ] ] }, { "sid": 96, "sent": "After polysome gradient separation C-terminally epitope-labeled Tsr3-3xHA was exclusively detectable in the low-density fraction (Figure 2E).", "section": "RESULTS", "ner": [ [ 6, 34, "polysome gradient separation", "experimental_method" ], [ 64, 73, "Tsr3-3xHA", "mutant" ] ] }, { "sid": 97, "sent": "Such distribution on a density gradient suggests that Tsr3 only interacts transiently with pre-40S subunits, which presumably explains why it was not characterized in pre-ribosome affinity purifications.", "section": "RESULTS", "ner": [ [ 5, 39, "distribution on a density gradient", "evidence" ], [ 54, 58, "Tsr3", "protein" ], [ 91, 107, "pre-40S subunits", "complex_assembly" ], [ 167, 202, "pre-ribosome affinity purifications", "experimental_method" ] ] }, { "sid": 98, "sent": "Structure of Tsr3", "section": "RESULTS", "ner": [ [ 0, 9, "Structure", "evidence" ], [ 13, 17, "Tsr3", "protein" ] ] }, { "sid": 99, "sent": "Searches for sequence homologs of S. cerevisiae Tsr3 (ScTsr3) by us and others revealed that the genomes of many archaea contain genes encoding Tsr3-like proteins.", "section": "RESULTS", "ner": [ [ 34, 47, "S. cerevisiae", "species" ], [ 48, 52, "Tsr3", "protein" ], [ 54, 60, "ScTsr3", "protein" ], [ 113, 120, "archaea", "taxonomy_domain" ], [ 144, 162, "Tsr3-like proteins", "protein_type" ] ] }, { "sid": 100, "sent": "However, these archaeal homologs are significantly smaller than ScTsr3 (\u223c190 aa in archaea vs. 313 aa in yeast) due to shortened N- and C-termini (Supplementary Figure S1A).", "section": "RESULTS", "ner": [ [ 15, 23, "archaeal", "taxonomy_domain" ], [ 64, 70, "ScTsr3", "protein" ], [ 83, 90, "archaea", "taxonomy_domain" ], [ 105, 110, "yeast", "taxonomy_domain" ] ] }, { "sid": 101, "sent": "To locate the domains most important for Tsr3 activity, ScTsr3 fragments of different lengths containing the highly conserved central part were expressed in a \u0394tsr3 mutant (Figure 3A) and analyzed by primer extension (Figure 3B) and Northern blotting (Figure 3C).", "section": "RESULTS", "ner": [ [ 41, 45, "Tsr3", "protein" ], [ 56, 62, "ScTsr3", "protein" ], [ 109, 125, "highly conserved", "protein_state" ], [ 144, 153, "expressed", "experimental_method" ], [ 159, 164, "\u0394tsr3", "mutant" ], [ 165, 171, "mutant", "protein_state" ], [ 200, 216, "primer extension", "experimental_method" ], [ 233, 250, "Northern blotting", "experimental_method" ] ] }, { "sid": 102, "sent": "N-terminal truncations of up to 45 aa and C-terminal truncations of up to 76 aa mediated acp modification as efficiently as the full-length protein and no significant increased levels of 20S pre-RNA were detected.", "section": "RESULTS", "ner": [ [ 11, 22, "truncations", "experimental_method" ], [ 32, 37, "45 aa", "residue_range" ], [ 53, 64, "truncations", "experimental_method" ], [ 74, 79, "76 aa", "residue_range" ], [ 89, 92, "acp", "chemical" ], [ 128, 139, "full-length", "protein_state" ], [ 187, 198, "20S pre-RNA", "chemical" ] ] }, { "sid": 103, "sent": "Even a Tsr3 fragment with a 90 aa C-terminal truncation showed a residual primer extension stop, whereas N-terminal truncations exceeding 46 aa almost completely abolished the primer extension arrest (Figure 3B).", "section": "RESULTS", "ner": [ [ 7, 11, "Tsr3", "protein" ], [ 28, 33, "90 aa", "residue_range" ], [ 138, 143, "46 aa", "residue_range" ] ] }, { "sid": 104, "sent": "Domain characterization of yeast Tsr3 and correlation of acp modification with late 18S rRNA processing steps. (A) Scheme of the TSR3 gene with truncation positions in the open reading frame.", "section": "FIG", "ner": [ [ 27, 32, "yeast", "taxonomy_domain" ], [ 33, 37, "Tsr3", "protein" ], [ 57, 60, "acp", "chemical" ], [ 84, 92, "18S rRNA", "chemical" ], [ 129, 133, "TSR3", "protein" ] ] }, { "sid": 105, "sent": "TSR3 fragments of different length were expressed under the native promotor from multicopy plasmids in a \u0394tsr3 deletion strain.", "section": "FIG", "ner": [ [ 0, 4, "TSR3", "protein" ], [ 105, 110, "\u0394tsr3", "mutant" ] ] }, { "sid": 106, "sent": "(B) Primer extension analysis of 18S rRNA acp modification in yeast cells expressing the indicated TSR3 fragments.", "section": "FIG", "ner": [ [ 4, 29, "Primer extension analysis", "experimental_method" ], [ 33, 41, "18S rRNA", "chemical" ], [ 42, 45, "acp", "chemical" ], [ 62, 67, "yeast", "taxonomy_domain" ], [ 99, 103, "TSR3", "protein" ] ] }, { "sid": 107, "sent": "N-terminal deletions of 36 or 45 amino acids and C-terminal deletions of 43 or 76 residues show a primer extension stop comparable to the wild type.", "section": "FIG", "ner": [ [ 11, 20, "deletions", "experimental_method" ], [ 24, 26, "36", "residue_range" ], [ 30, 32, "45", "residue_range" ], [ 60, 69, "deletions", "experimental_method" ], [ 73, 75, "43", "residue_range" ], [ 79, 81, "76", "residue_range" ], [ 98, 119, "primer extension stop", "evidence" ], [ 138, 147, "wild type", "protein_state" ] ] }, { "sid": 108, "sent": "Tsr3 fragments 37\u2013223 or 46\u2013223 cause a nearly complete loss of the arrest signal.", "section": "FIG", "ner": [ [ 0, 4, "Tsr3", "protein" ], [ 15, 21, "37\u2013223", "residue_range" ], [ 25, 31, "46\u2013223", "residue_range" ] ] }, { "sid": 109, "sent": "The box highlights the shortest Tsr3 fragment (aa 46\u2013270) with wild type activity (strong primer extension block). (C) Northern blot analysis of 20S pre-rRNA accumulation.", "section": "FIG", "ner": [ [ 32, 36, "Tsr3", "protein" ], [ 50, 56, "46\u2013270", "residue_range" ], [ 63, 72, "wild type", "protein_state" ], [ 90, 112, "primer extension block", "evidence" ], [ 119, 132, "Northern blot", "experimental_method" ], [ 145, 157, "20S pre-rRNA", "chemical" ] ] }, { "sid": 110, "sent": "A weak 20S rRNA signal, indicating normal processing, is observed for Tsr3 fragment 46\u2013270 (highlighted in a box) showing its functionality.", "section": "FIG", "ner": [ [ 7, 15, "20S rRNA", "chemical" ], [ 70, 74, "Tsr3", "protein" ], [ 84, 90, "46\u2013270", "residue_range" ] ] }, { "sid": 111, "sent": "Strong 20S rRNA accumulation similar to that of the \u0394tsr3 deletion is observed for Tsr3 fragments 37\u2013223 or 46\u2013223.", "section": "FIG", "ner": [ [ 52, 57, "\u0394tsr3", "mutant" ], [ 58, 66, "deletion", "experimental_method" ], [ 83, 87, "Tsr3", "protein" ], [ 98, 104, "37\u2013223", "residue_range" ], [ 108, 114, "46\u2013223", "residue_range" ] ] }, { "sid": 112, "sent": "Thus, the archaeal homologs correspond to the functional core of Tsr3.", "section": "RESULTS", "ner": [ [ 10, 18, "archaeal", "taxonomy_domain" ], [ 65, 69, "Tsr3", "protein" ] ] }, { "sid": 113, "sent": "In order to define the structural basis for Tsr3 function, homologs from thermophilic archaea were screened for crystallization.", "section": "RESULTS", "ner": [ [ 44, 48, "Tsr3", "protein" ], [ 73, 93, "thermophilic archaea", "taxonomy_domain" ], [ 112, 127, "crystallization", "experimental_method" ] ] }, { "sid": 114, "sent": "We focused on archaeal species containing a putative Nep1 homolog suggesting that these species are in principle capable of synthesizing N1-methyl-N3-acp-pseudouridine.", "section": "RESULTS", "ner": [ [ 14, 22, "archaeal", "taxonomy_domain" ], [ 53, 57, "Nep1", "protein" ], [ 137, 167, "N1-methyl-N3-acp-pseudouridine", "chemical" ] ] }, { "sid": 115, "sent": "Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46\u2013223).", "section": "RESULTS", "ner": [ [ 17, 25, "crystals", "evidence" ], [ 44, 48, "Tsr3", "protein" ], [ 71, 83, "crenarchaeal", "taxonomy_domain" ], [ 92, 115, "Vulcanisaeta distributa", "species" ], [ 117, 123, "VdTsr3", "protein" ], [ 129, 152, "Sulfolobus solfataricus", "species" ], [ 154, 160, "SsTsr3", "protein" ], [ 179, 185, "VdTsr3", "protein" ], [ 196, 202, "SsTsr3", "protein" ], [ 222, 228, "ScTsr3", "protein" ], [ 229, 240, "core region", "structure_element" ], [ 242, 248, "ScTsr3", "protein" ], [ 252, 258, "46\u2013223", "residue_range" ] ] }, { "sid": 116, "sent": "While for S. solfataricus the existence of a modified nucleotide of unknown chemical composition in the loop capping helix 31 of its 16S rRNA has been demonstrated, no information regarding rRNA modifications is yet available for V. distributa.", "section": "RESULTS", "ner": [ [ 10, 25, "S. solfataricus", "species" ], [ 54, 64, "nucleotide", "chemical" ], [ 104, 125, "loop capping helix 31", "structure_element" ], [ 133, 141, "16S rRNA", "chemical" ], [ 230, 243, "V. distributa", "species" ] ] }, { "sid": 117, "sent": "Crystals of VdTsr3 diffracted to a resolution of 1.6 \u00c5 whereas crystals of SsTsr3 diffracted to 2.25 \u00c5. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state.", "section": "RESULTS", "ner": [ [ 0, 8, "Crystals", "evidence" ], [ 12, 18, "VdTsr3", "protein" ], [ 63, 71, "crystals", "evidence" ], [ 75, 81, "SsTsr3", "protein" ], [ 121, 127, "VdTsr3", "protein" ], [ 145, 157, "crystallized", "experimental_method" ], [ 158, 173, "in complex with", "protein_state" ], [ 174, 184, "endogenous", "protein_state" ], [ 186, 193, "E. coli", "species" ], [ 195, 198, "SAM", "chemical" ], [ 231, 237, "SsTsr3", "protein" ], [ 238, 246, "crystals", "evidence" ], [ 276, 279, "apo", "protein_state" ] ] }, { "sid": 118, "sent": "The structure of VdTsr3 was solved ab initio, by single-wavelength anomalous diffraction phasing (Se-SAD) with Se containing derivatives (selenomethionine and seleno-substituted SAM).", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 17, 23, "VdTsr3", "protein" ], [ 49, 96, "single-wavelength anomalous diffraction phasing", "experimental_method" ], [ 98, 104, "Se-SAD", "experimental_method" ], [ 111, 113, "Se", "chemical" ], [ 138, 154, "selenomethionine", "chemical" ], [ 159, 181, "seleno-substituted SAM", "chemical" ] ] }, { "sid": 119, "sent": "The structure of SsTsr3 was solved by molecular replacement using VdTsr3 as a search model (see Supplementary Table S1 for data collection and refinement statistics).", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 17, 23, "SsTsr3", "protein" ], [ 38, 59, "molecular replacement", "experimental_method" ], [ 66, 72, "VdTsr3", "protein" ] ] }, { "sid": 120, "sent": "The structure of VdTsr3 can be divided into two domains (Figure 4A).", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 17, 23, "VdTsr3", "protein" ] ] }, { "sid": 121, "sent": "The N-terminal domain (aa 1\u201392) has a mixed \u03b1/\u03b2-structure centered around a five-stranded all-parallel \u03b2-sheet (Figure 4B) with the strand order \u03b25\u2191-\u03b23\u2191-\u03b24\u2191-\u03b21\u2191-\u03b22\u2191. The loops connecting \u03b21 and \u03b22, \u03b23 and \u03b24 and \u03b24 and \u03b25 include \u03b1-helices \u03b11, \u03b12 and \u03b13, respectively.", "section": "RESULTS", "ner": [ [ 4, 21, "N-terminal domain", "structure_element" ], [ 26, 30, "1\u201392", "residue_range" ], [ 44, 57, "\u03b1/\u03b2-structure", "structure_element" ], [ 76, 110, "five-stranded all-parallel \u03b2-sheet", "structure_element" ], [ 145, 148, "\u03b25\u2191", "structure_element" ], [ 149, 152, "\u03b23\u2191", "structure_element" ], [ 153, 156, "\u03b24\u2191", "structure_element" ], [ 157, 160, "\u03b21\u2191", "structure_element" ], [ 161, 164, "\u03b22\u2191", "structure_element" ], [ 170, 175, "loops", "structure_element" ], [ 187, 189, "\u03b21", "structure_element" ], [ 194, 196, "\u03b22", "structure_element" ], [ 198, 200, "\u03b23", "structure_element" ], [ 205, 207, "\u03b24", "structure_element" ], [ 212, 214, "\u03b24", "structure_element" ], [ 219, 221, "\u03b25", "structure_element" ], [ 230, 239, "\u03b1-helices", "structure_element" ], [ 240, 242, "\u03b11", "structure_element" ], [ 244, 246, "\u03b12", "structure_element" ], [ 251, 253, "\u03b13", "structure_element" ] ] }, { "sid": 122, "sent": "The loop connecting \u03b22 and \u03b23 contains a single turn of a 310-helix. Helices \u03b11 and \u03b12 are located on one side of the five-stranded \u03b2-sheet while \u03b13 packs against the opposite \u03b2-sheet surface.", "section": "RESULTS", "ner": [ [ 4, 8, "loop", "structure_element" ], [ 20, 22, "\u03b22", "structure_element" ], [ 27, 29, "\u03b23", "structure_element" ], [ 58, 67, "310-helix", "structure_element" ], [ 69, 76, "Helices", "structure_element" ], [ 77, 79, "\u03b11", "structure_element" ], [ 84, 86, "\u03b12", "structure_element" ], [ 118, 139, "five-stranded \u03b2-sheet", "structure_element" ], [ 146, 148, "\u03b13", "structure_element" ], [ 176, 183, "\u03b2-sheet", "structure_element" ] ] }, { "sid": 123, "sent": "The C-terminal domain (aa 93\u2013184) has a globular all \u03b1-helical structure comprising \u03b1-helices \u03b14 to \u03b19.", "section": "RESULTS", "ner": [ [ 4, 21, "C-terminal domain", "structure_element" ], [ 26, 32, "93\u2013184", "residue_range" ], [ 40, 72, "globular all \u03b1-helical structure", "structure_element" ], [ 84, 93, "\u03b1-helices", "structure_element" ], [ 94, 102, "\u03b14 to \u03b19", "structure_element" ] ] }, { "sid": 124, "sent": "Remarkably, the entire C-terminal domain (92 aa) of the protein is threaded through the loop which connects \u03b2-strand \u03b23 and \u03b1-helix \u03b12 of the N-terminal domain.", "section": "RESULTS", "ner": [ [ 23, 40, "C-terminal domain", "structure_element" ], [ 42, 47, "92 aa", "residue_range" ], [ 88, 92, "loop", "structure_element" ], [ 108, 116, "\u03b2-strand", "structure_element" ], [ 117, 119, "\u03b23", "structure_element" ], [ 124, 131, "\u03b1-helix", "structure_element" ], [ 132, 134, "\u03b12", "structure_element" ], [ 142, 159, "N-terminal domain", "structure_element" ] ] }, { "sid": 125, "sent": "Thus, the VdTsr3 structure contains a deep trefoil knot.", "section": "RESULTS", "ner": [ [ 10, 16, "VdTsr3", "protein" ], [ 17, 26, "structure", "evidence" ], [ 38, 55, "deep trefoil knot", "structure_element" ] ] }, { "sid": 126, "sent": "The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent C\u03b1 atoms of 1.1 \u00c5. The only significant difference in the global structure of the two proteins is the presence of an extended \u03b1-helix \u03b18 and the absence of \u03b1-helix \u03b19 in SsTsr3.", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 17, 23, "SsTsr3", "protein" ], [ 31, 34, "apo", "protein_state" ], [ 68, 74, "VdTsr3", "protein" ], [ 95, 99, "RMSD", "evidence" ], [ 180, 189, "structure", "evidence" ], [ 241, 248, "\u03b1-helix", "structure_element" ], [ 249, 251, "\u03b18", "structure_element" ], [ 260, 270, "absence of", "protein_state" ], [ 271, 278, "\u03b1-helix", "structure_element" ], [ 279, 281, "\u03b19", "structure_element" ], [ 285, 291, "SsTsr3", "protein" ] ] }, { "sid": 127, "sent": "Tsr3 has a fold similar to SPOUT-class RNA methyltransferases. (A) Cartoon representation of the X-ray structure of VdTsr3 in two orientations.", "section": "FIG", "ner": [ [ 0, 4, "Tsr3", "protein" ], [ 27, 61, "SPOUT-class RNA methyltransferases", "protein_type" ], [ 97, 112, "X-ray structure", "evidence" ], [ 116, 122, "VdTsr3", "protein" ] ] }, { "sid": 128, "sent": "\u03b2-strands are colored in crimson whereas \u03b1-helices in the N-terminal domain are colored light blue and \u03b1-helices in the C-terminal domain are colored dark blue.", "section": "FIG", "ner": [ [ 0, 9, "\u03b2-strands", "structure_element" ], [ 41, 50, "\u03b1-helices", "structure_element" ], [ 58, 75, "N-terminal domain", "structure_element" ], [ 103, 112, "\u03b1-helices", "structure_element" ], [ 120, 137, "C-terminal domain", "structure_element" ] ] }, { "sid": 129, "sent": "The bound S-adenosylmethionine is shown in a stick representation and colored by atom type.", "section": "FIG", "ner": [ [ 10, 30, "S-adenosylmethionine", "chemical" ] ] }, { "sid": 130, "sent": "A red arrow marks the location of the topological knot in the structure. (B) Secondary structure representation of the VdTsr3 structure.", "section": "FIG", "ner": [ [ 38, 54, "topological knot", "structure_element" ], [ 62, 71, "structure", "evidence" ], [ 119, 125, "VdTsr3", "protein" ], [ 126, 135, "structure", "evidence" ] ] }, { "sid": 131, "sent": "The color coding is the same as in (A). (C) Structural superposition of the X-ray structures of VdTsr3 in the SAM-bound state (red) and SsTsr3 (blue) in the apo state.", "section": "FIG", "ner": [ [ 44, 68, "Structural superposition", "experimental_method" ], [ 76, 92, "X-ray structures", "evidence" ], [ 96, 102, "VdTsr3", "protein" ], [ 110, 119, "SAM-bound", "protein_state" ], [ 136, 142, "SsTsr3", "protein" ], [ 157, 160, "apo", "protein_state" ] ] }, { "sid": 132, "sent": "The locations of the \u03b1-helix \u03b18 which is longer in SsTsr3 and of \u03b1-helix \u03b19 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)\u2014the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution.", "section": "FIG", "ner": [ [ 21, 28, "\u03b1-helix", "structure_element" ], [ 29, 31, "\u03b18", "structure_element" ], [ 51, 57, "SsTsr3", "protein" ], [ 65, 72, "\u03b1-helix", "structure_element" ], [ 73, 75, "\u03b19", "structure_element" ], [ 101, 107, "VdTsr3", "protein" ], [ 165, 173, "S. pombe", "species" ], [ 174, 179, "Trm10", "protein" ], [ 194, 227, "SPOUT-class RNA methyltransferase", "protein_type" ], [ 257, 261, "Tsr3", "protein" ], [ 266, 279, "superposition", "experimental_method" ], [ 287, 293, "VdTsr3", "protein" ], [ 298, 303, "Trm10", "protein" ], [ 304, 320, "X-ray structures", "evidence" ], [ 334, 359, "Analytical gel filtration", "experimental_method" ], [ 360, 368, "profiles", "evidence" ], [ 373, 379, "VdTsr3", "protein" ], [ 390, 396, "SsTsr3", "protein" ], [ 432, 441, "monomeric", "oligomeric_state" ] ] }, { "sid": 133, "sent": "Vd, Vulcanisaeta distributa; Ss, Sulfolobus solfataricus.", "section": "FIG", "ner": [ [ 0, 2, "Vd", "species" ], [ 4, 27, "Vulcanisaeta distributa", "species" ], [ 29, 31, "Ss", "species" ], [ 33, 56, "Sulfolobus solfataricus", "species" ] ] }, { "sid": 134, "sent": "Structure predictions suggested that Tsr3 might contain a so-called RLI domain which contains a \u2018bacterial like\u2019 ferredoxin fold and binds two iron-sulfur clusters through eight conserved cysteine residues.", "section": "RESULTS", "ner": [ [ 0, 21, "Structure predictions", "experimental_method" ], [ 37, 41, "Tsr3", "protein" ], [ 68, 78, "RLI domain", "structure_element" ], [ 97, 128, "bacterial like\u2019 ferredoxin fold", "structure_element" ], [ 178, 187, "conserved", "protein_state" ], [ 188, 196, "cysteine", "residue_name" ] ] }, { "sid": 135, "sent": "However, no structural similarity to an RLI-domain was detectable.", "section": "RESULTS", "ner": [ [ 40, 50, "RLI-domain", "structure_element" ] ] }, { "sid": 136, "sent": "This is in accordance with the functional analysis of alanine replacement mutations of cysteine residues in ScTsr3 (Supplementary Figure S3).", "section": "RESULTS", "ner": [ [ 54, 83, "alanine replacement mutations", "experimental_method" ], [ 87, 95, "cysteine", "residue_name" ], [ 108, 114, "ScTsr3", "protein" ] ] }, { "sid": 137, "sent": "The \u03b2-strand topology and the deep C-terminal trefoil knot of archaeal Tsr3 are the structural hallmarks of the SPOUT-class RNA-methyltransferase fold.", "section": "RESULTS", "ner": [ [ 4, 21, "\u03b2-strand topology", "structure_element" ], [ 46, 58, "trefoil knot", "structure_element" ], [ 62, 70, "archaeal", "taxonomy_domain" ], [ 71, 75, "Tsr3", "protein" ], [ 112, 145, "SPOUT-class RNA-methyltransferase", "protein_type" ] ] }, { "sid": 138, "sent": "The closest structural homolog identified in a DALI search is the tRNA methyltransferase Trm10 (DALI Z-score 6.8) which methylates the N1 nitrogen of G9/A9 in many archaeal and eukaryotic tRNAs by using SAM as the methyl group donor.", "section": "RESULTS", "ner": [ [ 47, 58, "DALI search", "experimental_method" ], [ 66, 88, "tRNA methyltransferase", "protein_type" ], [ 89, 94, "Trm10", "protein" ], [ 96, 108, "DALI Z-score", "evidence" ], [ 150, 152, "G9", "residue_name_number" ], [ 153, 155, "A9", "residue_name_number" ], [ 164, 172, "archaeal", "taxonomy_domain" ], [ 177, 187, "eukaryotic", "taxonomy_domain" ], [ 188, 193, "tRNAs", "chemical" ], [ 203, 206, "SAM", "chemical" ] ] }, { "sid": 139, "sent": "In comparison to Tsr3 the central \u03b2-sheet element of Trm10 is extended by one additional \u03b2-strand pairing to \u03b22.", "section": "RESULTS", "ner": [ [ 17, 21, "Tsr3", "protein" ], [ 34, 49, "\u03b2-sheet element", "structure_element" ], [ 53, 58, "Trm10", "protein" ], [ 89, 97, "\u03b2-strand", "structure_element" ], [ 109, 111, "\u03b22", "structure_element" ] ] }, { "sid": 140, "sent": "Furthermore, the trefoil knot of Trm10 is not as deep as that of Tsr3 (Figure 4D).", "section": "RESULTS", "ner": [ [ 17, 29, "trefoil knot", "structure_element" ], [ 33, 38, "Trm10", "protein" ], [ 65, 69, "Tsr3", "protein" ] ] }, { "sid": 141, "sent": "Interestingly, Nep1\u2014the enzyme preceding Tsr3 in the biosynthetic pathway for the synthesis of m1acp3\u03a8\u2014also belongs to the SPOUT-class of RNA methyltransferases.", "section": "RESULTS", "ner": [ [ 15, 19, "Nep1", "protein" ], [ 41, 45, "Tsr3", "protein" ], [ 95, 102, "m1acp3\u03a8", "chemical" ], [ 123, 160, "SPOUT-class of RNA methyltransferases", "protein_type" ] ] }, { "sid": 142, "sent": "However, the structural similarities between Nep1 and Tsr3 (DALI Z-score 4.4) are less pronounced than between Tsr3 and Trm10.", "section": "RESULTS", "ner": [ [ 45, 49, "Nep1", "protein" ], [ 54, 58, "Tsr3", "protein" ], [ 60, 72, "DALI Z-score", "evidence" ], [ 111, 115, "Tsr3", "protein" ], [ 120, 125, "Trm10", "protein" ] ] }, { "sid": 143, "sent": "Most SPOUT-class RNA-methyltransferases are homodimers.", "section": "RESULTS", "ner": [ [ 5, 39, "SPOUT-class RNA-methyltransferases", "protein_type" ], [ 44, 54, "homodimers", "oligomeric_state" ] ] }, { "sid": 144, "sent": "A notable exception is Trm10.", "section": "RESULTS", "ner": [ [ 23, 28, "Trm10", "protein" ] ] }, { "sid": 145, "sent": "Gel filtration experiments with both VdTsr3 and SsTsr3 (Figure 4E) showed that both proteins are monomeric in solution thereby extending the structural similarities to Trm10.", "section": "RESULTS", "ner": [ [ 0, 14, "Gel filtration", "experimental_method" ], [ 37, 43, "VdTsr3", "protein" ], [ 48, 54, "SsTsr3", "protein" ], [ 97, 106, "monomeric", "oligomeric_state" ], [ 168, 173, "Trm10", "protein" ] ] }, { "sid": 146, "sent": "So far, structural information is only available for one other enzyme that transfers the acp group from SAM to an RNA nucleotide.", "section": "RESULTS", "ner": [ [ 89, 92, "acp", "chemical" ], [ 104, 107, "SAM", "chemical" ], [ 114, 117, "RNA", "chemical" ], [ 118, 128, "nucleotide", "chemical" ] ] }, { "sid": 147, "sent": "This enzyme, Tyw2, is part of the biosynthesis pathway of wybutosine nucleotides in tRNAs.", "section": "RESULTS", "ner": [ [ 13, 17, "Tyw2", "protein" ], [ 58, 80, "wybutosine nucleotides", "chemical" ], [ 84, 89, "tRNAs", "chemical" ] ] }, { "sid": 148, "sent": "However, there are no structural similarities between Tsr3 and Tyw2, which contains an all-parallel \u03b2-sheet of a different topology and no knot structure.", "section": "RESULTS", "ner": [ [ 54, 58, "Tsr3", "protein" ], [ 63, 67, "Tyw2", "protein" ], [ 87, 107, "all-parallel \u03b2-sheet", "structure_element" ], [ 139, 153, "knot structure", "structure_element" ] ] }, { "sid": 149, "sent": "Instead, Tyw2 has a fold typical for the class-I-or Rossmann-fold class of methyltransferases (Supplementary Figure S5B).", "section": "RESULTS", "ner": [ [ 9, 13, "Tyw2", "protein" ], [ 41, 93, "class-I-or Rossmann-fold class of methyltransferases", "protein_type" ] ] }, { "sid": 150, "sent": "Cofactor binding of Tsr3", "section": "RESULTS", "ner": [ [ 20, 24, "Tsr3", "protein" ] ] }, { "sid": 151, "sent": "The SAM-binding site of Tsr3 is located in a deep crevice between the N- and C-terminal domains in the vicinity of the trefoil knot as typical for SPOUT-class RNA-methyltransferases (Figure 4A).", "section": "RESULTS", "ner": [ [ 4, 20, "SAM-binding site", "site" ], [ 24, 28, "Tsr3", "protein" ], [ 70, 95, "N- and C-terminal domains", "structure_element" ], [ 119, 131, "trefoil knot", "structure_element" ], [ 147, 181, "SPOUT-class RNA-methyltransferases", "protein_type" ] ] }, { "sid": 152, "sent": "The adenine base of the cofactor is recognized by hydrogen bonds between its N1 nitrogen and the backbone amide of L93 directly preceding \u03b25 as well as between its N6-amino group and the backbone carbonyl group of Y108 located in the loop connecting \u03b25 in the N-terminal and \u03b14 in the C-terminal domain (Figure 5A).", "section": "RESULTS", "ner": [ [ 4, 11, "adenine", "chemical" ], [ 50, 64, "hydrogen bonds", "bond_interaction" ], [ 115, 118, "L93", "residue_name_number" ], [ 138, 140, "\u03b25", "structure_element" ], [ 214, 218, "Y108", "residue_name_number" ], [ 234, 238, "loop", "structure_element" ], [ 250, 252, "\u03b25", "structure_element" ], [ 260, 270, "N-terminal", "structure_element" ], [ 275, 277, "\u03b14", "structure_element" ], [ 285, 302, "C-terminal domain", "structure_element" ] ] }, { "sid": 153, "sent": "Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (\u03b23), P47 and W73 (\u03b13) in the N-terminal domain as well as with L93, L110 (both in the loop connecting \u03b25 and \u03b14) and A115 (\u03b15) in the C-terminal domain.", "section": "RESULTS", "ner": [ [ 17, 24, "adenine", "chemical" ], [ 33, 36, "SAM", "chemical" ], [ 52, 84, "hydrophobic packing interactions", "bond_interaction" ], [ 109, 112, "L45", "residue_name_number" ], [ 114, 116, "\u03b23", "structure_element" ], [ 119, 122, "P47", "residue_name_number" ], [ 127, 130, "W73", "residue_name_number" ], [ 132, 134, "\u03b13", "structure_element" ], [ 143, 160, "N-terminal domain", "structure_element" ], [ 177, 180, "L93", "residue_name_number" ], [ 182, 186, "L110", "residue_name_number" ], [ 200, 204, "loop", "structure_element" ], [ 216, 218, "\u03b25", "structure_element" ], [ 223, 225, "\u03b14", "structure_element" ], [ 231, 235, "A115", "residue_name_number" ], [ 237, 239, "\u03b15", "structure_element" ], [ 248, 265, "C-terminal domain", "structure_element" ] ] }, { "sid": 154, "sent": "The ribose 2\u2032 and 3\u2032 hydroxyl groups of SAM are hydrogen bonded to the backbone carbonyl group of I69.", "section": "RESULTS", "ner": [ [ 4, 10, "ribose", "chemical" ], [ 40, 43, "SAM", "chemical" ], [ 48, 63, "hydrogen bonded", "bond_interaction" ], [ 98, 101, "I69", "residue_name_number" ] ] }, { "sid": 155, "sent": "The acp side chain of SAM is fixed in position by hydrogen bonding of its carboxylate group to the backbone amide and the side chain hydroxyl group of T19 in \u03b11 as well as the backbone amide group of T112 in \u03b14 (C-terminal domain).", "section": "RESULTS", "ner": [ [ 4, 7, "acp", "chemical" ], [ 22, 25, "SAM", "chemical" ], [ 50, 66, "hydrogen bonding", "bond_interaction" ], [ 151, 154, "T19", "residue_name_number" ], [ 158, 160, "\u03b11", "structure_element" ], [ 200, 204, "T112", "residue_name_number" ], [ 208, 210, "\u03b14", "structure_element" ], [ 212, 229, "C-terminal domain", "structure_element" ] ] }, { "sid": 156, "sent": "Most importantly, the methyl group of SAM is buried in a hydrophobic pocket formed by the sidechains of W73 and A76 both located in \u03b13 (Figure 5A and\u00a0B).", "section": "RESULTS", "ner": [ [ 38, 41, "SAM", "chemical" ], [ 57, 75, "hydrophobic pocket", "site" ], [ 104, 107, "W73", "residue_name_number" ], [ 112, 115, "A76", "residue_name_number" ], [ 132, 134, "\u03b13", "structure_element" ] ] }, { "sid": 157, "sent": "W73 is highly conserved in all known Tsr3 proteins, whereas A76 can be replaced by other hydrophobic amino acids.", "section": "RESULTS", "ner": [ [ 0, 3, "W73", "residue_name_number" ], [ 7, 23, "highly conserved", "protein_state" ], [ 37, 50, "Tsr3 proteins", "protein_type" ], [ 60, 63, "A76", "residue_name_number" ], [ 101, 112, "amino acids", "chemical" ] ] }, { "sid": 158, "sent": "Consequently, the accessibility of this methyl group for a nucleophilic attack is strongly reduced in comparison with RNA-methyltransferases such as Trm10 (Figure 5B, C).", "section": "RESULTS", "ner": [ [ 118, 140, "RNA-methyltransferases", "protein_type" ], [ 149, 154, "Trm10", "protein" ] ] }, { "sid": 159, "sent": "In contrast, the acp side chain of SAM is accessible for reactions in the Tsr3-bound state (Figure 5B).", "section": "RESULTS", "ner": [ [ 17, 20, "acp", "chemical" ], [ 35, 38, "SAM", "chemical" ], [ 74, 84, "Tsr3-bound", "protein_state" ] ] }, { "sid": 160, "sent": "SAM-binding by Tsr3.", "section": "FIG", "ner": [ [ 0, 3, "SAM", "chemical" ], [ 15, 19, "Tsr3", "protein" ] ] }, { "sid": 161, "sent": "(A) Close-up view of the SAM-binding pocket of VdTsr3.", "section": "FIG", "ner": [ [ 25, 43, "SAM-binding pocket", "site" ], [ 47, 53, "VdTsr3", "protein" ] ] }, { "sid": 162, "sent": "Nitrogen atoms are dark blue, oxygen atoms red, sulfur atoms orange, carbon atoms of the protein light blue and carbon atoms of SAM yellow.", "section": "FIG", "ner": [ [ 48, 54, "sulfur", "chemical" ], [ 128, 131, "SAM", "chemical" ] ] }, { "sid": 163, "sent": "Hydrogen bonds are indicated by dashed lines.", "section": "FIG", "ner": [ [ 0, 14, "Hydrogen bonds", "bond_interaction" ] ] }, { "sid": 164, "sent": "(B) Solvent accessibility of the acp group of SAM bound to VdTsr3.", "section": "FIG", "ner": [ [ 33, 36, "acp", "chemical" ], [ 46, 49, "SAM", "chemical" ], [ 50, 58, "bound to", "protein_state" ], [ 59, 65, "VdTsr3", "protein" ] ] }, { "sid": 165, "sent": "The solvent accessible surface of the protein is shown in semitransparent gray whereas SAM is show in a stick representation.", "section": "FIG", "ner": [ [ 87, 90, "SAM", "chemical" ] ] }, { "sid": 166, "sent": "A red arrow indicates the reactive CH2-moiety of the acp group. (C) Solvent accessibility of the SAM methyl group for SAM bound to the RNA methyltransferase Trm10.", "section": "FIG", "ner": [ [ 53, 56, "acp", "chemical" ], [ 97, 100, "SAM", "chemical" ], [ 118, 121, "SAM", "chemical" ], [ 122, 130, "bound to", "protein_state" ], [ 135, 156, "RNA methyltransferase", "protein_type" ], [ 157, 162, "Trm10", "protein" ] ] }, { "sid": 167, "sent": "Bound SAM was modelled based on the X-ray structure of the Trm10/SAH-complex (pdb4jwf).", "section": "FIG", "ner": [ [ 0, 5, "Bound", "protein_state" ], [ 6, 9, "SAM", "chemical" ], [ 36, 51, "X-ray structure", "evidence" ], [ 59, 68, "Trm10/SAH", "complex_assembly" ] ] }, { "sid": 168, "sent": "A red arrow indicates the SAM methyl group. (D) Binding of SAM analogs to SsTsr3.", "section": "FIG", "ner": [ [ 26, 29, "SAM", "chemical" ], [ 59, 62, "SAM", "chemical" ], [ 74, 80, "SsTsr3", "protein" ] ] }, { "sid": 169, "sent": "Tryptophan fluorescence quenching curves upon addition of SAM (blue), 5\u2032-methyl-thioadenosine (red) and SAH (black).", "section": "FIG", "ner": [ [ 0, 40, "Tryptophan fluorescence quenching curves", "evidence" ], [ 58, 61, "SAM", "chemical" ], [ 70, 93, "5\u2032-methyl-thioadenosine", "chemical" ], [ 104, 107, "SAH", "chemical" ] ] }, { "sid": 170, "sent": "(E) Binding of 14C-labeled SAM to SsTsr3.", "section": "FIG", "ner": [ [ 15, 30, "14C-labeled SAM", "chemical" ], [ 34, 40, "SsTsr3", "protein" ] ] }, { "sid": 171, "sent": "Radioactively labeled SAM is retained on a filter in the presence of SsTsr3.", "section": "FIG", "ner": [ [ 22, 25, "SAM", "chemical" ], [ 57, 68, "presence of", "protein_state" ], [ 69, 75, "SsTsr3", "protein" ] ] }, { "sid": 172, "sent": "Addition of unlabeled SAM competes with the binding of labeled SAM.", "section": "FIG", "ner": [ [ 22, 25, "SAM", "chemical" ], [ 63, 66, "SAM", "chemical" ] ] }, { "sid": 173, "sent": "A W66A-mutant of SsTsr3 (W73 in VdTsr3) does not bind SAM.", "section": "FIG", "ner": [ [ 2, 6, "W66A", "mutant" ], [ 7, 13, "mutant", "protein_state" ], [ 17, 23, "SsTsr3", "protein" ], [ 25, 28, "W73", "residue_name_number" ], [ 32, 38, "VdTsr3", "protein" ], [ 54, 57, "SAM", "chemical" ] ] }, { "sid": 174, "sent": "(F) Primer extension (upper left) shows a strongly reduced acp modification of yeast 18S rRNA in \u0394tsr3 cells expressing Tsr3-S62D, -E111A or \u2013W114A.", "section": "FIG", "ner": [ [ 4, 20, "Primer extension", "experimental_method" ], [ 59, 62, "acp", "chemical" ], [ 79, 84, "yeast", "taxonomy_domain" ], [ 85, 93, "18S rRNA", "chemical" ], [ 97, 102, "\u0394tsr3", "mutant" ], [ 120, 129, "Tsr3-S62D", "mutant" ], [ 131, 137, "-E111A", "mutant" ], [ 141, 147, "\u2013W114A", "mutant" ] ] }, { "sid": 175, "sent": "This correlates with a 20S pre-rRNA accumulation comparable to the \u0394tsr3 deletion (right: northern blot).", "section": "FIG", "ner": [ [ 23, 35, "20S pre-rRNA", "chemical" ], [ 67, 72, "\u0394tsr3", "mutant" ], [ 90, 103, "northern blot", "experimental_method" ] ] }, { "sid": 176, "sent": "3xHA tagged Tsr3 mutants are expressed comparable to the wild type as shown by western blot (lower left).", "section": "FIG", "ner": [ [ 0, 11, "3xHA tagged", "protein_state" ], [ 12, 16, "Tsr3", "protein" ], [ 17, 24, "mutants", "protein_state" ], [ 57, 66, "wild type", "protein_state" ], [ 79, 91, "western blot", "experimental_method" ] ] }, { "sid": 177, "sent": "Binding affinities for SAM and its analogs 5\u2032-methylthioadenosin and SAH to SsTsr3 were measured using tryptophan fluorescence quenching.", "section": "RESULTS", "ner": [ [ 0, 18, "Binding affinities", "evidence" ], [ 23, 26, "SAM", "chemical" ], [ 43, 64, "5\u2032-methylthioadenosin", "chemical" ], [ 69, 72, "SAH", "chemical" ], [ 76, 82, "SsTsr3", "protein" ], [ 103, 136, "tryptophan fluorescence quenching", "experimental_method" ] ] }, { "sid": 178, "sent": "VdTsr3 could not be used in these experiments since we could not purify it in a stable SAM-free form.", "section": "RESULTS", "ner": [ [ 0, 6, "VdTsr3", "protein" ], [ 80, 86, "stable", "protein_state" ], [ 87, 95, "SAM-free", "protein_state" ] ] }, { "sid": 179, "sent": "SsTsr3 bound SAM with a KD of 6.5 \u03bcM, which is similar to SAM-KD's reported for several SPOUT-class methyltransferases.", "section": "RESULTS", "ner": [ [ 0, 6, "SsTsr3", "protein" ], [ 7, 12, "bound", "protein_state" ], [ 13, 16, "SAM", "chemical" ], [ 24, 26, "KD", "evidence" ], [ 58, 66, "SAM-KD's", "evidence" ], [ 88, 118, "SPOUT-class methyltransferases", "protein_type" ] ] }, { "sid": 180, "sent": "5\u2032-methylthioadenosin\u2014the reaction product after the acp-transfer\u2014binds only \u223c2.5-fold weaker (KD = 16.7 \u03bcM) compared to SAM.", "section": "RESULTS", "ner": [ [ 0, 21, "5\u2032-methylthioadenosin", "chemical" ], [ 53, 56, "acp", "chemical" ], [ 121, 124, "SAM", "chemical" ] ] }, { "sid": 181, "sent": "S-adenosylhomocysteine which lacks the methyl group of SAM binds with significantly lower affinity (KD = 55.5 \u03bcM) (Figure 5D).", "section": "RESULTS", "ner": [ [ 0, 22, "S-adenosylhomocysteine", "chemical" ], [ 55, 58, "SAM", "chemical" ], [ 90, 98, "affinity", "evidence" ], [ 100, 102, "KD", "evidence" ] ] }, { "sid": 182, "sent": "This suggests that the hydrophobic interaction between SAM's methyl group and the hydrophobic pocket of Tsr3 is thermodynamically important for the interaction.", "section": "RESULTS", "ner": [ [ 23, 46, "hydrophobic interaction", "bond_interaction" ], [ 55, 58, "SAM", "chemical" ], [ 82, 100, "hydrophobic pocket", "site" ], [ 104, 108, "Tsr3", "protein" ] ] }, { "sid": 183, "sent": "On the other hand, the loss of hydrogen bonds between the acp sidechain carboxylate group and the protein appears to be thermodynamically less important but these hydrogen bonds might play a crucial role for the proper orientation of the cofactor side chain in the substrate binding pocket.", "section": "RESULTS", "ner": [ [ 31, 45, "hydrogen bonds", "bond_interaction" ], [ 58, 61, "acp", "chemical" ], [ 163, 177, "hydrogen bonds", "bond_interaction" ], [ 265, 289, "substrate binding pocket", "site" ] ] }, { "sid": 184, "sent": "Accordingly, a W66A-mutation (W73 in VdTsr3) of SsTsr3 significantly diminished SAM-binding in a filter binding assay compared to the wild type (Figure 5E).", "section": "RESULTS", "ner": [ [ 15, 19, "W66A", "mutant" ], [ 20, 28, "mutation", "experimental_method" ], [ 30, 33, "W73", "residue_name_number" ], [ 37, 43, "VdTsr3", "protein" ], [ 48, 54, "SsTsr3", "protein" ], [ 80, 91, "SAM-binding", "evidence" ], [ 97, 117, "filter binding assay", "experimental_method" ], [ 134, 143, "wild type", "protein_state" ] ] }, { "sid": 185, "sent": "Furthermore, a W to A mutation at the equivalent position W114 in ScTsr3 strongly reduced the in vivo acp transferase activity (Figure 5F).", "section": "RESULTS", "ner": [ [ 15, 30, "W to A mutation", "experimental_method" ], [ 58, 62, "W114", "residue_name_number" ], [ 66, 72, "ScTsr3", "protein" ], [ 102, 117, "acp transferase", "protein_type" ] ] }, { "sid": 186, "sent": "The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively).", "section": "RESULTS", "ner": [ [ 33, 36, "T19", "residue_name_number" ], [ 67, 70, "SAM", "chemical" ], [ 85, 94, "mutations", "experimental_method" ], [ 98, 101, "T17", "residue_name_number" ], [ 103, 106, "T19", "residue_name_number" ], [ 110, 116, "VdTsr3", "protein" ], [ 128, 129, "A", "residue_name" ], [ 133, 134, "D", "residue_name" ], [ 171, 191, "SAM-binding affinity", "evidence" ], [ 195, 201, "SsTsr3", "protein" ], [ 203, 205, "KD", "evidence" ] ] }, { "sid": 187, "sent": "Nevertheless, a mutation of the equivalent position S62 of ScTsr3 to D, but not to A, resulted in reduced acp modification in vivo, as shown by primer extension analysis (Figure 5F).", "section": "RESULTS", "ner": [ [ 16, 24, "mutation", "experimental_method" ], [ 52, 55, "S62", "residue_name_number" ], [ 59, 65, "ScTsr3", "protein" ], [ 69, 70, "D", "residue_name" ], [ 83, 84, "A", "residue_name" ], [ 106, 109, "acp", "chemical" ], [ 144, 169, "primer extension analysis", "experimental_method" ] ] }, { "sid": 188, "sent": "The acp-transfer reaction catalyzed by Tsr3 most likely requires the presence of a catalytic base in order to abstract a proton from the N3 imino group of the modified pseudouridine.", "section": "RESULTS", "ner": [ [ 4, 7, "acp", "chemical" ], [ 39, 43, "Tsr3", "protein" ], [ 168, 181, "pseudouridine", "chemical" ] ] }, { "sid": 189, "sent": "The side chain of D70 (VdTsr3) located in \u03b24 is \u223c5 \u00c5 away from the SAM sulfur atom.", "section": "RESULTS", "ner": [ [ 18, 21, "D70", "residue_name_number" ], [ 23, 29, "VdTsr3", "protein" ], [ 42, 44, "\u03b24", "structure_element" ], [ 67, 70, "SAM", "chemical" ] ] }, { "sid": 190, "sent": "This residue is conserved as D or E both in archaeal and eukaryotic Tsr3 homologs.", "section": "RESULTS", "ner": [ [ 16, 28, "conserved as", "protein_state" ], [ 29, 30, "D", "residue_name" ], [ 34, 35, "E", "residue_name" ], [ 44, 52, "archaeal", "taxonomy_domain" ], [ 57, 67, "eukaryotic", "taxonomy_domain" ], [ 68, 72, "Tsr3", "protein" ] ] }, { "sid": 191, "sent": "Mutations of the corresponding residue in SsTsr3 to A (D63) does not significantly alter the SAM-binding affinity of the protein (KD = 11.0 \u03bcM).", "section": "RESULTS", "ner": [ [ 0, 9, "Mutations", "experimental_method" ], [ 42, 48, "SsTsr3", "protein" ], [ 52, 53, "A", "residue_name" ], [ 55, 58, "D63", "residue_name_number" ], [ 93, 113, "SAM-binding affinity", "evidence" ], [ 130, 132, "KD", "evidence" ] ] }, { "sid": 192, "sent": "However, the mutation of the corresponding residue of ScTsr3 (E111A) leads to a significant decrease of the acp transferase activity in vivo (Figure 5F).", "section": "RESULTS", "ner": [ [ 13, 21, "mutation", "experimental_method" ], [ 54, 60, "ScTsr3", "protein" ], [ 62, 67, "E111A", "mutant" ], [ 108, 123, "acp transferase", "protein_type" ] ] }, { "sid": 193, "sent": "RNA-binding of Tsr3", "section": "RESULTS", "ner": [ [ 0, 3, "RNA", "chemical" ], [ 15, 19, "Tsr3", "protein" ] ] }, { "sid": 194, "sent": "Analysis of the electrostatic surface properties of VdTsr3 clearly identified positively charged surface patches in the vicinity of the SAM-binding site suggesting a putative RNA-binding site (Figure 6A).", "section": "RESULTS", "ner": [ [ 0, 48, "Analysis of the electrostatic surface properties", "experimental_method" ], [ 52, 58, "VdTsr3", "protein" ], [ 78, 112, "positively charged surface patches", "site" ], [ 136, 152, "SAM-binding site", "site" ], [ 175, 191, "RNA-binding site", "site" ] ] }, { "sid": 195, "sent": "Furthermore, a negatively charged MES-ion is found in the crystal structure of VdTsr3 complexed to the side chain of K22 in helix \u03b11.", "section": "RESULTS", "ner": [ [ 34, 37, "MES", "chemical" ], [ 58, 75, "crystal structure", "evidence" ], [ 79, 85, "VdTsr3", "protein" ], [ 86, 98, "complexed to", "protein_state" ], [ 117, 120, "K22", "residue_name_number" ], [ 124, 129, "helix", "structure_element" ], [ 130, 132, "\u03b11", "structure_element" ] ] }, { "sid": 196, "sent": "Its negatively charged sulfate group might mimic an RNA backbone phosphate.", "section": "RESULTS", "ner": [ [ 23, 30, "sulfate", "chemical" ], [ 52, 55, "RNA", "chemical" ] ] }, { "sid": 197, "sent": "Helix \u03b11 contains two more positively charged amino acids K17 and R25 as does the loop preceding it (R9).", "section": "RESULTS", "ner": [ [ 0, 5, "Helix", "structure_element" ], [ 6, 8, "\u03b11", "structure_element" ], [ 58, 61, "K17", "residue_name_number" ], [ 66, 69, "R25", "residue_name_number" ], [ 82, 86, "loop", "structure_element" ], [ 101, 103, "R9", "residue_name_number" ] ] }, { "sid": 198, "sent": "A second cluster of positively charged residues is found in or near helix \u03b13 (K74, R75, K82, R85 and K87).", "section": "RESULTS", "ner": [ [ 68, 73, "helix", "structure_element" ], [ 74, 76, "\u03b13", "structure_element" ], [ 78, 81, "K74", "residue_name_number" ], [ 83, 86, "R75", "residue_name_number" ], [ 88, 91, "K82", "residue_name_number" ], [ 93, 96, "R85", "residue_name_number" ], [ 101, 104, "K87", "residue_name_number" ] ] }, { "sid": 199, "sent": "Some of these amino acids are conserved between archaeal and eukaryotic Tsr3 (Supplementary Figure S1A).", "section": "RESULTS", "ner": [ [ 30, 39, "conserved", "protein_state" ], [ 48, 56, "archaeal", "taxonomy_domain" ], [ 61, 71, "eukaryotic", "taxonomy_domain" ], [ 72, 76, "Tsr3", "protein" ] ] }, { "sid": 200, "sent": "In the C-terminal domain, the surface exposed \u03b1-helices \u03b15 and \u03b17 carry a significant amount of positively charged amino acids.", "section": "RESULTS", "ner": [ [ 7, 24, "C-terminal domain", "structure_element" ], [ 46, 55, "\u03b1-helices", "structure_element" ], [ 56, 58, "\u03b15", "structure_element" ], [ 63, 65, "\u03b17", "structure_element" ] ] }, { "sid": 201, "sent": "A triple mutation of the conserved positively charged residues R60, K65 and R131 to A in ScTsr3 resulted in a protein with a significantly impaired acp transferase activity in vivo (Figure 6D) in line with an important functional role for these positively charged residues.", "section": "RESULTS", "ner": [ [ 2, 17, "triple mutation", "experimental_method" ], [ 25, 34, "conserved", "protein_state" ], [ 63, 66, "R60", "residue_name_number" ], [ 68, 71, "K65", "residue_name_number" ], [ 76, 80, "R131", "residue_name_number" ], [ 84, 85, "A", "residue_name" ], [ 89, 95, "ScTsr3", "protein" ], [ 148, 163, "acp transferase", "protein_type" ] ] }, { "sid": 202, "sent": "RNA-binding of Tsr3.", "section": "FIG", "ner": [ [ 15, 19, "Tsr3", "protein" ] ] }, { "sid": 203, "sent": "(A) Electrostatic charge distribution on the surface of VdTsr3.", "section": "FIG", "ner": [ [ 56, 62, "VdTsr3", "protein" ] ] }, { "sid": 204, "sent": "SAM is shown in a stick representation.", "section": "FIG", "ner": [ [ 0, 3, "SAM", "chemical" ] ] }, { "sid": 205, "sent": "Also shown in stick representation is a negatively charged MES ion.", "section": "FIG", "ner": [ [ 59, 62, "MES", "chemical" ] ] }, { "sid": 206, "sent": "Conserved basic amino acids are labeled. (B) Comparison of the secondary structures of helix 31 from the small ribosomal subunit rRNAs in S. cerevisiae and S. solfataricus with the location of the hypermodified nucleotide indicated in red.", "section": "FIG", "ner": [ [ 0, 9, "Conserved", "protein_state" ], [ 16, 27, "amino acids", "chemical" ], [ 87, 95, "helix 31", "structure_element" ], [ 129, 134, "rRNAs", "chemical" ], [ 138, 151, "S. cerevisiae", "species" ], [ 156, 171, "S. solfataricus", "species" ], [ 197, 210, "hypermodified", "protein_state" ], [ 211, 221, "nucleotide", "chemical" ] ] }, { "sid": 207, "sent": "For S. solfataricus the chemical identity of the hypermodified nucleotide is not known but the existence of NEP1 and TSR3 homologs suggest that it is indeed N1-methyl-N3-acp-pseudouridine.", "section": "FIG", "ner": [ [ 4, 19, "S. solfataricus", "species" ], [ 49, 62, "hypermodified", "protein_state" ], [ 63, 73, "nucleotide", "chemical" ], [ 108, 112, "NEP1", "protein" ], [ 117, 121, "TSR3", "protein" ], [ 157, 187, "N1-methyl-N3-acp-pseudouridine", "chemical" ] ] }, { "sid": 208, "sent": "(C) Binding of SsTsr3 to RNA.", "section": "FIG", "ner": [ [ 15, 21, "SsTsr3", "protein" ], [ 25, 28, "RNA", "chemical" ] ] }, { "sid": 209, "sent": "5\u2032-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer \u2013 see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer \u2013 red, 20mer_GC \u2013 blue).", "section": "FIG", "ner": [ [ 3, 15, "fluoresceine", "chemical" ], [ 24, 27, "RNA", "chemical" ], [ 73, 79, "native", "protein_state" ], [ 81, 86, "20mer", "oligomeric_state" ], [ 105, 115, "stabilized", "protein_state" ], [ 117, 125, "20mer_GC", "oligomeric_state" ], [ 135, 143, "helix 31", "structure_element" ], [ 175, 179, "rRNA", "chemical" ], [ 185, 200, "S. solfataricus", "species" ], [ 206, 238, "titrated with increasing amounts", "experimental_method" ], [ 242, 248, "SsTsr3", "protein" ], [ 272, 284, "fluoresceine", "chemical" ], [ 285, 308, "fluorescence anisotropy", "evidence" ], [ 339, 352, "binding curve", "evidence" ], [ 354, 359, "20mer", "oligomeric_state" ], [ 367, 375, "20mer_GC", "oligomeric_state" ] ] }, { "sid": 210, "sent": "Oligo-U9-RNA was used for comparison (black).", "section": "FIG", "ner": [ [ 0, 12, "Oligo-U9-RNA", "chemical" ] ] }, { "sid": 211, "sent": "The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in \u0394tsr3 yeast cells show a primer extension stop comparable to the wild type.", "section": "FIG", "ner": [ [ 4, 12, "20mer_GC", "oligomeric_state" ], [ 13, 16, "RNA", "chemical" ], [ 26, 34, "titrated", "experimental_method" ], [ 40, 46, "SsTsr3", "protein" ], [ 71, 74, "SAM", "chemical" ], [ 89, 96, "Mutants", "protein_state" ], [ 100, 106, "ScTsr3", "protein" ], [ 107, 110, "R60", "residue_name_number" ], [ 112, 115, "K65", "residue_name_number" ], [ 119, 123, "R131", "residue_name_number" ], [ 139, 142, "K17", "residue_name_number" ], [ 144, 147, "K22", "residue_name_number" ], [ 152, 155, "R91", "residue_name_number" ], [ 159, 165, "VdTsr3", "protein" ], [ 167, 176, "expressed", "experimental_method" ], [ 180, 185, "\u0394tsr3", "mutant" ], [ 186, 191, "yeast", "taxonomy_domain" ], [ 205, 226, "primer extension stop", "evidence" ], [ 245, 254, "wild type", "protein_state" ] ] }, { "sid": 212, "sent": "Combination of the three point mutations (R60A/K65A/R131A) leads to a strongly reduced acp modification of 18S rRNA.", "section": "FIG", "ner": [ [ 0, 40, "Combination of the three point mutations", "experimental_method" ], [ 42, 46, "R60A", "mutant" ], [ 47, 51, "K65A", "mutant" ], [ 52, 57, "R131A", "mutant" ], [ 87, 90, "acp", "chemical" ], [ 107, 115, "18S rRNA", "chemical" ] ] }, { "sid": 213, "sent": "In order to explore the RNA-ligand specificity of Tsr3 we titrated SsTsr3 prepared in RNase-free form with 5\u2032-fluoresceine-labeled RNA and determined the affinity by fluorescence anisotropy measurements.", "section": "RESULTS", "ner": [ [ 50, 54, "Tsr3", "protein" ], [ 58, 66, "titrated", "experimental_method" ], [ 67, 73, "SsTsr3", "protein" ], [ 86, 96, "RNase-free", "protein_state" ], [ 110, 122, "fluoresceine", "chemical" ], [ 131, 134, "RNA", "chemical" ], [ 154, 162, "affinity", "evidence" ], [ 166, 202, "fluorescence anisotropy measurements", "experimental_method" ] ] }, { "sid": 214, "sent": "SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 \u03bcM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 \u03bcM (Figure 6C).", "section": "RESULTS", "ner": [ [ 0, 6, "SsTsr3", "protein" ], [ 14, 17, "apo", "protein_state" ], [ 24, 29, "bound", "protein_state" ], [ 32, 37, "20mer", "oligomeric_state" ], [ 38, 41, "RNA", "chemical" ], [ 59, 67, "helix 31", "structure_element" ], [ 71, 86, "S. solfataricus", "species" ], [ 87, 95, "16S rRNA", "chemical" ], [ 115, 117, "KD", "evidence" ], [ 153, 160, "hairpin", "structure_element" ], [ 201, 209, "20mer-GC", "oligomeric_state" ], [ 218, 220, "KD", "evidence" ] ] }, { "sid": 215, "sent": "A single stranded oligoU-RNA bound with a 10-fold-reduced affinity (6.0 \u03bcM).", "section": "RESULTS", "ner": [ [ 18, 28, "oligoU-RNA", "chemical" ], [ 29, 34, "bound", "protein_state" ], [ 58, 66, "affinity", "evidence" ] ] }, { "sid": 216, "sent": "The presence of saturating amounts of SAM (2 mM) did not have a significant influence on the RNA-affinity of SsTsr3 (KD of 1.7 \u03bcM for the 20mer-GC-RNA) suggesting no cooperativity in substrate binding.", "section": "RESULTS", "ner": [ [ 38, 41, "SAM", "chemical" ], [ 93, 105, "RNA-affinity", "evidence" ], [ 109, 115, "SsTsr3", "protein" ], [ 117, 119, "KD", "evidence" ], [ 138, 146, "20mer-GC", "oligomeric_state" ], [ 147, 150, "RNA", "chemical" ] ] }, { "sid": 217, "sent": "U1191 is the only hypermodified base in the yeast 18S rRNA and is strongly conserved in eukaryotes.", "section": "DISCUSS", "ner": [ [ 0, 5, "U1191", "residue_name_number" ], [ 18, 31, "hypermodified", "protein_state" ], [ 44, 49, "yeast", "taxonomy_domain" ], [ 50, 58, "18S rRNA", "chemical" ], [ 66, 84, "strongly conserved", "protein_state" ], [ 88, 98, "eukaryotes", "taxonomy_domain" ] ] }, { "sid": 218, "sent": "The formation of 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3\u03a8) is very complex requiring three successive modification reactions involving one H/ACA snoRNP (snR35) and two protein enzymes (Nep1/Emg1 and Tsr3).", "section": "DISCUSS", "ner": [ [ 17, 67, "1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine", "chemical" ], [ 69, 76, "m1acp3\u03a8", "chemical" ], [ 158, 163, "H/ACA", "structure_element" ], [ 164, 170, "snoRNP", "complex_assembly" ], [ 172, 177, "snR35", "protein" ], [ 204, 208, "Nep1", "protein" ], [ 209, 213, "Emg1", "protein" ], [ 218, 222, "Tsr3", "protein" ] ] }, { "sid": 219, "sent": "This makes it unique in eukaryotic rRNA modification.", "section": "DISCUSS", "ner": [ [ 24, 34, "eukaryotic", "taxonomy_domain" ], [ 35, 39, "rRNA", "chemical" ] ] }, { "sid": 220, "sent": "The m1acp3\u03a8 base is located at the tip of helix 31 on the 18S rRNA (Supplementary Figure S1B) which, together with helices 18, 24, 34 and 44, contribute to building the decoding center of the small ribosomal subunit.", "section": "DISCUSS", "ner": [ [ 4, 11, "m1acp3\u03a8", "chemical" ], [ 42, 50, "helix 31", "structure_element" ], [ 58, 66, "18S rRNA", "chemical" ], [ 115, 140, "helices 18, 24, 34 and 44", "structure_element" ] ] }, { "sid": 221, "sent": "A similar modification (acp3U) was identified in Haloferax volcanii and corresponding modified nucleotides were also shown to occur in other archaea.", "section": "DISCUSS", "ner": [ [ 24, 29, "acp3U", "chemical" ], [ 49, 67, "Haloferax volcanii", "species" ], [ 95, 106, "nucleotides", "chemical" ], [ 141, 148, "archaea", "taxonomy_domain" ] ] }, { "sid": 222, "sent": "As shown here TSR3 encodes the transferase catalyzing the acp modification as the last step in the biosynthesis of m1acp3\u03a8 in yeast and human cells.", "section": "DISCUSS", "ner": [ [ 14, 18, "TSR3", "protein" ], [ 58, 61, "acp", "chemical" ], [ 115, 122, "m1acp3\u03a8", "chemical" ], [ 126, 131, "yeast", "taxonomy_domain" ], [ 136, 141, "human", "species" ] ] }, { "sid": 223, "sent": "Unexpectedly, archaeal Tsr3 has a structure similar to SPOUT-class RNA methyltransferases, and it is the first example for an enzyme of this class transferring an acp group, due to a modified SAM-binding pocket that exposes the acp instead of the methyl group of SAM to its RNA substrate.", "section": "DISCUSS", "ner": [ [ 14, 22, "archaeal", "taxonomy_domain" ], [ 23, 27, "Tsr3", "protein" ], [ 34, 43, "structure", "evidence" ], [ 55, 89, "SPOUT-class RNA methyltransferases", "protein_type" ], [ 163, 166, "acp", "chemical" ], [ 192, 210, "SAM-binding pocket", "site" ], [ 228, 231, "acp", "chemical" ], [ 263, 266, "SAM", "chemical" ], [ 274, 277, "RNA", "chemical" ] ] }, { "sid": 224, "sent": "Similar to the structurally unrelated Rossmann-fold Tyw2 acp transferase, the SAM methyl group of Tsr3 is bound in an inaccessible hydrophobic pocket whereas the acp side chain becomes accessible for a nucleophilic attack by the N3 of pseudouridine.", "section": "DISCUSS", "ner": [ [ 38, 72, "Rossmann-fold Tyw2 acp transferase", "protein_type" ], [ 78, 81, "SAM", "chemical" ], [ 98, 102, "Tsr3", "protein" ], [ 131, 149, "hydrophobic pocket", "site" ], [ 162, 165, "acp", "chemical" ], [ 235, 248, "pseudouridine", "chemical" ] ] }, { "sid": 225, "sent": "In contrast, in the structurally closely related RNA methyltransferase Trm10 the methyl group of the cofactor SAM is accessible whereas its acp side chain is buried inside the protein.", "section": "DISCUSS", "ner": [ [ 49, 70, "RNA methyltransferase", "protein_type" ], [ 71, 76, "Trm10", "protein" ], [ 110, 113, "SAM", "chemical" ], [ 140, 143, "acp", "chemical" ] ] }, { "sid": 226, "sent": "This suggests that enzymes with a SAM-dependent acp transferase activity might have evolved from SAM-dependent methyltransferases by slight modifications of the SAM-binding pocket.", "section": "DISCUSS", "ner": [ [ 34, 63, "SAM-dependent acp transferase", "protein_type" ], [ 97, 129, "SAM-dependent methyltransferases", "protein_type" ], [ 161, 179, "SAM-binding pocket", "site" ] ] }, { "sid": 227, "sent": "Thus, additional examples for acp transferase enzymes might be found with similarities to other structural classes of methyltransferases.", "section": "DISCUSS", "ner": [ [ 30, 45, "acp transferase", "protein_type" ], [ 118, 136, "methyltransferases", "protein_type" ] ] }, { "sid": 228, "sent": "In contrast to Nep1, the enzyme preceding Tsr3 in the m1acp3\u03a8 biosynthesis pathway, Tsr3 binds rather weakly and with little specificity to its isolated substrate RNA.", "section": "DISCUSS", "ner": [ [ 15, 19, "Nep1", "protein" ], [ 42, 46, "Tsr3", "protein" ], [ 54, 61, "m1acp3\u03a8", "chemical" ], [ 84, 88, "Tsr3", "protein" ], [ 163, 166, "RNA", "chemical" ] ] }, { "sid": 229, "sent": "This suggests that Tsr3 is not stably incorporated into pre-ribosomal particles and that its binding to the nascent ribosomal subunit possibly requires additional interactions with other pre-ribosomal components.", "section": "DISCUSS", "ner": [ [ 19, 23, "Tsr3", "protein" ], [ 56, 79, "pre-ribosomal particles", "complex_assembly" ], [ 116, 133, "ribosomal subunit", "complex_assembly" ] ] }, { "sid": 230, "sent": "Consistently, in sucrose gradient analysis, Tsr3 was found in low-molecular weight fractions rather than with pre-ribosome containing high-molecular weight fractions.", "section": "DISCUSS", "ner": [ [ 17, 42, "sucrose gradient analysis", "experimental_method" ], [ 44, 48, "Tsr3", "protein" ], [ 110, 122, "pre-ribosome", "complex_assembly" ] ] }, { "sid": 231, "sent": "In contrast to several enzymes that catalyze base specific modifications in rRNAs Tsr3 is not an essential protein.", "section": "DISCUSS", "ner": [ [ 76, 81, "rRNAs", "chemical" ], [ 82, 86, "Tsr3", "protein" ] ] }, { "sid": 232, "sent": "Typically, other small subunit rRNA methyltransferases as Dim1, Bud23 and Nep1/Emg1 carry dual functions, in ribosome biogenesis and rRNA modification, and it is their involvement in pre-RNA processing that is essential rather than their RNA-methylating activity (, discussed in 7).", "section": "DISCUSS", "ner": [ [ 17, 54, "small subunit rRNA methyltransferases", "protein_type" ], [ 58, 62, "Dim1", "protein" ], [ 64, 69, "Bud23", "protein" ], [ 74, 78, "Nep1", "protein" ], [ 79, 83, "Emg1", "protein" ], [ 133, 137, "rRNA", "chemical" ], [ 183, 190, "pre-RNA", "chemical" ] ] }, { "sid": 233, "sent": "In contrast, for several Tsr3 mutants (SAM-binding and cysteine mutations) we found a systematic correlation between the loss of acp modification and the efficiency of 18S rRNA maturation.", "section": "DISCUSS", "ner": [ [ 25, 29, "Tsr3", "protein" ], [ 39, 50, "SAM-binding", "protein_state" ], [ 55, 73, "cysteine mutations", "protein_state" ], [ 129, 132, "acp", "chemical" ], [ 168, 176, "18S rRNA", "chemical" ] ] }, { "sid": 234, "sent": "This demonstrates that, unlike the other small subunit rRNA base modifications, the acp modification is required for efficient pre-rRNA processing.", "section": "DISCUSS", "ner": [ [ 55, 59, "rRNA", "chemical" ], [ 84, 87, "acp", "chemical" ], [ 127, 135, "pre-rRNA", "chemical" ] ] }, { "sid": 235, "sent": "Recently, structural, functional, and CRAC (cross-linking and cDNA analysis) experiments of late assembly factors involved in cytoplasmic processing of 40S subunits, along with cryo-EM studies of the late pre-40S subunits have provided important insights into late pre-40S processing.", "section": "DISCUSS", "ner": [ [ 10, 88, "structural, functional, and CRAC (cross-linking and cDNA analysis) experiments", "experimental_method" ], [ 152, 164, "40S subunits", "complex_assembly" ], [ 177, 184, "cryo-EM", "experimental_method" ], [ 200, 204, "late", "protein_state" ], [ 205, 221, "pre-40S subunits", "complex_assembly" ], [ 265, 272, "pre-40S", "complex_assembly" ] ] }, { "sid": 236, "sent": "Apart from most of the ribosomal proteins, cytoplasmic pre-40S particles contain 20S rRNA and at least seven non-ribosomal proteins including the D-site endonuclease Nob1 as well as Tsr1, a putative GTPase and Rio2 which block the mRNA channel and the initiator tRNA binding site, respectively, thus preventing translation initiation.", "section": "DISCUSS", "ner": [ [ 55, 72, "pre-40S particles", "complex_assembly" ], [ 81, 89, "20S rRNA", "chemical" ], [ 109, 131, "non-ribosomal proteins", "protein_type" ], [ 146, 165, "D-site endonuclease", "protein_type" ], [ 166, 170, "Nob1", "protein" ], [ 182, 186, "Tsr1", "protein" ], [ 199, 205, "GTPase", "protein_type" ], [ 210, 214, "Rio2", "protein" ], [ 231, 243, "mRNA channel", "site" ], [ 252, 279, "initiator tRNA binding site", "site" ] ] }, { "sid": 237, "sent": "After structural changes, possibly driven by GTP hydrolysis, which go together with the formation of the decoding site, the 20S pre-rRNA becomes accessible for Nob1 cleavage at site D. This also involves joining of pre-40S and 60S subunits to 80S-like particles in a translation-like cycle promoted by eIF5B.", "section": "DISCUSS", "ner": [ [ 45, 48, "GTP", "chemical" ], [ 105, 118, "decoding site", "site" ], [ 124, 136, "20S pre-rRNA", "chemical" ], [ 160, 164, "Nob1", "protein" ], [ 177, 183, "site D", "site" ], [ 215, 222, "pre-40S", "complex_assembly" ], [ 227, 239, "60S subunits", "complex_assembly" ], [ 243, 261, "80S-like particles", "complex_assembly" ], [ 302, 307, "eIF5B", "protein" ] ] }, { "sid": 238, "sent": "The cleavage step most likely acts as a quality control check that ensures the proper 40S subunit assembly with only completely processed precursors.", "section": "DISCUSS", "ner": [ [ 86, 97, "40S subunit", "complex_assembly" ] ] }, { "sid": 239, "sent": "Finally, termination factor Rli1, an ATPase, promotes the dissociation of assembly factors and the 80S-like complex dissociates and releases the mature 40S subunit.", "section": "DISCUSS", "ner": [ [ 9, 27, "termination factor", "protein_type" ], [ 28, 32, "Rli1", "protein" ], [ 37, 43, "ATPase", "protein_type" ], [ 99, 115, "80S-like complex", "complex_assembly" ], [ 145, 151, "mature", "protein_state" ], [ 152, 163, "40S subunit", "complex_assembly" ] ] }, { "sid": 240, "sent": "Interestingly, differences in the level of acp modification were demonstrated for different steps of the cytoplasmic pre-40S subunit maturation after analyzing purified 20S pre-rRNAs using different purification bait proteins.", "section": "DISCUSS", "ner": [ [ 43, 46, "acp", "chemical" ], [ 117, 132, "pre-40S subunit", "complex_assembly" ], [ 169, 182, "20S pre-rRNAs", "chemical" ] ] }, { "sid": 241, "sent": "Early cytoplasmic pre-40S subunits still containing the ribosome assembly factors Tsr1, Ltv1, Enp1 and Rio2 were not or only partially acp modified.", "section": "DISCUSS", "ner": [ [ 18, 34, "pre-40S subunits", "complex_assembly" ], [ 56, 81, "ribosome assembly factors", "protein_type" ], [ 82, 86, "Tsr1", "protein" ], [ 88, 92, "Ltv1", "protein" ], [ 94, 98, "Enp1", "protein" ], [ 103, 107, "Rio2", "protein" ], [ 135, 147, "acp modified", "protein_state" ] ] }, { "sid": 242, "sent": "In contrast, late pre-40S subunits containing Nob1 and Rio1 or already associated with 60S subunits in 80S-like particles showed acp modification levels comparable to mature 40S subunits.", "section": "DISCUSS", "ner": [ [ 18, 34, "pre-40S subunits", "complex_assembly" ], [ 46, 50, "Nob1", "protein" ], [ 55, 59, "Rio1", "protein" ], [ 87, 99, "60S subunits", "complex_assembly" ], [ 103, 121, "80S-like particles", "complex_assembly" ], [ 129, 132, "acp", "chemical" ], [ 167, 173, "mature", "protein_state" ], [ 174, 186, "40S subunits", "complex_assembly" ] ] }, { "sid": 243, "sent": "Thus, the acp transfer to m1\u03a81191 occurs during the step at which Rio2 leaves the pre-40S particle.", "section": "DISCUSS", "ner": [ [ 10, 13, "acp", "chemical" ], [ 26, 33, "m1\u03a81191", "residue_name_number" ], [ 66, 70, "Rio2", "protein" ], [ 82, 98, "pre-40S particle", "complex_assembly" ] ] }, { "sid": 244, "sent": "These data and the finding that a missing acp modification hinders pre-20S rRNA processing, suggest that the acp modification together with the release of Rio2 promotes the formation of the decoding site and thus D-site cleavage by Nob1.", "section": "DISCUSS", "ner": [ [ 42, 45, "acp", "chemical" ], [ 67, 79, "pre-20S rRNA", "chemical" ], [ 109, 112, "acp", "chemical" ], [ 155, 159, "Rio2", "protein" ], [ 190, 203, "decoding site", "site" ], [ 213, 219, "D-site", "site" ], [ 232, 236, "Nob1", "protein" ] ] }, { "sid": 245, "sent": "The interrelation between acp modification and Rio2 release is also supported by CRAC analysis showing that Rio2 binds to helix 31 next to the \u03a81191 residue that receives the acp modification.", "section": "DISCUSS", "ner": [ [ 26, 29, "acp", "chemical" ], [ 47, 51, "Rio2", "protein" ], [ 81, 94, "CRAC analysis", "experimental_method" ], [ 108, 112, "Rio2", "protein" ], [ 122, 130, "helix 31", "structure_element" ], [ 143, 148, "\u03a81191", "residue_name_number" ], [ 175, 178, "acp", "chemical" ] ] }, { "sid": 246, "sent": "Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1\u03a81191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA.", "section": "DISCUSS", "ner": [ [ 11, 15, "Rio2", "protein" ], [ 44, 48, "Tsr3", "protein" ], [ 52, 60, "helix 31", "structure_element" ], [ 66, 69, "acp", "chemical" ], [ 104, 108, "Rio2", "protein" ], [ 129, 132, "acp", "chemical" ], [ 149, 156, "m1\u03a81191", "residue_name_number" ], [ 207, 215, "20S rRNA", "chemical" ], [ 238, 242, "Rio2", "protein" ], [ 246, 254, "helix 31", "structure_element" ], [ 288, 296, "pre-rRNA", "chemical" ] ] }, { "sid": 247, "sent": "In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3\u03a8 nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications.", "section": "DISCUSS", "ner": [ [ 27, 31, "Tsr3", "protein" ], [ 78, 81, "acp", "chemical" ], [ 95, 108, "hypermodified", "protein_state" ], [ 109, 116, "m1acp3\u03a8", "chemical" ], [ 117, 127, "nucleotide", "chemical" ], [ 140, 144, "1191", "residue_number" ], [ 146, 151, "yeast", "taxonomy_domain" ], [ 154, 158, "1248", "residue_number" ], [ 160, 166, "humans", "species" ], [ 171, 179, "18S rRNA", "chemical" ], [ 239, 249, "eukaryotic", "taxonomy_domain" ], [ 250, 278, "small ribosomal subunit rRNA", "chemical" ] ] }, { "sid": 248, "sent": "The current data together with the finding that acp modification takes place at the very last step in pre-40S subunit maturation indicate that the acp modification probably supports the formation of the decoding site and efficient 20S pre-rRNA D-site cleavage.", "section": "DISCUSS", "ner": [ [ 48, 51, "acp", "chemical" ], [ 102, 117, "pre-40S subunit", "complex_assembly" ], [ 147, 150, "acp", "chemical" ], [ 203, 216, "decoding site", "site" ], [ 231, 243, "20S pre-rRNA", "chemical" ], [ 244, 250, "D-site", "site" ] ] }, { "sid": 249, "sent": "Furthermore, our structural data unravelled how the regioselectivity of SAM-dependent group transfer reactions can be tuned by distinct small evolutionary adaptions of the ligand binding pocket of SAM-binding enzymes.", "section": "DISCUSS", "ner": [ [ 17, 32, "structural data", "evidence" ], [ 72, 75, "SAM", "chemical" ], [ 172, 193, "ligand binding pocket", "site" ], [ 197, 216, "SAM-binding enzymes", "protein_type" ] ] } ] }, "PMC4887326": { "annotations": [ { "sid": 0, "sent": "Structural insights into the regulatory mechanism of the Pseudomonas aeruginosa YfiBNR system", "section": "TITLE", "ner": [ [ 57, 79, "Pseudomonas aeruginosa", "species" ], [ 80, 86, "YfiBNR", "complex_assembly" ] ] }, { "sid": 1, "sent": "YfiBNR is a recently identified bis-(3\u2019-5\u2019)-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens.", "section": "ABSTRACT", "ner": [ [ 0, 6, "YfiBNR", "complex_assembly" ], [ 32, 62, "bis-(3\u2019-5\u2019)-cyclic dimeric GMP", "chemical" ], [ 64, 72, "c-di-GMP", "chemical" ] ] }, { "sid": 2, "sent": "In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production.", "section": "ABSTRACT", "ner": [ [ 28, 32, "YfiB", "protein" ], [ 93, 97, "YfiR", "protein" ], [ 127, 131, "YfiN", "protein" ], [ 209, 213, "YfiN", "protein" ], [ 224, 232, "c-di-GMP", "chemical" ] ] }, { "sid": 3, "sent": "Here, we report the crystal\u00a0structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry.", "section": "ABSTRACT", "ner": [ [ 20, 38, "crystal\u00a0structures", "evidence" ], [ 42, 46, "YfiB", "protein" ], [ 47, 52, "alone", "protein_state" ], [ 63, 69, "active", "protein_state" ], [ 70, 76, "mutant", "protein_state" ], [ 77, 85, "YfiBL43P", "mutant" ], [ 86, 100, "complexed with", "protein_state" ], [ 101, 105, "YfiR", "protein" ] ] }, { "sid": 4, "sent": "Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the\u00a0peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation.", "section": "ABSTRACT", "ner": [ [ 0, 19, "Structural analyses", "experimental_method" ], [ 53, 73, "compact conformation", "protein_state" ], [ 81, 88, "dimeric", "oligomeric_state" ], [ 89, 93, "YfiB", "protein" ], [ 94, 99, "alone", "protein_state" ], [ 101, 109, "YfiBL43P", "mutant" ], [ 119, 141, "stretched conformation", "protein_state" ], [ 151, 160, "activated", "protein_state" ], [ 161, 165, "YfiB", "protein" ], [ 183, 196, "peptidoglycan", "chemical" ], [ 198, 200, "PG", "chemical" ], [ 219, 223, "YfiR", "protein" ], [ 225, 233, "YfiBL43P", "mutant" ], [ 255, 272, "PG-binding pocket", "site" ], [ 289, 308, "PG binding affinity", "evidence" ], [ 314, 323, "wild-type", "protein_state" ], [ 324, 328, "YfiB", "protein" ], [ 384, 388, "YfiB", "protein" ] ] }, { "sid": 5, "sent": "In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation.", "section": "ABSTRACT", "ner": [ [ 17, 42, "crystallographic analyses", "experimental_method" ], [ 57, 61, "YfiR", "protein" ], [ 68, 78, "Vitamin B6", "chemical" ], [ 80, 83, "VB6", "chemical" ], [ 88, 93, "L-Trp", "chemical" ], [ 99, 116, "YfiB-binding site", "site" ], [ 131, 134, "VB6", "chemical" ], [ 139, 144, "L-Trp", "chemical" ], [ 164, 172, "YfiBL43P", "mutant" ] ] }, { "sid": 6, "sent": "Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system.", "section": "ABSTRACT", "ner": [ [ 13, 44, "structural and biochemical data", "evidence" ], [ 92, 98, "YfiBNR", "complex_assembly" ] ] }, { "sid": 7, "sent": "Bis-(3\u2019-5\u2019)-cyclic dimeric GMP (c-di-GMP) is a ubiquitous second messenger that bacteria use to facilitate behavioral adaptations to their ever-changing environment.", "section": "INTRO", "ner": [ [ 0, 30, "Bis-(3\u2019-5\u2019)-cyclic dimeric GMP", "chemical" ], [ 32, 40, "c-di-GMP", "chemical" ], [ 80, 88, "bacteria", "taxonomy_domain" ] ] }, { "sid": 8, "sent": "An increase in c-di-GMP promotes biofilm formation, and a decrease results in biofilm degradation (Boehm et al.,; Duerig et al.,; Hickman et al.,; Jenal,; Romling et al.,).", "section": "INTRO", "ner": [ [ 15, 23, "c-di-GMP", "chemical" ] ] }, { "sid": 9, "sent": "The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,).", "section": "INTRO", "ner": [ [ 4, 12, "c-di-GMP", "chemical" ], [ 74, 94, "diguanylate cyclases", "protein_type" ], [ 96, 100, "DGCs", "protein_type" ], [ 118, 126, "c-di-GMP", "chemical" ], [ 131, 149, "phosphodiesterases", "protein_type" ], [ 151, 155, "PDEs", "protein_type" ], [ 172, 180, "c-di-GMP", "chemical" ], [ 304, 321, "N-terminal domain", "structure_element" ], [ 400, 420, "relatively conserved", "protein_state" ], [ 421, 432, "GGDEF motif", "structure_element" ], [ 436, 440, "DGCs", "protein_type" ], [ 444, 462, "EAL/HD-GYP domains", "structure_element" ], [ 466, 470, "PDEs", "protein_type" ] ] }, { "sid": 10, "sent": "Intriguingly, studies in diverse species have revealed that a single bacterium can have dozens of DGCs and PDEs (Hickman et al.,; Kirillina et al.,; Kulasakara et al.,; Tamayo et al.,).", "section": "INTRO", "ner": [ [ 69, 78, "bacterium", "taxonomy_domain" ], [ 98, 102, "DGCs", "protein_type" ], [ 107, 111, "PDEs", "protein_type" ] ] }, { "sid": 11, "sent": "In Pseudomonas aeruginosa in particular, 42 genes containing putative DGCs and/or PDEs were identified (Kulasakara et al.,).", "section": "INTRO", "ner": [ [ 3, 25, "Pseudomonas aeruginosa", "species" ], [ 70, 74, "DGCs", "protein_type" ], [ 82, 86, "PDEs", "protein_type" ] ] }, { "sid": 12, "sent": "The functional role of a number of downstream effectors of c-di-GMP has been characterized as affecting exopolysaccharide (EPS) production, transcription, motility, and surface attachment (Caly et al.,; Camilli and Bassler,; Ha and O\u2019Toole,; Pesavento and Hengge,).", "section": "INTRO", "ner": [ [ 59, 67, "c-di-GMP", "chemical" ], [ 104, 121, "exopolysaccharide", "chemical" ], [ 123, 126, "EPS", "chemical" ] ] }, { "sid": 13, "sent": "However, due to the intricacy of c-di-GMP signaling networks and the diversity of experimental cues, the detailed mechanisms by which these signaling pathways specifically sense and integrate different inputs remain largely elusive.", "section": "INTRO", "ner": [ [ 33, 41, "c-di-GMP", "chemical" ] ] }, { "sid": 14, "sent": "Biofilm formation protects pathogenic bacteria from antibiotic treatment, and c-di-GMP-regulated biofilm formation has been extensively studied in P. aeruginosa (Evans,; Kirisits et al.,; Malone,; Reinhardt et al.,).", "section": "INTRO", "ner": [ [ 38, 46, "bacteria", "taxonomy_domain" ], [ 147, 160, "P. aeruginosa", "species" ] ] }, { "sid": 15, "sent": "In the lungs of cystic fibrosis (CF) patients, adherent biofilm\u00a0formation and the appearance of small colony variant (SCV) morphologies of P. aeruginosa correlate with prolonged persistence of infection and poor lung function (Govan and Deretic,; Haussler et al.,; Haussler et al.,; Parsek and Singh,; Smith et al.,).", "section": "INTRO", "ner": [ [ 139, 152, "P. aeruginosa", "species" ] ] }, { "sid": 16, "sent": "Recently, Malone and coworkers identified the tripartite c-di-GMP signaling module system YfiBNR (also known as AwsXRO (Beaumont et al.,; Giddens et al.,) or Tbp (Ueda and Wood,)) by genetic screening for mutants that displayed SCV phenotypes in P. aeruginosa PAO1 (Malone et al.,; Malone et al.,).", "section": "INTRO", "ner": [ [ 46, 56, "tripartite", "protein_state" ], [ 57, 65, "c-di-GMP", "chemical" ], [ 90, 96, "YfiBNR", "complex_assembly" ], [ 112, 118, "AwsXRO", "complex_assembly" ], [ 158, 161, "Tbp", "complex_assembly" ], [ 183, 200, "genetic screening", "experimental_method" ], [ 246, 264, "P. aeruginosa PAO1", "species" ] ] }, { "sid": 17, "sent": "The YfiBNR system contains three protein members and modulates intracellular c-di-GMP levels in response to signals received in the periplasm (Malone et al.,).", "section": "INTRO", "ner": [ [ 4, 10, "YfiBNR", "complex_assembly" ], [ 77, 85, "c-di-GMP", "chemical" ] ] }, { "sid": 18, "sent": "More recently, this system was also reported in other Gram-negative bacteria, such as Escherichia coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), Klebsiella pneumonia (Huertas et al.,) and Yersinia pestis (Ren et al.,).", "section": "INTRO", "ner": [ [ 54, 76, "Gram-negative bacteria", "taxonomy_domain" ], [ 86, 102, "Escherichia coli", "species" ], [ 165, 185, "Klebsiella pneumonia", "species" ], [ 208, 223, "Yersinia pestis", "species" ] ] }, { "sid": 19, "sent": "YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC\u2019s function (Giardina et al.,; Malone et al.,).", "section": "INTRO", "ner": [ [ 0, 4, "YfiN", "protein" ], [ 62, 83, "transmembrane helices", "structure_element" ], [ 99, 111, "Per-Arnt-Sim", "structure_element" ], [ 113, 116, "PAS", "structure_element" ], [ 161, 172, "HAMP domain", "structure_element" ], [ 208, 225, "histidine kinases", "protein_type" ], [ 227, 244, "adenylyl cyclases", "protein_type" ], [ 246, 282, "methyl-accepting chemotaxis proteins", "protein_type" ], [ 288, 300, "phosphatases", "protein_type" ], [ 319, 331, "GGDEF domain", "structure_element" ], [ 345, 348, "DGC", "protein_type" ] ] }, { "sid": 20, "sent": "YfiN is repressed by specific interaction between its periplasmic PAS domain and the periplasmic protein YfiR (Malone et al.,).", "section": "INTRO", "ner": [ [ 0, 4, "YfiN", "protein" ], [ 8, 20, "repressed by", "protein_state" ], [ 66, 76, "PAS domain", "structure_element" ], [ 105, 109, "YfiR", "protein" ] ] }, { "sid": 21, "sent": "YfiB is an OmpA/Pal-like outer-membrane lipoprotein (Parsons et al.,) that can activate YfiN by sequestering YfiR (Malone et al.,) in an unknown manner.", "section": "INTRO", "ner": [ [ 0, 4, "YfiB", "protein" ], [ 11, 24, "OmpA/Pal-like", "protein_type" ], [ 40, 51, "lipoprotein", "protein_type" ], [ 88, 92, "YfiN", "protein" ], [ 109, 113, "YfiR", "protein" ] ] }, { "sid": 22, "sent": "Whether YfiB directly recruits YfiR or recruits YfiR via a third partner is an open question.", "section": "INTRO", "ner": [ [ 8, 12, "YfiB", "protein" ], [ 31, 35, "YfiR", "protein" ], [ 48, 52, "YfiR", "protein" ] ] }, { "sid": 23, "sent": "After the sequestration of YfiR by YfiB, the c-di-GMP produced by activated YfiN increases the biosynthesis of the Pel and Psl EPSs, resulting in the appearance of the SCV phenotype, which indicates enhanced biofilm formation (Malone et al.,).", "section": "INTRO", "ner": [ [ 27, 31, "YfiR", "protein" ], [ 35, 39, "YfiB", "protein" ], [ 45, 53, "c-di-GMP", "chemical" ], [ 66, 75, "activated", "protein_state" ], [ 76, 80, "YfiN", "protein" ], [ 115, 118, "Pel", "chemical" ], [ 123, 126, "Psl", "chemical" ], [ 127, 131, "EPSs", "chemical" ] ] }, { "sid": 24, "sent": "It has been reported that the activation of YfiN may be induced by redox-driven misfolding of YfiR (Giardina et al.,; Malone et al.,; Malone et al.,).", "section": "INTRO", "ner": [ [ 44, 48, "YfiN", "protein" ], [ 94, 98, "YfiR", "protein" ] ] }, { "sid": 25, "sent": "It is also proposed that the sequestration of YfiR by YfiB can be induced by certain YfiB-mediated cell wall stress, and mutagenesis studies revealed a number of activation residues of YfiB that were located in close proximity to the predicted first helix of the periplasmic domain (Malone et al.,).", "section": "INTRO", "ner": [ [ 46, 50, "YfiR", "protein" ], [ 54, 58, "YfiB", "protein" ], [ 85, 89, "YfiB", "protein" ], [ 121, 140, "mutagenesis studies", "experimental_method" ], [ 162, 181, "activation residues", "structure_element" ], [ 185, 189, "YfiB", "protein" ], [ 234, 243, "predicted", "protein_state" ], [ 244, 255, "first helix", "structure_element" ], [ 263, 281, "periplasmic domain", "structure_element" ] ] }, { "sid": 26, "sent": "In addition, quorum sensing-related dephosphorylation of the PAS domain of YfiN may also be involved in the regulation (Ueda and Wood,; Xu et al.,).", "section": "INTRO", "ner": [ [ 61, 71, "PAS domain", "structure_element" ], [ 75, 79, "YfiN", "protein" ] ] }, { "sid": 27, "sent": "Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,).", "section": "INTRO", "ner": [ [ 24, 41, "crystal structure", "evidence" ], [ 45, 49, "YfiR", "protein" ], [ 62, 74, "non-oxidized", "protein_state" ], [ 83, 91, "oxidized", "protein_state" ], [ 136, 150, "disulfide bond", "ptm" ], [ 152, 157, "Cys71", "residue_name_number" ], [ 158, 164, "Cys110", "residue_name_number" ], [ 220, 226, "Cys145", "residue_name_number" ], [ 227, 233, "Cys152", "residue_name_number" ], [ 252, 258, "Cys145", "residue_name_number" ], [ 259, 265, "Cys152", "residue_name_number" ], [ 328, 332, "YfiR", "protein" ] ] }, { "sid": 28, "sent": "In the present study, we solved the crystal structures of an N-terminal truncated form of YfiB (34\u2013168) and YfiR in complex with an active mutant YfiBL43P.", "section": "INTRO", "ner": [ [ 36, 54, "crystal structures", "evidence" ], [ 72, 81, "truncated", "protein_state" ], [ 90, 94, "YfiB", "protein" ], [ 96, 102, "34\u2013168", "residue_range" ], [ 108, 112, "YfiR", "protein" ], [ 113, 128, "in complex with", "protein_state" ], [ 132, 138, "active", "protein_state" ], [ 139, 145, "mutant", "protein_state" ], [ 146, 154, "YfiBL43P", "mutant" ] ] }, { "sid": 29, "sent": "Most recently, Li and coworkers reported the crystal structures of YfiB (27\u2013168) alone and YfiRC71S in complex with YfiB (59\u2013168) (Li et al.,).", "section": "INTRO", "ner": [ [ 45, 63, "crystal structures", "evidence" ], [ 67, 71, "YfiB", "protein" ], [ 73, 79, "27\u2013168", "residue_range" ], [ 81, 86, "alone", "protein_state" ], [ 91, 99, "YfiRC71S", "mutant" ], [ 100, 115, "in complex with", "protein_state" ], [ 116, 120, "YfiB", "protein" ], [ 122, 128, "59\u2013168", "residue_range" ] ] }, { "sid": 30, "sent": "Compared with the reported complex structure, YfiBL43P in our YfiB-YfiR complex structure has additional visible N-terminal residues 44\u201358 that are shown to play essential roles in YfiB activation and biofilm formation.", "section": "INTRO", "ner": [ [ 46, 54, "YfiBL43P", "mutant" ], [ 62, 71, "YfiB-YfiR", "complex_assembly" ], [ 80, 89, "structure", "evidence" ], [ 133, 138, "44\u201358", "residue_range" ], [ 181, 185, "YfiB", "protein" ] ] }, { "sid": 31, "sent": "Therefore, we are able to visualize the detailed allosteric arrangement of the N-terminal structure of YfiB and its important role in YfiB-YfiR interaction.", "section": "INTRO", "ner": [ [ 103, 107, "YfiB", "protein" ], [ 134, 143, "YfiB-YfiR", "complex_assembly" ] ] }, { "sid": 32, "sent": "In addition, we found that the YfiBL43P shows a much higher PG-binding affinity than wild-type YfiB, most likely due to its more compact PG-binding pocket.", "section": "INTRO", "ner": [ [ 31, 39, "YfiBL43P", "mutant" ], [ 60, 79, "PG-binding affinity", "evidence" ], [ 85, 94, "wild-type", "protein_state" ], [ 95, 99, "YfiB", "protein" ], [ 137, 154, "PG-binding pocket", "site" ] ] }, { "sid": 33, "sent": "Moreover, we found that Vitamin B6 (VB6) or L-Trp can bind YfiR with an affinity in the ten millimolar range.", "section": "INTRO", "ner": [ [ 24, 34, "Vitamin B6", "chemical" ], [ 36, 39, "VB6", "chemical" ], [ 44, 49, "L-Trp", "chemical" ], [ 59, 63, "YfiR", "protein" ], [ 72, 80, "affinity", "evidence" ] ] }, { "sid": 34, "sent": "Together with functional data, these results provide new mechanistic insights into how activated YfiB sequesters YfiR and releases the suppression of YfiN. These findings may facilitate the development and optimization of anti-biofilm drugs for the treatment of chronic infections.", "section": "INTRO", "ner": [ [ 87, 96, "activated", "protein_state" ], [ 97, 101, "YfiB", "protein" ], [ 113, 117, "YfiR", "protein" ], [ 150, 154, "YfiN", "protein" ] ] }, { "sid": 35, "sent": "Overall structure of YfiB", "section": "RESULTS", "ner": [ [ 8, 17, "structure", "evidence" ], [ 21, 25, "YfiB", "protein" ] ] }, { "sid": 36, "sent": "We obtained two crystal forms of YfiB (residues 34\u2013168, lacking the signal peptide from residues 1\u201326 and periplasmic residues 27\u201333), crystal forms I and II, belonging to space groups P21 and P41, respectively.", "section": "RESULTS", "ner": [ [ 16, 29, "crystal forms", "evidence" ], [ 33, 37, "YfiB", "protein" ], [ 48, 54, "34\u2013168", "residue_range" ], [ 56, 63, "lacking", "protein_state" ], [ 68, 82, "signal peptide", "structure_element" ], [ 97, 101, "1\u201326", "residue_range" ], [ 127, 132, "27\u201333", "residue_range" ] ] }, { "sid": 37, "sent": "Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P ", "section": "FIG", "ner": [ [ 8, 17, "structure", "evidence" ], [ 21, 25, "YfiB", "protein" ], [ 43, 52, "structure", "evidence" ], [ 60, 64, "YfiB", "protein" ], [ 65, 72, "monomer", "oligomeric_state" ], [ 104, 108, "YfiB", "protein" ], [ 109, 116, "monomer", "oligomeric_state" ], [ 132, 162, "analytical ultracentrifugation", "experimental_method" ], [ 190, 199, "wild-type", "protein_state" ], [ 200, 204, "YfiB", "protein" ], [ 209, 217, "YfiBL43P", "mutant" ] ] }, { "sid": 38, "sent": "Two dimeric types of YfiB dimer. (A\u2013C) The \u201chead to head\u201d dimer.", "section": "FIG", "ner": [ [ 4, 11, "dimeric", "oligomeric_state" ], [ 21, 25, "YfiB", "protein" ], [ 26, 31, "dimer", "oligomeric_state" ], [ 44, 56, "head to head", "protein_state" ], [ 58, 63, "dimer", "oligomeric_state" ] ] }, { "sid": 39, "sent": "The \u201cback to back\u201d dimer.", "section": "FIG", "ner": [ [ 5, 17, "back to back", "protein_state" ], [ 19, 24, "dimer", "oligomeric_state" ] ] }, { "sid": 40, "sent": "(A) and (E) indicate the front views of the two dimers, (B) and (F) indicate the top views of the two dimers, and (C) and (D) indicate the details of the two dimeric interfaces", "section": "FIG", "ner": [ [ 48, 54, "dimers", "oligomeric_state" ], [ 102, 108, "dimers", "oligomeric_state" ], [ 158, 176, "dimeric interfaces", "site" ] ] }, { "sid": 41, "sent": "The crystal structure of YfiB monomer consists of a five-stranded \u03b2-sheet (\u03b21-2-5-3-4) flanked by five \u03b1-helices (\u03b11\u20135) on one side.", "section": "RESULTS", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 25, 29, "YfiB", "protein" ], [ 30, 37, "monomer", "oligomeric_state" ], [ 52, 73, "five-stranded \u03b2-sheet", "structure_element" ], [ 75, 85, "\u03b21-2-5-3-4", "structure_element" ], [ 98, 112, "five \u03b1-helices", "structure_element" ], [ 114, 118, "\u03b11\u20135", "structure_element" ] ] }, { "sid": 42, "sent": "In addition, there is a short helix turn connecting the \u03b24 strand and \u03b14 helix (Fig.\u00a01A and 1B).", "section": "RESULTS", "ner": [ [ 30, 40, "helix turn", "structure_element" ], [ 56, 65, "\u03b24 strand", "structure_element" ], [ 70, 78, "\u03b14 helix", "structure_element" ] ] }, { "sid": 43, "sent": "Each crystal form contains three different dimeric types of YfiB, two of which are present in both, suggesting that the rest of the dimeric types may result from crystal packing.", "section": "RESULTS", "ner": [ [ 43, 50, "dimeric", "oligomeric_state" ], [ 60, 64, "YfiB", "protein" ], [ 132, 139, "dimeric", "oligomeric_state" ] ] }, { "sid": 44, "sent": "Here, we refer to the two dimeric types as \u201chead to head\u201d and \u201cback to back\u201d according to the interacting mode (Fig.\u00a02A and 2E), with the total buried surface areas being 316.8 \u00c52 and 554.3 \u00c52, respectively.", "section": "RESULTS", "ner": [ [ 26, 33, "dimeric", "oligomeric_state" ], [ 44, 56, "head to head", "protein_state" ], [ 63, 75, "back to back", "protein_state" ] ] }, { "sid": 45, "sent": "The \u201chead to head\u201d dimer exhibits a clamp shape.", "section": "RESULTS", "ner": [ [ 5, 17, "head to head", "protein_state" ], [ 19, 24, "dimer", "oligomeric_state" ], [ 36, 47, "clamp shape", "protein_state" ] ] }, { "sid": 46, "sent": "The dimerization occurs mainly via hydrophobic interactions formed by A37 and I40 on the \u03b11 helices, L50 on the \u03b21 strands, and W55 on the \u03b22 strands of both molecules, making a hydrophobic interacting core (Fig.\u00a02A\u2013C).", "section": "RESULTS", "ner": [ [ 35, 59, "hydrophobic interactions", "bond_interaction" ], [ 70, 73, "A37", "residue_name_number" ], [ 78, 81, "I40", "residue_name_number" ], [ 89, 99, "\u03b11 helices", "structure_element" ], [ 101, 104, "L50", "residue_name_number" ], [ 112, 122, "\u03b21 strands", "structure_element" ], [ 128, 131, "W55", "residue_name_number" ], [ 139, 149, "\u03b22 strands", "structure_element" ], [ 178, 206, "hydrophobic interacting core", "site" ] ] }, { "sid": 47, "sent": "The \u201cback to back\u201d dimer presents a Y shape.", "section": "RESULTS", "ner": [ [ 5, 17, "back to back", "protein_state" ], [ 19, 24, "dimer", "oligomeric_state" ], [ 36, 43, "Y shape", "protein_state" ] ] }, { "sid": 48, "sent": "The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the \u03b12-\u03b13 loops and R116 and S120 on the \u03b14 helices of both molecules, resulting in a complex hydrogen bond network (Fig.\u00a02D\u2013F).", "section": "RESULTS", "ner": [ [ 4, 11, "dimeric", "oligomeric_state" ], [ 12, 45, "interaction is mainly hydrophilic", "bond_interaction" ], [ 102, 105, "N68", "residue_name_number" ], [ 107, 110, "L69", "residue_name_number" ], [ 112, 115, "D70", "residue_name_number" ], [ 120, 123, "R71", "residue_name_number" ], [ 131, 142, "\u03b12-\u03b13 loops", "structure_element" ], [ 147, 151, "R116", "residue_name_number" ], [ 156, 160, "S120", "residue_name_number" ], [ 168, 178, "\u03b14 helices", "structure_element" ], [ 221, 242, "hydrogen bond network", "site" ] ] }, { "sid": 49, "sent": "The YfiB-YfiR interaction", "section": "RESULTS", "ner": [ [ 4, 13, "YfiB-YfiR", "complex_assembly" ] ] }, { "sid": 50, "sent": "Overall structure of the YfiB-YfiR complex and the conserved surface in YfiR. (A) The overall structure of the YfiB-YfiR complex.", "section": "FIG", "ner": [ [ 8, 17, "structure", "evidence" ], [ 25, 34, "YfiB-YfiR", "complex_assembly" ], [ 51, 68, "conserved surface", "site" ], [ 72, 76, "YfiR", "protein" ], [ 94, 103, "structure", "evidence" ], [ 111, 120, "YfiB-YfiR", "complex_assembly" ] ] }, { "sid": 51, "sent": "The YfiBL43P molecules are shown in cyan and yellow.", "section": "FIG", "ner": [ [ 4, 12, "YfiBL43P", "mutant" ] ] }, { "sid": 52, "sent": "The YfiR molecules are shown in green and magenta.", "section": "FIG", "ner": [ [ 4, 8, "YfiR", "protein" ] ] }, { "sid": 53, "sent": "Two interacting regions are highlighted by red rectangles. (B) Structural superposition of apo YfiB and YfiR-bound YfiBL43P.", "section": "FIG", "ner": [ [ 63, 87, "Structural superposition", "experimental_method" ], [ 91, 94, "apo", "protein_state" ], [ 95, 99, "YfiB", "protein" ], [ 104, 114, "YfiR-bound", "protein_state" ], [ 115, 123, "YfiBL43P", "mutant" ] ] }, { "sid": 54, "sent": "To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34\u201370 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44\u201370 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P.", "section": "FIG", "ner": [ [ 38, 41, "apo", "protein_state" ], [ 42, 46, "YfiB", "protein" ], [ 51, 61, "YfiR-bound", "protein_state" ], [ 62, 70, "YfiBL43P", "mutant" ], [ 76, 79, "apo", "protein_state" ], [ 80, 84, "YfiB", "protein" ], [ 119, 124, "34\u201370", "residue_range" ], [ 155, 165, "YfiR-bound", "protein_state" ], [ 166, 174, "YfiBL43P", "mutant" ], [ 209, 214, "44\u201370", "residue_range" ], [ 279, 282, "apo", "protein_state" ], [ 283, 287, "YfiB", "protein" ], [ 292, 302, "YfiR-bound", "protein_state" ], [ 303, 311, "YfiBL43P", "mutant" ] ] }, { "sid": 55, "sent": "The residues proposed to contribute to YfiB activation are illustrated in sticks.", "section": "FIG", "ner": [ [ 39, 43, "YfiB", "protein" ] ] }, { "sid": 56, "sent": "The key residues in apo YfiB are shown in red and those in YfiBL43P are shown in blue. (D) Close-up views showing interactions in regions I and II.", "section": "FIG", "ner": [ [ 20, 23, "apo", "protein_state" ], [ 24, 28, "YfiB", "protein" ], [ 59, 67, "YfiBL43P", "mutant" ], [ 130, 146, "regions I and II", "structure_element" ] ] }, { "sid": 57, "sent": "YfiBL43P and YfiR are shown in cyan and green, respectively. (E and F) The conserved surface in YfiR contributes to the interaction with YfiB. (G) The residues of YfiR responsible for interacting with YfiB are shown in green sticks, and the proposed YfiN-interacting residues are shown in yellow sticks.", "section": "FIG", "ner": [ [ 0, 8, "YfiBL43P", "mutant" ], [ 13, 17, "YfiR", "protein" ], [ 75, 92, "conserved surface", "site" ], [ 96, 100, "YfiR", "protein" ], [ 137, 141, "YfiB", "protein" ], [ 151, 159, "residues", "structure_element" ], [ 163, 167, "YfiR", "protein" ], [ 201, 205, "YfiB", "protein" ], [ 250, 275, "YfiN-interacting residues", "site" ] ] }, { "sid": 58, "sent": "The red sticks, which represent the YfiB-interacting residues, are also responsible for the proposed interactions with YfiN", "section": "FIG", "ner": [ [ 36, 61, "YfiB-interacting residues", "site" ], [ 119, 123, "YfiN", "protein" ] ] }, { "sid": 59, "sent": "To gain structural insights into the YfiB-YfiR interaction, we co-expressed YfiB (residues 34\u2013168) and YfiR (residues 35\u2013190, lacking the signal peptide), but failed to obtain the complex, in accordance with a previous report in which no stable complex of YfiB-YfiR was observed (Malone et al.,).", "section": "RESULTS", "ner": [ [ 37, 46, "YfiB-YfiR", "complex_assembly" ], [ 63, 75, "co-expressed", "experimental_method" ], [ 76, 80, "YfiB", "protein" ], [ 91, 97, "34\u2013168", "residue_range" ], [ 103, 107, "YfiR", "protein" ], [ 118, 124, "35\u2013190", "residue_range" ], [ 126, 133, "lacking", "protein_state" ], [ 138, 152, "signal peptide", "structure_element" ], [ 235, 244, "no stable", "protein_state" ], [ 256, 265, "YfiB-YfiR", "complex_assembly" ] ] }, { "sid": 60, "sent": "It has been reported that single mutants of Q39, L43, F48 and W55 contribute to YfiB activation leading to the induction of the SCV phenotype in P. aeruginosa PAO1 (Malone et al.,).", "section": "RESULTS", "ner": [ [ 26, 43, "single mutants of", "experimental_method" ], [ 44, 47, "Q39", "residue_name_number" ], [ 49, 52, "L43", "residue_name_number" ], [ 54, 57, "F48", "residue_name_number" ], [ 62, 65, "W55", "residue_name_number" ], [ 80, 84, "YfiB", "protein" ], [ 145, 163, "P. aeruginosa PAO1", "species" ] ] }, { "sid": 61, "sent": "It is likely that these residues may be involved in the conformational changes of YfiB that are related to YfiR sequestration (Fig.\u00a03C).", "section": "RESULTS", "ner": [ [ 82, 86, "YfiB", "protein" ], [ 107, 111, "YfiR", "protein" ] ] }, { "sid": 62, "sent": "Therefore, we constructed two such single mutants of YfiB (YfiBL43P and YfiBF48S).", "section": "RESULTS", "ner": [ [ 14, 49, "constructed two such single mutants", "experimental_method" ], [ 53, 57, "YfiB", "protein" ], [ 59, 67, "YfiBL43P", "mutant" ], [ 72, 80, "YfiBF48S", "mutant" ] ] }, { "sid": 63, "sent": "As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 \u00c5 resolution by molecular replacement using YfiR and YfiB as models.", "section": "RESULTS", "ner": [ [ 33, 39, "stable", "protein_state" ], [ 40, 52, "complex with", "protein_state" ], [ 53, 57, "YfiR", "protein" ], [ 71, 82, "crystalized", "experimental_method" ], [ 83, 87, "YfiR", "protein" ], [ 88, 103, "in complex with", "protein_state" ], [ 108, 116, "YfiBL43P", "mutant" ], [ 117, 123, "mutant", "protein_state" ], [ 139, 148, "structure", "evidence" ], [ 173, 194, "molecular replacement", "experimental_method" ], [ 201, 205, "YfiR", "protein" ], [ 210, 214, "YfiB", "protein" ] ] }, { "sid": 64, "sent": "The YfiB-YfiR complex is a 2:2 heterotetramer (Fig.\u00a03A) in which the YfiR dimer is clamped by two separated YfiBL43P molecules with a total buried surface area of 3161.2 \u00c52.", "section": "RESULTS", "ner": [ [ 4, 13, "YfiB-YfiR", "complex_assembly" ], [ 31, 45, "heterotetramer", "oligomeric_state" ], [ 69, 73, "YfiR", "protein" ], [ 74, 79, "dimer", "oligomeric_state" ], [ 108, 116, "YfiBL43P", "mutant" ] ] }, { "sid": 65, "sent": "The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex.", "section": "RESULTS", "ner": [ [ 4, 8, "YfiR", "protein" ], [ 9, 14, "dimer", "oligomeric_state" ], [ 50, 62, "non-oxidized", "protein_state" ], [ 63, 67, "YfiR", "protein" ], [ 68, 73, "dimer", "oligomeric_state" ], [ 74, 79, "alone", "protein_state" ], [ 106, 112, "Cys145", "residue_name_number" ], [ 113, 119, "Cys152", "residue_name_number" ], [ 131, 146, "disulfide bonds", "ptm" ], [ 171, 176, "Cys71", "residue_name_number" ], [ 177, 183, "Cys110", "residue_name_number" ], [ 184, 198, "disulfide bond", "ptm" ], [ 238, 247, "YfiB-YfiR", "complex_assembly" ] ] }, { "sid": 66, "sent": "The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the \u03b11 helix (residues 34\u201343) is invisible on the electron density map, and the \u03b12 helix and \u03b21 and \u03b22 strands are rearranged to form a long loop containing two short \u03b1-helix turns (Fig.\u00a03B and 3C), thus embracing the YfiR dimer.", "section": "RESULTS", "ner": [ [ 42, 50, "YfiBL43P", "mutant" ], [ 92, 95, "D70", "residue_name_number" ], [ 148, 151, "apo", "protein_state" ], [ 152, 156, "YfiB", "protein" ], [ 178, 186, "\u03b11 helix", "structure_element" ], [ 197, 202, "34\u201343", "residue_range" ], [ 224, 244, "electron density map", "evidence" ], [ 254, 262, "\u03b12 helix", "structure_element" ], [ 267, 269, "\u03b21", "structure_element" ], [ 274, 284, "\u03b22 strands", "structure_element" ], [ 315, 319, "loop", "structure_element" ], [ 341, 354, "\u03b1-helix turns", "structure_element" ], [ 392, 396, "YfiR", "protein" ], [ 397, 402, "dimer", "oligomeric_state" ] ] }, { "sid": 67, "sent": "The observed changes in conformation of YfiB and the results of mutagenesis suggest a mechanism by which YfiB sequesters YfiR.", "section": "RESULTS", "ner": [ [ 40, 44, "YfiB", "protein" ], [ 64, 75, "mutagenesis", "experimental_method" ], [ 105, 109, "YfiB", "protein" ], [ 121, 125, "YfiR", "protein" ] ] }, { "sid": 68, "sent": "The YfiB-YfiR interface can be divided into two regions (Fig.\u00a03A and 3D).", "section": "RESULTS", "ner": [ [ 4, 23, "YfiB-YfiR interface", "site" ] ] }, { "sid": 69, "sent": "Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig.\u00a03D-I(i)).", "section": "RESULTS", "ner": [ [ 0, 8, "Region I", "structure_element" ], [ 57, 81, "hydrophilic interactions", "bond_interaction" ], [ 99, 102, "E45", "residue_name_number" ], [ 104, 107, "G47", "residue_name_number" ], [ 112, 115, "E53", "residue_name_number" ], [ 145, 149, "loop", "structure_element" ], [ 153, 157, "YfiB", "protein" ], [ 171, 174, "S57", "residue_name_number" ], [ 176, 179, "R60", "residue_name_number" ], [ 181, 184, "A89", "residue_name_number" ], [ 189, 193, "H177", "residue_name_number" ], [ 199, 203, "YfiR", "protein" ] ] }, { "sid": 70, "sent": "Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig.\u00a03D-I(ii)).", "section": "RESULTS", "ner": [ [ 20, 47, "hydrophobic anchoring sites", "site" ], [ 57, 65, "region I", "structure_element" ], [ 80, 83, "F48", "residue_name_number" ], [ 88, 91, "W55", "residue_name_number" ], [ 95, 99, "YfiB", "protein" ], [ 122, 139, "hydrophobic cores", "site" ], [ 212, 215, "S57", "residue_name_number" ], [ 216, 219, "Q88", "residue_name_number" ], [ 220, 223, "A89", "residue_name_number" ], [ 224, 227, "N90", "residue_name_number" ], [ 232, 235, "R60", "residue_name_number" ], [ 236, 240, "R175", "residue_name_number" ], [ 241, 245, "H177", "residue_name_number" ], [ 249, 253, "YfiR", "protein" ], [ 273, 276, "F57", "residue_name_number" ], [ 280, 284, "YfiB", "protein" ], [ 306, 324, "hydrophobic pocket", "site" ], [ 335, 339, "L166", "residue_name_number" ], [ 340, 344, "I169", "residue_name_number" ], [ 345, 349, "V176", "residue_name_number" ], [ 350, 354, "P178", "residue_name_number" ], [ 355, 359, "L181", "residue_name_number" ], [ 363, 367, "YfiR", "protein" ] ] }, { "sid": 71, "sent": "In region II, the side chains of R96, E98 and E157 from YfiB interact with the side chains of E163, S146 and R171 from YfiR, respectively.", "section": "RESULTS", "ner": [ [ 3, 12, "region II", "structure_element" ], [ 33, 36, "R96", "residue_name_number" ], [ 38, 41, "E98", "residue_name_number" ], [ 46, 50, "E157", "residue_name_number" ], [ 56, 60, "YfiB", "protein" ], [ 94, 98, "E163", "residue_name_number" ], [ 100, 104, "S146", "residue_name_number" ], [ 109, 113, "R171", "residue_name_number" ], [ 119, 123, "YfiR", "protein" ] ] }, { "sid": 72, "sent": "Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig.\u00a03D-II).", "section": "RESULTS", "ner": [ [ 33, 37, "I163", "residue_name_number" ], [ 42, 46, "V165", "residue_name_number" ], [ 52, 56, "YfiB", "protein" ], [ 62, 76, "hydrogen bonds", "bond_interaction" ], [ 101, 105, "L166", "residue_name_number" ], [ 110, 114, "A164", "residue_name_number" ], [ 120, 124, "YfiR", "protein" ], [ 162, 166, "P166", "residue_name_number" ], [ 172, 176, "YfiB", "protein" ], [ 210, 214, "R185", "residue_name_number" ], [ 220, 224, "YfiR", "protein" ] ] }, { "sid": 73, "sent": "These two regions contribute a robust hydrogen-bonding network to the YfiB-YfiR interface, resulting in a tightly bound complex.", "section": "RESULTS", "ner": [ [ 38, 62, "hydrogen-bonding network", "site" ], [ 70, 89, "YfiB-YfiR interface", "site" ] ] }, { "sid": 74, "sent": "Based on the observations that two separated YfiBL43P molecules form a 2:2 complex structure with YfiR dimer, we performed an analytical ultracentrifugation experiment to check the oligomeric states of wild-type YfiB and YfiBL43P.", "section": "RESULTS", "ner": [ [ 45, 53, "YfiBL43P", "mutant" ], [ 83, 92, "structure", "evidence" ], [ 98, 102, "YfiR", "protein" ], [ 103, 108, "dimer", "oligomeric_state" ], [ 126, 156, "analytical ultracentrifugation", "experimental_method" ], [ 202, 211, "wild-type", "protein_state" ], [ 212, 216, "YfiB", "protein" ], [ 221, 229, "YfiBL43P", "mutant" ] ] }, { "sid": 75, "sent": "The results showed that wild-type YfiB exists in both monomeric and dimeric states in solution, while YfiBL43P primarily adopts the monomer state in solution (Fig.\u00a01C\u2013D).", "section": "RESULTS", "ner": [ [ 24, 33, "wild-type", "protein_state" ], [ 34, 38, "YfiB", "protein" ], [ 54, 63, "monomeric", "oligomeric_state" ], [ 68, 75, "dimeric", "oligomeric_state" ], [ 102, 110, "YfiBL43P", "mutant" ], [ 132, 139, "monomer", "oligomeric_state" ] ] }, { "sid": 76, "sent": "This suggests that the N-terminus of YfiB plays an important role in forming the dimeric YfiB in solution and that the conformational change of residue L43 is associated with the stretch of the N-terminus and opening of the dimer.", "section": "RESULTS", "ner": [ [ 37, 41, "YfiB", "protein" ], [ 81, 88, "dimeric", "oligomeric_state" ], [ 89, 93, "YfiB", "protein" ], [ 152, 155, "L43", "residue_name_number" ], [ 224, 229, "dimer", "oligomeric_state" ] ] }, { "sid": 77, "sent": "Therefore, it is possible that both dimeric types might exist in solution.", "section": "RESULTS", "ner": [ [ 36, 43, "dimeric", "oligomeric_state" ] ] }, { "sid": 78, "sent": "For simplicity, we only discuss the \u201chead to head\u201d dimer in the following text.", "section": "RESULTS", "ner": [ [ 37, 49, "head to head", "protein_state" ], [ 51, 56, "dimer", "oligomeric_state" ] ] }, { "sid": 79, "sent": "The PG-binding site of YfiB", "section": "RESULTS", "ner": [ [ 4, 19, "PG-binding site", "site" ], [ 23, 27, "YfiB", "protein" ] ] }, { "sid": 80, "sent": "The PG-binding site in YfiB. (A) Structural superposition of the PG-binding sites of the H. influenzae Pal/PG-P complex and YfiR-bound YfiBL43P complexed with sulfate ions.", "section": "FIG", "ner": [ [ 4, 19, "PG-binding site", "site" ], [ 23, 27, "YfiB", "protein" ], [ 33, 57, "Structural superposition", "experimental_method" ], [ 65, 81, "PG-binding sites", "site" ], [ 89, 102, "H. influenzae", "species" ], [ 103, 111, "Pal/PG-P", "complex_assembly" ], [ 124, 134, "YfiR-bound", "protein_state" ], [ 135, 143, "YfiBL43P", "mutant" ], [ 144, 158, "complexed with", "protein_state" ], [ 159, 166, "sulfate", "chemical" ] ] }, { "sid": 81, "sent": "(B) Close-up view showing the key residues of Pal interacting with the m-Dap5 \u03b5-carboxylate group of PG-P. Pal is shown in wheat and PG-P is in magenta.", "section": "FIG", "ner": [ [ 46, 49, "Pal", "protein_type" ], [ 71, 91, "m-Dap5 \u03b5-carboxylate", "chemical" ], [ 101, 105, "PG-P", "chemical" ], [ 107, 110, "Pal", "protein_type" ], [ 133, 137, "PG-P", "chemical" ] ] }, { "sid": 82, "sent": "(C) Close-up view showing the key residues of YfiR-bound YfiBL43P interacting with a sulfate ion.", "section": "FIG", "ner": [ [ 46, 56, "YfiR-bound", "protein_state" ], [ 57, 65, "YfiBL43P", "mutant" ], [ 85, 92, "sulfate", "chemical" ] ] }, { "sid": 83, "sent": "YfiR-bound YfiBL43P is shown in cyan; the sulfate ion, in green; and the water molecule, in yellow. (D) Structural superposition of the PG-binding sites of apo YfiB and YfiR-bound YfiBL43P, the key residues are shown in stick.", "section": "FIG", "ner": [ [ 0, 10, "YfiR-bound", "protein_state" ], [ 11, 19, "YfiBL43P", "mutant" ], [ 42, 49, "sulfate", "chemical" ], [ 73, 78, "water", "chemical" ], [ 104, 128, "Structural superposition", "experimental_method" ], [ 136, 152, "PG-binding sites", "site" ], [ 156, 159, "apo", "protein_state" ], [ 160, 164, "YfiB", "protein" ], [ 169, 179, "YfiR-bound", "protein_state" ], [ 180, 188, "YfiBL43P", "mutant" ] ] }, { "sid": 84, "sent": "Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA", "section": "FIG", "ner": [ [ 0, 3, "Apo", "protein_state" ], [ 4, 8, "YfiB", "protein" ], [ 32, 42, "YfiR-bound", "protein_state" ], [ 43, 51, "YfiBL43P", "mutant" ], [ 71, 74, "MST", "experimental_method" ], [ 97, 115, "binding affinities", "evidence" ], [ 123, 127, "YfiB", "protein" ], [ 128, 137, "wild-type", "protein_state" ], [ 146, 154, "YfiBL43P", "mutant" ], [ 160, 162, "PG", "chemical" ], [ 172, 190, "sequence alignment", "experimental_method" ], [ 194, 207, "P. aeruginosa", "species" ], [ 212, 219, "E. coli", "species" ], [ 231, 235, "YfiB", "protein" ], [ 237, 240, "Pal", "protein_type" ], [ 249, 267, "periplasmic domain", "structure_element" ], [ 271, 275, "OmpA", "protein_type" ] ] }, { "sid": 85, "sent": "PG-associated lipoprotein (Pal) is highly conserved in Gram-negative bacteria and anchors to the outer membrane through an N-terminal lipid attachment and to PG layer through its periplasmic domain, which is implicated in maintaining outer membrane integrity.", "section": "RESULTS", "ner": [ [ 0, 25, "PG-associated lipoprotein", "protein_type" ], [ 27, 30, "Pal", "protein_type" ], [ 35, 51, "highly conserved", "protein_state" ], [ 55, 77, "Gram-negative bacteria", "taxonomy_domain" ], [ 158, 160, "PG", "chemical" ], [ 179, 197, "periplasmic domain", "structure_element" ] ] }, { "sid": 86, "sent": "Previous homology modeling studies suggested that YfiB contains a Pal-like PG-binding site (Parsons et al.,), and the mutation of two residues at this site, D102 and G105, reduces the ability for biofilm formation and surface attachment (Malone et al.,).", "section": "RESULTS", "ner": [ [ 9, 26, "homology modeling", "experimental_method" ], [ 50, 54, "YfiB", "protein" ], [ 66, 90, "Pal-like PG-binding site", "site" ], [ 118, 142, "mutation of two residues", "experimental_method" ], [ 157, 161, "D102", "residue_name_number" ], [ 166, 170, "G105", "residue_name_number" ] ] }, { "sid": 87, "sent": "In the YfiB-YfiR complex, one sulfate ion is found at the bottom of each YfiBL43P molecule (Fig.\u00a03A) and forms a strong hydrogen bond with D102 of YfiBL43P (Fig.\u00a04A and 4C).", "section": "RESULTS", "ner": [ [ 7, 16, "YfiB-YfiR", "complex_assembly" ], [ 30, 37, "sulfate", "chemical" ], [ 73, 81, "YfiBL43P", "mutant" ], [ 120, 133, "hydrogen bond", "bond_interaction" ], [ 139, 143, "D102", "residue_name_number" ], [ 147, 155, "YfiBL43P", "mutant" ] ] }, { "sid": 88, "sent": "Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-\u03b1-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 \u03f5-carboxylate group in the Pal/PG-P complex (Fig.\u00a04A).", "section": "RESULTS", "ner": [ [ 0, 24, "Structural superposition", "experimental_method" ], [ 33, 41, "YfiBL43P", "mutant" ], [ 46, 68, "Haemophilus influenzae", "species" ], [ 69, 72, "Pal", "protein_type" ], [ 73, 87, "complexed with", "protein_state" ], [ 101, 124, "peptidoglycan precursor", "chemical" ], [ 126, 130, "PG-P", "chemical" ], [ 133, 184, "UDP-N-acetylmuramyl-L-Ala-\u03b1-D-Glu-m-Dap-D-Ala-D-Ala", "chemical" ], [ 186, 191, "m-Dap", "chemical" ], [ 195, 215, "meso-diaminopimelate", "chemical" ], [ 271, 278, "sulfate", "chemical" ], [ 317, 337, "m-Dap5 \u03f5-carboxylate", "chemical" ], [ 351, 359, "Pal/PG-P", "complex_assembly" ] ] }, { "sid": 89, "sent": "In the Pal/PG-P complex structure, the m-Dap5 \u03f5-carboxylate group interacts with the side-chain atoms of D71 and the main-chain amide of D37 (Fig.\u00a04B).", "section": "RESULTS", "ner": [ [ 7, 15, "Pal/PG-P", "complex_assembly" ], [ 24, 33, "structure", "evidence" ], [ 39, 59, "m-Dap5 \u03f5-carboxylate", "chemical" ], [ 105, 108, "D71", "residue_name_number" ], [ 137, 140, "D37", "residue_name_number" ] ] }, { "sid": 90, "sent": "Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal).", "section": "RESULTS", "ner": [ [ 18, 28, "YfiR-bound", "protein_state" ], [ 29, 37, "YfiBL43P", "mutant" ], [ 38, 47, "structure", "evidence" ], [ 53, 60, "sulfate", "chemical" ], [ 104, 108, "D102", "residue_name_number" ], [ 127, 130, "D71", "residue_name_number" ], [ 134, 137, "Pal", "protein_type" ], [ 143, 147, "R117", "residue_name_number" ], [ 166, 169, "R86", "residue_name_number" ], [ 173, 176, "Pal", "protein_type" ], [ 206, 209, "N68", "residue_name_number" ], [ 228, 231, "D37", "residue_name_number" ], [ 235, 238, "Pal", "protein_type" ] ] }, { "sid": 91, "sent": "Moreover, a water molecule was found to bridge the sulfate ion and the side chains of N67 and D102, strengthening the hydrogen bond network (Fig.\u00a04C).", "section": "RESULTS", "ner": [ [ 12, 17, "water", "chemical" ], [ 51, 58, "sulfate", "chemical" ], [ 86, 89, "N67", "residue_name_number" ], [ 94, 98, "D102", "residue_name_number" ], [ 118, 139, "hydrogen bond network", "site" ] ] }, { "sid": 92, "sent": "In addition, sequence alignment of YfiB with Pal and the periplasmic domain of OmpA (proteins containing PG-binding site) showed that N68 and D102 are highly conserved (Fig.\u00a04G, blue stars), suggesting that these residues contribute to the PG-binding ability of YfiB.", "section": "RESULTS", "ner": [ [ 13, 31, "sequence alignment", "experimental_method" ], [ 35, 39, "YfiB", "protein" ], [ 45, 48, "Pal", "protein_type" ], [ 57, 75, "periplasmic domain", "structure_element" ], [ 79, 83, "OmpA", "protein_type" ], [ 105, 120, "PG-binding site", "site" ], [ 134, 137, "N68", "residue_name_number" ], [ 142, 146, "D102", "residue_name_number" ], [ 151, 167, "highly conserved", "protein_state" ], [ 262, 266, "YfiB", "protein" ] ] }, { "sid": 93, "sent": "Interestingly, superposition of apo YfiB with YfiR-bound YfiBL43P revealed that the PG-binding region is largely altered mainly due to different conformation of the N68 containing loop.", "section": "RESULTS", "ner": [ [ 15, 28, "superposition", "experimental_method" ], [ 32, 35, "apo", "protein_state" ], [ 36, 40, "YfiB", "protein" ], [ 46, 56, "YfiR-bound", "protein_state" ], [ 57, 65, "YfiBL43P", "mutant" ], [ 84, 101, "PG-binding region", "site" ], [ 135, 157, "different conformation", "protein_state" ], [ 165, 168, "N68", "residue_name_number" ], [ 180, 184, "loop", "structure_element" ] ] }, { "sid": 94, "sent": "Compared to YfiBL43P, the N68-containing loop of the apo YfiB flips away about 7 \u00c5, and D102 and R117 swing slightly outward; thus, the PG-binding pocket is enlarged with no sulfate ion or water bound (Fig.\u00a04D).", "section": "RESULTS", "ner": [ [ 12, 20, "YfiBL43P", "mutant" ], [ 26, 29, "N68", "residue_name_number" ], [ 41, 45, "loop", "structure_element" ], [ 53, 56, "apo", "protein_state" ], [ 57, 61, "YfiB", "protein" ], [ 88, 92, "D102", "residue_name_number" ], [ 97, 101, "R117", "residue_name_number" ], [ 136, 153, "PG-binding pocket", "site" ], [ 174, 181, "sulfate", "chemical" ], [ 189, 194, "water", "chemical" ] ] }, { "sid": 95, "sent": "Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively.", "section": "RESULTS", "ner": [ [ 32, 34, "PG", "chemical" ], [ 54, 62, "inactive", "protein_state" ], [ 63, 67, "YfiB", "protein" ], [ 97, 103, "active", "protein_state" ], [ 104, 108, "YfiB", "protein" ], [ 143, 168, "microscale thermophoresis", "experimental_method" ], [ 170, 173, "MST", "experimental_method" ], [ 196, 214, "binding affinities", "evidence" ], [ 218, 220, "PG", "chemical" ], [ 224, 233, "wild-type", "protein_state" ], [ 234, 238, "YfiB", "protein" ], [ 243, 251, "YfiBL43P", "mutant" ] ] }, { "sid": 96, "sent": "The results indicated that the PG-binding affinity of YfiBL43P is 65.5 \u03bcmol/L, which is about 16-fold stronger than that of wild-type YfiB (Kd = 1.1 mmol/L) (Fig.\u00a04E\u2013F).", "section": "RESULTS", "ner": [ [ 31, 50, "PG-binding affinity", "evidence" ], [ 54, 62, "YfiBL43P", "mutant" ], [ 124, 133, "wild-type", "protein_state" ], [ 134, 138, "YfiB", "protein" ], [ 140, 142, "Kd", "evidence" ] ] }, { "sid": 97, "sent": "As the experiment is performed in the absence of YfiR, it suggests that an increase in the PG-binding affinity of YfiB is not a result of YfiB-YfiR interaction and is highly coupled to the activation of YfiB characterized by a stretched N-terminal conformation.", "section": "RESULTS", "ner": [ [ 31, 48, "in the absence of", "protein_state" ], [ 49, 53, "YfiR", "protein" ], [ 91, 110, "PG-binding affinity", "evidence" ], [ 114, 118, "YfiB", "protein" ], [ 138, 147, "YfiB-YfiR", "complex_assembly" ], [ 203, 207, "YfiB", "protein" ], [ 227, 260, "stretched N-terminal conformation", "protein_state" ] ] }, { "sid": 98, "sent": "The conserved surface in YfiR is functional for binding YfiB and YfiN", "section": "RESULTS", "ner": [ [ 4, 21, "conserved surface", "site" ], [ 25, 29, "YfiR", "protein" ], [ 56, 60, "YfiB", "protein" ], [ 65, 69, "YfiN", "protein" ] ] }, { "sid": 99, "sent": "Calculation using the ConSurf Server (http://consurf.tau.ac.il/), which estimates the evolutionary conservation of amino acid positions and visualizes information on the structure surface, revealed a conserved surface on YfiR that contributes to the interaction with YfiB (Fig.\u00a03E and 3F).", "section": "RESULTS", "ner": [ [ 22, 36, "ConSurf Server", "experimental_method" ], [ 86, 111, "evolutionary conservation", "evidence" ], [ 170, 187, "structure surface", "site" ], [ 200, 217, "conserved surface", "site" ], [ 221, 225, "YfiR", "protein" ], [ 267, 271, "YfiB", "protein" ] ] }, { "sid": 100, "sent": "Interestingly, the majority of this conserved surface contributes to the interaction with YfiB (Fig.\u00a03E and 3F).", "section": "RESULTS", "ner": [ [ 36, 53, "conserved surface", "site" ], [ 90, 94, "YfiB", "protein" ] ] }, { "sid": 101, "sent": "Malone JG et al. have reported that F151, E163, I169 and Q187, located near the C-terminus of YfiR, comprise a putative YfiN binding site (Malone et al.,).", "section": "RESULTS", "ner": [ [ 36, 40, "F151", "residue_name_number" ], [ 42, 46, "E163", "residue_name_number" ], [ 48, 52, "I169", "residue_name_number" ], [ 57, 61, "Q187", "residue_name_number" ], [ 94, 98, "YfiR", "protein" ], [ 120, 137, "YfiN binding site", "site" ] ] }, { "sid": 102, "sent": "Interestingly, these residues are part of the conserved surface of YfiR (Fig.\u00a03G).", "section": "RESULTS", "ner": [ [ 46, 63, "conserved surface", "site" ], [ 67, 71, "YfiR", "protein" ] ] }, { "sid": 103, "sent": "F151, E163 and I169 form a hydrophobic core while, Q187 is located at the end of the \u03b16 helix.", "section": "RESULTS", "ner": [ [ 0, 4, "F151", "residue_name_number" ], [ 6, 10, "E163", "residue_name_number" ], [ 15, 19, "I169", "residue_name_number" ], [ 27, 43, "hydrophobic core", "site" ], [ 51, 55, "Q187", "residue_name_number" ], [ 85, 93, "\u03b16 helix", "structure_element" ] ] }, { "sid": 104, "sent": "E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig.\u00a03D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig.\u00a03D-I(ii)).", "section": "RESULTS", "ner": [ [ 0, 4, "E163", "residue_name_number" ], [ 9, 13, "I169", "residue_name_number" ], [ 18, 43, "YfiB-interacting residues", "site" ], [ 47, 51, "YfiR", "protein" ], [ 62, 66, "E163", "residue_name_number" ], [ 75, 88, "hydrogen bond", "bond_interaction" ], [ 94, 97, "R96", "residue_name_number" ], [ 101, 105, "YfiB", "protein" ], [ 123, 127, "I169", "residue_name_number" ], [ 155, 159, "L166", "residue_name_number" ], [ 160, 164, "I169", "residue_name_number" ], [ 165, 169, "V176", "residue_name_number" ], [ 170, 174, "P178", "residue_name_number" ], [ 175, 179, "L181", "residue_name_number" ], [ 180, 196, "hydrophobic core", "site" ], [ 211, 214, "F57", "residue_name_number" ], [ 218, 222, "YfiB", "protein" ] ] }, { "sid": 105, "sent": "Collectively, a part of the YfiB-YfiR interface overlaps with the proposed YfiR-YfiN interface, suggesting alteration in the association-disassociation equilibrium of YfiR-YfiN and hence the ability of YfiB to sequester YfiR.", "section": "RESULTS", "ner": [ [ 28, 47, "YfiB-YfiR interface", "site" ], [ 75, 94, "YfiR-YfiN interface", "site" ], [ 167, 171, "YfiR", "protein" ], [ 172, 176, "YfiN", "protein" ], [ 202, 206, "YfiB", "protein" ], [ 220, 224, "YfiR", "protein" ] ] }, { "sid": 106, "sent": "YfiR binds small molecules", "section": "RESULTS", "ner": [ [ 0, 4, "YfiR", "protein" ] ] }, { "sid": 107, "sent": "Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,).", "section": "RESULTS", "ner": [ [ 32, 36, "YfiR", "protein" ], [ 51, 55, "YfiB", "protein" ], [ 101, 105, "YfiN", "protein" ], [ 177, 181, "YfiR", "protein" ], [ 182, 192, "structures", "evidence" ], [ 205, 217, "non-oxidized", "protein_state" ], [ 226, 234, "oxidized", "protein_state" ], [ 270, 276, "Cys145", "residue_name_number" ], [ 277, 283, "Cys152", "residue_name_number" ], [ 284, 298, "disulfide bond", "ptm" ], [ 361, 365, "YfiR", "protein" ] ] }, { "sid": 108, "sent": "However, whether YfiR is involved in other regulatory mechanisms is still an open question.", "section": "RESULTS", "ner": [ [ 17, 21, "YfiR", "protein" ] ] }, { "sid": 109, "sent": "Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp.", "section": "FIG", "ner": [ [ 8, 18, "Structures", "evidence" ], [ 22, 31, "VB6-bound", "protein_state" ], [ 36, 45, "Trp-bound", "protein_state" ], [ 46, 50, "YfiR", "protein" ], [ 56, 69, "Superposition", "experimental_method" ], [ 85, 95, "structures", "evidence" ], [ 99, 108, "VB6-bound", "protein_state" ], [ 113, 122, "Trp-bound", "protein_state" ], [ 123, 127, "YfiR", "protein" ], [ 176, 180, "YfiR", "protein" ], [ 191, 194, "VB6", "chemical" ], [ 199, 204, "L-Trp", "chemical" ] ] }, { "sid": 110, "sent": "The electron densities of VB6 and Trp are countered at 3.0\u03c3 and 2.3\u03c3, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB", "section": "FIG", "ner": [ [ 4, 22, "electron densities", "evidence" ], [ 26, 29, "VB6", "chemical" ], [ 34, 37, "Trp", "chemical" ], [ 87, 101, "|Fo|-|Fc| maps", "evidence" ], [ 107, 120, "Superposition", "experimental_method" ], [ 128, 146, "hydrophobic pocket", "site" ], [ 150, 154, "YfiR", "protein" ], [ 160, 163, "VB6", "chemical" ], [ 165, 170, "L-Trp", "chemical" ], [ 175, 178, "F57", "residue_name_number" ], [ 182, 186, "YfiB", "protein" ] ] }, { "sid": 111, "sent": "Intriguingly, a Dali search (Holm and Rosenstrom,) indicated that the closest homologs of YfiR shared the characteristic of being able to bind several structurally similar small molecules, such as L-Trp, L-Phe, B-group vitamins and their analogs, encouraging us to test whether YfiR can recognize these molecules.", "section": "RESULTS", "ner": [ [ 16, 27, "Dali search", "experimental_method" ], [ 90, 94, "YfiR", "protein" ], [ 197, 202, "L-Trp", "chemical" ], [ 204, 209, "L-Phe", "chemical" ], [ 278, 282, "YfiR", "protein" ] ] }, { "sid": 112, "sent": "For this purpose, we co-crystallized YfiR or soaked YfiR crystals with different small molecules, including L-Trp and B-group vitamins.", "section": "RESULTS", "ner": [ [ 21, 36, "co-crystallized", "experimental_method" ], [ 37, 41, "YfiR", "protein" ], [ 45, 51, "soaked", "experimental_method" ], [ 52, 56, "YfiR", "protein" ], [ 57, 65, "crystals", "evidence" ], [ 108, 113, "L-Trp", "chemical" ] ] }, { "sid": 113, "sent": "Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig.\u00a05A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,).", "section": "RESULTS", "ner": [ [ 30, 52, "small-molecule density", "evidence" ], [ 60, 69, "VB6-bound", "protein_state" ], [ 74, 83, "Trp-bound", "protein_state" ], [ 84, 88, "YfiR", "protein" ], [ 89, 107, "crystal structures", "evidence" ], [ 138, 148, "structures", "evidence" ], [ 154, 158, "YfiR", "protein" ], [ 159, 165, "dimers", "oligomeric_state" ], [ 179, 187, "oxidized", "protein_state" ], [ 188, 192, "YfiR", "protein" ], [ 193, 202, "structure", "evidence" ], [ 221, 236, "disulfide bonds", "ptm" ] ] }, { "sid": 114, "sent": "Functional analysis of VB6 and L-Trp. (A and B) The effect of increasing concentrations of VB6 or L-Trp on YfiBL43P-induced attachment (bars).", "section": "FIG", "ner": [ [ 23, 26, "VB6", "chemical" ], [ 31, 36, "L-Trp", "chemical" ], [ 52, 87, "effect of increasing concentrations", "experimental_method" ], [ 91, 94, "VB6", "chemical" ], [ 98, 103, "L-Trp", "chemical" ], [ 107, 115, "YfiBL43P", "mutant" ] ] }, { "sid": 115, "sent": "The relative optical density is represented as curves.", "section": "FIG", "ner": [ [ 4, 28, "relative optical density", "evidence" ] ] }, { "sid": 116, "sent": "Wild-type YfiB is used as negative control.", "section": "FIG", "ner": [ [ 0, 9, "Wild-type", "protein_state" ], [ 10, 14, "YfiB", "protein" ] ] }, { "sid": 117, "sent": "(C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E\u2013G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR", "section": "FIG", "ner": [ [ 10, 17, "BIAcore", "experimental_method" ], [ 40, 58, "binding affinities", "evidence" ], [ 66, 69, "VB6", "chemical" ], [ 78, 83, "L-Trp", "chemical" ], [ 89, 93, "YfiR", "protein" ], [ 101, 104, "ITC", "experimental_method" ], [ 127, 136, "titration", "experimental_method" ], [ 144, 148, "YfiB", "protein" ], [ 149, 158, "wild-type", "protein_state" ], [ 182, 190, "YfiBL43P", "mutant" ], [ 191, 195, "F57A", "mutant" ], [ 201, 205, "YfiR", "protein" ] ] }, { "sid": 118, "sent": "Structural analyses revealed that the VB6 and L-Trp molecules are bound at the periphery of the YfiR dimer, but not at the dimer interface.", "section": "RESULTS", "ner": [ [ 0, 19, "Structural analyses", "experimental_method" ], [ 38, 41, "VB6", "chemical" ], [ 46, 51, "L-Trp", "chemical" ], [ 66, 74, "bound at", "protein_state" ], [ 96, 100, "YfiR", "protein" ], [ 101, 106, "dimer", "oligomeric_state" ], [ 123, 138, "dimer interface", "site" ] ] }, { "sid": 119, "sent": "Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig.\u00a05C).", "section": "RESULTS", "ner": [ [ 15, 18, "VB6", "chemical" ], [ 23, 28, "L-Trp", "chemical" ], [ 59, 77, "hydrophobic pocket", "site" ], [ 89, 93, "L166", "residue_name_number" ], [ 94, 98, "I169", "residue_name_number" ], [ 99, 103, "V176", "residue_name_number" ], [ 104, 108, "P178", "residue_name_number" ], [ 109, 113, "L181", "residue_name_number" ], [ 117, 121, "YfiR", "protein" ], [ 139, 153, "binding pocket", "site" ], [ 158, 161, "F57", "residue_name_number" ], [ 165, 169, "YfiB", "protein" ], [ 190, 199, "YfiB-YfiR", "complex_assembly" ] ] }, { "sid": 120, "sent": "To evaluate the importance of F57 in YfiBL43P-YfiR interaction, the binding affinities of YfiBL43P and YfiBL43P/F57A for YfiR were measured by isothermal titration calorimetry (ITC).", "section": "RESULTS", "ner": [ [ 30, 33, "F57", "residue_name_number" ], [ 37, 50, "YfiBL43P-YfiR", "complex_assembly" ], [ 68, 86, "binding affinities", "evidence" ], [ 90, 98, "YfiBL43P", "mutant" ], [ 103, 111, "YfiBL43P", "mutant" ], [ 112, 116, "F57A", "mutant" ], [ 121, 125, "YfiR", "protein" ], [ 143, 175, "isothermal titration calorimetry", "experimental_method" ], [ 177, 180, "ITC", "experimental_method" ] ] }, { "sid": 121, "sent": "The results showed Kd values of 1.4 \u00d7 10\u22127 mol/L and 5.3 \u00d7 10\u22127 mol/L for YfiBL43P and YfiBL43P/F57A, respectively, revealing that the YfiBL43P/F57A mutant caused a 3.8-fold reduction in the binding affinity compared with the YfiBL43P mutant (Fig.\u00a06F and 6G).", "section": "RESULTS", "ner": [ [ 19, 21, "Kd", "evidence" ], [ 74, 82, "YfiBL43P", "mutant" ], [ 87, 95, "YfiBL43P", "mutant" ], [ 96, 100, "F57A", "mutant" ], [ 135, 143, "YfiBL43P", "mutant" ], [ 144, 148, "F57A", "mutant" ], [ 149, 155, "mutant", "protein_state" ], [ 191, 207, "binding affinity", "evidence" ], [ 226, 234, "YfiBL43P", "mutant" ], [ 235, 241, "mutant", "protein_state" ] ] }, { "sid": 122, "sent": "In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 \u0394yfiB strain to trigger YfiBL43P-induced biofilm formation.", "section": "RESULTS", "ner": [ [ 66, 69, "VB6", "chemical" ], [ 74, 79, "L-Trp", "chemical" ], [ 81, 85, "yfiB", "gene" ], [ 90, 97, "deleted", "experimental_method" ], [ 103, 107, "PAO1", "species" ], [ 108, 117, "wild-type", "protein_state" ], [ 132, 152, "construct expressing", "experimental_method" ], [ 157, 165, "YfiBL43P", "mutant" ], [ 166, 172, "mutant", "protein_state" ], [ 177, 193, "transformed into", "experimental_method" ], [ 198, 202, "PAO1", "species" ], [ 203, 208, "\u0394yfiB", "mutant" ], [ 227, 235, "YfiBL43P", "mutant" ] ] }, { "sid": 123, "sent": "Growth and surface attachment assays were carried out for the yfiB-L43P strain in the presence of increasing concentrations of VB6 or L-Trp.", "section": "RESULTS", "ner": [ [ 0, 36, "Growth and surface attachment assays", "experimental_method" ], [ 62, 71, "yfiB-L43P", "mutant" ], [ 98, 123, "increasing concentrations", "experimental_method" ], [ 127, 130, "VB6", "chemical" ], [ 134, 139, "L-Trp", "chemical" ] ] }, { "sid": 124, "sent": "As shown in Fig.\u00a06A and 6B, the over-expression of YfiBL43P induced strong surface attachment and much slower growth of the yfiB-L43P strain, and as expected, a certain amount of VB6 or L-Trp (4\u20136 mmol/L for VB6 and 6\u201310 mmol/L for L-Trp) could reduce the surface attachment.", "section": "RESULTS", "ner": [ [ 32, 47, "over-expression", "experimental_method" ], [ 51, 59, "YfiBL43P", "mutant" ], [ 124, 133, "yfiB-L43P", "mutant" ], [ 179, 182, "VB6", "chemical" ], [ 186, 191, "L-Trp", "chemical" ], [ 208, 211, "VB6", "chemical" ], [ 232, 237, "L-Trp", "chemical" ] ] }, { "sid": 125, "sent": "Interestingly, at a concentration higher than 8 mmol/L, VB6 lost its ability to inhibit biofilm formation, implying that the VB6-involving regulatory mechanism is highly complicated and remains to be further investigated.", "section": "RESULTS", "ner": [ [ 56, 59, "VB6", "chemical" ], [ 125, 128, "VB6", "chemical" ] ] }, { "sid": 126, "sent": "Of note, both VB6 and L-Trp have been reported to correlate with biofilm formation in certain Gram-negative bacteria (Grubman et al.,; Shimazaki et al.,).", "section": "RESULTS", "ner": [ [ 14, 17, "VB6", "chemical" ], [ 22, 27, "L-Trp", "chemical" ], [ 94, 116, "Gram-negative bacteria", "taxonomy_domain" ] ] }, { "sid": 127, "sent": "In Helicobacter pylori in particular, VB6 biosynthetic enzymes act as novel virulence factors, and VB6 is required for full motility and virulence (Grubman et al.,).", "section": "RESULTS", "ner": [ [ 3, 22, "Helicobacter pylori", "species" ], [ 38, 41, "VB6", "chemical" ], [ 99, 102, "VB6", "chemical" ] ] }, { "sid": 128, "sent": "\u00a0In E.\u00a0coli,\u00a0mutants with decreased tryptophan synthesis show greater biofilm formation, and matured biofilm is degraded by L-tryptophan addition (Shimazaki et al.,).", "section": "RESULTS", "ner": [ [ 4, 11, "E.\u00a0coli", "species" ], [ 36, 46, "tryptophan", "chemical" ], [ 124, 136, "L-tryptophan", "chemical" ] ] }, { "sid": 129, "sent": "To answer the question whether competition of VB6 or L-Trp for the YfiB F57-binding pocket of YfiR plays an essential role in inhibiting biofilm formation, we measured the binding affinities of VB6 and L-Trp for YfiR via BIAcore experiments.", "section": "RESULTS", "ner": [ [ 46, 49, "VB6", "chemical" ], [ 53, 58, "L-Trp", "chemical" ], [ 67, 71, "YfiB", "protein" ], [ 72, 90, "F57-binding pocket", "site" ], [ 94, 98, "YfiR", "protein" ], [ 172, 190, "binding affinities", "evidence" ], [ 194, 197, "VB6", "chemical" ], [ 202, 207, "L-Trp", "chemical" ], [ 212, 216, "YfiR", "protein" ], [ 221, 228, "BIAcore", "experimental_method" ] ] }, { "sid": 130, "sent": "The results showed relatively weak Kd values of 35.2 mmol/L and 76.9 mmol/L for VB6 and L-Trp, respectively (Fig.\u00a06C and 6D).", "section": "RESULTS", "ner": [ [ 35, 37, "Kd", "evidence" ], [ 80, 83, "VB6", "chemical" ], [ 88, 93, "L-Trp", "chemical" ] ] }, { "sid": 131, "sent": "Based on our results, we concluded that VB6 or L-Trp can bind to YfiR, however, VB6 or L-Trp alone may have little effects in interrupting the YfiB-YfiR interaction, the mechanism by which VB6 or L-Trp inhibits biofilm formation remains unclear and requires further investigation.", "section": "RESULTS", "ner": [ [ 40, 43, "VB6", "chemical" ], [ 47, 52, "L-Trp", "chemical" ], [ 65, 69, "YfiR", "protein" ], [ 80, 83, "VB6", "chemical" ], [ 87, 92, "L-Trp", "chemical" ], [ 93, 98, "alone", "protein_state" ], [ 143, 152, "YfiB-YfiR", "complex_assembly" ], [ 189, 192, "VB6", "chemical" ], [ 196, 201, "L-Trp", "chemical" ] ] }, { "sid": 132, "sent": "Previous studies suggested that in response to cell stress, YfiB in the outer membrane sequesters the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus inducing the diguanylate cyclase activity of YfiN to allow c-di-GMP production (Giardina et al.,; Malone et al.,; Malone et al.,).", "section": "DISCUSS", "ner": [ [ 60, 64, "YfiB", "protein" ], [ 122, 126, "YfiR", "protein" ], [ 156, 160, "YfiN", "protein" ], [ 237, 241, "YfiN", "protein" ], [ 251, 259, "c-di-GMP", "chemical" ] ] }, { "sid": 133, "sent": "Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues.", "section": "DISCUSS", "ner": [ [ 20, 38, "crystal structures", "evidence" ], [ 42, 46, "YfiB", "protein" ], [ 47, 52, "alone", "protein_state" ], [ 60, 66, "active", "protein_state" ], [ 67, 73, "mutant", "protein_state" ], [ 74, 82, "YfiBL43P", "mutant" ], [ 83, 98, "in complex with", "protein_state" ], [ 99, 103, "YfiR", "protein" ], [ 121, 125, "YfiR", "protein" ], [ 138, 150, "complex with", "protein_state" ], [ 151, 155, "YfiB", "protein" ] ] }, { "sid": 134, "sent": "Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 \u00c5 in length.", "section": "DISCUSS", "ner": [ [ 4, 28, "structural data analysis", "experimental_method" ], [ 44, 53, "activated", "protein_state" ], [ 54, 58, "YfiB", "protein" ], [ 66, 84, "N-terminal portion", "structure_element" ], [ 121, 143, "stretched conformation", "protein_state" ], [ 162, 182, "compact conformation", "protein_state" ], [ 190, 193, "apo", "protein_state" ], [ 194, 198, "YfiB", "protein" ], [ 204, 207, "apo", "protein_state" ], [ 208, 212, "YfiB", "protein" ], [ 213, 222, "structure", "evidence" ], [ 256, 258, "34", "residue_number" ], [ 265, 285, "compact conformation", "protein_state" ] ] }, { "sid": 135, "sent": "In addition to the preceding 8 aa loop (from the lipid acceptor Cys26 to Gly34), the full length of the periplasmic portion of apo YfiB can reach approximately 60 \u00c5. It was reported that the distance between the outer membrane and the cell wall is approximately 50 \u00c5 and that the thickness of the PG layer is approximately 70 \u00c5 (Matias et al.,).", "section": "DISCUSS", "ner": [ [ 19, 33, "preceding 8 aa", "residue_range" ], [ 34, 38, "loop", "structure_element" ], [ 64, 78, "Cys26 to Gly34", "residue_range" ], [ 85, 96, "full length", "protein_state" ], [ 127, 130, "apo", "protein_state" ], [ 131, 135, "YfiB", "protein" ] ] }, { "sid": 136, "sent": "Thus, YfiB alone represents an inactive form that may only partially insert into the PG matrix.", "section": "DISCUSS", "ner": [ [ 6, 10, "YfiB", "protein" ], [ 11, 16, "alone", "protein_state" ], [ 31, 39, "inactive", "protein_state" ] ] }, { "sid": 137, "sent": "By contrast, YfiR-bound YfiBL43P (residues 44\u2013168) has a stretched conformation of approximately 55 \u00c5 in length.", "section": "DISCUSS", "ner": [ [ 13, 23, "YfiR-bound", "protein_state" ], [ 24, 32, "YfiBL43P", "mutant" ], [ 43, 49, "44\u2013168", "residue_range" ], [ 57, 79, "stretched conformation", "protein_state" ] ] }, { "sid": 138, "sent": "In addition to the 17 preceding intracellular residues (from the lipid acceptor Cys26 to Leu43), the length of the intracellular portion of active YfiB may extend over 100 \u00c5, assuming a fully stretched conformation.", "section": "DISCUSS", "ner": [ [ 19, 54, "17 preceding intracellular residues", "residue_range" ], [ 80, 94, "Cys26 to Leu43", "residue_range" ], [ 140, 146, "active", "protein_state" ], [ 147, 151, "YfiB", "protein" ], [ 186, 214, "fully stretched conformation", "protein_state" ] ] }, { "sid": 139, "sent": "Provided that the diameter of the widest part of the YfiB dimer is approximately 64 \u00c5, which is slightly smaller than the smallest diameter of the PG pore (70 \u00c5) (Meroueh et al.,), the YfiB dimer should be able to penetrate the PG layer.", "section": "DISCUSS", "ner": [ [ 53, 57, "YfiB", "protein" ], [ 58, 63, "dimer", "oligomeric_state" ], [ 185, 189, "YfiB", "protein" ], [ 190, 195, "dimer", "oligomeric_state" ] ] }, { "sid": 140, "sent": "Regulatory model of the YfiBNR tripartite system.", "section": "FIG", "ner": [ [ 24, 30, "YfiBNR", "complex_assembly" ], [ 31, 41, "tripartite", "protein_state" ] ] }, { "sid": 141, "sent": "The periplasmic domain of YfiB and the YfiB-YfiR complex are depicted according to the crystal structures.", "section": "FIG", "ner": [ [ 4, 22, "periplasmic domain", "structure_element" ], [ 26, 30, "YfiB", "protein" ], [ 39, 48, "YfiB-YfiR", "complex_assembly" ], [ 87, 105, "crystal structures", "evidence" ] ] }, { "sid": 142, "sent": "The lipid acceptor Cys26 is indicated as blue ball.", "section": "FIG", "ner": [ [ 19, 24, "Cys26", "residue_name_number" ] ] }, { "sid": 143, "sent": "The loop connecting Cys26 and Gly34 of YfiB is modeled.", "section": "FIG", "ner": [ [ 4, 8, "loop", "structure_element" ], [ 20, 25, "Cys26", "residue_name_number" ], [ 30, 35, "Gly34", "residue_name_number" ], [ 39, 43, "YfiB", "protein" ] ] }, { "sid": 144, "sent": "The PAS domain of YfiN is shown as pink oval.", "section": "FIG", "ner": [ [ 4, 14, "PAS domain", "structure_element" ], [ 18, 22, "YfiN", "protein" ] ] }, { "sid": 145, "sent": "Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN", "section": "FIG", "ner": [ [ 5, 14, "activated", "protein_state" ], [ 43, 50, "dimeric", "oligomeric_state" ], [ 51, 55, "YfiB", "protein" ], [ 74, 94, "compact conformation", "protein_state" ], [ 100, 122, "stretched conformation", "protein_state" ], [ 137, 155, "periplasmic domain", "structure_element" ], [ 163, 180, "membrane-anchored", "protein_state" ], [ 181, 185, "YfiB", "protein" ], [ 231, 235, "YfiR", "protein" ], [ 236, 241, "dimer", "oligomeric_state" ], [ 276, 280, "YfiN", "protein" ] ] }, { "sid": 146, "sent": "These results, together with our observation that activated YfiB has a much higher cell wall binding affinity, and previous mutagenesis data showing that (1) both PG binding and membrane anchoring are required for YfiB activity and (2) activating mutations possessing an altered N-terminal loop length are dominant over the loss of PG binding (Malone et al.,), suggest an updated regulatory model of the YfiBNR system (Fig.\u00a07).", "section": "DISCUSS", "ner": [ [ 50, 59, "activated", "protein_state" ], [ 60, 64, "YfiB", "protein" ], [ 83, 109, "cell wall binding affinity", "evidence" ], [ 163, 165, "PG", "chemical" ], [ 214, 218, "YfiB", "protein" ], [ 290, 294, "loop", "structure_element" ], [ 332, 334, "PG", "chemical" ], [ 404, 410, "YfiBNR", "complex_assembly" ] ] }, { "sid": 147, "sent": "In this model, in response to a particular cell stress that is yet to be identified, the dimeric YfiB is activated from a compact, inactive conformation to a stretched conformation, which possesses increased PG binding affinity.", "section": "DISCUSS", "ner": [ [ 89, 96, "dimeric", "oligomeric_state" ], [ 97, 101, "YfiB", "protein" ], [ 105, 114, "activated", "protein_state" ], [ 122, 129, "compact", "protein_state" ], [ 131, 139, "inactive", "protein_state" ], [ 140, 152, "conformation", "protein_state" ], [ 158, 180, "stretched conformation", "protein_state" ], [ 208, 210, "PG", "chemical" ] ] }, { "sid": 148, "sent": "This allows the C-terminal portion of the membrane-anchored YfiB to reach, bind and penetrate the cell wall and sequester the YfiR dimer.", "section": "DISCUSS", "ner": [ [ 16, 34, "C-terminal portion", "structure_element" ], [ 42, 59, "membrane-anchored", "protein_state" ], [ 60, 64, "YfiB", "protein" ], [ 126, 130, "YfiR", "protein" ], [ 131, 136, "dimer", "oligomeric_state" ] ] }, { "sid": 149, "sent": "The YfiBNR system provides a good example of a delicate homeostatic system that integrates multiple signals to regulate the c-di-GMP level.", "section": "DISCUSS", "ner": [ [ 4, 10, "YfiBNR", "complex_assembly" ], [ 124, 132, "c-di-GMP", "chemical" ] ] }, { "sid": 150, "sent": "Homologs of the YfiBNR system are functionally conserved in P. aeruginosa (Malone et al.,; Malone et al.,), E. coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), K. pneumonia (Huertas et al.,) and Y. pestis (Ren et al.,), where they affect c-di-GMP production and biofilm formation.", "section": "DISCUSS", "ner": [ [ 16, 22, "YfiBNR", "complex_assembly" ], [ 34, 56, "functionally conserved", "protein_state" ], [ 60, 73, "P. aeruginosa", "species" ], [ 108, 115, "E. coli", "species" ], [ 178, 190, "K. pneumonia", "species" ], [ 213, 222, "Y. pestis", "species" ], [ 256, 264, "c-di-GMP", "chemical" ] ] }, { "sid": 151, "sent": "The mechanism by which activated YfiB relieves the repression of YfiN may be applicable to the YfiBNR system in other bacteria and to analogous outside-in signaling for c-di-GMP production, which in turn may be relevant to the development of drugs that can circumvent complicated antibiotic resistance.", "section": "DISCUSS", "ner": [ [ 23, 32, "activated", "protein_state" ], [ 33, 37, "YfiB", "protein" ], [ 65, 69, "YfiN", "protein" ], [ 95, 101, "YfiBNR", "complex_assembly" ], [ 118, 126, "bacteria", "taxonomy_domain" ], [ 169, 177, "c-di-GMP", "chemical" ] ] } ] }, "PMC4831588": { "annotations": [ { "sid": 0, "sent": "X-ray Crystallographic Structures of a Trimer, Dodecamer, and Annular Pore Formed by an A\u03b217\u201336 \u03b2-Hairpin", "section": "TITLE", "ner": [ [ 0, 33, "X-ray Crystallographic Structures", "evidence" ], [ 39, 45, "Trimer", "oligomeric_state" ], [ 47, 56, "Dodecamer", "oligomeric_state" ], [ 62, 74, "Annular Pore", "site" ], [ 88, 90, "A\u03b2", "protein" ], [ 90, 95, "17\u201336", "residue_range" ], [ 96, 105, "\u03b2-Hairpin", "structure_element" ] ] }, { "sid": 1, "sent": "High-resolution structures of oligomers formed by the \u03b2-amyloid peptide A\u03b2 are needed to understand the molecular basis of Alzheimer\u2019s disease and develop therapies.", "section": "ABSTRACT", "ner": [ [ 16, 26, "structures", "evidence" ], [ 30, 39, "oligomers", "oligomeric_state" ], [ 54, 71, "\u03b2-amyloid peptide", "protein" ], [ 72, 74, "A\u03b2", "protein" ] ] }, { "sid": 2, "sent": "This paper presents the X-ray crystallographic structures of oligomers formed by a 20-residue peptide segment derived from A\u03b2.", "section": "ABSTRACT", "ner": [ [ 24, 57, "X-ray crystallographic structures", "evidence" ], [ 61, 70, "oligomers", "oligomeric_state" ], [ 83, 109, "20-residue peptide segment", "residue_range" ], [ 123, 125, "A\u03b2", "protein" ] ] }, { "sid": 3, "sent": "The development of a peptide in which A\u03b217\u201336 is stabilized as a \u03b2-hairpin is described, and the X-ray crystallographic structures of oligomers it forms are reported.", "section": "ABSTRACT", "ner": [ [ 38, 40, "A\u03b2", "protein" ], [ 40, 45, "17\u201336", "residue_range" ], [ 65, 74, "\u03b2-hairpin", "structure_element" ], [ 97, 130, "X-ray crystallographic structures", "evidence" ], [ 134, 143, "oligomers", "oligomeric_state" ] ] }, { "sid": 4, "sent": "Two covalent constraints act in tandem to stabilize the A\u03b217\u201336 peptide in a hairpin conformation: a \u03b4-linked ornithine turn connecting positions 17 and 36 to create a macrocycle and an intramolecular disulfide linkage between positions 24 and 29.", "section": "ABSTRACT", "ner": [ [ 56, 58, "A\u03b2", "protein" ], [ 58, 63, "17\u201336", "residue_range" ], [ 77, 84, "hairpin", "structure_element" ], [ 101, 109, "\u03b4-linked", "protein_state" ], [ 110, 119, "ornithine", "residue_name" ], [ 120, 124, "turn", "structure_element" ], [ 146, 148, "17", "residue_number" ], [ 153, 155, "36", "residue_number" ], [ 201, 218, "disulfide linkage", "ptm" ], [ 237, 239, "24", "residue_number" ], [ 244, 246, "29", "residue_number" ] ] }, { "sid": 5, "sent": "An N-methyl group at position 33 blocks uncontrolled aggregation.", "section": "ABSTRACT", "ner": [ [ 30, 32, "33", "residue_number" ] ] }, { "sid": 6, "sent": "The peptide readily crystallizes as a folded \u03b2-hairpin, which assembles hierarchically in the crystal lattice.", "section": "ABSTRACT", "ner": [ [ 12, 32, "readily crystallizes", "evidence" ], [ 38, 44, "folded", "protein_state" ], [ 45, 54, "\u03b2-hairpin", "structure_element" ], [ 94, 109, "crystal lattice", "evidence" ] ] }, { "sid": 7, "sent": "Three \u03b2-hairpin monomers assemble to form a triangular trimer, four trimers assemble in a tetrahedral arrangement to form a dodecamer, and five dodecamers pack together to form an annular pore.", "section": "ABSTRACT", "ner": [ [ 6, 15, "\u03b2-hairpin", "structure_element" ], [ 16, 24, "monomers", "oligomeric_state" ], [ 44, 54, "triangular", "protein_state" ], [ 55, 61, "trimer", "oligomeric_state" ], [ 68, 75, "trimers", "oligomeric_state" ], [ 124, 133, "dodecamer", "oligomeric_state" ], [ 144, 154, "dodecamers", "oligomeric_state" ], [ 180, 192, "annular pore", "site" ] ] }, { "sid": 8, "sent": "This hierarchical assembly provides a model, in which full-length A\u03b2 transitions from an unfolded monomer to a folded \u03b2-hairpin, which assembles to form oligomers that further pack to form an annular pore.", "section": "ABSTRACT", "ner": [ [ 54, 65, "full-length", "protein_state" ], [ 66, 68, "A\u03b2", "protein" ], [ 89, 97, "unfolded", "protein_state" ], [ 98, 105, "monomer", "oligomeric_state" ], [ 111, 117, "folded", "protein_state" ], [ 118, 127, "\u03b2-hairpin", "structure_element" ], [ 153, 162, "oligomers", "oligomeric_state" ], [ 192, 204, "annular pore", "site" ] ] }, { "sid": 9, "sent": "High-resolution structures of oligomers formed by the \u03b2-amyloid peptide A\u03b2 are desperately needed to understand the molecular basis of Alzheimer\u2019s disease and ultimately develop preventions or treatments.", "section": "INTRO", "ner": [ [ 16, 26, "structures", "evidence" ], [ 30, 39, "oligomers", "oligomeric_state" ], [ 54, 71, "\u03b2-amyloid peptide", "protein" ], [ 72, 74, "A\u03b2", "protein" ] ] }, { "sid": 10, "sent": "In Alzheimer\u2019s disease, monomeric A\u03b2 aggregates to form soluble low molecular weight oligomers, such as dimers, trimers, tetramers, hexamers, nonamers, and dodecamers, as well as high molecular weight aggregates, such as annular protofibrils.", "section": "INTRO", "ner": [ [ 24, 33, "monomeric", "oligomeric_state" ], [ 34, 36, "A\u03b2", "protein" ], [ 85, 94, "oligomers", "oligomeric_state" ], [ 104, 110, "dimers", "oligomeric_state" ], [ 112, 119, "trimers", "oligomeric_state" ], [ 121, 130, "tetramers", "oligomeric_state" ], [ 132, 140, "hexamers", "oligomeric_state" ], [ 142, 150, "nonamers", "oligomeric_state" ], [ 156, 166, "dodecamers", "oligomeric_state" ], [ 221, 241, "annular protofibrils", "complex_assembly" ] ] }, { "sid": 11, "sent": "Over the last two decades the role of A\u03b2 oligomers in the pathophysiology of Alzheimer\u2019s disease has begun to unfold.", "section": "INTRO", "ner": [ [ 38, 40, "A\u03b2", "protein" ], [ 41, 50, "oligomers", "oligomeric_state" ] ] }, { "sid": 12, "sent": "Mouse models for Alzheimer\u2019s disease have helped shape our current understanding about the A\u03b2 oligomerization that precedes neurodegeneration.", "section": "INTRO", "ner": [ [ 0, 5, "Mouse", "taxonomy_domain" ], [ 91, 93, "A\u03b2", "protein" ] ] }, { "sid": 13, "sent": "A\u03b2 isolated from the brains of young plaque-free Tg2576 mice forms a mixture of low molecular weight oligomers.", "section": "INTRO", "ner": [ [ 0, 2, "A\u03b2", "protein" ], [ 56, 60, "mice", "taxonomy_domain" ], [ 101, 110, "oligomers", "oligomeric_state" ] ] }, { "sid": 14, "sent": "A 56 kDa soluble oligomer identified by SDS-PAGE was found to be especially important within this mixture.", "section": "INTRO", "ner": [ [ 17, 25, "oligomer", "oligomeric_state" ], [ 40, 48, "SDS-PAGE", "experimental_method" ] ] }, { "sid": 15, "sent": "This oligomer was termed A\u03b2*56 and appears to be a dodecamer of A\u03b2.", "section": "INTRO", "ner": [ [ 5, 13, "oligomer", "oligomeric_state" ], [ 25, 30, "A\u03b2*56", "complex_assembly" ], [ 51, 60, "dodecamer", "oligomeric_state" ], [ 64, 66, "A\u03b2", "protein" ] ] }, { "sid": 16, "sent": "Purified A\u03b2*56 injected intercranially into healthy rats was found to impair memory, providing evidence that this A\u03b2 oligomer may cause memory loss in Alzheimer\u2019s disease.", "section": "INTRO", "ner": [ [ 9, 14, "A\u03b2*56", "complex_assembly" ], [ 15, 38, "injected intercranially", "experimental_method" ], [ 52, 56, "rats", "taxonomy_domain" ], [ 114, 116, "A\u03b2", "protein" ], [ 117, 125, "oligomer", "oligomeric_state" ] ] }, { "sid": 17, "sent": "Smaller oligomers with molecular weights consistent with trimers, hexamers, and nonamers were also identified within the mixture of low molecular weight oligomers.", "section": "INTRO", "ner": [ [ 8, 17, "oligomers", "oligomeric_state" ], [ 57, 64, "trimers", "oligomeric_state" ], [ 66, 74, "hexamers", "oligomeric_state" ], [ 80, 88, "nonamers", "oligomeric_state" ], [ 153, 162, "oligomers", "oligomeric_state" ] ] }, { "sid": 18, "sent": "Treatment of the mixture of low molecular weight oligomers with hexafluoroisopropanol resulted in the dissociation of the putative dodecamers, nonamers, and hexamers into trimers and monomers, suggesting that trimers may be the building block of the dodecamers, nonamers, and hexamers.", "section": "INTRO", "ner": [ [ 49, 58, "oligomers", "oligomeric_state" ], [ 64, 85, "hexafluoroisopropanol", "chemical" ], [ 131, 141, "dodecamers", "oligomeric_state" ], [ 143, 151, "nonamers", "oligomeric_state" ], [ 157, 165, "hexamers", "oligomeric_state" ], [ 171, 178, "trimers", "oligomeric_state" ], [ 183, 191, "monomers", "oligomeric_state" ], [ 209, 216, "trimers", "oligomeric_state" ], [ 250, 260, "dodecamers", "oligomeric_state" ], [ 262, 270, "nonamers", "oligomeric_state" ], [ 276, 284, "hexamers", "oligomeric_state" ] ] }, { "sid": 19, "sent": "Recently, A\u03b2 trimers and A\u03b2*56 were identified in the brains of cognitively normal humans and were found to increase with age.", "section": "INTRO", "ner": [ [ 10, 12, "A\u03b2", "protein" ], [ 13, 20, "trimers", "oligomeric_state" ], [ 25, 30, "A\u03b2*56", "complex_assembly" ], [ 83, 89, "humans", "species" ] ] }, { "sid": 20, "sent": "A type of large oligomers called annular protofibrils (APFs) have also been observed in the brains of transgenic mice and isolated from the brains of Alzheimer\u2019s patients.", "section": "INTRO", "ner": [ [ 16, 25, "oligomers", "oligomeric_state" ], [ 33, 53, "annular protofibrils", "complex_assembly" ], [ 55, 59, "APFs", "complex_assembly" ], [ 113, 117, "mice", "taxonomy_domain" ] ] }, { "sid": 21, "sent": "APFs were first discovered in vitro using chemically synthesized A\u03b2 that aggregated into porelike structures that could be observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM).", "section": "INTRO", "ner": [ [ 0, 4, "APFs", "complex_assembly" ], [ 42, 64, "chemically synthesized", "protein_state" ], [ 65, 67, "A\u03b2", "protein" ], [ 89, 108, "porelike structures", "structure_element" ], [ 135, 158, "atomic force microscopy", "experimental_method" ], [ 160, 163, "AFM", "experimental_method" ], [ 169, 201, "transmission electron microscopy", "experimental_method" ], [ 203, 206, "TEM", "experimental_method" ] ] }, { "sid": 22, "sent": "The sizes of APFs prepared in vitro vary among different studies.", "section": "INTRO", "ner": [ [ 13, 17, "APFs", "complex_assembly" ] ] }, { "sid": 23, "sent": "Lashuel et al. observed APFs with an outer diameter that ranged from 7\u201310 nm and an inner diameter that ranged from 1.5\u20132 nm, consistent with molecular weights of 150\u2013250 kDa.", "section": "INTRO", "ner": [ [ 24, 28, "APFs", "complex_assembly" ] ] }, { "sid": 24, "sent": "Quist et al. observed APFs with an outer diameter of 16 nm embedded in a lipid bilayer.", "section": "INTRO", "ner": [ [ 22, 26, "APFs", "complex_assembly" ] ] }, { "sid": 25, "sent": "Kayed et al. observed APFs with an outer diameter that ranged from 8\u201325 nm, which were composed of small spherical A\u03b2 oligomers, 3\u20135 nm in diameter.", "section": "INTRO", "ner": [ [ 22, 26, "APFs", "complex_assembly" ], [ 99, 114, "small spherical", "protein_state" ], [ 115, 117, "A\u03b2", "protein" ], [ 118, 127, "oligomers", "oligomeric_state" ] ] }, { "sid": 26, "sent": "Although the APFs in these studies differ in size, they share a similar annular morphology and appear to be composed of smaller oligomers.", "section": "INTRO", "ner": [ [ 13, 17, "APFs", "complex_assembly" ], [ 128, 137, "oligomers", "oligomeric_state" ] ] }, { "sid": 27, "sent": "APFs have also been observed in the brains of APP23 transgenic mice by immunofluorescence with an anti-APF antibody and were found to accumulate in neuronal processes and synapses.", "section": "INTRO", "ner": [ [ 0, 4, "APFs", "complex_assembly" ], [ 63, 67, "mice", "taxonomy_domain" ], [ 71, 89, "immunofluorescence", "experimental_method" ], [ 103, 106, "APF", "complex_assembly" ] ] }, { "sid": 28, "sent": "In a subsequent study, APFs were isolated from the brains of Alzheimer\u2019s patients by immunoprecipitation with an anti-APF antibody.", "section": "INTRO", "ner": [ [ 23, 27, "APFs", "complex_assembly" ], [ 85, 104, "immunoprecipitation", "experimental_method" ], [ 118, 121, "APF", "complex_assembly" ] ] }, { "sid": 29, "sent": "These APFs had an outer diameter that ranged from 11\u201314 nm and an inner diameter that ranged from 2.5\u20134 nm.", "section": "INTRO", "ner": [ [ 6, 10, "APFs", "complex_assembly" ] ] }, { "sid": 30, "sent": "Dimers of A\u03b2 have also been isolated from the brains of Alzheimer\u2019s patients.\u2212 A\u03b2 dimers inhibit long-term potentiation in mice and promote hyperphosphorylation of the microtubule-associated protein tau, leading to neuritic damage.", "section": "INTRO", "ner": [ [ 0, 6, "Dimers", "oligomeric_state" ], [ 10, 12, "A\u03b2", "protein" ], [ 79, 81, "A\u03b2", "protein" ], [ 82, 88, "dimers", "oligomeric_state" ], [ 123, 127, "mice", "taxonomy_domain" ], [ 140, 160, "hyperphosphorylation", "ptm" ], [ 168, 202, "microtubule-associated protein tau", "protein" ] ] }, { "sid": 31, "sent": "A\u03b2 dimers have only been isolated from human or transgenic mouse brains that contain the pathognomonic fibrillar A\u03b2 plaques associated with Alzheimer\u2019s disease.", "section": "INTRO", "ner": [ [ 0, 2, "A\u03b2", "protein" ], [ 3, 9, "dimers", "oligomeric_state" ], [ 39, 44, "human", "species" ], [ 59, 64, "mouse", "taxonomy_domain" ], [ 103, 112, "fibrillar", "protein_state" ], [ 113, 115, "A\u03b2", "protein" ] ] }, { "sid": 32, "sent": "Furthermore, the endogenous rise of A\u03b2 dimers in the brains of Tg2576 and J20 transgenic mice coincides with the deposition of A\u03b2 plaques.", "section": "INTRO", "ner": [ [ 36, 38, "A\u03b2", "protein" ], [ 39, 45, "dimers", "oligomeric_state" ], [ 89, 93, "mice", "taxonomy_domain" ], [ 127, 129, "A\u03b2", "protein" ] ] }, { "sid": 33, "sent": "These observations suggest that the A\u03b2 trimers, hexamers, dodecamers, and related assemblies may be associated with presymptomatic neurodegeneration, while A\u03b2 dimers are more closely associated with fibril formation and plaque deposition during symptomatic Alzheimer\u2019s disease.\u2212", "section": "INTRO", "ner": [ [ 36, 38, "A\u03b2", "protein" ], [ 39, 46, "trimers", "oligomeric_state" ], [ 48, 56, "hexamers", "oligomeric_state" ], [ 58, 68, "dodecamers", "oligomeric_state" ], [ 156, 158, "A\u03b2", "protein" ], [ 159, 165, "dimers", "oligomeric_state" ] ] }, { "sid": 34, "sent": "The approach of isolating and characterizing A\u03b2 oligomers has not provided any high-resolution structures of A\u03b2 oligomers.", "section": "INTRO", "ner": [ [ 45, 47, "A\u03b2", "protein" ], [ 48, 57, "oligomers", "oligomeric_state" ], [ 95, 105, "structures", "evidence" ], [ 109, 111, "A\u03b2", "protein" ], [ 112, 121, "oligomers", "oligomeric_state" ] ] }, { "sid": 35, "sent": "Techniques such as SDS-PAGE, TEM, and AFM have only provided information about the molecular weights, sizes, morphologies, and stoichiometry of A\u03b2 oligomers.", "section": "INTRO", "ner": [ [ 19, 27, "SDS-PAGE", "experimental_method" ], [ 29, 32, "TEM", "experimental_method" ], [ 38, 41, "AFM", "experimental_method" ], [ 144, 146, "A\u03b2", "protein" ], [ 147, 156, "oligomers", "oligomeric_state" ] ] }, { "sid": 36, "sent": "High-resolution structural studies of A\u03b2 have primarily focused on A\u03b2 fibrils and A\u03b2 monomers.", "section": "INTRO", "ner": [ [ 16, 34, "structural studies", "experimental_method" ], [ 38, 40, "A\u03b2", "protein" ], [ 67, 69, "A\u03b2", "protein" ], [ 70, 77, "fibrils", "oligomeric_state" ], [ 82, 84, "A\u03b2", "protein" ], [ 85, 93, "monomers", "oligomeric_state" ] ] }, { "sid": 37, "sent": "Solid-state NMR spectroscopy studies of A\u03b2 fibrils revealed that A\u03b2 fibrils are generally composed of extended networks of in-register parallel \u03b2-sheets.\u2212 X-ray crystallographic studies using fragments of A\u03b2 have provided additional information about how A\u03b2 fibrils pack.", "section": "INTRO", "ner": [ [ 0, 28, "Solid-state NMR spectroscopy", "experimental_method" ], [ 40, 42, "A\u03b2", "protein" ], [ 43, 50, "fibrils", "oligomeric_state" ], [ 65, 67, "A\u03b2", "protein" ], [ 68, 75, "fibrils", "oligomeric_state" ], [ 123, 152, "in-register parallel \u03b2-sheets", "structure_element" ], [ 155, 185, "X-ray crystallographic studies", "experimental_method" ], [ 205, 207, "A\u03b2", "protein" ], [ 255, 257, "A\u03b2", "protein" ], [ 258, 265, "fibrils", "oligomeric_state" ] ] }, { "sid": 38, "sent": "Solution-phase NMR and solid-state NMR have been used to study the structures of the A\u03b2 monomers within oligomeric assemblies.\u2212 A major finding from these studies is that oligomeric assemblies of A\u03b2 are primarily composed of antiparallel \u03b2-sheets.", "section": "INTRO", "ner": [ [ 0, 18, "Solution-phase NMR", "experimental_method" ], [ 23, 38, "solid-state NMR", "experimental_method" ], [ 67, 77, "structures", "evidence" ], [ 85, 87, "A\u03b2", "protein" ], [ 88, 96, "monomers", "oligomeric_state" ], [ 196, 198, "A\u03b2", "protein" ], [ 225, 246, "antiparallel \u03b2-sheets", "structure_element" ] ] }, { "sid": 39, "sent": "Many of these studies have reported the monomer subunit as adopting a \u03b2-hairpin conformation, in which the hydrophobic central and C-terminal regions form an antiparallel \u03b2-sheet.", "section": "INTRO", "ner": [ [ 40, 47, "monomer", "oligomeric_state" ], [ 48, 55, "subunit", "structure_element" ], [ 70, 79, "\u03b2-hairpin", "structure_element" ], [ 119, 126, "central", "structure_element" ], [ 131, 149, "C-terminal regions", "structure_element" ], [ 158, 178, "antiparallel \u03b2-sheet", "structure_element" ] ] }, { "sid": 40, "sent": "In 2008, Hoyer et al. reported the NMR structure of an A\u03b2 monomer bound to an artificial binding protein called an affibody (PDB 2OTK).", "section": "INTRO", "ner": [ [ 35, 38, "NMR", "experimental_method" ], [ 39, 48, "structure", "evidence" ], [ 55, 57, "A\u03b2", "protein" ], [ 58, 65, "monomer", "oligomeric_state" ], [ 66, 74, "bound to", "protein_state" ], [ 78, 104, "artificial binding protein", "chemical" ], [ 115, 123, "affibody", "chemical" ] ] }, { "sid": 41, "sent": "The structure revealed that monomeric A\u03b2 forms a \u03b2-hairpin when bound to the affibody.", "section": "INTRO", "ner": [ [ 4, 13, "structure", "evidence" ], [ 28, 37, "monomeric", "oligomeric_state" ], [ 38, 40, "A\u03b2", "protein" ], [ 49, 58, "\u03b2-hairpin", "structure_element" ], [ 64, 72, "bound to", "protein_state" ], [ 77, 85, "affibody", "chemical" ] ] }, { "sid": 42, "sent": "This A\u03b2 \u03b2-hairpin encompasses residues 17\u201337 and contains two \u03b2-strands comprising A\u03b217\u201324 and A\u03b230\u201337 connected by an A\u03b225\u201329 loop.", "section": "INTRO", "ner": [ [ 5, 7, "A\u03b2", "protein" ], [ 8, 17, "\u03b2-hairpin", "structure_element" ], [ 39, 44, "17\u201337", "residue_range" ], [ 62, 71, "\u03b2-strands", "structure_element" ], [ 83, 85, "A\u03b2", "protein" ], [ 85, 90, "17\u201324", "residue_range" ], [ 95, 97, "A\u03b2", "protein" ], [ 97, 102, "30\u201337", "residue_range" ], [ 119, 121, "A\u03b2", "protein" ], [ 121, 126, "25\u201329", "residue_range" ], [ 127, 131, "loop", "structure_element" ] ] }, { "sid": 43, "sent": "Sequestering A\u03b2 within the affibody prevents its fibrilization and reduces its neurotoxicity, providing evidence that the \u03b2-hairpin structure may contribute to the ability of A\u03b2 to form neurotoxic oligomers.", "section": "INTRO", "ner": [ [ 13, 15, "A\u03b2", "protein" ], [ 27, 35, "affibody", "chemical" ], [ 122, 131, "\u03b2-hairpin", "structure_element" ], [ 175, 177, "A\u03b2", "protein" ], [ 197, 206, "oligomers", "oligomeric_state" ] ] }, { "sid": 44, "sent": "In a related study, Sandberg et al. constrained A\u03b2 in a \u03b2-hairpin conformation by mutating residues A21 and A30 to cysteine and forming an intramolecular disulfide bond.", "section": "INTRO", "ner": [ [ 48, 50, "A\u03b2", "protein" ], [ 56, 65, "\u03b2-hairpin", "structure_element" ], [ 82, 90, "mutating", "experimental_method" ], [ 100, 103, "A21", "residue_name_number" ], [ 108, 111, "A30", "residue_name_number" ], [ 115, 123, "cysteine", "residue_name" ], [ 154, 168, "disulfide bond", "ptm" ] ] }, { "sid": 45, "sent": "Locking A\u03b2 into a \u03b2-hairpin structure resulted in the formation A\u03b2 oligomers, which were observed by size exclusion chromatography (SEC) and SDS-PAGE.", "section": "INTRO", "ner": [ [ 8, 10, "A\u03b2", "protein" ], [ 18, 27, "\u03b2-hairpin", "structure_element" ], [ 64, 66, "A\u03b2", "protein" ], [ 67, 76, "oligomers", "oligomeric_state" ], [ 101, 130, "size exclusion chromatography", "experimental_method" ], [ 132, 135, "SEC", "experimental_method" ], [ 141, 149, "SDS-PAGE", "experimental_method" ] ] }, { "sid": 46, "sent": "The oligomers with a molecular weight of \u223c100 kDa that were isolated by SEC were toxic toward neuronally derived SH-SY5Y cells.", "section": "INTRO", "ner": [ [ 4, 13, "oligomers", "oligomeric_state" ], [ 72, 75, "SEC", "experimental_method" ] ] }, { "sid": 47, "sent": "This study provides evidence for the role of \u03b2-hairpin structure in A\u03b2 oligomerization and neurotoxicity.", "section": "INTRO", "ner": [ [ 45, 54, "\u03b2-hairpin", "structure_element" ], [ 68, 70, "A\u03b2", "protein" ] ] }, { "sid": 48, "sent": "Inspired by these \u03b2-hairpin structures, our laboratory developed a macrocyclic \u03b2-sheet peptide derived from A\u03b217\u201336 designed to mimic an A\u03b2 \u03b2-hairpin and reported its X-ray crystallographic structure.", "section": "INTRO", "ner": [ [ 18, 27, "\u03b2-hairpin", "structure_element" ], [ 28, 38, "structures", "evidence" ], [ 79, 86, "\u03b2-sheet", "structure_element" ], [ 108, 110, "A\u03b2", "protein" ], [ 110, 115, "17\u201336", "residue_range" ], [ 137, 139, "A\u03b2", "protein" ], [ 140, 149, "\u03b2-hairpin", "structure_element" ], [ 167, 199, "X-ray crystallographic structure", "evidence" ] ] }, { "sid": 49, "sent": "This peptide (peptide 1) consists of two \u03b2-strands comprising A\u03b217\u201323 and A\u03b230\u201336 covalently linked by two \u03b4-linked ornithine (\u03b4Orn) \u03b2-turn mimics.", "section": "INTRO", "ner": [ [ 14, 23, "peptide 1", "mutant" ], [ 41, 50, "\u03b2-strands", "structure_element" ], [ 62, 64, "A\u03b2", "protein" ], [ 64, 69, "17\u201323", "residue_range" ], [ 74, 76, "A\u03b2", "protein" ], [ 76, 81, "30\u201336", "residue_range" ], [ 107, 115, "\u03b4-linked", "protein_state" ], [ 116, 125, "ornithine", "residue_name" ], [ 127, 131, "\u03b4Orn", "structure_element" ], [ 133, 139, "\u03b2-turn", "structure_element" ] ] }, { "sid": 50, "sent": "The \u03b4Orn that connects residues D23 and A30 replaces the A\u03b224\u201329 loop.", "section": "INTRO", "ner": [ [ 4, 8, "\u03b4Orn", "structure_element" ], [ 32, 35, "D23", "residue_name_number" ], [ 40, 43, "A30", "residue_name_number" ], [ 57, 59, "A\u03b2", "protein" ], [ 59, 64, "24\u201329", "residue_range" ], [ 65, 69, "loop", "structure_element" ] ] }, { "sid": 51, "sent": "The \u03b4Orn that connects residues L17 and V36 enforces \u03b2-hairpin structure.", "section": "INTRO", "ner": [ [ 4, 8, "\u03b4Orn", "structure_element" ], [ 32, 35, "L17", "residue_name_number" ], [ 40, 43, "V36", "residue_name_number" ], [ 53, 62, "\u03b2-hairpin", "structure_element" ] ] }, { "sid": 52, "sent": "We incorporated an N-methyl group at position G33 to prevent uncontrolled aggregation and precipitation of the peptide.", "section": "INTRO", "ner": [ [ 46, 49, "G33", "residue_name_number" ] ] }, { "sid": 53, "sent": "To improve the solubility of the peptide we replaced M35 with the hydrophilic isostere of methionine, ornithine (\u03b1-linked) (Figure 1B).", "section": "INTRO", "ner": [ [ 44, 52, "replaced", "experimental_method" ], [ 53, 56, "M35", "residue_name_number" ], [ 90, 100, "methionine", "residue_name" ], [ 102, 111, "ornithine", "residue_name" ], [ 113, 121, "\u03b1-linked", "protein_state" ] ] }, { "sid": 54, "sent": "The X-ray crystallographic structure of peptide 1 reveals that it folds to form a \u03b2-hairpin that assembles to form trimers and that the trimers further assemble to form hexamers and dodecamers.", "section": "INTRO", "ner": [ [ 4, 36, "X-ray crystallographic structure", "evidence" ], [ 40, 49, "peptide 1", "mutant" ], [ 82, 91, "\u03b2-hairpin", "structure_element" ], [ 115, 122, "trimers", "oligomeric_state" ], [ 136, 143, "trimers", "oligomeric_state" ], [ 169, 177, "hexamers", "oligomeric_state" ], [ 182, 192, "dodecamers", "oligomeric_state" ] ] }, { "sid": 55, "sent": "(A) Cartoon illustrating the design of peptides 1 and 2 and their relationship to an A\u03b217\u201336 \u03b2-hairpin.", "section": "FIG", "ner": [ [ 39, 55, "peptides 1 and 2", "chemical" ], [ 85, 87, "A\u03b2", "protein" ], [ 87, 92, "17\u201336", "residue_range" ], [ 93, 102, "\u03b2-hairpin", "structure_element" ] ] }, { "sid": 56, "sent": " (B) Chemical structure of peptide 1 illustrating A\u03b217\u201323 and A\u03b230\u201336, M35Orn, the N-methyl group, and the \u03b4-linked ornithine turns. (C) Chemical structure of peptide 2 illustrating A\u03b217\u201336, the N-methyl group, the disulfide bond across positions 24 and 29, and the \u03b4-linked ornithine turn.", "section": "FIG", "ner": [ [ 27, 36, "peptide 1", "mutant" ], [ 50, 52, "A\u03b2", "protein" ], [ 62, 64, "A\u03b2", "protein" ], [ 107, 115, "\u03b4-linked", "protein_state" ], [ 116, 125, "ornithine", "residue_name" ], [ 126, 131, "turns", "structure_element" ], [ 159, 168, "peptide 2", "mutant" ], [ 182, 184, "A\u03b2", "protein" ], [ 215, 229, "disulfide bond", "ptm" ], [ 247, 249, "24", "residue_number" ], [ 254, 256, "29", "residue_number" ], [ 266, 274, "\u03b4-linked", "protein_state" ], [ 275, 284, "ornithine", "residue_name" ], [ 285, 289, "turn", "structure_element" ] ] }, { "sid": 57, "sent": "Our design of peptide 1 omitted the A\u03b224\u201329 loop.", "section": "INTRO", "ner": [ [ 14, 23, "peptide 1", "mutant" ], [ 36, 38, "A\u03b2", "protein" ], [ 38, 43, "24\u201329", "residue_range" ], [ 44, 48, "loop", "structure_element" ] ] }, { "sid": 58, "sent": "To visualize the A\u03b224\u201329 loop, we performed replica-exchange molecular dynamics (REMD) simulations on A\u03b217\u201336 using the X-ray crystallographic coordinates of A\u03b217\u201323 and A\u03b230\u201336 from peptide 1.", "section": "INTRO", "ner": [ [ 17, 19, "A\u03b2", "protein" ], [ 19, 24, "24\u201329", "residue_range" ], [ 25, 29, "loop", "structure_element" ], [ 44, 79, "replica-exchange molecular dynamics", "experimental_method" ], [ 81, 85, "REMD", "experimental_method" ], [ 87, 98, "simulations", "experimental_method" ], [ 102, 104, "A\u03b2", "protein" ], [ 104, 109, "17\u201336", "residue_range" ], [ 120, 154, "X-ray crystallographic coordinates", "evidence" ], [ 158, 160, "A\u03b2", "protein" ], [ 160, 165, "17\u201323", "residue_range" ], [ 170, 172, "A\u03b2", "protein" ], [ 172, 177, "30\u201336", "residue_range" ], [ 183, 192, "peptide 1", "mutant" ] ] }, { "sid": 59, "sent": "These studies provided a working model for a trimer of A\u03b217\u201336 \u03b2-hairpins and demonstrated that the trimer should be capable of accommodating the A\u03b224\u201329 loop.", "section": "INTRO", "ner": [ [ 45, 51, "trimer", "oligomeric_state" ], [ 55, 57, "A\u03b2", "protein" ], [ 57, 62, "17\u201336", "residue_range" ], [ 63, 73, "\u03b2-hairpins", "structure_element" ], [ 100, 106, "trimer", "oligomeric_state" ], [ 146, 148, "A\u03b2", "protein" ], [ 148, 153, "24\u201329", "residue_range" ], [ 154, 158, "loop", "structure_element" ] ] }, { "sid": 60, "sent": "In the current study we set out to restore the A\u03b224\u201329 loop, reintroduce the methionine residue at position 35, and determine the X-ray crystallographic structures of oligomers that form.", "section": "INTRO", "ner": [ [ 35, 42, "restore", "experimental_method" ], [ 47, 49, "A\u03b2", "protein" ], [ 49, 54, "24\u201329", "residue_range" ], [ 55, 59, "loop", "structure_element" ], [ 61, 72, "reintroduce", "experimental_method" ], [ 77, 87, "methionine", "residue_name" ], [ 108, 110, "35", "residue_number" ], [ 130, 163, "X-ray crystallographic structures", "evidence" ], [ 167, 176, "oligomers", "oligomeric_state" ] ] }, { "sid": 61, "sent": "We designed peptide 2 as a homologue of peptide 1 that embodies these ideas.", "section": "INTRO", "ner": [ [ 12, 21, "peptide 2", "mutant" ], [ 40, 49, "peptide 1", "mutant" ] ] }, { "sid": 62, "sent": "Peptide 2 contains a methionine residue at position 35 and an A\u03b224\u201329 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C).", "section": "INTRO", "ner": [ [ 0, 9, "Peptide 2", "mutant" ], [ 21, 31, "methionine", "residue_name" ], [ 52, 54, "35", "residue_number" ], [ 62, 64, "A\u03b2", "protein" ], [ 64, 69, "24\u201329", "residue_range" ], [ 70, 74, "loop", "structure_element" ], [ 89, 91, "24", "residue_number" ], [ 96, 98, "29", "residue_number" ], [ 100, 103, "Val", "residue_name" ], [ 108, 111, "Gly", "residue_name" ], [ 113, 120, "mutated", "experimental_method" ], [ 124, 132, "cysteine", "residue_name" ], [ 149, 163, "disulfide bond", "ptm" ] ] }, { "sid": 63, "sent": "Here, we describe the development of peptide 2 and report the X-ray crystallographic structures of the trimer, dodecamer, and annular pore observed within the crystal structure.", "section": "INTRO", "ner": [ [ 37, 46, "peptide 2", "mutant" ], [ 62, 95, "X-ray crystallographic structures", "evidence" ], [ 103, 109, "trimer", "oligomeric_state" ], [ 111, 120, "dodecamer", "oligomeric_state" ], [ 126, 138, "annular pore", "site" ], [ 159, 176, "crystal structure", "evidence" ] ] }, { "sid": 64, "sent": "Development of Peptide 2", "section": "RESULTS", "ner": [ [ 15, 24, "Peptide 2", "mutant" ] ] }, { "sid": 65, "sent": "We developed peptide 2 from peptide 1 by an iterative process, in which we first attempted to restore the A\u03b224\u201329 loop without a disulfide linkage.", "section": "RESULTS", "ner": [ [ 13, 22, "peptide 2", "mutant" ], [ 28, 37, "peptide 1", "mutant" ], [ 106, 108, "A\u03b2", "protein" ], [ 108, 113, "24\u201329", "residue_range" ], [ 114, 118, "loop", "structure_element" ], [ 129, 146, "disulfide linkage", "ptm" ] ] }, { "sid": 66, "sent": "We envisioned peptide 3 as a homologue of peptide 1 with the A\u03b224\u201329 loop in place of the \u03b4Orn that connects D23 and A30 and p-iodophenylalanine (FI) in place of F19.", "section": "RESULTS", "ner": [ [ 14, 23, "peptide 3", "mutant" ], [ 42, 51, "peptide 1", "mutant" ], [ 61, 63, "A\u03b2", "protein" ], [ 63, 68, "24\u201329", "residue_range" ], [ 69, 73, "loop", "structure_element" ], [ 90, 94, "\u03b4Orn", "structure_element" ], [ 109, 112, "D23", "residue_name_number" ], [ 117, 120, "A30", "residue_name_number" ], [ 125, 144, "p-iodophenylalanine", "chemical" ], [ 146, 148, "FI", "chemical" ], [ 162, 165, "F19", "residue_name_number" ] ] }, { "sid": 67, "sent": "We routinely use p-iodophenylalanine to determine the X-ray crystallographic phases.", "section": "RESULTS", "ner": [ [ 17, 36, "p-iodophenylalanine", "chemical" ], [ 54, 83, "X-ray crystallographic phases", "evidence" ] ] }, { "sid": 68, "sent": "After determining the X-ray crystallographic structure of the p-iodophenylalanine variant we attempt to determine the structure of the native phenylalanine compound by isomorphous replacement.", "section": "RESULTS", "ner": [ [ 22, 54, "X-ray crystallographic structure", "evidence" ], [ 62, 81, "p-iodophenylalanine", "chemical" ], [ 118, 127, "structure", "evidence" ], [ 142, 155, "phenylalanine", "residue_name" ], [ 168, 191, "isomorphous replacement", "experimental_method" ] ] }, { "sid": 69, "sent": "Upon synthesizing peptide 3, we found that it formed an amorphous precipitate in most crystallization conditions screened and failed to afford crystals in any condition.", "section": "RESULTS", "ner": [ [ 18, 27, "peptide 3", "mutant" ], [ 143, 151, "crystals", "evidence" ] ] }, { "sid": 70, "sent": "We postulate that the loss of the \u03b4Orn constraint leads to conformational heterogeneity that prevents peptide 3 from crystallizing.", "section": "RESULTS", "ner": [ [ 34, 38, "\u03b4Orn", "structure_element" ], [ 102, 111, "peptide 3", "mutant" ] ] }, { "sid": 71, "sent": "To address this issue, we next incorporated a disulfide bond between residues 24 and 29 as a conformational constraint that serves as a surrogate for \u03b4Orn.", "section": "RESULTS", "ner": [ [ 46, 60, "disulfide bond", "ptm" ], [ 78, 80, "24", "residue_number" ], [ 85, 87, "29", "residue_number" ], [ 150, 154, "\u03b4Orn", "structure_element" ] ] }, { "sid": 72, "sent": "We designed peptide 4 to embody this idea, mutating Val24 and Gly29 to cysteine and forming an interstrand disulfide linkage.", "section": "RESULTS", "ner": [ [ 12, 21, "peptide 4", "mutant" ], [ 43, 51, "mutating", "experimental_method" ], [ 52, 57, "Val24", "residue_name_number" ], [ 62, 67, "Gly29", "residue_name_number" ], [ 71, 79, "cysteine", "residue_name" ], [ 107, 124, "disulfide linkage", "ptm" ] ] }, { "sid": 73, "sent": "We mutated these residues because they occupy the same position as the \u03b4Orn that connects D23 and A30 in peptide 1.", "section": "RESULTS", "ner": [ [ 3, 10, "mutated", "experimental_method" ], [ 71, 75, "\u03b4Orn", "structure_element" ], [ 90, 93, "D23", "residue_name_number" ], [ 98, 101, "A30", "residue_name_number" ], [ 105, 114, "peptide 1", "mutant" ] ] }, { "sid": 74, "sent": "Residues V24 and G29 form a non-hydrogen-bonded pair, which can readily accommodate disulfide linkages in antiparallel \u03b2-sheets.", "section": "RESULTS", "ner": [ [ 9, 12, "V24", "residue_name_number" ], [ 17, 20, "G29", "residue_name_number" ], [ 28, 52, "non-hydrogen-bonded pair", "bond_interaction" ], [ 84, 102, "disulfide linkages", "ptm" ], [ 106, 127, "antiparallel \u03b2-sheets", "structure_element" ] ] }, { "sid": 75, "sent": "Disulfide bonds across non-hydrogen-bonded pairs stabilize \u03b2-hairpins, while disulfide bonds across hydrogen-bonded pairs do not.", "section": "RESULTS", "ner": [ [ 0, 15, "Disulfide bonds", "ptm" ], [ 23, 48, "non-hydrogen-bonded pairs", "bond_interaction" ], [ 59, 69, "\u03b2-hairpins", "structure_element" ], [ 77, 92, "disulfide bonds", "ptm" ], [ 100, 121, "hydrogen-bonded pairs", "bond_interaction" ] ] }, { "sid": 76, "sent": "Although the disulfide bond between positions 24 and 29 helps stabilize the \u03b2-hairpin, it does not alter the charge or substantially change the hydrophobicity of the A\u03b217\u201336 \u03b2-hairpin.", "section": "RESULTS", "ner": [ [ 13, 27, "disulfide bond", "ptm" ], [ 46, 48, "24", "residue_number" ], [ 53, 55, "29", "residue_number" ], [ 76, 85, "\u03b2-hairpin", "structure_element" ], [ 166, 168, "A\u03b2", "protein" ], [ 168, 173, "17\u201336", "residue_range" ], [ 174, 183, "\u03b2-hairpin", "structure_element" ] ] }, { "sid": 77, "sent": "We were gratified to find that peptide 4 afforded crystals suitable for X-ray crystallography.", "section": "RESULTS", "ner": [ [ 31, 40, "peptide 4", "mutant" ], [ 50, 58, "crystals", "evidence" ], [ 72, 93, "X-ray crystallography", "experimental_method" ] ] }, { "sid": 78, "sent": "As the next step in the iterative process, we determined the X-ray crystallographic structure of this peptide (PDB 5HOW).", "section": "RESULTS", "ner": [ [ 46, 56, "determined", "experimental_method" ], [ 61, 93, "X-ray crystallographic structure", "evidence" ] ] }, { "sid": 79, "sent": "After determining the X-ray crystallographic structure of peptide 4 we reintroduced the native phenylalanine at position 19 and the methionine at position 35 to afford peptide 2.", "section": "RESULTS", "ner": [ [ 22, 54, "X-ray crystallographic structure", "evidence" ], [ 58, 67, "peptide 4", "mutant" ], [ 71, 83, "reintroduced", "experimental_method" ], [ 95, 108, "phenylalanine", "residue_name" ], [ 121, 123, "19", "residue_number" ], [ 132, 142, "methionine", "residue_name" ], [ 155, 157, "35", "residue_number" ], [ 168, 177, "peptide 2", "mutant" ] ] }, { "sid": 80, "sent": "We completed the iterative process\u2014from 1 to 3 to 4 to 2\u2014by successfully determining the X-ray crystallographic structure of peptide 2 (PDB 5HOX and 5HOY).", "section": "RESULTS", "ner": [ [ 89, 121, "X-ray crystallographic structure", "evidence" ], [ 125, 134, "peptide 2", "mutant" ] ] }, { "sid": 81, "sent": "The following sections describe the synthesis of peptides 2\u20134 and the X-ray crystallographic structure of peptide 2.", "section": "RESULTS", "ner": [ [ 49, 61, "peptides 2\u20134", "mutant" ], [ 70, 102, "X-ray crystallographic structure", "evidence" ], [ 106, 115, "peptide 2", "mutant" ] ] }, { "sid": 82, "sent": "Synthesis of Peptides 2\u20134", "section": "RESULTS", "ner": [ [ 13, 25, "Peptides 2\u20134", "mutant" ] ] }, { "sid": 83, "sent": "We synthesized peptides 2\u20134 by similar procedures to those we have developed for other macrocyclic peptides.", "section": "RESULTS", "ner": [ [ 15, 27, "peptides 2\u20134", "mutant" ] ] }, { "sid": 84, "sent": "In synthesizing peptides 2 and 4 we formed the disulfide linkage after macrolactamization and deprotection of the acid-labile side chain protecting groups.", "section": "RESULTS", "ner": [ [ 16, 32, "peptides 2 and 4", "mutant" ], [ 47, 64, "disulfide linkage", "ptm" ] ] }, { "sid": 85, "sent": "We used acid-stable Acm-protected cysteine residues at positions 24 and 29 and removed the Acm groups by oxidation with I2 in aqueous acetic acid to afford the disulfide linkage.", "section": "RESULTS", "ner": [ [ 8, 19, "acid-stable", "protein_state" ], [ 20, 33, "Acm-protected", "protein_state" ], [ 34, 42, "cysteine", "residue_name" ], [ 65, 67, "24", "residue_number" ], [ 72, 74, "29", "residue_number" ], [ 134, 145, "acetic acid", "chemical" ], [ 160, 177, "disulfide linkage", "ptm" ] ] }, { "sid": 86, "sent": "Peptides 2\u20134 were purified by RP-HPLC.", "section": "RESULTS", "ner": [ [ 0, 12, "Peptides 2\u20134", "mutant" ], [ 30, 37, "RP-HPLC", "experimental_method" ] ] }, { "sid": 87, "sent": "Crystallization, X-ray Crystallographic Data Collection, Data Processing, and Structure Determination of Peptides 2 and 4", "section": "RESULTS", "ner": [ [ 0, 15, "Crystallization", "experimental_method" ], [ 17, 55, "X-ray Crystallographic Data Collection", "experimental_method" ], [ 78, 101, "Structure Determination", "experimental_method" ], [ 105, 121, "Peptides 2 and 4", "mutant" ] ] }, { "sid": 88, "sent": "We screened crystallization conditions for peptide 4 in a 96-well-plate format using three different Hampton Research crystallization kits (Crystal Screen, Index, and PEG/Ion) with three ratios of peptide and mother liquor per condition (864 experiments).", "section": "RESULTS", "ner": [ [ 3, 38, "screened crystallization conditions", "experimental_method" ], [ 43, 52, "peptide 4", "mutant" ] ] }, { "sid": 89, "sent": "Peptide 4 afforded crystals in a single set of conditions containing HEPES buffer and Jeffamine M-600\u2014the same crystallization conditions that afforded crystals of peptide 1.", "section": "RESULTS", "ner": [ [ 0, 9, "Peptide 4", "mutant" ], [ 19, 27, "crystals", "evidence" ], [ 86, 101, "Jeffamine M-600", "chemical" ], [ 152, 160, "crystals", "evidence" ], [ 164, 173, "peptide 1", "mutant" ] ] }, { "sid": 90, "sent": "Peptide 2 also afforded crystals in these conditions.", "section": "RESULTS", "ner": [ [ 0, 9, "Peptide 2", "mutant" ], [ 24, 32, "crystals", "evidence" ] ] }, { "sid": 91, "sent": "We further optimized these conditions to rapidly (\u223c72 h) yield crystals suitable for X-ray crystallography.", "section": "RESULTS", "ner": [ [ 63, 71, "crystals", "evidence" ], [ 85, 106, "X-ray crystallography", "experimental_method" ] ] }, { "sid": 92, "sent": "The optimized conditions consist of 0.1 M HEPES at pH 6.4 with 31% Jeffamine M-600 for peptide 4 and 0.1 M HEPES pH 7.1 with 29% Jeffamine M-600 for peptide 2.", "section": "RESULTS", "ner": [ [ 42, 47, "HEPES", "chemical" ], [ 67, 82, "Jeffamine M-600", "chemical" ], [ 87, 96, "peptide 4", "mutant" ], [ 107, 112, "HEPES", "chemical" ], [ 129, 144, "Jeffamine M-600", "chemical" ], [ 149, 158, "peptide 2", "mutant" ] ] }, { "sid": 93, "sent": "Crystal diffraction data for peptides 4 and 2 were collected in-house with a Rigaku MicroMax 007HF X-ray diffractometer at 1.54 \u00c5 wavelength.", "section": "RESULTS", "ner": [ [ 0, 24, "Crystal diffraction data", "evidence" ], [ 29, 45, "peptides 4 and 2", "mutant" ] ] }, { "sid": 94, "sent": "Crystal diffraction data for peptide 2 were also collected at the Advanced Light Source at Lawrence Berkeley National Laboratory with a synchrotron source at 1.00 \u00c5 wavelength to achieve higher resolution.", "section": "RESULTS", "ner": [ [ 0, 24, "Crystal diffraction data", "evidence" ], [ 29, 38, "peptide 2", "mutant" ] ] }, { "sid": 95, "sent": "Data from peptides 4 and 2 suitable for refinement at 2.30 \u00c5 were obtained from the diffractometer; data from peptide 2 suitable for refinement at 1.90 \u00c5 were obtained from the synchrotron.", "section": "RESULTS", "ner": [ [ 10, 26, "peptides 4 and 2", "mutant" ], [ 110, 119, "peptide 2", "mutant" ] ] }, { "sid": 96, "sent": "Data for peptides 4 and 2 were scaled and merged using XDS.", "section": "RESULTS", "ner": [ [ 9, 25, "peptides 4 and 2", "mutant" ] ] }, { "sid": 97, "sent": "Phases for peptide 4 were determined by single-wavelength anomalous diffraction (SAD) phasing by using the coordinates of the iodine anomalous signal from p-iodophenylalanine.", "section": "RESULTS", "ner": [ [ 0, 6, "Phases", "evidence" ], [ 11, 20, "peptide 4", "mutant" ], [ 40, 79, "single-wavelength anomalous diffraction", "experimental_method" ], [ 81, 84, "SAD", "experimental_method" ], [ 86, 93, "phasing", "experimental_method" ], [ 126, 149, "iodine anomalous signal", "evidence" ], [ 155, 174, "p-iodophenylalanine", "chemical" ] ] }, { "sid": 98, "sent": "Phases for peptide 2 were determined by isomorphous replacement of peptide 4.", "section": "RESULTS", "ner": [ [ 0, 6, "Phases", "evidence" ], [ 11, 20, "peptide 2", "mutant" ], [ 40, 63, "isomorphous replacement", "experimental_method" ], [ 67, 76, "peptide 4", "mutant" ] ] }, { "sid": 99, "sent": "The structures of peptides 2 and 4 were solved and refined in the P6122 space group.", "section": "RESULTS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 18, 34, "peptides 2 and 4", "mutant" ], [ 40, 46, "solved", "experimental_method" ] ] }, { "sid": 100, "sent": "The asymmetric unit of each peptide consists of six monomers, arranged as two trimers.", "section": "RESULTS", "ner": [ [ 28, 35, "peptide", "chemical" ], [ 52, 60, "monomers", "oligomeric_state" ], [ 78, 85, "trimers", "oligomeric_state" ] ] }, { "sid": 101, "sent": "Peptides 2 and 4 form morphologically identical structures and assemblies in the crystal lattice.", "section": "RESULTS", "ner": [ [ 0, 16, "Peptides 2 and 4", "mutant" ], [ 81, 96, "crystal lattice", "evidence" ] ] }, { "sid": 102, "sent": "X-ray Crystallographic Structure of Peptide 2 and the Oligomers It Forms", "section": "RESULTS", "ner": [ [ 0, 32, "X-ray Crystallographic Structure", "evidence" ], [ 36, 45, "Peptide 2", "mutant" ], [ 54, 63, "Oligomers", "oligomeric_state" ] ] }, { "sid": 103, "sent": "The X-ray crystallographic structure of peptide 2 reveals that it folds to form a twisted \u03b2-hairpin comprising two \u03b2-strands connected by a loop (Figure 2A).", "section": "RESULTS", "ner": [ [ 4, 36, "X-ray crystallographic structure", "evidence" ], [ 40, 49, "peptide 2", "mutant" ], [ 82, 99, "twisted \u03b2-hairpin", "structure_element" ], [ 115, 124, "\u03b2-strands", "structure_element" ], [ 140, 144, "loop", "structure_element" ] ] }, { "sid": 104, "sent": "Eight residues make up each surface of the \u03b2-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface.", "section": "RESULTS", "ner": [ [ 43, 52, "\u03b2-hairpin", "structure_element" ], [ 54, 57, "L17", "residue_name_number" ], [ 59, 62, "F19", "residue_name_number" ], [ 64, 67, "A21", "residue_name_number" ], [ 69, 72, "D23", "residue_name_number" ], [ 74, 77, "A30", "residue_name_number" ], [ 79, 82, "I32", "residue_name_number" ], [ 84, 87, "L34", "residue_name_number" ], [ 93, 96, "V36", "residue_name_number" ], [ 118, 121, "V18", "residue_name_number" ], [ 123, 126, "F20", "residue_name_number" ], [ 128, 131, "E22", "residue_name_number" ], [ 133, 136, "C24", "residue_name_number" ], [ 138, 141, "C29", "residue_name_number" ], [ 143, 146, "I31", "residue_name_number" ], [ 148, 151, "G33", "residue_name_number" ], [ 157, 160, "M35", "residue_name_number" ] ] }, { "sid": 105, "sent": "The \u03b2-strands of the monomers in the asymmetric unit are virtually identical, differing primarily in rotamers of F20, E22, C24, C29, I31, and M35 (Figure S1).", "section": "RESULTS", "ner": [ [ 4, 13, "\u03b2-strands", "structure_element" ], [ 21, 29, "monomers", "oligomeric_state" ], [ 113, 116, "F20", "residue_name_number" ], [ 118, 121, "E22", "residue_name_number" ], [ 123, 126, "C24", "residue_name_number" ], [ 128, 131, "C29", "residue_name_number" ], [ 133, 136, "I31", "residue_name_number" ], [ 142, 145, "M35", "residue_name_number" ] ] }, { "sid": 106, "sent": "The disulfide linkages suffered radiation damage under synchrotron radiation.", "section": "RESULTS", "ner": [ [ 4, 22, "disulfide linkages", "ptm" ] ] }, { "sid": 107, "sent": "We refined three of the \u03b2-hairpins with intact disulfide linkages and three with thiols to represent cleaved disulfide linkages in the synchrotron data set (PDB 5HOX).", "section": "RESULTS", "ner": [ [ 3, 10, "refined", "experimental_method" ], [ 24, 34, "\u03b2-hairpins", "structure_element" ], [ 40, 46, "intact", "protein_state" ], [ 47, 65, "disulfide linkages", "ptm" ], [ 101, 108, "cleaved", "protein_state" ], [ 109, 127, "disulfide linkages", "ptm" ] ] }, { "sid": 108, "sent": "No evidence for cleavage of the disulfides was observed in the refinement of the data set collected on the X-ray diffractometer, and we refined all disulfide linkages as intact (PDB 5HOY).", "section": "RESULTS", "ner": [ [ 32, 42, "disulfides", "ptm" ], [ 63, 73, "refinement", "experimental_method" ], [ 136, 143, "refined", "experimental_method" ], [ 148, 166, "disulfide linkages", "ptm" ], [ 170, 176, "intact", "protein_state" ] ] }, { "sid": 109, "sent": "X-ray crystallographic structure of peptide 2 (PDB 5HOX, synchrotron data set). (A) X-ray crystallographic structure of a representative \u03b2-hairpin monomer formed by peptide 2. (B) Overlay of the six \u03b2-hairpin monomers in the asymmetric unit.", "section": "FIG", "ner": [ [ 36, 45, "peptide 2", "mutant" ], [ 84, 116, "X-ray crystallographic structure", "evidence" ], [ 137, 146, "\u03b2-hairpin", "structure_element" ], [ 147, 154, "monomer", "oligomeric_state" ], [ 165, 174, "peptide 2", "mutant" ], [ 180, 187, "Overlay", "experimental_method" ], [ 199, 208, "\u03b2-hairpin", "structure_element" ], [ 209, 217, "monomers", "oligomeric_state" ] ] }, { "sid": 110, "sent": "The \u03b2-hairpins are shown as cartoons to illustrate the differences in the A\u03b225\u201328 loops.", "section": "FIG", "ner": [ [ 4, 14, "\u03b2-hairpins", "structure_element" ], [ 74, 76, "A\u03b2", "protein" ], [ 76, 81, "25\u201328", "residue_range" ], [ 82, 87, "loops", "structure_element" ] ] }, { "sid": 111, "sent": "The A\u03b225\u201328 loops of the six monomers within the asymmetric unit vary substantially in backbone geometry and side chain rotamers (Figures 2B and S1).", "section": "RESULTS", "ner": [ [ 4, 6, "A\u03b2", "protein" ], [ 6, 11, "25\u201328", "residue_range" ], [ 12, 17, "loops", "structure_element" ], [ 29, 37, "monomers", "oligomeric_state" ] ] }, { "sid": 112, "sent": "The electron density for the loops is weak and diffuse compared to the electron density for the \u03b2-strands.", "section": "RESULTS", "ner": [ [ 4, 20, "electron density", "evidence" ], [ 29, 34, "loops", "structure_element" ], [ 71, 87, "electron density", "evidence" ], [ 96, 105, "\u03b2-strands", "structure_element" ] ] }, { "sid": 113, "sent": "The B values for the loops are large, indicating that the loops are dynamic and not well ordered.", "section": "RESULTS", "ner": [ [ 4, 12, "B values", "evidence" ], [ 21, 26, "loops", "structure_element" ], [ 58, 63, "loops", "structure_element" ] ] }, { "sid": 114, "sent": "Thus, the differences in backbone geometry and side chain rotamers among the loops are likely of little significance and should be interpreted with caution.", "section": "RESULTS", "ner": [ [ 77, 82, "loops", "structure_element" ] ] }, { "sid": 115, "sent": "Peptide 2 assembles into oligomers similar in morphology to those formed by peptide 1.", "section": "RESULTS", "ner": [ [ 0, 9, "Peptide 2", "mutant" ], [ 25, 34, "oligomers", "oligomeric_state" ], [ 76, 85, "peptide 1", "mutant" ] ] }, { "sid": 116, "sent": "Like peptide 1, peptide 2 forms a triangular trimer, and four trimers assemble to form a dodecamer.", "section": "RESULTS", "ner": [ [ 5, 14, "peptide 1", "mutant" ], [ 16, 25, "peptide 2", "mutant" ], [ 34, 44, "triangular", "protein_state" ], [ 45, 51, "trimer", "oligomeric_state" ], [ 62, 69, "trimers", "oligomeric_state" ], [ 89, 98, "dodecamer", "oligomeric_state" ] ] }, { "sid": 117, "sent": "In the higher-order assembly of the dodecamers formed by peptide 2 a new structure emerges, not seen in peptide 1, an annular pore consisting of five dodecamers.", "section": "RESULTS", "ner": [ [ 36, 46, "dodecamers", "oligomeric_state" ], [ 57, 66, "peptide 2", "mutant" ], [ 73, 82, "structure", "evidence" ], [ 104, 113, "peptide 1", "mutant" ], [ 118, 130, "annular pore", "site" ], [ 150, 160, "dodecamers", "oligomeric_state" ] ] }, { "sid": 118, "sent": "Trimer", "section": "RESULTS", "ner": [ [ 0, 6, "Trimer", "oligomeric_state" ] ] }, { "sid": 119, "sent": "Peptide 2 forms a trimer, much like that which we observed previously for peptide 1, in which three \u03b2-hairpins assemble to form an equilateral triangle (Figure 3A).", "section": "RESULTS", "ner": [ [ 0, 9, "Peptide 2", "mutant" ], [ 18, 24, "trimer", "oligomeric_state" ], [ 74, 83, "peptide 1", "mutant" ], [ 100, 110, "\u03b2-hairpins", "structure_element" ], [ 131, 151, "equilateral triangle", "structure_element" ] ] }, { "sid": 120, "sent": "The trimer maintains all of the same stabilizing contacts as those of peptide 1.", "section": "RESULTS", "ner": [ [ 4, 10, "trimer", "oligomeric_state" ], [ 70, 79, "peptide 1", "mutant" ] ] }, { "sid": 121, "sent": "Hydrogen bonding and hydrophobic interactions between residues on the \u03b2-strands comprising A\u03b217\u201323 and A\u03b230\u201336 stabilize the core of the trimer.", "section": "RESULTS", "ner": [ [ 0, 16, "Hydrogen bonding", "bond_interaction" ], [ 21, 45, "hydrophobic interactions", "bond_interaction" ], [ 70, 79, "\u03b2-strands", "structure_element" ], [ 91, 93, "A\u03b2", "protein" ], [ 93, 98, "17\u201323", "residue_range" ], [ 103, 105, "A\u03b2", "protein" ], [ 105, 110, "30\u201336", "residue_range" ], [ 125, 129, "core", "structure_element" ], [ 137, 143, "trimer", "oligomeric_state" ] ] }, { "sid": 122, "sent": "The disulfide bonds between residues 24 and 29 are adjacent to the structural core of the trimer and do not make any substantial intermolecular contacts.", "section": "RESULTS", "ner": [ [ 4, 19, "disulfide bonds", "ptm" ], [ 37, 39, "24", "residue_number" ], [ 44, 46, "29", "residue_number" ], [ 67, 82, "structural core", "structure_element" ], [ 90, 96, "trimer", "oligomeric_state" ] ] }, { "sid": 123, "sent": "Two crystallographically distinct trimers comprise the peptide portion of the asymmetric unit.", "section": "RESULTS", "ner": [ [ 34, 41, "trimers", "oligomeric_state" ], [ 55, 62, "peptide", "chemical" ] ] }, { "sid": 124, "sent": "The two trimers are almost identical in structure, differing slightly among side chain rotamers and loop conformations.", "section": "RESULTS", "ner": [ [ 8, 15, "trimers", "oligomeric_state" ], [ 100, 104, "loop", "structure_element" ] ] }, { "sid": 125, "sent": "X-ray crystallographic structure of the trimer formed by peptide 2. (A) Triangular trimer.", "section": "FIG", "ner": [ [ 0, 32, "X-ray crystallographic structure", "evidence" ], [ 40, 46, "trimer", "oligomeric_state" ], [ 57, 66, "peptide 2", "mutant" ], [ 72, 82, "Triangular", "protein_state" ], [ 83, 89, "trimer", "oligomeric_state" ] ] }, { "sid": 126, "sent": "The three water molecules in the center hole of the trimer are shown as spheres. (B) Detailed view of the intermolecular hydrogen bonds between the main chains of V18 and E22 and \u03b4Orn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The F20 face of the trimer, with key side chains as spheres.", "section": "FIG", "ner": [ [ 10, 15, "water", "chemical" ], [ 52, 58, "trimer", "oligomeric_state" ], [ 121, 135, "hydrogen bonds", "bond_interaction" ], [ 163, 166, "V18", "residue_name_number" ], [ 171, 174, "E22", "residue_name_number" ], [ 179, 183, "\u03b4Orn", "structure_element" ], [ 188, 191, "C24", "residue_name_number" ], [ 221, 231, "triangular", "protein_state" ], [ 232, 238, "trimer", "oligomeric_state" ], [ 248, 251, "F19", "residue_name_number" ], [ 264, 270, "trimer", "oligomeric_state" ], [ 319, 322, "F20", "residue_name_number" ], [ 335, 341, "trimer", "oligomeric_state" ] ] }, { "sid": 127, "sent": "A network of 18 intermolecular hydrogen bonds helps stabilize the trimer.", "section": "RESULTS", "ner": [ [ 31, 45, "hydrogen bonds", "bond_interaction" ], [ 66, 72, "trimer", "oligomeric_state" ] ] }, { "sid": 128, "sent": "At the corners of the trimer, the pairs of \u03b2-hairpin monomers form four hydrogen bonds: two between the main chains of V18 and E22 and two between \u03b4Orn and the main chain of C24 (Figure 3B).", "section": "RESULTS", "ner": [ [ 22, 28, "trimer", "oligomeric_state" ], [ 43, 52, "\u03b2-hairpin", "structure_element" ], [ 53, 61, "monomers", "oligomeric_state" ], [ 72, 86, "hydrogen bonds", "bond_interaction" ], [ 119, 122, "V18", "residue_name_number" ], [ 127, 130, "E22", "residue_name_number" ], [ 147, 151, "\u03b4Orn", "structure_element" ], [ 174, 177, "C24", "residue_name_number" ] ] }, { "sid": 129, "sent": "Three ordered water molecules fill the hole in the center of the trimer, hydrogen bonding to each other and to the main chain of F20 (Figure 3A).", "section": "RESULTS", "ner": [ [ 14, 19, "water", "chemical" ], [ 65, 71, "trimer", "oligomeric_state" ], [ 73, 89, "hydrogen bonding", "bond_interaction" ], [ 129, 132, "F20", "residue_name_number" ] ] }, { "sid": 130, "sent": "Hydrophobic contacts between residues at the three corners of the trimer, where the \u03b2-hairpins meet, further stabilize the trimer.", "section": "RESULTS", "ner": [ [ 0, 20, "Hydrophobic contacts", "bond_interaction" ], [ 66, 72, "trimer", "oligomeric_state" ], [ 84, 94, "\u03b2-hairpins", "structure_element" ], [ 123, 129, "trimer", "oligomeric_state" ] ] }, { "sid": 131, "sent": "At each corner, the side chains of residues L17, F19, and V36 of one \u03b2-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent \u03b2-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer.", "section": "RESULTS", "ner": [ [ 44, 47, "L17", "residue_name_number" ], [ 49, 52, "F19", "residue_name_number" ], [ 58, 61, "V36", "residue_name_number" ], [ 69, 78, "\u03b2-hairpin", "structure_element" ], [ 120, 123, "A21", "residue_name_number" ], [ 125, 128, "I32", "residue_name_number" ], [ 130, 133, "L34", "residue_name_number" ], [ 144, 147, "D23", "residue_name_number" ], [ 164, 173, "\u03b2-hairpin", "structure_element" ], [ 186, 205, "hydrophobic cluster", "site" ], [ 229, 249, "hydrophobic clusters", "site" ], [ 265, 284, "hydrophobic surface", "site" ], [ 304, 310, "trimer", "oligomeric_state" ] ] }, { "sid": 132, "sent": "The other face of the trimer displays a smaller hydrophobic surface, which includes the side chains of residues V18, F20, and I31 of the three \u03b2-hairpins (Figure 3D).", "section": "RESULTS", "ner": [ [ 22, 28, "trimer", "oligomeric_state" ], [ 48, 67, "hydrophobic surface", "site" ], [ 112, 115, "V18", "residue_name_number" ], [ 117, 120, "F20", "residue_name_number" ], [ 126, 129, "I31", "residue_name_number" ], [ 143, 153, "\u03b2-hairpins", "structure_element" ] ] }, { "sid": 133, "sent": "In subsequent discussion, we designate the former surface the \u201cF19 face\u201d and the latter surface the \u201cF20 face\u201d.", "section": "RESULTS", "ner": [ [ 63, 66, "F19", "residue_name_number" ], [ 101, 104, "F20", "residue_name_number" ] ] }, { "sid": 134, "sent": "Dodecamer", "section": "RESULTS", "ner": [ [ 0, 9, "Dodecamer", "oligomeric_state" ] ] }, { "sid": 135, "sent": "Four trimers assemble to form a dodecamer.", "section": "RESULTS", "ner": [ [ 5, 12, "trimers", "oligomeric_state" ], [ 32, 41, "dodecamer", "oligomeric_state" ] ] }, { "sid": 136, "sent": "The four trimers arrange in a tetrahedral fashion, creating a central cavity inside the dodecamer. Because each trimer is triangular, the resulting arrangement resembles an octahedron.", "section": "RESULTS", "ner": [ [ 9, 16, "trimers", "oligomeric_state" ], [ 30, 41, "tetrahedral", "protein_state" ], [ 62, 76, "central cavity", "site" ], [ 88, 97, "dodecamer", "oligomeric_state" ], [ 112, 118, "trimer", "oligomeric_state" ], [ 122, 132, "triangular", "protein_state" ], [ 173, 183, "octahedron", "protein_state" ] ] }, { "sid": 137, "sent": "Each of the 12 \u03b2-hairpins constitutes an edge of the octahedron, and the triangular trimers occupy four of the eight faces of the octahedron.", "section": "RESULTS", "ner": [ [ 15, 25, "\u03b2-hairpins", "structure_element" ], [ 53, 63, "octahedron", "protein_state" ], [ 73, 83, "triangular", "protein_state" ], [ 84, 91, "trimers", "oligomeric_state" ], [ 130, 140, "octahedron", "protein_state" ] ] }, { "sid": 138, "sent": "Figure 4A illustrates the octahedral shape of the dodecamer.", "section": "RESULTS", "ner": [ [ 26, 36, "octahedral", "protein_state" ], [ 50, 59, "dodecamer", "oligomeric_state" ] ] }, { "sid": 139, "sent": "Figure 4B illustrates the tetrahedral arrangement of the four trimers.", "section": "RESULTS", "ner": [ [ 26, 37, "tetrahedral", "protein_state" ], [ 62, 69, "trimers", "oligomeric_state" ] ] }, { "sid": 140, "sent": "X-ray crystallographic structure of the dodecamer formed by peptide 2. (A) View of the dodecamer that illustrates the octahedral shape. (B) View of the dodecamer that illustrates the tetrahedral arrangement of the four trimers that comprise the dodecamer. (C) View of two trimer subunits from inside the cavity of the dodecamer.", "section": "FIG", "ner": [ [ 40, 49, "dodecamer", "oligomeric_state" ], [ 60, 69, "peptide 2", "mutant" ], [ 87, 96, "dodecamer", "oligomeric_state" ], [ 118, 128, "octahedral", "protein_state" ], [ 152, 161, "dodecamer", "oligomeric_state" ], [ 183, 194, "tetrahedral", "protein_state" ], [ 219, 226, "trimers", "oligomeric_state" ], [ 245, 254, "dodecamer", "oligomeric_state" ], [ 272, 278, "trimer", "oligomeric_state" ], [ 279, 287, "subunits", "structure_element" ], [ 304, 310, "cavity", "site" ], [ 318, 327, "dodecamer", "oligomeric_state" ] ] }, { "sid": 141, "sent": "Residues L17, L34, and V36 are shown as spheres, illustrating the hydrophobic packing that occurs at the six vertices of the dodecamer. (D) Detailed view of one of the six vertices of the dodecamer.", "section": "FIG", "ner": [ [ 9, 12, "L17", "residue_name_number" ], [ 14, 17, "L34", "residue_name_number" ], [ 23, 26, "V36", "residue_name_number" ], [ 66, 85, "hydrophobic packing", "bond_interaction" ], [ 125, 134, "dodecamer", "oligomeric_state" ], [ 188, 197, "dodecamer", "oligomeric_state" ] ] }, { "sid": 142, "sent": "The F19 faces of the trimers line the interior of the dodecamer.", "section": "RESULTS", "ner": [ [ 4, 7, "F19", "residue_name_number" ], [ 21, 28, "trimers", "oligomeric_state" ], [ 54, 63, "dodecamer", "oligomeric_state" ] ] }, { "sid": 143, "sent": "At the six vertices, hydrophobic packing between the side chains of L17, L34, and V36 helps stabilize the dodecamer (Figures 4C and D).", "section": "RESULTS", "ner": [ [ 21, 40, "hydrophobic packing", "bond_interaction" ], [ 68, 71, "L17", "residue_name_number" ], [ 73, 76, "L34", "residue_name_number" ], [ 82, 85, "V36", "residue_name_number" ], [ 106, 115, "dodecamer", "oligomeric_state" ] ] }, { "sid": 144, "sent": "Salt bridges between the side chains of D23 and \u03b4Orn at the vertices further stabilize the dodecamer.", "section": "RESULTS", "ner": [ [ 40, 43, "D23", "residue_name_number" ], [ 48, 52, "\u03b4Orn", "structure_element" ], [ 91, 100, "dodecamer", "oligomeric_state" ] ] }, { "sid": 145, "sent": "Each of the six vertices includes two A\u03b225\u201328 loops that extend past the core of the dodecamer without making any substantial intermolecular contacts.", "section": "RESULTS", "ner": [ [ 38, 40, "A\u03b2", "protein" ], [ 40, 45, "25\u201328", "residue_range" ], [ 46, 51, "loops", "structure_element" ], [ 73, 77, "core", "structure_element" ], [ 85, 94, "dodecamer", "oligomeric_state" ] ] }, { "sid": 146, "sent": "The exterior of the dodecamer displays four F20 faces (Figure S3).", "section": "RESULTS", "ner": [ [ 20, 29, "dodecamer", "oligomeric_state" ], [ 44, 47, "F20", "residue_name_number" ] ] }, { "sid": 147, "sent": "In the crystal lattice, each F20 face of one dodecamer packs against an F20 face of another dodecamer.", "section": "RESULTS", "ner": [ [ 7, 22, "crystal lattice", "evidence" ], [ 29, 32, "F20", "residue_name_number" ], [ 45, 54, "dodecamer", "oligomeric_state" ], [ 72, 75, "F20", "residue_name_number" ], [ 92, 101, "dodecamer", "oligomeric_state" ] ] }, { "sid": 148, "sent": "Although the asymmetric unit comprises half a dodecamer, the crystal lattice may be thought of as being built of dodecamers.", "section": "RESULTS", "ner": [ [ 46, 55, "dodecamer", "oligomeric_state" ], [ 61, 76, "crystal lattice", "evidence" ], [ 113, 123, "dodecamers", "oligomeric_state" ] ] }, { "sid": 149, "sent": "The electron density map for the X-ray crystallographic structure of peptide 2 has long tubes of electron density inside the central cavity of the dodecamer.", "section": "RESULTS", "ner": [ [ 4, 24, "electron density map", "evidence" ], [ 33, 65, "X-ray crystallographic structure", "evidence" ], [ 69, 78, "peptide 2", "mutant" ], [ 97, 113, "electron density", "evidence" ], [ 125, 139, "central cavity", "site" ], [ 147, 156, "dodecamer", "oligomeric_state" ] ] }, { "sid": 150, "sent": "The shape and length of the electron density is consistent with the structure of Jeffamine M-600, which is an essential component of the crystallization conditions.", "section": "RESULTS", "ner": [ [ 28, 44, "electron density", "evidence" ], [ 68, 77, "structure", "evidence" ], [ 81, 96, "Jeffamine M-600", "chemical" ] ] }, { "sid": 151, "sent": "Jeffamine M-600 is a polypropylene glycol derivative with a 2-methoxyethoxy unit at one end and a 2-aminopropyl unit at the other end.", "section": "RESULTS", "ner": [ [ 0, 15, "Jeffamine M-600", "chemical" ] ] }, { "sid": 152, "sent": "Although Jeffamine M-600 is a heterogeneous mixture with varying chain lengths and stereochemistry, we modeled a single stereoisomer with nine propylene glycol units (n = 9) to fit the electron density.", "section": "RESULTS", "ner": [ [ 9, 24, "Jeffamine M-600", "chemical" ], [ 185, 201, "electron density", "evidence" ] ] }, { "sid": 153, "sent": "The Jeffamine M-600 appears to stabilize the dodecamer by occupying the central cavity and making hydrophobic contacts with residues lining the cavity (Figure S3).", "section": "RESULTS", "ner": [ [ 4, 19, "Jeffamine M-600", "chemical" ], [ 45, 54, "dodecamer", "oligomeric_state" ], [ 72, 86, "central cavity", "site" ], [ 98, 118, "hydrophobic contacts", "bond_interaction" ], [ 144, 150, "cavity", "site" ] ] }, { "sid": 154, "sent": "In a dodecamer formed by full-length A\u03b2, the hydrophobic C-terminal residues (A\u03b237\u201340 or A\u03b237\u201342) might play a similar role in filling the dodecamer and thus create a packed hydrophobic core within the central cavity of the dodecamer.", "section": "RESULTS", "ner": [ [ 5, 14, "dodecamer", "oligomeric_state" ], [ 25, 36, "full-length", "protein_state" ], [ 37, 39, "A\u03b2", "protein" ], [ 78, 80, "A\u03b2", "protein" ], [ 80, 85, "37\u201340", "residue_range" ], [ 89, 91, "A\u03b2", "protein" ], [ 91, 96, "37\u201342", "residue_range" ], [ 139, 148, "dodecamer", "oligomeric_state" ], [ 174, 190, "hydrophobic core", "site" ], [ 202, 216, "central cavity", "site" ], [ 224, 233, "dodecamer", "oligomeric_state" ] ] }, { "sid": 155, "sent": "Annular Pore", "section": "RESULTS", "ner": [ [ 0, 12, "Annular Pore", "site" ] ] }, { "sid": 156, "sent": "Five dodecamers assemble to form an annular porelike structure (Figure 5A).", "section": "RESULTS", "ner": [ [ 5, 15, "dodecamers", "oligomeric_state" ], [ 44, 52, "porelike", "structure_element" ] ] }, { "sid": 157, "sent": "Hydrophobic packing between the F20 faces of trimers displayed on the outer surface of each dodecamer stabilizes the porelike assembly.", "section": "RESULTS", "ner": [ [ 0, 19, "Hydrophobic packing", "bond_interaction" ], [ 32, 35, "F20", "residue_name_number" ], [ 45, 52, "trimers", "oligomeric_state" ], [ 92, 101, "dodecamer", "oligomeric_state" ] ] }, { "sid": 158, "sent": "Two morphologically distinct interactions between trimers occur at the interfaces of the five dodecamers: one in which the trimers are eclipsed (Figure 5B), and one in which the trimers are staggered (Figure 5C).", "section": "RESULTS", "ner": [ [ 50, 57, "trimers", "oligomeric_state" ], [ 71, 81, "interfaces", "site" ], [ 94, 104, "dodecamers", "oligomeric_state" ], [ 123, 130, "trimers", "oligomeric_state" ], [ 135, 143, "eclipsed", "protein_state" ], [ 178, 185, "trimers", "oligomeric_state" ], [ 190, 199, "staggered", "protein_state" ] ] }, { "sid": 159, "sent": "Hydrophobic packing between the side chains of F20, I31, and E22 stabilizes these interfaces (Figure 5D and E).", "section": "RESULTS", "ner": [ [ 0, 19, "Hydrophobic packing", "bond_interaction" ], [ 47, 50, "F20", "residue_name_number" ], [ 52, 55, "I31", "residue_name_number" ], [ 61, 64, "E22", "residue_name_number" ], [ 82, 92, "interfaces", "site" ] ] }, { "sid": 160, "sent": "The annular pore contains three eclipsed interfaces and two staggered interfaces.", "section": "RESULTS", "ner": [ [ 4, 16, "annular pore", "site" ], [ 32, 40, "eclipsed", "protein_state" ], [ 41, 51, "interfaces", "site" ], [ 60, 69, "staggered", "protein_state" ], [ 70, 80, "interfaces", "site" ] ] }, { "sid": 161, "sent": "The eclipsed interfaces occur between dodecamers 1 and 2, 1 and 5, and 3 and 4, as shown in Figure 5A.", "section": "RESULTS", "ner": [ [ 4, 12, "eclipsed", "protein_state" ], [ 13, 23, "interfaces", "site" ], [ 38, 56, "dodecamers 1 and 2", "structure_element" ], [ 58, 65, "1 and 5", "structure_element" ], [ 71, 78, "3 and 4", "structure_element" ] ] }, { "sid": 162, "sent": "The staggered interfaces occur between dodecamers 2 and 3 and 4 and 5.", "section": "RESULTS", "ner": [ [ 4, 13, "staggered", "protein_state" ], [ 14, 24, "interfaces", "site" ], [ 39, 57, "dodecamers 2 and 3", "structure_element" ], [ 62, 69, "4 and 5", "structure_element" ] ] }, { "sid": 163, "sent": "The annular pore is not completely flat, instead, adopting a slightly puckered shape, which accommodates the eclipsed and staggered interfaces.", "section": "RESULTS", "ner": [ [ 4, 16, "annular pore", "site" ], [ 109, 117, "eclipsed", "protein_state" ], [ 122, 131, "staggered", "protein_state" ], [ 132, 142, "interfaces", "site" ] ] }, { "sid": 164, "sent": "Ten A\u03b225\u201328 loops from the vertices of the five dodecamers line the hole in the center of the pore.", "section": "RESULTS", "ner": [ [ 4, 6, "A\u03b2", "protein" ], [ 6, 11, "25\u201328", "residue_range" ], [ 12, 17, "loops", "structure_element" ], [ 48, 58, "dodecamers", "oligomeric_state" ], [ 94, 98, "pore", "site" ] ] }, { "sid": 165, "sent": "The hydrophilic side chains of S26, N27, and K28 decorate the hole.", "section": "RESULTS", "ner": [ [ 31, 34, "S26", "residue_name_number" ], [ 36, 39, "N27", "residue_name_number" ], [ 45, 48, "K28", "residue_name_number" ] ] }, { "sid": 166, "sent": "X-ray crystallographic structure of the annular pore formed by peptide 2. (A) Annular porelike structure illustrating the relationship of the five dodecamers that form the pore (top view).", "section": "FIG", "ner": [ [ 0, 32, "X-ray crystallographic structure", "evidence" ], [ 40, 52, "annular pore", "site" ], [ 63, 72, "peptide 2", "mutant" ], [ 78, 94, "Annular porelike", "structure_element" ], [ 95, 104, "structure", "evidence" ], [ 147, 157, "dodecamers", "oligomeric_state" ], [ 172, 176, "pore", "site" ] ] }, { "sid": 167, "sent": " (B) Eclipsed interface between dodecamers 1 and 2 (side view).", "section": "FIG", "ner": [ [ 5, 23, "Eclipsed interface", "site" ], [ 32, 50, "dodecamers 1 and 2", "structure_element" ] ] }, { "sid": 168, "sent": "The same eclipsed interface also occurs between dodecamers 1 and 5 and 3 and 4. (C) Staggered interface between dodecamers 2 and 3 (side view).", "section": "FIG", "ner": [ [ 9, 27, "eclipsed interface", "site" ], [ 48, 66, "dodecamers 1 and 5", "structure_element" ], [ 71, 78, "3 and 4", "structure_element" ], [ 84, 103, "Staggered interface", "site" ], [ 112, 130, "dodecamers 2 and 3", "structure_element" ] ] }, { "sid": 169, "sent": "The same staggered interface also occurs between dodecamers 4 and 5. (D) Eclipsed interface between dodecamers 1 and 5 (top view).", "section": "FIG", "ner": [ [ 9, 28, "staggered interface", "site" ], [ 73, 91, "Eclipsed interface", "site" ], [ 100, 118, "dodecamers 1 and 5", "structure_element" ] ] }, { "sid": 170, "sent": "The annular pore is comparable in size to other large protein assemblies.", "section": "RESULTS", "ner": [ [ 4, 16, "annular pore", "site" ] ] }, { "sid": 171, "sent": "The diameter of the hole in the center of the pore is \u223c2 nm.", "section": "RESULTS", "ner": [ [ 46, 50, "pore", "site" ] ] }, { "sid": 172, "sent": "The thickness of the pore is \u223c5 nm, which is comparable to that of a lipid bilayer membrane.", "section": "RESULTS", "ner": [ [ 21, 25, "pore", "site" ] ] }, { "sid": 173, "sent": "It is important to note that the annular pore formed by peptide 2 is not a discrete unit in the crystal lattice.", "section": "RESULTS", "ner": [ [ 33, 45, "annular pore", "site" ], [ 56, 65, "peptide 2", "mutant" ], [ 96, 111, "crystal lattice", "evidence" ] ] }, { "sid": 174, "sent": "Rather, the crystal lattice is composed of conjoined annular pores in which all four F20 faces on the surface of each dodecamer contact F20 faces on other dodecamers (Figure S4).", "section": "RESULTS", "ner": [ [ 12, 27, "crystal lattice", "evidence" ], [ 53, 66, "annular pores", "site" ], [ 85, 88, "F20", "residue_name_number" ], [ 118, 127, "dodecamer", "oligomeric_state" ], [ 136, 139, "F20", "residue_name_number" ], [ 155, 165, "dodecamers", "oligomeric_state" ] ] }, { "sid": 175, "sent": "The crystal lattice shows how the dodecamers can further assemble to form larger structures.", "section": "RESULTS", "ner": [ [ 4, 19, "crystal lattice", "evidence" ], [ 34, 44, "dodecamers", "oligomeric_state" ] ] }, { "sid": 176, "sent": "Each dodecamer may be thought of as a tetravalent building block with the potential to assemble on all four faces to form higher-order supramolecular assemblies.", "section": "RESULTS", "ner": [ [ 5, 14, "dodecamer", "oligomeric_state" ] ] }, { "sid": 177, "sent": "The X-ray crystallographic study of peptide 2 described here provides high-resolution structures of oligomers formed by an A\u03b217\u201336 \u03b2-hairpin.", "section": "DISCUSS", "ner": [ [ 4, 32, "X-ray crystallographic study", "experimental_method" ], [ 36, 45, "peptide 2", "mutant" ], [ 86, 96, "structures", "evidence" ], [ 100, 109, "oligomers", "oligomeric_state" ], [ 123, 125, "A\u03b2", "protein" ], [ 125, 130, "17\u201336", "residue_range" ], [ 131, 140, "\u03b2-hairpin", "structure_element" ] ] }, { "sid": 178, "sent": "The crystallographic assembly of peptide 2 into a trimer, dodecamer, and annular pore provides a model for the assembly of the full-length A\u03b2 peptide to form oligomers.", "section": "DISCUSS", "ner": [ [ 4, 29, "crystallographic assembly", "evidence" ], [ 33, 42, "peptide 2", "mutant" ], [ 50, 56, "trimer", "oligomeric_state" ], [ 58, 67, "dodecamer", "oligomeric_state" ], [ 73, 85, "annular pore", "site" ], [ 127, 138, "full-length", "protein_state" ], [ 139, 141, "A\u03b2", "protein" ], [ 158, 167, "oligomers", "oligomeric_state" ] ] }, { "sid": 179, "sent": "In this model A\u03b2 folds to form a \u03b2-hairpin comprising the hydrophobic central and C-terminal regions.", "section": "DISCUSS", "ner": [ [ 14, 16, "A\u03b2", "protein" ], [ 33, 42, "\u03b2-hairpin", "structure_element" ], [ 70, 100, "central and C-terminal regions", "structure_element" ] ] }, { "sid": 180, "sent": "Three \u03b2-hairpins assemble to form a trimer, and four trimers assemble to form a dodecamer.", "section": "DISCUSS", "ner": [ [ 6, 16, "\u03b2-hairpins", "structure_element" ], [ 36, 42, "trimer", "oligomeric_state" ], [ 53, 60, "trimers", "oligomeric_state" ], [ 80, 89, "dodecamer", "oligomeric_state" ] ] }, { "sid": 181, "sent": "The dodecamers further assemble to form an annular pore (Figure 6).", "section": "DISCUSS", "ner": [ [ 4, 14, "dodecamers", "oligomeric_state" ], [ 43, 55, "annular pore", "site" ] ] }, { "sid": 182, "sent": "Model for the hierarchical assembly of an A\u03b2 \u03b2-hairpin into a trimer, dodecamer, and annular pore based on the crystallographic assembly of peptide 2.", "section": "FIG", "ner": [ [ 42, 44, "A\u03b2", "protein" ], [ 45, 54, "\u03b2-hairpin", "structure_element" ], [ 62, 68, "trimer", "oligomeric_state" ], [ 70, 79, "dodecamer", "oligomeric_state" ], [ 85, 97, "annular pore", "site" ], [ 140, 149, "peptide 2", "mutant" ] ] }, { "sid": 183, "sent": "Monomeric A\u03b2 folds to form a \u03b2-hairpin in which the hydrophobic central and C-terminal regions form an antiparallel \u03b2-sheet.", "section": "FIG", "ner": [ [ 0, 9, "Monomeric", "oligomeric_state" ], [ 10, 12, "A\u03b2", "protein" ], [ 29, 38, "\u03b2-hairpin", "structure_element" ], [ 64, 71, "central", "structure_element" ], [ 76, 94, "C-terminal regions", "structure_element" ], [ 103, 123, "antiparallel \u03b2-sheet", "structure_element" ] ] }, { "sid": 184, "sent": "Three \u03b2-hairpin monomers assemble to form a triangular trimer.", "section": "FIG", "ner": [ [ 6, 15, "\u03b2-hairpin", "structure_element" ], [ 16, 24, "monomers", "oligomeric_state" ], [ 44, 54, "triangular", "protein_state" ], [ 55, 61, "trimer", "oligomeric_state" ] ] }, { "sid": 185, "sent": "Four triangular trimers assemble to form a dodecamer.", "section": "FIG", "ner": [ [ 5, 15, "triangular", "protein_state" ], [ 16, 23, "trimers", "oligomeric_state" ], [ 43, 52, "dodecamer", "oligomeric_state" ] ] }, { "sid": 186, "sent": "Five dodecamers assemble to form an annular pore.", "section": "FIG", "ner": [ [ 5, 15, "dodecamers", "oligomeric_state" ], [ 36, 48, "annular pore", "site" ] ] }, { "sid": 187, "sent": "The molecular weights shown correspond to an A\u03b242 monomer (\u223c4.5 kDa), an A\u03b242 trimer (\u223c13.5 kDa), an A\u03b242 dodecamer (\u223c54 kDa), and an A\u03b242 annular pore composed of five dodecamers (\u223c270 kDa).", "section": "FIG", "ner": [ [ 45, 49, "A\u03b242", "protein" ], [ 50, 57, "monomer", "oligomeric_state" ], [ 73, 77, "A\u03b242", "protein" ], [ 78, 84, "trimer", "oligomeric_state" ], [ 101, 105, "A\u03b242", "protein" ], [ 106, 115, "dodecamer", "oligomeric_state" ], [ 134, 138, "A\u03b242", "protein" ], [ 139, 151, "annular pore", "site" ], [ 169, 179, "dodecamers", "oligomeric_state" ] ] }, { "sid": 188, "sent": "The model put forth in Figure 6 is consistent with the current understanding of endogenous A\u03b2 oligomerization and explains at atomic resolution many key observations about A\u03b2 oligomers.", "section": "DISCUSS", "ner": [ [ 91, 93, "A\u03b2", "protein" ], [ 172, 174, "A\u03b2", "protein" ], [ 175, 184, "oligomers", "oligomeric_state" ] ] }, { "sid": 189, "sent": "Two general types of endogenous A\u03b2 oligomers have been observed: A\u03b2 oligomers that occur on a pathway to fibrils, or \u201cfibrillar oligomers\u201d, and A\u03b2 oligomers that evade a fibrillar fate, or \u201cnonfibrillar oligomers\u201d.\u2212 Fibrillar oligomers accumulate in Alzheimer\u2019s disease later than nonfibrillar oligomers and coincide with the deposition of plaques.", "section": "DISCUSS", "ner": [ [ 32, 34, "A\u03b2", "protein" ], [ 35, 44, "oligomers", "oligomeric_state" ], [ 65, 67, "A\u03b2", "protein" ], [ 68, 77, "oligomers", "oligomeric_state" ], [ 105, 112, "fibrils", "oligomeric_state" ], [ 118, 127, "fibrillar", "protein_state" ], [ 128, 137, "oligomers", "oligomeric_state" ], [ 144, 146, "A\u03b2", "protein" ], [ 147, 156, "oligomers", "oligomeric_state" ], [ 170, 179, "fibrillar", "protein_state" ], [ 190, 202, "nonfibrillar", "protein_state" ], [ 203, 212, "oligomers", "oligomeric_state" ], [ 216, 225, "Fibrillar", "protein_state" ], [ 226, 235, "oligomers", "oligomeric_state" ], [ 281, 293, "nonfibrillar", "protein_state" ], [ 294, 303, "oligomers", "oligomeric_state" ] ] }, { "sid": 190, "sent": "Nonfibrillar oligomers accumulate early in Alzheimer\u2019s disease before plaque deposition.", "section": "DISCUSS", "ner": [ [ 0, 12, "Nonfibrillar", "protein_state" ], [ 13, 22, "oligomers", "oligomeric_state" ] ] }, { "sid": 191, "sent": "Fibrillar and nonfibrillar oligomers have structurally distinct characteristics, which are reflected in their reactivity with the fibril-specific OC antibody and the oligomer-specific A11 antibody.", "section": "DISCUSS", "ner": [ [ 0, 9, "Fibrillar", "protein_state" ], [ 14, 26, "nonfibrillar", "protein_state" ], [ 27, 36, "oligomers", "oligomeric_state" ], [ 166, 174, "oligomer", "oligomeric_state" ] ] }, { "sid": 192, "sent": "Fibrillar oligomers are recognized by the OC antibody but not the A11 antibody, whereas nonfibrillar oligomers are recognized by the A11 antibody but not the OC antibody.", "section": "DISCUSS", "ner": [ [ 0, 9, "Fibrillar", "protein_state" ], [ 10, 19, "oligomers", "oligomeric_state" ], [ 88, 100, "nonfibrillar", "protein_state" ], [ 101, 110, "oligomers", "oligomeric_state" ] ] }, { "sid": 193, "sent": "These criteria have been used to classify the A\u03b2 oligomers that accumulate in vivo.", "section": "DISCUSS", "ner": [ [ 46, 48, "A\u03b2", "protein" ], [ 49, 58, "oligomers", "oligomeric_state" ] ] }, { "sid": 194, "sent": "A\u03b2 dimers have been classified as fibrillar oligomers, whereas A\u03b2 trimers, A\u03b2*56, and APFs have been classified as nonfibrillar oligomers.", "section": "DISCUSS", "ner": [ [ 0, 2, "A\u03b2", "protein" ], [ 3, 9, "dimers", "oligomeric_state" ], [ 34, 43, "fibrillar", "protein_state" ], [ 44, 53, "oligomers", "oligomeric_state" ], [ 63, 65, "A\u03b2", "protein" ], [ 66, 73, "trimers", "oligomeric_state" ], [ 75, 80, "A\u03b2*56", "complex_assembly" ], [ 86, 90, "APFs", "complex_assembly" ], [ 115, 127, "nonfibrillar", "protein_state" ], [ 128, 137, "oligomers", "oligomeric_state" ] ] }, { "sid": 195, "sent": "Larson and Lesn\u00e9 proposed a model for the endogenous production of nonfibrillar oligomers that explains these observations.", "section": "DISCUSS", "ner": [ [ 67, 79, "nonfibrillar", "protein_state" ], [ 80, 89, "oligomers", "oligomeric_state" ] ] }, { "sid": 196, "sent": "In this model, folded A\u03b2 monomer assembles into a trimer, the trimer further assembles into hexamers and dodecamers, and the dodecamers further assemble to form annular protofibrils.", "section": "DISCUSS", "ner": [ [ 15, 21, "folded", "protein_state" ], [ 22, 24, "A\u03b2", "protein" ], [ 25, 32, "monomer", "oligomeric_state" ], [ 50, 56, "trimer", "oligomeric_state" ], [ 62, 68, "trimer", "oligomeric_state" ], [ 92, 100, "hexamers", "oligomeric_state" ], [ 105, 115, "dodecamers", "oligomeric_state" ], [ 125, 135, "dodecamers", "oligomeric_state" ], [ 161, 181, "annular protofibrils", "complex_assembly" ] ] }, { "sid": 197, "sent": "The hierarchical assembly of peptide 2 is consistent with this model; and the trimer, dodecamer, and annular pore formed by peptide 2 may share similarities to the trimers, A\u03b2*56, and APFs observed in vivo.", "section": "DISCUSS", "ner": [ [ 29, 38, "peptide 2", "mutant" ], [ 78, 84, "trimer", "oligomeric_state" ], [ 86, 95, "dodecamer", "oligomeric_state" ], [ 101, 113, "annular pore", "site" ], [ 124, 133, "peptide 2", "mutant" ], [ 164, 171, "trimers", "oligomeric_state" ], [ 173, 178, "A\u03b2*56", "complex_assembly" ], [ 184, 188, "APFs", "complex_assembly" ] ] }, { "sid": 198, "sent": "At this point, we can only speculate whether the trimer and dodecamer formed by peptide 2 share structural similarities to A\u03b2 trimers and A\u03b2*56, as little is known about the structure of A\u03b2 trimers and A\u03b2*56.", "section": "DISCUSS", "ner": [ [ 49, 55, "trimer", "oligomeric_state" ], [ 60, 69, "dodecamer", "oligomeric_state" ], [ 80, 89, "peptide 2", "mutant" ], [ 123, 125, "A\u03b2", "protein" ], [ 126, 133, "trimers", "oligomeric_state" ], [ 138, 143, "A\u03b2*56", "complex_assembly" ], [ 174, 183, "structure", "evidence" ], [ 187, 189, "A\u03b2", "protein" ], [ 190, 197, "trimers", "oligomeric_state" ], [ 202, 207, "A\u03b2*56", "complex_assembly" ] ] }, { "sid": 199, "sent": "The crystallographically observed annular pore formed by peptide 2 is morphologically similar to the APFs formed by full-length A\u03b2.", "section": "DISCUSS", "ner": [ [ 4, 33, "crystallographically observed", "evidence" ], [ 34, 46, "annular pore", "site" ], [ 57, 66, "peptide 2", "mutant" ], [ 101, 105, "APFs", "complex_assembly" ], [ 116, 127, "full-length", "protein_state" ], [ 128, 130, "A\u03b2", "protein" ] ] }, { "sid": 200, "sent": "The annular pore formed by peptide 2 is comparable in size to the APFs prepared in vitro or isolated from Alzheimer\u2019s brains (Figure 7 and Table 1).", "section": "DISCUSS", "ner": [ [ 4, 16, "annular pore", "site" ], [ 27, 36, "peptide 2", "mutant" ], [ 66, 70, "APFs", "complex_assembly" ] ] }, { "sid": 201, "sent": "The varying sizes of APFs formed by full-length A\u03b2 might result from differences in the number of oligomer subunits comprising each APF.", "section": "DISCUSS", "ner": [ [ 21, 25, "APFs", "complex_assembly" ], [ 36, 47, "full-length", "protein_state" ], [ 48, 50, "A\u03b2", "protein" ], [ 98, 106, "oligomer", "oligomeric_state" ], [ 107, 115, "subunits", "structure_element" ], [ 132, 135, "APF", "complex_assembly" ] ] }, { "sid": 202, "sent": "Although the annular pore formed by peptide 2 contains five dodecamer subunits, pores containing fewer or more subunits can easily be envisioned.", "section": "DISCUSS", "ner": [ [ 13, 25, "annular pore", "site" ], [ 36, 45, "peptide 2", "mutant" ], [ 60, 69, "dodecamer", "oligomeric_state" ], [ 70, 78, "subunits", "structure_element" ], [ 80, 85, "pores", "site" ], [ 111, 119, "subunits", "structure_element" ] ] }, { "sid": 203, "sent": "The dodecamers that comprise the annular pore exhibit two modes of assembly\u2014eclipsed interactions and staggered interactions between the F20 faces of trimers within dodecamers.", "section": "DISCUSS", "ner": [ [ 4, 14, "dodecamers", "oligomeric_state" ], [ 33, 45, "annular pore", "site" ], [ 76, 84, "eclipsed", "protein_state" ], [ 102, 111, "staggered", "protein_state" ], [ 137, 140, "F20", "residue_name_number" ], [ 150, 157, "trimers", "oligomeric_state" ], [ 165, 175, "dodecamers", "oligomeric_state" ] ] }, { "sid": 204, "sent": "These two modes of assembly might reflect a dynamic interaction between dodecamers, which could permit assemblies of more dodecamers into larger annular pores.", "section": "DISCUSS", "ner": [ [ 72, 82, "dodecamers", "oligomeric_state" ], [ 122, 132, "dodecamers", "oligomeric_state" ], [ 145, 158, "annular pores", "site" ] ] }, { "sid": 205, "sent": "Surface views of the annular pore formed by peptide 2. (A) Top view.", "section": "FIG", "ner": [ [ 21, 33, "annular pore", "site" ], [ 44, 53, "peptide 2", "mutant" ] ] }, { "sid": 206, "sent": "Annular Pores Formed by A\u03b2 and Peptide 2", "section": "TABLE", "ner": [ [ 0, 13, "Annular Pores", "site" ], [ 24, 26, "A\u03b2", "protein" ], [ 31, 40, "Peptide 2", "mutant" ] ] }, { "sid": 207, "sent": "annular pore\t \tsource\touter diameter\tinner diameter\tobservation\t \tmethod\t \tpeptide\u00a02\t \u223c11\u201312\u00a0nm\t\u223c2\u00a0nm\tX-ray\u00a0crystallography\t \tsynthetic A\u03b2\t7\u201310\u00a0nm\t1.5\u20132\u00a0nm\tTEM\t \tsynthetic A\u03b2\t16 nm\tnot reported\tAFM\t \tsynthetic A\u03b2\t8\u201325\u00a0nm\tnot reported\tTEM\t \tAlzheimer\u2019s brain\t11\u201314\u00a0nm\t2.5\u20134\u00a0nm\tTEM\t \t", "section": "TABLE", "ner": [ [ 0, 12, "annular pore", "site" ], [ 75, 82, "peptide", "chemical" ], [ 102, 123, "X-ray\u00a0crystallography", "experimental_method" ], [ 126, 135, "synthetic", "protein_state" ], [ 136, 138, "A\u03b2", "protein" ], [ 156, 159, "TEM", "experimental_method" ], [ 172, 174, "A\u03b2", "protein" ], [ 194, 197, "AFM", "experimental_method" ], [ 210, 212, "A\u03b2", "protein" ], [ 234, 237, "TEM", "experimental_method" ], [ 276, 279, "TEM", "experimental_method" ] ] }, { "sid": 208, "sent": "Dot blot analysis shows that peptide 2 is reactive toward the A11 antibody (Figure S5).", "section": "DISCUSS", "ner": [ [ 0, 8, "Dot blot", "experimental_method" ], [ 29, 38, "peptide 2", "mutant" ] ] }, { "sid": 209, "sent": "This reactivity suggests that peptide 2 forms oligomers in solution that share structural similarities to the nonfibrillar oligomers formed by full-length A\u03b2.", "section": "DISCUSS", "ner": [ [ 30, 39, "peptide 2", "mutant" ], [ 46, 55, "oligomers", "oligomeric_state" ], [ 110, 122, "nonfibrillar", "protein_state" ], [ 123, 132, "oligomers", "oligomeric_state" ], [ 143, 154, "full-length", "protein_state" ], [ 155, 157, "A\u03b2", "protein" ] ] }, { "sid": 210, "sent": "Further studies are needed to elucidate the species that peptide 2 forms in solution and to study their biological properties.", "section": "DISCUSS", "ner": [ [ 57, 66, "peptide 2", "mutant" ] ] }, { "sid": 211, "sent": "Preliminary attempts to study these species by SEC and SDS-PAGE have not provided a clear measure of the structures formed in solution.", "section": "DISCUSS", "ner": [ [ 47, 50, "SEC", "experimental_method" ], [ 55, 63, "SDS-PAGE", "experimental_method" ], [ 105, 115, "structures", "evidence" ] ] }, { "sid": 212, "sent": "The difficulty in studying the oligomers formed in solution may reflect the propensity of the dodecamer to assemble on all four F20 faces.", "section": "DISCUSS", "ner": [ [ 31, 40, "oligomers", "oligomeric_state" ], [ 94, 103, "dodecamer", "oligomeric_state" ], [ 128, 131, "F20", "residue_name_number" ] ] }, { "sid": 213, "sent": "The X-ray crystallographic structure and A11 reactivity of peptide 2 support the model proposed by Larsen and Lesn\u00e9 and suggest that \u03b2-hairpins constitute a fundamental building block for nonfibrillar oligomers.", "section": "DISCUSS", "ner": [ [ 4, 36, "X-ray crystallographic structure", "evidence" ], [ 59, 68, "peptide 2", "mutant" ], [ 133, 143, "\u03b2-hairpins", "structure_element" ], [ 188, 200, "nonfibrillar", "protein_state" ], [ 201, 210, "oligomers", "oligomeric_state" ] ] }, { "sid": 214, "sent": "What makes \u03b2-hairpins special is that three \u03b2-hairpins can nestle together to form trimers, stabilized by a network of hydrogen bonds and hydrophobic interactions.", "section": "DISCUSS", "ner": [ [ 11, 21, "\u03b2-hairpins", "structure_element" ], [ 44, 54, "\u03b2-hairpins", "structure_element" ], [ 83, 90, "trimers", "oligomeric_state" ], [ 119, 133, "hydrogen bonds", "bond_interaction" ], [ 138, 162, "hydrophobic interactions", "bond_interaction" ] ] }, { "sid": 215, "sent": "This mode of assembly is not unique to A\u03b2.", "section": "DISCUSS", "ner": [ [ 39, 41, "A\u03b2", "protein" ] ] }, { "sid": 216, "sent": "The foldon domain of bacteriophage T4 fibritin is composed of three \u03b2-hairpins that assemble into a triangular trimer similar to the triangular trimer formed by peptide 2.", "section": "DISCUSS", "ner": [ [ 4, 17, "foldon domain", "structure_element" ], [ 21, 37, "bacteriophage T4", "species" ], [ 38, 46, "fibritin", "protein" ], [ 68, 78, "\u03b2-hairpins", "structure_element" ], [ 100, 110, "triangular", "protein_state" ], [ 111, 117, "trimer", "oligomeric_state" ], [ 133, 143, "triangular", "protein_state" ], [ 144, 150, "trimer", "oligomeric_state" ], [ 161, 170, "peptide 2", "mutant" ] ] }, { "sid": 217, "sent": "Additionally, our research group has observed a similar assembly of a \u03b2-hairpin peptide derived from \u03b22-microglobulin.", "section": "DISCUSS", "ner": [ [ 70, 79, "\u03b2-hairpin", "structure_element" ], [ 101, 117, "\u03b22-microglobulin", "protein" ] ] }, { "sid": 218, "sent": "Although we began these studies with a relatively simple hypothesis\u2014that the trimers and dodecamers formed by peptide 1 could accommodate the A\u03b224\u201329 loop\u2014an even more exciting finding has emerged\u2014that the dodecamers can assemble to form annular pores.", "section": "CONCL", "ner": [ [ 77, 84, "trimers", "oligomeric_state" ], [ 89, 99, "dodecamers", "oligomeric_state" ], [ 110, 119, "peptide 1", "mutant" ], [ 142, 144, "A\u03b2", "protein" ], [ 144, 149, "24\u201329", "residue_range" ], [ 150, 154, "loop", "structure_element" ], [ 206, 216, "dodecamers", "oligomeric_state" ], [ 238, 251, "annular pores", "site" ] ] }, { "sid": 219, "sent": "This finding could not have been anticipated from the X-ray crystallographic structure of peptide 1 and reveals a new level of hierarchical assembly that recapitulates micrographic observations of annular protofibrils.", "section": "CONCL", "ner": [ [ 54, 86, "X-ray crystallographic structure", "evidence" ], [ 90, 99, "peptide 1", "mutant" ], [ 197, 217, "annular protofibrils", "complex_assembly" ] ] }, { "sid": 220, "sent": "The crystallographically observed dodecamer, in turn, recapitulates the observation of A\u03b2*56, which appears to be a dodecamer of A\u03b2.", "section": "CONCL", "ner": [ [ 4, 33, "crystallographically observed", "evidence" ], [ 34, 43, "dodecamer", "oligomeric_state" ], [ 87, 92, "A\u03b2*56", "complex_assembly" ], [ 116, 125, "dodecamer", "oligomeric_state" ], [ 129, 131, "A\u03b2", "protein" ] ] }, { "sid": 221, "sent": "The crystallographically observed trimer recapitulates the A\u03b2 trimers that are observed even before the onset of symptoms in Alzheimer\u2019s disease.", "section": "CONCL", "ner": [ [ 4, 33, "crystallographically observed", "evidence" ], [ 34, 40, "trimer", "oligomeric_state" ], [ 59, 61, "A\u03b2", "protein" ], [ 62, 69, "trimers", "oligomeric_state" ] ] }, { "sid": 222, "sent": "Our approach of constraining A\u03b217\u201336 into a \u03b2-hairpin conformation and blocking aggregation with an N-methyl group has allowed us to crystallize a large fragment of what is generally considered to be an uncrystallizable peptide.", "section": "CONCL", "ner": [ [ 29, 31, "A\u03b2", "protein" ], [ 31, 36, "17\u201336", "residue_range" ], [ 44, 53, "\u03b2-hairpin", "structure_element" ], [ 133, 144, "crystallize", "experimental_method" ] ] }, { "sid": 223, "sent": "We believe this iterative, \u201cbottom up\u201d approach of identifying the minimal modification required to crystallize A\u03b2 peptides will ultimately allow larger fragments of A\u03b2 to be crystallized, thus providing greater insights into the structures of A\u03b2 oligomers.", "section": "CONCL", "ner": [ [ 100, 111, "crystallize", "experimental_method" ], [ 112, 114, "A\u03b2", "protein" ], [ 166, 168, "A\u03b2", "protein" ], [ 175, 187, "crystallized", "experimental_method" ], [ 230, 240, "structures", "evidence" ], [ 244, 246, "A\u03b2", "protein" ], [ 247, 256, "oligomers", "oligomeric_state" ] ] } ] }, "PMC4848761": { "annotations": [ { "sid": 0, "sent": "Predictive features of ligand\u2010specific signaling through the estrogen receptor", "section": "TITLE", "ner": [ [ 61, 78, "estrogen receptor", "protein_type" ] ] }, { "sid": 1, "sent": "Some estrogen receptor\u2010\u03b1 (ER\u03b1)\u2010targeted breast cancer therapies such as tamoxifen have tissue\u2010selective or cell\u2010specific activities, while others have similar activities in different cell types.", "section": "ABSTRACT", "ner": [ [ 5, 24, "estrogen receptor\u2010\u03b1", "protein" ], [ 26, 29, "ER\u03b1", "protein" ], [ 72, 81, "tamoxifen", "chemical" ] ] }, { "sid": 2, "sent": "To identify biophysical determinants of cell\u2010specific signaling and breast cancer cell proliferation, we synthesized 241 ER\u03b1 ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ER\u03b1 activities and X\u2010ray crystallography.", "section": "ABSTRACT", "ner": [ [ 105, 116, "synthesized", "experimental_method" ], [ 121, 124, "ER\u03b1", "protein" ], [ 200, 222, "quantitative bioassays", "experimental_method" ], [ 237, 240, "ER\u03b1", "protein" ], [ 256, 277, "X\u2010ray crystallography", "experimental_method" ] ] }, { "sid": 3, "sent": "Ligands that regulate the dynamics and stability of the coactivator\u2010binding site in the C\u2010terminal ligand\u2010binding domain, called activation function\u20102 (AF\u20102), showed similar activity profiles in different cell types.", "section": "ABSTRACT", "ner": [ [ 56, 80, "coactivator\u2010binding site", "site" ], [ 99, 120, "ligand\u2010binding domain", "structure_element" ], [ 129, 150, "activation function\u20102", "structure_element" ], [ 152, 156, "AF\u20102", "structure_element" ] ] }, { "sid": 4, "sent": "Such ligands induced breast cancer cell proliferation in a manner that was predicted by the canonical recruitment of the coactivators NCOA1/2/3 and induction of the GREB1 proliferative gene.", "section": "ABSTRACT", "ner": [ [ 134, 143, "NCOA1/2/3", "protein" ], [ 165, 170, "GREB1", "protein" ] ] }, { "sid": 5, "sent": "For some ligand series, a single inter\u2010atomic distance in the ligand\u2010binding domain predicted their proliferative effects.", "section": "ABSTRACT", "ner": [ [ 33, 54, "inter\u2010atomic distance", "evidence" ], [ 62, 83, "ligand\u2010binding domain", "structure_element" ] ] }, { "sid": 6, "sent": "In contrast, the N\u2010terminal coactivator\u2010binding site, activation function\u20101 (AF\u20101), determined cell\u2010specific signaling induced by ligands that used alternate mechanisms to control cell proliferation.", "section": "ABSTRACT", "ner": [ [ 28, 52, "coactivator\u2010binding site", "site" ], [ 54, 75, "activation function\u20101", "structure_element" ], [ 77, 81, "AF\u20101", "structure_element" ] ] }, { "sid": 7, "sent": "Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ER\u03b1.", "section": "ABSTRACT", "ner": [ [ 20, 47, "systems structural analyses", "experimental_method" ], [ 53, 82, "quantitative chemical biology", "experimental_method" ], [ 162, 165, "ER\u03b1", "protein" ] ] }, { "sid": 8, "sent": "Many drugs are small\u2010molecule ligands of allosteric signaling proteins, including G protein\u2010coupled receptors (GPCRs) and nuclear receptors such as ER\u03b1.", "section": "INTRO", "ner": [ [ 82, 109, "G protein\u2010coupled receptors", "protein_type" ], [ 111, 116, "GPCRs", "protein_type" ], [ 122, 139, "nuclear receptors", "protein_type" ], [ 148, 151, "ER\u03b1", "protein" ] ] }, { "sid": 9, "sent": "Small\u2010molecule ligands control receptor activity by modulating recruitment of effector enzymes to distal regions of the receptor, relative to the ligand\u2010binding site.", "section": "INTRO", "ner": [ [ 146, 165, "ligand\u2010binding site", "site" ] ] }, { "sid": 10, "sent": "For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex\u00ae; AstraZeneca) or raloxifene (Evista\u00ae; Eli Lilly) (Fig\u00a01A) block the ER\u03b1\u2010mediated proliferative effects of the native estrogen, 17\u03b2\u2010estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et\u00a0al, 1996; Fisher et\u00a0al, 1998; McDonnell et\u00a0al, 2002; Jordan, 2003).", "section": "INTRO", "ner": [ [ 23, 51, "estrogen receptor modulators", "protein_type" ], [ 53, 58, "SERMs", "protein_type" ], [ 68, 77, "tamoxifen", "chemical" ], [ 79, 88, "Nolvadex\u00ae", "chemical" ], [ 106, 116, "raloxifene", "chemical" ], [ 118, 125, "Evista\u00ae", "chemical" ], [ 157, 160, "ER\u03b1", "protein" ], [ 206, 214, "estrogen", "chemical" ], [ 216, 229, "17\u03b2\u2010estradiol", "chemical" ], [ 231, 233, "E2", "chemical" ] ] }, { "sid": 11, "sent": "Allosteric control of ER\u03b1 activity", "section": "FIG", "ner": [ [ 22, 25, "ER\u03b1", "protein" ] ] }, { "sid": 12, "sent": "Chemical structures of some common ER\u03b1 ligands.", "section": "FIG", "ner": [ [ 35, 38, "ER\u03b1", "protein" ] ] }, { "sid": 13, "sent": "E2\u2010rings are numbered A\u2010D. The E\u2010ring is the common site of attachment for BSC found in many SERMS.", "section": "FIG", "ner": [ [ 0, 2, "E2", "chemical" ], [ 93, 98, "SERMS", "protein_type" ] ] }, { "sid": 14, "sent": "ER\u03b1 domain organization lettered, A\u2010F. DBD, DNA\u2010binding domain; LBD, ligand\u2010binding domain; AF, activation function", "section": "FIG", "ner": [ [ 0, 3, "ER\u03b1", "protein" ], [ 39, 42, "DBD", "structure_element" ], [ 44, 62, "DNA\u2010binding domain", "structure_element" ], [ 64, 67, "LBD", "structure_element" ], [ 69, 90, "ligand\u2010binding domain", "structure_element" ], [ 92, 94, "AF", "structure_element" ], [ 96, 115, "activation function", "structure_element" ] ] }, { "sid": 15, "sent": "Schematic illustration of the canonical ER\u03b1 signaling pathway.", "section": "FIG", "ner": [ [ 40, 43, "ER\u03b1", "protein" ] ] }, { "sid": 16, "sent": "Linear causality model for ER\u03b1\u2010mediated cell proliferation.", "section": "FIG", "ner": [ [ 27, 30, "ER\u03b1", "protein" ] ] }, { "sid": 17, "sent": "Branched causality model for ER\u03b1\u2010mediated cell proliferation.", "section": "FIG", "ner": [ [ 29, 32, "ER\u03b1", "protein" ] ] }, { "sid": 18, "sent": "ER\u03b1 contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA\u2010binding domain (DBD) that is connected by a flexible hinge region to the ligand\u2010binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig\u00a01B).", "section": "INTRO", "ner": [ [ 0, 3, "ER\u03b1", "protein" ], [ 13, 35, "structurally conserved", "protein_state" ], [ 36, 52, "globular domains", "structure_element" ], [ 60, 88, "nuclear receptor superfamily", "protein_type" ], [ 102, 120, "DNA\u2010binding domain", "structure_element" ], [ 122, 125, "DBD", "structure_element" ], [ 150, 158, "flexible", "protein_state" ], [ 159, 171, "hinge region", "structure_element" ], [ 179, 200, "ligand\u2010binding domain", "structure_element" ], [ 202, 205, "LBD", "structure_element" ], [ 219, 231, "unstructured", "protein_state" ], [ 232, 234, "AB", "structure_element" ], [ 239, 240, "F", "structure_element" ] ] }, { "sid": 19, "sent": "The LBD contains a ligand\u2010dependent coactivator\u2010binding site called activation function\u20102 (AF\u20102).", "section": "INTRO", "ner": [ [ 4, 7, "LBD", "structure_element" ], [ 36, 60, "coactivator\u2010binding site", "site" ], [ 68, 89, "activation function\u20102", "structure_element" ], [ 91, 95, "AF\u20102", "structure_element" ] ] }, { "sid": 20, "sent": "However, the agonist activity of SERMs derives from activation function\u20101 (AF\u20101)\u2014a coactivator recruitment site located in the AB domain (Berry et\u00a0al, 1990; Shang & Brown, 2002; Abot et\u00a0al, 2013).", "section": "INTRO", "ner": [ [ 33, 38, "SERMs", "protein_type" ], [ 52, 73, "activation function\u20101", "structure_element" ], [ 75, 79, "AF\u20101", "structure_element" ], [ 83, 111, "coactivator recruitment site", "site" ], [ 127, 129, "AB", "structure_element" ] ] }, { "sid": 21, "sent": "AF\u20101 and AF\u20102 bind distinct but overlapping sets of coregulators (Webb et\u00a0al, 1998; Endoh et\u00a0al, 1999; Delage\u2010Mourroux et\u00a0al, 2000; Yi et\u00a0al, 2015).", "section": "INTRO", "ner": [ [ 0, 4, "AF\u20101", "structure_element" ], [ 9, 13, "AF\u20102", "structure_element" ] ] }, { "sid": 22, "sent": "AF\u20102 binds the signature LxxLL motif peptides of coactivators such as NCOA1/2/3 (also known as SRC\u20101/2/3).", "section": "INTRO", "ner": [ [ 0, 4, "AF\u20102", "structure_element" ], [ 25, 36, "LxxLL motif", "structure_element" ], [ 70, 79, "NCOA1/2/3", "protein" ], [ 95, 104, "SRC\u20101/2/3", "protein" ] ] }, { "sid": 23, "sent": "AF\u20101 binds a separate surface on these coactivators (Webb et\u00a0al, 1998; Yi et\u00a0al, 2015).", "section": "INTRO", "ner": [ [ 0, 4, "AF\u20101", "structure_element" ] ] }, { "sid": 24, "sent": "Yet, it is unknown how different ER\u03b1 ligands control AF\u20101 through the LBD, and whether this inter\u2010domain communication is required for cell\u2010specific signaling or anti\u2010proliferative responses.", "section": "INTRO", "ner": [ [ 33, 36, "ER\u03b1", "protein" ], [ 53, 57, "AF\u20101", "structure_element" ], [ 70, 73, "LBD", "structure_element" ] ] }, { "sid": 25, "sent": "In the canonical model of the ER\u03b1 signaling pathway (Fig\u00a01C), E2\u2010bound ER\u03b1 forms a homodimer that binds DNA at estrogen\u2010response elements (EREs), recruits NCOA1/2/3 (Metivier et\u00a0al, 2003; Johnson & O'Malley, 2012), and activates the GREB1 gene, which is required for proliferation of ER\u03b1\u2010positive breast cancer cells (Ghosh et\u00a0al, 2000; Rae et\u00a0al, 2005; Deschenes et\u00a0al, 2007; Liu et\u00a0al, 2012; Srinivasan et\u00a0al, 2013).", "section": "INTRO", "ner": [ [ 30, 33, "ER\u03b1", "protein" ], [ 62, 70, "E2\u2010bound", "protein_state" ], [ 71, 74, "ER\u03b1", "protein" ], [ 83, 92, "homodimer", "oligomeric_state" ], [ 111, 137, "estrogen\u2010response elements", "site" ], [ 139, 143, "EREs", "site" ], [ 155, 164, "NCOA1/2/3", "protein" ], [ 233, 238, "GREB1", "protein" ], [ 284, 287, "ER\u03b1", "protein" ] ] }, { "sid": 26, "sent": "However, ER\u03b1\u2010mediated proliferative responses vary in a ligand\u2010dependent manner (Srinivasan et\u00a0al, 2013); thus, it is not known whether this canonical model is widely applicable across diverse ER\u03b1 ligands.", "section": "INTRO", "ner": [ [ 9, 12, "ER\u03b1", "protein" ], [ 193, 196, "ER\u03b1", "protein" ] ] }, { "sid": 27, "sent": "Our long\u2010term goal is to be able to predict proliferative or anti\u2010proliferative activity of a ligand in different tissues from its crystal structure by identifying different structural perturbations that lead to specific signaling outcomes.", "section": "INTRO", "ner": [ [ 131, 148, "crystal structure", "evidence" ] ] }, { "sid": 28, "sent": "The simplest response model for ligand\u2010specific proliferative effects is a linear causality model, where the degree of NCOA1/2/3 recruitment determines GREB1 expression, which in turn drives ligand\u2010specific cell proliferation (Fig\u00a01D).", "section": "INTRO", "ner": [ [ 119, 128, "NCOA1/2/3", "protein" ], [ 152, 157, "GREB1", "protein" ] ] }, { "sid": 29, "sent": "In this signaling model, multiple coregulator binding events and target genes (Won Jeong et\u00a0al, 2012; Nwachukwu et\u00a0al, 2014), LBD conformation, nucleocytoplasmic shuttling, the occupancy and dynamics of DNA binding, and other biophysical features could contribute independently to cell proliferation (Lickwar et\u00a0al, 2012).", "section": "INTRO", "ner": [ [ 126, 129, "LBD", "structure_element" ] ] }, { "sid": 30, "sent": "To test these signaling models, we profiled a diverse library of ER\u03b1 ligands using systems biology approaches to X\u2010ray crystallography and chemical biology (Srinivasan et\u00a0al, 2013), including a series of quantitative bioassays for ER\u03b1 function that were statistically robust and reproducible, based on the Z\u2019\u2010statistic (Fig\u00a0EV1A and B; see Materials and Methods).", "section": "INTRO", "ner": [ [ 65, 68, "ER\u03b1", "protein" ], [ 113, 134, "X\u2010ray crystallography", "experimental_method" ], [ 139, 155, "chemical biology", "experimental_method" ], [ 231, 234, "ER\u03b1", "protein" ], [ 306, 318, "Z\u2019\u2010statistic", "evidence" ] ] }, { "sid": 31, "sent": "We also determined the structures of 76 distinct ER\u03b1 LBD complexes bound to different ligand types, which allowed us to understand how diverse ligand scaffolds distort the active conformation of the ER\u03b1 LBD.", "section": "INTRO", "ner": [ [ 8, 18, "determined", "experimental_method" ], [ 23, 33, "structures", "evidence" ], [ 49, 52, "ER\u03b1", "protein" ], [ 53, 56, "LBD", "structure_element" ], [ 67, 75, "bound to", "protein_state" ], [ 172, 178, "active", "protein_state" ], [ 199, 202, "ER\u03b1", "protein" ], [ 203, 206, "LBD", "structure_element" ] ] }, { "sid": 32, "sent": "Our findings here indicate that specific structural perturbations can be tied to ligand\u2010selective domain usage and signaling patterns, thus providing a framework for structure\u2010based design of improved breast cancer therapeutics, and understanding the different phenotypic effects of environmental estrogens.", "section": "INTRO", "ner": [ [ 297, 306, "estrogens", "chemical" ] ] }, { "sid": 33, "sent": "High\u2010throughput screens for ER\u03b1 ligand profiling", "section": "FIG", "ner": [ [ 28, 31, "ER\u03b1", "protein" ] ] }, { "sid": 34, "sent": "Summary of ligand screening assays used to measure ER\u2010mediated activities.", "section": "FIG", "ner": [ [ 11, 34, "ligand screening assays", "experimental_method" ] ] }, { "sid": 35, "sent": "ERE, estrogen\u2010response element; Luc, luciferase reporter gene; M2H, mammalian 2\u2010hybrid; UAS, upstream\u2010activating sequence.", "section": "FIG", "ner": [ [ 0, 3, "ERE", "structure_element" ], [ 5, 30, "estrogen\u2010response element", "structure_element" ], [ 32, 35, "Luc", "experimental_method" ], [ 37, 61, "luciferase reporter gene", "experimental_method" ], [ 63, 66, "M2H", "experimental_method" ], [ 68, 86, "mammalian 2\u2010hybrid", "experimental_method" ], [ 88, 91, "UAS", "structure_element" ], [ 93, 121, "upstream\u2010activating sequence", "structure_element" ] ] }, { "sid": 36, "sent": "Strength of AF\u20101 signaling does not determine cell\u2010specific signaling", "section": "RESULTS", "ner": [ [ 12, 16, "AF\u20101", "structure_element" ] ] }, { "sid": 37, "sent": "To compare ER\u03b1 signaling induced by diverse ligand types, we synthesized and assayed a library of 241 ER\u03b1 ligands containing 19 distinct molecular scaffolds.", "section": "RESULTS", "ner": [ [ 11, 14, "ER\u03b1", "protein" ], [ 61, 84, "synthesized and assayed", "experimental_method" ], [ 102, 105, "ER\u03b1", "protein" ] ] }, { "sid": 38, "sent": "These include 15 indirect modulator series, which lack a SERM\u2010like side chain and modulate coactivator binding indirectly from the ligand\u2010binding pocket (Fig\u00a02A\u2013E; Dataset EV1) (Zheng et\u00a0al, 2012) (Zhu et\u00a0al, 2012) (Muthyala et\u00a0al, 2003; Seo et\u00a0al, 2006) (Srinivasan et\u00a0al, 2013) (Wang et\u00a0al, 2012) (Liao et\u00a0al, 2014) (Min et\u00a0al, 2013).", "section": "RESULTS", "ner": [ [ 50, 54, "lack", "protein_state" ], [ 57, 66, "SERM\u2010like", "protein_type" ], [ 131, 152, "ligand\u2010binding pocket", "site" ] ] }, { "sid": 39, "sent": "We also generated four direct modulator series with side chains designed to directly dislocate h12 and thereby completely occlude the AF\u20102 surface (Fig\u00a02C and E; Dataset EV1) (Kieser et\u00a0al, 2010).", "section": "RESULTS", "ner": [ [ 95, 98, "h12", "structure_element" ], [ 134, 146, "AF\u20102 surface", "site" ] ] }, { "sid": 40, "sent": "Ligand profiling using our quantitative bioassays revealed a wide range of ligand\u2010induced GREB1 expression, reporter gene activities, ER\u03b1\u2010coactivator interactions, and proliferative effects on MCF\u20107 breast cancer cells (Figs\u00a0EV1 and EV2A\u2013J).", "section": "RESULTS", "ner": [ [ 0, 16, "Ligand profiling", "experimental_method" ], [ 27, 49, "quantitative bioassays", "experimental_method" ], [ 90, 95, "GREB1", "protein" ], [ 134, 137, "ER\u03b1", "protein" ] ] }, { "sid": 41, "sent": "This wide variance enabled us to probe specific features of ER\u03b1 signaling using ligand class analyses, and identify signaling patterns shared by specific ligand series or scaffolds.", "section": "RESULTS", "ner": [ [ 60, 63, "ER\u03b1", "protein" ], [ 80, 101, "ligand class analyses", "experimental_method" ] ] }, { "sid": 42, "sent": "Classes of compounds in the ER\u03b1 ligand library", "section": "FIG", "ner": [ [ 28, 31, "ER\u03b1", "protein" ] ] }, { "sid": 43, "sent": "Structure of the E2\u2010bound ER\u03b1 LBD in complex with an NCOA2 peptide of (PDB 1GWR).", "section": "FIG", "ner": [ [ 0, 9, "Structure", "evidence" ], [ 17, 25, "E2\u2010bound", "protein_state" ], [ 26, 29, "ER\u03b1", "protein" ], [ 30, 33, "LBD", "structure_element" ], [ 34, 49, "in complex with", "protein_state" ], [ 53, 58, "NCOA2", "protein" ] ] }, { "sid": 44, "sent": "Structural details of the ER\u03b1 LBD bound to the indicated ligands.", "section": "FIG", "ner": [ [ 26, 29, "ER\u03b1", "protein" ], [ 30, 33, "LBD", "structure_element" ], [ 34, 42, "bound to", "protein_state" ] ] }, { "sid": 45, "sent": "Unlike E2 (PDB 1GWR), TAM is a direct modulator with a BSC that dislocates h12 to block the NCOA2\u2010binding site (PDB 3ERT).", "section": "FIG", "ner": [ [ 7, 9, "E2", "chemical" ], [ 22, 25, "TAM", "chemical" ], [ 75, 78, "h12", "structure_element" ], [ 92, 110, "NCOA2\u2010binding site", "site" ] ] }, { "sid": 46, "sent": "OBHS is an indirect modulator that dislocates the h11 C\u2010terminus to destabilize the h11\u2013h12 interface (PDB 4ZN9).", "section": "FIG", "ner": [ [ 0, 4, "OBHS", "chemical" ], [ 50, 53, "h11", "structure_element" ], [ 84, 101, "h11\u2013h12 interface", "site" ] ] }, { "sid": 47, "sent": "The ER\u03b1 ligand library contains 241 ligands representing 15 indirect modulator scaffolds, plus 4 direct modulator scaffolds.", "section": "FIG", "ner": [ [ 4, 7, "ER\u03b1", "protein" ] ] }, { "sid": 48, "sent": "ER\u03b1 ligands induced a range of agonist activity profiles", "section": "FIG", "ner": [ [ 0, 3, "ER\u03b1", "protein" ] ] }, { "sid": 49, "sent": "To this end, we compared the average ligand\u2010induced GREB1 mRNA levels in MCF\u20107 cells and 3\u00d7ERE\u2010Luc reporter gene activity in Ishikawa endometrial cancer cells (E\u2010Luc) or in HepG2 cells transfected with wild\u2010type ER\u03b1 (L\u2010Luc ER\u03b1\u2010WT) (Figs\u00a03A and EV2A\u2013C).", "section": "RESULTS", "ner": [ [ 52, 57, "GREB1", "protein" ], [ 89, 98, "3\u00d7ERE\u2010Luc", "experimental_method" ], [ 160, 165, "E\u2010Luc", "experimental_method" ], [ 202, 211, "wild\u2010type", "protein_state" ], [ 212, 215, "ER\u03b1", "protein" ], [ 217, 222, "L\u2010Luc", "experimental_method" ], [ 223, 226, "ER\u03b1", "protein" ], [ 227, 229, "WT", "protein_state" ] ] }, { "sid": 50, "sent": "Direct modulators showed significant differences in average activity between cell types except OBHS\u2010ASC analogs, which had similar low agonist activities in the three cell types.", "section": "RESULTS", "ner": [ [ 95, 103, "OBHS\u2010ASC", "chemical" ] ] }, { "sid": 51, "sent": "While it was known that direct modulators such as tamoxifen drive cell\u2010specific signaling, these experiments reveal that indirect modulators also drive cell\u2010specific signaling, since eight of fourteen classes showed significant differences in average activity (Figs\u00a03A and EV2A\u2013C).", "section": "RESULTS", "ner": [ [ 50, 59, "tamoxifen", "chemical" ] ] }, { "sid": 52, "sent": "Ligand\u2010specific signaling underlies ER\u03b1\u2010mediated cell proliferation", "section": "FIG", "ner": [ [ 36, 39, "ER\u03b1", "protein" ] ] }, { "sid": 53, "sent": "(A) Ligand\u2010specific ER\u03b1 activities in HepG2, Ishikawa and MCF\u20107 cells.", "section": "FIG", "ner": [ [ 20, 23, "ER\u03b1", "protein" ] ] }, { "sid": 54, "sent": "The ligand\u2010induced L\u2010Luc ER\u03b1\u2010WT and E\u2010Luc activities and GREB1 mRNA levels are shown by scaffold (mean\u00a0+\u00a0SD).", "section": "FIG", "ner": [ [ 19, 24, "L\u2010Luc", "experimental_method" ], [ 25, 28, "ER\u03b1", "protein" ], [ 29, 31, "WT", "protein_state" ], [ 36, 41, "E\u2010Luc", "experimental_method" ], [ 57, 62, "GREB1", "protein" ] ] }, { "sid": 55, "sent": "(B) Ligand class analysis of the L\u2010Luc ER\u03b1\u2010WT and ER\u03b1\u2010\u0394AB activities in HepG2 cells.", "section": "FIG", "ner": [ [ 11, 25, "class analysis", "experimental_method" ], [ 33, 38, "L\u2010Luc", "experimental_method" ], [ 39, 42, "ER\u03b1", "protein" ], [ 43, 45, "WT", "protein_state" ], [ 50, 57, "ER\u03b1\u2010\u0394AB", "mutant" ] ] }, { "sid": 56, "sent": "Significant sensitivity to AB domain deletion was determined by Student's t\u2010test (n\u00a0=\u00a0number of ligands per scaffold in Fig\u00a02).", "section": "FIG", "ner": [ [ 27, 29, "AB", "structure_element" ], [ 64, 80, "Student's t\u2010test", "experimental_method" ] ] }, { "sid": 57, "sent": "Correlation and regression analyses in a large test set.", "section": "FIG", "ner": [ [ 0, 35, "Correlation and regression analyses", "experimental_method" ] ] }, { "sid": 58, "sent": "In cluster 1, the first three comparisons (rows) showed significant positive correlations (F\u2010test for nonzero slope, P\u00a0\u2264\u00a00.05).", "section": "FIG", "ner": [ [ 91, 97, "F\u2010test", "experimental_method" ], [ 117, 118, "P", "evidence" ] ] }, { "sid": 59, "sent": "In cluster 2, only one of these comparisons revealed a significant positive correlation, while none was significant in cluster 3. +, statistically significant correlations gained by deletion of the AB or F domains.", "section": "FIG", "ner": [ [ 182, 190, "deletion", "experimental_method" ], [ 198, 200, "AB", "structure_element" ], [ 204, 205, "F", "structure_element" ] ] }, { "sid": 60, "sent": "\u2212, significant correlations lost upon deletion of AB or F domains.", "section": "FIG", "ner": [ [ 50, 52, "AB", "structure_element" ], [ 56, 57, "F", "structure_element" ] ] }, { "sid": 61, "sent": "Tamoxifen depends on AF\u20101 for its cell\u2010specific activity (Sakamoto et\u00a0al, 2002); therefore, we asked whether cell\u2010specific signaling observed here is due to a similar dependence on AF\u20101 for activity (Fig\u00a0EV1).", "section": "RESULTS", "ner": [ [ 0, 9, "Tamoxifen", "chemical" ], [ 21, 25, "AF\u20101", "structure_element" ], [ 181, 185, "AF\u20101", "structure_element" ] ] }, { "sid": 62, "sent": "To test this idea, we compared the average L\u2010Luc activities of each scaffold in HepG2 cells co\u2010transfected with wild\u2010type ER\u03b1 or with ER\u03b1 lacking the AB domain (Figs\u00a01B and EV1).", "section": "RESULTS", "ner": [ [ 35, 59, "average L\u2010Luc activities", "evidence" ], [ 92, 106, "co\u2010transfected", "experimental_method" ], [ 112, 121, "wild\u2010type", "protein_state" ], [ 122, 125, "ER\u03b1", "protein" ], [ 134, 137, "ER\u03b1", "protein" ], [ 138, 149, "lacking the", "protein_state" ], [ 150, 152, "AB", "structure_element" ] ] }, { "sid": 63, "sent": "While E2 showed similar L\u2010Luc ER\u03b1\u2010WT and ER\u03b1\u2010\u0394AB activities, tamoxifen showed complete loss of activity without the AB domain (Fig\u00a0EV1B).", "section": "RESULTS", "ner": [ [ 6, 8, "E2", "chemical" ], [ 24, 29, "L\u2010Luc", "experimental_method" ], [ 30, 33, "ER\u03b1", "protein" ], [ 34, 36, "WT", "protein_state" ], [ 41, 48, "ER\u03b1\u2010\u0394AB", "mutant" ], [ 61, 70, "tamoxifen", "chemical" ], [ 104, 111, "without", "protein_state" ], [ 116, 118, "AB", "structure_element" ] ] }, { "sid": 64, "sent": "Deletion of the AB domain significantly reduced the average L\u2010Luc activities of 14 scaffolds (Student's t\u2010test, P\u00a0\u2264\u00a00.05) (Fig\u00a03B).", "section": "RESULTS", "ner": [ [ 0, 11, "Deletion of", "experimental_method" ], [ 16, 18, "AB", "structure_element" ], [ 52, 76, "average L\u2010Luc activities", "evidence" ], [ 94, 110, "Student's t\u2010test", "experimental_method" ], [ 112, 113, "P", "evidence" ] ] }, { "sid": 65, "sent": "These \u201cAF\u20101\u2010sensitive\u201d activities were exhibited by both direct and indirect modulators, and were not limited to scaffolds that showed cell\u2010specific signaling (Fig\u00a03A and B).", "section": "RESULTS", "ner": [ [ 7, 11, "AF\u20101", "structure_element" ] ] }, { "sid": 66, "sent": "Thus, the strength of AF\u20101 signaling does not determine cell\u2010specific signaling.", "section": "RESULTS", "ner": [ [ 22, 26, "AF\u20101", "structure_element" ] ] }, { "sid": 67, "sent": "Identifying cell\u2010specific signaling clusters in ER\u03b1 ligand classes", "section": "RESULTS", "ner": [ [ 48, 51, "ER\u03b1", "protein" ] ] }, { "sid": 68, "sent": "For each ligand class or scaffold, we calculated the Pearson's correlation coefficient, r, for pairwise comparison of activity profiles in breast (GREB1), liver (L\u2010Luc), and endometrial cells (E\u2010Luc).", "section": "RESULTS", "ner": [ [ 53, 86, "Pearson's correlation coefficient", "evidence" ], [ 88, 89, "r", "evidence" ], [ 147, 152, "GREB1", "protein" ], [ 162, 167, "L\u2010Luc", "experimental_method" ], [ 193, 198, "E\u2010Luc", "experimental_method" ] ] }, { "sid": 69, "sent": "The value of r ranges from \u22121 to 1, and it defines the extent to which the data fit a straight line when compounds show similar agonist/antagonist activity profiles between cell types (Fig\u00a0EV3A).", "section": "RESULTS", "ner": [ [ 13, 14, "r", "evidence" ] ] }, { "sid": 70, "sent": "We also calculated the coefficient of determination, r 2, which describes the percentage of variance in a dependent variable such as proliferation that can be predicted by an independent variable such as GREB1 expression.", "section": "RESULTS", "ner": [ [ 23, 51, "coefficient of determination", "evidence" ], [ 53, 56, "r 2", "evidence" ], [ 204, 209, "GREB1", "protein" ] ] }, { "sid": 71, "sent": "We present both calculations as r 2 to readily compare signaling specificities using a heat map on which the red\u2013yellow palette indicates significant positive correlations (P\u00a0\u2264\u00a00.05, F\u2010test for nonzero slope), while the blue palette denotes negative correlations (Fig\u00a03C\u2013F).", "section": "RESULTS", "ner": [ [ 32, 35, "r 2", "evidence" ], [ 173, 174, "P", "evidence" ], [ 183, 189, "F\u2010test", "experimental_method" ] ] }, { "sid": 72, "sent": "The side chain of OBHS\u2010BSC analogs induces cell\u2010specific signaling", "section": "FIG", "ner": [ [ 18, 26, "OBHS\u2010BSC", "chemical" ] ] }, { "sid": 73, "sent": "Correlation analysis of OBHS versus OBHS\u2010BSC activity across cell types.", "section": "FIG", "ner": [ [ 24, 28, "OBHS", "chemical" ], [ 36, 44, "OBHS\u2010BSC", "chemical" ] ] }, { "sid": 74, "sent": "Correlation analysis of L\u2010Luc ER\u03b1\u2010\u0394AB activity versus endogenous ER\u03b1 activity of OBHS analogs.", "section": "FIG", "ner": [ [ 24, 29, "L\u2010Luc", "experimental_method" ], [ 30, 37, "ER\u03b1\u2010\u0394AB", "mutant" ], [ 65, 68, "ER\u03b1", "protein" ], [ 81, 85, "OBHS", "chemical" ] ] }, { "sid": 75, "sent": "In panel (D), L\u2010Luc ER\u03b1\u2010WT activity from panel (B) is shown for comparison.", "section": "FIG", "ner": [ [ 14, 19, "L\u2010Luc", "experimental_method" ], [ 20, 23, "ER\u03b1", "protein" ], [ 24, 26, "WT", "protein_state" ] ] }, { "sid": 76, "sent": "Correlation analysis of L\u2010Luc ER\u03b1\u2010\u0394F activity versus endogenous ER\u03b1 activities of OBHS analogs.", "section": "FIG", "ner": [ [ 24, 29, "L\u2010Luc", "experimental_method" ], [ 30, 36, "ER\u03b1\u2010\u0394F", "mutant" ], [ 64, 67, "ER\u03b1", "protein" ], [ 82, 86, "OBHS", "chemical" ] ] }, { "sid": 77, "sent": "Correlation analysis of MCF\u20107 cell proliferation versus NCOA2/3 recruitment or GREB1 levels observed in response to (G) OBHS\u2010N and (H) OBHS\u2010BSC analogs.", "section": "FIG", "ner": [ [ 56, 63, "NCOA2/3", "protein" ], [ 79, 84, "GREB1", "protein" ], [ 120, 126, "OBHS\u2010N", "chemical" ], [ 135, 143, "OBHS\u2010BSC", "chemical" ] ] }, { "sid": 78, "sent": "Scaffolds in cluster 1 exhibited strongly correlated GREB1 levels, E\u2010Luc and L\u2010Luc activity profiles across the three cell types (Fig\u00a03C lanes 1\u20134), suggesting these ligands use similar ER\u03b1 signaling pathways in the breast, endometrial, and liver cell types.", "section": "RESULTS", "ner": [ [ 53, 58, "GREB1", "protein" ], [ 67, 72, "E\u2010Luc", "experimental_method" ], [ 77, 82, "L\u2010Luc", "experimental_method" ], [ 186, 189, "ER\u03b1", "protein" ] ] }, { "sid": 79, "sent": "This cluster includes WAY\u2010C, OBHS, OBHS\u2010N, and triaryl\u2010ethylene analogs, all of which are indirect modulators.", "section": "RESULTS", "ner": [ [ 22, 27, "WAY\u2010C", "chemical" ], [ 29, 33, "OBHS", "chemical" ], [ 35, 41, "OBHS\u2010N", "chemical" ], [ 47, 63, "triaryl\u2010ethylene", "chemical" ] ] }, { "sid": 80, "sent": "This cluster includes two classes of direct modulators (cyclofenil\u2010ASC and WAY dimer), and six classes of indirect modulators (2,5\u2010DTP, 3,4\u2010DTP, S\u2010OBHS\u20102 and S\u2010OBHS\u20103, furan, and WAY\u2010D).", "section": "RESULTS", "ner": [ [ 56, 70, "cyclofenil\u2010ASC", "chemical" ], [ 75, 84, "WAY dimer", "chemical" ], [ 127, 134, "2,5\u2010DTP", "chemical" ], [ 136, 143, "3,4\u2010DTP", "chemical" ], [ 145, 153, "S\u2010OBHS\u20102", "chemical" ], [ 158, 166, "S\u2010OBHS\u20103", "chemical" ], [ 168, 173, "furan", "chemical" ], [ 179, 184, "WAY\u2010D", "chemical" ] ] }, { "sid": 81, "sent": "For example, 3,4\u2010DTP, furan, and S\u2010OBHS\u20102 drove positively correlated GREB1 levels and E\u2010Luc but not L\u2010Luc ER\u03b1\u2010WT activity (Fig\u00a03C lanes 5\u20137).", "section": "RESULTS", "ner": [ [ 13, 20, "3,4\u2010DTP", "chemical" ], [ 22, 27, "furan", "chemical" ], [ 33, 41, "S\u2010OBHS\u20102", "chemical" ], [ 70, 75, "GREB1", "protein" ], [ 87, 92, "E\u2010Luc", "experimental_method" ], [ 101, 106, "L\u2010Luc", "experimental_method" ], [ 107, 110, "ER\u03b1", "protein" ], [ 111, 113, "WT", "protein_state" ] ] }, { "sid": 82, "sent": "In\u00a0contrast, WAY dimer and WAY\u2010D analogs drove positively correlated GREB1 levels and L\u2010Luc ER\u03b1\u2010WT but not E\u2010Luc activity (Fig\u00a03C lanes 8 and 9).", "section": "RESULTS", "ner": [ [ 13, 22, "WAY dimer", "chemical" ], [ 27, 32, "WAY\u2010D", "chemical" ], [ 69, 74, "GREB1", "protein" ], [ 86, 91, "L\u2010Luc", "experimental_method" ], [ 92, 95, "ER\u03b1", "protein" ], [ 96, 98, "WT", "protein_state" ], [ 107, 112, "E\u2010Luc", "experimental_method" ] ] }, { "sid": 83, "sent": "This cluster includes two direct modulator scaffolds (OBHS\u2010ASC and OBHS\u2010BSC), and five indirect modulator scaffolds (A\u2010CD, cyclofenil, 3,4\u2010DTPD, imine, and imidazopyridine).", "section": "RESULTS", "ner": [ [ 54, 62, "OBHS\u2010ASC", "chemical" ], [ 67, 75, "OBHS\u2010BSC", "chemical" ], [ 117, 121, "A\u2010CD", "chemical" ], [ 123, 133, "cyclofenil", "chemical" ], [ 135, 143, "3,4\u2010DTPD", "chemical" ], [ 145, 150, "imine", "chemical" ], [ 156, 171, "imidazopyridine", "chemical" ] ] }, { "sid": 84, "sent": "These results suggest that addition of an extended side chain to an ER\u03b1 ligand scaffold is sufficient to induce cell\u2010specific signaling, where the relative activity profiles of the individual ligands change between cell types.", "section": "RESULTS", "ner": [ [ 68, 71, "ER\u03b1", "protein" ] ] }, { "sid": 85, "sent": "This is demonstrated by directly comparing the signaling specificities of matched OBHS (indirect modulator, cluster 1) and OBHS\u2010BSC analogs (direct modulator, cluster 3), which differ only in the basic side chain (Fig\u00a02E).", "section": "RESULTS", "ner": [ [ 82, 86, "OBHS", "chemical" ], [ 123, 131, "OBHS\u2010BSC", "chemical" ] ] }, { "sid": 86, "sent": "The activities of OBHS analogs were positively correlated across the three cell types, but the side chain of OBHS\u2010BSC analogs was sufficient to abolish these correlations (Figs\u00a03C lanes 1 and 19, and EV3A\u2013C).", "section": "RESULTS", "ner": [ [ 18, 22, "OBHS", "chemical" ], [ 109, 117, "OBHS\u2010BSC", "chemical" ] ] }, { "sid": 87, "sent": "Thus, examining the correlated patterns of ER\u03b1 activity within each scaffold demonstrates that an extended side chain is not required for cell\u2010specific signaling.", "section": "RESULTS", "ner": [ [ 43, 46, "ER\u03b1", "protein" ] ] }, { "sid": 88, "sent": "Modulation of signaling specificity by AF\u20101", "section": "RESULTS", "ner": [ [ 39, 43, "AF\u20101", "structure_element" ] ] }, { "sid": 89, "sent": "To evaluate the role of AF\u20101 and the F domain in ER\u03b1 signaling specificity, we compared activity of truncated ER\u03b1 constructs in HepG2 liver cells with endogenous ER\u03b1 activity in the other cell types.", "section": "RESULTS", "ner": [ [ 24, 28, "AF\u20101", "structure_element" ], [ 37, 38, "F", "structure_element" ], [ 49, 52, "ER\u03b1", "protein" ], [ 110, 113, "ER\u03b1", "protein" ], [ 162, 165, "ER\u03b1", "protein" ] ] }, { "sid": 90, "sent": "The positive correlation between the L\u2010Luc and E\u2010Luc activities or GREB1 levels induced by scaffolds in cluster 1 was generally retained without the AB domain, or the F domain (Fig\u00a03D lanes 1\u20134).", "section": "RESULTS", "ner": [ [ 37, 42, "L\u2010Luc", "experimental_method" ], [ 47, 52, "E\u2010Luc", "experimental_method" ], [ 67, 72, "GREB1", "protein" ], [ 149, 151, "AB", "structure_element" ], [ 167, 168, "F", "structure_element" ] ] }, { "sid": 91, "sent": "This demonstrates that the signaling specificities underlying these positive correlations are not modified by AF\u20101.", "section": "RESULTS", "ner": [ [ 110, 114, "AF\u20101", "structure_element" ] ] }, { "sid": 92, "sent": "OBHS analogs showed an average L\u2010Luc ER\u03b1\u2010\u0394AB activity of 3.2%\u00a0\u00b1\u00a03 (mean\u00a0+\u00a0SEM) relative to E2.", "section": "RESULTS", "ner": [ [ 0, 4, "OBHS", "chemical" ], [ 31, 36, "L\u2010Luc", "experimental_method" ], [ 37, 44, "ER\u03b1\u2010\u0394AB", "mutant" ], [ 91, 93, "E2", "chemical" ] ] }, { "sid": 93, "sent": "Despite this nearly complete lack of activity, the pattern of L\u2010Luc ER\u03b1\u2010\u0394AB activity was still highly correlated with the E\u2010Luc activity and GREB1 expression (Fig\u00a0EV3D and E), demonstrating that very small AF\u20102 activities can be amplified by AF\u20101 to produce robust signals.", "section": "RESULTS", "ner": [ [ 62, 67, "L\u2010Luc", "experimental_method" ], [ 68, 75, "ER\u03b1\u2010\u0394AB", "mutant" ], [ 122, 127, "E\u2010Luc", "experimental_method" ], [ 141, 146, "GREB1", "protein" ], [ 206, 210, "AF\u20102", "structure_element" ], [ 242, 246, "AF\u20101", "structure_element" ] ] }, { "sid": 94, "sent": "Similarly, deletion of the F domain did not abolish correlations between the L\u2010Luc and E\u2010Luc or GREB1 levels induced by OBHS analogs (Fig\u00a0EV3F).", "section": "RESULTS", "ner": [ [ 11, 22, "deletion of", "experimental_method" ], [ 27, 28, "F", "structure_element" ], [ 77, 82, "L\u2010Luc", "experimental_method" ], [ 87, 92, "E\u2010Luc", "experimental_method" ], [ 96, 101, "GREB1", "protein" ], [ 120, 124, "OBHS", "chemical" ] ] }, { "sid": 95, "sent": "These similar patterns of ligand activity in the wild\u2010type and deletion mutants suggest that AF\u20101 and the F domain purely amplify the AF\u20102 activities of ligands in cluster 1.", "section": "RESULTS", "ner": [ [ 49, 58, "wild\u2010type", "protein_state" ], [ 72, 79, "mutants", "protein_state" ], [ 93, 97, "AF\u20101", "structure_element" ], [ 106, 107, "F", "structure_element" ], [ 134, 138, "AF\u20102", "structure_element" ] ] }, { "sid": 96, "sent": "In contrast, AF\u20101 was a determinant of signaling specificity for scaffolds in cluster 2.", "section": "RESULTS", "ner": [ [ 13, 17, "AF\u20101", "structure_element" ] ] }, { "sid": 97, "sent": "Deletion of the AB or F domain altered correlations for six of the eight scaffolds in this cluster (2,5\u2010DTP, 3,4\u2010DTP, S\u2010OBHS\u20103, WAY\u2010D, WAY dimer, and cyclofenil\u2010ASC) (Fig\u00a03D lanes 5\u201312).", "section": "RESULTS", "ner": [ [ 0, 11, "Deletion of", "experimental_method" ], [ 16, 18, "AB", "structure_element" ], [ 22, 23, "F", "structure_element" ], [ 100, 107, "2,5\u2010DTP", "chemical" ], [ 109, 116, "3,4\u2010DTP", "chemical" ], [ 118, 126, "S\u2010OBHS\u20103", "chemical" ], [ 128, 133, "WAY\u2010D", "chemical" ], [ 135, 144, "WAY dimer", "chemical" ], [ 150, 164, "cyclofenil\u2010ASC", "chemical" ] ] }, { "sid": 98, "sent": "Comparing Fig\u00a03C and D, the + and \u2212 signs indicate where the deletion mutant assays led to a gain or loss of statically significant correlation, respectively.", "section": "RESULTS", "ner": [ [ 61, 83, "deletion mutant assays", "experimental_method" ] ] }, { "sid": 99, "sent": "Thus, in cluster 2, AF\u20101 substantially modulated the specificity of ligands with cell\u2010specific activity (Fig\u00a03D lanes 5\u201312).", "section": "RESULTS", "ner": [ [ 20, 24, "AF\u20101", "structure_element" ] ] }, { "sid": 100, "sent": "For ligands in cluster 3, we could not eliminate a role for AF\u20101 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig\u00a03C), and deletion of the AB or F domain rarely induced such correlations (Fig\u00a03D), except for A\u2010CD and OBHS\u2010ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E\u2010Luc activity and/or GREB1 levels (Fig\u00a03D lanes 13 and 18).", "section": "RESULTS", "ner": [ [ 60, 64, "AF\u20101", "structure_element" ], [ 183, 194, "deletion of", "experimental_method" ], [ 199, 201, "AB", "structure_element" ], [ 205, 206, "F", "structure_element" ], [ 268, 272, "A\u2010CD", "chemical" ], [ 277, 285, "OBHS\u2010ASC", "chemical" ], [ 301, 312, "deletion of", "experimental_method" ], [ 317, 319, "AB", "structure_element" ], [ 330, 331, "F", "structure_element" ], [ 373, 378, "E\u2010Luc", "experimental_method" ], [ 395, 400, "GREB1", "protein" ] ] }, { "sid": 101, "sent": "Thus, ligands in cluster 2 rely on AF\u20101 for both activity (Fig\u00a03B) and signaling specificity (Fig\u00a03D).", "section": "RESULTS", "ner": [ [ 35, 39, "AF\u20101", "structure_element" ] ] }, { "sid": 102, "sent": "Ligand\u2010specific control of GREB1 expression", "section": "RESULTS", "ner": [ [ 27, 32, "GREB1", "protein" ] ] }, { "sid": 103, "sent": "To determine whether ligand classes control expression of native ER\u03b1 target genes through the canonical linear signaling pathway, we performed pairwise linear regression analyses using ER\u03b1\u2013NCOA1/2/3 interactions in M2H assay as independent predictors of GREB1 expression (the dependent variable) (Figs\u00a0EV1 and EV2A, F\u2013H).", "section": "RESULTS", "ner": [ [ 65, 68, "ER\u03b1", "protein" ], [ 143, 178, "pairwise linear regression analyses", "experimental_method" ], [ 185, 198, "ER\u03b1\u2013NCOA1/2/3", "complex_assembly" ], [ 215, 224, "M2H assay", "experimental_method" ], [ 254, 259, "GREB1", "protein" ] ] }, { "sid": 104, "sent": "In cluster 1, the recruitment of NCOA1 and NCOA2 was highest for WAY\u2010C, followed by triaryl\u2010ethylene, OBHS\u2010N, and OBHS series, while for NCOA3, OBHS\u2010N compounds induced the most recruitment and OBHS ligands were inverse agonists (Fig\u00a0EV2F\u2013H).", "section": "RESULTS", "ner": [ [ 33, 38, "NCOA1", "protein" ], [ 43, 48, "NCOA2", "protein" ], [ 65, 70, "WAY\u2010C", "chemical" ], [ 84, 100, "triaryl\u2010ethylene", "chemical" ], [ 102, 108, "OBHS\u2010N", "chemical" ], [ 114, 118, "OBHS", "chemical" ], [ 137, 142, "NCOA3", "protein" ], [ 144, 150, "OBHS\u2010N", "chemical" ], [ 194, 198, "OBHS", "chemical" ] ] }, { "sid": 105, "sent": "The average induction of GREB1 by cluster 1 ligands showed greater variance, with a range between ~25 and ~75% for OBHS and a range from full agonist to inverse agonist for the others in cluster 1 (Fig\u00a0EV2A).", "section": "RESULTS", "ner": [ [ 25, 30, "GREB1", "protein" ], [ 115, 119, "OBHS", "chemical" ] ] }, { "sid": 106, "sent": "GREB1 levels induced by OBHS analogs were determined by recruitment of NCOA1 but not NCOA2/3 (Fig\u00a03E lane 1), suggesting that there may be alternate or preferential use of these coactivators by different classes.", "section": "RESULTS", "ner": [ [ 0, 5, "GREB1", "protein" ], [ 24, 28, "OBHS", "chemical" ], [ 71, 76, "NCOA1", "protein" ], [ 85, 92, "NCOA2/3", "protein" ] ] }, { "sid": 107, "sent": "However, in cluster 1, NCOA1/2/3 recruitment generally predicted GREB1 levels (Fig\u00a03E lanes 1\u20134), consistent with the canonical signaling model (Fig\u00a01D).", "section": "RESULTS", "ner": [ [ 23, 32, "NCOA1/2/3", "protein" ], [ 65, 70, "GREB1", "protein" ] ] }, { "sid": 108, "sent": "For clusters 2 and 3, GREB1 activity was generally not predicted by NCOA1/2/3 recruitment.", "section": "RESULTS", "ner": [ [ 22, 27, "GREB1", "protein" ], [ 68, 77, "NCOA1/2/3", "protein" ] ] }, { "sid": 109, "sent": "Direct modulators showed low NCOA1/2/3 recruitment (Fig\u00a0EV2F\u2013H), but only OBHS\u2010ASC analogs had NCOA2 recruitment profiles that predicted a full range of effects on GREB1 levels (Figs\u00a03E lanes 9, 11, 18\u201319, and EV2A).", "section": "RESULTS", "ner": [ [ 29, 38, "NCOA1/2/3", "protein" ], [ 74, 82, "OBHS\u2010ASC", "chemical" ], [ 95, 100, "NCOA2", "protein" ], [ 164, 169, "GREB1", "protein" ] ] }, { "sid": 110, "sent": "The indirect modulators in clusters 2 and 3 stimulated NCOA1/2/3 recruitment and GREB1 expression with substantial variance (Figs\u00a03A and EV2F\u2013H).", "section": "RESULTS", "ner": [ [ 55, 64, "NCOA1/2/3", "protein" ], [ 81, 86, "GREB1", "protein" ] ] }, { "sid": 111, "sent": "However, ligand\u2010induced GREB1 levels were generally not determined by NCOA1/2/3 recruitment (Fig\u00a03E lanes 5\u201319), consistent with an alternate causality model (Fig\u00a01E).", "section": "RESULTS", "ner": [ [ 24, 29, "GREB1", "protein" ], [ 70, 79, "NCOA1/2/3", "protein" ] ] }, { "sid": 112, "sent": "Out of 11 indirect modulator series in cluster 2 or 3, only the S\u2010OBHS\u20103 class had NCOA1/2/3 recruitment profiles that predicted GREB1 levels (Fig\u00a03E lane 12).", "section": "RESULTS", "ner": [ [ 64, 72, "S\u2010OBHS\u20103", "chemical" ], [ 83, 92, "NCOA1/2/3", "protein" ], [ 129, 134, "GREB1", "protein" ] ] }, { "sid": 113, "sent": "These results suggest that compounds that show cell\u2010specific signaling do not activate GREB1, or use coactivators other than NCOA1/2/3 to control GREB1 expression (Fig\u00a01E).", "section": "RESULTS", "ner": [ [ 87, 92, "GREB1", "protein" ], [ 125, 134, "NCOA1/2/3", "protein" ], [ 146, 151, "GREB1", "protein" ] ] }, { "sid": 114, "sent": "To determine mechanisms for ligand\u2010dependent control of breast cancer cell proliferation, we performed linear regression analyses across the 19 scaffolds using MCF\u20107 cell proliferation as the dependent variable, and the other activities as independent variables (Fig\u00a03F).", "section": "RESULTS", "ner": [ [ 103, 129, "linear regression analyses", "experimental_method" ] ] }, { "sid": 115, "sent": "In cluster 1, E\u2010Luc and L\u2010Luc activities, NCOA1/2/3 recruitment, and GREB1 levels generally predicted the proliferative response (Fig\u00a03F lanes 2\u20134).", "section": "RESULTS", "ner": [ [ 14, 19, "E\u2010Luc", "experimental_method" ], [ 24, 29, "L\u2010Luc", "experimental_method" ], [ 42, 51, "NCOA1/2/3", "protein" ], [ 69, 74, "GREB1", "protein" ] ] }, { "sid": 116, "sent": "With the OBHS\u2010N compounds, NCOA3 and GREB1 showed near perfect prediction of proliferation (Fig\u00a0EV3G), with unexplained variance similar to the noise in the assays.", "section": "RESULTS", "ner": [ [ 9, 15, "OBHS\u2010N", "chemical" ], [ 27, 32, "NCOA3", "protein" ], [ 37, 42, "GREB1", "protein" ] ] }, { "sid": 117, "sent": "The lack of significant predictors for OBHS analogs (Fig\u00a03F lane 1) reflects their small range of proliferative effects on MCF\u20107 cells (Fig\u00a0EV2I).", "section": "RESULTS", "ner": [ [ 39, 43, "OBHS", "chemical" ] ] }, { "sid": 118, "sent": "The significant correlations with GREB1 expression and NCOA1/2/3 recruitment observed in this cluster are consistent with the canonical signaling model (Fig\u00a01D), where NCOA1/2/3 recruitment determines GREB1 expression, which then drives proliferation.", "section": "RESULTS", "ner": [ [ 34, 39, "GREB1", "protein" ], [ 55, 64, "NCOA1/2/3", "protein" ], [ 168, 177, "NCOA1/2/3", "protein" ], [ 201, 206, "GREB1", "protein" ] ] }, { "sid": 119, "sent": "Despite this phenotypic variance, proliferation was not generally predicted by correlated NCOA1/2/3 recruitment and GREB1 induction (Figs\u00a03F lanes 5\u201319, and EV3H).", "section": "RESULTS", "ner": [ [ 90, 99, "NCOA1/2/3", "protein" ], [ 116, 121, "GREB1", "protein" ] ] }, { "sid": 120, "sent": "Out of 15 ligand series in these clusters, only 2,5\u2010DTP analogs induced a proliferative response that was predicted by GREB1 levels, which were not determined by NCOA1/2/3 recruitment (Fig\u00a03E and F lane 10).", "section": "RESULTS", "ner": [ [ 48, 55, "2,5\u2010DTP", "chemical" ], [ 119, 124, "GREB1", "protein" ], [ 162, 171, "NCOA1/2/3", "protein" ] ] }, { "sid": 121, "sent": "3,4\u2010DTP, cyclofenil, 3,4\u2010DTPD, and imidazopyridine analogs had NCOA1/3 recruitment profiles that predicted their proliferative effects, without determining GREB1 levels (Fig\u00a03E and F, lanes 5 and 14\u201316).", "section": "RESULTS", "ner": [ [ 0, 7, "3,4\u2010DTP", "chemical" ], [ 9, 19, "cyclofenil", "chemical" ], [ 21, 29, "3,4\u2010DTPD", "chemical" ], [ 35, 50, "imidazopyridine", "chemical" ], [ 63, 70, "NCOA1/3", "protein" ], [ 156, 161, "GREB1", "protein" ] ] }, { "sid": 122, "sent": "Similarly, S\u2010OBHS\u20103, cyclofenil\u2010ASC, and OBHS\u2010ASC had positively correlated NCOA1/2/3 recruitment and GREB1 levels, but none of these activities determined their proliferative effects (Fig\u00a03E and F lanes 11\u201312 and 18).", "section": "RESULTS", "ner": [ [ 11, 19, "S\u2010OBHS\u20103", "chemical" ], [ 21, 35, "cyclofenil\u2010ASC", "chemical" ], [ 41, 49, "OBHS\u2010ASC", "chemical" ], [ 76, 85, "NCOA1/2/3", "protein" ], [ 102, 107, "GREB1", "protein" ] ] }, { "sid": 123, "sent": "For ligands that show cell\u2010specific signaling, ER\u03b1\u2010mediated recruitment of other coregulators and activation of other target genes likely determine their proliferative effects on MCF\u20107 cells.", "section": "RESULTS", "ner": [ [ 47, 50, "ER\u03b1", "protein" ] ] }, { "sid": 124, "sent": "NCOA3 occupancy at GREB1 did not predict the proliferative response", "section": "RESULTS", "ner": [ [ 0, 5, "NCOA3", "protein" ], [ 19, 24, "GREB1", "protein" ] ] }, { "sid": 125, "sent": "We also questioned whether promoter occupancy by coactivators is statistically robust and reproducible for ligand class analysis using a chromatin immunoprecipitation (ChIP)\u2010based quantitative assay, and whether it has a better predictive power than the M2H assay.", "section": "RESULTS", "ner": [ [ 137, 199, "chromatin immunoprecipitation (ChIP)\u2010based quantitative assay,", "experimental_method" ], [ 254, 263, "M2H assay", "experimental_method" ] ] }, { "sid": 126, "sent": "ER\u03b1 and NCOA3 cycle on and off the GREB1 promoter (Nwachukwu et\u00a0al, 2014).", "section": "RESULTS", "ner": [ [ 0, 3, "ER\u03b1", "protein" ], [ 8, 13, "NCOA3", "protein" ], [ 35, 40, "GREB1", "protein" ] ] }, { "sid": 127, "sent": "Therefore, we first performed a time\u2010course study, and found that E2 and the WAY\u2010C analog, AAPII\u2010151\u20104, induced recruitment of NCOA3 to the GREB1 promoter in a temporal cycle that peaked after 45\u00a0min in MCF\u20107 cells (Fig\u00a04A).", "section": "RESULTS", "ner": [ [ 32, 49, "time\u2010course study", "experimental_method" ], [ 66, 68, "E2", "chemical" ], [ 77, 82, "WAY\u2010C", "chemical" ], [ 91, 102, "AAPII\u2010151\u20104", "chemical" ], [ 127, 132, "NCOA3", "protein" ], [ 140, 145, "GREB1", "protein" ] ] }, { "sid": 128, "sent": "At this time point, other WAY\u2010C analogs also induced recruitment of NCOA3 at this site to varying degrees (Fig\u00a04B).", "section": "RESULTS", "ner": [ [ 26, 31, "WAY\u2010C", "chemical" ], [ 68, 73, "NCOA3", "protein" ] ] }, { "sid": 129, "sent": "The Z\u2019 for this assay was 0.6, showing statistical robustness (see Materials and Methods).", "section": "RESULTS", "ner": [ [ 4, 6, "Z\u2019", "evidence" ] ] }, { "sid": 130, "sent": "We prepared biological replicates with different cell passage numbers and separately prepared samples, which showed r 2 of 0.81, demonstrating high reproducibility (Fig\u00a04C).", "section": "RESULTS", "ner": [ [ 116, 119, "r 2", "evidence" ] ] }, { "sid": 131, "sent": " NCOA3 occupancy at GREB1 is statistically robust but does not predict transcriptional activity", "section": "FIG", "ner": [ [ 1, 6, "NCOA3", "protein" ], [ 20, 25, "GREB1", "protein" ] ] }, { "sid": 132, "sent": "Kinetic ChIP assay examining recruitment of NCOA3 to the GREB1 gene in MCF\u20107 cells stimulated with E2 or the indicated WAY\u2010C analog.", "section": "FIG", "ner": [ [ 0, 18, "Kinetic ChIP assay", "experimental_method" ], [ 44, 49, "NCOA3", "protein" ], [ 57, 62, "GREB1", "protein" ], [ 99, 101, "E2", "chemical" ], [ 119, 124, "WAY\u2010C", "chemical" ] ] }, { "sid": 133, "sent": "NCOA3 occupancy at GREB1 was compared by ChIP assay 45\u00a0min after stimulation with vehicle, E2, or the WAY\u2010C analogs.", "section": "FIG", "ner": [ [ 0, 5, "NCOA3", "protein" ], [ 19, 24, "GREB1", "protein" ], [ 41, 51, "ChIP assay", "experimental_method" ], [ 91, 93, "E2", "chemical" ], [ 102, 107, "WAY\u2010C", "chemical" ] ] }, { "sid": 134, "sent": "In panel (B), the average recruitment of two biological replicates are shown as mean\u00a0+\u00a0SEM, and the Z\u2010score is indicated.", "section": "FIG", "ner": [ [ 100, 107, "Z\u2010score", "evidence" ] ] }, { "sid": 135, "sent": "In panel (C), correlation analysis was performed for two biological replicates.", "section": "FIG", "ner": [ [ 14, 34, "correlation analysis", "experimental_method" ] ] }, { "sid": 136, "sent": "Linear regression analyses comparing the ability of NCOA3 recruitment, measured by ChIP or M2H, to predict other agonist activities of WAY\u2010C analogs. *Significant positive correlation (F\u2010test for nonzero slope, P\u2010value).", "section": "FIG", "ner": [ [ 0, 26, "Linear regression analyses", "experimental_method" ], [ 52, 57, "NCOA3", "protein" ], [ 83, 87, "ChIP", "experimental_method" ], [ 91, 94, "M2H", "experimental_method" ], [ 135, 140, "WAY\u2010C", "chemical" ], [ 185, 191, "F\u2010test", "experimental_method" ], [ 211, 218, "P\u2010value", "evidence" ] ] }, { "sid": 137, "sent": "The M2H assay for NCOA3 recruitment broadly correlated with the other assays, and was predictive for GREB1 expression and cell proliferation (Fig\u00a03E).", "section": "RESULTS", "ner": [ [ 4, 13, "M2H assay", "experimental_method" ], [ 18, 23, "NCOA3", "protein" ], [ 101, 106, "GREB1", "protein" ] ] }, { "sid": 138, "sent": "However, the ChIP assays for WAY\u2010C\u2010induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY\u2010C activity profiles (Fig\u00a04D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2\u00a0=\u00a00.36 and P\u00a0=\u00a00.09 (F\u2010test for nonzero slope).", "section": "RESULTS", "ner": [ [ 13, 24, "ChIP assays", "experimental_method" ], [ 29, 34, "WAY\u2010C", "chemical" ], [ 58, 63, "NCOA3", "protein" ], [ 71, 76, "GREB1", "protein" ], [ 126, 131, "WAY\u2010C", "chemical" ], [ 202, 213, "ChIP assays", "experimental_method" ], [ 218, 223, "NCOA3", "protein" ], [ 240, 249, "M2H assay", "experimental_method" ], [ 290, 293, "r 2", "evidence" ], [ 305, 306, "P", "evidence" ], [ 315, 321, "F\u2010test", "experimental_method" ] ] }, { "sid": 139, "sent": "Thus, the simplified coactivator\u2010binding assay showed much greater predictive power than the ChIP assay for ligand\u2010specific effects on GREB1 expression and cell proliferation.", "section": "RESULTS", "ner": [ [ 21, 46, "coactivator\u2010binding assay", "experimental_method" ], [ 93, 103, "ChIP assay", "experimental_method" ], [ 135, 140, "GREB1", "protein" ] ] }, { "sid": 140, "sent": "ER\u03b2 activity is not an independent predictor of cell\u2010specific activity", "section": "RESULTS", "ner": [ [ 0, 3, "ER\u03b2", "protein" ] ] }, { "sid": 141, "sent": "One difference between MCF\u20107 breast cancer cells and Ishikawa endometrial cancer cells is the contribution of ER\u03b2 to estrogenic response, as Ishikawa cells may express ER\u03b2 (Bhat & Pezzuto, 2001).", "section": "RESULTS", "ner": [ [ 110, 113, "ER\u03b2", "protein" ], [ 168, 171, "ER\u03b2", "protein" ] ] }, { "sid": 142, "sent": "When overexpressed in MCF\u20107 cells, ER\u03b2 alters E2\u2010induced expression of only a subset of ER\u03b1\u2010target genes (Wu et\u00a0al, 2011), raising the possibility that ligand\u2010induced ER\u03b2 activity may contribute to E\u2010Luc activities, and thus underlie the lack of correlation between the E\u2010Luc and L\u2010Luc ER\u03b1\u2010WT activities or GREB1 levels induced by cell\u2010specific modulators in cluster 2 and cluster 3 (Fig\u00a03C).", "section": "RESULTS", "ner": [ [ 5, 18, "overexpressed", "experimental_method" ], [ 35, 38, "ER\u03b2", "protein" ], [ 46, 48, "E2", "chemical" ], [ 88, 91, "ER\u03b1", "protein" ], [ 167, 170, "ER\u03b2", "protein" ], [ 198, 203, "E\u2010Luc", "experimental_method" ], [ 270, 275, "E\u2010Luc", "experimental_method" ], [ 280, 285, "L\u2010Luc", "experimental_method" ], [ 286, 289, "ER\u03b1", "protein" ], [ 290, 292, "WT", "protein_state" ], [ 307, 312, "GREB1", "protein" ] ] }, { "sid": 143, "sent": "To test this idea, we determined the L\u2010Luc ER\u03b2 activity profiles of the ligands (Fig\u00a0EV1).", "section": "RESULTS", "ner": [ [ 37, 42, "L\u2010Luc", "experimental_method" ] ] }, { "sid": 144, "sent": "All direct modulator and two indirect modulator scaffolds (OBHS and S\u2010OBHS\u20103) lacked ER\u03b2 agonist activity.", "section": "RESULTS", "ner": [ [ 59, 63, "OBHS", "chemical" ], [ 68, 76, "S\u2010OBHS\u20103", "chemical" ] ] }, { "sid": 145, "sent": "For most scaffolds, L\u2010Luc ER\u03b2 and E\u2010Luc activities were not correlated, except for 2,5\u2010DTP and cyclofenil analogs, which showed moderate but significant correlations (Fig\u00a0EV4A).", "section": "RESULTS", "ner": [ [ 20, 25, "L\u2010Luc", "experimental_method" ], [ 34, 39, "E\u2010Luc", "experimental_method" ], [ 83, 90, "2,5\u2010DTP", "chemical" ], [ 95, 105, "cyclofenil", "chemical" ] ] }, { "sid": 146, "sent": "Nevertheless, the E\u2010Luc activities of both 2,5\u2010DTP and cyclofenil analogs were better predicted by their L\u2010Luc ER\u03b1\u2010WT than L\u2010Luc ER\u03b2 activities (Fig\u00a0EV4A and B).", "section": "RESULTS", "ner": [ [ 18, 23, "E\u2010Luc", "experimental_method" ], [ 43, 50, "2,5\u2010DTP", "chemical" ], [ 55, 65, "cyclofenil", "chemical" ], [ 105, 110, "L\u2010Luc", "experimental_method" ], [ 111, 114, "ER\u03b1", "protein" ], [ 115, 117, "WT", "protein_state" ], [ 123, 128, "L\u2010Luc", "experimental_method" ], [ 129, 132, "ER\u03b2", "protein" ] ] }, { "sid": 147, "sent": "ER\u03b2 activity is not an independent predictor of E\u2010Luc activity", "section": "FIG", "ner": [ [ 0, 3, "ER\u03b2", "protein" ], [ 48, 53, "E\u2010Luc", "experimental_method" ] ] }, { "sid": 148, "sent": "ER\u03b2 activity in HepG2 cells rarely correlates with E\u2010Luc activity.", "section": "FIG", "ner": [ [ 0, 3, "ER\u03b2", "protein" ], [ 51, 56, "E\u2010Luc", "experimental_method" ] ] }, { "sid": 149, "sent": "ER\u03b1 activity of 2,5\u2010DTP and cyclofenil analogs correlates with E\u2010Luc activity.", "section": "FIG", "ner": [ [ 0, 3, "ER\u03b1", "protein" ], [ 16, 23, "2,5\u2010DTP", "chemical" ], [ 28, 38, "cyclofenil", "chemical" ], [ 63, 68, "E\u2010Luc", "experimental_method" ] ] }, { "sid": 150, "sent": " Data information: The r 2 and P values for the indicated correlations are shown in both panels. *Significant positive correlation (F\u2010test for nonzero slope, P\u2010value)", "section": "FIG", "ner": [ [ 31, 39, "P values", "evidence" ], [ 132, 138, "F\u2010test", "experimental_method" ], [ 158, 165, "P\u2010value", "evidence" ] ] }, { "sid": 151, "sent": "To overcome barriers to crystallization of ER\u03b1 LBD complexes, we developed a conformation\u2010trapping X\u2010ray crystallography approach using the ER\u03b1\u2010Y537S mutation (Nettles et\u00a0al, 2008; Bruning et\u00a0al, 2010; Srinivasan et\u00a0al, 2013).", "section": "RESULTS", "ner": [ [ 24, 39, "crystallization", "experimental_method" ], [ 43, 46, "ER\u03b1", "protein" ], [ 47, 50, "LBD", "structure_element" ], [ 77, 120, "conformation\u2010trapping X\u2010ray crystallography", "experimental_method" ], [ 140, 149, "ER\u03b1\u2010Y537S", "mutant" ] ] }, { "sid": 152, "sent": "To further validate this approach, we solved the structure of the ER\u03b1\u2010Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild\u2010type and ER\u03b1\u2010Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix\u00a0Fig S1A and B) (Nettles et\u00a0al, 2008; Bruning et\u00a0al, 2010; Delfosse et\u00a0al, 2012).", "section": "RESULTS", "ner": [ [ 38, 44, "solved", "experimental_method" ], [ 49, 58, "structure", "evidence" ], [ 66, 75, "ER\u03b1\u2010Y537S", "mutant" ], [ 76, 79, "LBD", "structure_element" ], [ 80, 95, "in complex with", "protein_state" ], [ 96, 114, "diethylstilbestrol", "chemical" ], [ 116, 119, "DES", "chemical" ], [ 153, 162, "wild\u2010type", "protein_state" ], [ 167, 176, "ER\u03b1\u2010Y537S", "mutant" ], [ 177, 181, "LBDs", "structure_element" ], [ 241, 244, "h12", "structure_element" ] ] }, { "sid": 153, "sent": "Using this approach, we solved 76 ER\u03b1 LBD structures in the active conformation and bound to ligands studied here (Appendix\u00a0Fig S1C).", "section": "RESULTS", "ner": [ [ 24, 30, "solved", "experimental_method" ], [ 34, 37, "ER\u03b1", "protein" ], [ 38, 41, "LBD", "structure_element" ], [ 42, 52, "structures", "evidence" ], [ 60, 79, "active conformation", "protein_state" ], [ 84, 100, "bound to ligands", "protein_state" ] ] }, { "sid": 154, "sent": "Eleven of these structures have been published, while 65 are new, including the DES\u2010bound ER\u03b1\u2010Y537S LBD.", "section": "RESULTS", "ner": [ [ 16, 26, "structures", "evidence" ], [ 80, 89, "DES\u2010bound", "protein_state" ], [ 90, 99, "ER\u03b1\u2010Y537S", "mutant" ], [ 100, 103, "LBD", "structure_element" ] ] }, { "sid": 155, "sent": "We present 57 of these new structures here (Dataset EV2), while the remaining eight new structures bound to OBHS\u2010N analogs will be published elsewhere (S. Srinivasan et al, in preparation).", "section": "RESULTS", "ner": [ [ 27, 37, "structures", "evidence" ], [ 88, 98, "structures", "evidence" ], [ 99, 107, "bound to", "protein_state" ], [ 108, 114, "OBHS\u2010N", "chemical" ] ] }, { "sid": 156, "sent": "Examining many closely related structures allows us to visualize subtle structural differences, in effect using X\u2010ray crystallography as a systems biology tool.", "section": "RESULTS", "ner": [ [ 31, 41, "structures", "evidence" ], [ 112, 133, "X\u2010ray crystallography", "experimental_method" ] ] }, { "sid": 157, "sent": "The indirect modulator scaffolds in cluster 1 did not show cell\u2010specific signaling (Fig\u00a03C), but shared common structural perturbations that we designed to modulate h12 dynamics.", "section": "RESULTS", "ner": [ [ 165, 168, "h12", "structure_element" ] ] }, { "sid": 158, "sent": "Based on our original OBHS structure, the OBHS, OBHS\u2010N, and triaryl\u2010ethylene compounds were modified with h11\u2010directed pendant groups (Zheng et\u00a0al, 2012; Zhu et\u00a0al, 2012; Liao et\u00a0al, 2014).", "section": "RESULTS", "ner": [ [ 22, 26, "OBHS", "chemical" ], [ 27, 36, "structure", "evidence" ], [ 42, 46, "OBHS", "chemical" ], [ 48, 54, "OBHS\u2010N", "chemical" ], [ 60, 76, "triaryl\u2010ethylene", "chemical" ], [ 106, 109, "h11", "structure_element" ] ] }, { "sid": 159, "sent": "Superposing the LBDs based on the class of bound ligands provides an ensemble view of the structural variance and clarifies what part of the ligand\u2010binding pocket is differentially perturbed or targeted.", "section": "RESULTS", "ner": [ [ 0, 11, "Superposing", "experimental_method" ], [ 16, 20, "LBDs", "structure_element" ], [ 141, 162, "ligand\u2010binding pocket", "site" ] ] }, { "sid": 160, "sent": "The 24 structures containing OBHS, OBHS\u2010N, or triaryl\u2010ethylene analogs showed structural diversity in the same part of the scaffolds (Figs\u00a05A and EV5A), and the same region of the LBD\u2014the C\u2010terminal end of h11 (Figs\u00a05B and C, and EV5B), which in turn nudges h12 (Fig\u00a05C and D).", "section": "RESULTS", "ner": [ [ 7, 17, "structures", "evidence" ], [ 29, 33, "OBHS", "chemical" ], [ 35, 41, "OBHS\u2010N", "chemical" ], [ 46, 62, "triaryl\u2010ethylene", "chemical" ], [ 180, 183, "LBD", "structure_element" ], [ 206, 209, "h11", "structure_element" ], [ 258, 261, "h12", "structure_element" ] ] }, { "sid": 161, "sent": "We observed that the OBHS\u2010N analogs displaced h11 along a vector away from Leu354 in a region of h3 that is unaffected by the ligands, and toward the dimer interface.", "section": "RESULTS", "ner": [ [ 21, 27, "OBHS\u2010N", "chemical" ], [ 46, 49, "h11", "structure_element" ], [ 75, 81, "Leu354", "residue_name_number" ], [ 97, 99, "h3", "structure_element" ], [ 150, 165, "dimer interface", "site" ] ] }, { "sid": 162, "sent": "For the triaryl\u2010ethylene analogs, the displacement of h11 was in a perpendicular direction, away from Ile424 in h8 and toward h12.", "section": "RESULTS", "ner": [ [ 8, 24, "triaryl\u2010ethylene", "chemical" ], [ 54, 57, "h11", "structure_element" ], [ 102, 108, "Ile424", "residue_name_number" ], [ 112, 114, "h8", "structure_element" ], [ 126, 129, "h12", "structure_element" ] ] }, { "sid": 163, "sent": "Remarkably, these individual inter\u2010atomic distances showed a ligand class\u2010specific ability to significantly predict proliferative effects (Fig\u00a05E and F), demonstrating the feasibility of developing a minimal set of activity predictors from crystal structures.", "section": "RESULTS", "ner": [ [ 29, 51, "inter\u2010atomic distances", "evidence" ], [ 240, 258, "crystal structures", "evidence" ] ] }, { "sid": 164, "sent": "Structure\u2010class analysis of triaryl\u2010ethylene analogs.", "section": "FIG", "ner": [ [ 0, 24, "Structure\u2010class analysis", "experimental_method" ], [ 28, 44, "triaryl\u2010ethylene", "chemical" ] ] }, { "sid": 165, "sent": "Triaryl\u2010ethylene analogs bound to the superposed crystal structures of the ER\u03b1 LBD are shown.", "section": "FIG", "ner": [ [ 0, 16, "Triaryl\u2010ethylene", "chemical" ], [ 25, 33, "bound to", "protein_state" ], [ 38, 48, "superposed", "experimental_method" ], [ 49, 67, "crystal structures", "evidence" ], [ 75, 78, "ER\u03b1", "protein" ], [ 79, 82, "LBD", "structure_element" ] ] }, { "sid": 166, "sent": "Arrows indicate chemical variance in the orientation of the different h11\u2010directed ligand side groups (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF and 5DP0).", "section": "FIG", "ner": [ [ 70, 73, "h11", "structure_element" ] ] }, { "sid": 167, "sent": "Triaryl\u2010ethylene analogs induce variance of ER\u03b1 conformations at the C\u2010terminal region of h11.", "section": "FIG", "ner": [ [ 0, 16, "Triaryl\u2010ethylene", "chemical" ], [ 44, 47, "ER\u03b1", "protein" ], [ 90, 93, "h11", "structure_element" ] ] }, { "sid": 168, "sent": "Panel\u00a0(B) shows the crystal structure of a triaryl\u2010ethylene analog\u2010bound ER\u03b1 LBD (PDB 5DLR).", "section": "FIG", "ner": [ [ 20, 37, "crystal structure", "evidence" ], [ 43, 59, "triaryl\u2010ethylene", "chemical" ], [ 73, 76, "ER\u03b1", "protein" ], [ 77, 80, "LBD", "structure_element" ] ] }, { "sid": 169, "sent": "The h11\u2013h12 interface (circled) includes the C\u2010terminal part of h11.", "section": "FIG", "ner": [ [ 4, 21, "h11\u2013h12 interface", "site" ], [ 64, 67, "h11", "structure_element" ] ] }, { "sid": 170, "sent": "This region was expanded in panel (C), where the 10 triaryl\u2010ethylene analog\u2010bound ER\u03b1 LBD structures (see Datasets EV1 and EV2) were superposed to show variations in the h11 C\u2010terminus (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF, and 5DP0).", "section": "FIG", "ner": [ [ 52, 68, "triaryl\u2010ethylene", "chemical" ], [ 82, 85, "ER\u03b1", "protein" ], [ 86, 89, "LBD", "structure_element" ], [ 90, 100, "structures", "evidence" ], [ 133, 143, "superposed", "experimental_method" ], [ 170, 173, "h11", "structure_element" ] ] }, { "sid": 171, "sent": "ER\u03b1 LBDs in complex with diethylstilbestrol (DES) or a triaryl\u2010ethylene analog were superposed to show that the ligand\u2010induced difference in h11 conformation is transmitted to the C\u2010terminus of h12 (PDB 4ZN7, 5DMC).", "section": "FIG", "ner": [ [ 0, 3, "ER\u03b1", "protein" ], [ 4, 8, "LBDs", "structure_element" ], [ 9, 24, "in complex with", "protein_state" ], [ 25, 43, "diethylstilbestrol", "chemical" ], [ 45, 48, "DES", "chemical" ], [ 55, 71, "triaryl\u2010ethylene", "chemical" ], [ 84, 94, "superposed", "experimental_method" ], [ 141, 144, "h11", "structure_element" ], [ 194, 197, "h12", "structure_element" ] ] }, { "sid": 172, "sent": "Inter\u2010atomic distances predict the proliferative effects of specific ligand series.", "section": "FIG", "ner": [ [ 0, 22, "Inter\u2010atomic distances", "evidence" ] ] }, { "sid": 173, "sent": "Ile424\u2013His524 distance measured in the crystal structures correlates with the proliferative effect of triaryl\u2010ethylene analogs in MCF\u20107 cells.", "section": "FIG", "ner": [ [ 0, 6, "Ile424", "residue_name_number" ], [ 7, 13, "His524", "residue_name_number" ], [ 14, 22, "distance", "evidence" ], [ 39, 57, "crystal structures", "evidence" ], [ 102, 118, "triaryl\u2010ethylene", "chemical" ] ] }, { "sid": 174, "sent": "In contrast, the Leu354\u2013Leu525 distance correlates with the proliferative effects of OBHS\u2010N analogs in MCF\u20107 cells.", "section": "FIG", "ner": [ [ 17, 23, "Leu354", "residue_name_number" ], [ 24, 30, "Leu525", "residue_name_number" ], [ 31, 39, "distance", "evidence" ], [ 85, 91, "OBHS\u2010N", "chemical" ] ] }, { "sid": 175, "sent": "Structure\u2010class analysis of WAY\u2010C analogs.", "section": "FIG", "ner": [ [ 0, 24, "Structure\u2010class analysis", "experimental_method" ], [ 28, 33, "WAY\u2010C", "chemical" ] ] }, { "sid": 176, "sent": "WAY\u2010C side groups subtly nudge h12 Leu540.", "section": "FIG", "ner": [ [ 0, 5, "WAY\u2010C", "chemical" ], [ 31, 34, "h12", "structure_element" ], [ 35, 41, "Leu540", "residue_name_number" ] ] }, { "sid": 177, "sent": "ER\u03b1 LBD structures bound to 4 distinct WAY\u2010C analogs were superposed (PDB 4\u00a0IU7, 4IV4, 4IVW, 4IW6) (see Datasets EV1 and EV2).", "section": "FIG", "ner": [ [ 0, 3, "ER\u03b1", "protein" ], [ 4, 7, "LBD", "structure_element" ], [ 8, 18, "structures", "evidence" ], [ 19, 27, "bound to", "protein_state" ], [ 39, 44, "WAY\u2010C", "chemical" ], [ 58, 68, "superposed", "experimental_method" ] ] }, { "sid": 178, "sent": "Structure\u2010class analysis of indirect modulators", "section": "FIG", "ner": [ [ 0, 24, "Structure\u2010class analysis", "experimental_method" ] ] }, { "sid": 179, "sent": "Structure\u2010class analysis of indirect modulators in cluster 1.", "section": "FIG", "ner": [ [ 0, 24, "Structure\u2010class analysis", "experimental_method" ] ] }, { "sid": 180, "sent": "Crystal structures of the ER\u03b1 LBD bound to OBHS and OBHS\u2010N analogs were superposed.", "section": "FIG", "ner": [ [ 0, 18, "Crystal structures", "evidence" ], [ 26, 29, "ER\u03b1", "protein" ], [ 30, 33, "LBD", "structure_element" ], [ 34, 42, "bound to", "protein_state" ], [ 43, 47, "OBHS", "chemical" ], [ 52, 58, "OBHS\u2010N", "chemical" ], [ 72, 82, "superposed", "experimental_method" ] ] }, { "sid": 181, "sent": "Arrows indicate chemical variance in the orientation of the different h11\u2010directed ligand side groups.", "section": "FIG", "ner": [ [ 70, 73, "h11", "structure_element" ] ] }, { "sid": 182, "sent": "Panel\u00a0(B) shows the ligand\u2010induced conformational variation at the C\u2010terminal region of h11 (OBHS: PDB 4ZN9, 4ZNH, 4ZNS, 4ZNT, 4ZNU, 4ZNV, and 4ZNW; OBHS\u2010N: PDB 4ZUB, 4ZUC, 4ZWH, 4ZWK, 5BNU, 5BP6, 5BPR, and 5BQ4).", "section": "FIG", "ner": [ [ 88, 91, "h11", "structure_element" ], [ 93, 97, "OBHS", "chemical" ], [ 149, 155, "OBHS\u2010N", "chemical" ] ] }, { "sid": 183, "sent": "Structure\u2010class analysis of indirect modulators in clusters 2 and 3.", "section": "FIG", "ner": [ [ 0, 24, "Structure\u2010class analysis", "experimental_method" ] ] }, { "sid": 184, "sent": "Crystal structures of the ER\u03b1 LBD bound to ligands with cell\u2010specific activities were superposed.", "section": "FIG", "ner": [ [ 0, 18, "Crystal structures", "evidence" ], [ 26, 29, "ER\u03b1", "protein" ], [ 30, 33, "LBD", "structure_element" ], [ 34, 42, "bound to", "protein_state" ], [ 86, 96, "superposed", "experimental_method" ] ] }, { "sid": 185, "sent": "The bound ligands are shown, and arrows indicate considerable variation in the orientation of the different h3\u2010, h8\u2010, h11\u2010, or h12\u2010directed ligand side groups.", "section": "FIG", "ner": [ [ 108, 110, "h3", "structure_element" ], [ 113, 115, "h8", "structure_element" ], [ 118, 121, "h11", "structure_element" ], [ 127, 130, "h12", "structure_element" ] ] }, { "sid": 186, "sent": "As visualized in four LBD structures (Srinivasan et\u00a0al, 2013), WAY\u2010C analogs were designed with small substitutions that slightly nudge h12 Leu540, without exiting the ligand\u2010binding pocket (Fig\u00a05G and H).", "section": "RESULTS", "ner": [ [ 22, 25, "LBD", "structure_element" ], [ 26, 36, "structures", "evidence" ], [ 63, 68, "WAY\u2010C", "chemical" ], [ 136, 139, "h12", "structure_element" ], [ 140, 146, "Leu540", "residue_name_number" ], [ 168, 189, "ligand\u2010binding pocket", "site" ] ] }, { "sid": 187, "sent": "Therefore, changing h12 dynamics maintains the canonical signaling pathway defined by E2 (Fig\u00a01D) to support AF\u20102\u2010driven signaling and recruit NCOA1/2/3 for GREB1\u2010stimulated proliferation.", "section": "RESULTS", "ner": [ [ 20, 23, "h12", "structure_element" ], [ 86, 88, "E2", "chemical" ], [ 109, 113, "AF\u20102", "structure_element" ], [ 143, 152, "NCOA1/2/3", "protein" ], [ 157, 162, "GREB1", "protein" ] ] }, { "sid": 188, "sent": "Ligands with cell\u2010specific activity alter the shape of the AF\u20102 surface", "section": "RESULTS", "ner": [ [ 59, 71, "AF\u20102 surface", "site" ] ] }, { "sid": 189, "sent": "Direct modulators like tamoxifen drive AF\u20101\u2010dependent cell\u2010specific activity by completely occluding AF\u20102, but it is not known how indirect modulators produce cell\u2010specific ER\u03b1 activity.", "section": "RESULTS", "ner": [ [ 23, 32, "tamoxifen", "chemical" ], [ 39, 43, "AF\u20101", "structure_element" ], [ 101, 105, "AF\u20102", "structure_element" ], [ 173, 176, "ER\u03b1", "protein" ] ] }, { "sid": 190, "sent": "Therefore, we examined another 50 LBD structures containing ligands in clusters 2 and 3.", "section": "RESULTS", "ner": [ [ 34, 37, "LBD", "structure_element" ], [ 38, 48, "structures", "evidence" ] ] }, { "sid": 191, "sent": "These structures demonstrated that cell\u2010specific activity derived from altering the shape of the AF\u20102 surface without an extended side chain.", "section": "RESULTS", "ner": [ [ 6, 16, "structures", "evidence" ], [ 97, 109, "AF\u20102 surface", "site" ] ] }, { "sid": 192, "sent": "Ligands in cluster 2 and cluster 3 showed conformational heterogeneity in parts of the scaffold that were directed toward multiple regions of the receptor including h3, h8, h11, h12, and/or the \u03b2\u2010sheets (Fig\u00a0EV5C\u2013G).", "section": "RESULTS", "ner": [ [ 165, 167, "h3", "structure_element" ], [ 169, 171, "h8", "structure_element" ], [ 173, 176, "h11", "structure_element" ], [ 178, 181, "h12", "structure_element" ], [ 194, 202, "\u03b2\u2010sheets", "structure_element" ] ] }, { "sid": 193, "sent": "For instance, S\u2010OBHS\u20102 and S\u2010OBHS\u20103 analogs (Fig\u00a02) had similar ER\u03b1 activity profiles in the different cell types (Fig\u00a0EV2A\u2013C), but the 2\u2010 versus 3\u2010methyl substituted phenol rings altered the correlated signaling patterns in different cell types (Fig\u00a03B lanes 7 and 12).", "section": "RESULTS", "ner": [ [ 14, 22, "S\u2010OBHS\u20102", "chemical" ], [ 27, 35, "S\u2010OBHS\u20103", "chemical" ], [ 64, 67, "ER\u03b1", "protein" ] ] }, { "sid": 194, "sent": "This difference in ligand positioning altered the AF\u20102 surface via a shift in the N\u2010terminus of h12, which directly contacts the coactivator.", "section": "RESULTS", "ner": [ [ 50, 62, "AF\u20102 surface", "site" ], [ 96, 99, "h12", "structure_element" ] ] }, { "sid": 195, "sent": "This effect is evident in a single structure due to its 1 \u00c5 magnitude (Fig\u00a06A and B).", "section": "RESULTS", "ner": [ [ 35, 44, "structure", "evidence" ] ] }, { "sid": 196, "sent": "The shifts in h12 residues Asp538 and Leu539 led to rotation of the coactivator peptide (Fig\u00a06C).", "section": "RESULTS", "ner": [ [ 14, 17, "h12", "structure_element" ], [ 27, 33, "Asp538", "residue_name_number" ], [ 38, 44, "Leu539", "residue_name_number" ] ] }, { "sid": 197, "sent": "Thus, cell\u2010specific activity can stem from perturbation of the AF\u20102 surface without an\u00a0extended side chain, which presumably alters the receptor\u2013coregulator interaction profile.", "section": "RESULTS", "ner": [ [ 63, 75, "AF\u20102 surface", "site" ] ] }, { "sid": 198, "sent": "S\u2010OBHS\u20102/3 analogs subtly distort the AF\u20102 surface.", "section": "FIG", "ner": [ [ 0, 10, "S\u2010OBHS\u20102/3", "chemical" ], [ 38, 50, "AF\u20102 surface", "site" ] ] }, { "sid": 199, "sent": "Panel\u00a0(A) shows the crystal structure of an S\u2010OBHS\u20103\u2010bound ER\u03b1 LBD (PDB 5DUH).", "section": "FIG", "ner": [ [ 20, 37, "crystal structure", "evidence" ], [ 44, 58, "S\u2010OBHS\u20103\u2010bound", "protein_state" ], [ 59, 62, "ER\u03b1", "protein" ], [ 63, 66, "LBD", "structure_element" ] ] }, { "sid": 200, "sent": "The h3\u2013h12 interface (circled) at AF\u20102 (pink) was expanded in panels (B, C).", "section": "FIG", "ner": [ [ 4, 20, "h3\u2013h12 interface", "site" ], [ 34, 38, "AF\u20102", "structure_element" ] ] }, { "sid": 201, "sent": "The S\u2010OBHS\u20102/3\u2010bound ER\u03b1 LBDs were superposed to show shifts in h3 (panel B) and the NCOA2 peptide docked at the AF\u20102 surface (panel C).", "section": "FIG", "ner": [ [ 4, 20, "S\u2010OBHS\u20102/3\u2010bound", "protein_state" ], [ 21, 24, "ER\u03b1", "protein" ], [ 25, 29, "LBDs", "structure_element" ], [ 35, 45, "superposed", "experimental_method" ], [ 64, 66, "h3", "structure_element" ], [ 85, 90, "NCOA2", "protein" ], [ 113, 125, "AF\u20102 surface", "site" ] ] }, { "sid": 202, "sent": "Crystal structures show that 2,5\u2010DTP analogs shift h3 and h11 further apart compared to an A\u2010CD\u2010ring estrogen (PDB 4PPS, 5DRM, 5DRJ).", "section": "FIG", "ner": [ [ 0, 18, "Crystal structures", "evidence" ], [ 29, 36, "2,5\u2010DTP", "chemical" ], [ 51, 53, "h3", "structure_element" ], [ 58, 61, "h11", "structure_element" ], [ 101, 109, "estrogen", "chemical" ] ] }, { "sid": 203, "sent": "The 2F o\u2010F c electron density map and F o\u2010F c difference map of a 2,5\u2010DTP\u2010bound structure (PDB 5DRJ) were contoured at 1.0 sigma and \u00b1\u00a03.0 sigma, respectively.", "section": "FIG", "ner": [ [ 66, 79, "2,5\u2010DTP\u2010bound", "protein_state" ], [ 80, 89, "structure", "evidence" ] ] }, { "sid": 204, "sent": "Average (mean\u00a0+\u00a0SEM) \u03b1\u2010carbon distance measured from h3 Thr347 to h11 Leu525 of A\u2010CD\u2010, 2,5\u2010DTP\u2010, and 3,4\u2010DTPD\u2010bound ER\u03b1 LBDs.", "section": "FIG", "ner": [ [ 21, 38, "\u03b1\u2010carbon distance", "evidence" ], [ 53, 55, "h3", "structure_element" ], [ 56, 62, "Thr347", "residue_name_number" ], [ 66, 69, "h11", "structure_element" ], [ 70, 76, "Leu525", "residue_name_number" ], [ 80, 115, "A\u2010CD\u2010, 2,5\u2010DTP\u2010, and 3,4\u2010DTPD\u2010bound", "protein_state" ], [ 116, 119, "ER\u03b1", "protein" ], [ 120, 124, "LBDs", "structure_element" ] ] }, { "sid": 205, "sent": "*Two\u2010tailed Student's t\u2010test, P\u00a0=\u00a00.002 (PDB A\u2010CD: 5DI7, 5DID, 5DIE, 5DIG, and 4PPS; 2,5\u2010DTP: 4IWC, 5DRM, and 5DRJ; 3,4\u2010DTPD: 5DTV and 5DU5).", "section": "FIG", "ner": [ [ 12, 28, "Student's t\u2010test", "experimental_method" ], [ 30, 31, "P", "evidence" ], [ 45, 49, "A\u2010CD", "chemical" ], [ 85, 92, "2,5\u2010DTP", "chemical" ], [ 116, 124, "3,4\u2010DTPD", "chemical" ] ] }, { "sid": 206, "sent": "Crystal structures show that a 3,4\u2010DTPD analog shifts h3 (F) and the NCOA2 (G) peptide compared to an A\u2010CD\u2010ring estrogen (PDB 4PPS, 5DTV).", "section": "FIG", "ner": [ [ 0, 18, "Crystal structures", "evidence" ], [ 31, 39, "3,4\u2010DTPD", "chemical" ], [ 54, 56, "h3", "structure_element" ], [ 58, 59, "F", "structure_element" ], [ 69, 74, "NCOA2", "protein" ], [ 102, 106, "A\u2010CD", "chemical" ], [ 112, 120, "estrogen", "chemical" ] ] }, { "sid": 207, "sent": "Hierarchical clustering of ligand\u2010specific binding of 154 interacting peptides to the ER\u03b1 LBD was performed in triplicate by MARCoNI analysis.", "section": "FIG", "ner": [ [ 0, 23, "Hierarchical clustering", "experimental_method" ], [ 86, 89, "ER\u03b1", "protein" ], [ 90, 93, "LBD", "structure_element" ], [ 125, 141, "MARCoNI analysis", "experimental_method" ] ] }, { "sid": 208, "sent": "The 2,5\u2010DTP analogs showed perturbation of h11, as well as h3, which forms part of the AF\u20102 surface.", "section": "RESULTS", "ner": [ [ 4, 11, "2,5\u2010DTP", "chemical" ], [ 43, 46, "h11", "structure_element" ], [ 59, 61, "h3", "structure_element" ], [ 87, 99, "AF\u20102 surface", "site" ] ] }, { "sid": 209, "sent": "These compounds bind the LBD in an unusual fashion because they have a phenol\u2010to\u2010phenol length of ~12 \u00c5, which is longer than steroids and other prototypical ER\u03b1 agonists that are ~10 \u00c5 in length.", "section": "RESULTS", "ner": [ [ 25, 28, "LBD", "structure_element" ], [ 158, 161, "ER\u03b1", "protein" ] ] }, { "sid": 210, "sent": "One phenol pushed further toward h3 (Fig\u00a06D), while the other phenol pushed toward the C\u2010terminus of h11 to a greater extent than A\u2010CD\u2010ring estrogens (Nwachukwu et\u00a0al, 2014), which are close structural analogs of E2 that lack a B\u2010ring (Fig\u00a02).", "section": "RESULTS", "ner": [ [ 33, 35, "h3", "structure_element" ], [ 101, 104, "h11", "structure_element" ], [ 130, 134, "A\u2010CD", "chemical" ], [ 140, 149, "estrogens", "chemical" ], [ 213, 215, "E2", "chemical" ] ] }, { "sid": 211, "sent": "To quantify this difference, we compared the distance between \u03b1\u2010carbons at h3 Thr347 and h11 Leu525 in the set of structures containing 2,5\u2010DTP analogs (n\u00a0=\u00a03) or A\u2010CD\u2010ring analogs (n\u00a0=\u00a05) (Fig\u00a06E).", "section": "RESULTS", "ner": [ [ 45, 53, "distance", "evidence" ], [ 75, 77, "h3", "structure_element" ], [ 78, 84, "Thr347", "residue_name_number" ], [ 89, 92, "h11", "structure_element" ], [ 93, 99, "Leu525", "residue_name_number" ], [ 114, 124, "structures", "evidence" ], [ 136, 143, "2,5\u2010DTP", "chemical" ], [ 163, 167, "A\u2010CD", "chemical" ] ] }, { "sid": 212, "sent": "We observed a difference of 0.4 \u00c5 that was significant (two\u2010tailed Student's t\u2010test, P\u00a0=\u00a00.002) due to the very tight clustering of the 2,5\u2010DTP\u2010induced LBD conformation.", "section": "RESULTS", "ner": [ [ 67, 83, "Student's t\u2010test", "experimental_method" ], [ 85, 86, "P", "evidence" ], [ 136, 143, "2,5\u2010DTP", "chemical" ], [ 152, 155, "LBD", "structure_element" ] ] }, { "sid": 213, "sent": "The shifts in h3 suggest these compounds are positioned to alter coregulator preferences.", "section": "RESULTS", "ner": [ [ 14, 16, "h3", "structure_element" ] ] }, { "sid": 214, "sent": "The 2,5\u2010DTP and 3,4\u2010DTP scaffolds are isomeric, but with aryl groups at obtuse and acute angles, respectively (Fig\u00a02).", "section": "RESULTS", "ner": [ [ 4, 11, "2,5\u2010DTP", "chemical" ], [ 16, 23, "3,4\u2010DTP", "chemical" ] ] }, { "sid": 215, "sent": "The crystal structure of ER\u03b1 in complex with a 3,4\u2010DTP is unknown; however, we solved two crystal structures of ER\u03b1 bound to 3,4\u2010DTPD analogs and one structure containing a furan ligand\u2014all of which have a 3,4\u2010diaryl configuration (Fig\u00a02; Datasets EV1 and EV2).", "section": "RESULTS", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 25, 28, "ER\u03b1", "protein" ], [ 29, 44, "in complex with", "protein_state" ], [ 47, 54, "3,4\u2010DTP", "chemical" ], [ 79, 85, "solved", "experimental_method" ], [ 90, 108, "crystal structures", "evidence" ], [ 112, 115, "ER\u03b1", "protein" ], [ 116, 124, "bound to", "protein_state" ], [ 125, 133, "3,4\u2010DTPD", "chemical" ], [ 150, 159, "structure", "evidence" ], [ 173, 178, "furan", "chemical" ] ] }, { "sid": 216, "sent": "In these structures, the A\u2010ring mimetic of the 3,4\u2010DTPD scaffold bound h3 Glu353 as expected, but the other phenol wrapped around h3 to form a hydrogen bond with Thr347, indicating a change in binding epitopes in the ER\u03b1 ligand\u2010binding pocket (Fig\u00a06F).", "section": "RESULTS", "ner": [ [ 9, 19, "structures", "evidence" ], [ 47, 55, "3,4\u2010DTPD", "chemical" ], [ 71, 73, "h3", "structure_element" ], [ 74, 80, "Glu353", "residue_name_number" ], [ 130, 132, "h3", "structure_element" ], [ 143, 156, "hydrogen bond", "bond_interaction" ], [ 162, 168, "Thr347", "residue_name_number" ], [ 217, 220, "ER\u03b1", "protein" ], [ 221, 242, "ligand\u2010binding pocket", "site" ] ] }, { "sid": 217, "sent": "The 3,4\u2010DTPD analogs also induced a shift in h3 positioning, which translated again into a shift in the bound coactivator peptide (Fig\u00a06F).", "section": "RESULTS", "ner": [ [ 4, 12, "3,4\u2010DTPD", "chemical" ], [ 45, 47, "h3", "structure_element" ] ] }, { "sid": 218, "sent": "Therefore, these indirect modulators, including S\u2010OBHS\u20102, S\u2010OBHS\u20103, 2,5\u2010DTP, and 3,4\u2010DTPD analogs\u2014all of which show cell\u2010specific activity profiles\u2014induced shifts in h3 and h12 that were transmitted to the coactivator peptide via an altered AF\u20102 surface.", "section": "RESULTS", "ner": [ [ 48, 56, "S\u2010OBHS\u20102", "chemical" ], [ 58, 66, "S\u2010OBHS\u20103", "chemical" ], [ 68, 75, "2,5\u2010DTP", "chemical" ], [ 81, 89, "3,4\u2010DTPD", "chemical" ], [ 166, 168, "h3", "structure_element" ], [ 173, 176, "h12", "structure_element" ], [ 241, 253, "AF\u20102 surface", "site" ] ] }, { "sid": 219, "sent": "To test whether the AF\u20102 surface shows changes in shape in solution, we used the microarray assay for real\u2010time coregulator\u2013nuclear receptor interaction (MARCoNI) analysis (Aarts et\u00a0al, 2013).", "section": "RESULTS", "ner": [ [ 20, 32, "AF\u20102 surface", "site" ], [ 81, 152, "microarray assay for real\u2010time coregulator\u2013nuclear receptor interaction", "experimental_method" ], [ 154, 161, "MARCoNI", "experimental_method" ] ] }, { "sid": 220, "sent": "Here, the ligand\u2010dependent interactions of the ER\u03b1 LBD with over 150 distinct LxxLL motif peptides were assayed to define structural fingerprints for the AF\u20102 surface, in a manner similar to the use of phage display peptides as structural probes (Connor et\u00a0al, 2001).", "section": "RESULTS", "ner": [ [ 47, 50, "ER\u03b1", "protein" ], [ 51, 54, "LBD", "structure_element" ], [ 78, 89, "LxxLL motif", "structure_element" ], [ 154, 166, "AF\u20102 surface", "site" ], [ 202, 224, "phage display peptides", "experimental_method" ] ] }, { "sid": 221, "sent": "Despite the similar average activities of these ligand classes (Fig\u00a03A and B), 2,5\u2010DTP and 3,4\u2010DTP analogs displayed remarkably different peptide recruitment patterns (Fig\u00a06H), consistent with the structural analyses.", "section": "RESULTS", "ner": [ [ 79, 86, "2,5\u2010DTP", "chemical" ], [ 91, 98, "3,4\u2010DTP", "chemical" ], [ 197, 216, "structural analyses", "experimental_method" ] ] }, { "sid": 222, "sent": "Hierarchical clustering revealed that many of the 2,5\u2010DTP analogs recapitulated most of the peptide recruitment and dismissal patterns observed with E2 (Fig\u00a06H).", "section": "RESULTS", "ner": [ [ 0, 23, "Hierarchical clustering", "experimental_method" ], [ 50, 57, "2,5\u2010DTP", "chemical" ], [ 149, 151, "E2", "chemical" ] ] }, { "sid": 223, "sent": "However, there was a unique cluster of peptides that were recruited by E2 but not the 2,5\u2010DTP analogs.", "section": "RESULTS", "ner": [ [ 71, 73, "E2", "chemical" ], [ 86, 93, "2,5\u2010DTP", "chemical" ] ] }, { "sid": 224, "sent": "In contrast, 3,4\u2010DTP analogs dismissed most of the peptides from the AF\u20102 surface (Fig\u00a06H).", "section": "RESULTS", "ner": [ [ 13, 20, "3,4\u2010DTP", "chemical" ], [ 69, 81, "AF\u20102 surface", "site" ] ] }, { "sid": 225, "sent": "Thus, the isomeric attachment of diaryl groups to the thiophene core changed the AF\u20102 surface from inside the ligand\u2010binding pocket, as predicted by the crystal structures.", "section": "RESULTS", "ner": [ [ 54, 63, "thiophene", "chemical" ], [ 81, 93, "AF\u20102 surface", "site" ], [ 110, 131, "ligand\u2010binding pocket", "site" ], [ 153, 171, "crystal structures", "evidence" ] ] }, { "sid": 226, "sent": "Together, these findings suggest that without an extended side chain, cell\u2010specific activity stems from different coregulator recruitment profiles, due to unique ligand\u2010induced conformations of the AF\u20102 surface, in addition to differential usage of AF\u20101.", "section": "RESULTS", "ner": [ [ 198, 210, "AF\u20102 surface", "site" ], [ 249, 253, "AF\u20101", "structure_element" ] ] }, { "sid": 227, "sent": "Indirect modulators in cluster 1 avoid this by perturbing the h11\u2013h12 interface, and modulating the dynamics of h12 without changing the shape of AF\u20102 when stabilized.", "section": "RESULTS", "ner": [ [ 62, 79, "h11\u2013h12 interface", "site" ], [ 112, 115, "h12", "structure_element" ], [ 146, 150, "AF\u20102", "structure_element" ] ] }, { "sid": 228, "sent": "Our goal was to identify a minimal set of predictors that would link specific structural perturbations to ER\u03b1 signaling pathways that control cell\u2010specific signaling and proliferation.", "section": "DISCUSS", "ner": [ [ 106, 109, "ER\u03b1", "protein" ] ] }, { "sid": 229, "sent": "We found a very strong set of predictors, where ligands in cluster 1, defined by similar signaling across cell types, showed indirect modulation of h12 dynamics via the h11\u201312 interface or slight contact with h12.", "section": "DISCUSS", "ner": [ [ 148, 151, "h12", "structure_element" ], [ 169, 185, "h11\u201312 interface", "site" ], [ 209, 212, "h12", "structure_element" ] ] }, { "sid": 230, "sent": "This perturbation determined proliferation that correlated strongly with AF\u20102 activity, recruitment of NCOA1/2/3 family members, and induction of the GREB1 gene, consistent with the canonical ER\u03b1 signaling pathway (Fig\u00a01D).", "section": "DISCUSS", "ner": [ [ 73, 77, "AF\u20102", "structure_element" ], [ 103, 112, "NCOA1/2/3", "protein" ], [ 150, 155, "GREB1", "protein" ], [ 192, 195, "ER\u03b1", "protein" ] ] }, { "sid": 231, "sent": "For ligands in cluster 1, deletion of AF\u20101 reduced activity to varying degrees, but did not change the underlying signaling patterns established through AF\u20102.", "section": "DISCUSS", "ner": [ [ 26, 34, "deletion", "experimental_method" ], [ 38, 42, "AF\u20101", "structure_element" ], [ 153, 157, "AF\u20102", "structure_element" ] ] }, { "sid": 232, "sent": "In contrast, an extended side chain designed to directly reposition h12 and completely disrupt the AF\u20102 surface results in cell\u2010specific signaling.", "section": "DISCUSS", "ner": [ [ 68, 71, "h12", "structure_element" ], [ 99, 111, "AF\u20102 surface", "site" ] ] }, { "sid": 233, "sent": "Compared to cluster 1, the structural rules are less clear in clusters 2 and 3, but a number of indirect modulator classes perturbed the LBD conformation at the intersection of h3, the h12\u00a0N\u2010terminus, and the AF\u20102 surface.", "section": "DISCUSS", "ner": [ [ 137, 140, "LBD", "structure_element" ], [ 177, 179, "h3", "structure_element" ], [ 185, 188, "h12", "structure_element" ], [ 209, 221, "AF\u20102 surface", "site" ] ] }, { "sid": 234, "sent": "Ligands in these classes altered the shape of AF\u20102 to affect coregulator preferences.", "section": "DISCUSS", "ner": [ [ 46, 50, "AF\u20102", "structure_element" ] ] }, { "sid": 235, "sent": "For direct and indirect modulators in cluster 2 or 3, the canonical ER\u03b1 signaling pathway involving recruitment of NCOA1/2/3 and induction of GREB1 did not generally predict their proliferative effects, indicating an alternate causal model (Fig\u00a01E).", "section": "DISCUSS", "ner": [ [ 68, 71, "ER\u03b1", "protein" ], [ 115, 124, "NCOA1/2/3", "protein" ], [ 142, 147, "GREB1", "protein" ] ] }, { "sid": 236, "sent": "These principles outlined above provide a structural basis for how the ligand\u2013receptor interface leads to different signaling specificities through AF\u20101 and AF\u20102.", "section": "DISCUSS", "ner": [ [ 71, 96, "ligand\u2013receptor interface", "site" ], [ 148, 152, "AF\u20101", "structure_element" ], [ 157, 161, "AF\u20102", "structure_element" ] ] }, { "sid": 237, "sent": "It is noteworthy that regulation of h12 dynamics indirectly through h11 can virtually abolish AF\u20102 activity, and yet still drive robust transcriptional activity through AF\u20101, as demonstrated with the OBHS series.", "section": "DISCUSS", "ner": [ [ 36, 39, "h12", "structure_element" ], [ 68, 71, "h11", "structure_element" ], [ 94, 98, "AF\u20102", "structure_element" ], [ 169, 173, "AF\u20101", "structure_element" ], [ 200, 204, "OBHS", "chemical" ] ] }, { "sid": 238, "sent": "This finding can be explained by the fact that NCOA1/2/3 contain distinct binding sites for interaction with AF\u20101 and AF\u20102 (McInerney et\u00a0al, 1996; Webb et\u00a0al, 1998), which allows ligands to nucleate ER\u03b1\u2013NCOA1/2/3 interaction through AF\u20102, and reinforce this interaction with additional binding to AF\u20101.", "section": "DISCUSS", "ner": [ [ 47, 56, "NCOA1/2/3", "protein" ], [ 74, 87, "binding sites", "site" ], [ 109, 113, "AF\u20101", "structure_element" ], [ 118, 122, "AF\u20102", "structure_element" ], [ 199, 212, "ER\u03b1\u2013NCOA1/2/3", "complex_assembly" ], [ 233, 237, "AF\u20102", "structure_element" ], [ 297, 301, "AF\u20101", "structure_element" ] ] }, { "sid": 239, "sent": "Completely blocking AF\u20102 with an extended side chain or altering the shape of AF\u20102 changes the preference away from NCOA1/2/3 for determining GREB1 levels and proliferation of breast cancer cells.", "section": "DISCUSS", "ner": [ [ 20, 24, "AF\u20102", "structure_element" ], [ 78, 82, "AF\u20102", "structure_element" ], [ 116, 125, "NCOA1/2/3", "protein" ], [ 142, 147, "GREB1", "protein" ] ] }, { "sid": 240, "sent": "AF\u20102 blockade also allows AF\u20101 to function independently, which is important since AF\u20101 drives tissue\u2010selective effects in\u00a0vivo.", "section": "DISCUSS", "ner": [ [ 0, 4, "AF\u20102", "structure_element" ], [ 26, 30, "AF\u20101", "structure_element" ], [ 83, 87, "AF\u20101", "structure_element" ] ] }, { "sid": 241, "sent": "This was demonstrated with AF\u20101 knockout mice that show E2\u2010dependent vascular protection, but not uterine proliferation, thus highlighting the role of AF\u20101 in tissue\u2010selective or cell\u2010specific signaling (Billon\u2010Gales et\u00a0al, 2009; Abot et\u00a0al, 2013).", "section": "DISCUSS", "ner": [ [ 27, 31, "AF\u20101", "structure_element" ], [ 56, 58, "E2", "chemical" ], [ 151, 155, "AF\u20101", "structure_element" ] ] }, { "sid": 242, "sent": "Here, we examined many LBD structures and tested several variables that were not predictive, including ER\u03b2 activity, the strength of AF\u20101 signaling, and NCOA3 occupancy at the GREB1 gene.", "section": "DISCUSS", "ner": [ [ 23, 26, "LBD", "structure_element" ], [ 27, 37, "structures", "evidence" ], [ 103, 106, "ER\u03b2", "protein" ], [ 133, 137, "AF\u20101", "structure_element" ], [ 153, 158, "NCOA3", "protein" ], [ 176, 181, "GREB1", "protein" ] ] }, { "sid": 243, "sent": "Similarly, we visualized structures to identify patterns.", "section": "DISCUSS", "ner": [ [ 25, 35, "structures", "evidence" ] ] }, { "sid": 244, "sent": "For example, phage display was used to identify the androgen receptor interactome, which was cloned into an M2H library and used to identify clusters of ligand\u2010selective interactions (Norris et\u00a0al, 2009).", "section": "DISCUSS", "ner": [ [ 13, 26, "phage display", "experimental_method" ], [ 108, 111, "M2H", "experimental_method" ] ] }, { "sid": 245, "sent": "Also, we have used siRNA screening to identify a number of coregulators required for ER\u03b1\u2010mediated repression of the IL\u20106 gene (Nwachukwu et\u00a0al, 2014).", "section": "DISCUSS", "ner": [ [ 19, 34, "siRNA screening", "experimental_method" ], [ 85, 88, "ER\u03b1", "protein" ] ] }, { "sid": 246, "sent": "If we calculated inter\u2010atomic distance matrices containing 4,000 atoms per structure \u00d7\u00a076 ligand\u2013receptor complexes, we would have 3\u00a0\u00d7\u00a0105 predictions.", "section": "DISCUSS", "ner": [ [ 17, 47, "inter\u2010atomic distance matrices", "evidence" ] ] }, { "sid": 247, "sent": "We have identified atomic vectors for the OBHS\u2010N and triaryl\u2010ethylene classes that predict ligand response (Fig\u00a05E and\u00a0F).", "section": "DISCUSS", "ner": [ [ 19, 33, "atomic vectors", "evidence" ], [ 42, 48, "OBHS\u2010N", "chemical" ], [ 53, 69, "triaryl\u2010ethylene", "chemical" ] ] }, { "sid": 248, "sent": "Indeed, the most anti\u2010proliferative compound in the OBHS\u2010N series had a fulvestrant\u2010like profile across a battery of assays (S. Srinivasan et al, in preparation).", "section": "DISCUSS", "ner": [ [ 52, 58, "OBHS\u2010N", "chemical" ] ] }, { "sid": 249, "sent": "Secondly, our finding that WAY\u2010C compounds do not rely of AF\u20101 for signaling efficacy may derive from the slight contacts with h12 observed in crystal structures (Figs\u00a03B and 5H), unlike other compounds in cluster 1 that dislocate h11 and rely on AF\u20101 for signaling efficacy (Figs\u00a03B and 5C, and EV5B).", "section": "DISCUSS", "ner": [ [ 27, 32, "WAY\u2010C", "chemical" ], [ 58, 62, "AF\u20101", "structure_element" ], [ 127, 130, "h12", "structure_element" ], [ 143, 161, "crystal structures", "evidence" ], [ 231, 234, "h11", "structure_element" ], [ 247, 251, "AF\u20101", "structure_element" ] ] }, { "sid": 250, "sent": "Some of these ligands altered the shape of the AF\u20102 surface by perturbing the h3\u2013h12 interface, thus providing a route to new SERM\u2010like activity profiles by combining indirect and direct modulation of receptor structure.", "section": "DISCUSS", "ner": [ [ 47, 59, "AF\u20102 surface", "site" ], [ 78, 94, "h3\u2013h12 interface", "site" ] ] }, { "sid": 251, "sent": "Incorporation of statistical approaches to understand relationships between structure and signaling variables moves us toward predictive models for complex ER\u03b1\u2010mediated responses such as in\u00a0vivo uterine proliferation or tumor growth, and more generally toward structure\u2010based design for other allosteric drug targets including GPCRs and other nuclear receptors.", "section": "DISCUSS", "ner": [ [ 156, 159, "ER\u03b1", "protein" ], [ 327, 332, "GPCRs", "protein_type" ], [ 343, 360, "nuclear receptors", "protein_type" ] ] } ] }, "PMC4919469": { "annotations": [ { "sid": 0, "sent": "Investigation of the Interaction between Cdc42 and Its Effector TOCA1", "section": "TITLE", "ner": [ [ 41, 46, "Cdc42", "protein" ], [ 64, 69, "TOCA1", "protein" ] ] }, { "sid": 1, "sent": "Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42.", "section": "ABSTRACT", "ner": [ [ 0, 54, "Transducer of Cdc42-dependent actin assembly protein 1", "protein" ], [ 56, 61, "TOCA1", "protein" ], [ 85, 111, "Rho family small G protein", "protein_type" ], [ 112, 117, "Cdc42", "protein" ] ] }, { "sid": 2, "sent": "It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain.", "section": "ABSTRACT", "ner": [ [ 33, 38, "F-BAR", "structure_element" ], [ 59, 73, "Src homology 3", "structure_element" ], [ 75, 78, "SH3", "structure_element" ], [ 93, 128, "G protein-binding homology region 1", "structure_element" ], [ 130, 133, "HR1", "structure_element" ] ] }, { "sid": 3, "sent": "TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration.", "section": "ABSTRACT", "ner": [ [ 0, 5, "TOCA1", "protein" ], [ 17, 22, "Cdc42", "protein" ], [ 32, 37, "actin", "protein_type" ] ] }, { "sid": 4, "sent": "We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein.", "section": "ABSTRACT", "ner": [ [ 8, 14, "solved", "experimental_method" ], [ 19, 28, "structure", "evidence" ], [ 36, 39, "HR1", "structure_element" ], [ 50, 55, "TOCA1", "protein" ], [ 77, 92, "structural data", "evidence" ] ] }, { "sid": 5, "sent": "We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains.", "section": "ABSTRACT", "ner": [ [ 23, 28, "TOCA1", "protein" ], [ 29, 32, "HR1", "structure_element" ], [ 59, 63, "CIP4", "protein" ], [ 64, 67, "HR1", "structure_element" ], [ 136, 139, "HR1", "structure_element" ] ] }, { "sid": 6, "sent": "We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP.", "section": "ABSTRACT", "ner": [ [ 45, 49, "TOCA", "protein" ], [ 50, 53, "HR1", "structure_element" ], [ 64, 69, "Cdc42", "protein" ], [ 112, 117, "Cdc42", "protein" ], [ 126, 151, "G protein-binding regions", "site" ], [ 155, 160, "TOCA1", "protein" ], [ 181, 220, "Wiskott-Aldrich syndrome protein family", "protein_type" ], [ 222, 228, "N-WASP", "protein" ] ] }, { "sid": 7, "sent": "TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42.", "section": "ABSTRACT", "ner": [ [ 0, 5, "TOCA1", "protein" ], [ 12, 17, "Cdc42", "protein" ], [ 89, 95, "N-WASP", "protein" ], [ 96, 120, "G protein-binding region", "site" ], [ 125, 130, "Cdc42", "protein" ] ] }, { "sid": 8, "sent": "NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization.", "section": "ABSTRACT", "ner": [ [ 0, 3, "NMR", "experimental_method" ], [ 30, 50, "Cdc42-binding domain", "site" ], [ 56, 62, "N-WASP", "protein" ], [ 83, 88, "TOCA1", "protein" ], [ 89, 92, "HR1", "structure_element" ], [ 98, 103, "Cdc42", "protein" ], [ 117, 123, "N-WASP", "protein" ], [ 143, 148, "TOCA1", "protein" ], [ 149, 152, "HR1", "structure_element" ] ] }, { "sid": 9, "sent": "This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery.", "section": "ABSTRACT", "ner": [ [ 19, 24, "TOCA1", "protein" ], [ 36, 41, "Cdc42", "protein" ], [ 66, 71, "Cdc42", "protein" ], [ 140, 158, "binding affinities", "evidence" ], [ 203, 208, "TOCA1", "protein" ], [ 212, 218, "N-WASP", "protein" ] ] }, { "sid": 10, "sent": "The Ras superfamily of small GTPases comprises over 150 members that regulate a multitude of cellular processes in eukaryotes.", "section": "INTRO", "ner": [ [ 4, 19, "Ras superfamily", "protein_type" ], [ 23, 36, "small GTPases", "protein_type" ], [ 115, 125, "eukaryotes", "taxonomy_domain" ] ] }, { "sid": 11, "sent": "The superfamily can be divided into five families based on structural and functional similarities: Ras, Rho, Rab, Arf, and Ran.", "section": "INTRO", "ner": [ [ 99, 102, "Ras", "protein_type" ], [ 104, 107, "Rho", "protein_type" ], [ 109, 112, "Rab", "protein_type" ], [ 114, 117, "Arf", "protein_type" ], [ 123, 126, "Ran", "protein_type" ] ] }, { "sid": 12, "sent": "All members share a well defined core structure of \u223c20 kDa known as the G domain, which is responsible for guanine nucleotide binding.", "section": "INTRO", "ner": [ [ 72, 80, "G domain", "structure_element" ], [ 107, 125, "guanine nucleotide", "chemical" ] ] }, { "sid": 13, "sent": "These molecular switches cycle between active, GTP-bound, and inactive, GDP-bound, states with the help of auxiliary proteins.", "section": "INTRO", "ner": [ [ 39, 45, "active", "protein_state" ], [ 47, 56, "GTP-bound", "protein_state" ], [ 62, 70, "inactive", "protein_state" ], [ 72, 81, "GDP-bound", "protein_state" ] ] }, { "sid": 14, "sent": "The guanine nucleotide exchange factors mediate formation of the active state by promoting the dissociation of GDP, allowing GTP to bind.", "section": "INTRO", "ner": [ [ 4, 39, "guanine nucleotide exchange factors", "protein_type" ], [ 65, 71, "active", "protein_state" ], [ 111, 114, "GDP", "chemical" ], [ 125, 128, "GTP", "chemical" ] ] }, { "sid": 15, "sent": "The GTPase-activating proteins stimulate the rate of intrinsic GTP hydrolysis, mediating the return to the inactive state (reviewed in Ref.).", "section": "INTRO", "ner": [ [ 4, 30, "GTPase-activating proteins", "protein_type" ], [ 63, 66, "GTP", "chemical" ], [ 107, 115, "inactive", "protein_state" ] ] }, { "sid": 16, "sent": "The overall conformation of small G proteins in the active and inactive states is similar, but they differ significantly in two main regions known as switch I and switch II.", "section": "INTRO", "ner": [ [ 28, 44, "small G proteins", "protein_type" ], [ 52, 58, "active", "protein_state" ], [ 63, 71, "inactive", "protein_state" ], [ 150, 158, "switch I", "site" ], [ 163, 172, "switch II", "site" ] ] }, { "sid": 17, "sent": "These regions are responsible for \u201csensing\u201d the nucleotide state, with the GTP-bound state showing greater rigidity and the GDP-bound state adopting a more relaxed conformation (reviewed in Ref.).", "section": "INTRO", "ner": [ [ 75, 84, "GTP-bound", "protein_state" ], [ 124, 133, "GDP-bound", "protein_state" ] ] }, { "sid": 18, "sent": "In the active state, G proteins bind to an array of downstream effectors, through which they exert their extensive roles within the cell.", "section": "INTRO", "ner": [ [ 7, 13, "active", "protein_state" ], [ 21, 31, "G proteins", "protein_type" ] ] }, { "sid": 19, "sent": "The structures of more than 60 small G protein\u00b7effector complexes have been solved, and, not surprisingly, the switch regions have been implicated in a large proportion of the G protein-effector interactions (reviewed in Ref.).", "section": "INTRO", "ner": [ [ 4, 14, "structures", "evidence" ], [ 37, 46, "G protein", "protein_type" ], [ 76, 82, "solved", "experimental_method" ], [ 111, 125, "switch regions", "site" ], [ 176, 185, "G protein", "protein_type" ] ] }, { "sid": 20, "sent": "However, because each of the 150 members of the superfamily interacts with multiple effectors, there are still a huge number of known G protein-effector interactions that have not yet been studied structurally.", "section": "INTRO", "ner": [ [ 134, 143, "G protein", "protein_type" ] ] }, { "sid": 21, "sent": "The Rho family comprises 20 members, of which three, RhoA, Rac1, and Cdc42, have been relatively well studied.", "section": "INTRO", "ner": [ [ 4, 14, "Rho family", "protein_type" ], [ 53, 57, "RhoA", "protein" ], [ 59, 63, "Rac1", "protein" ], [ 69, 74, "Cdc42", "protein" ] ] }, { "sid": 22, "sent": "RhoA acts to rearrange existing actin structures to form stress fibers, whereas Rac1 and Cdc42 promote de novo actin polymerization to form lamellipodia and filopodia, respectively.", "section": "INTRO", "ner": [ [ 0, 4, "RhoA", "protein" ], [ 80, 84, "Rac1", "protein" ], [ 89, 94, "Cdc42", "protein" ] ] }, { "sid": 23, "sent": "A number of RhoA and Rac1 effector proteins, including the formins and members of the protein kinase C-related kinase (PRK)6 family, along with Cdc42 effectors, including the Wiskott-Aldrich syndrome (WASP) family and the transducer of Cdc42-dependent actin assembly (TOCA) family, have also been linked to the pathways that govern cytoskeletal dynamics.", "section": "INTRO", "ner": [ [ 12, 16, "RhoA", "protein" ], [ 21, 25, "Rac1", "protein" ], [ 86, 117, "protein kinase C-related kinase", "protein_type" ], [ 119, 122, "PRK", "protein_type" ], [ 123, 124, "6", "protein_type" ], [ 144, 149, "Cdc42", "protein" ], [ 175, 199, "Wiskott-Aldrich syndrome", "protein_type" ], [ 201, 205, "WASP", "protein_type" ], [ 236, 266, "Cdc42-dependent actin assembly", "protein_type" ], [ 268, 272, "TOCA", "protein_type" ] ] }, { "sid": 24, "sent": "Cdc42 effectors, TOCA1 and the ubiquitously expressed member of the WASP family, N-WASP, have been implicated in the regulation of actin polymerization downstream of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2).", "section": "INTRO", "ner": [ [ 0, 5, "Cdc42", "protein" ], [ 17, 22, "TOCA1", "protein" ], [ 68, 79, "WASP family", "protein_type" ], [ 81, 87, "N-WASP", "protein" ], [ 166, 171, "Cdc42", "protein" ], [ 176, 213, "phosphatidylinositol 4,5-bisphosphate", "chemical" ], [ 215, 224, "PI(4,5)P2", "chemical" ] ] }, { "sid": 25, "sent": "N-WASP exists in an autoinhibited conformation, which is released upon PI(4,5)P2 and Cdc42 binding or by other factors, such as phosphorylation.", "section": "INTRO", "ner": [ [ 0, 6, "N-WASP", "protein" ], [ 20, 46, "autoinhibited conformation", "protein_state" ], [ 71, 80, "PI(4,5)P2", "chemical" ], [ 85, 90, "Cdc42", "protein" ] ] }, { "sid": 26, "sent": "Following their release, the C-terminal regions of N-WASP are free to interact with G-actin and a known nucleator of actin assembly, the Arp2/3 complex.", "section": "INTRO", "ner": [ [ 29, 47, "C-terminal regions", "structure_element" ], [ 51, 57, "N-WASP", "protein" ], [ 84, 91, "G-actin", "protein_type" ], [ 137, 143, "Arp2/3", "complex_assembly" ] ] }, { "sid": 27, "sent": "The importance of TOCA1 in actin polymerization has been demonstrated in a range of in vitro and in vivo studies, but the exact role of TOCA1 in the many pathways involving actin assembly remains unclear.", "section": "INTRO", "ner": [ [ 18, 23, "TOCA1", "protein" ], [ 136, 141, "TOCA1", "protein" ] ] }, { "sid": 28, "sent": "The most widely studied role of TOCA1 is in membrane invagination and endocytosis, although it has also been implicated in filopodia formation, neurite elongation, transcriptional reprogramming via nuclear actin, and interaction with ZO-1 at tight junctions.", "section": "INTRO", "ner": [ [ 32, 37, "TOCA1", "protein" ], [ 206, 211, "actin", "protein_type" ], [ 234, 238, "ZO-1", "protein" ] ] }, { "sid": 29, "sent": "TOCA1 comprises an N-terminal F-BAR domain, a central homology region 1 (HR1) domain, and a C-terminal SH3 domain.", "section": "INTRO", "ner": [ [ 0, 5, "TOCA1", "protein" ], [ 30, 35, "F-BAR", "structure_element" ], [ 46, 71, "central homology region 1", "structure_element" ], [ 73, 76, "HR1", "structure_element" ], [ 103, 106, "SH3", "structure_element" ] ] }, { "sid": 30, "sent": "The F-BAR domain is a known dimerization, membrane-binding, and membrane-deforming module found in a number of cell signaling proteins.", "section": "INTRO", "ner": [ [ 4, 9, "F-BAR", "structure_element" ] ] }, { "sid": 31, "sent": "The TOCA1 SH3 domain has many known binding partners, including N-WASP and dynamin.", "section": "INTRO", "ner": [ [ 4, 9, "TOCA1", "protein" ], [ 10, 13, "SH3", "structure_element" ], [ 64, 70, "N-WASP", "protein" ], [ 75, 82, "dynamin", "protein" ] ] }, { "sid": 32, "sent": "The HR1 domain has been directly implicated in the interaction between TOCA1 and Cdc42, representing the first Cdc42-HR1 domain interaction to be identified.", "section": "INTRO", "ner": [ [ 4, 7, "HR1", "structure_element" ], [ 71, 76, "TOCA1", "protein" ], [ 81, 86, "Cdc42", "protein" ], [ 111, 116, "Cdc42", "protein" ], [ 117, 120, "HR1", "structure_element" ] ] }, { "sid": 33, "sent": "Other HR1 domains studied so far, including those from the PRK family, have been found to bind their cognate Rho family G protein-binding partner with high specificity and affinities in the nanomolar range.", "section": "INTRO", "ner": [ [ 6, 9, "HR1", "structure_element" ], [ 59, 69, "PRK family", "protein_type" ], [ 120, 129, "G protein", "protein_type" ], [ 172, 182, "affinities", "evidence" ] ] }, { "sid": 34, "sent": "The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices.", "section": "INTRO", "ner": [ [ 4, 14, "structures", "evidence" ], [ 22, 26, "PRK1", "protein" ], [ 27, 31, "HR1a", "structure_element" ], [ 42, 54, "complex with", "protein_state" ], [ 55, 59, "RhoA", "protein" ], [ 68, 72, "HR1b", "structure_element" ], [ 83, 95, "complex with", "protein_state" ], [ 96, 100, "Rac1", "protein" ], [ 115, 118, "HR1", "structure_element" ], [ 139, 164, "anti-parallel coiled-coil", "structure_element" ], [ 189, 198, "G protein", "protein_type" ], [ 224, 231, "helices", "structure_element" ] ] }, { "sid": 35, "sent": "Both of the G protein switch regions are involved in the interaction.", "section": "INTRO", "ner": [ [ 12, 36, "G protein switch regions", "site" ] ] }, { "sid": 36, "sent": "The coiled-coil fold is shared by the HR1 domain of the TOCA family protein, CIP4, and, based on sequence homology, by TOCA1 itself.", "section": "INTRO", "ner": [ [ 4, 20, "coiled-coil fold", "structure_element" ], [ 38, 41, "HR1", "structure_element" ], [ 56, 75, "TOCA family protein", "protein_type" ], [ 77, 81, "CIP4", "protein" ], [ 119, 124, "TOCA1", "protein" ] ] }, { "sid": 37, "sent": "These HR1 domains, however, show specificity for Cdc42, rather than RhoA or Rac1.", "section": "INTRO", "ner": [ [ 6, 9, "HR1", "structure_element" ], [ 49, 54, "Cdc42", "protein" ], [ 68, 72, "RhoA", "protein" ], [ 76, 80, "Rac1", "protein" ] ] }, { "sid": 38, "sent": "How different HR1 domain proteins distinguish their specific G protein partners remains only partially understood, and structural characterization of a novel G protein-HR1 domain interaction would add to the growing body of information pertaining to these protein complexes.", "section": "INTRO", "ner": [ [ 14, 17, "HR1", "structure_element" ], [ 61, 70, "G protein", "protein_type" ], [ 158, 167, "G protein", "protein_type" ], [ 168, 171, "HR1", "structure_element" ] ] }, { "sid": 39, "sent": "Furthermore, the biological function of the interaction between TOCA1 and Cdc42 remains poorly understood, and so far there has been no biophysical or structural insight.", "section": "INTRO", "ner": [ [ 64, 69, "TOCA1", "protein" ], [ 74, 79, "Cdc42", "protein" ] ] }, { "sid": 40, "sent": "The interactions of TOCA1 and N-WASP with Cdc42 as well as with each other have raised questions as to whether the two Cdc42 effectors can interact with a single molecule of Cdc42 simultaneously.", "section": "INTRO", "ner": [ [ 20, 25, "TOCA1", "protein" ], [ 30, 36, "N-WASP", "protein" ], [ 42, 47, "Cdc42", "protein" ], [ 119, 124, "Cdc42", "protein" ], [ 174, 179, "Cdc42", "protein" ] ] }, { "sid": 41, "sent": "There is some evidence for a ternary complex between Cdc42, N-WASP, and TOCA1, but there was no direct demonstration of simultaneous contacts between the two effectors and a single molecule of Cdc42.", "section": "INTRO", "ner": [ [ 53, 58, "Cdc42", "protein" ], [ 60, 66, "N-WASP", "protein" ], [ 72, 77, "TOCA1", "protein" ], [ 193, 198, "Cdc42", "protein" ] ] }, { "sid": 42, "sent": "Nonetheless, the substantial difference between the structures of the G protein-binding regions of the two effectors is intriguing and implies that they bind to Cdc42 quite differently, providing motivation for investigating the possibility that Cdc42 can bind both effectors concurrently.", "section": "INTRO", "ner": [ [ 52, 62, "structures", "evidence" ], [ 70, 95, "G protein-binding regions", "site" ], [ 161, 166, "Cdc42", "protein" ], [ 246, 251, "Cdc42", "protein" ] ] }, { "sid": 43, "sent": "WASP interacts with Cdc42 via a conserved, unstructured binding motif known as the Cdc42- and Rac-interactive binding region (CRIB), which forms an intermolecular \u03b2-sheet, expanding the anti-parallel \u03b22 and \u03b23 strands of Cdc42.", "section": "INTRO", "ner": [ [ 0, 4, "WASP", "protein_type" ], [ 20, 25, "Cdc42", "protein" ], [ 32, 41, "conserved", "protein_state" ], [ 43, 69, "unstructured binding motif", "structure_element" ], [ 83, 124, "Cdc42- and Rac-interactive binding region", "structure_element" ], [ 126, 130, "CRIB", "structure_element" ], [ 148, 170, "intermolecular \u03b2-sheet", "structure_element" ], [ 200, 217, "\u03b22 and \u03b23 strands", "structure_element" ], [ 221, 226, "Cdc42", "protein" ] ] }, { "sid": 44, "sent": "In contrast, the TOCA family proteins are thought to interact via the HR1 domain, which may form a triple coiled-coil with switch II of Rac1, like the HR1b domain of PRK1.", "section": "INTRO", "ner": [ [ 17, 37, "TOCA family proteins", "protein_type" ], [ 70, 73, "HR1", "structure_element" ], [ 99, 117, "triple coiled-coil", "structure_element" ], [ 123, 132, "switch II", "site" ], [ 136, 140, "Rac1", "protein" ], [ 151, 155, "HR1b", "structure_element" ], [ 166, 170, "PRK1", "protein" ] ] }, { "sid": 45, "sent": "Here, we present the solution NMR structure of the HR1 domain of TOCA1, providing the first structural data for this protein.", "section": "INTRO", "ner": [ [ 21, 33, "solution NMR", "experimental_method" ], [ 34, 43, "structure", "evidence" ], [ 51, 54, "HR1", "structure_element" ], [ 65, 70, "TOCA1", "protein" ], [ 92, 107, "structural data", "evidence" ] ] }, { "sid": 46, "sent": "We also present data pertaining to binding of the TOCA HR1 domain to Cdc42, which is the first biophysical description of an HR1 domain binding this particular Rho family small G protein.", "section": "INTRO", "ner": [ [ 50, 54, "TOCA", "protein_type" ], [ 55, 58, "HR1", "structure_element" ], [ 69, 74, "Cdc42", "protein" ], [ 125, 128, "HR1", "structure_element" ], [ 160, 186, "Rho family small G protein", "protein_type" ] ] }, { "sid": 47, "sent": "Finally, we investigate the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and N-WASP, contributing to our understanding of G protein-effector interactions as well as the roles of Cdc42, N-WASP, and TOCA1 in the pathways that govern actin dynamics.", "section": "INTRO", "ner": [ [ 62, 67, "Cdc42", "protein" ], [ 76, 101, "G protein-binding regions", "site" ], [ 105, 110, "TOCA1", "protein" ], [ 115, 121, "N-WASP", "protein" ], [ 160, 169, "G protein", "protein_type" ], [ 216, 221, "Cdc42", "protein" ], [ 223, 229, "N-WASP", "protein" ], [ 235, 240, "TOCA1", "protein" ], [ 269, 274, "actin", "protein_type" ] ] }, { "sid": 48, "sent": "Cdc42-TOCA1 Binding", "section": "RESULTS", "ner": [ [ 0, 5, "Cdc42", "protein" ], [ 6, 11, "TOCA1", "protein" ] ] }, { "sid": 49, "sent": "TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42\u00b7GTP\u03b3S but not to Cdc42\u00b7GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42.", "section": "RESULTS", "ner": [ [ 0, 5, "TOCA1", "protein" ], [ 24, 31, "Xenopus", "taxonomy_domain" ], [ 68, 73, "Cdc42", "protein" ], [ 124, 135, "Cdc42\u00b7GTP\u03b3S", "complex_assembly" ], [ 147, 156, "Cdc42\u00b7GDP", "complex_assembly" ], [ 163, 167, "Rac1", "protein" ], [ 172, 176, "RhoA", "protein" ], [ 206, 232, "Rho family binding modules", "site" ], [ 256, 259, "HR1", "structure_element" ], [ 270, 275, "TOCA1", "protein" ], [ 298, 303, "Cdc42", "protein" ] ] }, { "sid": 50, "sent": "The C. elegans TOCA1 orthologues also bind to Cdc42 via their consensus HR1 domain.", "section": "RESULTS", "ner": [ [ 4, 14, "C. elegans", "species" ], [ 15, 20, "TOCA1", "protein" ], [ 46, 51, "Cdc42", "protein" ], [ 72, 75, "HR1", "structure_element" ] ] }, { "sid": 51, "sent": "The HR1 domains from the PRK family bind their G protein partners with a high affinity, exhibiting a range of submicromolar dissociation constants (Kd) as low as 26 nm.", "section": "RESULTS", "ner": [ [ 4, 7, "HR1", "structure_element" ], [ 25, 35, "PRK family", "protein_type" ], [ 47, 56, "G protein", "protein_type" ], [ 124, 146, "dissociation constants", "evidence" ], [ 148, 150, "Kd", "evidence" ] ] }, { "sid": 52, "sent": "A Kd in the nanomolar range was therefore expected for the interaction of the TOCA1 HR1 domain with Cdc42.", "section": "RESULTS", "ner": [ [ 2, 4, "Kd", "evidence" ], [ 78, 83, "TOCA1", "protein" ], [ 84, 87, "HR1", "structure_element" ], [ 100, 105, "Cdc42", "protein" ] ] }, { "sid": 53, "sent": "We generated an X. tropicalis TOCA1 HR1 domain construct encompassing residues 330\u2013426.", "section": "RESULTS", "ner": [ [ 16, 29, "X. tropicalis", "species" ], [ 30, 35, "TOCA1", "protein" ], [ 36, 39, "HR1", "structure_element" ], [ 79, 86, "330\u2013426", "residue_range" ] ] }, { "sid": 54, "sent": "This region comprises the complete HR1 domain based on secondary structure predictions and sequence alignments with another TOCA family member, CIP4, whose structure has been determined.", "section": "RESULTS", "ner": [ [ 35, 38, "HR1", "structure_element" ], [ 91, 110, "sequence alignments", "experimental_method" ], [ 124, 135, "TOCA family", "protein_type" ], [ 144, 148, "CIP4", "protein" ], [ 156, 165, "structure", "evidence" ] ] }, { "sid": 55, "sent": "The interaction between [3H]GTP\u00b7Cdc42 and a C-terminally His-tagged TOCA1 HR1 domain construct was investigated using SPA.", "section": "RESULTS", "ner": [ [ 24, 37, "[3H]GTP\u00b7Cdc42", "complex_assembly" ], [ 57, 67, "His-tagged", "protein_state" ], [ 68, 73, "TOCA1", "protein" ], [ 74, 77, "HR1", "structure_element" ], [ 118, 121, "SPA", "experimental_method" ] ] }, { "sid": 56, "sent": "The binding isotherm for the interaction is shown in Fig. 1A, together with the Cdc42-PAK interaction as a positive control.", "section": "RESULTS", "ner": [ [ 4, 20, "binding isotherm", "evidence" ], [ 80, 85, "Cdc42", "protein" ], [ 86, 89, "PAK", "protein" ] ] }, { "sid": 57, "sent": "The binding of TOCA1 HR1 to Cdc42 was unexpectedly weak, with a Kd of >1 \u03bcm.", "section": "RESULTS", "ner": [ [ 15, 20, "TOCA1", "protein" ], [ 21, 24, "HR1", "structure_element" ], [ 28, 33, "Cdc42", "protein" ], [ 64, 66, "Kd", "evidence" ] ] }, { "sid": 58, "sent": "It was not possible to estimate the Kd more accurately using direct SPA experiments, because saturation could not be reached due to nonspecific signal at higher protein concentrations.", "section": "RESULTS", "ner": [ [ 36, 38, "Kd", "evidence" ], [ 68, 71, "SPA", "experimental_method" ] ] }, { "sid": 59, "sent": "The TOCA1 HR1-Cdc42 interaction is low affinity.", "section": "FIG", "ner": [ [ 4, 9, "TOCA1", "protein" ], [ 10, 13, "HR1", "structure_element" ], [ 14, 19, "Cdc42", "protein" ] ] }, { "sid": 60, "sent": " A, curves derived from direct binding assays in which the indicated concentrations of Cdc42\u03947Q61L\u00b7[3H]GTP were incubated with 30 nm GST-PAK or HR1-His6 in SPAs.", "section": "FIG", "ner": [ [ 24, 45, "direct binding assays", "experimental_method" ], [ 87, 106, "Cdc42\u03947Q61L\u00b7[3H]GTP", "complex_assembly" ], [ 112, 121, "incubated", "experimental_method" ], [ 133, 140, "GST-PAK", "mutant" ], [ 144, 152, "HR1-His6", "mutant" ], [ 156, 160, "SPAs", "experimental_method" ] ] }, { "sid": 61, "sent": "The SPA signal was corrected by subtraction of control data with no GST-PAK or HR1-His6.", "section": "FIG", "ner": [ [ 4, 7, "SPA", "experimental_method" ], [ 68, 75, "GST-PAK", "mutant" ], [ 79, 87, "HR1-His6", "mutant" ] ] }, { "sid": 62, "sent": "The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42\u03947Q61L\u00b7[3H]GTP or full-length Cdc42Q61L\u00b7[3H]GTP.", "section": "FIG", "ner": [ [ 26, 42, "binding isotherm", "evidence" ], [ 63, 65, "Kd", "evidence" ], [ 132, 147, "competition SPA", "experimental_method" ], [ 214, 217, "ACK", "protein" ], [ 218, 221, "GBD", "structure_element" ], [ 229, 232, "HR1", "structure_element" ], [ 244, 252, "titrated", "experimental_method" ], [ 264, 271, "GST-ACK", "mutant" ], [ 289, 308, "Cdc42\u03947Q61L\u00b7[3H]GTP", "complex_assembly" ], [ 312, 323, "full-length", "protein_state" ], [ 324, 341, "Cdc42Q61L\u00b7[3H]GTP", "complex_assembly" ] ] }, { "sid": 63, "sent": "The Kd values derived for the ACK GBD with Cdc42\u03947 and full-length Cdc42 were 0.032 \u00b1 0.01 and 0.011 \u00b1 0.01 \u03bcm, respectively.", "section": "FIG", "ner": [ [ 4, 6, "Kd", "evidence" ], [ 30, 33, "ACK", "protein" ], [ 34, 37, "GBD", "structure_element" ], [ 43, 50, "Cdc42\u03947", "mutant" ], [ 55, 66, "full-length", "protein_state" ], [ 67, 72, "Cdc42", "protein" ] ] }, { "sid": 64, "sent": "The Kd values derived for the TOCA1 HR1 with Cdc42\u03947 and full-length Cdc42 were 6.05 \u00b1 1.96 and 5.39 \u00b1 1.69 \u03bcm, respectively.", "section": "FIG", "ner": [ [ 4, 6, "Kd", "evidence" ], [ 30, 35, "TOCA1", "protein" ], [ 36, 39, "HR1", "structure_element" ], [ 45, 52, "Cdc42\u03947", "mutant" ], [ 57, 68, "full-length", "protein_state" ], [ 69, 74, "Cdc42", "protein" ] ] }, { "sid": 65, "sent": "It was possible that the low affinity observed was due to negative effects of immobilization of the HR1 domain, so other methods were employed, which utilized untagged proteins.", "section": "RESULTS", "ner": [ [ 100, 103, "HR1", "structure_element" ], [ 159, 167, "untagged", "protein_state" ] ] }, { "sid": 66, "sent": "Isothermal titration calorimetry was carried out, but no heat changes were observed at a range of concentrations and temperatures (data not shown), suggesting that the interaction is predominantly entropically driven.", "section": "RESULTS", "ner": [ [ 0, 32, "Isothermal titration calorimetry", "experimental_method" ] ] }, { "sid": 67, "sent": "Other G protein-HR1 domain interactions have also failed to show heat changes in our hands.7 Infrared interferometry with immobilized Cdc42 was also attempted but was unsuccessful for both TOCA1 HR1 and for the positive control, ACK.", "section": "RESULTS", "ner": [ [ 6, 15, "G protein", "protein_type" ], [ 16, 19, "HR1", "structure_element" ], [ 93, 116, "Infrared interferometry", "experimental_method" ], [ 122, 133, "immobilized", "protein_state" ], [ 134, 139, "Cdc42", "protein" ], [ 189, 194, "TOCA1", "protein" ], [ 195, 198, "HR1", "structure_element" ], [ 229, 232, "ACK", "protein" ] ] }, { "sid": 68, "sent": "The affinity was therefore determined using competition SPAs.", "section": "RESULTS", "ner": [ [ 4, 12, "affinity", "evidence" ], [ 44, 60, "competition SPAs", "experimental_method" ] ] }, { "sid": 69, "sent": "A complex of a GST fusion of the GBD of ACK, which binds with a high affinity to Cdc42, with radiolabeled [3H]GTP\u00b7Cdc42 was preformed, and the effect of increasing concentrations of untagged TOCA1 HR1 domain was examined.", "section": "RESULTS", "ner": [ [ 15, 25, "GST fusion", "experimental_method" ], [ 33, 36, "GBD", "structure_element" ], [ 40, 43, "ACK", "protein" ], [ 81, 86, "Cdc42", "protein" ], [ 106, 119, "[3H]GTP\u00b7Cdc42", "complex_assembly" ], [ 153, 178, "increasing concentrations", "experimental_method" ], [ 182, 190, "untagged", "protein_state" ], [ 191, 196, "TOCA1", "protein" ], [ 197, 200, "HR1", "structure_element" ] ] }, { "sid": 70, "sent": "Competition of GST-ACK GBD bound to [3H]GTP\u00b7Cdc42 by free ACK GBD was used as a control and to establish the value of background counts when Cdc42 is fully displaced.", "section": "RESULTS", "ner": [ [ 15, 22, "GST-ACK", "mutant" ], [ 23, 26, "GBD", "structure_element" ], [ 27, 35, "bound to", "protein_state" ], [ 36, 49, "[3H]GTP\u00b7Cdc42", "complex_assembly" ], [ 53, 57, "free", "protein_state" ], [ 58, 61, "ACK", "protein" ], [ 62, 65, "GBD", "structure_element" ], [ 141, 146, "Cdc42", "protein" ] ] }, { "sid": 71, "sent": "The data were fitted to a binding isotherm describing competition.", "section": "RESULTS", "ner": [ [ 26, 42, "binding isotherm", "evidence" ] ] }, { "sid": 72, "sent": "Free ACK competed with itself with an affinity of 32 nm, similar to the value obtained by direct binding of 23 nm.", "section": "RESULTS", "ner": [ [ 0, 4, "Free", "protein_state" ], [ 5, 8, "ACK", "protein" ], [ 38, 46, "affinity", "evidence" ], [ 90, 104, "direct binding", "experimental_method" ] ] }, { "sid": 73, "sent": "The TOCA1 HR1 domain also fully competed with the GST-ACK but bound with an affinity of 6 \u03bcm (Fig. 1, B and C), in agreement with the low affinity observed in the direct binding experiments.", "section": "RESULTS", "ner": [ [ 4, 9, "TOCA1", "protein" ], [ 10, 13, "HR1", "structure_element" ], [ 50, 57, "GST-ACK", "mutant" ], [ 62, 67, "bound", "protein_state" ], [ 76, 84, "affinity", "evidence" ], [ 138, 146, "affinity", "evidence" ], [ 163, 189, "direct binding experiments", "experimental_method" ] ] }, { "sid": 74, "sent": "The Cdc42 construct used in the binding assays has seven residues deleted from the C terminus to facilitate purification.", "section": "RESULTS", "ner": [ [ 4, 9, "Cdc42", "protein" ], [ 32, 46, "binding assays", "experimental_method" ], [ 51, 65, "seven residues", "residue_range" ], [ 66, 73, "deleted", "experimental_method" ] ] }, { "sid": 75, "sent": "These residues are not generally required for G protein-effector interactions, including the interaction between RhoA and the PRK1 HR1a domain.", "section": "RESULTS", "ner": [ [ 46, 55, "G protein", "protein_type" ], [ 113, 117, "RhoA", "protein" ], [ 126, 130, "PRK1", "protein" ], [ 131, 135, "HR1a", "structure_element" ] ] }, { "sid": 76, "sent": "In contrast, the C terminus of Rac1 contains a polybasic sequence, which is crucial for Rac1 binding to the HR1b domain from PRK1.", "section": "RESULTS", "ner": [ [ 31, 35, "Rac1", "protein" ], [ 88, 92, "Rac1", "protein" ], [ 108, 112, "HR1b", "structure_element" ], [ 125, 129, "PRK1", "protein" ] ] }, { "sid": 77, "sent": "As the observed affinity between TOCA1 HR1 and Cdc42 was much lower than expected, we reasoned that the C terminus of Cdc42 might be necessary for a high affinity interaction.", "section": "RESULTS", "ner": [ [ 16, 24, "affinity", "evidence" ], [ 33, 38, "TOCA1", "protein" ], [ 39, 42, "HR1", "structure_element" ], [ 47, 52, "Cdc42", "protein" ], [ 118, 123, "Cdc42", "protein" ], [ 154, 162, "affinity", "evidence" ] ] }, { "sid": 78, "sent": "The binding experiments were repeated with full-length [3H]GTP\u00b7Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd \u2248 5 \u03bcm; Fig. 1C).", "section": "RESULTS", "ner": [ [ 4, 23, "binding experiments", "experimental_method" ], [ 43, 54, "full-length", "protein_state" ], [ 55, 68, "[3H]GTP\u00b7Cdc42", "complex_assembly" ], [ 78, 86, "affinity", "evidence" ], [ 94, 97, "HR1", "structure_element" ], [ 109, 120, "full-length", "protein_state" ], [ 121, 126, "Cdc42", "protein" ], [ 146, 154, "affinity", "evidence" ], [ 159, 168, "truncated", "protein_state" ], [ 169, 174, "Cdc42", "protein" ], [ 176, 178, "Kd", "evidence" ] ] }, { "sid": 79, "sent": "Thus, the C-terminal region of Cdc42 is not required for maximal binding of TOCA1 HR1.", "section": "RESULTS", "ner": [ [ 10, 27, "C-terminal region", "structure_element" ], [ 31, 36, "Cdc42", "protein" ], [ 76, 81, "TOCA1", "protein" ], [ 82, 85, "HR1", "structure_element" ] ] }, { "sid": 80, "sent": "Another possible explanation for the low affinities observed was that the HR1 domain alone is not sufficient for maximal binding of the TOCA proteins to Cdc42 and that the other domains are required.", "section": "RESULTS", "ner": [ [ 41, 51, "affinities", "evidence" ], [ 74, 77, "HR1", "structure_element" ], [ 85, 90, "alone", "protein_state" ], [ 136, 149, "TOCA proteins", "protein_type" ], [ 153, 158, "Cdc42", "protein" ] ] }, { "sid": 81, "sent": "Indeed, GST pull-downs performed with in vitro translated human TOCA1 fragments had suggested that residues N-terminal to the HR1 domain may be required to stabilize the HR1 domain structure.", "section": "RESULTS", "ner": [ [ 8, 22, "GST pull-downs", "experimental_method" ], [ 58, 63, "human", "species" ], [ 64, 69, "TOCA1", "protein" ], [ 126, 129, "HR1", "structure_element" ], [ 170, 173, "HR1", "structure_element" ] ] }, { "sid": 82, "sent": "Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, \u03b2-PIX.", "section": "RESULTS", "ner": [ [ 18, 21, "BAR", "structure_element" ], [ 26, 29, "SH3", "structure_element" ], [ 86, 96, "G proteins", "protein_type" ], [ 107, 110, "BAR", "structure_element" ], [ 121, 130, "Arfaptin2", "protein" ], [ 140, 144, "Rac1", "protein" ], [ 149, 153, "Arl1", "protein" ], [ 165, 168, "SH3", "structure_element" ], [ 209, 213, "Rac1", "protein" ], [ 222, 256, "guanine nucleotide exchange factor", "protein" ], [ 258, 263, "\u03b2-PIX", "protein" ] ] }, { "sid": 83, "sent": "TOCA1 dimerizes via its F-BAR domain, which could also affect Cdc42 binding, for example by presenting two HR1 domains for Cdc42 interactions.", "section": "RESULTS", "ner": [ [ 0, 5, "TOCA1", "protein" ], [ 6, 11, "dimer", "oligomeric_state" ], [ 24, 29, "F-BAR", "structure_element" ], [ 62, 67, "Cdc42", "protein" ], [ 107, 110, "HR1", "structure_element" ], [ 123, 128, "Cdc42", "protein" ] ] }, { "sid": 84, "sent": "Various TOCA1 fragments (Fig. 2A) were therefore assessed for binding to full-length Cdc42 by direct SPA.", "section": "RESULTS", "ner": [ [ 8, 13, "TOCA1", "protein" ], [ 73, 84, "full-length", "protein_state" ], [ 85, 90, "Cdc42", "protein" ], [ 101, 104, "SPA", "experimental_method" ] ] }, { "sid": 85, "sent": "The isolated F-BAR domain showed no binding to full-length Cdc42 (Fig. 2B).", "section": "RESULTS", "ner": [ [ 13, 18, "F-BAR", "structure_element" ], [ 47, 58, "full-length", "protein_state" ], [ 59, 64, "Cdc42", "protein" ] ] }, { "sid": 86, "sent": "Full-length TOCA1 and \u0394SH3 TOCA1 bound with micromolar affinity (Fig. 2B), in a similar manner to the isolated HR1 domain (Fig. 1A).", "section": "RESULTS", "ner": [ [ 0, 11, "Full-length", "protein_state" ], [ 12, 17, "TOCA1", "protein" ], [ 22, 26, "\u0394SH3", "mutant" ], [ 27, 32, "TOCA1", "protein" ], [ 33, 38, "bound", "protein_state" ], [ 111, 114, "HR1", "structure_element" ] ] }, { "sid": 87, "sent": "The HR1-SH3 protein could not be purified to homogeneity as a fusion protein, so it was assayed in competition assays after cleavage of the His tag.", "section": "RESULTS", "ner": [ [ 4, 11, "HR1-SH3", "mutant" ], [ 99, 117, "competition assays", "experimental_method" ] ] }, { "sid": 88, "sent": "This construct competed with GST-ACK GBD to give a similar affinity to the HR1 domain alone (Kd = 4.6 \u00b1 4 \u03bcm; Fig. 2C).", "section": "RESULTS", "ner": [ [ 29, 36, "GST-ACK", "mutant" ], [ 37, 40, "GBD", "structure_element" ], [ 75, 78, "HR1", "structure_element" ], [ 86, 91, "alone", "protein_state" ], [ 93, 95, "Kd", "evidence" ] ] }, { "sid": 89, "sent": "Taken together, these data suggest that the TOCA1 HR1 domain is sufficient for maximal binding and that this binding is of a relatively low affinity compared with many other Cdc42\u00b7effector complexes.", "section": "RESULTS", "ner": [ [ 44, 49, "TOCA1", "protein" ], [ 50, 53, "HR1", "structure_element" ], [ 174, 179, "Cdc42", "protein" ] ] }, { "sid": 90, "sent": "The Cdc42-HR1 interaction is of low affinity in the context of full-length protein and in TOCA1 paralogues.", "section": "FIG", "ner": [ [ 4, 13, "Cdc42-HR1", "complex_assembly" ], [ 63, 74, "full-length", "protein_state" ], [ 90, 95, "TOCA1", "protein" ] ] }, { "sid": 91, "sent": " A, diagram illustrating the TOCA1 constructs assayed for Cdc42 binding.", "section": "FIG", "ner": [ [ 29, 34, "TOCA1", "protein" ], [ 58, 63, "Cdc42", "protein" ] ] }, { "sid": 92, "sent": "Domain boundaries are derived from secondary structure predictions; B, binding curves derived from direct binding assays, in which the indicated concentrations of Cdc42\u03947Q61L\u00b7[3H]GTP were incubated with 30 nm GST-ACK or His-tagged TOCA1 constructs, as indicated, in SPAs.", "section": "FIG", "ner": [ [ 71, 85, "binding curves", "evidence" ], [ 99, 120, "direct binding assays", "experimental_method" ], [ 163, 182, "Cdc42\u03947Q61L\u00b7[3H]GTP", "complex_assembly" ], [ 188, 197, "incubated", "experimental_method" ], [ 209, 216, "GST-ACK", "mutant" ], [ 220, 230, "His-tagged", "protein_state" ], [ 231, 236, "TOCA1", "protein" ], [ 266, 270, "SPAs", "experimental_method" ] ] }, { "sid": 93, "sent": "The SPA signal was corrected by subtraction of control data with no fusion protein.", "section": "FIG", "ner": [ [ 4, 7, "SPA", "experimental_method" ] ] }, { "sid": 94, "sent": "The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal.", "section": "FIG", "ner": [ [ 26, 42, "binding isotherm", "evidence" ], [ 63, 65, "Kd", "evidence" ] ] }, { "sid": 95, "sent": "C\u2013E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42\u03947Q61L\u00b7[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L\u00b7[3H]GTP.", "section": "FIG", "ner": [ [ 32, 47, "competition SPA", "experimental_method" ], [ 113, 118, "TOCA1", "protein" ], [ 119, 126, "HR1-SH3", "mutant" ], [ 137, 145, "titrated", "experimental_method" ], [ 157, 164, "GST-ACK", "mutant" ], [ 175, 194, "Cdc42\u03947Q61L\u00b7[3H]GTP", "complex_assembly" ], [ 202, 205, "HR1", "structure_element" ], [ 217, 220, "HR1", "structure_element" ], [ 230, 238, "titrated", "experimental_method" ], [ 250, 257, "GST-ACK", "mutant" ], [ 268, 287, "Cdc42FLQ61L\u00b7[3H]GTP", "complex_assembly" ] ] }, { "sid": 96, "sent": "The low affinity of the TOCA1 HR1-Cdc42 interaction raised the question of whether the other known Cdc42-binding TOCA family proteins, FBP17 and CIP4, also bind weakly.", "section": "RESULTS", "ner": [ [ 24, 29, "TOCA1", "protein" ], [ 30, 33, "HR1", "structure_element" ], [ 34, 39, "Cdc42", "protein" ], [ 99, 104, "Cdc42", "protein" ], [ 113, 133, "TOCA family proteins", "protein_type" ], [ 135, 140, "FBP17", "protein" ], [ 145, 149, "CIP4", "protein" ] ] }, { "sid": 97, "sent": "The HR1 domains from FBP17 and CIP4 were purified and assayed for Cdc42 binding in competition SPAs, analogous to those carried out with the TOCA1 HR1 domain.", "section": "RESULTS", "ner": [ [ 4, 7, "HR1", "structure_element" ], [ 21, 26, "FBP17", "protein" ], [ 31, 35, "CIP4", "protein" ], [ 41, 49, "purified", "experimental_method" ], [ 66, 71, "Cdc42", "protein" ], [ 83, 99, "competition SPAs", "experimental_method" ], [ 141, 146, "TOCA1", "protein" ], [ 147, 150, "HR1", "structure_element" ] ] }, { "sid": 98, "sent": "The affinities of both the FBP17 and CIP4 HR1 domains were also in the low micromolar range (10 and 5 \u03bcm, respectively) (Fig. 2, D and E), suggesting that low affinity interactions with Cdc42 are a common feature within the TOCA family.", "section": "RESULTS", "ner": [ [ 4, 14, "affinities", "evidence" ], [ 27, 32, "FBP17", "protein" ], [ 37, 41, "CIP4", "protein" ], [ 42, 45, "HR1", "structure_element" ], [ 186, 191, "Cdc42", "protein" ], [ 224, 235, "TOCA family", "protein_type" ] ] }, { "sid": 99, "sent": "Structure of the TOCA1 HR1 Domain", "section": "RESULTS", "ner": [ [ 0, 9, "Structure", "evidence" ], [ 17, 22, "TOCA1", "protein" ], [ 23, 26, "HR1", "structure_element" ] ] }, { "sid": 100, "sent": "Because the TOCA1 HR1 domain was sufficient for maximal Cdc42-binding, we used this construct for structural studies.", "section": "RESULTS", "ner": [ [ 12, 17, "TOCA1", "protein" ], [ 18, 21, "HR1", "structure_element" ], [ 56, 61, "Cdc42", "protein" ] ] }, { "sid": 101, "sent": "Initial experiments were performed with TOCA1 residues 324\u2013426, but we observed that the N terminus was cleaved during purification to yield a new N terminus at residue 330 (data not shown).", "section": "RESULTS", "ner": [ [ 40, 45, "TOCA1", "protein" ], [ 55, 62, "324\u2013426", "residue_range" ], [ 169, 172, "330", "residue_number" ] ] }, { "sid": 102, "sent": "We therefore engineered a construct comprising residues 330\u2013426 to produce the minimal, stable HR1 domain.", "section": "RESULTS", "ner": [ [ 56, 63, "330\u2013426", "residue_range" ], [ 79, 86, "minimal", "protein_state" ], [ 88, 94, "stable", "protein_state" ], [ 95, 98, "HR1", "structure_element" ] ] }, { "sid": 103, "sent": "2,778 non-degenerate NOE restraints were used in initial structure calculations (1,791 unambiguous and 987 ambiguous), derived from three-dimensional 15N-separated NOESY and 13C-separated NOESY experiments.", "section": "RESULTS", "ner": [ [ 21, 35, "NOE restraints", "evidence" ], [ 57, 79, "structure calculations", "experimental_method" ], [ 150, 169, "15N-separated NOESY", "experimental_method" ], [ 174, 193, "13C-separated NOESY", "experimental_method" ] ] }, { "sid": 104, "sent": "There were 1,845 unambiguous NOEs and 757 ambiguous NOEs after eight iterations.", "section": "RESULTS", "ner": [ [ 29, 33, "NOEs", "evidence" ], [ 52, 56, "NOEs", "evidence" ] ] }, { "sid": 105, "sent": "100 structures were calculated in the final iteration; the 50 lowest energy structures were water-refined; and of these, the 35 lowest energy structures were analyzed.", "section": "RESULTS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 20, 30, "calculated", "experimental_method" ], [ 76, 86, "structures", "evidence" ], [ 142, 152, "structures", "evidence" ] ] }, { "sid": 106, "sent": "Table 1 indicates that the HR1 domain structure is well defined by the NMR data.", "section": "RESULTS", "ner": [ [ 27, 30, "HR1", "structure_element" ], [ 38, 47, "structure", "evidence" ], [ 71, 74, "NMR", "experimental_method" ] ] }, { "sid": 107, "sent": "a , the average root mean square deviations for the ensemble \u00b1 S.D.", "section": "TABLE", "ner": [ [ 12, 47, "average root mean square deviations", "evidence" ] ] }, { "sid": 108, "sent": "b c, values for the structure that is closest to the mean.", "section": "TABLE", "ner": [ [ 24, 33, "structure", "evidence" ] ] }, { "sid": 109, "sent": "The structure closest to the mean is shown in Fig. 3A.", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ] ] }, { "sid": 110, "sent": "The two \u03b1-helices of the HR1 domain interact to form an anti-parallel coiled-coil with a slight left-handed twist, reminiscent of the HR1 domains of CIP4 (PDB code 2KE4) and PRK1 (PDB codes 1CXZ and 1URF).", "section": "RESULTS", "ner": [ [ 8, 17, "\u03b1-helices", "structure_element" ], [ 25, 28, "HR1", "structure_element" ], [ 56, 81, "anti-parallel coiled-coil", "structure_element" ], [ 134, 137, "HR1", "structure_element" ], [ 149, 153, "CIP4", "protein" ], [ 174, 178, "PRK1", "protein" ] ] }, { "sid": 111, "sent": "A sequence alignment illustrating the secondary structure elements of the TOCA1 and CIP4 HR1 domains and the HR1a and HR1b domains from PRK1 is shown in Fig. 3B.", "section": "RESULTS", "ner": [ [ 2, 20, "sequence alignment", "experimental_method" ], [ 74, 79, "TOCA1", "protein" ], [ 84, 88, "CIP4", "protein" ], [ 89, 92, "HR1", "structure_element" ], [ 109, 113, "HR1a", "structure_element" ], [ 118, 122, "HR1b", "structure_element" ], [ 136, 140, "PRK1", "protein" ] ] }, { "sid": 112, "sent": "The structure of the TOCA1 HR1 domain.", "section": "FIG", "ner": [ [ 4, 13, "structure", "evidence" ], [ 21, 26, "TOCA1", "protein" ], [ 27, 30, "HR1", "structure_element" ] ] }, { "sid": 113, "sent": " A, the backbone trace of the 35 lowest energy structures of the HR1 domain overlaid with the structure closest to the mean is shown alongside a schematic representation of the structure closest to the mean.", "section": "FIG", "ner": [ [ 17, 22, "trace", "evidence" ], [ 47, 57, "structures", "evidence" ], [ 65, 68, "HR1", "structure_element" ], [ 94, 103, "structure", "evidence" ], [ 177, 186, "structure", "evidence" ] ] }, { "sid": 114, "sent": "Flexible regions at the N and C termini (residues 330\u2013333 and 421\u2013426) are omitted for clarity.", "section": "FIG", "ner": [ [ 50, 57, "330\u2013333", "residue_range" ], [ 62, 69, "421\u2013426", "residue_range" ] ] }, { "sid": 115, "sent": "B, a sequence alignment of the HR1 domains from TOCA1, CIP4, and PRK1.", "section": "FIG", "ner": [ [ 5, 23, "sequence alignment", "experimental_method" ], [ 31, 34, "HR1", "structure_element" ], [ 48, 53, "TOCA1", "protein" ], [ 55, 59, "CIP4", "protein" ], [ 65, 69, "PRK1", "protein" ] ] }, { "sid": 116, "sent": "The secondary structure was deduced using Stride, based on the Ramachandran angles, and is indicated as follows: gray, turn; yellow, \u03b1-helix; blue, 310 helix; white, coil.", "section": "FIG", "ner": [ [ 42, 48, "Stride", "experimental_method" ], [ 63, 82, "Ramachandran angles", "evidence" ], [ 133, 140, "\u03b1-helix", "structure_element" ], [ 148, 157, "310 helix", "structure_element" ] ] }, { "sid": 117, "sent": "C, a close-up of the N-terminal region of TOCA1 HR1, indicating some of the NOEs defining its position with respect to the two \u03b1-helices.", "section": "FIG", "ner": [ [ 42, 47, "TOCA1", "protein" ], [ 48, 51, "HR1", "structure_element" ], [ 76, 80, "NOEs", "evidence" ], [ 127, 136, "\u03b1-helices", "structure_element" ] ] }, { "sid": 118, "sent": "Dotted lines, NOE restraints.", "section": "FIG", "ner": [ [ 14, 28, "NOE restraints", "evidence" ] ] }, { "sid": 119, "sent": "D, a close-up of the interhelix loop region showing some of the contacts between the loop and helix 1.", "section": "FIG", "ner": [ [ 21, 36, "interhelix loop", "structure_element" ], [ 85, 89, "loop", "structure_element" ], [ 94, 101, "helix 1", "structure_element" ] ] }, { "sid": 120, "sent": "In the HR1a domain of PRK1, a region N-terminal to helix 1 forms a short \u03b1-helix, which packs against both helices of the HR1 domain.", "section": "RESULTS", "ner": [ [ 7, 11, "HR1a", "structure_element" ], [ 22, 26, "PRK1", "protein" ], [ 51, 58, "helix 1", "structure_element" ], [ 67, 80, "short \u03b1-helix", "structure_element" ], [ 122, 125, "HR1", "structure_element" ] ] }, { "sid": 121, "sent": "This region of TOCA1 HR1 (residues 334\u2013340) is well defined in the family of structures (Fig. 3A) but does not form an \u03b1-helix.", "section": "RESULTS", "ner": [ [ 15, 20, "TOCA1", "protein" ], [ 21, 24, "HR1", "structure_element" ], [ 35, 42, "334\u2013340", "residue_range" ], [ 77, 87, "structures", "evidence" ], [ 119, 126, "\u03b1-helix", "structure_element" ] ] }, { "sid": 122, "sent": "It instead forms a series of turns, defined by NOE restraints observed between residues separated by one (residues 332\u2013334, 333\u2013335, etc.) or two (residues 337\u2013340) residues in the sequence and the \u03c6 and \u03c8 angles, assessed using Stride.", "section": "RESULTS", "ner": [ [ 47, 61, "NOE restraints", "evidence" ], [ 115, 122, "332\u2013334", "residue_range" ], [ 124, 131, "333\u2013335", "residue_range" ], [ 156, 163, "337\u2013340", "residue_range" ], [ 198, 212, "\u03c6 and \u03c8 angles", "evidence" ], [ 229, 235, "Stride", "experimental_method" ] ] }, { "sid": 123, "sent": "These turns cause the chain to reverse direction, allowing the N-terminal segment (residues 334\u2013340) to contact both helices of the HR1 domain.", "section": "RESULTS", "ner": [ [ 92, 99, "334\u2013340", "residue_range" ], [ 132, 135, "HR1", "structure_element" ] ] }, { "sid": 124, "sent": "Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1.", "section": "RESULTS", "ner": [ [ 11, 15, "NOEs", "evidence" ], [ 38, 45, "Leu-334", "residue_name_number" ], [ 47, 54, "Glu-335", "residue_name_number" ], [ 60, 67, "Asp-336", "residue_name_number" ], [ 73, 80, "Trp-413", "residue_name_number" ], [ 84, 91, "helix 2", "structure_element" ], [ 93, 100, "Leu-334", "residue_name_number" ], [ 106, 113, "Lys-409", "residue_name_number" ], [ 117, 124, "helix 2", "structure_element" ], [ 130, 137, "Phe-337", "residue_name_number" ], [ 142, 149, "Ser-338", "residue_name_number" ], [ 155, 162, "Arg-345", "residue_name_number" ], [ 164, 171, "Arg-348", "residue_name_number" ], [ 177, 184, "Leu-349", "residue_name_number" ], [ 188, 195, "helix 1", "structure_element" ] ] }, { "sid": 125, "sent": "The two \u03b1-helices of TOCA1 HR1 are separated by a long loop of 10 residues (residues 380\u2013389) that contains two short 310 helices (residues 381\u2013383 and 386\u2013389).", "section": "RESULTS", "ner": [ [ 8, 17, "\u03b1-helices", "structure_element" ], [ 21, 26, "TOCA1", "protein" ], [ 27, 30, "HR1", "structure_element" ], [ 55, 59, "loop", "structure_element" ], [ 85, 92, "380\u2013389", "residue_range" ], [ 112, 129, "short 310 helices", "structure_element" ], [ 140, 147, "381\u2013383", "residue_range" ], [ 152, 159, "386\u2013389", "residue_range" ] ] }, { "sid": 126, "sent": "Interestingly, side chains of residues within the loop region point back toward helix 1; for example, there are numerous distinct NOEs between the side chains of Asn-380 and Met-383 of the loop region and Tyr-377 and Val-376 of helix 1 (Fig. 3D).", "section": "RESULTS", "ner": [ [ 50, 61, "loop region", "structure_element" ], [ 80, 87, "helix 1", "structure_element" ], [ 162, 169, "Asn-380", "residue_name_number" ], [ 174, 181, "Met-383", "residue_name_number" ], [ 189, 200, "loop region", "structure_element" ], [ 205, 212, "Tyr-377", "residue_name_number" ], [ 217, 224, "Val-376", "residue_name_number" ], [ 228, 235, "helix 1", "structure_element" ] ] }, { "sid": 127, "sent": "The backbone NH and CH\u03b1 groups of Gly-384 and Asp-385 also show NOEs with the side chain of Tyr-377.", "section": "RESULTS", "ner": [ [ 34, 41, "Gly-384", "residue_name_number" ], [ 46, 53, "Asp-385", "residue_name_number" ], [ 92, 99, "Tyr-377", "residue_name_number" ] ] }, { "sid": 128, "sent": "Mapping the TOCA1 and Cdc42 Binding Interfaces", "section": "RESULTS", "ner": [ [ 12, 17, "TOCA1", "protein" ], [ 22, 46, "Cdc42 Binding Interfaces", "site" ] ] }, { "sid": 129, "sent": "The HR1TOCA1-Cdc42 interface was investigated using NMR spectroscopy.", "section": "RESULTS", "ner": [ [ 4, 28, "HR1TOCA1-Cdc42 interface", "site" ], [ 52, 68, "NMR spectroscopy", "experimental_method" ] ] }, { "sid": 130, "sent": "A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42\u03947Q61L\u00b7GMPPNP to map the Cdc42-binding surface.", "section": "RESULTS", "ner": [ [ 12, 20, "15N HSQC", "experimental_method" ], [ 49, 52, "15N", "chemical" ], [ 53, 60, "labeled", "protein_state" ], [ 61, 66, "TOCA1", "protein" ], [ 67, 70, "HR1", "structure_element" ], [ 85, 96, "presence of", "protein_state" ], [ 97, 122, "increasing concentrations", "experimental_method" ], [ 126, 135, "unlabeled", "protein_state" ], [ 136, 154, "Cdc42\u03947Q61L\u00b7GMPPNP", "complex_assembly" ], [ 166, 187, "Cdc42-binding surface", "site" ] ] }, { "sid": 131, "sent": "A comparison of the 15N HSQC spectra of free HR1 and HR1 in the presence of excess Cdc42 shows that although some peaks were shifted, several were much broader in the complex, and a considerable subset had disappeared (Fig. 4A).", "section": "RESULTS", "ner": [ [ 20, 28, "15N HSQC", "experimental_method" ], [ 29, 36, "spectra", "evidence" ], [ 40, 44, "free", "protein_state" ], [ 45, 48, "HR1", "structure_element" ], [ 53, 56, "HR1", "structure_element" ], [ 64, 75, "presence of", "protein_state" ], [ 83, 88, "Cdc42", "protein" ] ] }, { "sid": 132, "sent": "This behavior cannot be explained by the increase in molecular mass (from 12 to 33 kDa) when Cdc42 binds and is more likely to be due to conformational exchange.", "section": "RESULTS", "ner": [ [ 93, 98, "Cdc42", "protein" ] ] }, { "sid": 133, "sent": "Overall chemical shift perturbations (CSPs) were calculated for each residue, whereas those that had disappeared were assigned a shift change of 0.2 (Fig. 4B).", "section": "RESULTS", "ner": [ [ 8, 36, "chemical shift perturbations", "experimental_method" ], [ 38, 42, "CSPs", "experimental_method" ] ] }, { "sid": 134, "sent": "A peak that disappeared or had a CSP above the mean CSP for the spectrum was considered to be significantly affected.", "section": "RESULTS", "ner": [ [ 33, 36, "CSP", "experimental_method" ], [ 52, 55, "CSP", "experimental_method" ] ] }, { "sid": 135, "sent": "Mapping the binding surface of Cdc42 onto the TOCA1 HR1 domain.", "section": "FIG", "ner": [ [ 12, 27, "binding surface", "site" ], [ 31, 36, "Cdc42", "protein" ], [ 46, 51, "TOCA1", "protein" ], [ 52, 55, "HR1", "structure_element" ] ] }, { "sid": 136, "sent": " A, the 15N HSQC of 200 \u03bcm TOCA1 HR1 domain is shown in the free form (black) and in the presence of a 4-fold molar excess of Cdc42\u03947Q61L\u00b7GMPPNP (red).", "section": "FIG", "ner": [ [ 8, 16, "15N HSQC", "experimental_method" ], [ 27, 32, "TOCA1", "protein" ], [ 33, 36, "HR1", "structure_element" ], [ 60, 69, "free form", "protein_state" ], [ 89, 100, "presence of", "protein_state" ], [ 126, 144, "Cdc42\u03947Q61L\u00b7GMPPNP", "complex_assembly" ] ] }, { "sid": 137, "sent": "B, CSPs were calculated as described under \u201cExperimental Procedures\u201d and are shown for backbone and side chain NH groups.", "section": "FIG", "ner": [ [ 3, 7, "CSPs", "experimental_method" ] ] }, { "sid": 138, "sent": "The mean CSP is marked with a red line.", "section": "FIG", "ner": [ [ 9, 12, "CSP", "experimental_method" ] ] }, { "sid": 139, "sent": "Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.2 but were excluded when calculating the mean CSP and are indicated with open bars.", "section": "FIG", "ner": [ [ 33, 44, "presence of", "protein_state" ], [ 45, 50, "Cdc42", "protein" ], [ 67, 70, "CSP", "experimental_method" ], [ 122, 125, "CSP", "experimental_method" ] ] }, { "sid": 140, "sent": "Those that were not traceable due to spectral overlap were assigned a CSP of zero and are marked with an asterisk below the bar.", "section": "FIG", "ner": [ [ 70, 73, "CSP", "experimental_method" ] ] }, { "sid": 141, "sent": "Residues with affected side chain CSPs derived from 13C HSQCs are marked with green asterisks above the bars.", "section": "FIG", "ner": [ [ 34, 38, "CSPs", "experimental_method" ], [ 52, 61, "13C HSQCs", "experimental_method" ] ] }, { "sid": 142, "sent": "C, a schematic representation of the HR1 domain.", "section": "FIG", "ner": [ [ 37, 40, "HR1", "structure_element" ] ] }, { "sid": 143, "sent": "Residues with significantly affected backbone or side chain chemical shifts when Cdc42 bound and that are buried are colored dark blue, whereas those that are solvent-accessible are colored yellow.", "section": "FIG", "ner": [ [ 81, 92, "Cdc42 bound", "protein_state" ], [ 159, 177, "solvent-accessible", "protein_state" ] ] }, { "sid": 144, "sent": "Residues with significantly affected backbone and side chain groups that are solvent-accessible are colored red.", "section": "FIG", "ner": [ [ 77, 95, "solvent-accessible", "protein_state" ] ] }, { "sid": 145, "sent": "A close-up of the binding region is shown, with affected side chain heavy atoms shown as sticks.", "section": "FIG", "ner": [ [ 18, 32, "binding region", "site" ] ] }, { "sid": 146, "sent": "D, the G protein-binding region is marked in red onto structures of the HR1 domains as indicated.", "section": "FIG", "ner": [ [ 7, 31, "G protein-binding region", "site" ], [ 54, 64, "structures", "evidence" ], [ 72, 75, "HR1", "structure_element" ] ] }, { "sid": 147, "sent": "15N HSQC shift mapping experiments report on changes to amide groups, which are mainly inaccessible because they are buried inside the helices and are involved in hydrogen bonds.", "section": "RESULTS", "ner": [ [ 0, 22, "15N HSQC shift mapping", "experimental_method" ], [ 135, 142, "helices", "structure_element" ], [ 163, 177, "hydrogen bonds", "bond_interaction" ] ] }, { "sid": 148, "sent": "Therefore, 13C HSQC and methyl-selective SOFAST-HMQC experiments were also recorded on 15N,13C-labeled TOCA1 HR1 to yield more information on side chain involvement.", "section": "RESULTS", "ner": [ [ 11, 19, "13C HSQC", "experimental_method" ], [ 24, 52, "methyl-selective SOFAST-HMQC", "experimental_method" ], [ 87, 90, "15N", "chemical" ], [ 91, 94, "13C", "chemical" ], [ 95, 102, "labeled", "protein_state" ], [ 103, 108, "TOCA1", "protein" ], [ 109, 112, "HR1", "structure_element" ] ] }, { "sid": 149, "sent": "Side chains whose CH groups disappeared in the presence of Cdc42 are marked on the graph in Fig. 4B with green asterisks.", "section": "RESULTS", "ner": [ [ 47, 58, "presence of", "protein_state" ], [ 59, 64, "Cdc42", "protein" ] ] }, { "sid": 150, "sent": "TOCA1 residues whose signals were affected by Cdc42 binding were mapped onto the structure of TOCA1 HR1 (Fig. 4C).", "section": "RESULTS", "ner": [ [ 0, 5, "TOCA1", "protein" ], [ 46, 51, "Cdc42", "protein" ], [ 81, 90, "structure", "evidence" ], [ 94, 99, "TOCA1", "protein" ], [ 100, 103, "HR1", "structure_element" ] ] }, { "sid": 151, "sent": "The changes were localized to one end of the coiled-coil, and the binding site appeared to include residues from both \u03b1-helices and the loop region that joins them.", "section": "RESULTS", "ner": [ [ 45, 56, "coiled-coil", "structure_element" ], [ 66, 78, "binding site", "site" ], [ 118, 127, "\u03b1-helices", "structure_element" ], [ 136, 147, "loop region", "structure_element" ] ] }, { "sid": 152, "sent": "The residues in the interhelical loop and helix 1 that contact each other (Fig. 3D) show shift changes in their backbone NH and side chains in the presence of Cdc42.", "section": "RESULTS", "ner": [ [ 20, 37, "interhelical loop", "structure_element" ], [ 42, 49, "helix 1", "structure_element" ], [ 147, 158, "presence of", "protein_state" ], [ 159, 164, "Cdc42", "protein" ] ] }, { "sid": 153, "sent": "For example, the side chain of Asn-380 and the backbones of Val-376 and Tyr-377 were significantly affected but are all buried in the free TOCA1 HR1 structure, indicating that local conformational changes in the loop may facilitate complex formation.", "section": "RESULTS", "ner": [ [ 31, 38, "Asn-380", "residue_name_number" ], [ 60, 67, "Val-376", "residue_name_number" ], [ 72, 79, "Tyr-377", "residue_name_number" ], [ 134, 138, "free", "protein_state" ], [ 139, 144, "TOCA1", "protein" ], [ 145, 148, "HR1", "structure_element" ], [ 149, 158, "structure", "evidence" ], [ 212, 216, "loop", "structure_element" ] ] }, { "sid": 154, "sent": "The chemical shift mapping data indicate that the G protein-binding region of the TOCA1 HR1 domain is broadly similar to that of the CIP4 and PRK1 HR1 domains (Figs. 3B and 4D).", "section": "RESULTS", "ner": [ [ 4, 26, "chemical shift mapping", "experimental_method" ], [ 50, 74, "G protein-binding region", "site" ], [ 82, 87, "TOCA1", "protein" ], [ 88, 91, "HR1", "structure_element" ], [ 133, 137, "CIP4", "protein" ], [ 142, 146, "PRK1", "protein" ], [ 147, 150, "HR1", "structure_element" ] ] }, { "sid": 155, "sent": "The corresponding 15N and 13C NMR experiments were also recorded on 15N-Cdc42\u03947Q61L\u00b7GMPPNP or 15N/13C -Cdc42\u03947Q61L\u00b7GMPPNP in the presence of unlabeled HR1 domain.", "section": "RESULTS", "ner": [ [ 18, 21, "15N", "experimental_method" ], [ 26, 33, "13C NMR", "experimental_method" ], [ 68, 71, "15N", "chemical" ], [ 72, 90, "Cdc42\u03947Q61L\u00b7GMPPNP", "complex_assembly" ], [ 94, 97, "15N", "chemical" ], [ 98, 101, "13C", "chemical" ], [ 103, 121, "Cdc42\u03947Q61L\u00b7GMPPNP", "complex_assembly" ], [ 129, 140, "presence of", "protein_state" ], [ 141, 150, "unlabeled", "protein_state" ], [ 151, 154, "HR1", "structure_element" ] ] }, { "sid": 156, "sent": "The overall CSP was calculated for each residue.", "section": "RESULTS", "ner": [ [ 12, 15, "CSP", "experimental_method" ] ] }, { "sid": 157, "sent": "As was the case when labeled HR1 was observed, several peaks were shifted in the complex, but many disappeared, indicating exchange on an unfavorable, millisecond time scale (Fig. 5A).", "section": "RESULTS", "ner": [ [ 21, 28, "labeled", "protein_state" ], [ 29, 32, "HR1", "structure_element" ] ] }, { "sid": 158, "sent": "Detailed side chain data could not be obtained for all residues due to spectral overlap, but constant time 13C HSQC and methyl-selective SOFAST-HMQC experiments provided further information on certain well resolved side chains (marked with green asterisks in Fig. 5B).", "section": "RESULTS", "ner": [ [ 93, 115, "constant time 13C HSQC", "experimental_method" ], [ 120, 148, "methyl-selective SOFAST-HMQC", "experimental_method" ] ] }, { "sid": 159, "sent": "Mapping the binding surface of the HR1 domain onto Cdc42.", "section": "FIG", "ner": [ [ 12, 27, "binding surface", "site" ], [ 35, 38, "HR1", "structure_element" ], [ 51, 56, "Cdc42", "protein" ] ] }, { "sid": 160, "sent": " A, the 15N HSQC of Cdc42\u03947Q61L\u00b7GMPPNP is shown in its free form (black) and in the presence of excess TOCA1 HR1 domain (1:2.2, red).", "section": "FIG", "ner": [ [ 8, 16, "15N HSQC", "experimental_method" ], [ 20, 38, "Cdc42\u03947Q61L\u00b7GMPPNP", "complex_assembly" ], [ 55, 64, "free form", "protein_state" ], [ 84, 95, "presence of", "protein_state" ], [ 103, 108, "TOCA1", "protein" ], [ 109, 112, "HR1", "structure_element" ] ] }, { "sid": 161, "sent": "B, CSPs are shown for backbone NH groups.", "section": "FIG", "ner": [ [ 3, 7, "CSPs", "experimental_method" ] ] }, { "sid": 162, "sent": "The red line indicates the mean CSP, plus one S.D. Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.1 and are indicated with open bars.", "section": "FIG", "ner": [ [ 32, 35, "CSP", "experimental_method" ], [ 84, 95, "presence of", "protein_state" ], [ 96, 101, "Cdc42", "protein" ], [ 118, 121, "CSP", "experimental_method" ] ] }, { "sid": 163, "sent": "Residues with disappeared peaks in 13C HSQC experiments are marked on the chart with green asterisks.", "section": "FIG", "ner": [ [ 35, 43, "13C HSQC", "experimental_method" ] ] }, { "sid": 164, "sent": "C, the residues with significantly affected backbone and side chain groups are highlighted on an NMR structure of free Cdc42\u03947Q61L\u00b7GMPPNP; those that are buried are colored dark blue, whereas those that are solvent-accessible are colored red.", "section": "FIG", "ner": [ [ 97, 100, "NMR", "experimental_method" ], [ 101, 110, "structure", "evidence" ], [ 114, 118, "free", "protein_state" ], [ 119, 137, "Cdc42\u03947Q61L\u00b7GMPPNP", "complex_assembly" ], [ 207, 225, "solvent-accessible", "protein_state" ] ] }, { "sid": 165, "sent": "Residues with either side chain or backbone groups affected are colored blue if buried and yellow if solvent-accessible.", "section": "FIG", "ner": [ [ 101, 119, "solvent-accessible", "protein_state" ] ] }, { "sid": 166, "sent": "Residues without information from shift mapping are colored gray.", "section": "FIG", "ner": [ [ 34, 47, "shift mapping", "experimental_method" ] ] }, { "sid": 167, "sent": "The flexible switch regions are circled.", "section": "FIG", "ner": [ [ 4, 12, "flexible", "protein_state" ], [ 13, 27, "switch regions", "site" ] ] }, { "sid": 168, "sent": "As many of the peaks disappeared, the mean chemical shift change was relatively low, so a threshold of the mean plus one S.D. value was used to define a significant CSP.", "section": "RESULTS", "ner": [ [ 38, 64, "mean chemical shift change", "evidence" ], [ 165, 168, "CSP", "experimental_method" ] ] }, { "sid": 169, "sent": "Parts of the switch regions (Fig. 5, B and C) are invisible in NMR spectra recorded on free Cdc42 due to conformational exchange.", "section": "RESULTS", "ner": [ [ 13, 27, "switch regions", "site" ], [ 63, 66, "NMR", "experimental_method" ], [ 67, 74, "spectra", "evidence" ], [ 87, 91, "free", "protein_state" ], [ 92, 97, "Cdc42", "protein" ] ] }, { "sid": 170, "sent": "These switch regions become visible in Cdc42 and other small G protein\u00b7effector complexes due to a decrease in conformational freedom upon complex formation.", "section": "RESULTS", "ner": [ [ 6, 20, "switch regions", "site" ], [ 39, 44, "Cdc42", "protein" ], [ 61, 70, "G protein", "protein_type" ] ] }, { "sid": 171, "sent": "The switch regions of Cdc42 did not, however, become visible in the presence of the TOCA1 HR1 domain.", "section": "RESULTS", "ner": [ [ 4, 18, "switch regions", "site" ], [ 22, 27, "Cdc42", "protein" ], [ 68, 79, "presence of", "protein_state" ], [ 84, 89, "TOCA1", "protein" ], [ 90, 93, "HR1", "structure_element" ] ] }, { "sid": 172, "sent": "Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain.", "section": "RESULTS", "ner": [ [ 8, 14, "Ser-30", "residue_name_number" ], [ 18, 26, "switch I", "site" ], [ 31, 37, "Arg-66", "residue_name_number" ], [ 39, 45, "Arg-68", "residue_name_number" ], [ 47, 53, "Leu-70", "residue_name_number" ], [ 59, 65, "Ser-71", "residue_name_number" ], [ 69, 78, "switch II", "site" ], [ 94, 98, "free", "protein_state" ], [ 99, 104, "Cdc42", "protein" ], [ 126, 137, "presence of", "protein_state" ], [ 142, 145, "HR1", "structure_element" ] ] }, { "sid": 173, "sent": "This suggests that the switch regions are not rigidified in the HR1 complex and are still in conformational exchange.", "section": "RESULTS", "ner": [ [ 23, 37, "switch regions", "site" ], [ 64, 67, "HR1", "structure_element" ] ] }, { "sid": 174, "sent": "Nevertheless, mapping of the affected residues onto the NMR structure of free Cdc42\u03947Q61L\u00b7GMPPNP (Fig. 5C)8 shows that, although they are relatively widespread compared with changes in the HR1 domain, in general, they are on the face of the protein that includes the switches.", "section": "RESULTS", "ner": [ [ 56, 59, "NMR", "experimental_method" ], [ 60, 69, "structure", "evidence" ], [ 73, 77, "free", "protein_state" ], [ 78, 96, "Cdc42\u03947Q61L\u00b7GMPPNP", "complex_assembly" ], [ 189, 192, "HR1", "structure_element" ], [ 267, 275, "switches", "site" ] ] }, { "sid": 175, "sent": "Although the binding interface may be overestimated, this suggests that the switch regions are involved in binding to TOCA1.", "section": "RESULTS", "ner": [ [ 13, 30, "binding interface", "site" ], [ 76, 90, "switch regions", "site" ], [ 118, 123, "TOCA1", "protein" ] ] }, { "sid": 176, "sent": "Modeling the Cdc42\u00b7TOCA1 HR1 Complex", "section": "RESULTS", "ner": [ [ 13, 28, "Cdc42\u00b7TOCA1 HR1", "complex_assembly" ] ] }, { "sid": 177, "sent": "The Cdc42\u00b7HR1TOCA1 complex was not amenable to full structural analysis due to the weak interaction and the extensive exchange broadening seen in the NMR experiments.", "section": "RESULTS", "ner": [ [ 4, 18, "Cdc42\u00b7HR1TOCA1", "complex_assembly" ], [ 150, 153, "NMR", "experimental_method" ] ] }, { "sid": 178, "sent": "HADDOCK was therefore used to perform rigid body docking based on the structures of free HR1 domain and Cdc42 and ambiguous interaction restraints derived from the titration experiments described above.", "section": "RESULTS", "ner": [ [ 0, 7, "HADDOCK", "experimental_method" ], [ 38, 56, "rigid body docking", "experimental_method" ], [ 70, 80, "structures", "evidence" ], [ 84, 88, "free", "protein_state" ], [ 89, 92, "HR1", "structure_element" ], [ 104, 109, "Cdc42", "protein" ], [ 164, 185, "titration experiments", "experimental_method" ] ] }, { "sid": 179, "sent": "The orientation of the HR1 domain with respect to Cdc42 cannot be definitively concluded in the absence of unambiguous distance restraints; hence, HADDOCK produced a set of models in which the HR1 domain contacts the same surface on Cdc42 but is in various orientations with respect to Cdc42.", "section": "RESULTS", "ner": [ [ 23, 26, "HR1", "structure_element" ], [ 50, 55, "Cdc42", "protein" ], [ 147, 154, "HADDOCK", "experimental_method" ], [ 193, 196, "HR1", "structure_element" ], [ 233, 238, "Cdc42", "protein" ], [ 286, 291, "Cdc42", "protein" ] ] }, { "sid": 180, "sent": "The cluster with the lowest root mean square deviation from the lowest energy structure is assumed to be the best model.", "section": "RESULTS", "ner": [ [ 28, 54, "root mean square deviation", "evidence" ], [ 78, 87, "structure", "evidence" ] ] }, { "sid": 181, "sent": "By these criteria, in the best model, the HR1 domain is in a similar orientation to the HR1a domain of PRK1 bound to RhoA and the HR1b domain bound to Rac1.", "section": "RESULTS", "ner": [ [ 42, 45, "HR1", "structure_element" ], [ 88, 92, "HR1a", "structure_element" ], [ 103, 107, "PRK1", "protein" ], [ 108, 116, "bound to", "protein_state" ], [ 117, 121, "RhoA", "protein" ], [ 130, 134, "HR1b", "structure_element" ], [ 142, 150, "bound to", "protein_state" ], [ 151, 155, "Rac1", "protein" ] ] }, { "sid": 182, "sent": "A representative model from this cluster is shown in Fig. 6A alongside the Rac1-HR1b structure (PDB code 2RMK) in Fig. 6B.", "section": "RESULTS", "ner": [ [ 75, 84, "Rac1-HR1b", "complex_assembly" ], [ 85, 94, "structure", "evidence" ] ] }, { "sid": 183, "sent": "Model of Cdc42\u00b7HR1 complex.", "section": "FIG", "ner": [ [ 9, 18, "Cdc42\u00b7HR1", "complex_assembly" ] ] }, { "sid": 184, "sent": " A, a representative model of the Cdc42\u00b7HR1 complex from the cluster closest to the lowest energy model produced using HADDOCK.", "section": "FIG", "ner": [ [ 34, 43, "Cdc42\u00b7HR1", "complex_assembly" ], [ 119, 126, "HADDOCK", "experimental_method" ] ] }, { "sid": 185, "sent": "Residues of Cdc42 that are affected in the presence of the HR1 domain but are not in close proximity to it are colored in red and labeled.", "section": "FIG", "ner": [ [ 12, 17, "Cdc42", "protein" ], [ 43, 54, "presence of", "protein_state" ], [ 59, 62, "HR1", "structure_element" ] ] }, { "sid": 186, "sent": "B, structure of Rac1 in complex with the HR1b domain of PRK1 (PDB code 2RMK).", "section": "FIG", "ner": [ [ 3, 12, "structure", "evidence" ], [ 16, 20, "Rac1", "protein" ], [ 21, 36, "in complex with", "protein_state" ], [ 41, 45, "HR1b", "structure_element" ], [ 56, 60, "PRK1", "protein" ] ] }, { "sid": 187, "sent": "C, sequence alignment of RhoA, Cdc42 and Rac1.", "section": "FIG", "ner": [ [ 3, 21, "sequence alignment", "experimental_method" ], [ 25, 29, "RhoA", "protein" ], [ 31, 36, "Cdc42", "protein" ], [ 41, 45, "Rac1", "protein" ] ] }, { "sid": 188, "sent": "Contact residues of RhoA and Rac1 to PRK1 HR1a and HR1b, respectively, are colored cyan.", "section": "FIG", "ner": [ [ 20, 24, "RhoA", "protein" ], [ 29, 33, "Rac1", "protein" ], [ 37, 41, "PRK1", "protein" ], [ 42, 46, "HR1a", "structure_element" ], [ 51, 55, "HR1b", "structure_element" ] ] }, { "sid": 189, "sent": "Residues of Cdc42 that disappear or show chemical shift changes in the presence of TOCA1 are colored cyan if also identified as contacts in RhoA and Rac1 and yellow if they are not.", "section": "FIG", "ner": [ [ 12, 17, "Cdc42", "protein" ], [ 71, 82, "presence of", "protein_state" ], [ 83, 88, "TOCA1", "protein" ], [ 140, 144, "RhoA", "protein" ], [ 149, 153, "Rac1", "protein" ] ] }, { "sid": 190, "sent": "Residues equivalent to Rac1 and RhoA contact sites but that are invisible in free Cdc42 are gray.", "section": "FIG", "ner": [ [ 23, 27, "Rac1", "protein" ], [ 32, 36, "RhoA", "protein" ], [ 37, 50, "contact sites", "site" ], [ 77, 81, "free", "protein_state" ], [ 82, 87, "Cdc42", "protein" ] ] }, { "sid": 191, "sent": "D, regions of interest of the Cdc42\u00b7HR1 domain model.", "section": "FIG", "ner": [ [ 30, 39, "Cdc42\u00b7HR1", "complex_assembly" ] ] }, { "sid": 192, "sent": "The four lowest energy structures in the chosen HADDOCK cluster are shown overlaid, with the residues of interest shown as sticks and labeled.", "section": "FIG", "ner": [ [ 23, 33, "structures", "evidence" ], [ 48, 55, "HADDOCK", "experimental_method" ] ] }, { "sid": 193, "sent": "Cdc42 is shown in cyan, and TOCA1 is shown in purple.", "section": "FIG", "ner": [ [ 28, 33, "TOCA1", "protein" ] ] }, { "sid": 194, "sent": "A sequence alignment of RhoA, Cdc42, and Rac1 is shown in Fig. 6C.", "section": "RESULTS", "ner": [ [ 2, 20, "sequence alignment", "experimental_method" ], [ 24, 28, "RhoA", "protein" ], [ 30, 35, "Cdc42", "protein" ], [ 41, 45, "Rac1", "protein" ] ] }, { "sid": 195, "sent": "The RhoA and Rac1 contact residues in the switch regions are invisible in the spectra of Cdc42, but they are generally conserved between all three G proteins.", "section": "RESULTS", "ner": [ [ 4, 8, "RhoA", "protein" ], [ 13, 17, "Rac1", "protein" ], [ 42, 56, "switch regions", "site" ], [ 78, 85, "spectra", "evidence" ], [ 89, 94, "Cdc42", "protein" ], [ 119, 128, "conserved", "protein_state" ], [ 147, 157, "G proteins", "protein_type" ] ] }, { "sid": 196, "sent": "Several Cdc42 residues identified by chemical shift mapping are not in close contact in the Cdc42\u00b7TOCA1 model (Fig. 6A).", "section": "RESULTS", "ner": [ [ 8, 13, "Cdc42", "protein" ], [ 37, 59, "chemical shift mapping", "experimental_method" ], [ 92, 103, "Cdc42\u00b7TOCA1", "complex_assembly" ] ] }, { "sid": 197, "sent": "Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II.", "section": "RESULTS", "ner": [ [ 48, 54, "Thr-24", "residue_name_number" ], [ 54, 59, "Cdc42", "protein" ], [ 61, 68, "Leu-160", "residue_name_number" ], [ 68, 73, "Cdc42", "protein" ], [ 79, 86, "Lys-163", "residue_name_number" ], [ 86, 91, "Cdc42", "protein" ], [ 108, 116, "switch I", "site" ], [ 184, 190, "switch", "site" ], [ 198, 204, "Glu-95", "residue_name_number" ], [ 204, 209, "Cdc42", "protein" ], [ 214, 220, "Lys-96", "residue_name_number" ], [ 220, 225, "Cdc42", "protein" ], [ 237, 242, "helix", "structure_element" ], [ 250, 259, "switch II", "site" ] ] }, { "sid": 198, "sent": "Other residues that are affected in the Cdc42\u00b7TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42.", "section": "RESULTS", "ner": [ [ 40, 51, "Cdc42\u00b7TOCA1", "complex_assembly" ], [ 110, 114, "RhoA", "protein" ], [ 118, 122, "Rac1", "protein" ], [ 141, 146, "Gln-2", "residue_name_number" ], [ 146, 151, "Cdc42", "protein" ], [ 153, 159, "Lys-16", "residue_name_number" ], [ 159, 164, "Cdc42", "protein" ], [ 166, 172, "Thr-52", "residue_name_number" ], [ 172, 177, "Cdc42", "protein" ], [ 183, 189, "Arg-68", "residue_name_number" ], [ 189, 194, "Cdc42", "protein" ] ] }, { "sid": 199, "sent": "Lys-16Cdc42 is unlikely to be a contact residue because it is involved in nucleotide binding, but the others may represent specific Cdc42-TOCA1 contacts.", "section": "RESULTS", "ner": [ [ 0, 6, "Lys-16", "residue_name_number" ], [ 6, 11, "Cdc42", "protein" ], [ 132, 143, "Cdc42-TOCA1", "complex_assembly" ] ] }, { "sid": 200, "sent": "Competition between N-WASP and TOCA1", "section": "RESULTS", "ner": [ [ 20, 26, "N-WASP", "protein" ], [ 31, 36, "TOCA1", "protein" ] ] }, { "sid": 201, "sent": "From the known interactions and effects of the proteins in biological systems, it has been suggested that TOCA1 and N-WASP could bind Cdc42 simultaneously.", "section": "RESULTS", "ner": [ [ 106, 111, "TOCA1", "protein" ], [ 116, 122, "N-WASP", "protein" ], [ 134, 139, "Cdc42", "protein" ] ] }, { "sid": 202, "sent": "Studies in CHO cells indicated that a Cdc42\u00b7N-WASP\u00b7TOCA1 complex existed because FRET was observed between RFP-TOCA1 and GFP-N-WASP, and the efficiency was decreased when an N-WASP mutant was used that no longer binds Cdc42.", "section": "RESULTS", "ner": [ [ 38, 56, "Cdc42\u00b7N-WASP\u00b7TOCA1", "complex_assembly" ], [ 81, 85, "FRET", "evidence" ], [ 107, 110, "RFP", "chemical" ], [ 111, 116, "TOCA1", "protein" ], [ 121, 124, "GFP", "chemical" ], [ 125, 131, "N-WASP", "protein" ], [ 174, 180, "N-WASP", "protein" ], [ 181, 187, "mutant", "protein_state" ], [ 218, 223, "Cdc42", "protein" ] ] }, { "sid": 203, "sent": "An overlay of the HADDOCK model of the Cdc42\u00b7HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A).", "section": "RESULTS", "ner": [ [ 3, 10, "overlay", "experimental_method" ], [ 18, 25, "HADDOCK", "experimental_method" ], [ 26, 31, "model", "evidence" ], [ 39, 53, "Cdc42\u00b7HR1TOCA1", "complex_assembly" ], [ 70, 79, "structure", "evidence" ], [ 83, 88, "Cdc42", "protein" ], [ 89, 104, "in complex with", "protein_state" ], [ 109, 112, "GBD", "structure_element" ], [ 120, 126, "N-WASP", "protein" ], [ 138, 142, "WASP", "protein" ], [ 175, 178, "HR1", "structure_element" ], [ 183, 200, "GBD binding sites", "site" ] ] }, { "sid": 204, "sent": "Interestingly, the presence of the TOCA1 HR1 would not prevent the core CRIB of WASP from binding to Cdc42, although the regions C-terminal to the CRIB that are required for high affinity binding of WASP would interfere sterically with the TOCA1 HR1.", "section": "RESULTS", "ner": [ [ 19, 30, "presence of", "protein_state" ], [ 35, 40, "TOCA1", "protein" ], [ 41, 44, "HR1", "structure_element" ], [ 72, 76, "CRIB", "structure_element" ], [ 80, 84, "WASP", "protein" ], [ 101, 106, "Cdc42", "protein" ], [ 147, 151, "CRIB", "structure_element" ], [ 199, 203, "WASP", "protein" ], [ 240, 245, "TOCA1", "protein" ], [ 246, 249, "HR1", "structure_element" ] ] }, { "sid": 205, "sent": "A basic region in WASP including three lysines (residues 230\u2013232), N-terminal to the core CRIB, has been implicated in an electrostatic steering mechanism, and these residues would be free to bind in the presence of TOCA1 HR1 (Fig. 7A).", "section": "RESULTS", "ner": [ [ 18, 22, "WASP", "protein" ], [ 39, 46, "lysines", "residue_name" ], [ 57, 64, "230\u2013232", "residue_range" ], [ 90, 94, "CRIB", "structure_element" ], [ 204, 215, "presence of", "protein_state" ], [ 216, 221, "TOCA1", "protein" ], [ 222, 225, "HR1", "structure_element" ] ] }, { "sid": 206, "sent": "The N-WASP GBD displaces the TOCA1 HR1 domain.", "section": "FIG", "ner": [ [ 4, 10, "N-WASP", "protein" ], [ 11, 14, "GBD", "structure_element" ], [ 29, 34, "TOCA1", "protein" ], [ 35, 38, "HR1", "structure_element" ] ] }, { "sid": 207, "sent": " A, the model of the Cdc42\u00b7TOCA1 HR1 domain complex overlaid with the Cdc42-WASP structure.", "section": "FIG", "ner": [ [ 21, 32, "Cdc42\u00b7TOCA1", "complex_assembly" ], [ 33, 36, "HR1", "structure_element" ], [ 70, 80, "Cdc42-WASP", "complex_assembly" ], [ 81, 90, "structure", "evidence" ] ] }, { "sid": 208, "sent": "Cdc42 is shown in green, and TOCA1 is shown in purple.", "section": "FIG", "ner": [ [ 29, 34, "TOCA1", "protein" ] ] }, { "sid": 209, "sent": "The core CRIB region of WASP is shown in red, whereas its basic region is shown in orange and the C-terminal region required for maximal affinity is shown in cyan.", "section": "FIG", "ner": [ [ 9, 13, "CRIB", "structure_element" ], [ 24, 28, "WASP", "protein" ] ] }, { "sid": 210, "sent": "A semitransparent surface representation of Cdc42 and WASP is shown overlaid with the schematic.", "section": "FIG", "ner": [ [ 44, 49, "Cdc42", "protein" ], [ 54, 58, "WASP", "protein" ] ] }, { "sid": 211, "sent": "B, competition SPA experiments carried out with indicated concentrations of the N-WASP GBD construct titrated into 30 nm GST-ACK or GST-WASP GBD and 30 nm Cdc42\u03947Q61L\u00b7[3H]GTP.", "section": "FIG", "ner": [ [ 3, 18, "competition SPA", "experimental_method" ], [ 80, 86, "N-WASP", "protein" ], [ 87, 90, "GBD", "structure_element" ], [ 101, 109, "titrated", "experimental_method" ], [ 121, 128, "GST-ACK", "mutant" ], [ 132, 140, "GST-WASP", "mutant" ], [ 141, 144, "GBD", "structure_element" ], [ 155, 174, "Cdc42\u03947Q61L\u00b7[3H]GTP", "complex_assembly" ] ] }, { "sid": 212, "sent": "C, Selected regions of the 15N HSQC of 145 \u03bcm Cdc42\u03947Q61L\u00b7GMPPNP with the indicated ratios of the TOCA1 HR1 domain, the N-WASP GBD, or both, showing that the TOCA HR1 domain does not displace the N-WASP GBD.", "section": "FIG", "ner": [ [ 27, 35, "15N HSQC", "experimental_method" ], [ 46, 64, "Cdc42\u03947Q61L\u00b7GMPPNP", "complex_assembly" ], [ 98, 103, "TOCA1", "protein" ], [ 104, 107, "HR1", "structure_element" ], [ 120, 126, "N-WASP", "protein" ], [ 127, 130, "GBD", "structure_element" ], [ 158, 162, "TOCA", "protein" ], [ 163, 166, "HR1", "structure_element" ], [ 196, 202, "N-WASP", "protein" ], [ 203, 206, "GBD", "structure_element" ] ] }, { "sid": 213, "sent": "D, selected regions of the 15N HSQC of 600 \u03bcm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP.", "section": "FIG", "ner": [ [ 27, 35, "15N HSQC", "experimental_method" ], [ 46, 51, "TOCA1", "protein" ], [ 52, 55, "HR1", "structure_element" ], [ 63, 78, "in complex with", "protein_state" ], [ 79, 84, "Cdc42", "protein" ], [ 92, 99, "absence", "protein_state" ], [ 104, 115, "presence of", "protein_state" ], [ 120, 126, "N-WASP", "protein" ], [ 127, 130, "GBD", "structure_element" ], [ 156, 161, "Cdc42", "protein" ], [ 171, 174, "HR1", "structure_element" ], [ 185, 191, "N-WASP", "protein" ] ] }, { "sid": 214, "sent": "An N-WASP GBD construct was produced, and its affinity for Cdc42 was measured by competition SPA (Fig. 7B).", "section": "RESULTS", "ner": [ [ 3, 9, "N-WASP", "protein" ], [ 10, 13, "GBD", "structure_element" ], [ 46, 54, "affinity", "evidence" ], [ 59, 64, "Cdc42", "protein" ], [ 81, 96, "competition SPA", "experimental_method" ] ] }, { "sid": 215, "sent": "The Kd that was determined (37 nm) is consistent with the previously reported affinity.", "section": "RESULTS", "ner": [ [ 4, 6, "Kd", "evidence" ], [ 78, 86, "affinity", "evidence" ] ] }, { "sid": 216, "sent": "Unlabeled N-WASP GBD was titrated into 15N-Cdc42\u03947Q61L\u00b7GMPPNP, and the backbone NH groups were monitored using HSQCs (Fig. 7C).", "section": "RESULTS", "ner": [ [ 0, 9, "Unlabeled", "protein_state" ], [ 10, 16, "N-WASP", "protein" ], [ 17, 20, "GBD", "structure_element" ], [ 25, 33, "titrated", "experimental_method" ], [ 39, 42, "15N", "chemical" ], [ 43, 61, "Cdc42\u03947Q61L\u00b7GMPPNP", "complex_assembly" ], [ 111, 116, "HSQCs", "experimental_method" ] ] }, { "sid": 217, "sent": "Unlabeled HR1TOCA1 was then added to the Cdc42\u00b7N-WASP complex, and no changes were seen, suggesting that the N-WASP GBD was not displaced even in the presence of a 5-fold excess of HR1TOCA1.", "section": "RESULTS", "ner": [ [ 0, 9, "Unlabeled", "protein_state" ], [ 10, 13, "HR1", "structure_element" ], [ 13, 18, "TOCA1", "protein" ], [ 41, 53, "Cdc42\u00b7N-WASP", "complex_assembly" ], [ 109, 115, "N-WASP", "protein" ], [ 116, 119, "GBD", "structure_element" ], [ 150, 161, "presence of", "protein_state" ], [ 181, 184, "HR1", "structure_element" ], [ 184, 189, "TOCA1", "protein" ] ] }, { "sid": 218, "sent": "These experiments were recorded at sufficiently high protein concentrations (145 \u03bcm Cdc42, 145 \u03bcm N-WASP GBD, 725 \u03bcm TOCA1 HR1 domain) to be far in excess of the Kd values of the individual interactions (TOCA1 Kd \u2248 5 \u03bcm, N-WASP Kd = 37 nm).", "section": "RESULTS", "ner": [ [ 84, 89, "Cdc42", "protein" ], [ 98, 104, "N-WASP", "protein" ], [ 105, 108, "GBD", "structure_element" ], [ 117, 122, "TOCA1", "protein" ], [ 123, 126, "HR1", "structure_element" ], [ 162, 164, "Kd", "evidence" ], [ 204, 209, "TOCA1", "protein" ], [ 210, 212, "Kd", "evidence" ], [ 221, 227, "N-WASP", "protein" ], [ 228, 230, "Kd", "evidence" ] ] }, { "sid": 219, "sent": "A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C).", "section": "RESULTS", "ner": [ [ 20, 24, "HSQC", "experimental_method" ], [ 49, 52, "15N", "chemical" ], [ 53, 58, "Cdc42", "protein" ], [ 59, 64, "alone", "protein_state" ], [ 73, 84, "presence of", "protein_state" ], [ 85, 90, "TOCA1", "protein" ], [ 91, 94, "HR1", "structure_element" ], [ 96, 102, "N-WASP", "protein" ], [ 103, 106, "GBD", "structure_element" ], [ 132, 139, "spectra", "evidence" ], [ 147, 158, "presence of", "protein_state" ], [ 159, 165, "N-WASP", "protein" ], [ 177, 188, "presence of", "protein_state" ], [ 194, 200, "N-WASP", "protein" ], [ 205, 210, "TOCA1", "protein" ], [ 211, 214, "HR1", "structure_element" ] ] }, { "sid": 220, "sent": "Furthermore, 15N-TOCA1 HR1 was monitored in the presence of unlabeled Cdc42\u03947Q61L\u00b7GMPPNP (1:1) before and after the addition of 0.25 and 1.0 eq of unlabeled N-WASP GBD.", "section": "RESULTS", "ner": [ [ 13, 16, "15N", "chemical" ], [ 17, 22, "TOCA1", "protein" ], [ 23, 26, "HR1", "structure_element" ], [ 48, 59, "presence of", "protein_state" ], [ 60, 69, "unlabeled", "protein_state" ], [ 70, 88, "Cdc42\u03947Q61L\u00b7GMPPNP", "complex_assembly" ], [ 147, 156, "unlabeled", "protein_state" ], [ 157, 163, "N-WASP", "protein" ], [ 164, 167, "GBD", "structure_element" ] ] }, { "sid": 221, "sent": "The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D).", "section": "RESULTS", "ner": [ [ 4, 12, "spectrum", "evidence" ], [ 18, 24, "N-WASP", "protein" ], [ 29, 34, "TOCA1", "protein" ], [ 79, 83, "free", "protein_state" ], [ 84, 87, "HR1", "structure_element" ], [ 108, 116, "spectrum", "evidence" ], [ 124, 135, "presence of", "protein_state" ], [ 147, 153, "N-WASP", "protein" ], [ 183, 188, "TOCA1", "protein" ], [ 189, 192, "HR1", "structure_element" ], [ 193, 197, "free", "protein_state" ], [ 202, 209, "complex", "protein_state" ], [ 210, 217, "spectra", "evidence" ] ] }, { "sid": 222, "sent": "When in fast exchange, the NMR signal represents a population-weighted average between free and bound states, so the intermediate spectrum indicates that the population comprises a mixture of free and bound HR1 domain.", "section": "RESULTS", "ner": [ [ 27, 30, "NMR", "experimental_method" ], [ 87, 91, "free", "protein_state" ], [ 96, 101, "bound", "protein_state" ], [ 130, 138, "spectrum", "evidence" ], [ 192, 196, "free", "protein_state" ], [ 201, 206, "bound", "protein_state" ], [ 207, 210, "HR1", "structure_element" ] ] }, { "sid": 223, "sent": "Again, the experiments were recorded on protein samples far in excess of the individual Kd values (600 \u03bcm each protein).", "section": "RESULTS", "ner": [ [ 88, 90, "Kd", "evidence" ] ] }, { "sid": 224, "sent": "These data indicate that the HR1 domain is displaced from Cdc42 by N-WASP and that a ternary complex comprising TOCA1 HR1, N-WASP GBD, and Cdc42 is not formed.", "section": "RESULTS", "ner": [ [ 29, 32, "HR1", "structure_element" ], [ 58, 63, "Cdc42", "protein" ], [ 67, 73, "N-WASP", "protein" ], [ 112, 117, "TOCA1", "protein" ], [ 118, 121, "HR1", "structure_element" ], [ 123, 129, "N-WASP", "protein" ], [ 130, 133, "GBD", "structure_element" ], [ 139, 144, "Cdc42", "protein" ] ] }, { "sid": 225, "sent": "Taken together, the data in Fig. 7, C and D, indicate unidirectional competition for Cdc42 binding in which the N-WASP GBD displaces TOCA1 HR1 but not vice versa.", "section": "RESULTS", "ner": [ [ 85, 90, "Cdc42", "protein" ], [ 112, 118, "N-WASP", "protein" ], [ 119, 122, "GBD", "structure_element" ], [ 133, 138, "TOCA1", "protein" ], [ 139, 142, "HR1", "structure_element" ] ] }, { "sid": 226, "sent": "To extend these studies to a more complex system and to assess the ability of TOCA1 HR1 to compete with full-length N-WASP, pyrene actin assays were employed.", "section": "RESULTS", "ner": [ [ 78, 83, "TOCA1", "protein" ], [ 84, 87, "HR1", "structure_element" ], [ 104, 115, "full-length", "protein_state" ], [ 116, 122, "N-WASP", "protein" ], [ 124, 143, "pyrene actin assays", "experimental_method" ] ] }, { "sid": 227, "sent": "These assays, described in detail elsewhere, were carried out using pyrene actin-supplemented Xenopus extracts into which exogenous TOCA1 HR1 domain or N-WASP GBD was added, to assess their effects on actin polymerization.", "section": "RESULTS", "ner": [ [ 68, 80, "pyrene actin", "chemical" ], [ 94, 101, "Xenopus", "taxonomy_domain" ], [ 132, 137, "TOCA1", "protein" ], [ 138, 141, "HR1", "structure_element" ], [ 152, 158, "N-WASP", "protein" ], [ 159, 162, "GBD", "structure_element" ], [ 201, 206, "actin", "protein_type" ] ] }, { "sid": 228, "sent": "Actin polymerization in all cases was initiated by the addition of PI(4,5)P2-containing liposomes.", "section": "RESULTS", "ner": [ [ 0, 5, "Actin", "protein_type" ], [ 67, 76, "PI(4,5)P2", "chemical" ] ] }, { "sid": 229, "sent": "Actin polymerization triggered by the addition of PI(4,5)P2-containing liposomes has previously been shown to depend on TOCA1 and N-WASP.", "section": "RESULTS", "ner": [ [ 0, 5, "Actin", "protein_type" ], [ 50, 59, "PI(4,5)P2", "chemical" ], [ 120, 125, "TOCA1", "protein" ], [ 130, 136, "N-WASP", "protein" ] ] }, { "sid": 230, "sent": "Endogenous N-WASP is present at \u223c100 nm in Xenopus extracts, whereas TOCA1 is present at a 10-fold lower concentration than N-WASP.", "section": "RESULTS", "ner": [ [ 11, 17, "N-WASP", "protein" ], [ 43, 50, "Xenopus", "taxonomy_domain" ], [ 69, 74, "TOCA1", "protein" ], [ 124, 130, "N-WASP", "protein" ] ] }, { "sid": 231, "sent": "The addition of the isolated N-WASP GBD significantly inhibited the polymerization of actin at concentrations as low as 100 nm and completely abolished polymerization at higher concentrations (Fig. 8).", "section": "RESULTS", "ner": [ [ 4, 12, "addition", "experimental_method" ], [ 29, 35, "N-WASP", "protein" ], [ 36, 39, "GBD", "structure_element" ], [ 86, 91, "actin", "protein_type" ] ] }, { "sid": 232, "sent": "The GBD presumably acts as a dominant negative, sequestering endogenous Cdc42 and preventing endogenous full-length N-WASP from binding and becoming activated.", "section": "RESULTS", "ner": [ [ 4, 7, "GBD", "structure_element" ], [ 72, 77, "Cdc42", "protein" ], [ 93, 103, "endogenous", "protein_state" ], [ 104, 115, "full-length", "protein_state" ], [ 116, 122, "N-WASP", "protein" ] ] }, { "sid": 233, "sent": "The addition of the TOCA1 HR1 domain to 100 \u03bcm had no significant effect on the rate of actin polymerization or maximum fluorescence.", "section": "RESULTS", "ner": [ [ 4, 12, "addition", "experimental_method" ], [ 20, 25, "TOCA1", "protein" ], [ 26, 29, "HR1", "structure_element" ], [ 88, 93, "actin", "protein_type" ], [ 112, 132, "maximum fluorescence", "evidence" ] ] }, { "sid": 234, "sent": "This is consistent with endogenous N-WASP, activated by other components of the assay, outcompeting the TOCA1 HR1 domain for Cdc42 binding.", "section": "RESULTS", "ner": [ [ 24, 34, "endogenous", "protein_state" ], [ 35, 41, "N-WASP", "protein" ], [ 104, 109, "TOCA1", "protein" ], [ 110, 113, "HR1", "structure_element" ], [ 125, 130, "Cdc42", "protein" ] ] }, { "sid": 235, "sent": "Actin polymerization downstream of Cdc42\u00b7N-WASP\u00b7TOCA1 is inhibited by excess N-WASP GBD but not by the TOCA1 HR1 domain.", "section": "FIG", "ner": [ [ 35, 53, "Cdc42\u00b7N-WASP\u00b7TOCA1", "complex_assembly" ], [ 57, 66, "inhibited", "protein_state" ], [ 77, 83, "N-WASP", "protein" ], [ 84, 87, "GBD", "structure_element" ], [ 103, 108, "TOCA1", "protein" ], [ 109, 112, "HR1", "structure_element" ] ] }, { "sid": 236, "sent": "Fluorescence curves show actin polymerization in the presence of increasing concentrations of N-WASP GBD or TOCA1 HR1 domain as indicated.", "section": "FIG", "ner": [ [ 0, 19, "Fluorescence curves", "evidence" ], [ 53, 64, "presence of", "protein_state" ], [ 65, 90, "increasing concentrations", "experimental_method" ], [ 94, 100, "N-WASP", "protein" ], [ 101, 104, "GBD", "structure_element" ], [ 108, 113, "TOCA1", "protein" ], [ 114, 117, "HR1", "structure_element" ] ] }, { "sid": 237, "sent": "The Cdc42-TOCA1 Interaction", "section": "DISCUSS", "ner": [ [ 4, 9, "Cdc42", "protein" ], [ 10, 15, "TOCA1", "protein" ] ] }, { "sid": 238, "sent": "The TOCA1 HR1 domain alone is sufficient for Cdc42 binding in vitro, yet the affinity of the TOCA1 HR1 domain for Cdc42 is remarkably low (Kd \u2248 5 \u03bcm).", "section": "DISCUSS", "ner": [ [ 4, 9, "TOCA1", "protein" ], [ 10, 13, "HR1", "structure_element" ], [ 21, 26, "alone", "protein_state" ], [ 45, 50, "Cdc42", "protein" ], [ 77, 85, "affinity", "evidence" ], [ 93, 98, "TOCA1", "protein" ], [ 99, 102, "HR1", "structure_element" ], [ 114, 119, "Cdc42", "protein" ], [ 139, 141, "Kd", "evidence" ] ] }, { "sid": 239, "sent": "This is over 100 times lower than that of the N-WASP GBD (Kd = 37 nm) and considerably lower than other known G protein-HR1 domain interactions.", "section": "DISCUSS", "ner": [ [ 46, 52, "N-WASP", "protein" ], [ 53, 56, "GBD", "structure_element" ], [ 58, 60, "Kd", "evidence" ], [ 110, 119, "G protein", "protein_type" ], [ 120, 123, "HR1", "structure_element" ] ] }, { "sid": 240, "sent": "The polybasic tract within the C-terminal region of Cdc42 does not appear to be required for binding to TOCA1, which is in contrast to the interaction between Rac1 and the HR1b domain of PRK1 but more similar to the PRK1 HR1a-RhoA interaction.", "section": "DISCUSS", "ner": [ [ 31, 48, "C-terminal region", "structure_element" ], [ 52, 57, "Cdc42", "protein" ], [ 104, 109, "TOCA1", "protein" ], [ 159, 163, "Rac1", "protein" ], [ 172, 176, "HR1b", "structure_element" ], [ 187, 191, "PRK1", "protein" ], [ 216, 220, "PRK1", "protein" ], [ 221, 225, "HR1a", "structure_element" ], [ 226, 230, "RhoA", "protein" ] ] }, { "sid": 241, "sent": "A single binding interface on both the HR1 domain and Cdc42 can be concluded from the data presented here.", "section": "DISCUSS", "ner": [ [ 9, 26, "binding interface", "site" ], [ 39, 42, "HR1", "structure_element" ], [ 54, 59, "Cdc42", "protein" ] ] }, { "sid": 242, "sent": "Furthermore, the interfaces are comparable with those of other G protein-HR1 interactions (Fig. 4), and the lowest energy model produced in rigid body docking resembles previously studied G protein\u00b7HR1 complexes (Fig. 6).", "section": "DISCUSS", "ner": [ [ 17, 27, "interfaces", "site" ], [ 63, 72, "G protein", "protein_type" ], [ 73, 76, "HR1", "structure_element" ], [ 122, 127, "model", "evidence" ], [ 140, 158, "rigid body docking", "experimental_method" ], [ 188, 201, "G protein\u00b7HR1", "complex_assembly" ] ] }, { "sid": 243, "sent": "It seems, therefore, that the interaction, despite its relatively low affinity, is specific and sterically similar to other HR1 domain-G protein interactions.", "section": "DISCUSS", "ner": [ [ 124, 127, "HR1", "structure_element" ], [ 135, 144, "G protein", "protein_type" ] ] }, { "sid": 244, "sent": "The TOCA1 HR1 domain is a left-handed coiled-coil comparable with other known HR1 domains.", "section": "DISCUSS", "ner": [ [ 4, 9, "TOCA1", "protein" ], [ 10, 13, "HR1", "structure_element" ], [ 38, 49, "coiled-coil", "structure_element" ], [ 78, 81, "HR1", "structure_element" ] ] }, { "sid": 245, "sent": "A short region N-terminal to the coiled-coil exhibits a series of turns and contacts residues of both helices of the coiled-coil (Fig. 3).", "section": "DISCUSS", "ner": [ [ 33, 44, "coiled-coil", "structure_element" ], [ 117, 128, "coiled-coil", "structure_element" ] ] }, { "sid": 246, "sent": "The corresponding sequence in CIP4 also includes a series of turns but is flexible, whereas in the HR1a domain of PRK1, the equivalent region adopts an \u03b1-helical structure that packs against the coiled-coil.", "section": "DISCUSS", "ner": [ [ 30, 34, "CIP4", "protein" ], [ 99, 103, "HR1a", "structure_element" ], [ 114, 118, "PRK1", "protein" ], [ 152, 171, "\u03b1-helical structure", "structure_element" ], [ 195, 206, "coiled-coil", "structure_element" ] ] }, { "sid": 247, "sent": "The contacts between the N-terminal region and the coiled-coil are predominantly hydrophobic in both cases, but sequence-specific contacts do not appear to be conserved.", "section": "DISCUSS", "ner": [ [ 4, 12, "contacts", "bond_interaction" ], [ 51, 62, "coiled-coil", "structure_element" ], [ 81, 92, "hydrophobic", "bond_interaction" ] ] }, { "sid": 248, "sent": "This region is distant from the G protein-binding interface of the HR1 domains, so the structural differences may relate to the structure and regulation of these domains rather than their G protein interactions.", "section": "DISCUSS", "ner": [ [ 32, 59, "G protein-binding interface", "site" ], [ 67, 70, "HR1", "structure_element" ], [ 188, 197, "G protein", "protein_type" ] ] }, { "sid": 249, "sent": "The interhelical loops of TOCA1 and CIP4 differ from the same region in the HR1 domains of PRK1 in that they are longer and contain two short stretches of 310-helix.", "section": "DISCUSS", "ner": [ [ 4, 22, "interhelical loops", "structure_element" ], [ 26, 31, "TOCA1", "protein" ], [ 36, 40, "CIP4", "protein" ], [ 76, 79, "HR1", "structure_element" ], [ 91, 95, "PRK1", "protein" ], [ 155, 164, "310-helix", "structure_element" ] ] }, { "sid": 250, "sent": "This region lies within the G protein-binding surface of all of the HR1 domains (Fig. 4D).", "section": "DISCUSS", "ner": [ [ 28, 53, "G protein-binding surface", "site" ], [ 68, 71, "HR1", "structure_element" ] ] }, { "sid": 251, "sent": "TOCA1 and CIP4 both bind weakly to Cdc42, whereas the HR1a domain of PRK1 binds tightly to RhoA and Rac1, and the HR1b domain binds to Rac1.", "section": "DISCUSS", "ner": [ [ 0, 5, "TOCA1", "protein" ], [ 10, 14, "CIP4", "protein" ], [ 35, 40, "Cdc42", "protein" ], [ 54, 58, "HR1a", "structure_element" ], [ 69, 73, "PRK1", "protein" ], [ 91, 95, "RhoA", "protein" ], [ 100, 104, "Rac1", "protein" ], [ 114, 118, "HR1b", "structure_element" ], [ 135, 139, "Rac1", "protein" ] ] }, { "sid": 252, "sent": "The structural features shared by TOCA1 and CIP4 may therefore be related to Cdc42 binding specificity and the low affinities.", "section": "DISCUSS", "ner": [ [ 34, 39, "TOCA1", "protein" ], [ 44, 48, "CIP4", "protein" ], [ 77, 82, "Cdc42", "protein" ] ] }, { "sid": 253, "sent": "In free TOCA1, the side chains of the interhelical region make extensive contacts with residues in helix 1.", "section": "DISCUSS", "ner": [ [ 3, 7, "free", "protein_state" ], [ 8, 13, "TOCA1", "protein" ], [ 38, 57, "interhelical region", "structure_element" ], [ 99, 106, "helix 1", "structure_element" ] ] }, { "sid": 254, "sent": "Many of these residues are significantly affected in the presence of Cdc42, so it is likely that the conformation of this loop is altered in the Cdc42 complex.", "section": "DISCUSS", "ner": [ [ 57, 68, "presence of", "protein_state" ], [ 69, 74, "Cdc42", "protein" ], [ 122, 126, "loop", "structure_element" ], [ 145, 150, "Cdc42", "protein" ] ] }, { "sid": 255, "sent": "These observations therefore provide a molecular mechanism whereby mutation of Met383-Gly384-Asp385 to Ile383-Ser384-Thr385 abolishes TOCA1 binding to Cdc42.", "section": "DISCUSS", "ner": [ [ 67, 75, "mutation", "experimental_method" ], [ 79, 85, "Met383", "residue_name_number" ], [ 86, 92, "Gly384", "residue_name_number" ], [ 93, 99, "Asp385", "residue_name_number" ], [ 103, 109, "Ile383", "residue_name_number" ], [ 110, 116, "Ser384", "residue_name_number" ], [ 117, 123, "Thr385", "residue_name_number" ], [ 134, 139, "TOCA1", "protein" ], [ 151, 156, "Cdc42", "protein" ] ] }, { "sid": 256, "sent": "The lowest energy model produced by HADDOCK using ambiguous interaction restraints from the titration data resembled the NMR structures of RhoA and Rac1 in complex with their HR1 domain partners.", "section": "DISCUSS", "ner": [ [ 18, 23, "model", "evidence" ], [ 36, 43, "HADDOCK", "experimental_method" ], [ 92, 101, "titration", "evidence" ], [ 121, 124, "NMR", "experimental_method" ], [ 125, 135, "structures", "evidence" ], [ 139, 143, "RhoA", "protein" ], [ 148, 152, "Rac1", "protein" ], [ 153, 168, "in complex with", "protein_state" ], [ 175, 178, "HR1", "structure_element" ] ] }, { "sid": 257, "sent": "For example, Phe-56Cdc42, which is not visible in free Cdc42 or Cdc42\u00b7HR1TOCA1, is close to the TOCA1 HR1 (Fig. 6A).", "section": "DISCUSS", "ner": [ [ 13, 19, "Phe-56", "residue_name_number" ], [ 19, 24, "Cdc42", "protein" ], [ 50, 54, "free", "protein_state" ], [ 55, 60, "Cdc42", "protein" ], [ 64, 78, "Cdc42\u00b7HR1TOCA1", "complex_assembly" ], [ 96, 101, "TOCA1", "protein" ], [ 102, 105, "HR1", "structure_element" ] ] }, { "sid": 258, "sent": "Phe-56Cdc42, which is a Trp in both Rac1 and RhoA (Fig. 6C), is thought to pack behind switch I when Cdc42 interacts with ACK, maintaining the switch in a binding-competent orientation.", "section": "DISCUSS", "ner": [ [ 0, 6, "Phe-56", "residue_name_number" ], [ 6, 11, "Cdc42", "protein" ], [ 24, 27, "Trp", "residue_name" ], [ 36, 40, "Rac1", "protein" ], [ 45, 49, "RhoA", "protein" ], [ 87, 95, "switch I", "site" ], [ 101, 106, "Cdc42", "protein" ], [ 122, 125, "ACK", "protein" ] ] }, { "sid": 259, "sent": "This residue has also been identified as important for Cdc42-WASP binding.", "section": "DISCUSS", "ner": [ [ 55, 60, "Cdc42", "protein" ], [ 61, 65, "WASP", "protein" ] ] }, { "sid": 260, "sent": "Phe-56Cdc42 is therefore likely to be involved in the Cdc42-TOCA1 interaction, probably by stabilizing the position of switch I.", "section": "DISCUSS", "ner": [ [ 0, 6, "Phe-56", "residue_name_number" ], [ 6, 11, "Cdc42", "protein" ], [ 54, 59, "Cdc42", "protein" ], [ 60, 65, "TOCA1", "protein" ], [ 119, 127, "switch I", "site" ] ] }, { "sid": 261, "sent": "Some residues that are affected in the Cdc42\u00b7HR1TOCA1 complex but do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) may contact HR1TOCA1 directly (Fig. 6D).", "section": "DISCUSS", "ner": [ [ 39, 53, "Cdc42\u00b7HR1TOCA1", "complex_assembly" ], [ 107, 111, "RhoA", "protein" ], [ 115, 119, "Rac1", "protein" ], [ 142, 145, "HR1", "structure_element" ], [ 145, 150, "TOCA1", "protein" ] ] }, { "sid": 262, "sent": "Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42\u00b7ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil.", "section": "DISCUSS", "ner": [ [ 0, 5, "Gln-2", "residue_name_number" ], [ 5, 10, "Cdc42", "protein" ], [ 71, 80, "Cdc42\u00b7ACK", "complex_assembly" ], [ 99, 106, "Val-376", "residue_name_number" ], [ 106, 111, "TOCA1", "protein" ], [ 116, 123, "Asn-380", "residue_name_number" ], [ 123, 128, "TOCA1", "protein" ], [ 180, 197, "interhelical loop", "structure_element" ], [ 206, 217, "first helix", "structure_element" ], [ 225, 230, "TOCA1", "protein" ], [ 231, 242, "coiled-coil", "structure_element" ] ] }, { "sid": 263, "sent": "Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1.", "section": "DISCUSS", "ner": [ [ 0, 6, "Thr-52", "residue_name_number" ], [ 6, 11, "Cdc42", "protein" ], [ 74, 77, "ACK", "protein" ], [ 109, 112, "HR1", "structure_element" ], [ 112, 117, "TOCA1", "protein" ], [ 118, 125, "helix 1", "structure_element" ], [ 140, 147, "Lys-372", "residue_name_number" ], [ 147, 152, "TOCA1", "protein" ], [ 189, 193, "Rac1", "protein" ], [ 197, 203, "Asn-52", "residue_name_number" ], [ 203, 207, "Rac1", "protein" ] ] }, { "sid": 264, "sent": "N52T is one of a combination of seven residues found to confer ACK binding on Rac1 and so may represent a specific Cdc42-effector contact residue.", "section": "DISCUSS", "ner": [ [ 0, 4, "N52T", "mutant" ], [ 63, 66, "ACK", "protein" ], [ 78, 82, "Rac1", "protein" ], [ 115, 120, "Cdc42", "protein" ] ] }, { "sid": 265, "sent": "The position equivalent to Lys-372TOCA1 in PRK1 is Glu-58HR1a or Gln-151HR1b.", "section": "DISCUSS", "ner": [ [ 27, 34, "Lys-372", "residue_name_number" ], [ 34, 39, "TOCA1", "protein" ], [ 43, 47, "PRK1", "protein" ], [ 51, 57, "Glu-58", "residue_name_number" ], [ 57, 61, "HR1a", "structure_element" ], [ 65, 72, "Gln-151", "residue_name_number" ], [ 72, 76, "HR1b", "structure_element" ] ] }, { "sid": 266, "sent": "Thr-52Cdc42-Lys-372TOCA1 may therefore represent a specific Cdc42-HR1TOCA1 contact.", "section": "DISCUSS", "ner": [ [ 0, 6, "Thr-52", "residue_name_number" ], [ 6, 11, "Cdc42", "protein" ], [ 12, 19, "Lys-372", "residue_name_number" ], [ 19, 24, "TOCA1", "protein" ], [ 60, 65, "Cdc42", "protein" ], [ 66, 69, "HR1", "structure_element" ], [ 69, 74, "TOCA1", "protein" ] ] }, { "sid": 267, "sent": "Arg-68Cdc42 of switch II is positioned close to Glu-395TOCA1 (Fig. 6D), suggesting a direct electrostatic contact between switch II of Cdc42 and helix 2 of the HR1 domain.", "section": "DISCUSS", "ner": [ [ 0, 6, "Arg-68", "residue_name_number" ], [ 6, 11, "Cdc42", "protein" ], [ 15, 24, "switch II", "site" ], [ 48, 55, "Glu-395", "residue_name_number" ], [ 55, 60, "TOCA1", "protein" ], [ 122, 131, "switch II", "site" ], [ 135, 140, "Cdc42", "protein" ], [ 145, 152, "helix 2", "structure_element" ], [ 160, 163, "HR1", "structure_element" ] ] }, { "sid": 268, "sent": "The equivalent Arg in Rac1 and RhoA is pointing away from the HR1 domains of PRK1.", "section": "DISCUSS", "ner": [ [ 15, 18, "Arg", "residue_name" ], [ 22, 26, "Rac1", "protein" ], [ 31, 35, "RhoA", "protein" ], [ 62, 65, "HR1", "structure_element" ], [ 77, 81, "PRK1", "protein" ] ] }, { "sid": 269, "sent": "The importance of this residue in the Cdc42-TOCA1 interaction remains unclear, although its mutation reduces binding to RhoGAP, suggesting that it can be involved in Cdc42 interactions.", "section": "DISCUSS", "ner": [ [ 38, 43, "Cdc42", "protein" ], [ 44, 49, "TOCA1", "protein" ], [ 92, 100, "mutation", "experimental_method" ], [ 120, 126, "RhoGAP", "protein" ], [ 166, 171, "Cdc42", "protein" ] ] }, { "sid": 270, "sent": "The solution structure of the TOCA1 HR1 domain presented here, along with the model of the HR1TOCA1\u00b7Cdc42 complex is consistent with a conserved mode of binding across the known HR1 domain-Rho family interactions, despite their differing affinities.", "section": "DISCUSS", "ner": [ [ 4, 22, "solution structure", "evidence" ], [ 30, 35, "TOCA1", "protein" ], [ 36, 39, "HR1", "structure_element" ], [ 91, 105, "HR1TOCA1\u00b7Cdc42", "complex_assembly" ], [ 178, 181, "HR1", "structure_element" ] ] }, { "sid": 271, "sent": "The weak binding prevented detailed structural and thermodynamic studies of the complex.", "section": "DISCUSS", "ner": [ [ 36, 72, "structural and thermodynamic studies", "experimental_method" ] ] }, { "sid": 272, "sent": "Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding.", "section": "DISCUSS", "ner": [ [ 13, 31, "structural studies", "experimental_method" ], [ 39, 44, "TOCA1", "protein" ], [ 45, 48, "HR1", "structure_element" ], [ 71, 93, "chemical shift mapping", "experimental_method" ], [ 161, 166, "Cdc42", "protein" ], [ 167, 170, "HR1", "structure_element" ], [ 170, 175, "TOCA1", "protein" ], [ 180, 184, "RhoA", "protein" ], [ 185, 189, "Rac1", "protein" ], [ 190, 193, "HR1", "structure_element" ], [ 193, 197, "PRK1", "protein" ] ] }, { "sid": 273, "sent": "We have previously postulated that the inherent flexibility of HR1 domains contributes to their ability to bind to different Rho family G proteins, with Rho-binding HR1 domains displaying increased flexibility, reflected in their lower melting temperatures (Tm) and Rac binders being more rigid.", "section": "DISCUSS", "ner": [ [ 63, 66, "HR1", "structure_element" ], [ 125, 146, "Rho family G proteins", "protein_type" ], [ 165, 168, "HR1", "structure_element" ], [ 236, 256, "melting temperatures", "evidence" ], [ 258, 260, "Tm", "evidence" ], [ 266, 269, "Rac", "protein_type" ] ] }, { "sid": 274, "sent": "The Tm of the TOCA1 HR1 domain is 61.9 \u00b0C (data not shown), which is the highest Tm that we have measured for an HR1 domain thus far.", "section": "DISCUSS", "ner": [ [ 4, 6, "Tm", "evidence" ], [ 14, 19, "TOCA1", "protein" ], [ 20, 23, "HR1", "structure_element" ], [ 81, 83, "Tm", "evidence" ], [ 113, 116, "HR1", "structure_element" ] ] }, { "sid": 275, "sent": "As such, the ability of the TOCA1 HR1 domain to bind to Cdc42 (a close relative of Rac1 rather than RhoA) fits this trend.", "section": "DISCUSS", "ner": [ [ 28, 33, "TOCA1", "protein" ], [ 34, 37, "HR1", "structure_element" ], [ 56, 61, "Cdc42", "protein" ], [ 83, 87, "Rac1", "protein" ], [ 100, 104, "RhoA", "protein" ] ] }, { "sid": 276, "sent": "An investigation into the local motions, particularly in the G protein-binding regions, may offer further insight into the differential specificities and affinities of G protein-HR1 domain interactions.", "section": "DISCUSS", "ner": [ [ 61, 86, "G protein-binding regions", "site" ], [ 168, 177, "G protein", "protein_type" ], [ 178, 181, "HR1", "structure_element" ] ] }, { "sid": 277, "sent": "The low affinity of the Cdc42-HR1TOCA1 interaction is consistent with a tightly spatially and temporally regulated pathway, requiring combinatorial signals leading to a series of coincident weak interactions that elicit full activation.", "section": "DISCUSS", "ner": [ [ 24, 29, "Cdc42", "protein" ], [ 30, 33, "HR1", "structure_element" ], [ 33, 38, "TOCA1", "protein" ] ] }, { "sid": 278, "sent": "The HR1 domains from other TOCA family members, CIP4 and FBP17, also bind at low micromolar affinities to Cdc42, so the low affinity interaction appears to be commonplace among this family of HR1 domain proteins, in contrast to the PRK family.", "section": "DISCUSS", "ner": [ [ 4, 7, "HR1", "structure_element" ], [ 27, 46, "TOCA family members", "protein_type" ], [ 48, 52, "CIP4", "protein" ], [ 57, 62, "FBP17", "protein" ], [ 106, 111, "Cdc42", "protein" ], [ 192, 211, "HR1 domain proteins", "protein_type" ], [ 232, 242, "PRK family", "protein_type" ] ] }, { "sid": 279, "sent": "The low affinity of the HR1TOCA1-Cdc42 interaction in the context of the physiological concentration of TOCA1 in Xenopus extracts (\u223c10 nm) suggests that binding between TOCA1 and Cdc42 is likely to occur in vivo only when TOCA1 is at high local concentrations and membrane-localized and therefore in close proximity to activated Cdc42.", "section": "DISCUSS", "ner": [ [ 24, 32, "HR1TOCA1", "structure_element" ], [ 33, 38, "Cdc42", "protein" ], [ 104, 109, "TOCA1", "protein" ], [ 113, 120, "Xenopus", "taxonomy_domain" ], [ 169, 174, "TOCA1", "protein" ], [ 179, 184, "Cdc42", "protein" ], [ 222, 227, "TOCA1", "protein" ], [ 319, 328, "activated", "protein_state" ], [ 329, 334, "Cdc42", "protein" ] ] }, { "sid": 280, "sent": "Evidence suggests that the TOCA family of proteins are recruited to the membrane via an interaction between their F-BAR domain and specific signaling lipids.", "section": "DISCUSS", "ner": [ [ 27, 38, "TOCA family", "protein_type" ], [ 114, 119, "F-BAR", "structure_element" ] ] }, { "sid": 281, "sent": "For example, electrostatic interactions between the F-BAR domain and the membrane are required for TOCA1 recruitment to membrane vesicles and tubules, and TOCA1-dependent actin polymerization is known to depend specifically on PI(4,5)P2.", "section": "DISCUSS", "ner": [ [ 13, 39, "electrostatic interactions", "bond_interaction" ], [ 52, 57, "F-BAR", "structure_element" ], [ 99, 104, "TOCA1", "protein" ], [ 155, 160, "TOCA1", "protein" ], [ 227, 236, "PI(4,5)P2", "chemical" ] ] }, { "sid": 282, "sent": "Furthermore, the isolated F-BAR domain of FBP17 has been shown to induce membrane tubulation of brain liposomes and BAR domain proteins that promote tubulation cluster on membranes at high densities.", "section": "DISCUSS", "ner": [ [ 17, 25, "isolated", "experimental_method" ], [ 26, 31, "F-BAR", "structure_element" ], [ 42, 47, "FBP17", "protein" ], [ 116, 119, "BAR", "structure_element" ] ] }, { "sid": 283, "sent": "Once at the membrane, high local concentrations of TOCA1 could exceed the Kd of F-BAR dimerization (likely to be comparable with that of the FCHo2 F-BAR domain (2.5 \u03bcm)) and that of the Cdc42-HR1TOCA1 interaction.", "section": "DISCUSS", "ner": [ [ 51, 56, "TOCA1", "protein" ], [ 74, 76, "Kd", "evidence" ], [ 80, 85, "F-BAR", "structure_element" ], [ 86, 91, "dimer", "oligomeric_state" ], [ 141, 146, "FCHo2", "protein" ], [ 147, 152, "F-BAR", "structure_element" ], [ 186, 191, "Cdc42", "protein" ], [ 192, 195, "HR1", "structure_element" ], [ 195, 200, "TOCA1", "protein" ] ] }, { "sid": 284, "sent": "Cdc42-HR1TOCA1 binding would then be favorable, as long as coincident activation of Cdc42 had occurred, leading to stabilization of TOCA1 at the membrane and downstream activation of N-WASP.", "section": "DISCUSS", "ner": [ [ 0, 5, "Cdc42", "protein" ], [ 6, 9, "HR1", "structure_element" ], [ 9, 14, "TOCA1", "protein" ], [ 84, 89, "Cdc42", "protein" ], [ 132, 137, "TOCA1", "protein" ], [ 183, 189, "N-WASP", "protein" ] ] }, { "sid": 285, "sent": "It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced.", "section": "DISCUSS", "ner": [ [ 28, 32, "WASP", "protein_type" ], [ 37, 43, "N-WASP", "protein" ], [ 73, 79, "folded", "protein_state" ], [ 81, 89, "inactive", "protein_state" ], [ 95, 103, "unfolded", "protein_state" ], [ 105, 111, "active", "protein_state" ], [ 128, 136, "affinity", "evidence" ], [ 140, 145, "Cdc42", "protein" ], [ 154, 162, "unfolded", "protein_state" ], [ 163, 167, "WASP", "protein_type" ] ] }, { "sid": 286, "sent": "The unfolded, high affinity state of WASP is represented by a short peptide, the GBD, which binds with a low nanomolar affinity to Cdc42.", "section": "DISCUSS", "ner": [ [ 4, 12, "unfolded", "protein_state" ], [ 37, 41, "WASP", "protein_type" ], [ 68, 75, "peptide", "chemical" ], [ 81, 84, "GBD", "structure_element" ], [ 131, 136, "Cdc42", "protein" ] ] }, { "sid": 287, "sent": "In contrast, the best estimate of the affinity of full-length WASP for Cdc42 is low micromolar.", "section": "DISCUSS", "ner": [ [ 38, 46, "affinity", "evidence" ], [ 50, 61, "full-length", "protein_state" ], [ 62, 66, "WASP", "protein_type" ], [ 71, 76, "Cdc42", "protein" ] ] }, { "sid": 288, "sent": "In the inactive state of WASP, the actin- and Arp2/3-binding VCA domain contacts the GBD, competing for Cdc42 binding.", "section": "DISCUSS", "ner": [ [ 7, 15, "inactive", "protein_state" ], [ 25, 29, "WASP", "protein_type" ], [ 46, 52, "Arp2/3", "complex_assembly" ], [ 61, 64, "VCA", "structure_element" ], [ 85, 88, "GBD", "structure_element" ], [ 104, 109, "Cdc42", "protein" ] ] }, { "sid": 289, "sent": "The high affinity of Cdc42 for the unfolded, active form pushes the equilibrium in favor of (N-)WASP activation.", "section": "DISCUSS", "ner": [ [ 21, 26, "Cdc42", "protein" ], [ 35, 43, "unfolded", "protein_state" ], [ 45, 51, "active", "protein_state" ], [ 92, 100, "(N-)WASP", "protein" ] ] }, { "sid": 290, "sent": "Binding of PI(4,5)P2 to the basic region just N-terminal to the GBD further favors the active conformation.", "section": "DISCUSS", "ner": [ [ 11, 20, "PI(4,5)P2", "chemical" ], [ 64, 67, "GBD", "structure_element" ], [ 87, 93, "active", "protein_state" ] ] }, { "sid": 291, "sent": "A substantial body of data has illuminated the complex regulation of WASP/N-WASP proteins, and current evidence suggests that these allosteric activation mechanisms and oligomerization combine to regulate WASP activity, allowing the synchronization and integration of multiple potential activation signals (reviewed in Ref.).", "section": "DISCUSS", "ner": [ [ 69, 89, "WASP/N-WASP proteins", "protein_type" ], [ 205, 209, "WASP", "protein_type" ] ] }, { "sid": 292, "sent": "We envisage that TOCA1 is first recruited to the appropriate membrane in response to PI(4,5)P2 via its F-BAR domain, where the local increase in concentration favors F-BAR-mediated dimerization of TOCA1.", "section": "DISCUSS", "ner": [ [ 17, 22, "TOCA1", "protein" ], [ 85, 94, "PI(4,5)P2", "chemical" ], [ 103, 108, "F-BAR", "structure_element" ], [ 166, 171, "F-BAR", "structure_element" ], [ 181, 186, "dimer", "oligomeric_state" ], [ 197, 202, "TOCA1", "protein" ] ] }, { "sid": 293, "sent": "Cdc42 is activated in response to co-incident signals and can then bind to TOCA1, further stabilizing TOCA1 at the membrane.", "section": "DISCUSS", "ner": [ [ 0, 5, "Cdc42", "protein" ], [ 75, 80, "TOCA1", "protein" ], [ 102, 107, "TOCA1", "protein" ] ] }, { "sid": 294, "sent": "TOCA1 can then recruit N-WASP via an interaction between its SH3 domain and the N-WASP proline-rich region.", "section": "DISCUSS", "ner": [ [ 0, 5, "TOCA1", "protein" ], [ 23, 29, "N-WASP", "protein" ], [ 61, 64, "SH3", "structure_element" ], [ 80, 86, "N-WASP", "protein" ], [ 87, 106, "proline-rich region", "structure_element" ] ] }, { "sid": 295, "sent": "The recruitment of N-WASP alone and of the N-WASP\u00b7WIP complex by TOCA1 and FBP17 has been demonstrated.", "section": "DISCUSS", "ner": [ [ 19, 25, "N-WASP", "protein" ], [ 26, 31, "alone", "protein_state" ], [ 43, 53, "N-WASP\u00b7WIP", "complex_assembly" ], [ 65, 70, "TOCA1", "protein" ], [ 75, 80, "FBP17", "protein" ] ] }, { "sid": 296, "sent": "WIP inhibits the activation of N-WASP by Cdc42, an effect that is reversed by TOCA1.", "section": "DISCUSS", "ner": [ [ 0, 3, "WIP", "protein" ], [ 31, 37, "N-WASP", "protein" ], [ 41, 46, "Cdc42", "protein" ], [ 78, 83, "TOCA1", "protein" ] ] }, { "sid": 297, "sent": "It may therefore be envisaged that WIP and TOCA1 exert opposing allosteric effects on N-WASP, with TOCA1 favoring the unfolded, active conformation of N-WASP and increasing its affinity for Cdc42.", "section": "DISCUSS", "ner": [ [ 35, 38, "WIP", "protein" ], [ 43, 48, "TOCA1", "protein" ], [ 86, 92, "N-WASP", "protein" ], [ 99, 104, "TOCA1", "protein" ], [ 118, 126, "unfolded", "protein_state" ], [ 128, 134, "active", "protein_state" ], [ 151, 157, "N-WASP", "protein" ], [ 190, 195, "Cdc42", "protein" ] ] }, { "sid": 298, "sent": "TOCA1 may also activate N-WASP by effective oligomerization because clustering of TOCA1 at the membrane following coincident interactions with PI(4,5)P2 and Cdc42 would in turn lead to clustering of N-WASP, in addition to pushing the equilibrium toward the unfolded, active state.", "section": "DISCUSS", "ner": [ [ 0, 5, "TOCA1", "protein" ], [ 24, 30, "N-WASP", "protein" ], [ 82, 87, "TOCA1", "protein" ], [ 143, 152, "PI(4,5)P2", "chemical" ], [ 157, 162, "Cdc42", "protein" ], [ 199, 205, "N-WASP", "protein" ], [ 257, 265, "unfolded", "protein_state" ], [ 267, 273, "active", "protein_state" ] ] }, { "sid": 299, "sent": "In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases.", "section": "DISCUSS", "ner": [ [ 23, 34, "full-length", "protein_state" ], [ 35, 40, "TOCA1", "protein" ], [ 45, 51, "N-WASP", "protein" ], [ 79, 89, "affinities", "evidence" ], [ 94, 100, "active", "protein_state" ], [ 101, 106, "Cdc42", "protein" ], [ 119, 127, "unfolded", "protein_state" ], [ 129, 135, "active", "protein_state" ], [ 154, 162, "affinity", "evidence" ], [ 166, 172, "N-WASP", "protein" ], [ 177, 182, "Cdc42", "protein" ] ] }, { "sid": 300, "sent": "Our binding data suggest that TOCA1 HR1 binding is not allosterically regulated, and our NMR data, along with the high stability of TOCA1 HR1, suggest that there is no widespread conformational change in the presence of Cdc42.", "section": "DISCUSS", "ner": [ [ 4, 16, "binding data", "evidence" ], [ 30, 35, "TOCA1", "protein" ], [ 36, 39, "HR1", "structure_element" ], [ 89, 92, "NMR", "experimental_method" ], [ 119, 128, "stability", "protein_state" ], [ 132, 137, "TOCA1", "protein" ], [ 138, 141, "HR1", "structure_element" ], [ 208, 219, "presence of", "protein_state" ], [ 220, 225, "Cdc42", "protein" ] ] }, { "sid": 301, "sent": "As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42.", "section": "DISCUSS", "ner": [ [ 3, 14, "full-length", "protein_state" ], [ 15, 20, "TOCA1", "protein" ], [ 29, 37, "isolated", "protein_state" ], [ 38, 41, "HR1", "structure_element" ], [ 54, 59, "Cdc42", "protein" ], [ 89, 95, "N-WASP", "protein" ], [ 96, 101, "Cdc42", "protein" ], [ 142, 148, "N-WASP", "protein" ], [ 149, 152, "GBD", "structure_element" ], [ 179, 184, "TOCA1", "protein" ], [ 185, 188, "HR1", "structure_element" ], [ 193, 198, "Cdc42", "protein" ] ] }, { "sid": 302, "sent": "A combination of allosteric activation by PI(4,5)P2, activated Cdc42 and TOCA1, and oligomeric activation implemented by TOCA1 would lead to full activation of N-WASP and downstream actin polymerization.", "section": "DISCUSS", "ner": [ [ 42, 51, "PI(4,5)P2", "chemical" ], [ 53, 62, "activated", "protein_state" ], [ 63, 68, "Cdc42", "protein" ], [ 73, 78, "TOCA1", "protein" ], [ 121, 126, "TOCA1", "protein" ], [ 141, 156, "full activation", "protein_state" ], [ 160, 166, "N-WASP", "protein" ] ] }, { "sid": 303, "sent": "In such an array of molecules localized to a discrete region of the membrane, it is plausible that WASP could bind to a second Cdc42 molecule rather than displacing TOCA1 from its cognate Cdc42.", "section": "DISCUSS", "ner": [ [ 99, 103, "WASP", "protein" ], [ 127, 132, "Cdc42", "protein" ], [ 165, 170, "TOCA1", "protein" ], [ 188, 193, "Cdc42", "protein" ] ] }, { "sid": 304, "sent": "Our NMR and affinity data, however, are consistent with displacement of the TOCA1 HR1 by the N-WASP GBD.", "section": "DISCUSS", "ner": [ [ 4, 7, "NMR", "experimental_method" ], [ 12, 25, "affinity data", "evidence" ], [ 76, 81, "TOCA1", "protein" ], [ 82, 85, "HR1", "structure_element" ], [ 93, 99, "N-WASP", "protein" ], [ 100, 103, "GBD", "structure_element" ] ] }, { "sid": 305, "sent": "Furthermore, TOCA1 is required for Cdc42-mediated activation of N-WASP\u00b7WIP, implying that it may not be possible for Cdc42 to bind and activate N-WASP prior to TOCA1-Cdc42 binding.", "section": "DISCUSS", "ner": [ [ 13, 18, "TOCA1", "protein" ], [ 35, 40, "Cdc42", "protein" ], [ 64, 74, "N-WASP\u00b7WIP", "complex_assembly" ], [ 117, 122, "Cdc42", "protein" ], [ 144, 150, "N-WASP", "protein" ], [ 160, 165, "TOCA1", "protein" ], [ 166, 171, "Cdc42", "protein" ] ] }, { "sid": 306, "sent": "The commonly used MGD \u2192 IST (Cdc42-binding deficient) mutant of TOCA1 has a reduced ability to activate the N-WASP\u00b7WIP complex, further indicating the importance of the Cdc42-HR1TOCA1 interaction prior to downstream activation of N-WASP.", "section": "DISCUSS", "ner": [ [ 18, 27, "MGD \u2192 IST", "mutant" ], [ 29, 52, "Cdc42-binding deficient", "protein_state" ], [ 64, 69, "TOCA1", "protein" ], [ 108, 118, "N-WASP\u00b7WIP", "complex_assembly" ], [ 169, 174, "Cdc42", "protein" ], [ 175, 178, "HR1", "structure_element" ], [ 178, 183, "TOCA1", "protein" ], [ 230, 236, "N-WASP", "protein" ] ] }, { "sid": 307, "sent": "In light of this, we favor an \u201ceffector handover\u201d scheme whereby TOCA1 interacts with Cdc42 prior to N-WASP activation, after which N-WASP displaces TOCA1 from its bound Cdc42 in order to be fully activated rather than binding a second Cdc42 molecule.", "section": "DISCUSS", "ner": [ [ 65, 70, "TOCA1", "protein" ], [ 86, 91, "Cdc42", "protein" ], [ 101, 107, "N-WASP", "protein" ], [ 132, 138, "N-WASP", "protein" ], [ 149, 154, "TOCA1", "protein" ], [ 164, 169, "bound", "protein_state" ], [ 170, 175, "Cdc42", "protein" ], [ 191, 206, "fully activated", "protein_state" ], [ 236, 241, "Cdc42", "protein" ] ] }, { "sid": 308, "sent": "Potentially, the TOCA1-Cdc42 interaction functions to position N-WASP and Cdc42 such that they are poised to interact with high affinity.", "section": "DISCUSS", "ner": [ [ 17, 22, "TOCA1", "protein" ], [ 23, 28, "Cdc42", "protein" ], [ 63, 69, "N-WASP", "protein" ], [ 74, 79, "Cdc42", "protein" ] ] }, { "sid": 309, "sent": "The concomitant release of TOCA1 from Cdc42 while still bound to N-WASP presumably enhances the ability of TOCA1 to further activate N-WASP\u00b7WIP-induced actin polymerization.", "section": "DISCUSS", "ner": [ [ 27, 32, "TOCA1", "protein" ], [ 38, 43, "Cdc42", "protein" ], [ 56, 64, "bound to", "protein_state" ], [ 65, 71, "N-WASP", "protein" ], [ 107, 112, "TOCA1", "protein" ], [ 133, 143, "N-WASP\u00b7WIP", "complex_assembly" ] ] }, { "sid": 310, "sent": "There is an advantage to such an effector handover, in that N-WASP would only be robustly recruited when F-BAR domains are already present.", "section": "DISCUSS", "ner": [ [ 60, 66, "N-WASP", "protein" ], [ 105, 110, "F-BAR", "structure_element" ] ] }, { "sid": 311, "sent": "Hence, actin polymerization cannot occur until F-BAR domains are poised for membrane distortion.", "section": "DISCUSS", "ner": [ [ 47, 52, "F-BAR", "structure_element" ] ] }, { "sid": 312, "sent": "Our model of the Cdc42\u00b7HR1TOCA1 complex indicates a mechanism by which such a handover could take place (Fig. 9) because it shows that the effector binding sites only partially overlap on Cdc42.", "section": "DISCUSS", "ner": [ [ 17, 31, "Cdc42\u00b7HR1TOCA1", "complex_assembly" ], [ 139, 161, "effector binding sites", "site" ], [ 188, 193, "Cdc42", "protein" ] ] }, { "sid": 313, "sent": "The lysine residues thought to be involved in an electrostatic steering mechanism in WASP-Cdc42 binding are conserved in N-WASP and would be able to interact with Cdc42 even when the TOCA1 HR1 domain is already bound.", "section": "DISCUSS", "ner": [ [ 4, 10, "lysine", "residue_name" ], [ 85, 89, "WASP", "protein" ], [ 90, 95, "Cdc42", "protein" ], [ 121, 127, "N-WASP", "protein" ], [ 163, 168, "Cdc42", "protein" ], [ 183, 188, "TOCA1", "protein" ], [ 189, 192, "HR1", "structure_element" ], [ 211, 216, "bound", "protein_state" ] ] }, { "sid": 314, "sent": "It has been postulated that the initial interactions between this basic region and Cdc42 could stabilize the active conformation of WASP, leading to high affinity binding between the core CRIB and Cdc42.", "section": "DISCUSS", "ner": [ [ 83, 88, "Cdc42", "protein" ], [ 109, 115, "active", "protein_state" ], [ 132, 136, "WASP", "protein" ], [ 188, 192, "CRIB", "structure_element" ], [ 197, 202, "Cdc42", "protein" ] ] }, { "sid": 315, "sent": "The region C-terminal to the core CRIB, required for maximal affinity binding, would then fully displace the TOCA1 HR1.", "section": "DISCUSS", "ner": [ [ 34, 38, "CRIB", "structure_element" ], [ 109, 114, "TOCA1", "protein" ], [ 115, 118, "HR1", "structure_element" ] ] }, { "sid": 316, "sent": "A simplified model of the early stages of Cdc42\u00b7N-WASP\u00b7TOCA1-dependent actin polymerization.", "section": "FIG", "ner": [ [ 42, 60, "Cdc42\u00b7N-WASP\u00b7TOCA1", "complex_assembly" ] ] }, { "sid": 317, "sent": " Step 1, TOCA1 is recruited to the membrane via its F-BAR domain and/or Cdc42 interactions.", "section": "FIG", "ner": [ [ 9, 14, "TOCA1", "protein" ], [ 52, 57, "F-BAR", "structure_element" ], [ 72, 77, "Cdc42", "protein" ] ] }, { "sid": 318, "sent": "F-BAR oligomerization is expected to occur following membrane binding, but a single monomer is shown for clarity.", "section": "FIG", "ner": [ [ 84, 91, "monomer", "oligomeric_state" ] ] }, { "sid": 319, "sent": "Step 2, N-WASP exists in an inactive, folded conformation.", "section": "FIG", "ner": [ [ 8, 14, "N-WASP", "protein" ], [ 28, 36, "inactive", "protein_state" ], [ 38, 44, "folded", "protein_state" ] ] }, { "sid": 320, "sent": "The TOCA1 SH3 domain interacts with N-WASP, causing an activatory allosteric effect.", "section": "FIG", "ner": [ [ 4, 9, "TOCA1", "protein" ], [ 10, 13, "SH3", "structure_element" ], [ 36, 42, "N-WASP", "protein" ] ] }, { "sid": 321, "sent": "The HR1TOCA1-Cdc42 and SH3TOCA1-N-WASP interactions position Cdc42 and N-WASP for binding.", "section": "FIG", "ner": [ [ 4, 7, "HR1", "structure_element" ], [ 23, 26, "SH3", "structure_element" ], [ 61, 66, "Cdc42", "protein" ], [ 71, 77, "N-WASP", "protein" ] ] }, { "sid": 322, "sent": "Step 3, electrostatic interactions between Cdc42 and the basic region upstream of the CRIB initiate Cdc42\u00b7N-WASP binding.", "section": "FIG", "ner": [ [ 8, 34, "electrostatic interactions", "bond_interaction" ], [ 43, 48, "Cdc42", "protein" ], [ 86, 90, "CRIB", "structure_element" ], [ 100, 112, "Cdc42\u00b7N-WASP", "complex_assembly" ] ] }, { "sid": 323, "sent": "Step 4, the core CRIB binds with high affinity while the region C-terminal to the CRIB displaces the TOCA1 HR1 domain and increases the affinity of the N-WASP-Cdc42 interaction further.", "section": "FIG", "ner": [ [ 17, 21, "CRIB", "structure_element" ], [ 82, 86, "CRIB", "structure_element" ], [ 101, 106, "TOCA1", "protein" ], [ 107, 110, "HR1", "structure_element" ], [ 152, 158, "N-WASP", "protein" ] ] }, { "sid": 324, "sent": "The VCA domain is released for downstream interactions, and actin polymerization proceeds.", "section": "FIG", "ner": [ [ 4, 7, "VCA", "structure_element" ] ] }, { "sid": 325, "sent": "WH1, WASP homology 1 domain; PP, proline-rich region; VCA, verprolin homology, cofilin homology, acidic region.", "section": "FIG", "ner": [ [ 5, 27, "WASP homology 1 domain", "structure_element" ], [ 29, 31, "PP", "structure_element" ], [ 33, 52, "proline-rich region", "structure_element" ], [ 54, 57, "VCA", "structure_element" ], [ 59, 110, "verprolin homology, cofilin homology, acidic region", "structure_element" ] ] }, { "sid": 326, "sent": "In conclusion, the data presented here show that the TOCA1 HR1 domain is sufficient for Cdc42 binding in vitro and that the interaction is of micromolar affinity, lower than that of other G protein-HR1 domain interactions.", "section": "DISCUSS", "ner": [ [ 53, 58, "TOCA1", "protein" ], [ 59, 62, "HR1", "structure_element" ], [ 88, 93, "Cdc42", "protein" ], [ 188, 197, "G protein", "protein_type" ], [ 198, 201, "HR1", "structure_element" ] ] }, { "sid": 327, "sent": "The analogous HR1 domains from other TOCA1 family members, FBP17 and CIP4, also exhibit micromolar affinity for Cdc42.", "section": "DISCUSS", "ner": [ [ 14, 17, "HR1", "structure_element" ], [ 37, 49, "TOCA1 family", "protein_type" ], [ 59, 64, "FBP17", "protein" ], [ 69, 73, "CIP4", "protein" ], [ 112, 117, "Cdc42", "protein" ] ] }, { "sid": 328, "sent": "A role for the TOCA1-, FBP17-, and CIP4-Cdc42 interactions in the recruitment of these proteins to the membrane therefore appears unlikely.", "section": "DISCUSS", "ner": [ [ 15, 20, "TOCA1", "protein" ], [ 23, 28, "FBP17", "protein" ], [ 35, 39, "CIP4", "protein" ], [ 40, 45, "Cdc42", "protein" ] ] }, { "sid": 329, "sent": "Instead, our findings agree with earlier suggestions that the F-BAR domain is responsible for membrane recruitment.", "section": "DISCUSS", "ner": [ [ 62, 67, "F-BAR", "structure_element" ] ] }, { "sid": 330, "sent": "The role of the Cdc42-TOCA1 interaction remains somewhat elusive, but it may serve to position activated Cdc42 and N-WASP to allow full activation of N-WASP and as such serve to couple F-BAR-mediated membrane deformation with N-WASP activation.", "section": "DISCUSS", "ner": [ [ 16, 21, "Cdc42", "protein" ], [ 22, 27, "TOCA1", "protein" ], [ 95, 104, "activated", "protein_state" ], [ 105, 110, "Cdc42", "protein" ], [ 115, 121, "N-WASP", "protein" ], [ 131, 146, "full activation", "protein_state" ], [ 150, 156, "N-WASP", "protein" ], [ 185, 190, "F-BAR", "structure_element" ], [ 226, 232, "N-WASP", "protein" ] ] }, { "sid": 331, "sent": "We envisage a complex interplay of equilibria between free and bound, active and inactive Cdc42, TOCA family, and WASP family proteins, facilitating a tightly spatially and temporally regulated pathway requiring numerous simultaneous events in order to achieve appropriate and robust activation of the downstream pathway.", "section": "DISCUSS", "ner": [ [ 54, 58, "free", "protein_state" ], [ 63, 68, "bound", "protein_state" ], [ 70, 76, "active", "protein_state" ], [ 81, 89, "inactive", "protein_state" ], [ 90, 95, "Cdc42", "protein" ], [ 97, 108, "TOCA family", "protein_type" ], [ 114, 118, "WASP", "protein_type" ] ] }, { "sid": 332, "sent": "Our data are therefore easily reconciled with the dynamic instability models described in relation to the formation of endocytic vesicles and with the current data pertaining to the complex activation of WASP/N-WASP pathways by allosteric and oligomeric effects.", "section": "DISCUSS", "ner": [ [ 204, 208, "WASP", "protein_type" ], [ 209, 215, "N-WASP", "protein" ] ] }, { "sid": 333, "sent": "It is clear from the data presented here that TOCA1 and N-WASP do not bind Cdc42 simultaneously and that N-WASP is likely to outcompete TOCA1 for Cdc42 binding.", "section": "DISCUSS", "ner": [ [ 46, 51, "TOCA1", "protein" ], [ 56, 62, "N-WASP", "protein" ], [ 75, 80, "Cdc42", "protein" ], [ 105, 111, "N-WASP", "protein" ], [ 136, 141, "TOCA1", "protein" ], [ 146, 151, "Cdc42", "protein" ] ] }, { "sid": 334, "sent": "We therefore postulate an effector handover mechanism based on current evidence surrounding WASP/N-WASP activation and our model of the Cdc42\u00b7HR1TOCA1 complex.", "section": "DISCUSS", "ner": [ [ 92, 96, "WASP", "protein" ], [ 97, 103, "N-WASP", "protein" ], [ 136, 150, "Cdc42\u00b7HR1TOCA1", "complex_assembly" ] ] }, { "sid": 335, "sent": "The displacement of the TOCA1 HR1 domain from Cdc42 by N-WASP may represent a unidirectional step in the pathway of Cdc42\u00b7N-WASP\u00b7TOCA1-dependent actin assembly.", "section": "DISCUSS", "ner": [ [ 24, 29, "TOCA1", "protein" ], [ 30, 33, "HR1", "structure_element" ], [ 46, 51, "Cdc42", "protein" ], [ 55, 61, "N-WASP", "protein" ], [ 116, 134, "Cdc42\u00b7N-WASP\u00b7TOCA1", "complex_assembly" ] ] } ] }, "PMC4833862": { "annotations": [ { "sid": 0, "sent": "The dynamic organization of fungal acetyl-CoA carboxylase", "section": "TITLE", "ner": [ [ 4, 11, "dynamic", "protein_state" ], [ 28, 34, "fungal", "taxonomy_domain" ], [ 35, 57, "acetyl-CoA carboxylase", "protein_type" ] ] }, { "sid": 1, "sent": "Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer.", "section": "ABSTRACT", "ner": [ [ 0, 23, "Acetyl-CoA carboxylases", "protein_type" ], [ 25, 29, "ACCs", "protein_type" ], [ 91, 94, "ATP", "chemical" ], [ 122, 132, "acetyl-CoA", "chemical" ], [ 136, 147, "malonyl-CoA", "chemical" ] ] }, { "sid": 2, "sent": "Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized.", "section": "ABSTRACT", "ner": [ [ 0, 10, "Eukaryotic", "taxonomy_domain" ], [ 11, 15, "ACCs", "protein_type" ], [ 20, 45, "single-chain multienzymes", "protein_type" ], [ 72, 85, "non-catalytic", "protein_state" ], [ 86, 100, "central domain", "structure_element" ], [ 102, 104, "CD", "structure_element" ], [ 121, 124, "ACC", "protein_type" ] ] }, { "sid": 3, "sent": "Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization.", "section": "ABSTRACT", "ner": [ [ 19, 36, "crystal structure", "evidence" ], [ 44, 49, "yeast", "taxonomy_domain" ], [ 50, 53, "ACC", "protein_type" ], [ 54, 56, "CD", "structure_element" ] ] }, { "sid": 4, "sent": "A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD.", "section": "ABSTRACT", "ner": [ [ 2, 17, "regulatory loop", "structure_element" ], [ 28, 42, "phosphorylated", "protein_state" ], [ 65, 85, "phosphorylation site", "site" ], [ 89, 95, "fungal", "taxonomy_domain" ], [ 96, 99, "ACC", "protein_type" ], [ 146, 148, "CD", "structure_element" ] ] }, { "sid": 5, "sent": "Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture.", "section": "ABSTRACT", "ner": [ [ 14, 19, "yeast", "taxonomy_domain" ], [ 20, 22, "CD", "structure_element" ], [ 23, 32, "structure", "evidence" ], [ 78, 94, "larger fragments", "mutant" ], [ 101, 107, "intact", "protein_state" ], [ 108, 112, "ACCs", "protein_type" ], [ 162, 169, "dynamic", "protein_state" ], [ 170, 176, "fungal", "taxonomy_domain" ], [ 177, 180, "ACC", "protein_type" ] ] }, { "sid": 6, "sent": "In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.", "section": "ABSTRACT", "ner": [ [ 23, 35, "carboxylases", "protein_type" ], [ 133, 135, "CD", "structure_element" ], [ 142, 157, "phosphorylation", "ptm" ] ] }, { "sid": 7, "sent": " Acetyl-CoA carboxylases are central regulatory hubs of fatty acid metabolism and are important targets for drug development in obesity and cancer.", "section": "ABSTRACT", "ner": [ [ 1, 24, "Acetyl-CoA carboxylases", "protein_type" ] ] }, { "sid": 8, "sent": "Here, the authors demonstrate that the regulation of these highly dynamic enzymes in fungi is governed by a mechanism based on phosphorylation-dependent conformational variability.", "section": "ABSTRACT", "ner": [ [ 59, 73, "highly dynamic", "protein_state" ], [ 74, 81, "enzymes", "protein_type" ], [ 85, 90, "fungi", "taxonomy_domain" ], [ 127, 142, "phosphorylation", "ptm" ] ] }, { "sid": 9, "sent": "Biotin-dependent acetyl-CoA carboxylases (ACCs) are essential enzymes that catalyse the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This reaction provides the committed activated substrate for the biosynthesis of fatty acids via fatty-acid synthase.", "section": "INTRO", "ner": [ [ 0, 40, "Biotin-dependent acetyl-CoA carboxylases", "protein_type" ], [ 42, 46, "ACCs", "protein_type" ], [ 88, 91, "ATP", "chemical" ], [ 119, 129, "acetyl-CoA", "chemical" ], [ 133, 144, "malonyl-CoA", "chemical" ], [ 227, 238, "fatty acids", "chemical" ], [ 243, 262, "fatty-acid synthase", "protein_type" ] ] }, { "sid": 10, "sent": "By catalysing this rate-limiting step in fatty-acid biosynthesis, ACC plays a key role in anabolic metabolism.", "section": "INTRO", "ner": [ [ 66, 69, "ACC", "protein_type" ] ] }, { "sid": 11, "sent": "ACC inhibition and knock-out studies show the potential of targeting ACC for treatment of the metabolic syndrome.", "section": "INTRO", "ner": [ [ 0, 36, "ACC inhibition and knock-out studies", "experimental_method" ], [ 69, 72, "ACC", "protein_type" ] ] }, { "sid": 12, "sent": "Furthermore, elevated ACC activity is observed in malignant tumours.", "section": "INTRO", "ner": [ [ 22, 25, "ACC", "protein_type" ] ] }, { "sid": 13, "sent": "A direct link between ACC and cancer is provided by cancer-associated mutations in the breast cancer susceptibility gene 1 (BRCA1), which relieve inhibitory interactions of BRCA1 with ACC.", "section": "INTRO", "ner": [ [ 22, 25, "ACC", "protein_type" ], [ 70, 79, "mutations", "mutant" ], [ 87, 122, "breast cancer susceptibility gene 1", "protein" ], [ 124, 129, "BRCA1", "protein" ], [ 173, 178, "BRCA1", "protein" ], [ 184, 187, "ACC", "protein_type" ] ] }, { "sid": 14, "sent": "Thus, ACC is a relevant drug target for type 2 diabetes and cancer.", "section": "INTRO", "ner": [ [ 6, 9, "ACC", "protein_type" ] ] }, { "sid": 15, "sent": "Microbial ACCs are also the principal target of antifungal and antibiotic compounds, such as Soraphen A.", "section": "INTRO", "ner": [ [ 0, 9, "Microbial", "taxonomy_domain" ], [ 10, 14, "ACCs", "protein_type" ], [ 93, 103, "Soraphen A", "chemical" ] ] }, { "sid": 16, "sent": "The principal functional protein components of ACCs have been described already in the late 1960s for Escherichia coli (E. coli) ACC: Biotin carboxylase (BC) catalyses the ATP-dependent carboxylation of a biotin moiety, which is covalently linked to the biotin carboxyl carrier protein (BCCP).", "section": "INTRO", "ner": [ [ 47, 51, "ACCs", "protein_type" ], [ 102, 118, "Escherichia coli", "species" ], [ 120, 127, "E. coli", "species" ], [ 129, 132, "ACC", "protein_type" ], [ 134, 152, "Biotin carboxylase", "protein_type" ], [ 154, 156, "BC", "protein_type" ], [ 172, 175, "ATP", "chemical" ], [ 205, 211, "biotin", "chemical" ], [ 254, 285, "biotin carboxyl carrier protein", "protein_type" ], [ 287, 291, "BCCP", "protein_type" ] ] }, { "sid": 17, "sent": "Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits.", "section": "INTRO", "ner": [ [ 0, 19, "Carboxyltransferase", "protein_type" ], [ 21, 23, "CT", "protein_type" ], [ 49, 57, "carboxyl", "chemical" ], [ 69, 82, "carboxybiotin", "chemical" ], [ 86, 96, "acetyl-CoA", "chemical" ], [ 106, 117, "malonyl-CoA", "chemical" ], [ 119, 130, "Prokaryotic", "taxonomy_domain" ], [ 131, 135, "ACCs", "protein_type" ], [ 140, 149, "transient", "protein_state" ], [ 175, 177, "BC", "protein_type" ], [ 179, 181, "CT", "protein_type" ], [ 186, 190, "BCCP", "protein_type" ] ] }, { "sid": 18, "sent": "Eukaryotic ACCs, instead, are multienzymes, which integrate all functional components into a single polypeptide chain of \u223c2,300 amino acids.", "section": "INTRO", "ner": [ [ 0, 10, "Eukaryotic", "taxonomy_domain" ], [ 11, 15, "ACCs", "protein_type" ], [ 30, 42, "multienzymes", "protein_type" ] ] }, { "sid": 19, "sent": "Human ACC occurs in two closely related isoforms, ACC1 and 2, located in the cytosol and at the outer mitochondrial membrane, respectively.", "section": "INTRO", "ner": [ [ 0, 5, "Human", "species" ], [ 6, 9, "ACC", "protein_type" ], [ 40, 48, "isoforms", "protein_state" ], [ 50, 54, "ACC1", "protein" ], [ 59, 60, "2", "protein" ] ] }, { "sid": 20, "sent": "In addition to the canonical ACC components, eukaryotic ACCs contain two non-catalytic regions, the large central domain (CD) and the BC\u2013CT interaction domain (BT).", "section": "INTRO", "ner": [ [ 29, 43, "ACC components", "structure_element" ], [ 45, 55, "eukaryotic", "taxonomy_domain" ], [ 56, 60, "ACCs", "protein_type" ], [ 73, 86, "non-catalytic", "protein_state" ], [ 87, 94, "regions", "structure_element" ], [ 106, 120, "central domain", "structure_element" ], [ 122, 124, "CD", "structure_element" ], [ 134, 158, "BC\u2013CT interaction domain", "structure_element" ], [ 160, 162, "BT", "structure_element" ] ] }, { "sid": 21, "sent": "The CD comprises one-third of the protein and is a unique feature of eukaryotic ACCs without homologues in other proteins.", "section": "INTRO", "ner": [ [ 4, 6, "CD", "structure_element" ], [ 51, 68, "unique feature of", "protein_state" ], [ 69, 79, "eukaryotic", "taxonomy_domain" ], [ 80, 84, "ACCs", "protein_type" ] ] }, { "sid": 22, "sent": "The function of this domain remains poorly characterized, although phosphorylation of several serine residues in the CD regulates ACC activity.", "section": "INTRO", "ner": [ [ 67, 82, "phosphorylation", "ptm" ], [ 94, 100, "serine", "residue_name" ], [ 117, 119, "CD", "structure_element" ], [ 130, 133, "ACC", "protein_type" ] ] }, { "sid": 23, "sent": "The BT domain has been visualized in bacterial carboxylases, where it mediates contacts between \u03b1- and \u03b2-subunits.", "section": "INTRO", "ner": [ [ 4, 6, "BT", "structure_element" ], [ 37, 46, "bacterial", "taxonomy_domain" ], [ 47, 59, "carboxylases", "protein_type" ], [ 96, 98, "\u03b1-", "structure_element" ], [ 103, 113, "\u03b2-subunits", "structure_element" ] ] }, { "sid": 24, "sent": "Structural studies on the functional architecture of intact ACCs have been hindered by their huge size and pronounced dynamics, as well as the transient assembly mode of bacterial ACCs.", "section": "INTRO", "ner": [ [ 0, 18, "Structural studies", "experimental_method" ], [ 53, 59, "intact", "protein_state" ], [ 60, 64, "ACCs", "protein_type" ], [ 143, 152, "transient", "protein_state" ], [ 170, 179, "bacterial", "taxonomy_domain" ], [ 180, 184, "ACCs", "protein_type" ] ] }, { "sid": 25, "sent": "However, crystal structures of individual components or domains from prokaryotic and eukaryotic ACCs, respectively, have been solved.", "section": "INTRO", "ner": [ [ 9, 27, "crystal structures", "evidence" ], [ 69, 80, "prokaryotic", "taxonomy_domain" ], [ 85, 95, "eukaryotic", "taxonomy_domain" ], [ 96, 100, "ACCs", "protein_type" ] ] }, { "sid": 26, "sent": "The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components.", "section": "INTRO", "ner": [ [ 4, 27, "structure determination", "experimental_method" ], [ 35, 46, "holoenzymes", "protein_state" ], [ 50, 59, "bacterial", "taxonomy_domain" ], [ 60, 89, "biotin-dependent carboxylases", "protein_type" ], [ 97, 101, "lack", "protein_state" ], [ 121, 123, "CD", "structure_element" ], [ 137, 157, "pyruvate carboxylase", "protein_type" ], [ 159, 161, "PC", "protein_type" ], [ 164, 189, "propionyl-CoA carboxylase", "protein_type" ], [ 191, 224, "3-methyl-crotonyl-CoA carboxylase", "protein_type" ], [ 231, 262, "long-chain acyl-CoA carboxylase", "protein_type" ] ] }, { "sid": 27, "sent": "In these structures, the BC and CT active sites are at distances between 40 and 80\u2009\u00c5, such that substrate transfer could be mediated solely by the mobility of the flexibly tethered BCCP.", "section": "INTRO", "ner": [ [ 9, 19, "structures", "evidence" ], [ 25, 27, "BC", "protein_type" ], [ 32, 34, "CT", "protein_type" ], [ 35, 47, "active sites", "site" ], [ 163, 180, "flexibly tethered", "protein_state" ], [ 181, 185, "BCCP", "protein_type" ] ] }, { "sid": 28, "sent": "Human ACC1 is regulated allosterically, via specific protein\u2013protein interactions, and by reversible phosphorylation.", "section": "INTRO", "ner": [ [ 0, 5, "Human", "species" ], [ 6, 10, "ACC1", "protein" ], [ 14, 38, "regulated allosterically", "protein_state" ], [ 101, 116, "phosphorylation", "ptm" ] ] }, { "sid": 29, "sent": "Dynamic polymerization of human ACC1 is linked to increased activity and is regulated allosterically by the activator citrate and the inhibitor palmitate, or by binding of the small protein MIG-12 (ref.).", "section": "INTRO", "ner": [ [ 26, 31, "human", "species" ], [ 32, 36, "ACC1", "protein" ], [ 76, 100, "regulated allosterically", "protein_state" ], [ 118, 125, "citrate", "chemical" ], [ 144, 153, "palmitate", "chemical" ], [ 190, 196, "MIG-12", "protein" ] ] }, { "sid": 30, "sent": "Human ACC1 is further regulated by specific phosphorylation-dependent binding of BRCA1 to Ser1263 in the CD.", "section": "INTRO", "ner": [ [ 0, 5, "Human", "species" ], [ 6, 10, "ACC1", "protein" ], [ 44, 59, "phosphorylation", "ptm" ], [ 81, 86, "BRCA1", "protein" ], [ 90, 97, "Ser1263", "residue_name_number" ], [ 105, 107, "CD", "structure_element" ] ] }, { "sid": 31, "sent": "BRCA1 binds only to the phosphorylated form of ACC1 and prevents ACC activation by phosphatase-mediated dephosphorylation.", "section": "INTRO", "ner": [ [ 0, 5, "BRCA1", "protein" ], [ 24, 38, "phosphorylated", "protein_state" ], [ 47, 51, "ACC1", "protein" ], [ 65, 68, "ACC", "protein_type" ], [ 83, 94, "phosphatase", "protein_type" ] ] }, { "sid": 32, "sent": "Furthermore, phosphorylation by AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA) leads to a decrease in ACC1 activity.", "section": "INTRO", "ner": [ [ 13, 28, "phosphorylation", "ptm" ], [ 32, 60, "AMP-activated protein kinase", "protein" ], [ 62, 66, "AMPK", "protein" ], [ 72, 101, "cAMP-dependent protein kinase", "protein" ], [ 103, 106, "PKA", "protein" ], [ 131, 135, "ACC1", "protein" ] ] }, { "sid": 33, "sent": "AMPK phosphorylates ACC1 in vitro at Ser80, Ser1201 and Ser1216 and PKA at Ser78 and Ser1201.", "section": "INTRO", "ner": [ [ 0, 4, "AMPK", "protein" ], [ 20, 24, "ACC1", "protein" ], [ 37, 42, "Ser80", "residue_name_number" ], [ 44, 51, "Ser1201", "residue_name_number" ], [ 56, 63, "Ser1216", "residue_name_number" ], [ 68, 71, "PKA", "protein" ], [ 75, 80, "Ser78", "residue_name_number" ], [ 85, 92, "Ser1201", "residue_name_number" ] ] }, { "sid": 34, "sent": "However, regulatory effects on ACC1 activity are mainly mediated by phosphorylation of Ser80 and Ser1201 (refs).", "section": "INTRO", "ner": [ [ 31, 35, "ACC1", "protein" ], [ 68, 83, "phosphorylation", "ptm" ], [ 87, 92, "Ser80", "residue_name_number" ], [ 97, 104, "Ser1201", "residue_name_number" ] ] }, { "sid": 35, "sent": "Phosphorylated Ser80, which is highly conserved only in higher eukaryotes, presumably binds into the Soraphen A-binding pocket.", "section": "INTRO", "ner": [ [ 0, 14, "Phosphorylated", "protein_state" ], [ 15, 20, "Ser80", "residue_name_number" ], [ 31, 47, "highly conserved", "protein_state" ], [ 56, 73, "higher eukaryotes", "taxonomy_domain" ], [ 101, 126, "Soraphen A-binding pocket", "site" ] ] }, { "sid": 36, "sent": "The regulatory Ser1201 shows only moderate conservation across higher eukaryotes, while the phosphorylated Ser1216 is highly conserved across all eukaryotes.", "section": "INTRO", "ner": [ [ 15, 22, "Ser1201", "residue_name_number" ], [ 34, 55, "moderate conservation", "protein_state" ], [ 63, 80, "higher eukaryotes", "taxonomy_domain" ], [ 92, 106, "phosphorylated", "protein_state" ], [ 107, 114, "Ser1216", "residue_name_number" ], [ 118, 134, "highly conserved", "protein_state" ], [ 146, 156, "eukaryotes", "taxonomy_domain" ] ] }, { "sid": 37, "sent": "However, no effect of Ser1216 phosphorylation on ACC activity has been reported in higher eukaryotes.", "section": "INTRO", "ner": [ [ 22, 29, "Ser1216", "residue_name_number" ], [ 30, 45, "phosphorylation", "ptm" ], [ 49, 52, "ACC", "protein_type" ], [ 83, 100, "higher eukaryotes", "taxonomy_domain" ] ] }, { "sid": 38, "sent": "For fungal ACC, neither spontaneous nor inducible polymerization has been detected despite considerable sequence conservation to human ACC1.", "section": "INTRO", "ner": [ [ 4, 10, "fungal", "taxonomy_domain" ], [ 11, 14, "ACC", "protein_type" ], [ 129, 134, "human", "species" ], [ 135, 139, "ACC1", "protein" ] ] }, { "sid": 39, "sent": "The BRCA1-interacting phosphoserine position is not conserved in fungal ACC, and no other phospho-dependent protein\u2013protein interactions of fungal ACC have been described.", "section": "INTRO", "ner": [ [ 4, 9, "BRCA1", "protein" ], [ 22, 35, "phosphoserine", "residue_name" ], [ 48, 61, "not conserved", "protein_state" ], [ 65, 71, "fungal", "taxonomy_domain" ], [ 72, 75, "ACC", "protein_type" ], [ 140, 146, "fungal", "taxonomy_domain" ], [ 147, 150, "ACC", "protein_type" ] ] }, { "sid": 40, "sent": "In yeast ACC, phosphorylation sites have been identified at Ser2, Ser735, Ser1148, Ser1157 and Ser1162 (ref.).", "section": "INTRO", "ner": [ [ 3, 8, "yeast", "taxonomy_domain" ], [ 9, 12, "ACC", "protein_type" ], [ 14, 35, "phosphorylation sites", "site" ], [ 60, 64, "Ser2", "residue_name_number" ], [ 66, 72, "Ser735", "residue_name_number" ], [ 74, 81, "Ser1148", "residue_name_number" ], [ 83, 90, "Ser1157", "residue_name_number" ], [ 95, 102, "Ser1162", "residue_name_number" ] ] }, { "sid": 41, "sent": "Of these, only Ser1157 is highly conserved in fungal ACC and aligns to Ser1216 in human ACC1.", "section": "INTRO", "ner": [ [ 15, 22, "Ser1157", "residue_name_number" ], [ 26, 42, "highly conserved", "protein_state" ], [ 46, 52, "fungal", "taxonomy_domain" ], [ 53, 56, "ACC", "protein_type" ], [ 61, 70, "aligns to", "experimental_method" ], [ 71, 78, "Ser1216", "residue_name_number" ], [ 82, 87, "human", "species" ], [ 88, 92, "ACC1", "protein" ] ] }, { "sid": 42, "sent": "Its phosphorylation by the AMPK homologue SNF1 results in strongly reduced ACC activity.", "section": "INTRO", "ner": [ [ 4, 19, "phosphorylation", "ptm" ], [ 27, 31, "AMPK", "protein" ], [ 42, 46, "SNF1", "protein" ], [ 75, 78, "ACC", "protein_type" ] ] }, { "sid": 43, "sent": "Despite the outstanding relevance of ACC in primary metabolism and disease, the dynamic organization and regulation of the giant eukaryotic, and in particular fungal ACC, remain poorly characterized.", "section": "INTRO", "ner": [ [ 37, 40, "ACC", "protein_type" ], [ 129, 139, "eukaryotic", "taxonomy_domain" ], [ 159, 165, "fungal", "taxonomy_domain" ], [ 166, 169, "ACC", "protein_type" ] ] }, { "sid": 44, "sent": "Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a).", "section": "INTRO", "ner": [ [ 20, 29, "structure", "evidence" ], [ 33, 57, "Saccharomyces cerevisiae", "species" ], [ 59, 62, "Sce", "species" ], [ 64, 67, "ACC", "protein_type" ], [ 68, 70, "CD", "structure_element" ], [ 105, 115, "structures", "evidence" ], [ 119, 124, "human", "species" ], [ 126, 129, "Hsa", "species" ], [ 131, 134, "ACC", "protein_type" ], [ 135, 137, "CD", "structure_element" ], [ 142, 158, "larger fragments", "mutant" ], [ 162, 168, "fungal", "taxonomy_domain" ], [ 169, 172, "ACC", "protein_type" ], [ 178, 201, "Chaetomium thermophilum", "species" ], [ 203, 206, "Cth", "species" ] ] }, { "sid": 45, "sent": "Integrating these data with small-angle X-ray scattering (SAXS) and electron microscopy (EM) observations yield a comprehensive representation of the dynamic structure and regulation of fungal ACC.", "section": "INTRO", "ner": [ [ 28, 56, "small-angle X-ray scattering", "experimental_method" ], [ 58, 62, "SAXS", "experimental_method" ], [ 68, 87, "electron microscopy", "experimental_method" ], [ 89, 91, "EM", "experimental_method" ], [ 186, 192, "fungal", "taxonomy_domain" ], [ 193, 196, "ACC", "protein_type" ] ] }, { "sid": 46, "sent": "The organization of the yeast ACC CD", "section": "RESULTS", "ner": [ [ 24, 29, "yeast", "taxonomy_domain" ], [ 30, 33, "ACC", "protein_type" ], [ 34, 36, "CD", "structure_element" ] ] }, { "sid": 47, "sent": "First, we focused on structure determination of the 82-kDa CD.", "section": "RESULTS", "ner": [ [ 21, 44, "structure determination", "experimental_method" ], [ 59, 61, "CD", "structure_element" ] ] }, { "sid": 48, "sent": "The crystal structure of the CD of SceACC (SceCD) was determined at 3.0\u2009\u00c5 resolution by experimental phasing and refined to Rwork/Rfree=0.20/0.24 (Table 1).", "section": "RESULTS", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 29, 31, "CD", "structure_element" ], [ 35, 41, "SceACC", "protein" ], [ 43, 46, "Sce", "species" ], [ 46, 48, "CD", "structure_element" ], [ 88, 108, "experimental phasing", "experimental_method" ], [ 113, 120, "refined", "experimental_method" ], [ 124, 129, "Rwork", "evidence" ], [ 130, 135, "Rfree", "evidence" ] ] }, { "sid": 49, "sent": "The overall extent of the SceCD is 70 by 75\u2009\u00c5 (Fig. 1b and Supplementary Fig. 1a,b), and the attachment points of the N-terminal 26-residue linker to the BCCP domain and the C-terminal CT domain are separated by 46\u2009\u00c5 (the N- and C termini are indicated with spheres in Fig. 1b).", "section": "RESULTS", "ner": [ [ 26, 29, "Sce", "species" ], [ 29, 31, "CD", "structure_element" ], [ 129, 146, "26-residue linker", "structure_element" ], [ 154, 158, "BCCP", "structure_element" ], [ 185, 187, "CT", "structure_element" ] ] }, { "sid": 50, "sent": "SceCD comprises four distinct domains, an N-terminal \u03b1-helical domain (CDN), and a central four-helix bundle linker domain (CDL), followed by two \u03b1\u2013\u03b2-fold C-terminal domains (CDC1/CDC2).", "section": "RESULTS", "ner": [ [ 0, 3, "Sce", "species" ], [ 3, 5, "CD", "structure_element" ], [ 53, 69, "\u03b1-helical domain", "structure_element" ], [ 71, 74, "CDN", "structure_element" ], [ 91, 122, "four-helix bundle linker domain", "structure_element" ], [ 124, 127, "CDL", "structure_element" ], [ 146, 173, "\u03b1\u2013\u03b2-fold C-terminal domains", "structure_element" ], [ 175, 179, "CDC1", "structure_element" ], [ 180, 184, "CDC2", "structure_element" ] ] }, { "sid": 51, "sent": "CDN adopts a letter C shape, where one of the ends is a regular four-helix bundle (N\u03b13-6), the other end is a helical hairpin (N\u03b18,9) and the bridging region comprises six helices (N\u03b11,2,7,10\u201312).", "section": "RESULTS", "ner": [ [ 0, 3, "CDN", "structure_element" ], [ 20, 27, "C shape", "protein_state" ], [ 56, 81, "regular four-helix bundle", "structure_element" ], [ 83, 88, "N\u03b13-6", "structure_element" ], [ 110, 125, "helical hairpin", "structure_element" ], [ 127, 132, "N\u03b18,9", "structure_element" ], [ 142, 157, "bridging region", "structure_element" ], [ 172, 179, "helices", "structure_element" ], [ 181, 194, "N\u03b11,2,7,10\u201312", "structure_element" ] ] }, { "sid": 52, "sent": "CDL is composed of a small, irregular four-helix bundle (L\u03b11\u20134) and tightly interacts with the open face of CDC1 via an interface of 1,300\u2009\u00c52 involving helices L\u03b13 and L\u03b14.", "section": "RESULTS", "ner": [ [ 0, 3, "CDL", "structure_element" ], [ 21, 55, "small, irregular four-helix bundle", "structure_element" ], [ 57, 62, "L\u03b11\u20134", "structure_element" ], [ 108, 112, "CDC1", "structure_element" ], [ 120, 129, "interface", "site" ], [ 152, 159, "helices", "structure_element" ], [ 160, 163, "L\u03b13", "structure_element" ], [ 168, 171, "L\u03b14", "structure_element" ] ] }, { "sid": 53, "sent": "CDL does not interact with CDN apart from the covalent linkage and forms only a small contact to CDC2 via a loop between L\u03b12/\u03b13 and the N-terminal end of L\u03b11, with an interface area of 400\u2009\u00c52.", "section": "RESULTS", "ner": [ [ 0, 3, "CDL", "structure_element" ], [ 27, 30, "CDN", "structure_element" ], [ 97, 101, "CDC2", "structure_element" ], [ 108, 112, "loop", "structure_element" ], [ 121, 127, "L\u03b12/\u03b13", "structure_element" ], [ 154, 157, "L\u03b11", "structure_element" ], [ 167, 176, "interface", "site" ] ] }, { "sid": 54, "sent": "CDC1/CDC2 share a common fold; they are composed of six-stranded \u03b2-sheets flanked on one side by two long, bent helices inserted between strands \u03b23/\u03b24 and \u03b24/\u03b25.", "section": "RESULTS", "ner": [ [ 0, 4, "CDC1", "structure_element" ], [ 5, 9, "CDC2", "structure_element" ], [ 52, 73, "six-stranded \u03b2-sheets", "structure_element" ], [ 101, 119, "long, bent helices", "structure_element" ], [ 137, 144, "strands", "structure_element" ], [ 145, 150, "\u03b23/\u03b24", "structure_element" ], [ 155, 160, "\u03b24/\u03b25", "structure_element" ] ] }, { "sid": 55, "sent": "CDC2 is extended at its C terminus by an additional \u03b2-strand and an irregular \u03b2-hairpin.", "section": "RESULTS", "ner": [ [ 0, 4, "CDC2", "structure_element" ], [ 8, 16, "extended", "protein_state" ], [ 52, 60, "\u03b2-strand", "structure_element" ], [ 68, 87, "irregular \u03b2-hairpin", "structure_element" ] ] }, { "sid": 56, "sent": "On the basis of a root mean square deviation of main chain atom positions of 2.2\u2009\u00c5, CDC1/CDC2 are structurally more closely related to each other than to any other protein (Fig. 1c); they may thus have evolved by duplication.", "section": "RESULTS", "ner": [ [ 18, 44, "root mean square deviation", "evidence" ], [ 84, 88, "CDC1", "structure_element" ], [ 89, 93, "CDC2", "structure_element" ] ] }, { "sid": 57, "sent": "Close structural homologues could not be found for the CDN or the CDC domains.", "section": "RESULTS", "ner": [ [ 55, 58, "CDN", "structure_element" ], [ 66, 69, "CDC", "structure_element" ] ] }, { "sid": 58, "sent": "A regulatory loop mediates interdomain interactions", "section": "RESULTS", "ner": [ [ 2, 17, "regulatory loop", "structure_element" ] ] }, { "sid": 59, "sent": "To define the functional state of insect-cell-expressed ACC variants, we employed mass spectrometry (MS) for phosphorylation site detection.", "section": "RESULTS", "ner": [ [ 34, 55, "insect-cell-expressed", "experimental_method" ], [ 56, 59, "ACC", "protein_type" ], [ 82, 99, "mass spectrometry", "experimental_method" ], [ 101, 103, "MS", "experimental_method" ], [ 109, 139, "phosphorylation site detection", "experimental_method" ] ] }, { "sid": 60, "sent": "In insect-cell-expressed full-length SceACC, the highly conserved Ser1157 is the only fully occupied phosphorylation site with functional relevance in S. cerevisiae.", "section": "RESULTS", "ner": [ [ 3, 24, "insect-cell-expressed", "experimental_method" ], [ 25, 36, "full-length", "protein_state" ], [ 37, 43, "SceACC", "protein" ], [ 49, 65, "highly conserved", "protein_state" ], [ 66, 73, "Ser1157", "residue_name_number" ], [ 86, 100, "fully occupied", "protein_state" ], [ 101, 121, "phosphorylation site", "site" ], [ 151, 164, "S. cerevisiae", "species" ] ] }, { "sid": 61, "sent": "Additional phosphorylation was detected for Ser2101 and Tyr2179; however, these sites are neither conserved across fungal ACC nor natively phosphorylated in yeast.", "section": "RESULTS", "ner": [ [ 11, 26, "phosphorylation", "ptm" ], [ 44, 51, "Ser2101", "residue_name_number" ], [ 56, 63, "Tyr2179", "residue_name_number" ], [ 90, 107, "neither conserved", "protein_state" ], [ 115, 121, "fungal", "taxonomy_domain" ], [ 122, 125, "ACC", "protein_type" ], [ 126, 153, "nor natively phosphorylated", "protein_state" ], [ 157, 162, "yeast", "taxonomy_domain" ] ] }, { "sid": 62, "sent": "MS analysis of dissolved crystals confirmed the phosphorylated state of Ser1157 also in SceCD crystals.", "section": "RESULTS", "ner": [ [ 0, 2, "MS", "experimental_method" ], [ 15, 33, "dissolved crystals", "experimental_method" ], [ 48, 62, "phosphorylated", "protein_state" ], [ 72, 79, "Ser1157", "residue_name_number" ], [ 88, 91, "Sce", "species" ], [ 91, 93, "CD", "structure_element" ], [ 94, 102, "crystals", "evidence" ] ] }, { "sid": 63, "sent": "The SceCD structure thus authentically represents the state of SceACC, where the enzyme is inhibited by SNF1-dependent phosphorylation.", "section": "RESULTS", "ner": [ [ 4, 7, "Sce", "species" ], [ 7, 9, "CD", "structure_element" ], [ 10, 19, "structure", "evidence" ], [ 63, 69, "SceACC", "protein" ], [ 81, 87, "enzyme", "protein" ], [ 91, 100, "inhibited", "protein_state" ], [ 104, 134, "SNF1-dependent phosphorylation", "ptm" ] ] }, { "sid": 64, "sent": "In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands \u03b22 and \u03b23 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here.", "section": "RESULTS", "ner": [ [ 7, 10, "Sce", "species" ], [ 10, 12, "CD", "structure_element" ], [ 13, 30, "crystal structure", "evidence" ], [ 36, 50, "phosphorylated", "protein_state" ], [ 51, 58, "Ser1157", "residue_name_number" ], [ 72, 101, "regulatory 36-amino-acid loop", "structure_element" ], [ 110, 117, "strands", "structure_element" ], [ 118, 120, "\u03b22", "structure_element" ], [ 125, 127, "\u03b23", "structure_element" ], [ 131, 135, "CDC1", "structure_element" ], [ 179, 193, "less-conserved", "protein_state" ], [ 194, 215, "phosphorylation sites", "site" ], [ 217, 224, "Ser1148", "residue_name_number" ], [ 229, 236, "Ser1162", "residue_name_number" ], [ 251, 256, "yeast", "taxonomy_domain" ] ] }, { "sid": 65, "sent": "This regulatory loop wedges between the CDC1 and CDC2 domains and provides the largest contribution to the interdomain interface.", "section": "RESULTS", "ner": [ [ 5, 20, "regulatory loop", "structure_element" ], [ 40, 44, "CDC1", "structure_element" ], [ 49, 53, "CDC2", "structure_element" ], [ 107, 128, "interdomain interface", "site" ] ] }, { "sid": 66, "sent": "The N-terminal region of the regulatory loop also directly contacts the C-terminal region of CDC2 leading into CT.", "section": "RESULTS", "ner": [ [ 29, 44, "regulatory loop", "structure_element" ], [ 93, 97, "CDC2", "structure_element" ], [ 111, 113, "CT", "structure_element" ] ] }, { "sid": 67, "sent": "Phosphoserine 1157 is tightly bound by two highly conserved arginines (Arg1173 and Arg1260) of CDC1 (Fig. 1d).", "section": "RESULTS", "ner": [ [ 0, 18, "Phosphoserine 1157", "residue_name_number" ], [ 43, 59, "highly conserved", "protein_state" ], [ 60, 69, "arginines", "residue_name" ], [ 71, 78, "Arg1173", "residue_name_number" ], [ 83, 90, "Arg1260", "residue_name_number" ], [ 95, 99, "CDC1", "structure_element" ] ] }, { "sid": 68, "sent": "Already the binding of phosphorylated Ser1157 apparently stabilizes the regulatory loop conformation; the accessory phosphorylation sites Ser1148 and Ser1162 in the same loop may further modulate the strength of interaction between the regulatory loop and the CDC1 and CDC2 domains.", "section": "RESULTS", "ner": [ [ 23, 37, "phosphorylated", "protein_state" ], [ 38, 45, "Ser1157", "residue_name_number" ], [ 72, 87, "regulatory loop", "structure_element" ], [ 116, 137, "phosphorylation sites", "site" ], [ 138, 145, "Ser1148", "residue_name_number" ], [ 150, 157, "Ser1162", "residue_name_number" ], [ 165, 174, "same loop", "structure_element" ], [ 236, 251, "regulatory loop", "structure_element" ], [ 260, 264, "CDC1", "structure_element" ], [ 269, 273, "CDC2", "structure_element" ] ] }, { "sid": 69, "sent": "Phosphorylation of the regulatory loop thus determines interdomain interactions of CDC1 and CDC2, suggesting that it may exert its regulatory function by modifying the overall structure and dynamics of the CD.", "section": "RESULTS", "ner": [ [ 0, 15, "Phosphorylation", "ptm" ], [ 23, 38, "regulatory loop", "structure_element" ], [ 83, 87, "CDC1", "structure_element" ], [ 92, 96, "CDC2", "structure_element" ], [ 206, 208, "CD", "structure_element" ] ] }, { "sid": 70, "sent": "The functional role of Ser1157 was confirmed by an activity assay based on the incorporation of radioactive carbonate into acid non-volatile material.", "section": "RESULTS", "ner": [ [ 23, 30, "Ser1157", "residue_name_number" ], [ 51, 65, "activity assay", "experimental_method" ] ] }, { "sid": 71, "sent": "Phosphorylated SceACC shows only residual activity (kcat=0.4\u00b10.2\u2009s\u22121, s.d. based on five replicate measurements), which increases 16-fold (kcat=6.5\u00b10.3\u2009s\u22121) after dephosphorylation with \u03bb protein phosphatase.", "section": "RESULTS", "ner": [ [ 0, 14, "Phosphorylated", "protein_state" ], [ 15, 21, "SceACC", "protein" ], [ 52, 56, "kcat", "evidence" ], [ 139, 143, "kcat", "evidence" ], [ 186, 207, "\u03bb protein phosphatase", "protein_type" ] ] }, { "sid": 72, "sent": "The values obtained for dephosphorylated SceACC are comparable to earlier measurements of non-phosphorylated yeast ACC expressed in E. coli.", "section": "RESULTS", "ner": [ [ 24, 40, "dephosphorylated", "protein_state" ], [ 41, 47, "SceACC", "protein" ], [ 90, 108, "non-phosphorylated", "protein_state" ], [ 109, 114, "yeast", "taxonomy_domain" ], [ 115, 118, "ACC", "protein_type" ], [ 119, 131, "expressed in", "experimental_method" ], [ 132, 139, "E. coli", "species" ] ] }, { "sid": 73, "sent": "The variable CD is conserved between yeast and human", "section": "RESULTS", "ner": [ [ 13, 15, "CD", "structure_element" ], [ 19, 28, "conserved", "protein_state" ], [ 37, 42, "yeast", "taxonomy_domain" ], [ 47, 52, "human", "species" ] ] }, { "sid": 74, "sent": "To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a).", "section": "RESULTS", "ner": [ [ 31, 37, "fungal", "taxonomy_domain" ], [ 42, 47, "human", "species" ], [ 48, 51, "ACC", "protein_type" ], [ 52, 54, "CD", "structure_element" ], [ 59, 83, "determined the structure", "experimental_method" ], [ 89, 94, "human", "species" ], [ 95, 108, "ACC1 fragment", "mutant" ], [ 128, 130, "BT", "structure_element" ], [ 135, 137, "CD", "structure_element" ], [ 147, 155, "HsaBT-CD", "mutant" ], [ 162, 167, "lacks", "protein_state" ], [ 179, 183, "BCCP", "structure_element" ] ] }, { "sid": 75, "sent": "An experimentally phased map was obtained at 3.7\u2009\u00c5 resolution for a cadmium-derivatized crystal and was interpreted by a poly-alanine model (Fig. 1e and Table 1).", "section": "RESULTS", "ner": [ [ 3, 28, "experimentally phased map", "evidence" ], [ 68, 75, "cadmium", "chemical" ] ] }, { "sid": 76, "sent": "Each of the four CD domains in HsaBT-CD individually resembles the corresponding SceCD domain; however, human and yeast CDs exhibit distinct overall structures.", "section": "RESULTS", "ner": [ [ 17, 19, "CD", "structure_element" ], [ 31, 39, "HsaBT-CD", "mutant" ], [ 81, 84, "Sce", "species" ], [ 84, 86, "CD", "structure_element" ], [ 104, 109, "human", "species" ], [ 114, 119, "yeast", "taxonomy_domain" ], [ 120, 123, "CDs", "structure_element" ], [ 149, 159, "structures", "evidence" ] ] }, { "sid": 77, "sent": "In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30\u00b0 based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c).", "section": "RESULTS", "ner": [ [ 45, 48, "Sce", "species" ], [ 48, 50, "CD", "structure_element" ], [ 88, 91, "CDL", "structure_element" ], [ 96, 100, "CDC1", "structure_element" ], [ 117, 125, "HsaBT-CD", "mutant" ], [ 135, 140, "human", "species" ], [ 141, 144, "CDL", "structure_element" ], [ 145, 149, "CDC1", "structure_element" ], [ 187, 200, "superposition", "experimental_method" ], [ 204, 209, "human", "species" ], [ 214, 219, "yeast", "taxonomy_domain" ], [ 220, 224, "CDC2", "structure_element" ] ] }, { "sid": 78, "sent": "As a result, the N terminus of CDL at helix L\u03b11, which connects to CDN, is shifted by 12\u2009\u00c5. Remarkably, CDN of HsaBT-CD adopts a completely different orientation compared with SceCD.", "section": "RESULTS", "ner": [ [ 31, 34, "CDL", "structure_element" ], [ 38, 43, "helix", "structure_element" ], [ 44, 47, "L\u03b11", "structure_element" ], [ 67, 70, "CDN", "structure_element" ], [ 104, 107, "CDN", "structure_element" ], [ 111, 119, "HsaBT-CD", "mutant" ], [ 176, 179, "Sce", "species" ], [ 179, 181, "CD", "structure_element" ] ] }, { "sid": 79, "sent": "With CDL/CDC1 superposed, CDN in HsaBT-CD is rotated by 160\u00b0 around a hinge at the connection of CDN/CDL (Supplementary Fig. 1d).", "section": "RESULTS", "ner": [ [ 5, 8, "CDL", "structure_element" ], [ 9, 13, "CDC1", "structure_element" ], [ 14, 24, "superposed", "experimental_method" ], [ 26, 29, "CDN", "structure_element" ], [ 33, 41, "HsaBT-CD", "mutant" ], [ 70, 75, "hinge", "structure_element" ], [ 97, 100, "CDN", "structure_element" ], [ 101, 104, "CDL", "structure_element" ] ] }, { "sid": 80, "sent": "This rotation displaces the N terminus of CDN in HsaBT-CD by 51\u2009\u00c5 compared with SceCD, resulting in a separation of the attachment points of the N-terminal linker to the BCCP domain and the C-terminal CT domain by 67\u2009\u00c5 (the attachment points are indicated with spheres in Fig. 1e).", "section": "RESULTS", "ner": [ [ 42, 45, "CDN", "structure_element" ], [ 49, 57, "HsaBT-CD", "mutant" ], [ 80, 83, "Sce", "species" ], [ 83, 85, "CD", "structure_element" ], [ 156, 162, "linker", "structure_element" ], [ 170, 181, "BCCP domain", "structure_element" ], [ 201, 203, "CT", "structure_element" ] ] }, { "sid": 81, "sent": "The BT domain of HsaBT-CD consists of a helix that is surrounded at its N terminus by an antiparallel eight-stranded \u03b2-barrel.", "section": "RESULTS", "ner": [ [ 4, 6, "BT", "structure_element" ], [ 17, 25, "HsaBT-CD", "mutant" ], [ 40, 45, "helix", "structure_element" ], [ 89, 125, "antiparallel eight-stranded \u03b2-barrel", "structure_element" ] ] }, { "sid": 82, "sent": "It resembles the BT of propionyl-CoA carboxylase; only the four C-terminal strands of the \u03b2-barrel are slightly tilted.", "section": "RESULTS", "ner": [ [ 17, 19, "BT", "structure_element" ], [ 23, 48, "propionyl-CoA carboxylase", "protein_type" ], [ 75, 98, "strands of the \u03b2-barrel", "structure_element" ] ] }, { "sid": 83, "sent": "On the basis of MS analysis of insect-cell-expressed human full-length ACC, Ser80 shows the highest degree of phosphorylation (90%).", "section": "RESULTS", "ner": [ [ 16, 18, "MS", "experimental_method" ], [ 31, 52, "insect-cell-expressed", "experimental_method" ], [ 53, 58, "human", "species" ], [ 59, 70, "full-length", "protein_state" ], [ 71, 74, "ACC", "protein_type" ], [ 76, 81, "Ser80", "residue_name_number" ], [ 110, 125, "phosphorylation", "ptm" ] ] }, { "sid": 84, "sent": "Ser29 and Ser1263, implicated in insulin-dependent phosphorylation and BRCA1 binding, respectively, are phosphorylated at intermediate levels (40%).", "section": "RESULTS", "ner": [ [ 0, 5, "Ser29", "residue_name_number" ], [ 10, 17, "Ser1263", "residue_name_number" ], [ 33, 66, "insulin-dependent phosphorylation", "ptm" ], [ 71, 76, "BRCA1", "protein" ], [ 104, 118, "phosphorylated", "protein_state" ] ] }, { "sid": 85, "sent": "The highly conserved Ser1216 (corresponding to S. cerevisiae Ser1157), as well as Ser1201, both in the regulatory loop discussed above, are not phosphorylated.", "section": "RESULTS", "ner": [ [ 4, 20, "highly conserved", "protein_state" ], [ 21, 28, "Ser1216", "residue_name_number" ], [ 47, 60, "S. cerevisiae", "species" ], [ 61, 68, "Ser1157", "residue_name_number" ], [ 82, 89, "Ser1201", "residue_name_number" ], [ 103, 118, "regulatory loop", "structure_element" ], [ 140, 158, "not phosphorylated", "protein_state" ] ] }, { "sid": 86, "sent": "However, residual phosphorylation levels were detected for Ser1204 (7%) and Ser1218 (7%) in the same loop.", "section": "RESULTS", "ner": [ [ 18, 33, "phosphorylation", "ptm" ], [ 59, 66, "Ser1204", "residue_name_number" ], [ 76, 83, "Ser1218", "residue_name_number" ], [ 96, 105, "same loop", "structure_element" ] ] }, { "sid": 87, "sent": "MS analysis of the HsaBT-CD crystallization sample reveals partial proteolytic digestion of the regulatory loop.", "section": "RESULTS", "ner": [ [ 0, 2, "MS", "experimental_method" ], [ 19, 27, "HsaBT-CD", "mutant" ], [ 28, 50, "crystallization sample", "evidence" ], [ 96, 111, "regulatory loop", "structure_element" ] ] }, { "sid": 88, "sent": "Accordingly, most of this loop is not represented in the HsaBT-CD crystal structure.", "section": "RESULTS", "ner": [ [ 21, 30, "this loop", "structure_element" ], [ 57, 65, "HsaBT-CD", "mutant" ], [ 66, 83, "crystal structure", "evidence" ] ] }, { "sid": 89, "sent": "The absence of the regulatory loop might be linked to the less-restrained interface of CDL/CDC1 and CDC2 and altered relative orientations of these domains.", "section": "RESULTS", "ner": [ [ 4, 14, "absence of", "protein_state" ], [ 19, 34, "regulatory loop", "structure_element" ], [ 58, 73, "less-restrained", "protein_state" ], [ 74, 83, "interface", "site" ], [ 87, 90, "CDL", "structure_element" ], [ 91, 95, "CDC1", "structure_element" ], [ 100, 104, "CDC2", "structure_element" ], [ 148, 155, "domains", "structure_element" ] ] }, { "sid": 90, "sent": "Besides the regulatory loop, also the phosphopeptide target region for BRCA1 interaction is not resolved presumably because of pronounced flexibility.", "section": "RESULTS", "ner": [ [ 12, 27, "regulatory loop", "structure_element" ], [ 38, 66, "phosphopeptide target region", "site" ], [ 71, 76, "BRCA1", "protein" ] ] }, { "sid": 91, "sent": "At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region.", "section": "RESULTS", "ner": [ [ 16, 24, "isolated", "experimental_method" ], [ 25, 30, "yeast", "taxonomy_domain" ], [ 35, 40, "human", "species" ], [ 41, 43, "CD", "structure_element" ], [ 49, 68, "structural analysis", "experimental_method" ], [ 108, 114, "hinges", "structure_element" ], [ 156, 174, "CDN/CDL connection", "structure_element" ], [ 216, 219, "CDL", "structure_element" ], [ 220, 224, "CDC1", "structure_element" ], [ 229, 233, "CDC2", "structure_element" ], [ 257, 272, "phosphorylation", "ptm" ], [ 280, 295, "regulatory loop", "structure_element" ] ] }, { "sid": 92, "sent": "The integration of CD into the fungal ACC multienzyme", "section": "RESULTS", "ner": [ [ 19, 21, "CD", "structure_element" ], [ 31, 37, "fungal", "taxonomy_domain" ], [ 38, 53, "ACC multienzyme", "protein_type" ] ] }, { "sid": 93, "sent": "To further obtain insights into the functional architecture of fungal ACC, we characterized larger multidomain fragments up to the intact enzymes.", "section": "RESULTS", "ner": [ [ 63, 69, "fungal", "taxonomy_domain" ], [ 70, 73, "ACC", "protein_type" ], [ 92, 120, "larger multidomain fragments", "mutant" ], [ 131, 137, "intact", "protein_state" ], [ 138, 145, "enzymes", "protein" ] ] }, { "sid": 94, "sent": "Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5\u2009\u00c5 (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2\u2009\u00c5 resolution (CthCD-CT; Figs 1a and 2, Table 1).", "section": "RESULTS", "ner": [ [ 6, 27, "molecular replacement", "experimental_method" ], [ 37, 43, "fungal", "taxonomy_domain" ], [ 44, 47, "ACC", "protein_type" ], [ 48, 50, "CD", "structure_element" ], [ 55, 57, "CT", "structure_element" ], [ 78, 88, "structures", "evidence" ], [ 94, 101, "variant", "mutant" ], [ 113, 116, "Cth", "species" ], [ 116, 118, "CT", "structure_element" ], [ 123, 127, "CDC1", "structure_element" ], [ 128, 132, "CDC2", "structure_element" ], [ 136, 153, "two crystal forms", "evidence" ], [ 187, 202, "CthCD-CTCter1/2", "mutant" ], [ 235, 238, "Cth", "species" ], [ 238, 240, "CT", "structure_element" ], [ 262, 264, "CD", "structure_element" ], [ 286, 294, "CthCD-CT", "mutant" ] ] }, { "sid": 95, "sent": "No crystals diffracting to sufficient resolution were obtained for larger BC-containing fragments, or for full-length Cth or SceACC.", "section": "RESULTS", "ner": [ [ 67, 97, "larger BC-containing fragments", "mutant" ], [ 106, 117, "full-length", "protein_state" ], [ 118, 121, "Cth", "species" ], [ 125, 131, "SceACC", "protein" ] ] }, { "sid": 96, "sent": "To improve crystallizability, we generated \u0394BCCP variants of full-length ACC, which, based on SAXS analysis, preserve properties of intact ACC (Supplementary Table 1 and Supplementary Fig. 2a\u2013c).", "section": "RESULTS", "ner": [ [ 3, 28, "improve crystallizability", "experimental_method" ], [ 33, 42, "generated", "experimental_method" ], [ 43, 57, "\u0394BCCP variants", "mutant" ], [ 61, 72, "full-length", "protein_state" ], [ 73, 76, "ACC", "protein_type" ], [ 94, 107, "SAXS analysis", "experimental_method" ], [ 132, 138, "intact", "protein_state" ], [ 139, 142, "ACC", "protein_type" ] ] }, { "sid": 97, "sent": "For Cth\u0394BCCP, crystals diffracting to 8.4\u2009\u00c5 resolution were obtained.", "section": "RESULTS", "ner": [ [ 4, 12, "Cth\u0394BCCP", "mutant" ], [ 14, 22, "crystals", "evidence" ] ] }, { "sid": 98, "sent": "However, molecular replacement did not reveal a unique positioning of the BC domain.", "section": "RESULTS", "ner": [ [ 9, 30, "molecular replacement", "experimental_method" ], [ 74, 76, "BC", "structure_element" ] ] }, { "sid": 99, "sent": "Owing to the limited resolution the discussion of structures of CthCD-CT and Cth\u0394BCCP is restricted to the analysis of domain localization.", "section": "RESULTS", "ner": [ [ 50, 60, "structures", "evidence" ], [ 64, 72, "CthCD-CT", "mutant" ], [ 77, 85, "Cth\u0394BCCP", "mutant" ] ] }, { "sid": 100, "sent": "Still, these structures contribute considerably to the visualization of an intrinsically dynamic fungal ACC.", "section": "RESULTS", "ner": [ [ 7, 23, "these structures", "evidence" ], [ 89, 96, "dynamic", "protein_state" ], [ 97, 103, "fungal", "taxonomy_domain" ], [ 104, 107, "ACC", "protein_type" ] ] }, { "sid": 101, "sent": "In all these crystal structures, the CT domains build a canonical head-to-tail dimer, with active sites formed by contributions from both protomers (Fig. 2 and Supplementary Fig. 3a).", "section": "RESULTS", "ner": [ [ 13, 31, "crystal structures", "evidence" ], [ 37, 39, "CT", "structure_element" ], [ 66, 78, "head-to-tail", "protein_state" ], [ 79, 84, "dimer", "oligomeric_state" ], [ 91, 103, "active sites", "site" ], [ 138, 147, "protomers", "oligomeric_state" ] ] }, { "sid": 102, "sent": "The connection of CD and CT is provided by a 10-residue peptide stretch, which links the N terminus of CT to the irregular \u03b2-hairpin/\u03b2-strand extension of CDC2 (Supplementary Fig. 3b).", "section": "RESULTS", "ner": [ [ 4, 14, "connection", "structure_element" ], [ 18, 20, "CD", "structure_element" ], [ 25, 27, "CT", "structure_element" ], [ 45, 71, "10-residue peptide stretch", "residue_range" ], [ 103, 105, "CT", "structure_element" ], [ 113, 151, "irregular \u03b2-hairpin/\u03b2-strand extension", "structure_element" ], [ 155, 159, "CDC2", "structure_element" ] ] }, { "sid": 103, "sent": "The connecting region is remarkably similar in isolated CD and CthCD-CTCter structures, indicating inherent conformational stability.", "section": "RESULTS", "ner": [ [ 4, 21, "connecting region", "structure_element" ], [ 47, 55, "isolated", "protein_state" ], [ 56, 58, "CD", "structure_element" ], [ 63, 75, "CthCD-CTCter", "mutant" ], [ 76, 86, "structures", "evidence" ] ] }, { "sid": 104, "sent": "CD/CT contacts are only formed in direct vicinity of the covalent linkage and involve the \u03b2-hairpin extension of CDC2 as well as the loop between strands \u03b22/\u03b23 of the CT N-lobe, which contains a conserved RxxGxN motif.", "section": "RESULTS", "ner": [ [ 0, 2, "CD", "structure_element" ], [ 3, 5, "CT", "structure_element" ], [ 90, 109, "\u03b2-hairpin extension", "structure_element" ], [ 113, 117, "CDC2", "structure_element" ], [ 133, 137, "loop", "structure_element" ], [ 146, 159, "strands \u03b22/\u03b23", "structure_element" ], [ 167, 176, "CT N-lobe", "structure_element" ], [ 195, 204, "conserved", "protein_state" ], [ 205, 217, "RxxGxN motif", "structure_element" ] ] }, { "sid": 105, "sent": "The neighbouring loop on the CT side (between CT \u03b21/\u03b22) is displaced by 2.5\u2009\u00c5 compared to isolated CT structures (Supplementary Fig. 3c).", "section": "RESULTS", "ner": [ [ 17, 21, "loop", "structure_element" ], [ 29, 31, "CT", "structure_element" ], [ 46, 48, "CT", "structure_element" ], [ 49, 51, "\u03b21", "structure_element" ], [ 52, 54, "\u03b22", "structure_element" ], [ 90, 98, "isolated", "protein_state" ], [ 99, 101, "CT", "structure_element" ], [ 102, 112, "structures", "evidence" ] ] }, { "sid": 106, "sent": "On the basis of an interface area of \u223c600\u2009\u00c52 and its edge-to-edge connection characteristics, the interface between CT and CD might be classified as conformationally variable.", "section": "RESULTS", "ner": [ [ 98, 107, "interface", "site" ], [ 116, 118, "CT", "structure_element" ], [ 123, 125, "CD", "structure_element" ] ] }, { "sid": 107, "sent": "Indeed, the comparison of the positioning of eight instances of the C-terminal part of CD relative to CT in crystal structures determined here, reveals flexible interdomain linking (Fig. 3a).", "section": "RESULTS", "ner": [ [ 87, 89, "CD", "structure_element" ], [ 102, 104, "CT", "structure_element" ], [ 108, 126, "crystal structures", "evidence" ], [ 127, 137, "determined", "experimental_method" ] ] }, { "sid": 108, "sent": "The CDC2/CT interface acts as a true hinge with observed rotation up to 16\u00b0, which results in a translocation of the distal end of CDC2 by 8\u2009\u00c5.", "section": "RESULTS", "ner": [ [ 4, 21, "CDC2/CT interface", "site" ], [ 32, 42, "true hinge", "structure_element" ], [ 131, 135, "CDC2", "structure_element" ] ] }, { "sid": 109, "sent": "The interface between CDC2 and CDL/CDC1, which is mediated by the phosphorylated regulatory loop in the SceCD structure, is less variable than the CD\u2013CT junction, and permits only limited rotation and tilting (Fig. 3b).", "section": "RESULTS", "ner": [ [ 4, 13, "interface", "site" ], [ 22, 26, "CDC2", "structure_element" ], [ 31, 34, "CDL", "structure_element" ], [ 35, 39, "CDC1", "structure_element" ], [ 66, 80, "phosphorylated", "protein_state" ], [ 81, 96, "regulatory loop", "structure_element" ], [ 104, 107, "Sce", "species" ], [ 107, 109, "CD", "structure_element" ], [ 110, 119, "structure", "evidence" ], [ 147, 161, "CD\u2013CT junction", "structure_element" ] ] }, { "sid": 110, "sent": "Analysis of the impact of phosphorylation on the interface between CDC2 and CDL/CDC1 in CthACC variant structures is precluded by the limited crystallographic resolution.", "section": "RESULTS", "ner": [ [ 26, 41, "phosphorylation", "ptm" ], [ 49, 58, "interface", "site" ], [ 67, 71, "CDC2", "structure_element" ], [ 76, 79, "CDL", "structure_element" ], [ 80, 84, "CDC1", "structure_element" ], [ 88, 102, "CthACC variant", "mutant" ], [ 103, 113, "structures", "evidence" ] ] }, { "sid": 111, "sent": "However, MS analysis of CthCD-CT and Cth\u0394BCCP constructs revealed between 60 and 70% phosphorylation of Ser1170 (corresponding to SceACC Ser1157).", "section": "RESULTS", "ner": [ [ 9, 11, "MS", "experimental_method" ], [ 24, 32, "CthCD-CT", "mutant" ], [ 37, 45, "Cth\u0394BCCP", "mutant" ], [ 85, 100, "phosphorylation", "ptm" ], [ 104, 111, "Ser1170", "residue_name_number" ], [ 130, 136, "SceACC", "protein" ], [ 137, 144, "Ser1157", "residue_name_number" ] ] }, { "sid": 112, "sent": "The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23\u2009\u00c5 (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of Cth\u0394BCCP, denoted as CthCD-CT1/2 and Cth\u0394BCCP1, respectively).", "section": "RESULTS", "ner": [ [ 4, 7, "CDN", "structure_element" ], [ 39, 42, "CDL", "structure_element" ], [ 43, 47, "CDC1", "structure_element" ], [ 112, 122, "structures", "evidence" ], [ 126, 129, "Sce", "species" ], [ 129, 131, "CD", "structure_element" ], [ 140, 163, "larger CthACC fragments", "mutant" ], [ 165, 168, "CDN", "structure_element" ], [ 257, 266, "protomers", "oligomeric_state" ], [ 270, 278, "CthCD-CT", "mutant" ], [ 287, 295, "protomer", "oligomeric_state" ], [ 299, 307, "Cth\u0394BCCP", "mutant" ], [ 320, 331, "CthCD-CT1/2", "mutant" ], [ 336, 345, "Cth\u0394BCCP1", "mutant" ] ] }, { "sid": 113, "sent": "In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70\u00b0 (Fig. 4b, observed in the second protomer of Cth\u0394BCCP, denoted as Cth\u0394BCCP2) and 160\u00b0 (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40\u2009\u00c5, respectively.", "section": "RESULTS", "ner": [ [ 13, 16, "CDN", "structure_element" ], [ 35, 41, "hinges", "structure_element" ], [ 68, 71, "CDN", "structure_element" ], [ 72, 75, "CDL", "structure_element" ], [ 116, 124, "protomer", "oligomeric_state" ], [ 128, 136, "Cth\u0394BCCP", "mutant" ], [ 149, 158, "Cth\u0394BCCP2", "mutant" ], [ 191, 194, "Sce", "species" ], [ 194, 196, "CD", "structure_element" ], [ 229, 240, "anchor site", "site" ], [ 249, 260, "BCCP linker", "structure_element" ] ] }, { "sid": 114, "sent": "Conformational variability in the CD thus contributes considerably to variations in the spacing between the BC and CT domains, and may extend to distance variations beyond the mobility range of the flexibly tethered BCCP.", "section": "RESULTS", "ner": [ [ 34, 36, "CD", "structure_element" ], [ 108, 110, "BC", "structure_element" ], [ 115, 117, "CT", "structure_element" ], [ 198, 215, "flexibly tethered", "protein_state" ], [ 216, 220, "BCCP", "structure_element" ] ] }, { "sid": 115, "sent": "On the basis of the occurrence of related conformational changes between fungal and human ACC fragments, the observed set of conformations may well represent general states present in all eukaryotic ACCs.", "section": "RESULTS", "ner": [ [ 73, 79, "fungal", "taxonomy_domain" ], [ 84, 89, "human", "species" ], [ 90, 103, "ACC fragments", "mutant" ], [ 188, 198, "eukaryotic", "taxonomy_domain" ], [ 199, 203, "ACCs", "protein_type" ] ] }, { "sid": 116, "sent": "Large-scale conformational variability of fungal ACC", "section": "RESULTS", "ner": [ [ 42, 48, "fungal", "taxonomy_domain" ], [ 49, 52, "ACC", "protein_type" ] ] }, { "sid": 117, "sent": "To obtain a comprehensive view of fungal ACC dynamics in solution, we employed SAXS and EM.", "section": "RESULTS", "ner": [ [ 34, 40, "fungal", "taxonomy_domain" ], [ 41, 44, "ACC", "protein_type" ], [ 54, 65, "in solution", "protein_state" ], [ 79, 83, "SAXS", "experimental_method" ], [ 88, 90, "EM", "experimental_method" ] ] }, { "sid": 118, "sent": "SAXS analysis of CthACC agrees with a dimeric state and an elongated shape with a maximum extent of 350\u2009\u00c5 (Supplementary Table 1).", "section": "RESULTS", "ner": [ [ 0, 4, "SAXS", "experimental_method" ], [ 17, 23, "CthACC", "protein" ], [ 38, 45, "dimeric", "oligomeric_state" ], [ 59, 74, "elongated shape", "protein_state" ] ] }, { "sid": 119, "sent": "The smooth appearance of scattering curves and derived distance distributions might indicate substantial interdomain flexibility (Supplementary Fig. 2a\u2013c).", "section": "RESULTS", "ner": [ [ 25, 42, "scattering curves", "evidence" ], [ 47, 77, "derived distance distributions", "evidence" ] ] }, { "sid": 120, "sent": "Direct observation of individual full-length CthACC particles, according to MS results predominantly in a phosphorylated low-activity state, in negative stain EM reveals a large set of conformations from rod-like extended to U-shaped particles.", "section": "RESULTS", "ner": [ [ 33, 44, "full-length", "protein_state" ], [ 45, 51, "CthACC", "protein" ], [ 52, 61, "particles", "evidence" ], [ 76, 78, "MS", "experimental_method" ], [ 106, 120, "phosphorylated", "protein_state" ], [ 121, 139, "low-activity state", "protein_state" ], [ 144, 161, "negative stain EM", "experimental_method" ], [ 204, 221, "rod-like extended", "protein_state" ], [ 225, 233, "U-shaped", "protein_state" ], [ 234, 243, "particles", "evidence" ] ] }, { "sid": 121, "sent": "Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC\u2013BCCP\u2013CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer.", "section": "RESULTS", "ner": [ [ 0, 14, "Class averages", "evidence" ], [ 28, 88, "maximum-likelihood-based two-dimensional (2D) classification", "experimental_method" ], [ 109, 116, "dimeric", "oligomeric_state" ], [ 117, 119, "CT", "structure_element" ], [ 135, 139, "full", "protein_state" ], [ 140, 150, "BC\u2013BCCP\u2013CD", "mutant" ], [ 170, 178, "protomer", "oligomeric_state" ], [ 230, 232, "BC", "structure_element" ], [ 233, 235, "CD", "structure_element" ], [ 260, 262, "CT", "structure_element" ], [ 263, 268, "dimer", "oligomeric_state" ] ] }, { "sid": 122, "sent": "They identify the connections between CDN/CDL and between CDC2/CT as major contributors to conformational heterogeneity (Supplementary Fig. 4a,b).", "section": "RESULTS", "ner": [ [ 38, 41, "CDN", "structure_element" ], [ 42, 45, "CDL", "structure_element" ], [ 58, 62, "CDC2", "structure_element" ], [ 63, 65, "CT", "structure_element" ] ] }, { "sid": 123, "sent": "The flexibility in the CDC2/CT hinge appears substantially larger than the variations observed in the set of crystal structures.", "section": "RESULTS", "ner": [ [ 23, 36, "CDC2/CT hinge", "structure_element" ], [ 109, 127, "crystal structures", "evidence" ] ] }, { "sid": 124, "sent": "The BC domain is not completely disordered, but laterally attached to BT/CDN in a generally conserved position, albeit with increased flexibility.", "section": "RESULTS", "ner": [ [ 4, 6, "BC", "structure_element" ], [ 70, 72, "BT", "structure_element" ], [ 73, 76, "CDN", "structure_element" ], [ 82, 110, "generally conserved position", "protein_state" ] ] }, { "sid": 125, "sent": "Surprisingly, in both the linear and U-shaped conformations, the approximate distances between the BC and CT active sites would remain larger than 110\u2009\u00c5. These observed distances are considerably larger than in static structures of any other related biotin-dependent carboxylase.", "section": "RESULTS", "ner": [ [ 26, 59, "linear and U-shaped conformations", "protein_state" ], [ 99, 101, "BC", "structure_element" ], [ 106, 108, "CT", "structure_element" ], [ 109, 121, "active sites", "site" ], [ 211, 217, "static", "protein_state" ], [ 218, 228, "structures", "evidence" ], [ 250, 278, "biotin-dependent carboxylase", "protein_type" ] ] }, { "sid": 126, "sent": "Furthermore, based on an average length of the BCCP\u2013CD linker in fungal ACC of 26 amino acids, mobility of the BCCP alone would not be sufficient to bridge the active sites of BC and CT.", "section": "RESULTS", "ner": [ [ 47, 61, "BCCP\u2013CD linker", "structure_element" ], [ 65, 71, "fungal", "taxonomy_domain" ], [ 72, 75, "ACC", "protein_type" ], [ 79, 93, "26 amino acids", "residue_range" ], [ 111, 115, "BCCP", "structure_element" ], [ 160, 172, "active sites", "site" ], [ 176, 178, "BC", "structure_element" ], [ 183, 185, "CT", "structure_element" ] ] }, { "sid": 127, "sent": "The most relevant candidate site for mediating such additional flexibility and permitting an extended set of conformations is the CDC1/CDC2 interface, which is rigidified by the Ser1157-phosphorylated regulatory loop, as depicted in the SceCD crystal structure.", "section": "RESULTS", "ner": [ [ 130, 149, "CDC1/CDC2 interface", "site" ], [ 178, 185, "Ser1157", "residue_name_number" ], [ 186, 200, "phosphorylated", "protein_state" ], [ 201, 216, "regulatory loop", "structure_element" ], [ 237, 240, "Sce", "species" ], [ 240, 242, "CD", "structure_element" ], [ 243, 260, "crystal structure", "evidence" ] ] }, { "sid": 128, "sent": "Altogether, the architecture of fungal ACC is based on the central dimeric CT domain (Fig. 4d).", "section": "DISCUSS", "ner": [ [ 32, 38, "fungal", "taxonomy_domain" ], [ 39, 42, "ACC", "protein_type" ], [ 67, 74, "dimeric", "oligomeric_state" ], [ 75, 77, "CT", "structure_element" ] ] }, { "sid": 129, "sent": "The CD consists of four distinct subdomains and acts as a tether from the CT to the mobile BCCP and an oriented BC domain.", "section": "DISCUSS", "ner": [ [ 4, 6, "CD", "structure_element" ], [ 33, 43, "subdomains", "structure_element" ], [ 74, 76, "CT", "structure_element" ], [ 84, 90, "mobile", "protein_state" ], [ 91, 95, "BCCP", "structure_element" ], [ 103, 111, "oriented", "protein_state" ], [ 112, 114, "BC", "structure_element" ] ] }, { "sid": 130, "sent": "The CD has no direct role in substrate recognition or catalysis but contributes to the regulation of all eukaryotic ACCs.", "section": "DISCUSS", "ner": [ [ 4, 6, "CD", "structure_element" ], [ 105, 115, "eukaryotic", "taxonomy_domain" ], [ 116, 120, "ACCs", "protein_type" ] ] }, { "sid": 131, "sent": "In higher eukaryotic ACCs, regulation via phosphorylation is achieved by combining the effects of phosphorylation at Ser80, Ser1201 and Ser1263.", "section": "DISCUSS", "ner": [ [ 3, 20, "higher eukaryotic", "taxonomy_domain" ], [ 21, 25, "ACCs", "protein_type" ], [ 42, 57, "phosphorylation", "ptm" ], [ 98, 113, "phosphorylation", "ptm" ], [ 117, 122, "Ser80", "residue_name_number" ], [ 124, 131, "Ser1201", "residue_name_number" ], [ 136, 143, "Ser1263", "residue_name_number" ] ] }, { "sid": 132, "sent": "In fungal ACC, however, Ser1157 in the regulatory loop of the CD is the only phosphorylation site that has been demonstrated to be both phosphorylated in vivo and involved in the regulation of ACC activity.", "section": "DISCUSS", "ner": [ [ 3, 9, "fungal", "taxonomy_domain" ], [ 10, 13, "ACC", "protein_type" ], [ 24, 31, "Ser1157", "residue_name_number" ], [ 39, 54, "regulatory loop", "structure_element" ], [ 62, 64, "CD", "structure_element" ], [ 77, 97, "phosphorylation site", "site" ], [ 136, 150, "phosphorylated", "protein_state" ], [ 193, 196, "ACC", "protein_type" ] ] }, { "sid": 133, "sent": "In its phosphorylated state, the regulatory loop containing Ser1157 wedges between CDC1/CDC2 and presumably limits the conformational freedom at this interdomain interface.", "section": "DISCUSS", "ner": [ [ 7, 21, "phosphorylated", "protein_state" ], [ 33, 48, "regulatory loop", "structure_element" ], [ 60, 67, "Ser1157", "residue_name_number" ], [ 83, 87, "CDC1", "structure_element" ], [ 88, 92, "CDC2", "structure_element" ], [ 119, 141, "conformational freedom", "protein_state" ], [ 150, 171, "interdomain interface", "site" ] ] }, { "sid": 134, "sent": "However, flexibility at this hinge may be required for full ACC activity, as the distances between the BCCP anchor points and the active sites of BC and CT observed here are such large that mobility of the BCCP alone is not sufficient for substrate transfer.", "section": "DISCUSS", "ner": [ [ 29, 34, "hinge", "structure_element" ], [ 55, 72, "full ACC activity", "protein_state" ], [ 103, 121, "BCCP anchor points", "structure_element" ], [ 130, 142, "active sites", "site" ], [ 146, 148, "BC", "structure_element" ], [ 153, 155, "CT", "structure_element" ], [ 206, 210, "BCCP", "structure_element" ] ] }, { "sid": 135, "sent": "The current data thus suggest that regulation of fungal ACC is mediated by controlling the dynamics of the unique CD, rather than directly affecting catalytic turnover at the active sites of BC and CT.", "section": "DISCUSS", "ner": [ [ 49, 55, "fungal", "taxonomy_domain" ], [ 56, 59, "ACC", "protein_type" ], [ 107, 113, "unique", "protein_state" ], [ 114, 116, "CD", "structure_element" ], [ 175, 187, "active sites", "site" ], [ 191, 193, "BC", "structure_element" ], [ 198, 200, "CT", "structure_element" ] ] }, { "sid": 136, "sent": "A comparison between fungal and human ACC will help to further discriminate mechanistic differences that contribute to the extended control and polymerization of human ACC.", "section": "DISCUSS", "ner": [ [ 21, 27, "fungal", "taxonomy_domain" ], [ 32, 37, "human", "species" ], [ 38, 41, "ACC", "protein_type" ], [ 162, 167, "human", "species" ], [ 168, 171, "ACC", "protein_type" ] ] }, { "sid": 137, "sent": "Most recently, a crystal structure of near full-length non-phosphorylated ACC from S. cerevisae (lacking only 21 N-terminal amino acids, here denoted as flACC) was published by Wei and Tong.", "section": "DISCUSS", "ner": [ [ 17, 34, "crystal structure", "evidence" ], [ 38, 54, "near full-length", "protein_state" ], [ 55, 73, "non-phosphorylated", "protein_state" ], [ 74, 77, "ACC", "protein_type" ], [ 83, 95, "S. cerevisae", "species" ], [ 97, 109, "lacking only", "protein_state" ], [ 110, 112, "21", "residue_range" ], [ 153, 158, "flACC", "protein" ] ] }, { "sid": 138, "sent": "In flACC, the ACC dimer obeys twofold symmetry and assembles in a triangular architecture with dimeric BC domains (Supplementary Fig. 5a).", "section": "DISCUSS", "ner": [ [ 3, 8, "flACC", "protein" ], [ 14, 17, "ACC", "protein_type" ], [ 18, 23, "dimer", "oligomeric_state" ], [ 66, 89, "triangular architecture", "protein_state" ], [ 95, 102, "dimeric", "oligomeric_state" ], [ 103, 105, "BC", "structure_element" ] ] }, { "sid": 139, "sent": "In their study, mutational data indicate a requirement for BC dimerization for catalytic activity.", "section": "DISCUSS", "ner": [ [ 16, 31, "mutational data", "experimental_method" ] ] }, { "sid": 140, "sent": "The transition from the elongated open shape, observed in our experiments, towards a compact triangular shape is based on an intricate interplay of several hinge-bending motions in the CD (Fig. 4d).", "section": "DISCUSS", "ner": [ [ 24, 44, "elongated open shape", "protein_state" ], [ 85, 109, "compact triangular shape", "protein_state" ], [ 185, 187, "CD", "structure_element" ] ] }, { "sid": 141, "sent": "Comparison of flACC with our Cth\u0394BCCP structure reveals the CDC2/CT hinge as a major contributor to conformational flexibility (Supplementary Fig. 5b,c).", "section": "DISCUSS", "ner": [ [ 0, 10, "Comparison", "experimental_method" ], [ 14, 19, "flACC", "protein" ], [ 29, 37, "Cth\u0394BCCP", "mutant" ], [ 38, 47, "structure", "evidence" ], [ 60, 73, "CDC2/CT hinge", "structure_element" ] ] }, { "sid": 142, "sent": "In flACC, CDC2 rotates \u223c120\u00b0 with respect to the CT domain.", "section": "DISCUSS", "ner": [ [ 3, 8, "flACC", "protein" ], [ 10, 14, "CDC2", "structure_element" ], [ 49, 51, "CT", "structure_element" ] ] }, { "sid": 143, "sent": "A second hinge can be identified between CDC1/CDC2.", "section": "DISCUSS", "ner": [ [ 2, 14, "second hinge", "structure_element" ], [ 41, 45, "CDC1", "structure_element" ], [ 46, 50, "CDC2", "structure_element" ] ] }, { "sid": 144, "sent": "On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30\u00b0 relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d).", "section": "DISCUSS", "ner": [ [ 18, 31, "superposition", "experimental_method" ], [ 35, 39, "CDC2", "structure_element" ], [ 41, 45, "CDC1", "structure_element" ], [ 53, 67, "phosphorylated", "protein_state" ], [ 68, 71, "Sce", "species" ], [ 71, 73, "CD", "structure_element" ], [ 104, 108, "CDC1", "structure_element" ], [ 116, 134, "non-phosphorylated", "protein_state" ], [ 135, 140, "flACC", "protein" ], [ 207, 225, "non-phosphorylated", "protein_state" ], [ 226, 234, "HsaBT-CD", "mutant" ] ] }, { "sid": 145, "sent": "When inspecting all individual protomer and fragment structures in their study, Wei and Tong also identify the CDN/CDC1 connection as a highly flexible hinge, in agreement with our observations.", "section": "DISCUSS", "ner": [ [ 5, 15, "inspecting", "experimental_method" ], [ 31, 39, "protomer", "oligomeric_state" ], [ 44, 52, "fragment", "mutant" ], [ 53, 63, "structures", "evidence" ], [ 111, 130, "CDN/CDC1 connection", "structure_element" ], [ 136, 151, "highly flexible", "protein_state" ], [ 152, 157, "hinge", "structure_element" ] ] }, { "sid": 146, "sent": "The only bona fide regulatory phophorylation site of fungal ACC in the regulatory loop is directly participating in CDC1/CDC2 domain interactions and thus stabilizes the hinge conformation.", "section": "DISCUSS", "ner": [ [ 19, 29, "regulatory", "protein_state" ], [ 30, 49, "phophorylation site", "site" ], [ 53, 59, "fungal", "taxonomy_domain" ], [ 60, 63, "ACC", "protein_type" ], [ 71, 86, "regulatory loop", "structure_element" ], [ 116, 120, "CDC1", "structure_element" ], [ 121, 125, "CDC2", "structure_element" ], [ 170, 188, "hinge conformation", "structure_element" ] ] }, { "sid": 147, "sent": "In flACC, the regulatory loop is mostly disordered, illustrating the increased flexibility due to the absence of the phosphoryl group.", "section": "DISCUSS", "ner": [ [ 3, 8, "flACC", "protein" ], [ 14, 29, "regulatory loop", "structure_element" ], [ 33, 50, "mostly disordered", "protein_state" ], [ 117, 127, "phosphoryl", "chemical" ] ] }, { "sid": 148, "sent": "Only in three out of eight observed protomers a short peptide stretch (including Ser1157) was modelled.", "section": "DISCUSS", "ner": [ [ 36, 45, "protomers", "oligomeric_state" ], [ 48, 61, "short peptide", "structure_element" ], [ 81, 88, "Ser1157", "residue_name_number" ], [ 94, 102, "modelled", "evidence" ] ] }, { "sid": 149, "sent": "In those instances the Ser1157 residue is located at a distance of 14\u201320\u2009\u00c5 away from the location of the phosphorylated serine observed here, based on superposition of either CDC1 or CDC2.", "section": "DISCUSS", "ner": [ [ 23, 30, "Ser1157", "residue_name_number" ], [ 105, 119, "phosphorylated", "protein_state" ], [ 120, 126, "serine", "residue_name" ], [ 151, 164, "superposition", "experimental_method" ], [ 175, 179, "CDC1", "structure_element" ], [ 183, 187, "CDC2", "structure_element" ] ] }, { "sid": 150, "sent": "Applying the conformation of the CDC1/CDC2 hinge observed in SceCD on flACC leads to CDN sterically clashing with CDC2 and BT/CDN clashing with CT (Supplementary Fig. 6a,b).", "section": "DISCUSS", "ner": [ [ 0, 8, "Applying", "experimental_method" ], [ 33, 48, "CDC1/CDC2 hinge", "structure_element" ], [ 61, 64, "Sce", "species" ], [ 64, 66, "CD", "structure_element" ], [ 70, 75, "flACC", "protein" ], [ 85, 88, "CDN", "structure_element" ], [ 114, 118, "CDC2", "structure_element" ], [ 123, 125, "BT", "structure_element" ], [ 126, 129, "CDN", "structure_element" ], [ 144, 146, "CT", "structure_element" ] ] }, { "sid": 151, "sent": "Thus, in accordance with the results presented here, phosphorylation of Ser1157 in SceACC most likely limits flexibility in the CDC1/CDC2 hinge such that activation through BC dimerization is not possible (Fig. 4d), which however does not exclude intermolecular dimerization.", "section": "DISCUSS", "ner": [ [ 53, 68, "phosphorylation", "ptm" ], [ 72, 79, "Ser1157", "residue_name_number" ], [ 83, 89, "SceACC", "protein" ], [ 128, 143, "CDC1/CDC2 hinge", "structure_element" ], [ 173, 175, "BC", "structure_element" ] ] }, { "sid": 152, "sent": "In addition, EM micrographs of phosphorylated and dephosphorylated SceACC display for both samples mainly elongated and U-shaped conformations and reveal no apparent differences in particle shape distributions (Supplementary Fig. 7).", "section": "DISCUSS", "ner": [ [ 13, 15, "EM", "experimental_method" ], [ 16, 27, "micrographs", "evidence" ], [ 31, 45, "phosphorylated", "protein_state" ], [ 50, 66, "dephosphorylated", "protein_state" ], [ 67, 73, "SceACC", "protein" ], [ 106, 142, "elongated and U-shaped conformations", "protein_state" ], [ 181, 209, "particle shape distributions", "evidence" ] ] }, { "sid": 153, "sent": "This implicates that the triangular shape with dimeric BC domains has a low population also in the active form, even though a biasing influence of grid preparation cannot be excluded completely.", "section": "DISCUSS", "ner": [ [ 25, 41, "triangular shape", "protein_state" ], [ 47, 54, "dimeric", "oligomeric_state" ], [ 55, 57, "BC", "structure_element" ], [ 99, 110, "active form", "protein_state" ] ] }, { "sid": 154, "sent": "Large-scale conformational variability has also been observed in most other carrier protein-based multienzymes, including polyketide and fatty-acid synthases (with the exception of fungal-type fatty-acid synthases), non-ribosomal peptide synthetases and the pyruvate dehydrogenase complexes, although based on completely different architectures.", "section": "DISCUSS", "ner": [ [ 76, 110, "carrier protein-based multienzymes", "protein_type" ], [ 122, 157, "polyketide and fatty-acid synthases", "protein_type" ], [ 181, 213, "fungal-type fatty-acid synthases", "protein_type" ], [ 216, 249, "non-ribosomal peptide synthetases", "protein_type" ], [ 258, 290, "pyruvate dehydrogenase complexes", "protein_type" ] ] }, { "sid": 155, "sent": "Together, this structural information suggests that variable carrier protein tethering is not sufficient for efficient substrate transfer and catalysis in any of these systems.", "section": "DISCUSS", "ner": [ [ 15, 37, "structural information", "evidence" ] ] }, { "sid": 156, "sent": "The determination of a set of crystal structures of SceACC in two states, unphosphorylated and phosphorylated at the major regulatory site Ser1157, provides a unique depiction of multienzyme regulation by post-translational modification (Fig. 4d).", "section": "DISCUSS", "ner": [ [ 4, 29, "determination of a set of", "experimental_method" ], [ 30, 48, "crystal structures", "evidence" ], [ 52, 58, "SceACC", "protein" ], [ 74, 90, "unphosphorylated", "protein_state" ], [ 95, 109, "phosphorylated", "protein_state" ], [ 117, 138, "major regulatory site", "site" ], [ 139, 146, "Ser1157", "residue_name_number" ] ] }, { "sid": 157, "sent": "The phosphorylated regulatory loop binds to an allosteric site at the interface of two non-catalytic domains and restricts conformational freedom at several hinges in the dynamic ACC.", "section": "DISCUSS", "ner": [ [ 4, 18, "phosphorylated", "protein_state" ], [ 19, 34, "regulatory loop", "structure_element" ], [ 47, 62, "allosteric site", "site" ], [ 70, 79, "interface", "site" ], [ 87, 100, "non-catalytic", "protein_state" ], [ 157, 163, "hinges", "structure_element" ], [ 171, 178, "dynamic", "protein_state" ], [ 179, 182, "ACC", "protein_type" ] ] }, { "sid": 158, "sent": "It disfavours the adoption of a rare, compact conformation, in which intramolecular dimerization of the BC domains results in catalytic turnover.", "section": "DISCUSS", "ner": [ [ 32, 58, "rare, compact conformation", "protein_state" ], [ 104, 106, "BC", "structure_element" ] ] }, { "sid": 159, "sent": "The regulation of activity thus results from restrained large-scale conformational dynamics rather than a direct or indirect influence on active site structure.", "section": "DISCUSS", "ner": [ [ 138, 159, "active site structure", "site" ] ] }, { "sid": 160, "sent": "To our best knowledge, ACC is the first multienzyme for which such a phosphorylation-dependent mechanical control mechanism has been visualized.", "section": "DISCUSS", "ner": [ [ 23, 26, "ACC", "protein_type" ], [ 40, 51, "multienzyme", "protein_type" ], [ 69, 84, "phosphorylation", "ptm" ] ] }, { "sid": 161, "sent": "However, the example of ACC now demonstrates the possibility of regulating activity by controlled dynamics of non-enzymatic linker regions also in other families of carrier-dependent multienzymes.", "section": "DISCUSS", "ner": [ [ 24, 27, "ACC", "protein_type" ], [ 110, 138, "non-enzymatic linker regions", "structure_element" ], [ 165, 195, "carrier-dependent multienzymes", "protein_type" ] ] }, { "sid": 162, "sent": "The phosphorylated central domain of yeast ACC.", "section": "FIG", "ner": [ [ 4, 18, "phosphorylated", "protein_state" ], [ 19, 33, "central domain", "structure_element" ], [ 37, 42, "yeast", "taxonomy_domain" ], [ 43, 46, "ACC", "protein_type" ] ] }, { "sid": 163, "sent": "(a) Schematic overview of the domain organization of eukaryotic ACCs.", "section": "FIG", "ner": [ [ 53, 63, "eukaryotic", "taxonomy_domain" ], [ 64, 68, "ACCs", "protein_type" ] ] }, { "sid": 164, "sent": "Crystallized constructs are indicated.", "section": "FIG", "ner": [ [ 0, 23, "Crystallized constructs", "evidence" ] ] }, { "sid": 165, "sent": "(b) Cartoon representation of the SceCD crystal structure.", "section": "FIG", "ner": [ [ 34, 37, "Sce", "species" ], [ 37, 39, "CD", "structure_element" ], [ 40, 57, "crystal structure", "evidence" ] ] }, { "sid": 166, "sent": "CDN is linked by a four-helix bundle (CDL) to two \u03b1\u2013\u03b2-fold domains (CDC1 and CDC2).", "section": "FIG", "ner": [ [ 0, 3, "CDN", "structure_element" ], [ 19, 36, "four-helix bundle", "structure_element" ], [ 38, 41, "CDL", "structure_element" ], [ 46, 66, "two \u03b1\u2013\u03b2-fold domains", "structure_element" ], [ 68, 72, "CDC1", "structure_element" ], [ 77, 81, "CDC2", "structure_element" ] ] }, { "sid": 167, "sent": "The regulatory loop is shown as bold cartoon, and the phosphorylated Ser1157 is marked by a red triangle.", "section": "FIG", "ner": [ [ 4, 19, "regulatory loop", "structure_element" ], [ 54, 68, "phosphorylated", "protein_state" ], [ 69, 76, "Ser1157", "residue_name_number" ] ] }, { "sid": 168, "sent": "(c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group.", "section": "FIG", "ner": [ [ 4, 17, "Superposition", "experimental_method" ], [ 21, 25, "CDC1", "structure_element" ], [ 30, 34, "CDC2", "structure_element" ], [ 43, 59, "highly conserved", "protein_state" ], [ 60, 65, "folds", "structure_element" ], [ 75, 90, "regulatory loop", "structure_element" ], [ 100, 114, "phosphorylated", "protein_state" ], [ 115, 122, "Ser1157", "residue_name_number" ], [ 155, 159, "CDC1", "structure_element" ], [ 164, 168, "CDC2", "structure_element" ], [ 174, 183, "conserved", "protein_state" ], [ 193, 200, "Arg1173", "residue_name_number" ], [ 205, 212, "Arg1260", "residue_name_number" ], [ 228, 238, "phosphoryl", "chemical" ] ] }, { "sid": 169, "sent": "(e) Structural overview of HsaBT-CD.", "section": "FIG", "ner": [ [ 27, 35, "HsaBT-CD", "mutant" ] ] }, { "sid": 170, "sent": "The attachment points to the N-terminal BCCP domain and the C-terminal CT domain are indicated with spheres.", "section": "FIG", "ner": [ [ 40, 44, "BCCP", "structure_element" ], [ 71, 73, "CT", "structure_element" ] ] }, { "sid": 171, "sent": "Architecture of the CD\u2013CT core of fungal ACC.", "section": "FIG", "ner": [ [ 20, 22, "CD", "structure_element" ], [ 23, 25, "CT", "structure_element" ], [ 34, 40, "fungal", "taxonomy_domain" ], [ 41, 44, "ACC", "protein_type" ] ] }, { "sid": 172, "sent": "Cartoon representation of crystal structures of multidomain constructs of CthACC.", "section": "FIG", "ner": [ [ 26, 44, "crystal structures", "evidence" ], [ 48, 70, "multidomain constructs", "mutant" ], [ 74, 80, "CthACC", "protein" ] ] }, { "sid": 173, "sent": "One protomer is shown in colour and one in grey.", "section": "FIG", "ner": [ [ 4, 12, "protomer", "oligomeric_state" ] ] }, { "sid": 174, "sent": "Individual domains are labelled; the active site of CT and the position of the conserved regulatory phosphoserine site based on SceCD are indicated by an asterisk and a triangle, respectively.", "section": "FIG", "ner": [ [ 37, 48, "active site", "site" ], [ 52, 54, "CT", "structure_element" ], [ 79, 88, "conserved", "protein_state" ], [ 89, 99, "regulatory", "protein_state" ], [ 100, 118, "phosphoserine site", "site" ], [ 128, 131, "Sce", "species" ], [ 131, 133, "CD", "structure_element" ] ] }, { "sid": 175, "sent": "Variability of the connections of CDC2 to CT and CDC1 in fungal ACC.", "section": "FIG", "ner": [ [ 34, 38, "CDC2", "structure_element" ], [ 42, 44, "CT", "structure_element" ], [ 49, 53, "CDC1", "structure_element" ], [ 57, 63, "fungal", "taxonomy_domain" ], [ 64, 67, "ACC", "protein_type" ] ] }, { "sid": 176, "sent": "(a) Hinge properties of the CDC2\u2013CT connection analysed by a CT-based superposition of eight instances of the CDC2-CT segment.", "section": "FIG", "ner": [ [ 4, 9, "Hinge", "structure_element" ], [ 28, 46, "CDC2\u2013CT connection", "structure_element" ], [ 61, 83, "CT-based superposition", "experimental_method" ], [ 110, 125, "CDC2-CT segment", "mutant" ] ] }, { "sid": 177, "sent": "For clarity, only one protomer of CthCD-CTCter1 is shown in full colour as reference.", "section": "FIG", "ner": [ [ 22, 30, "protomer", "oligomeric_state" ], [ 34, 47, "CthCD-CTCter1", "mutant" ] ] }, { "sid": 178, "sent": "For other instances, CDC2 domains are shown in transparent tube representation with only one helix each highlighted.", "section": "FIG", "ner": [ [ 21, 25, "CDC2", "structure_element" ] ] }, { "sid": 179, "sent": "The range of hinge bending is indicated and the connection points between CDC2 and CT (blue) as well as between CDC1 and CDC2 (green and grey) are marked as spheres.", "section": "FIG", "ner": [ [ 74, 78, "CDC2", "structure_element" ], [ 83, 85, "CT", "structure_element" ], [ 112, 116, "CDC1", "structure_element" ], [ 121, 125, "CDC2", "structure_element" ] ] }, { "sid": 180, "sent": "(b) The interdomain interface of CDC1 and CDC2 exhibits only limited plasticity.", "section": "FIG", "ner": [ [ 8, 29, "interdomain interface", "site" ], [ 33, 37, "CDC1", "structure_element" ], [ 42, 46, "CDC2", "structure_element" ] ] }, { "sid": 181, "sent": "Representation as in a, but the CDC1 and CDC2 are superposed based on CDC2.", "section": "FIG", "ner": [ [ 32, 36, "CDC1", "structure_element" ], [ 41, 45, "CDC2", "structure_element" ], [ 50, 60, "superposed", "experimental_method" ], [ 70, 74, "CDC2", "structure_element" ] ] }, { "sid": 182, "sent": "One protomer of Cth\u0394BCCP is shown in colour, the CDL domains are omitted for clarity and the position of the phosphorylated serine based on SceCD is indicated with a red triangle.", "section": "FIG", "ner": [ [ 4, 12, "protomer", "oligomeric_state" ], [ 16, 24, "Cth\u0394BCCP", "mutant" ], [ 49, 52, "CDL", "structure_element" ], [ 109, 123, "phosphorylated", "protein_state" ], [ 124, 130, "serine", "residue_name" ], [ 140, 143, "Sce", "species" ], [ 143, 145, "CD", "structure_element" ] ] }, { "sid": 183, "sent": "The connection points from CDC1 to CDC2 and to CDL are represented by green spheres.", "section": "FIG", "ner": [ [ 27, 31, "CDC1", "structure_element" ], [ 35, 39, "CDC2", "structure_element" ], [ 47, 50, "CDL", "structure_element" ] ] }, { "sid": 184, "sent": "The conformational dynamics of fungal ACC.", "section": "FIG", "ner": [ [ 31, 37, "fungal", "taxonomy_domain" ], [ 38, 41, "ACC", "protein_type" ] ] }, { "sid": 185, "sent": "(a\u2013c) Large-scale conformational variability of the CDN domain relative to the CDL/CDC1 domain.", "section": "FIG", "ner": [ [ 52, 55, "CDN", "structure_element" ], [ 79, 82, "CDL", "structure_element" ], [ 83, 87, "CDC1", "structure_element" ] ] }, { "sid": 186, "sent": "CthCD-CT1 (in colour) serves as reference, the compared structures (as indicated, numbers after construct name differentiate between individual protomers) are shown in grey.", "section": "FIG", "ner": [ [ 0, 9, "CthCD-CT1", "mutant" ], [ 47, 66, "compared structures", "experimental_method" ], [ 144, 153, "protomers", "oligomeric_state" ] ] }, { "sid": 187, "sent": "Domains other than CDN and CDL/CDC1 are omitted for clarity.", "section": "FIG", "ner": [ [ 19, 22, "CDN", "structure_element" ], [ 27, 30, "CDL", "structure_element" ], [ 31, 35, "CDC1", "structure_element" ] ] }, { "sid": 188, "sent": "The domains are labelled and the distances between the N termini of CDN (spheres) in the compared structures are indicated.", "section": "FIG", "ner": [ [ 68, 71, "CDN", "structure_element" ] ] }, { "sid": 189, "sent": "(d) Schematic model of fungal ACC showing the intrinsic, regulated flexibility of CD in the phosphorylated inhibited or the non-phosphorylated activated state.", "section": "FIG", "ner": [ [ 23, 29, "fungal", "taxonomy_domain" ], [ 30, 33, "ACC", "protein_type" ], [ 82, 84, "CD", "structure_element" ], [ 92, 106, "phosphorylated", "protein_state" ], [ 107, 116, "inhibited", "protein_state" ], [ 124, 142, "non-phosphorylated", "protein_state" ], [ 143, 152, "activated", "protein_state" ] ] }, { "sid": 190, "sent": "Flexibility of the CDC2/CT and CDN/CDL hinges is illustrated by arrows.", "section": "FIG", "ner": [ [ 19, 23, "CDC2", "structure_element" ], [ 24, 26, "CT", "structure_element" ], [ 31, 34, "CDN", "structure_element" ], [ 35, 38, "CDL", "structure_element" ], [ 39, 45, "hinges", "structure_element" ] ] }, { "sid": 191, "sent": "The Ser1157 phosphorylation site and the regulatory loop are schematically indicated in magenta.", "section": "FIG", "ner": [ [ 4, 11, "Ser1157", "residue_name_number" ], [ 12, 27, "phosphorylation", "ptm" ], [ 41, 56, "regulatory loop", "structure_element" ] ] } ] }, "PMC4880283": { "annotations": [ { "sid": 0, "sent": "Crystal Structures of Putative Sugar Kinases from Synechococcus Elongatus PCC 7942 and Arabidopsis Thaliana", "section": "TITLE", "ner": [ [ 0, 18, "Crystal Structures", "evidence" ], [ 31, 44, "Sugar Kinases", "protein_type" ], [ 50, 82, "Synechococcus Elongatus PCC 7942", "species" ], [ 87, 107, "Arabidopsis Thaliana", "species" ] ] }, { "sid": 1, "sent": "The genome of the Synechococcus elongatus strain PCC 7942 encodes a putative sugar kinase (SePSK), which shares 44.9% sequence identity with the xylulose kinase-1 (AtXK-1) from Arabidopsis thaliana.", "section": "ABSTRACT", "ner": [ [ 18, 57, "Synechococcus elongatus strain PCC 7942", "species" ], [ 77, 89, "sugar kinase", "protein_type" ], [ 91, 96, "SePSK", "protein" ], [ 145, 162, "xylulose kinase-1", "protein" ], [ 164, 170, "AtXK-1", "protein" ], [ 177, 197, "Arabidopsis thaliana", "species" ] ] }, { "sid": 2, "sent": "Sequence alignment suggests that both kinases belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases.", "section": "ABSTRACT", "ner": [ [ 0, 18, "Sequence alignment", "experimental_method" ], [ 38, 45, "kinases", "protein_type" ], [ 60, 98, "ribulokinase-like carbohydrate kinases", "protein_type" ], [ 116, 148, "FGGY family carbohydrate kinases", "protein_type" ] ] }, { "sid": 3, "sent": "Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates.", "section": "ABSTRACT", "ner": [ [ 8, 14, "solved", "experimental_method" ], [ 19, 29, "structures", "evidence" ], [ 33, 38, "SePSK", "protein" ], [ 43, 49, "AtXK-1", "protein" ], [ 64, 67, "apo", "protein_state" ], [ 78, 93, "in complex with", "protein_state" ], [ 94, 104, "nucleotide", "chemical" ] ] }, { "sid": 4, "sent": "The two kinases exhibit nearly identical overall architecture, with both kinases possessing ATP hydrolysis activity in the absence of substrates.", "section": "ABSTRACT", "ner": [ [ 73, 80, "kinases", "protein_type" ], [ 92, 95, "ATP", "chemical" ], [ 123, 144, "absence of substrates", "protein_state" ] ] }, { "sid": 5, "sent": "In addition, our enzymatic assays suggested that SePSK has the capability to phosphorylate D-ribulose.", "section": "ABSTRACT", "ner": [ [ 17, 33, "enzymatic assays", "experimental_method" ], [ 49, 54, "SePSK", "protein" ], [ 91, 101, "D-ribulose", "chemical" ] ] }, { "sid": 6, "sent": "In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK.", "section": "ABSTRACT", "ner": [ [ 50, 55, "SePSK", "protein" ], [ 60, 66, "solved", "experimental_method" ], [ 71, 80, "structure", "evidence" ], [ 84, 89, "SePSK", "protein" ], [ 90, 105, "in complex with", "protein_state" ], [ 106, 116, "D-ribulose", "chemical" ], [ 141, 166, "substrate binding pockets", "site" ], [ 170, 175, "SePSK", "protein" ] ] }, { "sid": 7, "sent": "Using mutation and activity analysis, we further verified the key residues important for its catalytic activity.", "section": "ABSTRACT", "ner": [ [ 6, 36, "mutation and activity analysis", "experimental_method" ] ] }, { "sid": 8, "sent": "Moreover, our structural comparison with other family members suggests that there are major conformational changes in SePSK upon substrate binding, facilitating the catalytic process.", "section": "ABSTRACT", "ner": [ [ 14, 35, "structural comparison", "experimental_method" ], [ 118, 123, "SePSK", "protein" ] ] }, { "sid": 9, "sent": "Together, these results provide important information for a more detailed understanding of the cofactor and substrate binding mode as well as the catalytic mechanism of SePSK, and possible similarities with its plant homologue AtXK-1.", "section": "ABSTRACT", "ner": [ [ 169, 174, "SePSK", "protein" ], [ 211, 216, "plant", "taxonomy_domain" ], [ 227, 233, "AtXK-1", "protein" ] ] }, { "sid": 10, "sent": "Carbohydrates are essential cellular compounds involved in the metabolic processes present in all organisms.", "section": "INTRO", "ner": [ [ 0, 13, "Carbohydrates", "chemical" ] ] }, { "sid": 11, "sent": "Phosphorylation is one of the various pivotal modifications of carbohydrates, and is catalyzed by specific sugar kinases.", "section": "INTRO", "ner": [ [ 0, 15, "Phosphorylation", "ptm" ], [ 63, 76, "carbohydrates", "chemical" ], [ 107, 120, "sugar kinases", "protein_type" ] ] }, { "sid": 12, "sent": "These kinases exhibit considerable differences in their folding pattern and substrate specificity.", "section": "INTRO", "ner": [ [ 6, 13, "kinases", "protein_type" ] ] }, { "sid": 13, "sent": "Based on sequence analysis, they can be divided into four families, namely HSP 70_NBD family, FGGY family, Mer_B like family and Parm_like family.", "section": "INTRO", "ner": [ [ 9, 26, "sequence analysis", "experimental_method" ], [ 75, 92, "HSP 70_NBD family", "protein_type" ], [ 94, 105, "FGGY family", "protein_type" ], [ 107, 124, "Mer_B like family", "protein_type" ], [ 129, 145, "Parm_like family", "protein_type" ] ] }, { "sid": 14, "sent": "The FGGY family carbohydrate kinases contain different types of sugar kinases, all of which possess different catalytic substrates with preferences for short-chained sugar substrates, ranging from triose to heptose.", "section": "INTRO", "ner": [ [ 4, 36, "FGGY family carbohydrate kinases", "protein_type" ], [ 64, 77, "sugar kinases", "protein_type" ], [ 166, 171, "sugar", "chemical" ], [ 197, 203, "triose", "chemical" ], [ 207, 214, "heptose", "chemical" ] ] }, { "sid": 15, "sent": "These sugar substrates include L-ribulose, erythritol, L-fuculose, D-glycerol, D-gluconate, L-xylulose, D-ribulose, L-rhamnulose and D-xylulose.", "section": "INTRO", "ner": [ [ 6, 11, "sugar", "chemical" ], [ 31, 41, "L-ribulose", "chemical" ], [ 43, 53, "erythritol", "chemical" ], [ 55, 65, "L-fuculose", "chemical" ], [ 67, 77, "D-glycerol", "chemical" ], [ 79, 90, "D-gluconate", "chemical" ], [ 92, 102, "L-xylulose", "chemical" ], [ 104, 114, "D-ribulose", "chemical" ], [ 116, 128, "L-rhamnulose", "chemical" ], [ 133, 143, "D-xylulose", "chemical" ] ] }, { "sid": 16, "sent": "Structures reported in the Protein Data Bank of the FGGY family carbohydrate kinases exhibit a similar overall architecture containing two protein domains, one of which is responsible for the binding of substrate, while the second is used for binding cofactor ATP.", "section": "INTRO", "ner": [ [ 0, 10, "Structures", "evidence" ], [ 52, 84, "FGGY family carbohydrate kinases", "protein_type" ], [ 260, 263, "ATP", "chemical" ] ] }, { "sid": 17, "sent": "While the binding pockets for substrates are at the same position, each FGGY family carbohydrate kinases uses different substrate-binding residues, resulting in high substrate specificity.", "section": "INTRO", "ner": [ [ 10, 25, "binding pockets", "site" ], [ 72, 104, "FGGY family carbohydrate kinases", "protein_type" ], [ 120, 146, "substrate-binding residues", "site" ] ] }, { "sid": 18, "sent": "Synpcc7942_2462 from the cyanobacteria Synechococcus elongatus PCC 7942 encodes a putative sugar kinase (SePSK), and this kinase contains 426 amino acids.", "section": "INTRO", "ner": [ [ 0, 15, "Synpcc7942_2462", "gene" ], [ 25, 38, "cyanobacteria", "taxonomy_domain" ], [ 39, 71, "Synechococcus elongatus PCC 7942", "species" ], [ 91, 103, "sugar kinase", "protein_type" ], [ 105, 110, "SePSK", "protein" ], [ 122, 128, "kinase", "protein_type" ], [ 138, 141, "426", "residue_range" ] ] }, { "sid": 19, "sent": "The At2g21370 gene product from Arabidopsis thaliana, xylulose kinase-1 (AtXK-1), whose mature form contains 436 amino acids, is located in the chloroplast (ChloroP 1.1 Server).", "section": "INTRO", "ner": [ [ 4, 13, "At2g21370", "gene" ], [ 32, 52, "Arabidopsis thaliana", "species" ], [ 54, 71, "xylulose kinase-1", "protein" ], [ 73, 79, "AtXK-1", "protein" ], [ 88, 99, "mature form", "protein_state" ], [ 109, 112, "436", "residue_range" ] ] }, { "sid": 20, "sent": "SePSK and AtXK-1 display a sequence identity of 44.9%, and belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases.", "section": "INTRO", "ner": [ [ 0, 5, "SePSK", "protein" ], [ 10, 16, "AtXK-1", "protein" ], [ 73, 111, "ribulokinase-like carbohydrate kinases", "protein_type" ], [ 129, 161, "FGGY family carbohydrate kinases", "protein_type" ] ] }, { "sid": 21, "sent": "Members of this sub-family are responsible for the phosphorylation of sugars similar to L-ribulose and D-ribulose.", "section": "INTRO", "ner": [ [ 51, 66, "phosphorylation", "ptm" ], [ 70, 76, "sugars", "chemical" ], [ 88, 98, "L-ribulose", "chemical" ], [ 103, 113, "D-ribulose", "chemical" ] ] }, { "sid": 22, "sent": "The sequence and the substrate specificity of ribulokinase-like carbohydrate kinases are different, but they share the common folding feature with two domains.", "section": "INTRO", "ner": [ [ 46, 84, "ribulokinase-like carbohydrate kinases", "protein_type" ] ] }, { "sid": 23, "sent": "Domain I exhibits a ribonuclease H-like folding pattern, and is responsible for the substrate binding, while domain II possesses an actin-like ATPase domain that binds cofactor ATP.", "section": "INTRO", "ner": [ [ 0, 8, "Domain I", "structure_element" ], [ 20, 55, "ribonuclease H-like folding pattern", "structure_element" ], [ 109, 118, "domain II", "structure_element" ], [ 132, 156, "actin-like ATPase domain", "structure_element" ], [ 177, 180, "ATP", "chemical" ] ] }, { "sid": 24, "sent": "Two possible xylulose kinases (xylulose kinase-1: XK-1 and xylulose kinase-2: XK-2) from Arabidopsis thaliana were previously proposed.", "section": "INTRO", "ner": [ [ 13, 29, "xylulose kinases", "protein_type" ], [ 31, 48, "xylulose kinase-1", "protein" ], [ 50, 54, "XK-1", "protein" ], [ 59, 76, "xylulose kinase-2", "protein" ], [ 78, 82, "XK-2", "protein" ], [ 89, 109, "Arabidopsis thaliana", "species" ] ] }, { "sid": 25, "sent": "It was shown that XK-2 (At5g49650) located in the cytosol is indeed xylulose kinase.", "section": "INTRO", "ner": [ [ 18, 22, "XK-2", "protein" ], [ 24, 33, "At5g49650", "gene" ], [ 68, 83, "xylulose kinase", "protein_type" ] ] }, { "sid": 26, "sent": "However, the function of XK-1 (At2g21370) inside the chloroplast stroma has remained unknown.", "section": "INTRO", "ner": [ [ 25, 29, "XK-1", "protein" ], [ 31, 40, "At2g21370", "gene" ] ] }, { "sid": 27, "sent": "SePSK from Synechococcus elongatus strain PCC 7942 is the homolog of AtXK-1, though its physiological function and substrates remain unclear.", "section": "INTRO", "ner": [ [ 0, 5, "SePSK", "protein" ], [ 11, 50, "Synechococcus elongatus strain PCC 7942", "species" ], [ 69, 75, "AtXK-1", "protein" ] ] }, { "sid": 28, "sent": "In order to obtain functional and structural information about these two proteins, here we reported the crystal structures of SePSK and AtXK-1.", "section": "INTRO", "ner": [ [ 104, 122, "crystal structures", "evidence" ], [ 126, 131, "SePSK", "protein" ], [ 136, 142, "AtXK-1", "protein" ] ] }, { "sid": 29, "sent": "Our findings provide new details of the catalytic mechanism of SePSK and lay the foundation for future studies into its homologs in eukaryotes.", "section": "INTRO", "ner": [ [ 63, 68, "SePSK", "protein" ], [ 132, 142, "eukaryotes", "taxonomy_domain" ] ] }, { "sid": 30, "sent": "Overall structures of apo-SePSK and apo-AtXK-1", "section": "RESULTS", "ner": [ [ 8, 18, "structures", "evidence" ], [ 22, 25, "apo", "protein_state" ], [ 26, 31, "SePSK", "protein" ], [ 36, 39, "apo", "protein_state" ], [ 40, 46, "AtXK-1", "protein" ] ] }, { "sid": 31, "sent": "The attempt to solve the SePSK structure by molecular replacement method failed with ribulokinase from Bacillus halodurans (PDB code: 3QDK, 15.7% sequence identity) as an initial model.", "section": "RESULTS", "ner": [ [ 25, 30, "SePSK", "protein" ], [ 31, 40, "structure", "evidence" ], [ 44, 72, "molecular replacement method", "experimental_method" ], [ 85, 97, "ribulokinase", "protein" ], [ 103, 122, "Bacillus halodurans", "species" ] ] }, { "sid": 32, "sent": "We therefore used single isomorphous replacement anomalous scattering method (SIRAS) for successful solution of the apo-SePSK structure at a resolution of 2.3 \u00c5. Subsequently, the apo-SePSK structure was used as molecular replacement model to solve all other structures identified in this study.", "section": "RESULTS", "ner": [ [ 18, 76, "single isomorphous replacement anomalous scattering method", "experimental_method" ], [ 78, 83, "SIRAS", "experimental_method" ], [ 116, 119, "apo", "protein_state" ], [ 120, 125, "SePSK", "protein" ], [ 126, 135, "structure", "evidence" ], [ 180, 183, "apo", "protein_state" ], [ 184, 189, "SePSK", "protein" ], [ 190, 199, "structure", "evidence" ], [ 212, 239, "molecular replacement model", "experimental_method" ], [ 259, 269, "structures", "evidence" ] ] }, { "sid": 33, "sent": "Our structural analysis showed that apo-SePSK consists of one SePSK protein molecule in an asymmetric unit.", "section": "RESULTS", "ner": [ [ 4, 23, "structural analysis", "experimental_method" ], [ 36, 39, "apo", "protein_state" ], [ 40, 45, "SePSK", "protein" ], [ 62, 67, "SePSK", "protein" ] ] }, { "sid": 34, "sent": "The amino-acid residues were traced from Val2 to His419, except for the Met1 residue and the seven residues at the C-termini.", "section": "RESULTS", "ner": [ [ 41, 45, "Val2", "residue_name_number" ], [ 49, 55, "His419", "residue_name_number" ], [ 72, 76, "Met1", "residue_name_number" ] ] }, { "sid": 35, "sent": "Apo-SePSK contains two domains referred to further on as domain I and domain II (Fig 1A).", "section": "RESULTS", "ner": [ [ 0, 3, "Apo", "protein_state" ], [ 4, 9, "SePSK", "protein" ], [ 57, 65, "domain I", "structure_element" ], [ 70, 79, "domain II", "structure_element" ] ] }, { "sid": 36, "sent": "Domain I consists of non-contiguous portions of the polypeptide chains (aa.", "section": "RESULTS", "ner": [ [ 0, 8, "Domain I", "structure_element" ] ] }, { "sid": 37, "sent": "2\u2013228 and aa.", "section": "RESULTS", "ner": [ [ 0, 5, "2\u2013228", "residue_range" ] ] }, { "sid": 38, "sent": "402\u2013419), exhibiting 11 \u03b1-helices and 11 \u03b2-sheets.", "section": "RESULTS", "ner": [ [ 0, 7, "402\u2013419", "residue_range" ], [ 24, 33, "\u03b1-helices", "structure_element" ], [ 41, 49, "\u03b2-sheets", "structure_element" ] ] }, { "sid": 39, "sent": "Among all these structural elements, \u03b14/\u03b15/\u03b111/\u03b118, \u03b23/\u03b22/\u03b21/\u03b26/\u03b219/\u03b220/\u03b217 and \u03b121/\u03b132 form three patches, referred to as A1, B1 and A2, exhibiting the core region.", "section": "RESULTS", "ner": [ [ 37, 39, "\u03b14", "structure_element" ], [ 40, 42, "\u03b15", "structure_element" ], [ 43, 46, "\u03b111", "structure_element" ], [ 47, 50, "\u03b118", "structure_element" ], [ 52, 54, "\u03b23", "structure_element" ], [ 55, 57, "\u03b22", "structure_element" ], [ 58, 60, "\u03b21", "structure_element" ], [ 61, 63, "\u03b26", "structure_element" ], [ 64, 67, "\u03b219", "structure_element" ], [ 68, 71, "\u03b220", "structure_element" ], [ 72, 75, "\u03b217", "structure_element" ], [ 80, 83, "\u03b121", "structure_element" ], [ 84, 87, "\u03b132", "structure_element" ], [ 123, 125, "A1", "structure_element" ], [ 127, 129, "B1", "structure_element" ], [ 134, 136, "A2", "structure_element" ], [ 153, 164, "core region", "structure_element" ] ] }, { "sid": 40, "sent": "In addition, four \u03b2-sheets (\u03b27, \u03b210, \u03b212 and \u03b216) and five \u03b1-helices (\u03b18, \u03b19, \u03b113, \u03b114 and \u03b115) flank the left side of the core region.", "section": "RESULTS", "ner": [ [ 18, 26, "\u03b2-sheets", "structure_element" ], [ 28, 30, "\u03b27", "structure_element" ], [ 32, 35, "\u03b210", "structure_element" ], [ 37, 40, "\u03b212", "structure_element" ], [ 45, 48, "\u03b216", "structure_element" ], [ 59, 68, "\u03b1-helices", "structure_element" ], [ 70, 72, "\u03b18", "structure_element" ], [ 74, 76, "\u03b19", "structure_element" ], [ 78, 81, "\u03b113", "structure_element" ], [ 83, 86, "\u03b114", "structure_element" ], [ 91, 94, "\u03b115", "structure_element" ], [ 123, 134, "core region", "structure_element" ] ] }, { "sid": 41, "sent": "Domain II is comprised of aa.", "section": "RESULTS", "ner": [ [ 0, 9, "Domain II", "structure_element" ] ] }, { "sid": 42, "sent": "229\u2013401 and classified into B2 (\u03b231/\u03b229/\u03b222/\u03b223/\u03b225/\u03b224) and A3 (\u03b126/\u03b127/\u03b128/\u03b130) (Fig 1A and S1 Fig).", "section": "RESULTS", "ner": [ [ 0, 7, "229\u2013401", "residue_range" ], [ 28, 30, "B2", "structure_element" ], [ 32, 35, "\u03b231", "structure_element" ], [ 36, 39, "\u03b229", "structure_element" ], [ 40, 43, "\u03b222", "structure_element" ], [ 44, 47, "\u03b223", "structure_element" ], [ 48, 51, "\u03b225", "structure_element" ], [ 52, 55, "\u03b224", "structure_element" ], [ 61, 63, "A3", "structure_element" ], [ 65, 68, "\u03b126", "structure_element" ], [ 69, 72, "\u03b127", "structure_element" ], [ 73, 76, "\u03b128", "structure_element" ], [ 77, 80, "\u03b130", "structure_element" ] ] }, { "sid": 43, "sent": "In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (\u03b1/\u03b2/\u03b1/\u03b2/\u03b1) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig).", "section": "RESULTS", "ner": [ [ 7, 12, "SePSK", "protein" ], [ 13, 22, "structure", "evidence" ], [ 24, 26, "B1", "structure_element" ], [ 31, 33, "B2", "structure_element" ], [ 52, 54, "A1", "structure_element" ], [ 56, 58, "A2", "structure_element" ], [ 63, 65, "A3", "structure_element" ], [ 81, 90, "structure", "evidence" ], [ 101, 103, "A1", "structure_element" ], [ 104, 106, "B1", "structure_element" ], [ 107, 109, "A2", "structure_element" ], [ 110, 112, "B2", "structure_element" ], [ 113, 115, "A3", "structure_element" ], [ 117, 118, "\u03b1", "structure_element" ], [ 119, 120, "\u03b2", "structure_element" ], [ 121, 122, "\u03b1", "structure_element" ], [ 123, 124, "\u03b2", "structure_element" ], [ 125, 126, "\u03b1", "structure_element" ], [ 186, 218, "FGGY family carbohydrate kinases", "protein_type" ] ] }, { "sid": 44, "sent": "The overall folding of SePSK resembles a clip, with A2 of domain I acting as a hinge region.", "section": "RESULTS", "ner": [ [ 23, 28, "SePSK", "protein" ], [ 52, 54, "A2", "structure_element" ], [ 58, 66, "domain I", "structure_element" ], [ 79, 91, "hinge region", "structure_element" ] ] }, { "sid": 45, "sent": "Overall structures of SePSK and AtXK-1.", "section": "FIG", "ner": [ [ 8, 18, "structures", "evidence" ], [ 22, 27, "SePSK", "protein" ], [ 32, 38, "AtXK-1", "protein" ] ] }, { "sid": 46, "sent": "(A) Three-dimensional structure of apo-SePSK.", "section": "FIG", "ner": [ [ 22, 31, "structure", "evidence" ], [ 35, 38, "apo", "protein_state" ], [ 39, 44, "SePSK", "protein" ] ] }, { "sid": 47, "sent": "The secondary structural elements are indicated (\u03b1-helix: cyan, \u03b2-sheet: yellow).", "section": "FIG", "ner": [ [ 49, 56, "\u03b1-helix", "structure_element" ], [ 64, 71, "\u03b2-sheet", "structure_element" ] ] }, { "sid": 48, "sent": "(B) Three-dimensional structure of apo-AtXK-1.", "section": "FIG", "ner": [ [ 22, 31, "structure", "evidence" ], [ 35, 38, "apo", "protein_state" ], [ 39, 45, "AtXK-1", "protein" ] ] }, { "sid": 49, "sent": "The secondary structural elements are indicated (\u03b1-helix: green, \u03b2-sheet: wheat).", "section": "FIG", "ner": [ [ 49, 56, "\u03b1-helix", "structure_element" ], [ 65, 72, "\u03b2-sheet", "structure_element" ] ] }, { "sid": 50, "sent": "Apo-AtXK-1 exhibits a folding pattern similar to that of SePSK in line with their high sequence identity (Fig 1B and S1 Fig).", "section": "RESULTS", "ner": [ [ 0, 3, "Apo", "protein_state" ], [ 4, 10, "AtXK-1", "protein" ], [ 57, 62, "SePSK", "protein" ] ] }, { "sid": 51, "sent": "However, superposition of structures of AtXK-1 and SePSK shows some differences, especially at the loop regions.", "section": "RESULTS", "ner": [ [ 9, 22, "superposition", "experimental_method" ], [ 26, 36, "structures", "evidence" ], [ 40, 46, "AtXK-1", "protein" ], [ 51, 56, "SePSK", "protein" ], [ 99, 111, "loop regions", "structure_element" ] ] }, { "sid": 52, "sent": "A considerable difference is found in the loop3 linking \u03b23 and \u03b14, which is stretched out in the AtXK-1 structure, while in the SePSK structure, it is bent back towards the inner part.", "section": "RESULTS", "ner": [ [ 42, 47, "loop3", "structure_element" ], [ 56, 58, "\u03b23", "structure_element" ], [ 63, 65, "\u03b14", "structure_element" ], [ 97, 103, "AtXK-1", "protein" ], [ 104, 113, "structure", "evidence" ], [ 128, 133, "SePSK", "protein" ], [ 134, 143, "structure", "evidence" ] ] }, { "sid": 53, "sent": "The corresponding residues between these two structures (SePSK-Lys35 and AtXK-1-Lys48) have a distance of 15.4 \u00c5 (S3 Fig).", "section": "RESULTS", "ner": [ [ 45, 55, "structures", "evidence" ], [ 57, 62, "SePSK", "protein" ], [ 63, 68, "Lys35", "residue_name_number" ], [ 73, 79, "AtXK-1", "protein" ], [ 80, 85, "Lys48", "residue_name_number" ] ] }, { "sid": 54, "sent": "Activity assays of SePSK and AtXK-1", "section": "RESULTS", "ner": [ [ 0, 15, "Activity assays", "experimental_method" ], [ 19, 24, "SePSK", "protein" ], [ 29, 35, "AtXK-1", "protein" ] ] }, { "sid": 55, "sent": "In order to understand the function of these two kinases, we performed structural comparison using Dali server.", "section": "RESULTS", "ner": [ [ 71, 92, "structural comparison", "experimental_method" ], [ 99, 110, "Dali server", "experimental_method" ] ] }, { "sid": 56, "sent": "The structures most closely related to SePSK are xylulose kinase, glycerol kinase and ribulose kinase, implying that SePSK and AtXK-1 might function similarly to these kinases.", "section": "RESULTS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 39, 44, "SePSK", "protein" ], [ 49, 64, "xylulose kinase", "protein_type" ], [ 66, 81, "glycerol kinase", "protein_type" ], [ 86, 101, "ribulose kinase", "protein_type" ], [ 117, 122, "SePSK", "protein" ], [ 127, 133, "AtXK-1", "protein" ], [ 168, 175, "kinases", "protein_type" ] ] }, { "sid": 57, "sent": "We first tested whether both enzymes possessed ATP hydrolysis activity in the absence of substrates.", "section": "RESULTS", "ner": [ [ 47, 50, "ATP", "chemical" ], [ 78, 88, "absence of", "protein_state" ] ] }, { "sid": 58, "sent": "As shown in Fig 2A, both SePSK and AtXK-1 exhibited ATP hydrolysis activity.", "section": "RESULTS", "ner": [ [ 25, 30, "SePSK", "protein" ], [ 35, 41, "AtXK-1", "protein" ], [ 52, 55, "ATP", "chemical" ] ] }, { "sid": 59, "sent": "This finding is in agreement with a previous result showing that xylulose kinase (PDB code: 2ITM) possessed ATP hydrolysis activity without adding substrate.", "section": "RESULTS", "ner": [ [ 65, 80, "xylulose kinase", "protein_type" ], [ 108, 111, "ATP", "chemical" ] ] }, { "sid": 60, "sent": "To further identify the actual substrate of SePSK and AtXK-1, five different sugar molecules, including D-ribulose, L-ribulose, D-xylulose, L-xylulose and Glycerol, were used in enzymatic activity assays.", "section": "RESULTS", "ner": [ [ 44, 49, "SePSK", "protein" ], [ 54, 60, "AtXK-1", "protein" ], [ 104, 114, "D-ribulose", "chemical" ], [ 116, 126, "L-ribulose", "chemical" ], [ 128, 138, "D-xylulose", "chemical" ], [ 140, 150, "L-xylulose", "chemical" ], [ 155, 163, "Glycerol", "chemical" ], [ 178, 203, "enzymatic activity assays", "experimental_method" ] ] }, { "sid": 61, "sent": "As shown in Fig 2B, the ATP hydrolysis activity of SePSK greatly increased upon adding D-ribulose than adding other potential substrates, suggesting that it has D-ribulose kinase activity.", "section": "RESULTS", "ner": [ [ 24, 27, "ATP", "chemical" ], [ 51, 56, "SePSK", "protein" ], [ 87, 97, "D-ribulose", "chemical" ], [ 161, 178, "D-ribulose kinase", "protein_type" ] ] }, { "sid": 62, "sent": "In contrary, limited increasing of ATP hydrolysis activity was detected for AtXK-1 upon addition of D-ribulose (Fig 2C), despite its structural similarity with SePSK.", "section": "RESULTS", "ner": [ [ 35, 38, "ATP", "chemical" ], [ 76, 82, "AtXK-1", "protein" ], [ 100, 110, "D-ribulose", "chemical" ], [ 160, 165, "SePSK", "protein" ] ] }, { "sid": 63, "sent": "The enzymatic activity assays of SePSK and AtXK-1.", "section": "FIG", "ner": [ [ 4, 29, "enzymatic activity assays", "experimental_method" ], [ 33, 38, "SePSK", "protein" ], [ 43, 49, "AtXK-1", "protein" ] ] }, { "sid": 64, "sent": "(A) The ATP hydrolysis activity of SePSK and AtXK-1.", "section": "FIG", "ner": [ [ 8, 11, "ATP", "chemical" ], [ 35, 40, "SePSK", "protein" ], [ 45, 51, "AtXK-1", "protein" ] ] }, { "sid": 65, "sent": "Both SePSK and AtXK-1 showed ATP hydrolysis activity in the absence of substrate.", "section": "FIG", "ner": [ [ 5, 10, "SePSK", "protein" ], [ 15, 21, "AtXK-1", "protein" ], [ 29, 32, "ATP", "chemical" ], [ 60, 70, "absence of", "protein_state" ] ] }, { "sid": 66, "sent": "While the ATP hydrolysis activity of SePSK greatly increases upon addition of D-ribulose (DR).", "section": "FIG", "ner": [ [ 10, 13, "ATP", "chemical" ], [ 37, 42, "SePSK", "protein" ], [ 78, 88, "D-ribulose", "chemical" ], [ 90, 92, "DR", "chemical" ] ] }, { "sid": 67, "sent": "(B) The ATP hydrolysis activity of SePSK with addition of five different substrates.", "section": "FIG", "ner": [ [ 8, 11, "ATP", "chemical" ], [ 35, 40, "SePSK", "protein" ] ] }, { "sid": 68, "sent": "The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK.", "section": "FIG", "ner": [ [ 19, 21, "DR", "chemical" ], [ 23, 33, "D-ribulose", "chemical" ], [ 36, 38, "LR", "chemical" ], [ 40, 50, "L-ribulose", "chemical" ], [ 53, 55, "DX", "chemical" ], [ 57, 67, "D-xylulose", "chemical" ], [ 70, 72, "LX", "chemical" ], [ 74, 84, "L-xylulose", "chemical" ], [ 90, 93, "GLY", "chemical" ], [ 95, 103, "Glycerol", "chemical" ], [ 114, 117, "ATP", "chemical" ], [ 141, 146, "SePSK", "protein" ], [ 151, 157, "AtXK-1", "protein" ], [ 174, 184, "D-ribulose", "chemical" ], [ 194, 197, "ATP", "chemical" ], [ 221, 230, "wild-type", "protein_state" ], [ 232, 234, "WT", "protein_state" ], [ 263, 268, "SePSK", "protein" ] ] }, { "sid": 69, "sent": "Three single-site mutants of SePSK are D8A-SePSK, T11A-SePSK and D221A-SePSK.", "section": "FIG", "ner": [ [ 29, 34, "SePSK", "protein" ], [ 39, 42, "D8A", "mutant" ], [ 43, 48, "SePSK", "protein" ], [ 50, 54, "T11A", "mutant" ], [ 55, 60, "SePSK", "protein" ], [ 65, 70, "D221A", "mutant" ], [ 71, 76, "SePSK", "protein" ] ] }, { "sid": 70, "sent": "The ATP hydrolysis activity measured via luminescent ADP-Glo assay (Promega).", "section": "FIG", "ner": [ [ 4, 7, "ATP", "chemical" ], [ 41, 66, "luminescent ADP-Glo assay", "experimental_method" ] ] }, { "sid": 71, "sent": "To understand the catalytic mechanism of SePSK, we performed structural comparisons among xylulose kinase, glycerol kinase, ribulose kinase and SePSK.", "section": "RESULTS", "ner": [ [ 41, 46, "SePSK", "protein" ], [ 61, 83, "structural comparisons", "experimental_method" ], [ 90, 105, "xylulose kinase", "protein_type" ], [ 107, 122, "glycerol kinase", "protein_type" ], [ 124, 139, "ribulose kinase", "protein_type" ], [ 144, 149, "SePSK", "protein" ] ] }, { "sid": 72, "sent": "Our results suggested that three conserved residues (D8, T11 and D221 of SePSK) play an important role in SePSK function.", "section": "RESULTS", "ner": [ [ 53, 55, "D8", "residue_name_number" ], [ 57, 60, "T11", "residue_name_number" ], [ 65, 69, "D221", "residue_name_number" ], [ 73, 78, "SePSK", "protein" ], [ 106, 111, "SePSK", "protein" ] ] }, { "sid": 73, "sent": "Mutations of the corresponding residue in xylulose kinase and glycerol kinase from Escherichia coli greatly reduced their activity.", "section": "RESULTS", "ner": [ [ 0, 9, "Mutations", "experimental_method" ], [ 42, 57, "xylulose kinase", "protein_type" ], [ 62, 77, "glycerol kinase", "protein_type" ], [ 83, 99, "Escherichia coli", "species" ] ] }, { "sid": 74, "sent": "To identify the function of these three residues of SePSK, we constructed D8A, T11A and D221A mutants.", "section": "RESULTS", "ner": [ [ 52, 57, "SePSK", "protein" ], [ 74, 77, "D8A", "mutant" ], [ 79, 83, "T11A", "mutant" ], [ 88, 93, "D221A", "mutant" ], [ 94, 101, "mutants", "protein_state" ] ] }, { "sid": 75, "sent": "Using enzymatic activity assays, we found that all of these mutants exhibit much lower activity of ATP hydrolysis after adding D-ribulose than that of wild type, indicating the possibility that these three residues are involved in the catalytic process of phosphorylation D-ribulose and are vital for the function of SePSK (Fig 2D).", "section": "RESULTS", "ner": [ [ 6, 31, "enzymatic activity assays", "experimental_method" ], [ 99, 102, "ATP", "chemical" ], [ 127, 137, "D-ribulose", "chemical" ], [ 151, 160, "wild type", "protein_state" ], [ 256, 271, "phosphorylation", "ptm" ], [ 272, 282, "D-ribulose", "chemical" ], [ 317, 322, "SePSK", "protein" ] ] }, { "sid": 76, "sent": "SePSK and AtXK-1 possess a similar ATP binding site", "section": "RESULTS", "ner": [ [ 0, 5, "SePSK", "protein" ], [ 10, 16, "AtXK-1", "protein" ], [ 35, 51, "ATP binding site", "site" ] ] }, { "sid": 77, "sent": "To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 \u00c5 and 1.8 \u00c5, respectively.", "section": "RESULTS", "ner": [ [ 39, 44, "SePSK", "protein" ], [ 49, 55, "AtXK-1", "protein" ], [ 56, 71, "in complex with", "protein_state" ], [ 72, 75, "ATP", "chemical" ], [ 80, 86, "soaked", "experimental_method" ], [ 91, 94, "apo", "protein_state" ], [ 95, 103, "crystals", "evidence" ], [ 137, 140, "ATP", "chemical" ], [ 159, 169, "structures", "evidence" ], [ 173, 178, "SePSK", "protein" ], [ 183, 189, "AtXK-1", "protein" ], [ 190, 200, "bound with", "protein_state" ], [ 201, 204, "ATP", "chemical" ] ] }, { "sid": 78, "sent": "In both structures, a strong electron density was found in the conserved ATP binding pocket, but can only be fitted with an ADP molecule (S4 Fig).", "section": "RESULTS", "ner": [ [ 8, 18, "structures", "evidence" ], [ 29, 45, "electron density", "evidence" ], [ 63, 72, "conserved", "protein_state" ], [ 73, 91, "ATP binding pocket", "site" ], [ 124, 127, "ADP", "chemical" ] ] }, { "sid": 79, "sent": "Thus the two structures were named ADP-SePSK and ADP-AtXK-1, respectively.", "section": "RESULTS", "ner": [ [ 13, 23, "structures", "evidence" ], [ 35, 44, "ADP-SePSK", "complex_assembly" ], [ 49, 59, "ADP-AtXK-1", "complex_assembly" ] ] }, { "sid": 80, "sent": "The extremely weak electron densities of ATP \u03b3-phosphate in both structures suggest that the \u03b3-phosphate group of ATP is either flexible or hydrolyzed by SePSK and AtXK-1.", "section": "RESULTS", "ner": [ [ 19, 37, "electron densities", "evidence" ], [ 47, 56, "phosphate", "chemical" ], [ 65, 75, "structures", "evidence" ], [ 95, 104, "phosphate", "chemical" ], [ 114, 117, "ATP", "chemical" ], [ 154, 159, "SePSK", "protein" ], [ 164, 170, "AtXK-1", "protein" ] ] }, { "sid": 81, "sent": "This result was consistent with our enzymatic activity assays where SePSK and AtXK-1 showed ATP hydrolysis activity without adding any substrates (Fig 2A and 2C).", "section": "RESULTS", "ner": [ [ 36, 61, "enzymatic activity assays", "experimental_method" ], [ 68, 73, "SePSK", "protein" ], [ 78, 84, "AtXK-1", "protein" ], [ 92, 95, "ATP", "chemical" ] ] }, { "sid": 82, "sent": "To avoid hydrolysis of ATP, we soaked the crystals of apo-SePSK and apo-AtXK-1 into the reservoir adding AMP-PNP.", "section": "RESULTS", "ner": [ [ 23, 26, "ATP", "chemical" ], [ 31, 37, "soaked", "experimental_method" ], [ 42, 50, "crystals", "evidence" ], [ 54, 57, "apo", "protein_state" ], [ 58, 63, "SePSK", "protein" ], [ 68, 71, "apo", "protein_state" ], [ 72, 78, "AtXK-1", "protein" ], [ 105, 112, "AMP-PNP", "chemical" ] ] }, { "sid": 83, "sent": "However, we found that the electron densities of \u03b3-phosphate group of AMP-PNP (AMP-PNP \u03b3-phosphate) are still weak in the AMP-PNP-SePSK and AMP-PNP-AtXK-1 structures, suggesting high flexibility of ATP-\u03b3-phosphate.", "section": "RESULTS", "ner": [ [ 27, 45, "electron densities", "evidence" ], [ 51, 60, "phosphate", "chemical" ], [ 70, 77, "AMP-PNP", "chemical" ], [ 79, 86, "AMP-PNP", "chemical" ], [ 89, 98, "phosphate", "chemical" ], [ 122, 135, "AMP-PNP-SePSK", "complex_assembly" ], [ 140, 154, "AMP-PNP-AtXK-1", "complex_assembly" ], [ 155, 165, "structures", "evidence" ], [ 198, 201, "ATP", "chemical" ], [ 204, 213, "phosphate", "chemical" ] ] }, { "sid": 84, "sent": "The \u03b3-phosphate group of ATP is transferred to the sugar substrate during the reaction process, so this flexibility might be important for the ability of these kinases.", "section": "RESULTS", "ner": [ [ 6, 15, "phosphate", "chemical" ], [ 25, 28, "ATP", "chemical" ], [ 51, 56, "sugar", "chemical" ], [ 160, 167, "kinases", "protein_type" ] ] }, { "sid": 85, "sent": "The overall structures as well as the coordination modes of ADP and AMP-PNP in the AMP-PNP-AtXK-1, ADP-AtXK-1, ADP-SePSK and AMP-PNP-SePSK structures are nearly identical (S5 Fig), therefore the structure of AMP-PNP-SePSK is used here to describe the structural details and to compare with those of other family members.", "section": "RESULTS", "ner": [ [ 12, 22, "structures", "evidence" ], [ 60, 63, "ADP", "chemical" ], [ 68, 75, "AMP-PNP", "chemical" ], [ 83, 97, "AMP-PNP-AtXK-1", "complex_assembly" ], [ 99, 109, "ADP-AtXK-1", "complex_assembly" ], [ 111, 120, "ADP-SePSK", "complex_assembly" ], [ 125, 138, "AMP-PNP-SePSK", "complex_assembly" ], [ 139, 149, "structures", "evidence" ], [ 195, 204, "structure", "evidence" ], [ 208, 221, "AMP-PNP-SePSK", "complex_assembly" ] ] }, { "sid": 86, "sent": "As shown in Fig 3A, one SePSK protein molecule is in an asymmetric unit with one AMP-PNP molecule.", "section": "RESULTS", "ner": [ [ 24, 29, "SePSK", "protein" ], [ 81, 88, "AMP-PNP", "chemical" ] ] }, { "sid": 87, "sent": "The AMP-PNP is bound at the domain II, where it fits well inside a positively charged groove.", "section": "RESULTS", "ner": [ [ 4, 11, "AMP-PNP", "chemical" ], [ 28, 37, "domain II", "structure_element" ], [ 67, 92, "positively charged groove", "site" ] ] }, { "sid": 88, "sent": "The AMP-PNP binding pocket consists of four \u03b1-helices (\u03b126, \u03b128, \u03b127 and \u03b130) and forms a shape resembling a half-fist (Fig 3A and 3B).", "section": "RESULTS", "ner": [ [ 4, 26, "AMP-PNP binding pocket", "site" ], [ 39, 53, "four \u03b1-helices", "structure_element" ], [ 55, 58, "\u03b126", "structure_element" ], [ 60, 63, "\u03b128", "structure_element" ], [ 65, 68, "\u03b127", "structure_element" ], [ 73, 76, "\u03b130", "structure_element" ], [ 90, 118, "shape resembling a half-fist", "protein_state" ] ] }, { "sid": 89, "sent": "The head group of the AMP-PNP is embedded in a pocket surrounded by Trp383, Asn380, Gly376 and Gly377.", "section": "RESULTS", "ner": [ [ 22, 29, "AMP-PNP", "chemical" ], [ 47, 53, "pocket", "site" ], [ 68, 74, "Trp383", "residue_name_number" ], [ 76, 82, "Asn380", "residue_name_number" ], [ 84, 90, "Gly376", "residue_name_number" ], [ 95, 101, "Gly377", "residue_name_number" ] ] }, { "sid": 90, "sent": "The purine ring of AMP-PNP is positioned in parallel to the indole ring of Trp383.", "section": "RESULTS", "ner": [ [ 19, 26, "AMP-PNP", "chemical" ], [ 75, 81, "Trp383", "residue_name_number" ] ] }, { "sid": 91, "sent": "In addition, it is hydrogen-bonded with the side chain amide of Asn380 (Fig 3B).", "section": "RESULTS", "ner": [ [ 19, 34, "hydrogen-bonded", "bond_interaction" ], [ 64, 70, "Asn380", "residue_name_number" ] ] }, { "sid": 92, "sent": "The tail of AMP-PNP points to the hinge region of SePSK, and its \u03b1-phosphate and \u03b2-phosphate groups are stabilized by Gly376 and Ser243, respectively.", "section": "RESULTS", "ner": [ [ 12, 19, "AMP-PNP", "chemical" ], [ 34, 46, "hinge region", "structure_element" ], [ 50, 55, "SePSK", "protein" ], [ 67, 76, "phosphate", "chemical" ], [ 83, 92, "phosphate", "chemical" ], [ 118, 124, "Gly376", "residue_name_number" ], [ 129, 135, "Ser243", "residue_name_number" ] ] }, { "sid": 93, "sent": "Together, this structure clearly shows that the AMP-PNP-\u03b2-phosphate is sticking out of the ATP binding pocket, thus the \u03b3-phosphate group is at the empty space between domain I and domain II and is unconstrained in its movement by the protein.", "section": "RESULTS", "ner": [ [ 15, 24, "structure", "evidence" ], [ 48, 55, "AMP-PNP", "chemical" ], [ 58, 67, "phosphate", "chemical" ], [ 91, 109, "ATP binding pocket", "site" ], [ 122, 131, "phosphate", "chemical" ], [ 168, 176, "domain I", "structure_element" ], [ 181, 190, "domain II", "structure_element" ] ] }, { "sid": 94, "sent": "Structure of SePSK in complex with AMP-PNP.", "section": "FIG", "ner": [ [ 0, 9, "Structure", "evidence" ], [ 13, 18, "SePSK", "protein" ], [ 19, 34, "in complex with", "protein_state" ], [ 35, 42, "AMP-PNP", "chemical" ] ] }, { "sid": 95, "sent": "(A) The electron density of AMP-PNP.", "section": "FIG", "ner": [ [ 8, 24, "electron density", "evidence" ], [ 28, 35, "AMP-PNP", "chemical" ] ] }, { "sid": 96, "sent": "The SePSK structure is shown in the electrostatic potential surface mode.", "section": "FIG", "ner": [ [ 4, 9, "SePSK", "protein" ], [ 10, 19, "structure", "evidence" ] ] }, { "sid": 97, "sent": "The AMP-PNP is depicted as sticks with its \u01c0Fo\u01c0-\u01c0Fc\u01c0 map contoured at 3 \u03c3 shown as cyan mesh.", "section": "FIG", "ner": [ [ 4, 11, "AMP-PNP", "chemical" ], [ 43, 56, "\u01c0Fo\u01c0-\u01c0Fc\u01c0 map", "evidence" ] ] }, { "sid": 98, "sent": "(B) The AMP-PNP binding pocket.", "section": "FIG", "ner": [ [ 8, 30, "AMP-PNP binding pocket", "site" ] ] }, { "sid": 99, "sent": "The head of AMP-PNP is sandwiched by four residues (Leu293, Gly376, Gly377 and Trp383).", "section": "FIG", "ner": [ [ 12, 19, "AMP-PNP", "chemical" ], [ 23, 36, "sandwiched by", "bond_interaction" ], [ 52, 58, "Leu293", "residue_name_number" ], [ 60, 66, "Gly376", "residue_name_number" ], [ 68, 74, "Gly377", "residue_name_number" ], [ 79, 85, "Trp383", "residue_name_number" ] ] }, { "sid": 100, "sent": "The four \u03b1-helices (\u03b126, \u03b128, \u03b127 and \u03b130) are labeled in red.", "section": "FIG", "ner": [ [ 9, 18, "\u03b1-helices", "structure_element" ], [ 20, 23, "\u03b126", "structure_element" ], [ 25, 28, "\u03b128", "structure_element" ], [ 30, 33, "\u03b127", "structure_element" ], [ 38, 41, "\u03b130", "structure_element" ] ] }, { "sid": 101, "sent": "The AMP-PNP and coordinated residues are shown as sticks.", "section": "FIG", "ner": [ [ 4, 11, "AMP-PNP", "chemical" ] ] }, { "sid": 102, "sent": "The potential substrate binding site in SePSK", "section": "RESULTS", "ner": [ [ 14, 36, "substrate binding site", "site" ], [ 40, 45, "SePSK", "protein" ] ] }, { "sid": 103, "sent": "The results from our activity assays suggested that SePSK has D-ribulose kinase activity.", "section": "RESULTS", "ner": [ [ 21, 36, "activity assays", "experimental_method" ], [ 52, 57, "SePSK", "protein" ], [ 62, 79, "D-ribulose kinase", "protein_type" ] ] }, { "sid": 104, "sent": "To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved.", "section": "RESULTS", "ner": [ [ 53, 58, "SePSK", "protein" ], [ 63, 73, "D-ribulose", "chemical" ], [ 79, 82, "apo", "protein_state" ], [ 83, 88, "SePSK", "protein" ], [ 89, 114, "crystals were soaked into", "experimental_method" ], [ 119, 128, "reservoir", "experimental_method" ], [ 140, 150, "D-ribulose", "chemical" ], [ 152, 155, "RBL", "chemical" ], [ 165, 174, "RBL-SePSK", "complex_assembly" ], [ 175, 184, "structure", "evidence" ], [ 189, 195, "solved", "experimental_method" ] ] }, { "sid": 105, "sent": "As shown in S6 Fig, two residual electron densities are visible in domain I, which can be interpreted as two D-ribulose molecules with reasonable fit.", "section": "RESULTS", "ner": [ [ 33, 51, "electron densities", "evidence" ], [ 67, 75, "domain I", "structure_element" ], [ 109, 119, "D-ribulose", "chemical" ] ] }, { "sid": 106, "sent": "As shown in Fig 4A, the nearest distance between the carbon skeleton of two D-ribulose molecules are approx.", "section": "RESULTS", "ner": [ [ 76, 86, "D-ribulose", "chemical" ] ] }, { "sid": 107, "sent": "7.1 \u00c5 (RBL1-C4 and RBL2-C1).", "section": "RESULTS", "ner": [ [ 7, 11, "RBL1", "residue_name_number" ], [ 19, 23, "RBL2", "residue_name_number" ] ] }, { "sid": 108, "sent": "RBL1 is located in the pocket consisting of \u03b121 and the loop between \u03b26 and \u03b27.", "section": "RESULTS", "ner": [ [ 0, 4, "RBL1", "residue_name_number" ], [ 23, 29, "pocket", "site" ], [ 44, 47, "\u03b121", "structure_element" ], [ 56, 60, "loop", "structure_element" ], [ 69, 78, "\u03b26 and \u03b27", "structure_element" ] ] }, { "sid": 109, "sent": "The O4 and O5 of RBL1 are coordinated with the side chain carboxyl group of Asp221.", "section": "RESULTS", "ner": [ [ 17, 21, "RBL1", "residue_name_number" ], [ 26, 42, "coordinated with", "bond_interaction" ], [ 76, 82, "Asp221", "residue_name_number" ] ] }, { "sid": 110, "sent": "Furthermore, the O2 of RBL1 interacts with the main chain amide nitrogen of Ser72 (Fig 4B).", "section": "RESULTS", "ner": [ [ 23, 27, "RBL1", "residue_name_number" ], [ 28, 42, "interacts with", "bond_interaction" ], [ 76, 81, "Ser72", "residue_name_number" ] ] }, { "sid": 111, "sent": "This pocket is at a similar position of substrate binding site of other sugar kinase, such as L-ribulokinase (PDB code: 3QDK) (S7 Fig).", "section": "RESULTS", "ner": [ [ 5, 11, "pocket", "site" ], [ 40, 62, "substrate binding site", "site" ], [ 72, 84, "sugar kinase", "protein_type" ], [ 94, 108, "L-ribulokinase", "protein" ] ] }, { "sid": 112, "sent": "However, structural comparison shows that the substrate ligating residues between the two structures are not strictly conserved.", "section": "RESULTS", "ner": [ [ 9, 30, "structural comparison", "experimental_method" ], [ 90, 100, "structures", "evidence" ], [ 105, 127, "not strictly conserved", "protein_state" ] ] }, { "sid": 113, "sent": "Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig).", "section": "RESULTS", "ner": [ [ 13, 23, "structures", "evidence" ], [ 50, 54, "RBL1", "residue_name_number" ], [ 58, 67, "RBL-SePSK", "complex_assembly" ], [ 68, 77, "structure", "evidence" ], [ 82, 87, "Ser72", "residue_name_number" ], [ 89, 95, "Asp221", "residue_name_number" ], [ 100, 106, "Ser222", "residue_name_number" ], [ 140, 150, "L-ribulose", "chemical" ], [ 156, 170, "L-ribulokinase", "protein" ], [ 175, 180, "Ala96", "residue_name_number" ], [ 182, 188, "Lys208", "residue_name_number" ], [ 190, 196, "Asp274", "residue_name_number" ], [ 201, 207, "Glu329", "residue_name_number" ] ] }, { "sid": 114, "sent": "Glu329 in 3QDK has no counterpart in RBL-SePSK structure.", "section": "RESULTS", "ner": [ [ 0, 6, "Glu329", "residue_name_number" ], [ 37, 46, "RBL-SePSK", "complex_assembly" ], [ 47, 56, "structure", "evidence" ] ] }, { "sid": 115, "sent": "In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two \u03b1-helices (\u03b19 and \u03b113) of SePSK.", "section": "RESULTS", "ner": [ [ 22, 28, "Lys208", "residue_name_number" ], [ 32, 46, "L-ribulokinase", "protein" ], [ 78, 84, "Lys163", "residue_name_number" ], [ 89, 98, "RBL-SePSK", "complex_assembly" ], [ 99, 108, "structure", "evidence" ], [ 114, 127, "hydrogen bond", "bond_interaction" ], [ 131, 137, "Lys163", "residue_name_number" ], [ 192, 201, "\u03b1-helices", "structure_element" ], [ 203, 205, "\u03b19", "structure_element" ], [ 210, 213, "\u03b113", "structure_element" ], [ 218, 223, "SePSK", "protein" ] ] }, { "sid": 116, "sent": "The binding of D-ribulose (RBL) with SePSK.", "section": "FIG", "ner": [ [ 15, 25, "D-ribulose", "chemical" ], [ 27, 30, "RBL", "chemical" ], [ 37, 42, "SePSK", "protein" ] ] }, { "sid": 117, "sent": "(A) The electrostatic potential surface map of RBL-SePSK and a zoom-in view of RBL binding site.", "section": "FIG", "ner": [ [ 8, 43, "electrostatic potential surface map", "evidence" ], [ 47, 56, "RBL-SePSK", "complex_assembly" ], [ 79, 95, "RBL binding site", "site" ] ] }, { "sid": 118, "sent": "The RBL1 and RBL2 are depicted as sticks. (B) Interaction of two D-ribulose molecules (RBL1 and RBL2) with SePSK.", "section": "FIG", "ner": [ [ 4, 8, "RBL1", "residue_name_number" ], [ 13, 17, "RBL2", "residue_name_number" ], [ 65, 75, "D-ribulose", "chemical" ], [ 87, 91, "RBL1", "residue_name_number" ], [ 96, 100, "RBL2", "residue_name_number" ], [ 107, 112, "SePSK", "protein" ] ] }, { "sid": 119, "sent": "The RBL molecules (carbon atoms colored yellow) and amino acid residues of SePSK (carbon atoms colored green) involved in RBL interaction are shown as sticks.", "section": "FIG", "ner": [ [ 4, 7, "RBL", "chemical" ], [ 75, 80, "SePSK", "protein" ], [ 122, 125, "RBL", "chemical" ] ] }, { "sid": 120, "sent": "The hydrogen bonds are indicated by the black dashed lines and the numbers near the dashed lines are the distances (\u00c5). (C) The binding affinity assays of SePSK with D-ribulose.", "section": "FIG", "ner": [ [ 4, 18, "hydrogen bonds", "bond_interaction" ], [ 128, 151, "binding affinity assays", "experimental_method" ], [ 155, 160, "SePSK", "protein" ], [ 166, 176, "D-ribulose", "chemical" ] ] }, { "sid": 121, "sent": "Single-cycle kinetic data are reflecting the interaction of SePSK and D8A-SePSK with D-ribulose.", "section": "FIG", "ner": [ [ 0, 25, "Single-cycle kinetic data", "experimental_method" ], [ 60, 65, "SePSK", "protein" ], [ 70, 73, "D8A", "mutant" ], [ 74, 79, "SePSK", "protein" ], [ 85, 95, "D-ribulose", "chemical" ] ] }, { "sid": 122, "sent": "It shows two experimental sensorgrams after minus the empty sensorgrams.", "section": "FIG", "ner": [ [ 26, 37, "sensorgrams", "evidence" ], [ 60, 71, "sensorgrams", "evidence" ] ] }, { "sid": 123, "sent": "The original data is shown as black curve, and the fitted data is shown as different color (wild type SePSK: red curve, D8A-SePSK: green curve).", "section": "FIG", "ner": [ [ 92, 101, "wild type", "protein_state" ], [ 102, 107, "SePSK", "protein" ], [ 120, 123, "D8A", "mutant" ], [ 124, 129, "SePSK", "protein" ] ] }, { "sid": 124, "sent": "Dissociation rate constant of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively.", "section": "FIG", "ner": [ [ 0, 26, "Dissociation rate constant", "evidence" ], [ 30, 39, "wild type", "protein_state" ], [ 44, 47, "D8A", "mutant" ], [ 48, 53, "SePSK", "protein" ] ] }, { "sid": 125, "sent": "The binding pocket of RBL2 with relatively weak electron density is near the N-terminal region of SePSK and is negatively charged.", "section": "RESULTS", "ner": [ [ 4, 18, "binding pocket", "site" ], [ 22, 26, "RBL2", "residue_name_number" ], [ 48, 64, "electron density", "evidence" ], [ 98, 103, "SePSK", "protein" ] ] }, { "sid": 126, "sent": "The side chain of Asp8 interacts strongly with O3 and O4 of RBL2.", "section": "RESULTS", "ner": [ [ 18, 22, "Asp8", "residue_name_number" ], [ 23, 46, "interacts strongly with", "bond_interaction" ], [ 60, 64, "RBL2", "residue_name_number" ] ] }, { "sid": 127, "sent": "The hydroxyl group of Ser12 coordinates with O2 of RBL2.", "section": "RESULTS", "ner": [ [ 22, 27, "Ser12", "residue_name_number" ], [ 28, 44, "coordinates with", "bond_interaction" ], [ 51, 55, "RBL2", "residue_name_number" ] ] }, { "sid": 128, "sent": "The backbone amide nitrogens of Gly13 and Arg15 also keep hydrogen bonds with RBL2 (Fig 4B).", "section": "RESULTS", "ner": [ [ 32, 37, "Gly13", "residue_name_number" ], [ 42, 47, "Arg15", "residue_name_number" ], [ 58, 72, "hydrogen bonds", "bond_interaction" ], [ 78, 82, "RBL2", "residue_name_number" ] ] }, { "sid": 129, "sent": "Structural comparison of SePSK and AtXK-1 showed that while the RBL1 binding pocket is conserved, the RBL2 pocket is disrupted in AtXK-1 structure, despite the fact that the residues interacting with RBL2 are highly conserved between the two proteins.", "section": "RESULTS", "ner": [ [ 0, 21, "Structural comparison", "experimental_method" ], [ 25, 30, "SePSK", "protein" ], [ 35, 41, "AtXK-1", "protein" ], [ 64, 83, "RBL1 binding pocket", "site" ], [ 87, 96, "conserved", "protein_state" ], [ 102, 113, "RBL2 pocket", "site" ], [ 130, 136, "AtXK-1", "protein" ], [ 137, 146, "structure", "evidence" ], [ 200, 204, "RBL2", "residue_name_number" ], [ 209, 225, "highly conserved", "protein_state" ] ] }, { "sid": 130, "sent": "In the RBL-SePSK structure, a 2.6 \u00c5 hydrogen bond is present between RBL2 and Ser12 (Fig 4B), while in the AtXK-1 structure this hydrogen bond with the corresponding residue (Ser22) is broken.", "section": "RESULTS", "ner": [ [ 7, 16, "RBL-SePSK", "complex_assembly" ], [ 17, 26, "structure", "evidence" ], [ 36, 49, "hydrogen bond", "bond_interaction" ], [ 69, 73, "RBL2", "residue_name_number" ], [ 78, 83, "Ser12", "residue_name_number" ], [ 107, 113, "AtXK-1", "protein" ], [ 114, 123, "structure", "evidence" ], [ 129, 142, "hydrogen bond", "bond_interaction" ], [ 175, 180, "Ser22", "residue_name_number" ] ] }, { "sid": 131, "sent": "This break is probably induced by the conformational change of the two \u03b2-sheets (\u03b21 and \u03b22), with the result that the linking loop (loop 1) is located further away from the RBL2 binding site.", "section": "RESULTS", "ner": [ [ 71, 79, "\u03b2-sheets", "structure_element" ], [ 81, 83, "\u03b21", "structure_element" ], [ 88, 90, "\u03b22", "structure_element" ], [ 118, 130, "linking loop", "structure_element" ], [ 132, 138, "loop 1", "structure_element" ], [ 173, 190, "RBL2 binding site", "site" ] ] }, { "sid": 132, "sent": "This change might be the reason that AtXK-1 only shows limited increasing in its ATP hydrolysis ability upon adding D-ribulose as a substrate after comparing with SePSK (Fig 2C).", "section": "RESULTS", "ner": [ [ 37, 43, "AtXK-1", "protein" ], [ 81, 84, "ATP", "chemical" ], [ 116, 126, "D-ribulose", "chemical" ], [ 163, 168, "SePSK", "protein" ] ] }, { "sid": 133, "sent": "Our SePSK structure shows that the Asp8 residue forms strong hydrogen bond with RBL2 (Fig 4B).", "section": "RESULTS", "ner": [ [ 4, 9, "SePSK", "protein" ], [ 10, 19, "structure", "evidence" ], [ 35, 39, "Asp8", "residue_name_number" ], [ 61, 74, "hydrogen bond", "bond_interaction" ], [ 80, 84, "RBL2", "residue_name_number" ] ] }, { "sid": 134, "sent": "In addition, our enzymatic assays indicated that Asp8 is important for the activity of SePSK (Fig 2D).", "section": "RESULTS", "ner": [ [ 17, 33, "enzymatic assays", "experimental_method" ], [ 49, 53, "Asp8", "residue_name_number" ], [ 87, 92, "SePSK", "protein" ] ] }, { "sid": 135, "sent": "To further verified this result, we measured the binding affinity for D-ribulose of both wild type (WT) and D8A mutant of SePSK using a surface plasmon resonance method.", "section": "RESULTS", "ner": [ [ 49, 65, "binding affinity", "evidence" ], [ 70, 80, "D-ribulose", "chemical" ], [ 89, 98, "wild type", "protein_state" ], [ 100, 102, "WT", "protein_state" ], [ 108, 111, "D8A", "mutant" ], [ 112, 118, "mutant", "protein_state" ], [ 122, 127, "SePSK", "protein" ], [ 136, 168, "surface plasmon resonance method", "experimental_method" ] ] }, { "sid": 136, "sent": "The results showed that the affinity of D8A-SePSK with D-ribulose is weaker than that of WT with a reduction of approx.", "section": "RESULTS", "ner": [ [ 28, 36, "affinity", "evidence" ], [ 40, 43, "D8A", "mutant" ], [ 44, 49, "SePSK", "protein" ], [ 55, 65, "D-ribulose", "chemical" ], [ 89, 91, "WT", "protein_state" ] ] }, { "sid": 137, "sent": "Dissociation rate constant (Kd) of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively.", "section": "RESULTS", "ner": [ [ 0, 26, "Dissociation rate constant", "evidence" ], [ 28, 30, "Kd", "evidence" ], [ 35, 44, "wild type", "protein_state" ], [ 49, 52, "D8A", "mutant" ], [ 53, 58, "SePSK", "protein" ] ] }, { "sid": 138, "sent": "The results implied that the second RBL binding site plays a role in the D-ribulose kinase function of SePSK.", "section": "RESULTS", "ner": [ [ 29, 52, "second RBL binding site", "site" ], [ 73, 90, "D-ribulose kinase", "protein_type" ], [ 103, 108, "SePSK", "protein" ] ] }, { "sid": 139, "sent": "However, considering the high concentration of D-ribulose used for crystal soaking, as well as the relatively weak electron density of RBL2, it is also possible that the second binding site of D-ribulose in SePSK is an artifact.", "section": "RESULTS", "ner": [ [ 47, 57, "D-ribulose", "chemical" ], [ 67, 82, "crystal soaking", "experimental_method" ], [ 115, 131, "electron density", "evidence" ], [ 135, 139, "RBL2", "residue_name_number" ], [ 170, 189, "second binding site", "site" ], [ 193, 203, "D-ribulose", "chemical" ], [ 207, 212, "SePSK", "protein" ] ] }, { "sid": 140, "sent": "Simulated conformational change of SePSK during the catalytic process", "section": "RESULTS", "ner": [ [ 35, 40, "SePSK", "protein" ] ] }, { "sid": 141, "sent": "It was reported earlier that the crossing angle between the domain I and domain II in FGGY family carbohydrate kinases is different.", "section": "RESULTS", "ner": [ [ 60, 68, "domain I", "structure_element" ], [ 73, 82, "domain II", "structure_element" ], [ 86, 118, "FGGY family carbohydrate kinases", "protein_type" ] ] }, { "sid": 142, "sent": "In addition, this difference may be caused by the binding of substrates and/or ATP.", "section": "RESULTS", "ner": [ [ 79, 82, "ATP", "chemical" ] ] }, { "sid": 143, "sent": "As reported previously, members of the sugar kinase family undergo a conformational change to narrow the crossing angle between two domains and reduce the distance between substrate and ATP in order to facilitate the catalytic reaction of phosphorylation of sugar substrates.", "section": "RESULTS", "ner": [ [ 39, 51, "sugar kinase", "protein_type" ], [ 186, 189, "ATP", "chemical" ], [ 239, 254, "phosphorylation", "ptm" ] ] }, { "sid": 144, "sent": "After comparing structures of apo-SePSK, RBL-SePSK and AMP-PNP-SePSK, we noticed that these structures presented here are similar.", "section": "RESULTS", "ner": [ [ 16, 26, "structures", "evidence" ], [ 30, 33, "apo", "protein_state" ], [ 34, 39, "SePSK", "protein" ], [ 41, 50, "RBL-SePSK", "complex_assembly" ], [ 55, 68, "AMP-PNP-SePSK", "complex_assembly" ], [ 92, 102, "structures", "evidence" ] ] }, { "sid": 145, "sent": "Superposing the structures of RBL-SePSK and AMP-PNP-SePSK, the results show that the nearest distance between AMP-PNP \u03b3-phosphate and RBL1/RBL2 is 7.5 \u00c5 (RBL1-O5)/6.7 \u00c5 (RBL2-O1) (S8 Fig).", "section": "RESULTS", "ner": [ [ 0, 11, "Superposing", "experimental_method" ], [ 16, 26, "structures", "evidence" ], [ 30, 39, "RBL-SePSK", "complex_assembly" ], [ 44, 57, "AMP-PNP-SePSK", "complex_assembly" ], [ 110, 117, "AMP-PNP", "chemical" ], [ 120, 129, "phosphate", "chemical" ], [ 134, 138, "RBL1", "residue_name_number" ], [ 139, 143, "RBL2", "residue_name_number" ], [ 154, 158, "RBL1", "residue_name_number" ], [ 170, 174, "RBL2", "residue_name_number" ] ] }, { "sid": 146, "sent": "This distance is too long to transfer the \u03b3-phosphate group from ATP to the substrate.", "section": "RESULTS", "ner": [ [ 44, 53, "phosphate", "chemical" ], [ 65, 68, "ATP", "chemical" ] ] }, { "sid": 147, "sent": "Since the two domains of SePSK are widely separated in this structure, we hypothesize that our structures of SePSK represent its open form, and that a conformational rearrangement must occur to switch to the closed state in order to facilitate the catalytic process of phosphorylation of sugar substrates.", "section": "RESULTS", "ner": [ [ 25, 30, "SePSK", "protein" ], [ 60, 69, "structure", "evidence" ], [ 95, 105, "structures", "evidence" ], [ 109, 114, "SePSK", "protein" ], [ 129, 133, "open", "protein_state" ], [ 208, 214, "closed", "protein_state" ], [ 269, 284, "phosphorylation", "ptm" ] ] }, { "sid": 148, "sent": "For studying such potential conformational change, a simulation on the Hingeprot Server was performed to predict the movement of different SePSK domains.", "section": "RESULTS", "ner": [ [ 53, 63, "simulation", "experimental_method" ], [ 71, 87, "Hingeprot Server", "experimental_method" ], [ 139, 144, "SePSK", "protein" ] ] }, { "sid": 149, "sent": "The results showed that domain I and domain II are closer to each other with Ala228 and Thr401 in A2 as Hinge-residues.", "section": "RESULTS", "ner": [ [ 24, 32, "domain I", "structure_element" ], [ 37, 46, "domain II", "structure_element" ], [ 77, 83, "Ala228", "residue_name_number" ], [ 88, 94, "Thr401", "residue_name_number" ], [ 98, 100, "A2", "structure_element" ], [ 104, 118, "Hinge-residues", "structure_element" ] ] }, { "sid": 150, "sent": "Based on the above results, SePSK is divided into two rigid parts.", "section": "RESULTS", "ner": [ [ 28, 33, "SePSK", "protein" ] ] }, { "sid": 151, "sent": "The domain I of RBL-SePSK (aa. 1\u2013228, aa. 402\u2013421) and the domain II of AMP-PNP-SePSK (aa. 229\u2013401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ).", "section": "RESULTS", "ner": [ [ 4, 12, "domain I", "structure_element" ], [ 16, 25, "RBL-SePSK", "complex_assembly" ], [ 31, 36, "1\u2013228", "residue_range" ], [ 42, 49, "402\u2013421", "residue_range" ], [ 59, 68, "domain II", "structure_element" ], [ 72, 85, "AMP-PNP-SePSK", "complex_assembly" ], [ 91, 98, "229\u2013401", "residue_range" ], [ 105, 115, "superposed", "experimental_method" ], [ 121, 131, "structures", "evidence" ], [ 143, 146, "apo", "protein_state" ], [ 147, 153, "AtXK-1", "protein" ], [ 155, 158, "apo", "protein_state" ], [ 159, 164, "SePSK", "protein" ], [ 166, 181, "xylulose kinase", "protein_type" ], [ 187, 212, "Lactobacillus acidophilus", "species" ], [ 238, 242, "S58W", "mutant" ], [ 243, 249, "mutant", "protein_state" ], [ 258, 273, "glycerol kinase", "protein_type" ], [ 279, 295, "Escherichia coli", "species" ] ] }, { "sid": 152, "sent": "The results of superposition displayed different crossing angle between these two domains.", "section": "RESULTS", "ner": [ [ 15, 28, "superposition", "experimental_method" ] ] }, { "sid": 153, "sent": "After superposition, the distances of AMP-PNP \u03b3-phosphate and the fifth hydroxyl group of RBL1 are 7.9 \u00c5 (superposed with AtXK-1), 7.4 \u00c5 (superposed with SePSK), 6.6 \u00c5 (superposed with 3LL3) and 6.1 \u00c5 (superposed with 1GLJ).", "section": "RESULTS", "ner": [ [ 6, 19, "superposition", "experimental_method" ], [ 38, 45, "AMP-PNP", "chemical" ], [ 48, 57, "phosphate", "chemical" ], [ 90, 94, "RBL1", "residue_name_number" ], [ 106, 116, "superposed", "experimental_method" ], [ 122, 128, "AtXK-1", "protein" ], [ 138, 148, "superposed", "experimental_method" ], [ 154, 159, "SePSK", "protein" ], [ 169, 179, "superposed", "experimental_method" ], [ 202, 212, "superposed", "experimental_method" ] ] }, { "sid": 154, "sent": "Meanwhile, the distances of AMP-PNP \u03b3-phosphate and the first hydroxyl group of RBL2 are 7.2 \u00c5 (superposed with AtXK-1), 6.7 \u00c5 (superposed with SePSK), 3.7 \u00c5 (superposed with 3LL3), until AMP-PNP \u03b3-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5).", "section": "RESULTS", "ner": [ [ 28, 35, "AMP-PNP", "chemical" ], [ 38, 47, "phosphate", "chemical" ], [ 80, 84, "RBL2", "residue_name_number" ], [ 96, 106, "superposed", "experimental_method" ], [ 112, 118, "AtXK-1", "protein" ], [ 128, 138, "superposed", "experimental_method" ], [ 144, 149, "SePSK", "protein" ], [ 159, 169, "superposed", "experimental_method" ], [ 188, 195, "AMP-PNP", "chemical" ], [ 198, 207, "phosphate", "chemical" ], [ 223, 227, "RBL2", "residue_name_number" ], [ 234, 247, "superposition", "experimental_method" ] ] }, { "sid": 155, "sent": "This distance between RBL2 and AMP-PNP-\u03b3-phosphate is close enough to facilitate phosphate transferring.", "section": "RESULTS", "ner": [ [ 22, 26, "RBL2", "residue_name_number" ], [ 31, 38, "AMP-PNP", "chemical" ], [ 41, 50, "phosphate", "chemical" ], [ 81, 90, "phosphate", "chemical" ] ] }, { "sid": 156, "sent": "Together, our superposition results provided snapshots of the conformational changes at different catalytic stages of SePSK and potentially revealed the closed form of SePSK.", "section": "RESULTS", "ner": [ [ 14, 27, "superposition", "experimental_method" ], [ 118, 123, "SePSK", "protein" ], [ 153, 159, "closed", "protein_state" ], [ 168, 173, "SePSK", "protein" ] ] }, { "sid": 157, "sent": "Simulated conformational change of SePSK during the catalytic process.", "section": "FIG", "ner": [ [ 35, 40, "SePSK", "protein" ] ] }, { "sid": 158, "sent": "The structures are shown as cartoon and the ligands are shown as sticks.", "section": "FIG", "ner": [ [ 4, 14, "structures", "evidence" ] ] }, { "sid": 159, "sent": "Domain I from D-ribulose-SePSK (green) and Domain II from AMP-PNP-SePSK (cyan) are superposed with apo-AtXK-1 (1st), apo-SePSK (2nd), 3LL3 (3rd) and 1GLJ (4th), respectively.", "section": "FIG", "ner": [ [ 0, 8, "Domain I", "structure_element" ], [ 14, 30, "D-ribulose-SePSK", "complex_assembly" ], [ 43, 52, "Domain II", "structure_element" ], [ 58, 71, "AMP-PNP-SePSK", "complex_assembly" ], [ 83, 93, "superposed", "experimental_method" ], [ 99, 102, "apo", "protein_state" ], [ 103, 109, "AtXK-1", "protein" ], [ 117, 120, "apo", "protein_state" ], [ 121, 126, "SePSK", "protein" ] ] }, { "sid": 160, "sent": "The numbers near the black dashed lines show the distances (\u00c5) between two nearest atoms of RBL and AMP-PNP.", "section": "FIG", "ner": [ [ 92, 95, "RBL", "chemical" ], [ 100, 107, "AMP-PNP", "chemical" ] ] }, { "sid": 161, "sent": "In summary, our structural and enzymatic analyses provide evidence that SePSK shows D-ribulose kinase activity, and exhibits the conserved features of FGGY family carbohydrate kinases.", "section": "RESULTS", "ner": [ [ 16, 49, "structural and enzymatic analyses", "experimental_method" ], [ 72, 77, "SePSK", "protein" ], [ 84, 101, "D-ribulose kinase", "protein_type" ], [ 151, 183, "FGGY family carbohydrate kinases", "protein_type" ] ] }, { "sid": 162, "sent": "Three conserved residues in SePSK were identified to be essential for this function.", "section": "RESULTS", "ner": [ [ 6, 15, "conserved", "site" ], [ 28, 33, "SePSK", "protein" ] ] }, { "sid": 163, "sent": "Our results provide the detailed information about the interaction of SePSK with ATP and substrates.", "section": "RESULTS", "ner": [ [ 70, 75, "SePSK", "protein" ], [ 81, 84, "ATP", "chemical" ] ] }, { "sid": 164, "sent": "Moreover, structural superposition results enable us to visualize the conformational change of SePSK during the catalytic process.", "section": "RESULTS", "ner": [ [ 10, 34, "structural superposition", "experimental_method" ], [ 95, 100, "SePSK", "protein" ] ] }, { "sid": 165, "sent": "In conclusion, our results provide important information for a more detailed understanding of the mechanisms of SePSK and other members of FGGY family carbohydrate kinases.", "section": "RESULTS", "ner": [ [ 112, 117, "SePSK", "protein" ], [ 139, 171, "FGGY family carbohydrate kinases", "protein_type" ] ] } ] }, "PMC4832331": { "annotations": [ { "sid": 0, "sent": "Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA", "section": "TITLE", "ner": [ [ 29, 45, "Escherichia coli", "species" ], [ 46, 67, "lysine decarboxylases", "protein_type" ], [ 119, 130, "AAA+ ATPase", "protein_type" ], [ 131, 135, "RavA", "protein" ] ] }, { "sid": 1, "sent": "The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role.", "section": "ABSTRACT", "ner": [ [ 4, 13, "inducible", "protein_state" ], [ 14, 34, "lysine decarboxylase", "protein_type" ], [ 35, 39, "LdcI", "protein" ], [ 56, 71, "enterobacterial", "taxonomy_domain" ], [ 72, 99, "acid stress response enzyme", "protein_type" ], [ 108, 112, "LdcC", "protein" ] ] }, { "sid": 2, "sent": "A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation.", "section": "ABSTRACT", "ner": [ [ 43, 51, "decamers", "oligomeric_state" ], [ 59, 75, "Escherichia coli", "species" ], [ 76, 80, "LdcI", "protein" ], [ 90, 98, "hexamers", "oligomeric_state" ], [ 106, 117, "AAA+ ATPase", "protein_type" ], [ 118, 122, "RavA", "protein" ] ] }, { "sid": 3, "sent": "Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer.", "section": "ABSTRACT", "ner": [ [ 26, 44, "pseudoatomic model", "evidence" ], [ 52, 61, "LdcI-RavA", "complex_assembly" ], [ 80, 104, "cryo-electron microscopy", "experimental_method" ], [ 105, 108, "map", "evidence" ], [ 113, 131, "crystal structures", "evidence" ], [ 138, 146, "inactive", "protein_state" ], [ 147, 151, "LdcI", "protein" ], [ 152, 159, "decamer", "oligomeric_state" ], [ 166, 170, "RavA", "protein" ], [ 171, 178, "monomer", "oligomeric_state" ] ] }, { "sid": 4, "sent": "We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity.", "section": "ABSTRACT", "ner": [ [ 15, 39, "cryo-electron microscopy", "experimental_method" ], [ 40, 58, "3D reconstructions", "evidence" ], [ 66, 73, "E. coli", "species" ], [ 74, 78, "LdcI", "protein" ], [ 83, 87, "LdcC", "protein" ], [ 96, 108, "improved map", "evidence" ], [ 116, 120, "LdcI", "protein" ], [ 121, 129, "bound to", "protein_state" ], [ 134, 145, "LARA domain", "structure_element" ], [ 149, 153, "RavA", "protein" ], [ 158, 168, "pH optimal", "protein_state" ] ] }, { "sid": 5, "sent": "Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding.", "section": "ABSTRACT", "ner": [ [ 0, 10, "Comparison", "experimental_method" ], [ 46, 56, "structures", "evidence" ], [ 86, 90, "LdcI", "protein" ], [ 95, 99, "LdcC", "protein" ], [ 124, 151, "acid stress response enzyme", "protein_type" ], [ 174, 178, "RavA", "protein" ], [ 234, 246, "pH-dependent", "protein_state" ], [ 278, 282, "RavA", "protein" ] ] }, { "sid": 6, "sent": "Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions.", "section": "ABSTRACT", "ner": [ [ 0, 27, "Multiple sequence alignment", "experimental_method" ], [ 41, 62, "phylogenetic analysis", "experimental_method" ], [ 84, 98, "enterobacteria", "taxonomy_domain" ], [ 134, 154, "lysine decarboxylase", "protein_type" ], [ 191, 195, "RavA", "protein" ] ] }, { "sid": 7, "sent": "Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the \u03b1-family of pyridoxal-5\u2032-phosphate (PLP)-dependent enzymes.", "section": "INTRO", "ner": [ [ 0, 15, "Enterobacterial", "taxonomy_domain" ], [ 16, 25, "inducible", "protein_state" ], [ 26, 40, "decarboxylases", "protein_type" ], [ 44, 49, "basic", "protein_state" ], [ 50, 61, "amino acids", "chemical" ], [ 62, 68, "lysine", "residue_name" ], [ 70, 78, "arginine", "residue_name" ], [ 83, 92, "ornithine", "residue_name" ], [ 145, 153, "\u03b1-family", "protein_type" ], [ 157, 179, "pyridoxal-5\u2032-phosphate", "chemical" ], [ 181, 184, "PLP", "chemical" ] ] }, { "sid": 8, "sent": "They counteract acid stress experienced by the bacterium in the host digestive and urinary tract, and in particular in the extremely acidic stomach.", "section": "INTRO", "ner": [ [ 47, 56, "bacterium", "taxonomy_domain" ] ] }, { "sid": 9, "sent": "Each decarboxylase is induced by an excess of the target amino acid and a specific range of extracellular pH, and works in conjunction with a cognate inner membrane antiporter.", "section": "INTRO", "ner": [ [ 5, 18, "decarboxylase", "protein_type" ], [ 57, 67, "amino acid", "chemical" ], [ 150, 175, "inner membrane antiporter", "protein_type" ] ] }, { "sid": 10, "sent": "Decarboxylation of the amino acid into a polyamine is catalysed by a PLP cofactor in a multistep reaction that consumes a cytoplasmic proton and produces a CO2 molecule passively diffusing out of the cell, while the polyamine is excreted by the antiporter in exchange for a new amino acid substrate.", "section": "INTRO", "ner": [ [ 23, 33, "amino acid", "chemical" ], [ 41, 50, "polyamine", "chemical" ], [ 69, 72, "PLP", "chemical" ], [ 134, 140, "proton", "chemical" ], [ 156, 159, "CO2", "chemical" ], [ 216, 225, "polyamine", "chemical" ], [ 245, 255, "antiporter", "protein_type" ], [ 278, 288, "amino acid", "chemical" ] ] }, { "sid": 11, "sent": "Consequently, these enzymes buffer both the bacterial cytoplasm and the local extracellular environment.", "section": "INTRO", "ner": [ [ 44, 53, "bacterial", "taxonomy_domain" ] ] }, { "sid": 12, "sent": "These amino acid decarboxylases are therefore called acid stress inducible or biodegradative to distinguish them from their biosynthetic lysine and ornithine decarboxylase paralogs catalysing the same reaction but responsible for the polyamine production at neutral pH.", "section": "INTRO", "ner": [ [ 6, 31, "amino acid decarboxylases", "protein_type" ], [ 65, 74, "inducible", "protein_state" ], [ 78, 92, "biodegradative", "protein_state" ], [ 124, 136, "biosynthetic", "protein_state" ], [ 137, 171, "lysine and ornithine decarboxylase", "protein_type" ], [ 234, 243, "polyamine", "chemical" ], [ 258, 268, "neutral pH", "protein_state" ] ] }, { "sid": 13, "sent": "Inducible enterobacterial amino acid decarboxylases have been intensively studied since the early 1940 because the ability of bacteria to withstand acid stress can be linked to their pathogenicity in humans.", "section": "INTRO", "ner": [ [ 0, 9, "Inducible", "protein_state" ], [ 10, 25, "enterobacterial", "taxonomy_domain" ], [ 26, 51, "amino acid decarboxylases", "protein_type" ], [ 126, 134, "bacteria", "taxonomy_domain" ], [ 200, 206, "humans", "species" ] ] }, { "sid": 14, "sent": "In particular, the inducible lysine decarboxylase LdcI (or CadA) attracts attention due to its broad pH range of activity and its capacity to promote survival and growth of pathogenic enterobacteria such as Salmonella enterica serovar Typhimurium, Vibrio cholerae and Vibrio vulnificus under acidic conditions.", "section": "INTRO", "ner": [ [ 19, 28, "inducible", "protein_state" ], [ 29, 49, "lysine decarboxylase", "protein_type" ], [ 50, 54, "LdcI", "protein" ], [ 59, 63, "CadA", "protein" ], [ 95, 109, "broad pH range", "protein_state" ], [ 184, 198, "enterobacteria", "taxonomy_domain" ], [ 207, 246, "Salmonella enterica serovar Typhimurium", "species" ], [ 248, 263, "Vibrio cholerae", "species" ], [ 268, 285, "Vibrio vulnificus", "species" ] ] }, { "sid": 15, "sent": "Furthermore, both LdcI and the biosynthetic lysine decarboxylase LdcC of uropathogenic Escherichia coli (UPEC) appear to play an important role in increased resistance of this pathogen to nitrosative stress produced by nitric oxide and other damaging reactive nitrogen intermediates accumulating during the course of urinary tract infections (UTI).", "section": "INTRO", "ner": [ [ 18, 22, "LdcI", "protein" ], [ 31, 43, "biosynthetic", "protein_state" ], [ 44, 64, "lysine decarboxylase", "protein_type" ], [ 65, 69, "LdcC", "protein" ], [ 73, 103, "uropathogenic Escherichia coli", "species" ], [ 105, 109, "UPEC", "species" ], [ 219, 231, "nitric oxide", "chemical" ] ] }, { "sid": 16, "sent": "This effect is attributed to cadaverine, the diamine produced by decarboxylation of lysine by LdcI and LdcC, that was shown to enhance UPEC colonisation of the bladder.", "section": "INTRO", "ner": [ [ 29, 39, "cadaverine", "chemical" ], [ 84, 90, "lysine", "residue_name" ], [ 94, 98, "LdcI", "protein" ], [ 103, 107, "LdcC", "protein" ], [ 135, 139, "UPEC", "species" ] ] }, { "sid": 17, "sent": "In addition, the biosynthetic E. coli lysine decarboxylase LdcC, long thought to be constitutively expressed in low amounts, was demonstrated to be strongly upregulated by fluoroquinolones via their induction of RpoS. A direct correlation between the level of cadaverine and the resistance of E. coli to these antibiotics commonly used as a first-line treatment of UTI could be established.", "section": "INTRO", "ner": [ [ 17, 29, "biosynthetic", "protein_state" ], [ 30, 37, "E. coli", "species" ], [ 38, 58, "lysine decarboxylase", "protein_type" ], [ 59, 63, "LdcC", "protein" ], [ 172, 188, "fluoroquinolones", "chemical" ], [ 212, 216, "RpoS", "protein" ], [ 260, 270, "cadaverine", "chemical" ], [ 293, 300, "E. coli", "species" ] ] }, { "sid": 18, "sent": "Both acid pH and cadaverine induce closure of outer membrane porins thereby contributing to bacterial protection from acid stress, but also from certain antibiotics, by reduction in membrane permeability.", "section": "INTRO", "ner": [ [ 5, 12, "acid pH", "protein_state" ], [ 17, 27, "cadaverine", "chemical" ], [ 61, 67, "porins", "protein_type" ], [ 92, 101, "bacterial", "taxonomy_domain" ] ] }, { "sid": 19, "sent": "The crystal structure of the E. coli LdcI as well as its low resolution characterisation by electron microscopy (EM) showed that it is a decamer made of two pentameric rings.", "section": "INTRO", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 29, 36, "E. coli", "species" ], [ 37, 41, "LdcI", "protein" ], [ 92, 111, "electron microscopy", "experimental_method" ], [ 113, 115, "EM", "experimental_method" ], [ 137, 144, "decamer", "oligomeric_state" ], [ 157, 167, "pentameric", "oligomeric_state" ], [ 168, 173, "rings", "structure_element" ] ] }, { "sid": 20, "sent": "Each monomer is composed of three domains \u2013 an N-terminal wing domain (residues 1\u2013129), a PLP-binding core domain (residues 130\u2013563), and a C-terminal domain (CTD, residues 564\u2013715).", "section": "INTRO", "ner": [ [ 5, 12, "monomer", "oligomeric_state" ], [ 58, 69, "wing domain", "structure_element" ], [ 80, 85, "1\u2013129", "residue_range" ], [ 90, 113, "PLP-binding core domain", "structure_element" ], [ 124, 131, "130\u2013563", "residue_range" ], [ 140, 157, "C-terminal domain", "structure_element" ], [ 159, 162, "CTD", "structure_element" ], [ 173, 180, "564\u2013715", "residue_range" ] ] }, { "sid": 21, "sent": "Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery.", "section": "INTRO", "ner": [ [ 0, 8, "Monomers", "oligomeric_state" ], [ 37, 49, "core domains", "structure_element" ], [ 55, 73, "2-fold symmetrical", "protein_state" ], [ 74, 80, "dimers", "oligomeric_state" ], [ 99, 111, "active sites", "site" ], [ 133, 166, "toroidal D5-symmetrical structure", "structure_element" ], [ 179, 183, "wing", "structure_element" ], [ 188, 199, "core domain", "structure_element" ], [ 224, 236, "central pore", "structure_element" ], [ 247, 251, "CTDs", "structure_element" ] ] }, { "sid": 22, "sent": "Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response.", "section": "INTRO", "ner": [ [ 33, 40, "E. coli", "species" ], [ 41, 52, "AAA+ ATPase", "protein_type" ], [ 53, 57, "RavA", "protein" ], [ 130, 134, "LdcI", "protein" ], [ 169, 173, "LdcC", "protein" ], [ 203, 213, "pentameric", "oligomeric_state" ], [ 214, 219, "rings", "structure_element" ], [ 227, 231, "LdcI", "protein" ], [ 260, 269, "hexameric", "oligomeric_state" ], [ 270, 275, "rings", "structure_element" ], [ 279, 283, "RavA", "protein" ], [ 341, 350, "bacterium", "taxonomy_domain" ] ] }, { "sid": 23, "sent": "Furthermore, we recently solved the structure of the E. coli LdcI-RavA complex by cryo-electron microscopy (cryoEM) and combined it with the crystal structures of the individual proteins.", "section": "INTRO", "ner": [ [ 25, 45, "solved the structure", "experimental_method" ], [ 53, 60, "E. coli", "species" ], [ 61, 70, "LdcI-RavA", "complex_assembly" ], [ 82, 106, "cryo-electron microscopy", "experimental_method" ], [ 108, 114, "cryoEM", "experimental_method" ], [ 141, 159, "crystal structures", "evidence" ] ] }, { "sid": 24, "sent": "This allowed us to make a pseudoatomic model of the whole assembly, underpinned by a cryoEM map of the LdcI-LARA complex (with LARA standing for LdcI associating domain of RavA), and to identify conformational rearrangements and specific elements essential for complex formation.", "section": "INTRO", "ner": [ [ 26, 44, "pseudoatomic model", "evidence" ], [ 85, 91, "cryoEM", "experimental_method" ], [ 92, 95, "map", "evidence" ], [ 103, 112, "LdcI-LARA", "complex_assembly" ], [ 127, 131, "LARA", "structure_element" ], [ 145, 176, "LdcI associating domain of RavA", "structure_element" ] ] }, { "sid": 25, "sent": "The main determinants of the LdcI-RavA cage assembly appeared to be the N-terminal loop of the LARA domain of RavA and the C-terminal \u03b2-sheet of LdcI.", "section": "INTRO", "ner": [ [ 29, 38, "LdcI-RavA", "complex_assembly" ], [ 83, 87, "loop", "structure_element" ], [ 95, 106, "LARA domain", "structure_element" ], [ 110, 114, "RavA", "protein" ], [ 134, 141, "\u03b2-sheet", "structure_element" ], [ 145, 149, "LdcI", "protein" ] ] }, { "sid": 26, "sent": "In spite of this wealth of structural information, the fact that LdcC does not interact with RavA, although the two lysine decarboxylases are 69% identical and 84% similar, and the physiological significance of the absence of this interaction remained unexplored.", "section": "INTRO", "ner": [ [ 27, 49, "structural information", "evidence" ], [ 65, 69, "LdcC", "protein" ], [ 93, 97, "RavA", "protein" ], [ 116, 137, "lysine decarboxylases", "protein_type" ] ] }, { "sid": 27, "sent": "To solve this discrepancy, in the present work we provided a three-dimensional (3D) cryoEM reconstruction of LdcC and compared it with the available LdcI and LdcI-RavA structures.", "section": "INTRO", "ner": [ [ 84, 90, "cryoEM", "experimental_method" ], [ 91, 105, "reconstruction", "evidence" ], [ 109, 113, "LdcC", "protein" ], [ 149, 153, "LdcI", "protein" ], [ 158, 167, "LdcI-RavA", "complex_assembly" ], [ 168, 178, "structures", "evidence" ] ] }, { "sid": 28, "sent": "Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2).", "section": "INTRO", "ner": [ [ 15, 19, "LdcI", "protein" ], [ 20, 38, "crystal structures", "evidence" ], [ 56, 63, "high pH", "protein_state" ], [ 84, 92, "inactive", "protein_state" ], [ 94, 99, "LdcIi", "protein" ], [ 101, 107, "pH 8.5", "protein_state" ], [ 122, 128, "cryoEM", "experimental_method" ], [ 129, 144, "reconstructions", "evidence" ], [ 148, 157, "LdcI-RavA", "complex_assembly" ], [ 162, 171, "LdcI-LARA", "complex_assembly" ], [ 185, 202, "acidic pH optimal", "protein_state" ], [ 279, 296, "3D reconstruction", "evidence" ], [ 304, 308, "LdcI", "protein" ], [ 312, 321, "active pH", "protein_state" ], [ 323, 328, "LdcIa", "protein" ], [ 330, 336, "pH 6.2", "protein_state" ] ] }, { "sid": 29, "sent": "This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal \u03b2-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal \u03b2-sheets in question.", "section": "INTRO", "ner": [ [ 51, 65, "biodegradative", "protein_state" ], [ 74, 86, "biosynthetic", "protein_state" ], [ 87, 108, "lysine decarboxylases", "protein_type" ], [ 166, 178, "pH-dependent", "protein_state" ], [ 201, 205, "RavA", "protein" ], [ 240, 257, "RavA binding site", "site" ], [ 289, 296, "\u03b2-sheet", "structure_element" ], [ 300, 304, "LdcI", "protein" ], [ 364, 382, "LdcI-LdcC chimeras", "mutant" ], [ 392, 404, "interchanged", "experimental_method" ], [ 420, 428, "\u03b2-sheets", "structure_element" ] ] }, { "sid": 30, "sent": "Finally, we performed multiple sequence alignment of 22 lysine decarboxylases from Enterobacteriaceae containing the ravA-viaA operon in their genome.", "section": "INTRO", "ner": [ [ 22, 49, "multiple sequence alignment", "experimental_method" ], [ 56, 77, "lysine decarboxylases", "protein_type" ], [ 83, 101, "Enterobacteriaceae", "taxonomy_domain" ], [ 117, 133, "ravA-viaA operon", "gene" ] ] }, { "sid": 31, "sent": "Remarkably, this analysis revealed that several specific residues in the above-mentioned \u03b2-sheet, independently of the rest of the protein sequence, are sufficient to define if a particular lysine decarboxylase should be classified as an \u201cLdcC-like\u201d or an \u201cLdcI-like\u201d.", "section": "INTRO", "ner": [ [ 48, 65, "specific residues", "structure_element" ], [ 89, 96, "\u03b2-sheet", "structure_element" ], [ 190, 210, "lysine decarboxylase", "protein_type" ], [ 239, 248, "LdcC-like", "protein_type" ], [ 257, 266, "LdcI-like", "protein_type" ] ] }, { "sid": 32, "sent": "This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA.", "section": "INTRO", "ner": [ [ 56, 60, "RavA", "protein" ], [ 103, 118, "enterobacterial", "taxonomy_domain" ], [ 119, 139, "lysine decarboxylase", "protein_type" ], [ 156, 183, "high degree of conservation", "protein_state" ], [ 192, 214, "small structural motif", "structure_element" ], [ 279, 293, "biodegradative", "protein_state" ], [ 294, 309, "enterobacterial", "taxonomy_domain" ], [ 310, 331, "lysine decarboxylases", "protein_type" ], [ 340, 351, "AAA+ ATPase", "protein_type" ], [ 352, 356, "RavA", "protein" ] ] }, { "sid": 33, "sent": "CryoEM 3D reconstructions of LdcC, LdcIa and LdcI-LARA", "section": "RESULTS", "ner": [ [ 0, 6, "CryoEM", "experimental_method" ], [ 7, 25, "3D reconstructions", "evidence" ], [ 29, 33, "LdcC", "protein" ], [ 35, 40, "LdcIa", "protein" ], [ 45, 54, "LdcI-LARA", "complex_assembly" ] ] }, { "sid": 34, "sent": "In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity \u2013 a 5.5\u2009\u00c5 resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1\u2009\u00c5 resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2).", "section": "RESULTS", "ner": [ [ 73, 79, "cryoEM", "experimental_method" ], [ 80, 95, "reconstructions", "evidence" ], [ 103, 110, "E. coli", "species" ], [ 111, 132, "lysine decarboxylases", "protein_type" ], [ 136, 146, "pH optimal", "protein_state" ], [ 197, 203, "cryoEM", "experimental_method" ], [ 204, 207, "map", "evidence" ], [ 215, 219, "LdcC", "protein" ], [ 221, 227, "pH 7.5", "protein_state" ], [ 341, 347, "cryoEM", "experimental_method" ], [ 348, 351, "map", "evidence" ], [ 359, 364, "LdcIa", "protein" ], [ 367, 373, "pH 6.2", "protein_state" ] ] }, { "sid": 35, "sent": "In addition, we improved our earlier cryoEM map of the LdcI-LARA complex from 7.5\u2009\u00c5 to 6.2\u2009\u00c5 resolution (Figs 1E,F and S3).", "section": "RESULTS", "ner": [ [ 37, 43, "cryoEM", "experimental_method" ], [ 44, 47, "map", "evidence" ], [ 55, 64, "LdcI-LARA", "complex_assembly" ] ] }, { "sid": 36, "sent": "Based on these reconstructions, reliable pseudoatomic models of the three assemblies were obtained by flexible fitting of either the crystal structure of LdcIi or a derived structural homology model of LdcC (Table S1).", "section": "RESULTS", "ner": [ [ 15, 30, "reconstructions", "evidence" ], [ 41, 60, "pseudoatomic models", "evidence" ], [ 102, 121, "flexible fitting of", "experimental_method" ], [ 133, 150, "crystal structure", "evidence" ], [ 154, 159, "LdcIi", "protein" ], [ 173, 198, "structural homology model", "experimental_method" ], [ 202, 206, "LdcC", "protein" ] ] }, { "sid": 37, "sent": "Significant differences between these pseudoatomic models can be interpreted as movements between specific biological states of the proteins as described below.", "section": "RESULTS", "ner": [ [ 38, 57, "pseudoatomic models", "evidence" ] ] }, { "sid": 38, "sent": "The wing domains as a stable anchor at the center of the double-ring", "section": "RESULTS", "ner": [ [ 4, 16, "wing domains", "structure_element" ], [ 57, 68, "double-ring", "structure_element" ] ] }, { "sid": 39, "sent": "As a first step of a comparative analysis, we superimposed the three cryoEM reconstructions (LdcIa, LdcI-LARA and LdcC) and the crystal structure of the LdcIi decamer (Fig. 2 and Movie S1).", "section": "RESULTS", "ner": [ [ 46, 58, "superimposed", "experimental_method" ], [ 69, 75, "cryoEM", "experimental_method" ], [ 76, 91, "reconstructions", "evidence" ], [ 93, 98, "LdcIa", "protein" ], [ 100, 109, "LdcI-LARA", "complex_assembly" ], [ 114, 118, "LdcC", "protein" ], [ 128, 145, "crystal structure", "evidence" ], [ 153, 158, "LdcIi", "protein" ], [ 159, 166, "decamer", "oligomeric_state" ] ] }, { "sid": 40, "sent": "This superposition reveals that the densities lining the central hole of the toroid are roughly at the same location, while the rest of the structure exhibits noticeable changes.", "section": "RESULTS", "ner": [ [ 5, 18, "superposition", "experimental_method" ], [ 36, 45, "densities", "evidence" ], [ 57, 69, "central hole", "structure_element" ], [ 77, 83, "toroid", "structure_element" ], [ 140, 149, "structure", "evidence" ] ] }, { "sid": 41, "sent": "Specifically, at the center of the double-ring the wing domains of the subunits provide the conserved basis for the assembly with the lowest root mean square deviation (RMSD) (between 1.4 and 2\u2009\u00c5 for the C\u03b1 atoms only), whereas the peripheral CTDs containing the RavA binding interface manifest the highest RMSD (up to 4.2\u2009\u00c5) (Table S2).", "section": "RESULTS", "ner": [ [ 35, 46, "double-ring", "structure_element" ], [ 51, 63, "wing domains", "structure_element" ], [ 92, 101, "conserved", "protein_state" ], [ 134, 167, "lowest root mean square deviation", "evidence" ], [ 169, 173, "RMSD", "evidence" ], [ 243, 247, "CTDs", "structure_element" ], [ 263, 285, "RavA binding interface", "site" ], [ 307, 311, "RMSD", "evidence" ] ] }, { "sid": 42, "sent": "In addition, the wing domains of all structures are very similar, with the RMSD after optimal rigid body alignment (RMSDmin) less than 1.1\u2009\u00c5. Thus, taking the limited resolution of the cryoEM maps into account, we consider that the wing domains of all the four structures are essentially identical and that in the present study the RMSD of less than 2\u2009\u00c5 can serve as a baseline below which differences may be assumed as insignificant.", "section": "RESULTS", "ner": [ [ 17, 29, "wing domains", "structure_element" ], [ 37, 47, "structures", "evidence" ], [ 75, 79, "RMSD", "evidence" ], [ 116, 123, "RMSDmin", "evidence" ], [ 185, 191, "cryoEM", "experimental_method" ], [ 192, 196, "maps", "evidence" ], [ 232, 244, "wing domains", "structure_element" ], [ 261, 271, "structures", "evidence" ], [ 332, 336, "RMSD", "evidence" ] ] }, { "sid": 43, "sent": "This preservation of the central part of the double-ring assembly may help the enzymes to maintain their decameric state upon activation and incorporation into the LdcI-RavA cage.", "section": "RESULTS", "ner": [ [ 25, 37, "central part", "structure_element" ], [ 105, 114, "decameric", "oligomeric_state" ], [ 164, 173, "LdcI-RavA", "complex_assembly" ] ] }, { "sid": 44, "sent": "The core domain and the active site rearrangements upon pH-dependent enzyme activation and LARA binding", "section": "RESULTS", "ner": [ [ 4, 15, "core domain", "structure_element" ], [ 24, 35, "active site", "site" ], [ 56, 68, "pH-dependent", "protein_state" ] ] }, { "sid": 45, "sent": "Both visual inspection (Fig. 2) and RMSD calculations (Table S2) show that globally the three structures at active pH (LdcIa, LdcI-LARA and LdcC) are more similar to each other than to the structure determined at high pH conditions (LdcIi).", "section": "RESULTS", "ner": [ [ 5, 22, "visual inspection", "experimental_method" ], [ 36, 53, "RMSD calculations", "experimental_method" ], [ 94, 104, "structures", "evidence" ], [ 108, 117, "active pH", "protein_state" ], [ 119, 124, "LdcIa", "protein" ], [ 126, 135, "LdcI-LARA", "complex_assembly" ], [ 140, 144, "LdcC", "protein" ], [ 213, 220, "high pH", "protein_state" ], [ 233, 238, "LdcIi", "protein" ] ] }, { "sid": 46, "sent": "The decameric enzyme is built of five dimers associating into a 5-fold symmetrical double-ring (two monomers making a dimer are delineated in Fig. 1).", "section": "RESULTS", "ner": [ [ 4, 13, "decameric", "oligomeric_state" ], [ 38, 44, "dimers", "oligomeric_state" ], [ 64, 94, "5-fold symmetrical double-ring", "structure_element" ], [ 100, 108, "monomers", "oligomeric_state" ], [ 118, 123, "dimer", "oligomeric_state" ] ] }, { "sid": 47, "sent": "As common for the \u03b1 family of the PLP-dependent decarboxylases, dimerization is required for the enzymatic activity because the active site is buried in the dimer interface (Fig. 3A,B).", "section": "RESULTS", "ner": [ [ 18, 26, "\u03b1 family", "protein_type" ], [ 34, 62, "PLP-dependent decarboxylases", "protein_type" ], [ 128, 139, "active site", "site" ], [ 157, 172, "dimer interface", "site" ] ] }, { "sid": 48, "sent": "This interface is formed essentially by the core domains with some contribution of the CTDs.", "section": "RESULTS", "ner": [ [ 5, 14, "interface", "site" ], [ 44, 56, "core domains", "structure_element" ], [ 87, 91, "CTDs", "structure_element" ] ] }, { "sid": 49, "sent": "The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184\u2013417) flanked by two smaller subdomains rich in partly disordered loops \u2013 the linker region (residues 130\u2013183) and the subdomain 4 (residues 418\u2013563).", "section": "RESULTS", "ner": [ [ 4, 15, "core domain", "structure_element" ], [ 32, 53, "PLP-binding subdomain", "structure_element" ], [ 55, 61, "PLP-SD", "structure_element" ], [ 72, 79, "184\u2013417", "residue_range" ], [ 104, 114, "subdomains", "structure_element" ], [ 123, 140, "partly disordered", "protein_state" ], [ 141, 146, "loops", "structure_element" ], [ 153, 166, "linker region", "structure_element" ], [ 177, 184, "130\u2013183", "residue_range" ], [ 194, 205, "subdomain 4", "structure_element" ], [ 216, 223, "418\u2013563", "residue_range" ] ] }, { "sid": 50, "sent": "Zooming in the variations in the PLP-SD shows that most of the structural changes concern displacements in the active site (Fig. 3C\u2013F).", "section": "RESULTS", "ner": [ [ 33, 39, "PLP-SD", "structure_element" ], [ 111, 122, "active site", "site" ] ] }, { "sid": 51, "sent": "The most conspicuous differences between the PLP-SDs can be linked to the pH-dependent activation of the enzymes.", "section": "RESULTS", "ner": [ [ 45, 52, "PLP-SDs", "structure_element" ], [ 74, 86, "pH-dependent", "protein_state" ] ] }, { "sid": 52, "sent": "The resolution of the cryoEM maps does not allow modeling the position of the PLP moiety and calls for caution in detailed mechanistic interpretations in terms of individual amino acids.", "section": "RESULTS", "ner": [ [ 22, 28, "cryoEM", "experimental_method" ], [ 29, 33, "maps", "evidence" ], [ 78, 81, "PLP", "chemical" ], [ 174, 185, "amino acids", "chemical" ] ] }, { "sid": 53, "sent": "In particular, transition from LdcIi to LdcI-LARA involves ~3.5\u2009\u00c5 and ~4.5\u2009\u00c5 shifts away from the 5-fold axis in the active site \u03b1-helices spanning residues 218\u2013232 and 246\u2013254 respectively (Fig. 3C\u2013E).", "section": "RESULTS", "ner": [ [ 31, 36, "LdcIi", "protein" ], [ 40, 49, "LdcI-LARA", "complex_assembly" ], [ 117, 128, "active site", "site" ], [ 129, 138, "\u03b1-helices", "structure_element" ], [ 157, 164, "218\u2013232", "residue_range" ], [ 169, 176, "246\u2013254", "residue_range" ] ] }, { "sid": 54, "sent": "Between these two extremes, the PLP-SDs of LdcIa and LdcC are similar both in the context of the decamer (Fig. 3F) and in terms of RMSDmin\u2009=\u20090.9\u2009\u00c5, which probably reflects the fact that, at the optimal pH, these lysine decarboxylases have a similar enzymatic activity.", "section": "RESULTS", "ner": [ [ 32, 39, "PLP-SDs", "structure_element" ], [ 43, 48, "LdcIa", "protein" ], [ 53, 57, "LdcC", "protein" ], [ 97, 104, "decamer", "oligomeric_state" ], [ 131, 138, "RMSDmin", "evidence" ], [ 194, 204, "optimal pH", "protein_state" ], [ 212, 233, "lysine decarboxylases", "protein_type" ] ] }, { "sid": 55, "sent": "In addition, our earlier biochemical observation that the enzymatic activity of LdcIa is unaffected by RavA binding is consistent with the relatively small changes undergone by the active site upon transition from LdcIa to LdcI-LARA.", "section": "RESULTS", "ner": [ [ 25, 48, "biochemical observation", "experimental_method" ], [ 80, 85, "LdcIa", "protein" ], [ 103, 107, "RavA", "protein" ], [ 181, 192, "active site", "site" ], [ 214, 219, "LdcIa", "protein" ], [ 223, 232, "LdcI-LARA", "complex_assembly" ] ] }, { "sid": 56, "sent": "Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.).", "section": "RESULTS", "ner": [ [ 47, 64, "crystal structure", "evidence" ], [ 68, 73, "LdcIi", "protein" ], [ 91, 100, "inducible", "protein_state" ], [ 101, 123, "arginine decarboxylase", "protein_type" ], [ 124, 128, "AdiA", "protein" ], [ 138, 155, "high conservation", "protein_state" ], [ 163, 188, "PLP-coordinating residues", "site" ], [ 206, 242, "patch of negatively charged residues", "site" ], [ 254, 273, "active site channel", "site" ], [ 289, 301, "binding site", "site" ], [ 317, 327, "amino acid", "chemical" ] ] }, { "sid": 57, "sent": "Rearrangements of the ppGpp binding pocket upon pH-dependent enzyme activation and LARA binding", "section": "RESULTS", "ner": [ [ 22, 42, "ppGpp binding pocket", "site" ], [ 48, 60, "pH-dependent", "protein_state" ], [ 83, 87, "LARA", "structure_element" ] ] }, { "sid": 58, "sent": "An inhibitor of the LdcI and LdcC activity, the stringent response alarmone ppGpp, is known to bind at the interface between neighboring monomers within each ring (Fig. S4).", "section": "RESULTS", "ner": [ [ 20, 24, "LdcI", "protein" ], [ 29, 33, "LdcC", "protein" ], [ 48, 75, "stringent response alarmone", "chemical" ], [ 76, 81, "ppGpp", "chemical" ], [ 107, 116, "interface", "site" ], [ 137, 145, "monomers", "oligomeric_state" ], [ 158, 162, "ring", "structure_element" ] ] }, { "sid": 59, "sent": "The ppGpp binding pocket is made up by residues from all domains and is located approximately 30\u2009\u00c5 away from the PLP moiety.", "section": "RESULTS", "ner": [ [ 4, 24, "ppGpp binding pocket", "site" ], [ 113, 116, "PLP", "chemical" ] ] }, { "sid": 60, "sent": "Whereas the crystal structure of the ppGpp-LdcIi was solved to 2\u2009\u00c5 resolution, only a 4.1\u2009\u00c5 resolution structure of the ppGpp-free LdcIi could be obtained.", "section": "RESULTS", "ner": [ [ 12, 29, "crystal structure", "evidence" ], [ 37, 48, "ppGpp-LdcIi", "complex_assembly" ], [ 53, 59, "solved", "experimental_method" ], [ 103, 112, "structure", "evidence" ], [ 120, 130, "ppGpp-free", "protein_state" ], [ 131, 136, "LdcIi", "protein" ] ] }, { "sid": 61, "sent": "At this resolution, the apo-LdcIi and ppGpp-LdcIi structures (both solved at pH 8.5) appeared indistinguishable except for the presence of ppGpp (Fig. S11 in ref. ).", "section": "RESULTS", "ner": [ [ 24, 27, "apo", "protein_state" ], [ 28, 33, "LdcIi", "protein" ], [ 38, 49, "ppGpp-LdcIi", "complex_assembly" ], [ 50, 60, "structures", "evidence" ], [ 77, 83, "pH 8.5", "protein_state" ], [ 139, 144, "ppGpp", "chemical" ] ] }, { "sid": 62, "sent": "Thus, we speculated that inhibition of LdcI by ppGpp would be accompanied by a transduction of subtle structural changes at the level of individual amino acid side chains between the ppGpp binding pocket and the active site of the enzyme.", "section": "RESULTS", "ner": [ [ 39, 43, "LdcI", "protein" ], [ 47, 52, "ppGpp", "chemical" ], [ 148, 158, "amino acid", "chemical" ], [ 183, 203, "ppGpp binding pocket", "site" ], [ 212, 223, "active site", "site" ] ] }, { "sid": 63, "sent": "All our current cryoEM reconstructions of the lysine decarboxylases were obtained in the absence of ppGpp in order to be closer to the active state of the enzymes under study.", "section": "RESULTS", "ner": [ [ 16, 22, "cryoEM", "experimental_method" ], [ 23, 38, "reconstructions", "evidence" ], [ 46, 67, "lysine decarboxylases", "protein_type" ], [ 89, 99, "absence of", "protein_state" ], [ 100, 105, "ppGpp", "chemical" ], [ 135, 141, "active", "protein_state" ] ] }, { "sid": 64, "sent": "While differences in the ppGpp binding site could indeed be visualized (Fig. S4), the level of resolution warns against speculations about their significance.", "section": "RESULTS", "ner": [ [ 25, 43, "ppGpp binding site", "site" ] ] }, { "sid": 65, "sent": "The fact that interaction with RavA reduces the ppGpp affinity for LdcI despite the long distance of ~30\u2009\u00c5 between the LARA domain binding site and the closest ppGpp binding pocket (Fig. S5) seems to favor an allosteric regulation mechanism.", "section": "RESULTS", "ner": [ [ 31, 35, "RavA", "protein" ], [ 48, 53, "ppGpp", "chemical" ], [ 67, 71, "LdcI", "protein" ], [ 119, 143, "LARA domain binding site", "site" ], [ 160, 180, "ppGpp binding pocket", "site" ] ] }, { "sid": 66, "sent": "Interestingly, although a number of ppGpp binding residues are strictly conserved between LdcI and AdiA that also forms decamers at low pH optimal for its arginine decarboxylase activity, no ppGpp regulation of AdiA could be demonstrated.", "section": "RESULTS", "ner": [ [ 36, 58, "ppGpp binding residues", "site" ], [ 63, 81, "strictly conserved", "protein_state" ], [ 90, 94, "LdcI", "protein" ], [ 99, 103, "AdiA", "protein" ], [ 120, 128, "decamers", "oligomeric_state" ], [ 132, 146, "low pH optimal", "protein_state" ], [ 155, 177, "arginine decarboxylase", "protein_type" ], [ 191, 196, "ppGpp", "chemical" ], [ 211, 215, "AdiA", "protein" ] ] }, { "sid": 67, "sent": "Swinging and stretching of the CTDs upon pH-dependent LdcI activation and LARA binding", "section": "RESULTS", "ner": [ [ 31, 35, "CTDs", "structure_element" ], [ 41, 53, "pH-dependent", "protein_state" ], [ 54, 58, "LdcI", "protein" ], [ 74, 78, "LARA", "structure_element" ] ] }, { "sid": 68, "sent": "Inspection of the superimposed decameric structures (Figs 2 and S6) suggests a depiction of the wing domains as an anchor around which the peripheral CTDs swing.", "section": "RESULTS", "ner": [ [ 18, 30, "superimposed", "experimental_method" ], [ 31, 40, "decameric", "oligomeric_state" ], [ 41, 51, "structures", "evidence" ], [ 96, 108, "wing domains", "structure_element" ], [ 150, 154, "CTDs", "structure_element" ] ] }, { "sid": 69, "sent": "This swinging movement seems to be mediated by the core domains and is accompanied by a stretching of the whole LdcI subunits attracted by the RavA magnets.", "section": "RESULTS", "ner": [ [ 51, 63, "core domains", "structure_element" ], [ 112, 116, "LdcI", "protein" ], [ 117, 125, "subunits", "structure_element" ], [ 143, 147, "RavA", "protein" ] ] }, { "sid": 70, "sent": "Indeed, all CTDs have very similar structures (RMSDmin <1\u2009\u00c5).", "section": "RESULTS", "ner": [ [ 12, 16, "CTDs", "structure_element" ], [ 47, 54, "RMSDmin", "evidence" ] ] }, { "sid": 71, "sent": "Yet the superposition of the decamers lays bare a progressive movement of the CTD as a whole upon enzyme activation by pH and the binding of LARA.", "section": "RESULTS", "ner": [ [ 8, 21, "superposition", "experimental_method" ], [ 29, 37, "decamers", "oligomeric_state" ], [ 78, 81, "CTD", "structure_element" ], [ 141, 145, "LARA", "structure_element" ] ] }, { "sid": 72, "sent": "The LdcIi monomer is the most compact, whereas LdcIa and especially LdcI-LARA gradually extend their CTDs towards the LARA domain of RavA (Figs 2 and 4).", "section": "RESULTS", "ner": [ [ 4, 9, "LdcIi", "protein" ], [ 10, 17, "monomer", "oligomeric_state" ], [ 25, 37, "most compact", "protein_state" ], [ 47, 52, "LdcIa", "protein" ], [ 68, 77, "LdcI-LARA", "complex_assembly" ], [ 78, 94, "gradually extend", "protein_state" ], [ 101, 105, "CTDs", "structure_element" ], [ 118, 129, "LARA domain", "structure_element" ], [ 133, 137, "RavA", "protein" ] ] }, { "sid": 73, "sent": "These small but noticeable swinging and stretching (up to ~4\u2009\u00c5) may be related to the incorporation of the LdcI decamer into the LdcI-RavA cage.", "section": "RESULTS", "ner": [ [ 107, 111, "LdcI", "protein" ], [ 112, 119, "decamer", "oligomeric_state" ], [ 129, 138, "LdcI-RavA", "complex_assembly" ] ] }, { "sid": 74, "sent": "The C-terminal \u03b2-sheet of a lysine decarboxylase as a major determinant of the interaction with RavA", "section": "RESULTS", "ner": [ [ 15, 22, "\u03b2-sheet", "structure_element" ], [ 28, 48, "lysine decarboxylase", "protein_type" ], [ 96, 100, "RavA", "protein" ] ] }, { "sid": 75, "sent": "In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded \u03b2-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases.", "section": "RESULTS", "ner": [ [ 54, 59, "LdcIi", "protein" ], [ 68, 72, "LARA", "structure_element" ], [ 73, 91, "crystal structures", "evidence" ], [ 101, 110, "LdcI-LARA", "complex_assembly" ], [ 111, 117, "cryoEM", "experimental_method" ], [ 118, 125, "density", "evidence" ], [ 149, 158, "LdcI-RavA", "complex_assembly" ], [ 201, 221, "two-stranded \u03b2-sheet", "structure_element" ], [ 229, 233, "LdcI", "protein" ], [ 247, 253, "cryoEM", "experimental_method" ], [ 254, 258, "maps", "evidence" ], [ 263, 282, "pseudoatomic models", "evidence" ], [ 355, 364, "inducible", "protein_state" ], [ 373, 385, "constitutive", "protein_state" ], [ 386, 407, "lysine decarboxylases", "protein_type" ] ] }, { "sid": 76, "sent": "Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant \u03b2-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal \u03b2-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal \u03b2-sheet of LdcI) (Fig. 5A\u2013C).", "section": "RESULTS", "ner": [ [ 137, 141, "RavA", "protein" ], [ 159, 166, "swapped", "experimental_method" ], [ 180, 188, "\u03b2-sheets", "structure_element" ], [ 228, 236, "chimeras", "mutant" ], [ 245, 250, "LdcIC", "mutant" ], [ 257, 261, "LdcI", "protein" ], [ 282, 289, "\u03b2-sheet", "structure_element" ], [ 293, 297, "LdcC", "protein" ], [ 303, 308, "LdcCI", "mutant" ], [ 315, 319, "LdcC", "protein" ], [ 340, 347, "\u03b2-sheet", "structure_element" ], [ 351, 355, "LdcI", "protein" ] ] }, { "sid": 77, "sent": "Both constructs could be purified and could form decamers visually indistinguishable from the wild-type proteins.", "section": "RESULTS", "ner": [ [ 0, 15, "Both constructs", "mutant" ], [ 49, 57, "decamers", "oligomeric_state" ], [ 94, 103, "wild-type", "protein_state" ] ] }, { "sid": 78, "sent": "As expected, binding of LdcI to RavA was completely abolished by this procedure and no LdcIC-RavA complex could be detected.", "section": "RESULTS", "ner": [ [ 24, 28, "LdcI", "protein" ], [ 32, 36, "RavA", "protein" ], [ 87, 97, "LdcIC-RavA", "complex_assembly" ] ] }, { "sid": 79, "sent": "On the contrary, introduction of the C-terminal \u03b2-sheet of LdcI into LdcC led to an assembly of the LdcCI-RavA complex.", "section": "RESULTS", "ner": [ [ 17, 29, "introduction", "experimental_method" ], [ 48, 55, "\u03b2-sheet", "structure_element" ], [ 59, 63, "LdcI", "protein" ], [ 69, 73, "LdcC", "protein" ], [ 100, 110, "LdcCI-RavA", "complex_assembly" ] ] }, { "sid": 80, "sent": "On the negative stain EM grid, the chimeric cages appeared less rigid than the native LdcI-RavA, which probably means that the environment of the \u03b2-sheet contributes to the efficiency of the interaction and the stability of the entire architecture (Fig. 5D\u2013F).", "section": "RESULTS", "ner": [ [ 7, 29, "negative stain EM grid", "experimental_method" ], [ 35, 43, "chimeric", "protein_state" ], [ 79, 85, "native", "protein_state" ], [ 86, 95, "LdcI-RavA", "complex_assembly" ], [ 146, 153, "\u03b2-sheet", "structure_element" ] ] }, { "sid": 81, "sent": "The C-terminal \u03b2-sheet of a lysine decarboxylase is a highly conserved signature allowing to distinguish between LdcI and LdcC", "section": "RESULTS", "ner": [ [ 15, 22, "\u03b2-sheet", "structure_element" ], [ 28, 48, "lysine decarboxylase", "protein_type" ], [ 54, 70, "highly conserved", "protein_state" ], [ 113, 117, "LdcI", "protein" ], [ 122, 126, "LdcC", "protein" ] ] }, { "sid": 82, "sent": "Alignment of the primary sequences of the E. coli LdcI and LdcC shows that some amino acid residues of the C-terminal \u03b2-sheet are the same in the two proteins, whereas others are notably different in chemical nature.", "section": "RESULTS", "ner": [ [ 0, 34, "Alignment of the primary sequences", "experimental_method" ], [ 42, 49, "E. coli", "species" ], [ 50, 54, "LdcI", "protein" ], [ 59, 63, "LdcC", "protein" ], [ 118, 125, "\u03b2-sheet", "structure_element" ] ] }, { "sid": 83, "sent": "Importantly, most of the amino acid differences between the two enzymes are located in this very region.", "section": "RESULTS", "ner": [ [ 92, 103, "very region", "structure_element" ] ] }, { "sid": 84, "sent": "Thus, to advance beyond our experimental confirmation of the C-terminal \u03b2-sheet as a major determinant of the capacity of a particular lysine decarboxylase to form a cage with RavA, we set out to investigate whether certain residues in this \u03b2-sheet are conserved in lysine decarboxylases of different enterobacteria that have the ravA-viaA operon in their genome.", "section": "RESULTS", "ner": [ [ 72, 79, "\u03b2-sheet", "structure_element" ], [ 135, 155, "lysine decarboxylase", "protein_type" ], [ 176, 180, "RavA", "protein" ], [ 216, 232, "certain residues", "structure_element" ], [ 241, 248, "\u03b2-sheet", "structure_element" ], [ 253, 262, "conserved", "protein_state" ], [ 266, 287, "lysine decarboxylases", "protein_type" ], [ 301, 315, "enterobacteria", "taxonomy_domain" ], [ 330, 346, "ravA-viaA operon", "gene" ] ] }, { "sid": 85, "sent": "We inspected the genetic environment of lysine decarboxylases from 22 enterobacterial species referenced in the NCBI database, corrected the gene annotation where necessary (Tables S3 and S4), and performed multiple sequence alignment coupled to a phylogenetic analysis (see Methods).", "section": "RESULTS", "ner": [ [ 3, 36, "inspected the genetic environment", "experimental_method" ], [ 40, 61, "lysine decarboxylases", "protein_type" ], [ 70, 85, "enterobacterial", "taxonomy_domain" ], [ 207, 234, "multiple sequence alignment", "experimental_method" ], [ 248, 269, "phylogenetic analysis", "experimental_method" ] ] }, { "sid": 86, "sent": "First of all, consensus sequence for the entire lysine decarboxylase family was derived.", "section": "RESULTS", "ner": [ [ 14, 32, "consensus sequence", "evidence" ], [ 48, 68, "lysine decarboxylase", "protein_type" ] ] }, { "sid": 87, "sent": "Second, the phylogenetic analysis clearly split the lysine decarboxylases into two groups (Fig. 6A).", "section": "RESULTS", "ner": [ [ 12, 33, "phylogenetic analysis", "experimental_method" ], [ 52, 73, "lysine decarboxylases", "protein_type" ] ] }, { "sid": 88, "sent": "All lysine decarboxylases predicted to be \u201cLdcI-like\u201d or biodegradable based on their genetic environment, as for example their organization in an operon with a gene encoding the CadB antiporter (see Methods), were found in one group, whereas all enzymes predicted as \u201cLdcC-like\u201d or biosynthetic partitioned into another group.", "section": "RESULTS", "ner": [ [ 4, 25, "lysine decarboxylases", "protein_type" ], [ 43, 52, "LdcI-like", "protein_type" ], [ 57, 70, "biodegradable", "protein_state" ], [ 179, 183, "CadB", "protein" ], [ 184, 194, "antiporter", "protein_type" ], [ 247, 254, "enzymes", "protein_type" ], [ 269, 278, "LdcC-like", "protein_type" ], [ 283, 295, "biosynthetic", "protein_state" ] ] }, { "sid": 89, "sent": "Thus, consensus sequences could also be determined for each of the two groups (Figs 6B,C and S7).", "section": "RESULTS", "ner": [ [ 6, 25, "consensus sequences", "evidence" ] ] }, { "sid": 90, "sent": "Inspection of these consensus sequences revealed important differences between the groups regarding charge, size and hydrophobicity of several residues precisely at the level of the C-terminal \u03b2-sheet that is responsible for the interaction with RavA (Fig. 6B\u2013D).", "section": "RESULTS", "ner": [ [ 20, 39, "consensus sequences", "evidence" ], [ 193, 200, "\u03b2-sheet", "structure_element" ], [ 246, 250, "RavA", "protein" ] ] }, { "sid": 91, "sent": "For example, in our previous study, site-directed mutations identified Y697 as critically required for the RavA binding.", "section": "RESULTS", "ner": [ [ 36, 59, "site-directed mutations", "experimental_method" ], [ 71, 75, "Y697", "residue_name_number" ], [ 107, 111, "RavA", "protein" ] ] }, { "sid": 92, "sent": "Our current analysis shows that Y697 is strictly conserved in the \u201cLdcI-like\u201d group whereas the \u201cLdcC-like\u201d enzymes always have a lysine in this position; it also uncovers several other residues potentially essential for the interaction with RavA which can now be addressed by site-directed mutagenesis.", "section": "RESULTS", "ner": [ [ 32, 36, "Y697", "residue_name_number" ], [ 40, 58, "strictly conserved", "protein_state" ], [ 67, 76, "LdcI-like", "protein_type" ], [ 97, 106, "LdcC-like", "protein_type" ], [ 116, 127, "always have", "protein_state" ], [ 130, 136, "lysine", "residue_name" ], [ 242, 246, "RavA", "protein" ], [ 277, 302, "site-directed mutagenesis", "experimental_method" ] ] }, { "sid": 93, "sent": "The third and most remarkable finding was that exactly the same separation into \u201cLdcI-like\u201d and \u201cLdcC\u201d-like groups can be obtained based on a comparison of the C-terminal \u03b2-sheets only, without taking the rest of the primary sequence into account.", "section": "RESULTS", "ner": [ [ 81, 90, "LdcI-like", "protein_type" ], [ 97, 107, "LdcC\u201d-like", "protein_type" ], [ 171, 179, "\u03b2-sheets", "structure_element" ] ] }, { "sid": 94, "sent": "Therefore the C-terminal \u03b2-sheet emerges as being a highly conserved signature sequence, sufficient to unambiguously discriminate between the \u201cLdcI-like\u201d and \u201cLdcC-like\u201d enterobacterial lysine decarboxylases independently of any other information (Figs 6 and S7).", "section": "RESULTS", "ner": [ [ 25, 32, "\u03b2-sheet", "structure_element" ], [ 52, 68, "highly conserved", "protein_state" ], [ 69, 87, "signature sequence", "structure_element" ], [ 143, 152, "LdcI-like", "protein_type" ], [ 159, 168, "LdcC-like", "protein_type" ], [ 170, 185, "enterobacterial", "taxonomy_domain" ], [ 186, 207, "lysine decarboxylases", "protein_type" ] ] }, { "sid": 95, "sent": "Our structures show that this motif is not involved in the enzymatic activity or the oligomeric state of the proteins.", "section": "RESULTS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 25, 35, "this motif", "structure_element" ] ] }, { "sid": 96, "sent": "Thus, enterobacteria identified here (Fig. 6, Table S4) appear to exert evolutionary pressure on the biodegradative lysine decarboxylase towards the RavA binding.", "section": "RESULTS", "ner": [ [ 6, 20, "enterobacteria", "taxonomy_domain" ], [ 101, 115, "biodegradative", "protein_state" ], [ 116, 136, "lysine decarboxylase", "protein_type" ], [ 149, 153, "RavA", "protein" ] ] }, { "sid": 97, "sent": "One of the elucidated roles of the LdcI-RavA cage is to maintain LdcI activity under conditions of enterobacterial starvation by preventing LdcI inhibition by the stringent response alarmone ppGpp.", "section": "RESULTS", "ner": [ [ 35, 44, "LdcI-RavA", "complex_assembly" ], [ 65, 69, "LdcI", "protein" ], [ 99, 114, "enterobacterial", "taxonomy_domain" ], [ 140, 144, "LdcI", "protein" ], [ 163, 190, "stringent response alarmone", "chemical" ], [ 191, 196, "ppGpp", "chemical" ] ] }, { "sid": 98, "sent": "Furthermore, the recently documented interaction of both LdcI and RavA with specific subunits of the respiratory complex I, together with the unanticipated link between RavA and maturation of numerous iron-sulfur proteins, tend to suggest an additional intriguing function for this 3.5\u2009MDa assembly.", "section": "RESULTS", "ner": [ [ 57, 61, "LdcI", "protein" ], [ 66, 70, "RavA", "protein" ], [ 85, 93, "subunits", "structure_element" ], [ 101, 122, "respiratory complex I", "protein_type" ], [ 169, 173, "RavA", "protein" ], [ 201, 221, "iron-sulfur proteins", "protein_type" ] ] }, { "sid": 99, "sent": "The conformational rearrangements of LdcI upon enzyme activation and RavA binding revealed in this work, and our amazing finding that the molecular determinant of the LdcI-RavA interaction is the one that straightforwardly determines if a particular enterobacterial lysine decarboxylase belongs to \u201cLdcI-like\u201d or \u201cLdcC-like\u201d proteins, should give a new impetus to functional studies of the unique LdcI-RavA cage.", "section": "RESULTS", "ner": [ [ 37, 41, "LdcI", "protein" ], [ 69, 73, "RavA", "protein" ], [ 167, 176, "LdcI-RavA", "complex_assembly" ], [ 250, 265, "enterobacterial", "taxonomy_domain" ], [ 266, 286, "lysine decarboxylase", "protein_type" ], [ 299, 308, "LdcI-like", "protein_type" ], [ 314, 323, "LdcC-like", "protein_type" ], [ 397, 406, "LdcI-RavA", "complex_assembly" ] ] }, { "sid": 100, "sent": "Besides, the structures and the pseudoatomic models of the active ppGpp-free states of both the biodegradative and the biosynthetic E. coli lysine decarboxylases offer an additional tool for analysis of their role in UPEC infectivity.", "section": "RESULTS", "ner": [ [ 13, 23, "structures", "evidence" ], [ 32, 51, "pseudoatomic models", "evidence" ], [ 59, 65, "active", "protein_state" ], [ 66, 76, "ppGpp-free", "protein_state" ], [ 96, 110, "biodegradative", "protein_state" ], [ 119, 131, "biosynthetic", "protein_state" ], [ 132, 139, "E. coli", "species" ], [ 140, 161, "lysine decarboxylases", "protein_type" ], [ 217, 221, "UPEC", "species" ] ] }, { "sid": 101, "sent": "Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development.", "section": "RESULTS", "ner": [ [ 18, 21, "apo", "protein_state" ], [ 22, 26, "LdcI", "protein" ], [ 31, 42, "ppGpp-LdcIi", "complex_assembly" ], [ 43, 61, "crystal structures", "evidence" ], [ 67, 73, "cryoEM", "experimental_method" ], [ 74, 89, "reconstructions", "evidence" ], [ 179, 200, "lysine decarboxylases", "protein_type" ], [ 212, 226, "enterobacteria", "taxonomy_domain" ], [ 264, 282, "Enterobacteriaceae", "taxonomy_domain" ], [ 301, 321, "lysine decarboxylase", "protein_type" ], [ 325, 344, "Eikenella corrodens", "species" ] ] }, { "sid": 102, "sent": "Finally, cadaverine being an important platform chemical for the production of industrial polymers such as nylon, structural information is valuable for optimisation of bacterial lysine decarboxylases used for its production in biotechnology.", "section": "RESULTS", "ner": [ [ 9, 19, "cadaverine", "chemical" ], [ 169, 178, "bacterial", "taxonomy_domain" ], [ 179, 200, "lysine decarboxylases", "protein_type" ] ] }, { "sid": 103, "sent": "3D cryoEM reconstructions of LdcC, LdcI-LARA and LdcIa.", "section": "FIG", "ner": [ [ 3, 9, "cryoEM", "experimental_method" ], [ 10, 25, "reconstructions", "evidence" ], [ 29, 33, "LdcC", "protein" ], [ 35, 44, "LdcI-LARA", "complex_assembly" ], [ 49, 54, "LdcIa", "protein" ] ] }, { "sid": 104, "sent": "(A,C,E) cryoEM map of the LdcC (A), LdcIa (C) and LdcI-LARA (E) decamers with one protomer in light grey.", "section": "FIG", "ner": [ [ 8, 14, "cryoEM", "experimental_method" ], [ 15, 18, "map", "evidence" ], [ 26, 30, "LdcC", "protein" ], [ 36, 41, "LdcIa", "protein" ], [ 50, 59, "LdcI-LARA", "complex_assembly" ], [ 64, 72, "decamers", "oligomeric_state" ], [ 82, 90, "protomer", "oligomeric_state" ] ] }, { "sid": 105, "sent": "In the rest of the protomers, the wing, core and C-terminal domains are colored from light to dark in shades of green for LdcC (A), pink for LdcIa (C) and blue for LdcI in LdcI-LARA (E).", "section": "FIG", "ner": [ [ 19, 28, "protomers", "oligomeric_state" ], [ 34, 38, "wing", "structure_element" ], [ 40, 44, "core", "structure_element" ], [ 49, 67, "C-terminal domains", "structure_element" ], [ 122, 126, "LdcC", "protein" ], [ 141, 146, "LdcIa", "protein" ], [ 164, 168, "LdcI", "protein" ], [ 172, 181, "LdcI-LARA", "complex_assembly" ] ] }, { "sid": 106, "sent": " In (E), the LARA domain density is shown in dark grey.", "section": "FIG", "ner": [ [ 13, 24, "LARA domain", "structure_element" ] ] }, { "sid": 107, "sent": "Two monomers making a dimer are delineated.", "section": "FIG", "ner": [ [ 4, 12, "monomers", "oligomeric_state" ], [ 22, 27, "dimer", "oligomeric_state" ] ] }, { "sid": 108, "sent": "Scale bar 50\u2009\u00c5. (B,D,F) One protomer from the cryoEM map of the LdcC (B), LdcIa (D) and LdcI-LARA (F) in light grey with the pseudoatomic model represented as cartoons and colored as the densities in (A,C,E).", "section": "FIG", "ner": [ [ 28, 36, "protomer", "oligomeric_state" ], [ 46, 52, "cryoEM", "experimental_method" ], [ 53, 56, "map", "evidence" ], [ 64, 68, "LdcC", "protein" ], [ 74, 79, "LdcIa", "protein" ], [ 88, 97, "LdcI-LARA", "complex_assembly" ], [ 125, 143, "pseudoatomic model", "evidence" ] ] }, { "sid": 109, "sent": "Superposition of the pseudoatomic models of LdcC, LdcI from LdcI-LARA and LdcIa colored as in Fig. 1, and the crystal structure of LdcIi in shades of yellow.", "section": "FIG", "ner": [ [ 0, 13, "Superposition", "experimental_method" ], [ 21, 40, "pseudoatomic models", "evidence" ], [ 44, 48, "LdcC", "protein" ], [ 50, 54, "LdcI", "protein" ], [ 60, 69, "LdcI-LARA", "complex_assembly" ], [ 74, 79, "LdcIa", "protein" ], [ 110, 127, "crystal structure", "evidence" ], [ 131, 136, "LdcIi", "protein" ] ] }, { "sid": 110, "sent": "Only one of the two rings of the double toroid is shown for clarity.", "section": "FIG", "ner": [ [ 20, 25, "rings", "structure_element" ], [ 33, 46, "double toroid", "structure_element" ] ] }, { "sid": 111, "sent": "The dashed circle indicates the central region that remains virtually unchanged between all the structures, while the periphery undergoes visible movements.", "section": "FIG", "ner": [ [ 40, 46, "region", "structure_element" ], [ 96, 106, "structures", "evidence" ] ] }, { "sid": 112, "sent": "Conformational rearrangements in the enzyme active site.", "section": "FIG", "ner": [ [ 44, 55, "active site", "site" ] ] }, { "sid": 113, "sent": "(A) LdcIi crystal structure, with one ring represented as a grey surface and the second as a cartoon.", "section": "FIG", "ner": [ [ 4, 9, "LdcIi", "protein" ], [ 10, 27, "crystal structure", "evidence" ], [ 38, 42, "ring", "structure_element" ] ] }, { "sid": 114, "sent": "A monomer with its PLP cofactor is delineated.", "section": "FIG", "ner": [ [ 2, 9, "monomer", "oligomeric_state" ], [ 19, 22, "PLP", "chemical" ] ] }, { "sid": 115, "sent": "The PLP moieties of the cartoon ring are shown in red.", "section": "FIG", "ner": [ [ 4, 7, "PLP", "chemical" ], [ 32, 36, "ring", "structure_element" ] ] }, { "sid": 116, "sent": " (B) The LdcIi dimer extracted from the crystal structure of the decamer.", "section": "FIG", "ner": [ [ 9, 14, "LdcIi", "protein" ], [ 15, 20, "dimer", "oligomeric_state" ], [ 40, 57, "crystal structure", "evidence" ], [ 65, 72, "decamer", "oligomeric_state" ] ] }, { "sid": 117, "sent": "One monomer is colored in shades of yellow as in Figs 1 and 2, while the monomer related by C2 symmetry is grey.", "section": "FIG", "ner": [ [ 4, 11, "monomer", "oligomeric_state" ], [ 73, 80, "monomer", "oligomeric_state" ] ] }, { "sid": 118, "sent": "The PLP is red.", "section": "FIG", "ner": [ [ 4, 7, "PLP", "chemical" ] ] }, { "sid": 119, "sent": "The active site is boxed.", "section": "FIG", "ner": [ [ 4, 15, "active site", "site" ] ] }, { "sid": 120, "sent": "Stretching of the LdcI monomer upon pH-dependent enzyme activation and LARA binding.", "section": "FIG", "ner": [ [ 18, 22, "LdcI", "protein" ], [ 23, 30, "monomer", "oligomeric_state" ], [ 36, 48, "pH-dependent", "protein_state" ], [ 71, 75, "LARA", "structure_element" ] ] }, { "sid": 121, "sent": "(A\u2013C) A slice through the pseudoatomic models of the LdcI monomers extracted from the superimposed decamers (Fig. 2) The rectangle indicates the regions enlarged in (D\u2013F).", "section": "FIG", "ner": [ [ 26, 45, "pseudoatomic models", "evidence" ], [ 53, 57, "LdcI", "protein" ], [ 58, 66, "monomers", "oligomeric_state" ], [ 86, 98, "superimposed", "experimental_method" ], [ 99, 107, "decamers", "oligomeric_state" ] ] }, { "sid": 122, "sent": "(A) compares LdcIi (yellow) and LdcIa (pink), (B) compares LdcIa (pink) and LdcI-LARA (blue), and (C) compares LdcIi (yellow), LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to show the progressive stretching described in the text.", "section": "FIG", "ner": [ [ 13, 18, "LdcIi", "protein" ], [ 32, 37, "LdcIa", "protein" ], [ 59, 64, "LdcIa", "protein" ], [ 76, 85, "LdcI-LARA", "complex_assembly" ], [ 111, 116, "LdcIi", "protein" ], [ 127, 132, "LdcIa", "protein" ], [ 144, 153, "LdcI-LARA", "complex_assembly" ] ] }, { "sid": 123, "sent": "The cryoEM density of the LARA domain is represented as a grey surface to show the position of the binding site and the direction of the movement.", "section": "FIG", "ner": [ [ 4, 10, "cryoEM", "experimental_method" ], [ 11, 18, "density", "evidence" ], [ 26, 37, "LARA domain", "structure_element" ], [ 99, 111, "binding site", "site" ] ] }, { "sid": 124, "sent": "(D\u2013F) Inserts zooming at the CTD part in proximity of the LARA binding site.", "section": "FIG", "ner": [ [ 29, 32, "CTD", "structure_element" ], [ 58, 75, "LARA binding site", "site" ] ] }, { "sid": 125, "sent": "Analysis of the LdcIC and LdcCI chimeras.", "section": "FIG", "ner": [ [ 16, 21, "LdcIC", "mutant" ], [ 26, 31, "LdcCI", "mutant" ], [ 32, 40, "chimeras", "mutant" ] ] }, { "sid": 126, "sent": "(A) A slice through the pseudoatomic models of the LdcIa (purple) and LdcC (green) monomers extracted from the superimposed decamers (Fig. 2). (B) The C-terminal \u03b2-sheet in LdcIa and LdcC enlarged from (A,C) Exchanged primary sequences (capital letters) and their immediate vicinity (lower case letters) colored as in (A,B), with the corresponding secondary structure elements and the amino acid numbering shown.", "section": "FIG", "ner": [ [ 24, 43, "pseudoatomic models", "evidence" ], [ 51, 56, "LdcIa", "protein" ], [ 70, 74, "LdcC", "protein" ], [ 83, 91, "monomers", "oligomeric_state" ], [ 111, 123, "superimposed", "experimental_method" ], [ 124, 132, "decamers", "oligomeric_state" ], [ 162, 169, "\u03b2-sheet", "structure_element" ], [ 173, 178, "LdcIa", "protein" ], [ 183, 187, "LdcC", "protein" ] ] }, { "sid": 127, "sent": "(D,E) A gallery of negative stain EM images of (D) the wild type LdcI-RavA cage and (E) the LdcCI-RavA cage-like particles. (F) Some representative class averages of the LdcCI-RavA cage-like particles.", "section": "FIG", "ner": [ [ 55, 64, "wild type", "protein_state" ], [ 65, 74, "LdcI-RavA", "complex_assembly" ], [ 92, 122, "LdcCI-RavA cage-like particles", "mutant" ], [ 170, 200, "LdcCI-RavA cage-like particles", "mutant" ] ] }, { "sid": 128, "sent": "Sequence analysis of enterobacterial lysine decarboxylases.", "section": "FIG", "ner": [ [ 0, 17, "Sequence analysis", "experimental_method" ], [ 21, 36, "enterobacterial", "taxonomy_domain" ], [ 37, 58, "lysine decarboxylases", "protein_type" ] ] }, { "sid": 129, "sent": "(A) Maximum likelihood tree with the \u201cLdcC-like\u201d and the \u201cLdcI-like\u201d groups highlighted in green and pink, respectively.", "section": "FIG", "ner": [ [ 4, 27, "Maximum likelihood tree", "evidence" ], [ 38, 47, "LdcC-like", "protein_type" ], [ 58, 67, "LdcI-like", "protein_type" ] ] }, { "sid": 130, "sent": "(B) Analysis of consensus \u201cLdcI-like\u201d and \u201cLdcC-like\u201d sequences around the first and second C-terminal \u03b2-strands.", "section": "FIG", "ner": [ [ 27, 36, "LdcI-like", "protein_type" ], [ 43, 52, "LdcC-like", "protein_type" ], [ 103, 112, "\u03b2-strands", "structure_element" ] ] }, { "sid": 131, "sent": "Numbering as in E. coli.", "section": "FIG", "ner": [ [ 16, 23, "E. coli", "species" ] ] }, { "sid": 132, "sent": " (C) Signature sequences of LdcI and LdcC in the C-terminal \u03b2-sheet.", "section": "FIG", "ner": [ [ 28, 32, "LdcI", "protein" ], [ 37, 41, "LdcC", "protein" ], [ 60, 67, "\u03b2-sheet", "structure_element" ] ] }, { "sid": 133, "sent": "Polarity differences are highlighted. (D) Position and nature of these differences at the surface of the respective cryoEM maps with the color code as in B. See also Fig. S7 and Tables S3 and S4.", "section": "FIG", "ner": [ [ 116, 122, "cryoEM", "experimental_method" ], [ 123, 127, "maps", "evidence" ] ] } ] }, "PMC4852598": { "annotations": [ { "sid": 0, "sent": "Structural basis for Mep2 ammonium transceptor activation by phosphorylation", "section": "TITLE", "ner": [ [ 21, 25, "Mep2", "protein_type" ], [ 26, 46, "ammonium transceptor", "protein_type" ], [ 61, 76, "phosphorylation", "ptm" ] ] }, { "sid": 1, "sent": "Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development.", "section": "ABSTRACT", "ner": [ [ 0, 13, "Mep2 proteins", "protein_type" ], [ 18, 24, "fungal", "taxonomy_domain" ], [ 25, 37, "transceptors", "protein_type" ], [ 69, 77, "ammonium", "chemical" ], [ 89, 95, "fungal", "taxonomy_domain" ] ] }, { "sid": 2, "sent": "Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear.", "section": "ABSTRACT", "ner": [ [ 0, 4, "Mep2", "protein_type" ], [ 38, 53, "phosphorylation", "ptm" ] ] }, { "sid": 3, "sent": "Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations.", "section": "ABSTRACT", "ner": [ [ 15, 39, "X-ray crystal structures", "evidence" ], [ 47, 51, "Mep2", "protein_type" ], [ 69, 93, "Saccharomyces cerevisiae", "species" ], [ 98, 114, "Candida albicans", "species" ], [ 170, 182, "transporters", "protein_type" ], [ 187, 205, "not phosphorylated", "protein_state" ], [ 221, 227, "closed", "protein_state" ], [ 229, 237, "inactive", "protein_state" ] ] }, { "sid": 4, "sent": "Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif.", "section": "ABSTRACT", "ner": [ [ 16, 20, "open", "protein_state" ], [ 21, 30, "bacterial", "taxonomy_domain" ], [ 31, 52, "ammonium transporters", "protein_type" ], [ 54, 72, "non-phosphorylated", "protein_state" ], [ 73, 77, "Mep2", "protein_type" ], [ 97, 114, "cytoplasmic loops", "structure_element" ], [ 123, 140, "C-terminal region", "structure_element" ], [ 142, 145, "CTR", "structure_element" ], [ 174, 178, "exit", "site" ], [ 186, 193, "channel", "site" ], [ 215, 219, "His2", "residue_name_number" ], [ 227, 241, "twin-His motif", "structure_element" ] ] }, { "sid": 5, "sent": "The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket \u223c30\u2009\u00c5 away from the channel exit.", "section": "ABSTRACT", "ner": [ [ 4, 24, "phosphorylation site", "site" ], [ 32, 35, "CTR", "structure_element" ], [ 39, 57, "solvent accessible", "protein_state" ], [ 75, 100, "negatively charged pocket", "site" ], [ 121, 133, "channel exit", "site" ] ] }, { "sid": 6, "sent": "The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR.", "section": "ABSTRACT", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 25, 50, "phosphorylation-mimicking", "protein_state" ], [ 51, 64, "Mep2 variants", "mutant" ], [ 70, 81, "C. albicans", "species" ], [ 121, 130, "conserved", "protein_state" ], [ 172, 175, "CTR", "structure_element" ] ] }, { "sid": 7, "sent": "The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.", "section": "ABSTRACT", "ner": [ [ 58, 68, "eukaryotic", "taxonomy_domain" ], [ 69, 77, "ammonium", "chemical" ], [ 91, 106, "phosphorylation", "ptm" ] ] }, { "sid": 8, "sent": " Mep2 proteins are tightly regulated fungal ammonium transporters.", "section": "ABSTRACT", "ner": [ [ 1, 14, "Mep2 proteins", "protein_type" ], [ 37, 43, "fungal", "taxonomy_domain" ], [ 44, 65, "ammonium transporters", "protein_type" ] ] }, { "sid": 9, "sent": "Here, the authors report the crystal structures of closed states of Mep2 proteins and propose a model for their regulation by comparing them with the open ammonium transporters of bacteria.", "section": "ABSTRACT", "ner": [ [ 29, 47, "crystal structures", "evidence" ], [ 51, 57, "closed", "protein_state" ], [ 68, 81, "Mep2 proteins", "protein_type" ], [ 123, 145, "by comparing them with", "experimental_method" ], [ 150, 154, "open", "protein_state" ], [ 155, 176, "ammonium transporters", "protein_type" ], [ 180, 188, "bacteria", "taxonomy_domain" ] ] }, { "sid": 10, "sent": "Transceptors are membrane proteins that function not only as transporters but also as receptors/sensors during nutrient sensing to activate downstream signalling pathways.", "section": "INTRO", "ner": [ [ 0, 12, "Transceptors", "protein_type" ], [ 17, 34, "membrane proteins", "protein_type" ] ] }, { "sid": 11, "sent": "A common feature of transceptors is that they are induced when cells are starved for their substrate.", "section": "INTRO", "ner": [ [ 20, 32, "transceptors", "protein_type" ] ] }, { "sid": 12, "sent": "While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter).", "section": "INTRO", "ner": [ [ 39, 63, "Saccharomyces cerevisiae", "species" ], [ 64, 76, "transceptors", "protein_type" ], [ 81, 90, "phosphate", "chemical" ], [ 92, 97, "Pho84", "protein" ], [ 100, 111, "amino acids", "chemical" ], [ 113, 117, "Gap1", "protein" ], [ 123, 131, "ammonium", "chemical" ], [ 133, 137, "Mep2", "protein" ], [ 140, 152, "transceptors", "protein_type" ], [ 166, 183, "higher eukaryotes", "taxonomy_domain" ], [ 210, 219, "mammalian", "taxonomy_domain" ], [ 220, 225, "SNAT2", "protein" ], [ 226, 248, "amino-acid transporter", "protein_type" ], [ 257, 262, "GLUT2", "protein" ], [ 263, 282, "glucose transporter", "protein_type" ] ] }, { "sid": 13, "sent": "One of the most important unresolved questions in the field is how the transceptors couple to downstream signalling pathways.", "section": "INTRO", "ner": [ [ 71, 83, "transceptors", "protein_type" ] ] }, { "sid": 14, "sent": "One hypothesis is that downstream signalling is dependent on a specific conformation of the transporter.", "section": "INTRO", "ner": [ [ 92, 103, "transporter", "protein_type" ] ] }, { "sid": 15, "sent": "Mep2 (methylammonium (MA) permease) proteins are ammonium transceptors that are ubiquitous in fungi.", "section": "INTRO", "ner": [ [ 0, 4, "Mep2", "protein_type" ], [ 5, 44, "(methylammonium (MA) permease) proteins", "protein_type" ], [ 49, 70, "ammonium transceptors", "protein_type" ], [ 94, 99, "fungi", "taxonomy_domain" ] ] }, { "sid": 16, "sent": "They belong to the Amt/Mep/Rh family of transporters that are present in all kingdoms of life and they take up ammonium from the extracellular environment.", "section": "INTRO", "ner": [ [ 19, 52, "Amt/Mep/Rh family of transporters", "protein_type" ], [ 73, 93, "all kingdoms of life", "taxonomy_domain" ], [ 111, 119, "ammonium", "chemical" ] ] }, { "sid": 17, "sent": "Fungi typically have more than one Mep paralogue, for example, Mep1-3 in S. cerevisiae.", "section": "INTRO", "ner": [ [ 0, 5, "Fungi", "taxonomy_domain" ], [ 35, 38, "Mep", "protein_type" ], [ 63, 69, "Mep1-3", "protein" ], [ 73, 86, "S. cerevisiae", "species" ] ] }, { "sid": 18, "sent": "Of these, only Mep2 proteins function as ammonium receptors/sensors in fungal development.", "section": "INTRO", "ner": [ [ 15, 28, "Mep2 proteins", "protein_type" ], [ 41, 49, "ammonium", "chemical" ], [ 71, 77, "fungal", "taxonomy_domain" ] ] }, { "sid": 19, "sent": "Under conditions of nitrogen limitation, Mep2 initiates a signalling cascade that results in a switch from the yeast form to filamentous (pseudohyphal) growth that may be required for fungal pathogenicity.", "section": "INTRO", "ner": [ [ 41, 45, "Mep2", "protein" ], [ 184, 190, "fungal", "taxonomy_domain" ] ] }, { "sid": 20, "sent": "As is the case for other transceptors, it is not clear how Mep2 interacts with downstream signalling partners, but the protein kinase A and mitogen-activated protein kinase pathways have been proposed as downstream effectors of Mep2 (refs).", "section": "INTRO", "ner": [ [ 25, 37, "transceptors", "protein_type" ], [ 59, 63, "Mep2", "protein" ], [ 228, 232, "Mep2", "protein" ] ] }, { "sid": 21, "sent": "Compared with Mep1 and Mep3, Mep2 is highly expressed and functions as a low-capacity, high-affinity transporter in the uptake of MA.", "section": "INTRO", "ner": [ [ 14, 18, "Mep1", "protein" ], [ 23, 27, "Mep3", "protein" ], [ 29, 33, "Mep2", "protein" ], [ 37, 53, "highly expressed", "protein_state" ], [ 130, 132, "MA", "chemical" ] ] }, { "sid": 22, "sent": "In addition, Mep2 is also important for uptake of ammonium produced by growth on other nitrogen sources.", "section": "INTRO", "ner": [ [ 13, 17, "Mep2", "protein" ], [ 50, 58, "ammonium", "chemical" ], [ 87, 95, "nitrogen", "chemical" ] ] }, { "sid": 23, "sent": "With the exception of the human RhCG structure, no structural information is available for eukaryotic ammonium transporters.", "section": "INTRO", "ner": [ [ 26, 31, "human", "species" ], [ 32, 36, "RhCG", "protein" ], [ 37, 46, "structure", "evidence" ], [ 91, 101, "eukaryotic", "taxonomy_domain" ], [ 102, 123, "ammonium transporters", "protein_type" ] ] }, { "sid": 24, "sent": "By contrast, several bacterial Amt orthologues have been characterized in detail via high-resolution crystal structures and a number of molecular dynamics (MD) studies.", "section": "INTRO", "ner": [ [ 21, 30, "bacterial", "taxonomy_domain" ], [ 31, 34, "Amt", "protein_type" ], [ 101, 119, "crystal structures", "evidence" ], [ 136, 154, "molecular dynamics", "experimental_method" ], [ 156, 158, "MD", "experimental_method" ] ] }, { "sid": 25, "sent": "All the solved structures including that of RhCG are very similar, establishing the basic architecture of ammonium transporters.", "section": "INTRO", "ner": [ [ 15, 25, "structures", "evidence" ], [ 44, 48, "RhCG", "protein" ], [ 106, 127, "ammonium transporters", "protein_type" ] ] }, { "sid": 26, "sent": "The proteins form stable trimers, with each monomer having 11 transmembrane (TM) helices and a central channel for the transport of ammonium.", "section": "INTRO", "ner": [ [ 18, 24, "stable", "protein_state" ], [ 25, 32, "trimers", "oligomeric_state" ], [ 44, 51, "monomer", "oligomeric_state" ], [ 62, 75, "transmembrane", "structure_element" ], [ 77, 79, "TM", "structure_element" ], [ 81, 88, "helices", "structure_element" ], [ 95, 110, "central channel", "site" ], [ 132, 140, "ammonium", "chemical" ] ] }, { "sid": 27, "sent": "All structures show the transporters in open conformations.", "section": "INTRO", "ner": [ [ 4, 14, "structures", "evidence" ], [ 24, 36, "transporters", "protein_type" ], [ 40, 44, "open", "protein_state" ] ] }, { "sid": 28, "sent": "Where earlier studies favoured the transport of ammonia gas, recent data and theoretical considerations suggest that Amt/Mep proteins are instead active, electrogenic transporters of either NH4+ (uniport) or NH3/H+ (symport).", "section": "INTRO", "ner": [ [ 48, 55, "ammonia", "chemical" ], [ 117, 133, "Amt/Mep proteins", "protein_type" ], [ 146, 152, "active", "protein_state" ], [ 154, 179, "electrogenic transporters", "protein_type" ], [ 190, 194, "NH4+", "chemical" ], [ 208, 211, "NH3", "chemical" ], [ 212, 214, "H+", "chemical" ] ] }, { "sid": 29, "sent": "A highly conserved pair of channel-lining histidine residues dubbed the twin-His motif may serve as a proton relay system while NH3 moves through the channel during NH3/H+ symport.", "section": "INTRO", "ner": [ [ 2, 18, "highly conserved", "protein_state" ], [ 27, 34, "channel", "site" ], [ 42, 51, "histidine", "residue_name" ], [ 72, 86, "twin-His motif", "structure_element" ], [ 128, 131, "NH3", "chemical" ], [ 150, 157, "channel", "site" ], [ 165, 168, "NH3", "chemical" ], [ 169, 171, "H+", "chemical" ] ] }, { "sid": 30, "sent": "Ammonium transport is tightly regulated.", "section": "INTRO", "ner": [ [ 0, 8, "Ammonium", "chemical" ] ] }, { "sid": 31, "sent": "In animals, this is due to toxicity of elevated intracellular ammonium levels, whereas for microorganisms ammonium is a preferred nitrogen source.", "section": "INTRO", "ner": [ [ 3, 10, "animals", "taxonomy_domain" ], [ 62, 70, "ammonium", "chemical" ], [ 91, 105, "microorganisms", "taxonomy_domain" ], [ 106, 114, "ammonium", "chemical" ] ] }, { "sid": 32, "sent": "In bacteria, amt genes are present in an operon with glnK, encoding a PII-like signal transduction class protein.", "section": "INTRO", "ner": [ [ 3, 11, "bacteria", "taxonomy_domain" ], [ 13, 16, "amt", "gene" ], [ 53, 57, "glnK", "gene" ], [ 70, 112, "PII-like signal transduction class protein", "protein_type" ] ] }, { "sid": 33, "sent": "By binding tightly to Amt proteins without inducing a conformational change in the transporter, GlnK sterically blocks ammonium conductance when nitrogen levels are sufficient.", "section": "INTRO", "ner": [ [ 22, 34, "Amt proteins", "protein_type" ], [ 83, 94, "transporter", "protein_type" ], [ 96, 100, "GlnK", "protein_type" ], [ 119, 127, "ammonium", "chemical" ] ] }, { "sid": 34, "sent": "Under conditions of nitrogen limitation, GlnK becomes uridylated, blocking its ability to bind and inhibit Amt proteins.", "section": "INTRO", "ner": [ [ 20, 28, "nitrogen", "chemical" ], [ 41, 45, "GlnK", "protein_type" ], [ 54, 64, "uridylated", "protein_state" ], [ 107, 119, "Amt proteins", "protein_type" ] ] }, { "sid": 35, "sent": "Importantly, eukaryotes do not have GlnK orthologues and have a different mechanism for regulation of ammonium transport activity.", "section": "INTRO", "ner": [ [ 13, 23, "eukaryotes", "taxonomy_domain" ], [ 36, 40, "GlnK", "protein_type" ], [ 102, 110, "ammonium", "chemical" ] ] }, { "sid": 36, "sent": "In plants, transporter phosphorylation and dephosphorylation are known to regulate activity.", "section": "INTRO", "ner": [ [ 3, 9, "plants", "taxonomy_domain" ], [ 11, 22, "transporter", "protein_type" ], [ 23, 38, "phosphorylation", "ptm" ], [ 43, 60, "dephosphorylation", "ptm" ] ] }, { "sid": 37, "sent": "In S. cerevisiae, phosphorylation of Ser457 within the C-terminal region (CTR) in the cytoplasm was recently proposed to cause Mep2 opening, possibly via inducing a conformational change.", "section": "INTRO", "ner": [ [ 3, 16, "S. cerevisiae", "species" ], [ 18, 33, "phosphorylation", "ptm" ], [ 37, 43, "Ser457", "residue_name_number" ], [ 55, 72, "C-terminal region", "structure_element" ], [ 74, 77, "CTR", "structure_element" ], [ 127, 131, "Mep2", "protein_type" ] ] }, { "sid": 38, "sent": "To elucidate the mechanism of Mep2 transport regulation, we present here X-ray crystal structures of the Mep2 transceptors from S. cerevisiae and C. albicans.", "section": "INTRO", "ner": [ [ 30, 34, "Mep2", "protein_type" ], [ 73, 97, "X-ray crystal structures", "evidence" ], [ 105, 122, "Mep2 transceptors", "protein_type" ], [ 128, 141, "S. cerevisiae", "species" ], [ 146, 157, "C. albicans", "species" ] ] }, { "sid": 39, "sent": "The structures are similar to each other but show considerable differences to all other ammonium transporter structures.", "section": "INTRO", "ner": [ [ 4, 14, "structures", "evidence" ], [ 88, 108, "ammonium transporter", "protein_type" ], [ 109, 119, "structures", "evidence" ] ] }, { "sid": 40, "sent": "The most striking difference is the fact that the Mep2 proteins have closed conformations.", "section": "INTRO", "ner": [ [ 50, 63, "Mep2 proteins", "protein_type" ], [ 69, 75, "closed", "protein_state" ] ] }, { "sid": 41, "sent": "The putative phosphorylation site is solvent accessible and located in a negatively charged pocket \u223c30\u2009\u00c5 away from the channel exit.", "section": "INTRO", "ner": [ [ 13, 33, "phosphorylation site", "site" ], [ 37, 55, "solvent accessible", "protein_state" ], [ 73, 98, "negatively charged pocket", "site" ], [ 119, 131, "channel exit", "site" ] ] }, { "sid": 42, "sent": "The channels of phosphorylation-mimicking mutants of C. albicans Mep2 are still closed but show large conformational changes within a conserved part of the CTR.", "section": "INTRO", "ner": [ [ 4, 12, "channels", "site" ], [ 16, 49, "phosphorylation-mimicking mutants", "protein_state" ], [ 53, 64, "C. albicans", "species" ], [ 65, 69, "Mep2", "protein" ], [ 80, 86, "closed", "protein_state" ], [ 134, 143, "conserved", "protein_state" ], [ 156, 159, "CTR", "structure_element" ] ] }, { "sid": 43, "sent": "Together with a structure of a C-terminal Mep2 variant lacking the segment containing the phosphorylation site, the results allow us to propose a structural model for phosphorylation-based regulation of eukaryotic ammonium transport.", "section": "INTRO", "ner": [ [ 16, 25, "structure", "evidence" ], [ 42, 54, "Mep2 variant", "mutant" ], [ 55, 62, "lacking", "protein_state" ], [ 67, 74, "segment", "structure_element" ], [ 90, 110, "phosphorylation site", "site" ], [ 203, 213, "eukaryotic", "taxonomy_domain" ], [ 214, 222, "ammonium", "chemical" ] ] }, { "sid": 44, "sent": "General architecture of Mep2 ammonium transceptors", "section": "RESULTS", "ner": [ [ 24, 28, "Mep2", "protein_type" ], [ 29, 50, "ammonium transceptors", "protein_type" ] ] }, { "sid": 45, "sent": "The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2\u2009\u00c5 resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section).", "section": "RESULTS", "ner": [ [ 4, 8, "Mep2", "protein" ], [ 20, 33, "S. cerevisiae", "species" ], [ 35, 41, "ScMep2", "protein" ], [ 47, 60, "overexpressed", "experimental_method" ], [ 64, 77, "S. cerevisiae", "species" ], [ 103, 126, "structure determination", "experimental_method" ], [ 130, 151, "X-ray crystallography", "experimental_method" ], [ 186, 207, "molecular replacement", "experimental_method" ], [ 209, 211, "MR", "experimental_method" ], [ 222, 237, "archaebacterial", "taxonomy_domain" ], [ 238, 243, "Amt-1", "protein" ], [ 244, 253, "structure", "evidence" ] ] }, { "sid": 46, "sent": "Given that the modest resolution of the structure and the limited detergent stability of ScMep2 would likely complicate structure\u2013function studies, several other fungal Mep2 orthologues were subsequently overexpressed and screened for diffraction-quality crystals.", "section": "RESULTS", "ner": [ [ 40, 49, "structure", "evidence" ], [ 89, 95, "ScMep2", "protein" ], [ 120, 146, "structure\u2013function studies", "experimental_method" ], [ 162, 168, "fungal", "taxonomy_domain" ], [ 169, 173, "Mep2", "protein_type" ], [ 204, 234, "overexpressed and screened for", "experimental_method" ], [ 255, 263, "crystals", "evidence" ] ] }, { "sid": 47, "sent": "Of these, Mep2 from C. albicans (CaMep2) showed superior stability in relatively harsh detergents such as nonyl-glucoside, allowing structure determination in two different crystal forms to high resolution (up to 1.5\u2009\u00c5).", "section": "RESULTS", "ner": [ [ 10, 14, "Mep2", "protein" ], [ 20, 31, "C. albicans", "species" ], [ 33, 39, "CaMep2", "protein" ], [ 132, 155, "structure determination", "experimental_method" ], [ 173, 186, "crystal forms", "evidence" ] ] }, { "sid": 48, "sent": "Despite different crystal packing (Supplementary Table 1), the two CaMep2 structures are identical to each other and very similar to ScMep2 (C\u03b1 r.m.s.d.", "section": "RESULTS", "ner": [ [ 67, 73, "CaMep2", "protein" ], [ 74, 84, "structures", "evidence" ], [ 133, 139, "ScMep2", "protein" ], [ 144, 152, "r.m.s.d.", "evidence" ] ] }, { "sid": 49, "sent": "(root mean square deviation)=0.7\u2009\u00c5 for 434 residues), with the main differences confined to the N terminus and the CTR (Fig. 1).", "section": "RESULTS", "ner": [ [ 1, 27, "root mean square deviation", "evidence" ], [ 115, 118, "CTR", "structure_element" ] ] }, { "sid": 50, "sent": "Electron density is visible for the entire polypeptide chains, with the exception of the C-terminal 43 (ScMep2) and 25 residues (CaMep2), which are poorly conserved and presumably disordered.", "section": "RESULTS", "ner": [ [ 0, 16, "Electron density", "evidence" ], [ 100, 102, "43", "residue_range" ], [ 104, 110, "ScMep2", "protein" ], [ 116, 118, "25", "residue_range" ], [ 129, 135, "CaMep2", "protein" ], [ 148, 164, "poorly conserved", "protein_state" ], [ 180, 190, "disordered", "protein_state" ] ] }, { "sid": 51, "sent": "Both Mep2 proteins show the archetypal trimeric assemblies in which each monomer consists of 11 TM helices surrounding a central pore.", "section": "RESULTS", "ner": [ [ 5, 18, "Mep2 proteins", "protein_type" ], [ 39, 47, "trimeric", "oligomeric_state" ], [ 73, 80, "monomer", "oligomeric_state" ], [ 96, 106, "TM helices", "structure_element" ], [ 121, 133, "central pore", "structure_element" ] ] }, { "sid": 52, "sent": "Important functional features such as the extracellular ammonium binding site, the Phe gate and the twin-His motif within the hydrophobic channel are all very similar to those present in the bacterial transporters and RhCG.", "section": "RESULTS", "ner": [ [ 56, 77, "ammonium binding site", "site" ], [ 83, 91, "Phe gate", "site" ], [ 100, 114, "twin-His motif", "structure_element" ], [ 126, 145, "hydrophobic channel", "site" ], [ 191, 200, "bacterial", "taxonomy_domain" ], [ 201, 213, "transporters", "protein_type" ], [ 218, 222, "RhCG", "protein" ] ] }, { "sid": 53, "sent": "In the remainder of the manuscript, we will specifically discuss CaMep2 due to the superior resolution of the structure.", "section": "RESULTS", "ner": [ [ 65, 71, "CaMep2", "protein" ], [ 110, 119, "structure", "evidence" ] ] }, { "sid": 54, "sent": "Unless specifically stated, the drawn conclusions also apply to ScMep2.", "section": "RESULTS", "ner": [ [ 64, 70, "ScMep2", "protein" ] ] }, { "sid": 55, "sent": "While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (C\u03b1 r.m.s.d. with Amt-1=1.4\u2009\u00c5 for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR.", "section": "RESULTS", "ner": [ [ 34, 38, "Mep2", "protein" ], [ 65, 76, "prokaryotic", "taxonomy_domain" ], [ 77, 89, "transporters", "protein_type" ], [ 94, 102, "r.m.s.d.", "evidence" ], [ 108, 113, "Amt-1", "protein" ], [ 190, 209, "intracellular loops", "structure_element" ], [ 211, 215, "ICLs", "structure_element" ], [ 217, 221, "ICL1", "structure_element" ], [ 226, 230, "ICL3", "structure_element" ], [ 240, 243, "CTR", "structure_element" ] ] }, { "sid": 56, "sent": "The N termini of the Mep2 proteins are \u223c20\u201325 residues longer compared with their bacterial counterparts (Figs 1 and 2), substantially increasing the size of the extracellular domain.", "section": "RESULTS", "ner": [ [ 21, 34, "Mep2 proteins", "protein_type" ], [ 40, 45, "20\u201325", "residue_range" ], [ 82, 91, "bacterial", "taxonomy_domain" ], [ 162, 182, "extracellular domain", "structure_element" ] ] }, { "sid": 57, "sent": "Moreover, the N terminus of one monomer interacts with the extended extracellular loop ECL5 of a neighbouring monomer.", "section": "RESULTS", "ner": [ [ 32, 39, "monomer", "oligomeric_state" ], [ 68, 86, "extracellular loop", "structure_element" ], [ 87, 91, "ECL5", "structure_element" ], [ 110, 117, "monomer", "oligomeric_state" ] ] }, { "sid": 58, "sent": "Together with additional, smaller differences in other extracellular loops, these changes generate a distinct vestibule leading to the ammonium binding site that is much more pronounced than in the bacterial proteins.", "section": "RESULTS", "ner": [ [ 55, 74, "extracellular loops", "structure_element" ], [ 110, 119, "vestibule", "structure_element" ], [ 135, 156, "ammonium binding site", "site" ], [ 198, 207, "bacterial", "taxonomy_domain" ] ] }, { "sid": 59, "sent": "The N-terminal vestibule and the resulting inter-monomer interactions likely increase the stability of the Mep2 trimer, in support of data for plant AMT proteins.", "section": "RESULTS", "ner": [ [ 15, 24, "vestibule", "structure_element" ], [ 49, 56, "monomer", "oligomeric_state" ], [ 107, 111, "Mep2", "protein" ], [ 112, 118, "trimer", "oligomeric_state" ], [ 143, 148, "plant", "taxonomy_domain" ], [ 149, 161, "AMT proteins", "protein_type" ] ] }, { "sid": 60, "sent": "However, given that an N-terminal deletion mutant (2-27\u0394) grows as well as wild-type (WT) Mep2 on minimal ammonium medium (Fig. 3 and Supplementary Fig. 1), the importance of the N terminus for Mep2 activity is not clear.", "section": "RESULTS", "ner": [ [ 34, 49, "deletion mutant", "protein_state" ], [ 51, 56, "2-27\u0394", "mutant" ], [ 75, 84, "wild-type", "protein_state" ], [ 86, 88, "WT", "protein_state" ], [ 90, 94, "Mep2", "protein" ], [ 106, 114, "ammonium", "chemical" ], [ 194, 198, "Mep2", "protein" ] ] }, { "sid": 61, "sent": "Mep2 channels are closed by a two-tier channel block", "section": "RESULTS", "ner": [ [ 0, 4, "Mep2", "protein" ], [ 5, 13, "channels", "site" ], [ 18, 24, "closed", "protein_state" ], [ 39, 52, "channel block", "structure_element" ] ] }, { "sid": 62, "sent": "The largest differences between the Mep2 structures and the other known ammonium transporter structures are located on the intracellular side of the membrane.", "section": "RESULTS", "ner": [ [ 36, 40, "Mep2", "protein" ], [ 41, 51, "structures", "evidence" ], [ 72, 92, "ammonium transporter", "protein_type" ], [ 93, 103, "structures", "evidence" ] ] }, { "sid": 63, "sent": "In the vicinity of the Mep2 channel exit, the cytoplasmic end of TM2 has unwound, generating a longer ICL1 even though there are no insertions in this region compared to the bacterial proteins (Figs 2 and 4).", "section": "RESULTS", "ner": [ [ 23, 27, "Mep2", "protein" ], [ 28, 40, "channel exit", "site" ], [ 65, 68, "TM2", "structure_element" ], [ 102, 106, "ICL1", "structure_element" ], [ 174, 183, "bacterial", "taxonomy_domain" ] ] }, { "sid": 64, "sent": "ICL1 has also moved inwards relative to its position in the bacterial Amts.", "section": "RESULTS", "ner": [ [ 0, 4, "ICL1", "structure_element" ], [ 60, 69, "bacterial", "taxonomy_domain" ], [ 70, 74, "Amts", "protein_type" ] ] }, { "sid": 65, "sent": "The largest backbone movements of equivalent residues within ICL1 are \u223c10\u2009\u00c5, markedly affecting the conserved basic RxK motif (Fig. 4).", "section": "RESULTS", "ner": [ [ 61, 65, "ICL1", "structure_element" ], [ 100, 109, "conserved", "protein_state" ], [ 110, 115, "basic", "protein_state" ], [ 116, 125, "RxK motif", "structure_element" ] ] }, { "sid": 66, "sent": "The head group of Arg54 has moved \u223c11\u2009\u00c5 relative to that in Amt-1, whereas the shift of the head group of the variable Lys55 residue is almost 20\u2009\u00c5. The side chain of Lys56 in the basic motif points in an opposite direction in the Mep2 structures compared with that of, for example, Amt-1 (Fig. 4).", "section": "RESULTS", "ner": [ [ 18, 23, "Arg54", "residue_name_number" ], [ 60, 65, "Amt-1", "protein" ], [ 119, 124, "Lys55", "residue_name_number" ], [ 167, 172, "Lys56", "residue_name_number" ], [ 180, 185, "basic", "protein_state" ], [ 186, 191, "motif", "structure_element" ], [ 231, 235, "Mep2", "protein" ], [ 236, 246, "structures", "evidence" ], [ 283, 288, "Amt-1", "protein" ] ] }, { "sid": 67, "sent": "In addition to changing the RxK motif, the movement of ICL1 has another, crucial functional consequence.", "section": "RESULTS", "ner": [ [ 28, 37, "RxK motif", "structure_element" ], [ 55, 59, "ICL1", "structure_element" ] ] }, { "sid": 68, "sent": "At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the \u025b2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5).", "section": "RESULTS", "ner": [ [ 25, 28, "TM1", "structure_element" ], [ 67, 87, "relatively conserved", "protein_state" ], [ 88, 93, "Tyr49", "residue_name_number" ], [ 95, 100, "Tyr53", "residue_name_number" ], [ 104, 110, "ScMep2", "protein" ], [ 127, 140, "hydrogen bond", "bond_interaction" ], [ 174, 194, "absolutely conserved", "protein_state" ], [ 195, 201, "His342", "residue_name_number" ], [ 209, 223, "twin-His motif", "structure_element" ], [ 225, 231, "His348", "residue_name_number" ], [ 235, 241, "ScMep2", "protein" ], [ 256, 263, "channel", "site" ] ] }, { "sid": 69, "sent": "In bacterial Amt proteins, this Tyr side chain is rotated \u223c4\u2009\u00c5 away as a result of the different conformation of TM1, leaving the channel open and the histidine available for its putative role in substrate transport (Supplementary Fig. 2).", "section": "RESULTS", "ner": [ [ 3, 12, "bacterial", "taxonomy_domain" ], [ 13, 25, "Amt proteins", "protein_type" ], [ 32, 35, "Tyr", "residue_name" ], [ 113, 116, "TM1", "structure_element" ], [ 130, 137, "channel", "site" ], [ 138, 142, "open", "protein_state" ], [ 151, 160, "histidine", "residue_name" ] ] }, { "sid": 70, "sent": "Compared with ICL1, the backbone conformational changes observed for the neighbouring ICL2 are smaller, but large shifts are nevertheless observed for the conserved residues Glu140 and Arg141 (Fig. 4).", "section": "RESULTS", "ner": [ [ 14, 18, "ICL1", "structure_element" ], [ 86, 90, "ICL2", "structure_element" ], [ 155, 164, "conserved", "protein_state" ], [ 174, 180, "Glu140", "residue_name_number" ], [ 185, 191, "Arg141", "residue_name_number" ] ] }, { "sid": 71, "sent": "Finally, the important ICL3 linking the pseudo-symmetrical halves (TM1-5 and TM6-10) of the transporter is also shifted up to \u223c10\u2009\u00c5 and forms an additional barrier that closes the channel on the cytoplasmic side (Fig. 5).", "section": "RESULTS", "ner": [ [ 23, 27, "ICL3", "structure_element" ], [ 40, 65, "pseudo-symmetrical halves", "structure_element" ], [ 67, 72, "TM1-5", "structure_element" ], [ 77, 83, "TM6-10", "structure_element" ], [ 92, 103, "transporter", "protein_type" ], [ 180, 187, "channel", "site" ] ] }, { "sid": 72, "sent": "This two-tier channel block likely ensures that very little ammonium transport will take place under nitrogen-sufficient conditions.", "section": "RESULTS", "ner": [ [ 14, 27, "channel block", "structure_element" ], [ 60, 68, "ammonium", "chemical" ], [ 101, 109, "nitrogen", "chemical" ] ] }, { "sid": 73, "sent": "The closed state of the channel might also explain why no density, which could correspond to ammonium (or water), is observed in the hydrophobic part of the Mep2 channel close to the twin-His motif.", "section": "RESULTS", "ner": [ [ 4, 10, "closed", "protein_state" ], [ 24, 31, "channel", "site" ], [ 55, 65, "no density", "evidence" ], [ 93, 101, "ammonium", "chemical" ], [ 106, 111, "water", "chemical" ], [ 157, 161, "Mep2", "protein" ], [ 162, 169, "channel", "site" ], [ 183, 197, "twin-His motif", "structure_element" ] ] }, { "sid": 74, "sent": "Significantly, this is also true for ScMep2, which was crystallized in the presence of 0.2\u2009M ammonium ions (see Methods section).", "section": "RESULTS", "ner": [ [ 37, 43, "ScMep2", "protein" ], [ 55, 67, "crystallized", "experimental_method" ], [ 93, 101, "ammonium", "chemical" ] ] }, { "sid": 75, "sent": "The final region in Mep2 that shows large differences compared with the bacterial transporters is the CTR.", "section": "RESULTS", "ner": [ [ 20, 24, "Mep2", "protein" ], [ 72, 81, "bacterial", "taxonomy_domain" ], [ 82, 94, "transporters", "protein_type" ], [ 102, 105, "CTR", "structure_element" ] ] }, { "sid": 76, "sent": "In Mep2, the CTR has moved away and makes relatively few contacts with the main body of the transporter, generating a more elongated protein (Figs 1 and 4).", "section": "RESULTS", "ner": [ [ 3, 7, "Mep2", "protein" ], [ 13, 16, "CTR", "structure_element" ], [ 75, 84, "main body", "structure_element" ], [ 92, 103, "transporter", "protein_type" ], [ 123, 132, "elongated", "protein_state" ] ] }, { "sid": 77, "sent": "By contrast, in the structures of bacterial proteins, the CTR is docked tightly onto the N-terminal half of the transporters (corresponding to TM1-5), resulting in a more compact structure.", "section": "RESULTS", "ner": [ [ 20, 30, "structures", "evidence" ], [ 34, 43, "bacterial", "taxonomy_domain" ], [ 58, 61, "CTR", "structure_element" ], [ 89, 104, "N-terminal half", "structure_element" ], [ 112, 124, "transporters", "protein_type" ], [ 143, 148, "TM1-5", "structure_element" ], [ 171, 178, "compact", "protein_state" ], [ 179, 188, "structure", "evidence" ] ] }, { "sid": 78, "sent": "This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2).", "section": "RESULTS", "ner": [ [ 49, 70, "universally conserved", "protein_state" ], [ 91, 94, "CTR", "structure_element" ], [ 105, 111, "Arg415", "residue_name_number" ], [ 112, 115, "370", "residue_number" ], [ 118, 124, "Glu421", "residue_name_number" ], [ 125, 128, "376", "residue_number" ], [ 131, 137, "Gly424", "residue_name_number" ], [ 138, 141, "379", "residue_number" ], [ 144, 150, "Asp426", "residue_name_number" ], [ 151, 154, "381", "residue_number" ], [ 160, 167, "Tyr 435", "residue_name_number" ], [ 168, 171, "390", "residue_number" ], [ 176, 182, "CaMep2", "protein" ], [ 183, 188, "Amt-1", "protein" ] ] }, { "sid": 79, "sent": "These residues include those of the \u2018ExxGxD' motif, which when mutated generate inactive transporters.", "section": "RESULTS", "ner": [ [ 36, 50, "\u2018ExxGxD' motif", "structure_element" ], [ 63, 70, "mutated", "experimental_method" ], [ 80, 88, "inactive", "protein_state" ], [ 89, 101, "transporters", "protein_type" ] ] }, { "sid": 80, "sent": "In Amt-1 and other bacterial ammonium transporters, these CTR residues interact with residues within the N-terminal half of the protein.", "section": "RESULTS", "ner": [ [ 3, 8, "Amt-1", "protein" ], [ 19, 28, "bacterial", "taxonomy_domain" ], [ 29, 50, "ammonium transporters", "protein_type" ], [ 58, 61, "CTR", "structure_element" ], [ 105, 120, "N-terminal half", "structure_element" ] ] }, { "sid": 81, "sent": "On one side, the Tyr390 hydroxyl in Amt-1 is hydrogen bonded with the side chain of the conserved His185 at the C-terminal end of loop ICL3.", "section": "RESULTS", "ner": [ [ 17, 23, "Tyr390", "residue_name_number" ], [ 36, 41, "Amt-1", "protein" ], [ 45, 60, "hydrogen bonded", "bond_interaction" ], [ 88, 97, "conserved", "protein_state" ], [ 98, 104, "His185", "residue_name_number" ], [ 130, 134, "loop", "structure_element" ], [ 135, 139, "ICL3", "structure_element" ] ] }, { "sid": 82, "sent": "At the other end of ICL3, the backbone carbonyl groups of Gly172 and Lys173 are hydrogen bonded to the side chain of Arg370.", "section": "RESULTS", "ner": [ [ 20, 24, "ICL3", "structure_element" ], [ 58, 64, "Gly172", "residue_name_number" ], [ 69, 75, "Lys173", "residue_name_number" ], [ 80, 95, "hydrogen bonded", "bond_interaction" ], [ 117, 123, "Arg370", "residue_name_number" ] ] }, { "sid": 83, "sent": "Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71).", "section": "RESULTS", "ner": [ [ 31, 39, "modelled", "experimental_method" ], [ 47, 53, "active", "protein_state" ], [ 55, 73, "non-phosphorylated", "protein_state" ], [ 74, 79, "plant", "taxonomy_domain" ], [ 80, 89, "AtAmt-1;1", "protein" ], [ 90, 99, "structure", "evidence" ], [ 114, 118, "Y467", "residue_name_number" ], [ 119, 123, "H239", "residue_name_number" ], [ 128, 132, "D458", "residue_name_number" ], [ 133, 136, "K71", "residue_name_number" ] ] }, { "sid": 84, "sent": "The result of these interactions is that the CTR \u2018hugs' the N-terminal half of the transporters (Fig. 4).", "section": "RESULTS", "ner": [ [ 45, 48, "CTR", "structure_element" ], [ 60, 75, "N-terminal half", "structure_element" ], [ 83, 95, "transporters", "protein_type" ] ] }, { "sid": 85, "sent": "Also noteworthy is Asp381, the side chain of which interacts strongly with the positive dipole on the N-terminal end of TM2.", "section": "RESULTS", "ner": [ [ 19, 25, "Asp381", "residue_name_number" ], [ 120, 123, "TM2", "structure_element" ] ] }, { "sid": 86, "sent": "This interaction in the centre of the protein may be particularly important to stabilize the open conformations of ammonium transporters.", "section": "RESULTS", "ner": [ [ 93, 97, "open", "protein_state" ], [ 115, 136, "ammonium transporters", "protein_type" ] ] }, { "sid": 87, "sent": "In the Mep2 structures, none of the interactions mentioned above are present.", "section": "RESULTS", "ner": [ [ 7, 11, "Mep2", "protein" ], [ 12, 22, "structures", "evidence" ] ] }, { "sid": 88, "sent": "Phosphorylation target site is at the periphery of Mep2", "section": "RESULTS", "ner": [ [ 0, 27, "Phosphorylation target site", "site" ], [ 51, 55, "Mep2", "protein" ] ] }, { "sid": 89, "sent": "Recently Boeckstaens et al. provided evidence that Ser457 in ScMep2 (corresponding to Ser453 in CaMep2) is phosphorylated by the TORC1 effector kinase Npr1 under nitrogen-limiting conditions.", "section": "RESULTS", "ner": [ [ 51, 57, "Ser457", "residue_name_number" ], [ 61, 67, "ScMep2", "protein" ], [ 86, 92, "Ser453", "residue_name_number" ], [ 96, 102, "CaMep2", "protein" ], [ 107, 121, "phosphorylated", "protein_state" ], [ 129, 150, "TORC1 effector kinase", "protein_type" ], [ 151, 155, "Npr1", "protein" ], [ 162, 170, "nitrogen", "chemical" ] ] }, { "sid": 90, "sent": "In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3\u0394 strain (triple mep\u0394) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1).", "section": "RESULTS", "ner": [ [ 7, 17, "absence of", "protein_state" ], [ 18, 22, "Npr1", "protein" ], [ 24, 39, "plasmid-encoded", "experimental_method" ], [ 40, 42, "WT", "protein_state" ], [ 43, 47, "Mep2", "protein" ], [ 53, 66, "S. cerevisiae", "species" ], [ 67, 74, "mep1-3\u0394", "mutant" ], [ 83, 94, "triple mep\u0394", "mutant" ], [ 143, 151, "ammonium", "chemical" ], [ 173, 184, "transporter", "protein_type" ], [ 188, 196, "inactive", "protein_state" ] ] }, { "sid": 91, "sent": "Conversely, the phosphorylation-mimicking S457D variant is active both in the triple mep\u0394 background and in a triple mep\u0394 npr1\u0394 strain (Fig. 3).", "section": "RESULTS", "ner": [ [ 16, 41, "phosphorylation-mimicking", "protein_state" ], [ 42, 47, "S457D", "mutant" ], [ 59, 65, "active", "protein_state" ], [ 78, 89, "triple mep\u0394", "mutant" ], [ 110, 127, "triple mep\u0394 npr1\u0394", "mutant" ] ] }, { "sid": 92, "sent": "Mutation of other potential phosphorylation sites in the CTR did not support growth in the npr1\u0394 background.", "section": "RESULTS", "ner": [ [ 0, 8, "Mutation", "experimental_method" ], [ 28, 49, "phosphorylation sites", "site" ], [ 57, 60, "CTR", "structure_element" ], [ 91, 96, "npr1\u0394", "mutant" ] ] }, { "sid": 93, "sent": "Collectively, these data suggest that phosphorylation of Ser457 opens the Mep2 channel to allow ammonium uptake.", "section": "RESULTS", "ner": [ [ 38, 53, "phosphorylation", "ptm" ], [ 57, 63, "Ser457", "residue_name_number" ], [ 57, 63, "Ser457", "residue_name_number" ], [ 74, 78, "Mep2", "protein" ], [ 79, 86, "channel", "site" ], [ 96, 104, "ammonium", "chemical" ] ] }, { "sid": 94, "sent": "Ser457 is located in a part of the CTR that is conserved in a subgroup of Mep2 proteins, but which is not present in bacterial proteins (Fig. 2).", "section": "RESULTS", "ner": [ [ 0, 6, "Ser457", "residue_name_number" ], [ 35, 38, "CTR", "structure_element" ], [ 47, 56, "conserved", "protein_state" ], [ 74, 87, "Mep2 proteins", "protein_type" ], [ 117, 126, "bacterial", "taxonomy_domain" ] ] }, { "sid": 95, "sent": "This segment (residues 450\u2013457 in ScMep2 and 446\u2013453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3).", "section": "RESULTS", "ner": [ [ 5, 12, "segment", "structure_element" ], [ 23, 30, "450\u2013457", "residue_range" ], [ 34, 40, "ScMep2", "protein" ], [ 45, 52, "446\u2013453", "residue_range" ], [ 56, 62, "CaMep2", "protein" ], [ 78, 104, "autoinhibitory (AI) region", "structure_element" ], [ 132, 139, "removal", "experimental_method" ], [ 153, 159, "active", "protein_state" ], [ 160, 171, "transporter", "protein_type" ], [ 179, 189, "absence of", "protein_state" ], [ 190, 194, "Npr1", "protein" ] ] }, { "sid": 96, "sent": "Where is the AI region and the Npr1 phosphorylation site located? Our structures reveal that surprisingly, the AI region is folded back onto the CTR and is not located near the centre of the trimer as expected from the bacterial structures (Fig. 4).", "section": "RESULTS", "ner": [ [ 13, 22, "AI region", "structure_element" ], [ 31, 35, "Npr1", "protein" ], [ 36, 56, "phosphorylation site", "site" ], [ 70, 80, "structures", "evidence" ], [ 111, 120, "AI region", "structure_element" ], [ 145, 148, "CTR", "structure_element" ], [ 191, 197, "trimer", "oligomeric_state" ], [ 219, 228, "bacterial", "taxonomy_domain" ], [ 229, 239, "structures", "evidence" ] ] }, { "sid": 97, "sent": "The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6).", "section": "RESULTS", "ner": [ [ 4, 13, "AI region", "structure_element" ], [ 52, 55, "TM2", "structure_element" ], [ 60, 63, "TM4", "structure_element" ], [ 88, 97, "main body", "structure_element" ], [ 105, 116, "transporter", "protein_type" ], [ 126, 129, "CTR", "structure_element" ], [ 189, 195, "Val447", "residue_name_number" ], [ 197, 203, "Asp449", "residue_name_number" ], [ 205, 211, "Pro450", "residue_name_number" ], [ 216, 222, "Arg452", "residue_name_number" ] ] }, { "sid": 98, "sent": "The AI regions have very similar conformations in CaMep2 and ScMep2, despite considerable differences in the rest of the CTR (Fig. 6).", "section": "RESULTS", "ner": [ [ 4, 14, "AI regions", "structure_element" ], [ 50, 56, "CaMep2", "protein" ], [ 61, 67, "ScMep2", "protein" ], [ 121, 124, "CTR", "structure_element" ] ] }, { "sid": 99, "sent": "Strikingly, the Npr1 target serine residue is located at the periphery of the trimer, far away (\u223c30\u2009\u00c5) from any channel exit (Fig. 6).", "section": "RESULTS", "ner": [ [ 16, 34, "Npr1 target serine", "site" ], [ 78, 84, "trimer", "oligomeric_state" ], [ 112, 124, "channel exit", "site" ] ] }, { "sid": 100, "sent": "Despite its location at the periphery of the trimer, the electron density for the serine is well defined in both Mep2 structures and corresponds to the non-phosphorylated state (Fig. 6).", "section": "RESULTS", "ner": [ [ 45, 51, "trimer", "oligomeric_state" ], [ 57, 73, "electron density", "evidence" ], [ 82, 88, "serine", "residue_name" ], [ 113, 117, "Mep2", "protein" ], [ 118, 128, "structures", "evidence" ], [ 152, 170, "non-phosphorylated", "protein_state" ] ] }, { "sid": 101, "sent": "This makes sense since the proteins were expressed in rich medium and confirms the recent suggestion by Boeckstaens et al. that the non-phosphorylated form of Mep2 corresponds to the inactive state.", "section": "RESULTS", "ner": [ [ 132, 150, "non-phosphorylated", "protein_state" ], [ 159, 163, "Mep2", "protein" ], [ 183, 191, "inactive", "protein_state" ] ] }, { "sid": 102, "sent": "For ScMep2, Ser457 is the most C-terminal residue for which electron density is visible, indicating that the region beyond Ser457 is disordered.", "section": "RESULTS", "ner": [ [ 4, 10, "ScMep2", "protein" ], [ 12, 18, "Ser457", "residue_name_number" ], [ 60, 76, "electron density", "evidence" ], [ 123, 129, "Ser457", "residue_name_number" ], [ 133, 143, "disordered", "protein_state" ] ] }, { "sid": 103, "sent": "In CaMep2, the visible part of the sequence extends for two residues beyond Ser453 (Fig. 6).", "section": "RESULTS", "ner": [ [ 3, 9, "CaMep2", "protein" ], [ 76, 82, "Ser453", "residue_name_number" ] ] }, { "sid": 104, "sent": "The peripheral location and disorder of the CTR beyond the kinase target site should facilitate the phosphorylation by Npr1.", "section": "RESULTS", "ner": [ [ 28, 36, "disorder", "protein_state" ], [ 44, 47, "CTR", "structure_element" ], [ 59, 77, "kinase target site", "site" ], [ 100, 115, "phosphorylation", "ptm" ], [ 119, 123, "Npr1", "protein" ] ] }, { "sid": 105, "sent": "The disordered part of the CTR is not conserved in ammonium transporters (Fig. 2), suggesting that it is not important for transport.", "section": "RESULTS", "ner": [ [ 4, 14, "disordered", "protein_state" ], [ 27, 30, "CTR", "structure_element" ], [ 34, 47, "not conserved", "protein_state" ], [ 51, 72, "ammonium transporters", "protein_type" ] ] }, { "sid": 106, "sent": "Interestingly, a ScMep2 457\u0394 truncation mutant in which a His-tag directly follows Ser457 is highly expressed but has low activity (Fig. 3 and Supplementary Fig. 1b), suggesting that the His-tag interferes with phosphorylation by Npr1.", "section": "RESULTS", "ner": [ [ 17, 23, "ScMep2", "protein" ], [ 24, 28, "457\u0394", "mutant" ], [ 29, 46, "truncation mutant", "protein_state" ], [ 83, 89, "Ser457", "residue_name_number" ], [ 118, 130, "low activity", "protein_state" ], [ 211, 226, "phosphorylation", "ptm" ], [ 230, 234, "Npr1", "protein" ] ] }, { "sid": 107, "sent": "The same mutant lacking the His-tag has WT properties (Supplementary Fig. 1b), confirming that the region following the phosphorylation site is dispensable for function.", "section": "RESULTS", "ner": [ [ 9, 15, "mutant", "mutant" ], [ 16, 35, "lacking the His-tag", "protein_state" ], [ 40, 42, "WT", "protein_state" ], [ 120, 140, "phosphorylation site", "site" ] ] }, { "sid": 108, "sent": "Mep2 lacking the AI region is conformationally heterogeneous", "section": "RESULTS", "ner": [ [ 0, 4, "Mep2", "protein" ], [ 5, 12, "lacking", "protein_state" ], [ 17, 26, "AI region", "structure_element" ], [ 30, 60, "conformationally heterogeneous", "protein_state" ] ] }, { "sid": 109, "sent": "Given that Ser457/453 is far from any channel exit (Fig. 6), the crucial question is how phosphorylation opens the Mep2 channel to generate an active transporter.", "section": "RESULTS", "ner": [ [ 11, 17, "Ser457", "residue_name_number" ], [ 18, 21, "453", "residue_number" ], [ 38, 50, "channel exit", "site" ], [ 89, 104, "phosphorylation", "ptm" ], [ 115, 119, "Mep2", "protein" ], [ 120, 127, "channel", "site" ], [ 143, 149, "active", "protein_state" ], [ 150, 161, "transporter", "protein_type" ] ] }, { "sid": 110, "sent": "Boeckstaens et al. proposed that phosphorylation does not affect channel activity directly, but instead relieves inhibition by the AI region.", "section": "RESULTS", "ner": [ [ 33, 48, "phosphorylation", "ptm" ], [ 131, 140, "AI region", "structure_element" ] ] }, { "sid": 111, "sent": "The data behind this hypothesis is the observation that a ScMep2 449-485\u0394 deletion mutant lacking the AI region is highly active in MA uptake both in the triple mep\u0394 and triple mep\u0394 npr1\u0394 backgrounds, implying that this Mep2 variant has a constitutively open channel.", "section": "RESULTS", "ner": [ [ 58, 64, "ScMep2", "protein" ], [ 65, 73, "449-485\u0394", "mutant" ], [ 74, 89, "deletion mutant", "protein_state" ], [ 90, 97, "lacking", "protein_state" ], [ 102, 111, "AI region", "structure_element" ], [ 115, 128, "highly active", "protein_state" ], [ 132, 134, "MA", "chemical" ], [ 154, 165, "triple mep\u0394", "mutant" ], [ 170, 187, "triple mep\u0394 npr1\u0394", "mutant" ], [ 220, 232, "Mep2 variant", "mutant" ], [ 239, 258, "constitutively open", "protein_state" ], [ 259, 266, "channel", "site" ] ] }, { "sid": 112, "sent": "We obtained a similar result for ammonium uptake by the 446\u0394 mutant (Fig. 3), supporting the data from Marini et al. We then constructed and purified the analogous CaMep2 442\u0394 truncation mutant and determined the crystal structure using data to 3.4\u2009\u00c5 resolution.", "section": "RESULTS", "ner": [ [ 56, 60, "446\u0394", "mutant" ], [ 61, 67, "mutant", "protein_state" ], [ 125, 149, "constructed and purified", "experimental_method" ], [ 164, 170, "CaMep2", "protein" ], [ 171, 175, "442\u0394", "mutant" ], [ 176, 193, "truncation mutant", "protein_state" ], [ 198, 208, "determined", "experimental_method" ], [ 213, 230, "crystal structure", "evidence" ] ] }, { "sid": 113, "sent": "The structure shows that removal of the AI region markedly increases the dynamics of the cytoplasmic parts of the transporter.", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 25, 35, "removal of", "experimental_method" ], [ 40, 49, "AI region", "structure_element" ], [ 89, 106, "cytoplasmic parts", "structure_element" ], [ 114, 125, "transporter", "protein_type" ] ] }, { "sid": 114, "sent": "This is not unexpected given the fact that the AI region bridges the CTR and the main body of Mep2 (Fig. 6).", "section": "RESULTS", "ner": [ [ 47, 56, "AI region", "structure_element" ], [ 69, 72, "CTR", "structure_element" ], [ 81, 90, "main body", "structure_element" ], [ 94, 98, "Mep2", "protein" ] ] }, { "sid": 115, "sent": "Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442\u0394 mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7).", "section": "RESULTS", "ner": [ [ 0, 7, "Density", "evidence" ], [ 12, 16, "ICL3", "structure_element" ], [ 25, 28, "CTR", "structure_element" ], [ 44, 50, "Arg415", "residue_name_number" ], [ 69, 73, "442\u0394", "mutant" ], [ 74, 80, "mutant", "protein_state" ], [ 90, 97, "density", "evidence" ], [ 112, 116, "ICLs", "structure_element" ], [ 127, 131, "ICL1", "structure_element" ], [ 176, 185, "structure", "evidence" ] ] }, { "sid": 116, "sent": "Interestingly, however, the Tyr49-His342 hydrogen bond that closes the channel in the WT protein is still present (Fig. 7 and Supplementary Fig. 2).", "section": "RESULTS", "ner": [ [ 28, 33, "Tyr49", "residue_name_number" ], [ 34, 40, "His342", "residue_name_number" ], [ 41, 54, "hydrogen bond", "bond_interaction" ], [ 86, 88, "WT", "protein_state" ] ] }, { "sid": 117, "sent": "Why then does this mutant appear to be constitutively active? We propose two possibilities.", "section": "RESULTS", "ner": [ [ 54, 60, "active", "protein_state" ] ] }, { "sid": 118, "sent": "The first one is that the open state is disfavoured by crystallization because of lower stability or due to crystal packing constraints.", "section": "RESULTS", "ner": [ [ 26, 30, "open", "protein_state" ], [ 55, 70, "crystallization", "experimental_method" ] ] }, { "sid": 119, "sent": "The second possibility is that the Tyr\u2013His hydrogen bond has to be disrupted by the incoming substrate to open the channel.", "section": "RESULTS", "ner": [ [ 35, 56, "Tyr\u2013His hydrogen bond", "site" ], [ 106, 110, "open", "protein_state" ] ] }, { "sid": 120, "sent": "The latter model would fit well with the NH3/H+ symport model in which the proton is relayed by the twin-His motif.", "section": "RESULTS", "ner": [ [ 41, 44, "NH3", "chemical" ], [ 45, 47, "H+", "chemical" ], [ 100, 114, "twin-His motif", "structure_element" ] ] }, { "sid": 121, "sent": "The importance of the Tyr\u2013His hydrogen bond is underscored by the fact that its removal in the ScMep2 Y53A mutant results in a constitutively active transporter (Fig. 3).", "section": "RESULTS", "ner": [ [ 22, 43, "Tyr\u2013His hydrogen bond", "site" ], [ 80, 87, "removal", "experimental_method" ], [ 95, 101, "ScMep2", "protein" ], [ 102, 106, "Y53A", "mutant" ], [ 107, 113, "mutant", "protein_state" ], [ 127, 148, "constitutively active", "protein_state" ], [ 149, 160, "transporter", "protein_type" ] ] }, { "sid": 122, "sent": "Phosphorylation causes a conformational change in the CTR", "section": "RESULTS", "ner": [ [ 0, 15, "Phosphorylation", "ptm" ], [ 54, 57, "CTR", "structure_element" ] ] }, { "sid": 123, "sent": "Do the Mep2 structures provide any clues regarding the potential effect of phosphorylation?", "section": "RESULTS", "ner": [ [ 7, 11, "Mep2", "protein" ], [ 12, 22, "structures", "evidence" ], [ 75, 90, "phosphorylation", "ptm" ] ] }, { "sid": 124, "sent": "The side-chain hydroxyl of Ser457/453 is located in a well-defined electronegative pocket that is solvent accessible (Fig. 6).", "section": "RESULTS", "ner": [ [ 27, 33, "Ser457", "residue_name_number" ], [ 34, 37, "453", "residue_number" ], [ 67, 89, "electronegative pocket", "site" ], [ 98, 116, "solvent accessible", "protein_state" ] ] }, { "sid": 125, "sent": "The closest atoms to the serine hydroxyl group are the backbone carbonyl atoms of Asp419, Glu420 and Glu421, which are 3\u20134\u2009\u00c5 away.", "section": "RESULTS", "ner": [ [ 25, 31, "serine", "residue_name" ], [ 82, 88, "Asp419", "residue_name_number" ], [ 90, 96, "Glu420", "residue_name_number" ], [ 101, 107, "Glu421", "residue_name_number" ] ] }, { "sid": 126, "sent": "We therefore predict that phosphorylation of Ser453 will result in steric clashes as well as electrostatic repulsion, which in turn might cause substantial conformational changes within the CTR.", "section": "RESULTS", "ner": [ [ 26, 41, "phosphorylation", "ptm" ], [ 45, 51, "Ser453", "residue_name_number" ], [ 190, 193, "CTR", "structure_element" ] ] }, { "sid": 127, "sent": "To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed \u2018DD mutant'), using data to a resolution of 2.4\u2009\u00c5. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6).", "section": "RESULTS", "ner": [ [ 28, 38, "determined", "experimental_method" ], [ 43, 52, "structure", "evidence" ], [ 60, 85, "phosphorylation-mimicking", "protein_state" ], [ 86, 97, "R452D/S453D", "mutant" ], [ 125, 134, "DD mutant", "mutant" ], [ 179, 201, "additional mutation of", "experimental_method" ], [ 206, 214, "arginine", "residue_name" ], [ 229, 249, "phosphorylation site", "site" ], [ 342, 351, "phosphate", "chemical" ], [ 423, 432, "AI region", "structure_element" ], [ 442, 451, "main body", "structure_element" ], [ 459, 470, "transporter", "protein_type" ] ] }, { "sid": 128, "sent": "The ammonium uptake activity of the S. cerevisiae version of the DD mutant is the same as that of WT Mep2 and the S453D mutant, indicating that the mutations do not affect transporter functionality in the triple mep\u0394 background (Fig. 3).", "section": "RESULTS", "ner": [ [ 4, 12, "ammonium", "chemical" ], [ 36, 49, "S. cerevisiae", "species" ], [ 65, 74, "DD mutant", "mutant" ], [ 98, 100, "WT", "protein_state" ], [ 101, 105, "Mep2", "protein" ], [ 114, 119, "S453D", "mutant" ], [ 120, 126, "mutant", "protein_state" ], [ 205, 216, "triple mep\u0394", "mutant" ] ] }, { "sid": 129, "sent": "Unexpectedly, the AI segment containing the mutated residues has only undergone a slight shift compared with the WT protein (Fig. 8 and Supplementary Fig. 3).", "section": "RESULTS", "ner": [ [ 18, 28, "AI segment", "structure_element" ], [ 113, 115, "WT", "protein_state" ] ] }, { "sid": 130, "sent": "By contrast, the conserved part of the CTR has undergone a large conformational change involving formation of a 12-residue-long \u03b1-helix from Leu427 to Asp438.", "section": "RESULTS", "ner": [ [ 17, 26, "conserved", "protein_state" ], [ 39, 42, "CTR", "structure_element" ], [ 112, 135, "12-residue-long \u03b1-helix", "structure_element" ], [ 141, 157, "Leu427 to Asp438", "residue_range" ] ] }, { "sid": 131, "sent": "In addition, residues Glu420-Leu423 including Glu421 of the ExxGxD motif are now disordered (Fig. 8 and Supplementary Fig. 3).", "section": "RESULTS", "ner": [ [ 22, 35, "Glu420-Leu423", "residue_range" ], [ 46, 52, "Glu421", "residue_name_number" ], [ 60, 72, "ExxGxD motif", "structure_element" ], [ 81, 91, "disordered", "protein_state" ] ] }, { "sid": 132, "sent": "This is the first time a large conformational change has been observed in an ammonium transporter as a result of a mutation, and confirms previous hypotheses that phosphorylation causes structural changes in the CTR.", "section": "RESULTS", "ner": [ [ 77, 97, "ammonium transporter", "protein_type" ], [ 115, 123, "mutation", "experimental_method" ], [ 163, 178, "phosphorylation", "ptm" ], [ 212, 215, "CTR", "structure_element" ] ] }, { "sid": 133, "sent": "To exclude the possibility that the additional R452D mutation is responsible for the observed changes, we also determined the structure of the \u2018single D' S453D mutant.", "section": "RESULTS", "ner": [ [ 47, 52, "R452D", "mutant" ], [ 111, 121, "determined", "experimental_method" ], [ 126, 135, "structure", "evidence" ], [ 144, 152, "single D", "mutant" ], [ 154, 159, "S453D", "mutant" ], [ 160, 166, "mutant", "protein_state" ] ] }, { "sid": 134, "sent": "As shown in Supplementary Fig. 4, the consequence of the single D mutation is very similar to that of the DD substitution, with conformational changes and increased dynamics confined to the conserved part of the CTR (Supplementary Fig. 4).", "section": "RESULTS", "ner": [ [ 57, 65, "single D", "mutant" ], [ 66, 74, "mutation", "experimental_method" ], [ 106, 121, "DD substitution", "mutant" ], [ 190, 199, "conserved", "protein_state" ], [ 212, 215, "CTR", "structure_element" ] ] }, { "sid": 135, "sent": "To supplement the crystal structures, we also performed modelling and MD studies of WT CaMep2, the DD mutant and phosphorylated protein (S453J).", "section": "RESULTS", "ner": [ [ 18, 36, "crystal structures", "evidence" ], [ 56, 65, "modelling", "experimental_method" ], [ 70, 72, "MD", "experimental_method" ], [ 84, 86, "WT", "protein_state" ], [ 87, 93, "CaMep2", "protein" ], [ 99, 108, "DD mutant", "mutant" ], [ 113, 127, "phosphorylated", "protein_state" ], [ 137, 142, "S453J", "mutant" ] ] }, { "sid": 136, "sent": "In the WT structure, the acidic residues Asp419, Glu420 and Glu421 are within hydrogen bonding distance of Ser453.", "section": "RESULTS", "ner": [ [ 7, 9, "WT", "protein_state" ], [ 10, 19, "structure", "evidence" ], [ 41, 47, "Asp419", "residue_name_number" ], [ 49, 55, "Glu420", "residue_name_number" ], [ 60, 66, "Glu421", "residue_name_number" ], [ 78, 94, "hydrogen bonding", "bond_interaction" ], [ 107, 113, "Ser453", "residue_name_number" ] ] }, { "sid": 137, "sent": "After 200\u2009ns of MD simulation, the interactions between these residues and Ser453 remain intact.", "section": "RESULTS", "ner": [ [ 16, 18, "MD", "experimental_method" ], [ 19, 29, "simulation", "experimental_method" ], [ 75, 81, "Ser453", "residue_name_number" ] ] }, { "sid": 138, "sent": "The protein backbone has an average r.m.s.d. of only \u223c3\u2009\u00c5 during the 200-ns simulation, indicating that the protein is stable.", "section": "RESULTS", "ner": [ [ 36, 44, "r.m.s.d.", "evidence" ], [ 76, 86, "simulation", "experimental_method" ], [ 119, 125, "stable", "protein_state" ] ] }, { "sid": 139, "sent": "There is flexibility in the side chains of the acidic residues so that they are able to form stable hydrogen bonds with Ser453.", "section": "RESULTS", "ner": [ [ 93, 99, "stable", "protein_state" ], [ 100, 114, "hydrogen bonds", "bond_interaction" ], [ 120, 126, "Ser453", "residue_name_number" ] ] }, { "sid": 140, "sent": "In particular, persistent hydrogen bonds are observed between the Ser453 hydroxyl group and the acidic group of Glu420, and also between the amine group of Ser453 and the backbone carbonyl of Glu420 (Supplementary Fig. 5).", "section": "RESULTS", "ner": [ [ 26, 40, "hydrogen bonds", "bond_interaction" ], [ 66, 72, "Ser453", "residue_name_number" ], [ 112, 118, "Glu420", "residue_name_number" ], [ 156, 162, "Ser453", "residue_name_number" ], [ 192, 198, "Glu420", "residue_name_number" ] ] }, { "sid": 141, "sent": "The DD mutant is also stable during the simulations, but the average backbone r.m.s.d of \u223c3.6\u2009\u00c5 suggests slightly more conformational flexibility than WT.", "section": "RESULTS", "ner": [ [ 4, 13, "DD mutant", "mutant" ], [ 22, 28, "stable", "protein_state" ], [ 40, 51, "simulations", "experimental_method" ], [ 78, 85, "r.m.s.d", "evidence" ], [ 151, 153, "WT", "protein_state" ] ] }, { "sid": 142, "sent": "As the simulation proceeds, the side chains of the acidic residues move away from Asp452 and Asp453, presumably to avoid electrostatic repulsion.", "section": "RESULTS", "ner": [ [ 7, 17, "simulation", "experimental_method" ], [ 82, 88, "Asp452", "residue_name_number" ], [ 93, 99, "Asp453", "residue_name_number" ] ] }, { "sid": 143, "sent": "For example, the distance between the Asp453 acidic oxygens and the Glu420 acidic oxygens increases from \u223c7 to >22\u2009\u00c5 after 200\u2009ns simulations, and thus these residues are not interacting.", "section": "RESULTS", "ner": [ [ 17, 25, "distance", "evidence" ], [ 38, 44, "Asp453", "residue_name_number" ], [ 68, 74, "Glu420", "residue_name_number" ], [ 130, 141, "simulations", "experimental_method" ] ] }, { "sid": 144, "sent": "The protein is structurally stable throughout the simulation with little deviation in the other parts of the protein.", "section": "RESULTS", "ner": [ [ 15, 34, "structurally stable", "protein_state" ], [ 50, 60, "simulation", "experimental_method" ] ] }, { "sid": 145, "sent": "Finally, the S453J mutant is also stable throughout the 200-ns simulation and has an average backbone deviation of \u223c3.8\u2009\u00c5, which is similar to the DD mutant.", "section": "RESULTS", "ner": [ [ 13, 18, "S453J", "mutant" ], [ 19, 25, "mutant", "protein_state" ], [ 34, 40, "stable", "protein_state" ], [ 63, 73, "simulation", "experimental_method" ], [ 147, 156, "DD mutant", "mutant" ] ] }, { "sid": 146, "sent": "The movement of the acidic residues away from Arg452 and Sep453 is more pronounced in this simulation in comparison with the movement away from Asp452 and Asp453 in the DD mutant.", "section": "RESULTS", "ner": [ [ 46, 52, "Arg452", "residue_name_number" ], [ 57, 63, "Sep453", "residue_name_number" ], [ 91, 101, "simulation", "experimental_method" ], [ 144, 150, "Asp452", "residue_name_number" ], [ 155, 161, "Asp453", "residue_name_number" ], [ 169, 178, "DD mutant", "mutant" ] ] }, { "sid": 147, "sent": "The distance between the phosphate of Sep453 and the acidic oxygen atoms of Glu420 is initially \u223c11\u2009\u00c5, but increases to >30\u2009\u00c5 after 200\u2009ns.", "section": "RESULTS", "ner": [ [ 4, 12, "distance", "evidence" ], [ 25, 34, "phosphate", "chemical" ], [ 38, 44, "Sep453", "residue_name_number" ], [ 76, 82, "Glu420", "residue_name_number" ] ] }, { "sid": 148, "sent": "The short helix formed by residues Leu427 to Asp438 unravels during the simulations to a disordered state.", "section": "RESULTS", "ner": [ [ 4, 15, "short helix", "structure_element" ], [ 35, 51, "Leu427 to Asp438", "residue_range" ], [ 72, 83, "simulations", "experimental_method" ], [ 89, 99, "disordered", "protein_state" ] ] }, { "sid": 149, "sent": "Thus, the MD simulations support the notion from the crystal structures that phosphorylation generates conformational changes in the conserved part of the CTR.", "section": "RESULTS", "ner": [ [ 10, 12, "MD", "experimental_method" ], [ 13, 24, "simulations", "experimental_method" ], [ 53, 71, "crystal structures", "evidence" ], [ 77, 92, "phosphorylation", "ptm" ], [ 133, 142, "conserved", "protein_state" ], [ 155, 158, "CTR", "structure_element" ] ] }, { "sid": 150, "sent": "However, the conformational changes for the phosphomimetic mutants in the crystals are confined to the CTR (Fig. 8), and the channels are still closed (Supplementary Fig. 2).", "section": "RESULTS", "ner": [ [ 44, 66, "phosphomimetic mutants", "mutant" ], [ 74, 82, "crystals", "evidence" ], [ 103, 106, "CTR", "structure_element" ], [ 125, 133, "channels", "site" ], [ 144, 150, "closed", "protein_state" ] ] }, { "sid": 151, "sent": "One possible explanation is that the mutants do not accurately mimic a phosphoserine, but the observation that the S453D and DD mutants are fully active in the absence of Npr1 suggests that the mutations do mimic the effect of phosphorylation (Fig. 3).", "section": "RESULTS", "ner": [ [ 37, 44, "mutants", "mutant" ], [ 71, 84, "phosphoserine", "residue_name" ], [ 115, 120, "S453D", "mutant" ], [ 125, 135, "DD mutants", "mutant" ], [ 140, 152, "fully active", "protein_state" ], [ 160, 170, "absence of", "protein_state" ], [ 171, 175, "Npr1", "protein" ], [ 194, 203, "mutations", "experimental_method" ], [ 227, 242, "phosphorylation", "ptm" ] ] }, { "sid": 152, "sent": "The fact that the S453D structure was obtained in the presence of 10\u2009mM ammonium ions suggests that the crystallization process favours closed states of the Mep2 channels.", "section": "RESULTS", "ner": [ [ 18, 23, "S453D", "mutant" ], [ 24, 33, "structure", "evidence" ], [ 72, 80, "ammonium", "chemical" ], [ 104, 119, "crystallization", "experimental_method" ], [ 136, 142, "closed", "protein_state" ], [ 157, 161, "Mep2", "protein" ], [ 162, 170, "channels", "site" ] ] }, { "sid": 153, "sent": "Knowledge about ammonium transporter structure has been obtained from experimental and theoretical studies on bacterial family members.", "section": "DISCUSS", "ner": [ [ 16, 36, "ammonium transporter", "protein_type" ], [ 37, 46, "structure", "evidence" ], [ 110, 119, "bacterial", "taxonomy_domain" ] ] }, { "sid": 154, "sent": "In addition, a number of biochemical and genetic studies are available for bacterial, fungal and plant proteins.", "section": "DISCUSS", "ner": [ [ 25, 56, "biochemical and genetic studies", "experimental_method" ], [ 75, 84, "bacterial", "taxonomy_domain" ], [ 86, 92, "fungal", "taxonomy_domain" ], [ 97, 102, "plant", "taxonomy_domain" ] ] }, { "sid": 155, "sent": "These efforts have advanced our knowledge considerably but have not yet yielded atomic-level answers to several important mechanistic questions, including how ammonium transport is regulated in eukaryotes and the mechanism of ammonium signalling.", "section": "DISCUSS", "ner": [ [ 159, 167, "ammonium", "chemical" ], [ 194, 204, "eukaryotes", "taxonomy_domain" ], [ 226, 234, "ammonium", "chemical" ] ] }, { "sid": 156, "sent": "In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open.", "section": "DISCUSS", "ner": [ [ 3, 23, "Arabidopsis thaliana", "species" ], [ 24, 31, "Amt-1;1", "protein" ], [ 33, 48, "phosphorylation", "ptm" ], [ 56, 59, "CTR", "structure_element" ], [ 68, 72, "T460", "residue_name_number" ], [ 98, 106, "ammonium", "chemical" ], [ 158, 176, "non-phosphorylated", "protein_state" ], [ 191, 196, "plant", "taxonomy_domain" ], [ 197, 208, "transporter", "protein_type" ], [ 212, 216, "open", "protein_state" ] ] }, { "sid": 157, "sent": "Interestingly, phosphomimetic mutations introduced into one monomer inactivate the entire trimer, indicating that (i) heterotrimerization occurs and (ii) the CTR mediates allosteric regulation of ammonium transport activity via phosphorylation.", "section": "DISCUSS", "ner": [ [ 15, 39, "phosphomimetic mutations", "mutant" ], [ 60, 67, "monomer", "oligomeric_state" ], [ 90, 96, "trimer", "oligomeric_state" ], [ 158, 161, "CTR", "structure_element" ], [ 196, 204, "ammonium", "chemical" ], [ 228, 243, "phosphorylation", "ptm" ] ] }, { "sid": 158, "sent": "Owing to the lack of structural information for plant AMTs, the details of channel closure and inter-monomer crosstalk are not yet clear.", "section": "DISCUSS", "ner": [ [ 48, 53, "plant", "taxonomy_domain" ], [ 54, 58, "AMTs", "protein_type" ], [ 75, 82, "channel", "site" ] ] }, { "sid": 159, "sent": "Contrasting with the plant transporters, the inactive states of Mep2 proteins under conditions of high ammonium are non-phosphorylated, with channels that are closed on the cytoplasmic side.", "section": "DISCUSS", "ner": [ [ 21, 26, "plant", "taxonomy_domain" ], [ 27, 39, "transporters", "protein_type" ], [ 45, 53, "inactive", "protein_state" ], [ 64, 77, "Mep2 proteins", "protein_type" ], [ 103, 111, "ammonium", "chemical" ], [ 116, 134, "non-phosphorylated", "protein_state" ], [ 141, 149, "channels", "site" ], [ 159, 165, "closed", "protein_state" ] ] }, { "sid": 160, "sent": "The reason why similar transporters such as A. thaliana Amt-1;1 and Mep2 are regulated in opposite ways by phosphorylation (inactivation in plants and activation in fungi) is not known.", "section": "DISCUSS", "ner": [ [ 23, 35, "transporters", "protein_type" ], [ 44, 55, "A. thaliana", "species" ], [ 56, 63, "Amt-1;1", "protein" ], [ 68, 72, "Mep2", "protein" ], [ 107, 122, "phosphorylation", "ptm" ], [ 124, 136, "inactivation", "protein_state" ], [ 140, 146, "plants", "taxonomy_domain" ], [ 151, 161, "activation", "protein_state" ], [ 165, 170, "fungi", "taxonomy_domain" ] ] }, { "sid": 161, "sent": "In fungi, preventing ammonium entry via channel closure in ammonium transporters would be one way to alleviate ammonium toxicity, in addition to ammonium excretion via Ato transporters and amino-acid secretion.", "section": "DISCUSS", "ner": [ [ 3, 8, "fungi", "taxonomy_domain" ], [ 21, 29, "ammonium", "chemical" ], [ 59, 80, "ammonium transporters", "protein_type" ], [ 111, 119, "ammonium", "chemical" ], [ 145, 153, "ammonium", "chemical" ], [ 168, 171, "Ato", "protein_type" ], [ 172, 184, "transporters", "protein_type" ] ] }, { "sid": 162, "sent": "By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3.", "section": "DISCUSS", "ner": [ [ 25, 35, "structures", "evidence" ], [ 39, 45, "closed", "protein_state" ], [ 46, 67, "ammonium transporters", "protein_type" ], [ 72, 81, "comparing", "experimental_method" ], [ 88, 98, "structures", "evidence" ], [ 108, 124, "permanently open", "protein_state" ], [ 125, 134, "bacterial", "taxonomy_domain" ], [ 165, 169, "Mep2", "protein_type" ], [ 170, 177, "channel", "site" ], [ 220, 223, "CTR", "structure_element" ], [ 228, 232, "ICL1", "structure_element" ], [ 237, 241, "ICL3", "structure_element" ] ] }, { "sid": 163, "sent": "More specifically, the close interactions between the CTR and ICL1/ICL3 present in open transporters are disrupted, causing ICL3 to move outwards and block the channel (Figs 4 and 9a).", "section": "DISCUSS", "ner": [ [ 54, 57, "CTR", "structure_element" ], [ 62, 66, "ICL1", "structure_element" ], [ 67, 71, "ICL3", "structure_element" ], [ 83, 87, "open", "protein_state" ], [ 88, 100, "transporters", "protein_type" ], [ 124, 128, "ICL3", "structure_element" ], [ 160, 167, "channel", "site" ] ] }, { "sid": 164, "sent": "In addition, ICL1 has shifted inwards to contribute to the channel closure by engaging His2 from the twin-His motif via hydrogen bonding with a highly conserved tyrosine hydroxyl group.", "section": "DISCUSS", "ner": [ [ 13, 17, "ICL1", "structure_element" ], [ 59, 66, "channel", "site" ], [ 87, 91, "His2", "residue_name_number" ], [ 101, 115, "twin-His motif", "structure_element" ], [ 120, 136, "hydrogen bonding", "bond_interaction" ], [ 144, 160, "highly conserved", "protein_state" ], [ 161, 169, "tyrosine", "residue_name" ] ] }, { "sid": 165, "sent": "Upon phosphorylation by the Npr1 kinase in response to nitrogen limitation, the region around the conserved ExxGxD motif undergoes a conformational change that opens the channel (Fig. 9).", "section": "DISCUSS", "ner": [ [ 5, 20, "phosphorylation", "ptm" ], [ 28, 32, "Npr1", "protein" ], [ 33, 39, "kinase", "protein_type" ], [ 55, 63, "nitrogen", "chemical" ], [ 98, 107, "conserved", "protein_state" ], [ 108, 120, "ExxGxD motif", "structure_element" ], [ 170, 177, "channel", "site" ] ] }, { "sid": 166, "sent": "Importantly, the structural similarities in the TM parts of Mep2 and AfAmt-1 (Fig. 5a) suggest that channel opening/closure does not require substantial changes in the residues lining the channel.", "section": "DISCUSS", "ner": [ [ 17, 40, "structural similarities", "evidence" ], [ 48, 56, "TM parts", "structure_element" ], [ 60, 64, "Mep2", "protein" ], [ 69, 76, "AfAmt-1", "protein" ], [ 100, 107, "channel", "site" ], [ 188, 195, "channel", "site" ] ] }, { "sid": 167, "sent": "How exactly the channel opens and whether opening is intra-monomeric are still open questions; it is possible that the change in the CTR may disrupt its interactions with ICL3 of the neighbouring monomer (Fig. 9b), which could result in opening of the neighbouring channel via inward movement of its ICL3.", "section": "DISCUSS", "ner": [ [ 16, 23, "channel", "site" ], [ 79, 83, "open", "protein_state" ], [ 133, 136, "CTR", "structure_element" ], [ 171, 175, "ICL3", "structure_element" ], [ 196, 203, "monomer", "oligomeric_state" ], [ 265, 272, "channel", "site" ], [ 300, 304, "ICL3", "structure_element" ] ] }, { "sid": 168, "sent": "Owing to the crosstalk between monomers, a single phosphorylation event might lead to opening of the entire trimer, although this has not yet been tested (Fig. 9b).", "section": "DISCUSS", "ner": [ [ 31, 39, "monomers", "oligomeric_state" ], [ 50, 65, "phosphorylation", "ptm" ], [ 108, 114, "trimer", "oligomeric_state" ] ] }, { "sid": 169, "sent": "Whether or not Mep2 channel opening requires, in addition to phosphorylation, disruption of the Tyr\u2013His2 interaction by the ammonium substrate is not yet clear.", "section": "DISCUSS", "ner": [ [ 15, 19, "Mep2", "protein_type" ], [ 20, 27, "channel", "site" ], [ 61, 76, "phosphorylation", "ptm" ], [ 96, 116, "Tyr\u2013His2 interaction", "site" ], [ 124, 132, "ammonium", "chemical" ] ] }, { "sid": 170, "sent": "Is our model for opening and closing of Mep2 channels valid for other eukaryotic ammonium transporters? Our structural data support previous studies and clarify the central role of the CTR and cytoplasmic loops in the transition between closed and open states.", "section": "DISCUSS", "ner": [ [ 40, 44, "Mep2", "protein" ], [ 45, 53, "channels", "site" ], [ 70, 80, "eukaryotic", "taxonomy_domain" ], [ 81, 102, "ammonium transporters", "protein_type" ], [ 108, 123, "structural data", "evidence" ], [ 185, 188, "CTR", "structure_element" ], [ 193, 210, "cytoplasmic loops", "structure_element" ], [ 237, 243, "closed", "protein_state" ], [ 248, 252, "open", "protein_state" ] ] }, { "sid": 171, "sent": "However, even the otherwise highly similar Mep2 proteins of S. cerevisiae and C. albicans have different structures for their CTRs (Fig. 1 and Supplementary Fig. 6).", "section": "DISCUSS", "ner": [ [ 43, 56, "Mep2 proteins", "protein_type" ], [ 60, 73, "S. cerevisiae", "species" ], [ 78, 89, "C. albicans", "species" ], [ 105, 115, "structures", "evidence" ], [ 126, 130, "CTRs", "structure_element" ] ] }, { "sid": 172, "sent": "In addition, the AI region of the CTR containing the Npr1 kinase site is conserved in only a subset of fungal transporters, suggesting that the details of the structural changes underpinning regulation vary.", "section": "DISCUSS", "ner": [ [ 17, 26, "AI region", "structure_element" ], [ 34, 37, "CTR", "structure_element" ], [ 53, 69, "Npr1 kinase site", "site" ], [ 73, 82, "conserved", "protein_state" ], [ 103, 109, "fungal", "taxonomy_domain" ], [ 110, 122, "transporters", "protein_type" ] ] }, { "sid": 173, "sent": "Nevertheless, given the central role of absolutely conserved residues within the ICL1-ICL3-CTR interaction network (Fig. 4), we propose that the structural basics of fungal ammonium transporter activation are conserved.", "section": "DISCUSS", "ner": [ [ 40, 60, "absolutely conserved", "protein_state" ], [ 81, 114, "ICL1-ICL3-CTR interaction network", "site" ], [ 166, 172, "fungal", "taxonomy_domain" ], [ 173, 181, "ammonium", "chemical" ], [ 209, 218, "conserved", "protein_state" ] ] }, { "sid": 174, "sent": "The fact that Mep2 orthologues of distantly related fungi are fully functional in ammonium transport and signalling in S. cerevisiae supports this notion.", "section": "DISCUSS", "ner": [ [ 14, 18, "Mep2", "protein_type" ], [ 52, 57, "fungi", "taxonomy_domain" ], [ 82, 90, "ammonium", "chemical" ], [ 119, 132, "S. cerevisiae", "species" ] ] }, { "sid": 175, "sent": "It should also be noted that the tyrosine residue interacting with His2 is highly conserved in fungal Mep2 orthologues, suggesting that the Tyr\u2013His2 hydrogen bond might be a general way to close Mep2 proteins.", "section": "DISCUSS", "ner": [ [ 33, 41, "tyrosine", "residue_name" ], [ 67, 71, "His2", "residue_name_number" ], [ 75, 91, "highly conserved", "protein_state" ], [ 95, 101, "fungal", "taxonomy_domain" ], [ 102, 106, "Mep2", "protein_type" ], [ 140, 162, "Tyr\u2013His2 hydrogen bond", "site" ], [ 189, 194, "close", "protein_state" ], [ 195, 208, "Mep2 proteins", "protein_type" ] ] }, { "sid": 176, "sent": "With regards to plant AMTs, it has been proposed that phosphorylation at T460 generates conformational changes that would close the neighbouring pore via the C terminus.", "section": "DISCUSS", "ner": [ [ 16, 21, "plant", "taxonomy_domain" ], [ 22, 26, "AMTs", "protein_type" ], [ 54, 69, "phosphorylation", "ptm" ], [ 73, 77, "T460", "residue_name_number" ], [ 145, 149, "pore", "site" ], [ 158, 168, "C terminus", "structure_element" ] ] }, { "sid": 177, "sent": "This assumption was based partly on a homology model for Amt-1;1 based on the (open) archaebacterial AfAmt-1 structure, which suggested that the C terminus of Amt-1;1 would extend further to the neighbouring monomer.", "section": "DISCUSS", "ner": [ [ 38, 52, "homology model", "experimental_method" ], [ 57, 64, "Amt-1;1", "protein" ], [ 79, 83, "open", "protein_state" ], [ 85, 100, "archaebacterial", "taxonomy_domain" ], [ 101, 108, "AfAmt-1", "protein" ], [ 109, 118, "structure", "evidence" ], [ 145, 155, "C terminus", "structure_element" ], [ 159, 166, "Amt-1;1", "protein" ], [ 208, 215, "monomer", "oligomeric_state" ] ] }, { "sid": 178, "sent": "Our Mep2 structures show that this assumption may not be correct (Fig. 4 and Supplementary Fig. 6).", "section": "DISCUSS", "ner": [ [ 4, 8, "Mep2", "protein" ], [ 9, 19, "structures", "evidence" ] ] }, { "sid": 179, "sent": "In addition, the considerable differences between structurally resolved CTR domains means that the exact environment of T460 in Amt-1;1 is also not known (Supplementary Fig. 6).", "section": "DISCUSS", "ner": [ [ 72, 75, "CTR", "structure_element" ], [ 120, 124, "T460", "residue_name_number" ], [ 128, 135, "Amt-1;1", "protein" ] ] }, { "sid": 180, "sent": "Based on the available structural information, we consider it more likely that phosphorylation-mediated pore closure in Amt-1;1 is intra-monomeric, via disruption of the interactions between the CTR and ICL1/ICL3 (for example, Y467-H239 and D458-K71).", "section": "DISCUSS", "ner": [ [ 23, 45, "structural information", "evidence" ], [ 120, 127, "Amt-1;1", "protein" ], [ 195, 198, "CTR", "structure_element" ], [ 203, 207, "ICL1", "structure_element" ], [ 208, 212, "ICL3", "structure_element" ], [ 227, 231, "Y467", "residue_name_number" ], [ 232, 236, "H239", "residue_name_number" ], [ 241, 245, "D458", "residue_name_number" ], [ 246, 249, "K71", "residue_name_number" ] ] }, { "sid": 181, "sent": "There is generally no equivalent for CaMep2 Tyr49 in plant AMTs, indicating that a Tyr\u2013His2 hydrogen bond as observed in Mep2 may not contribute to the closed state in plant transporters.", "section": "DISCUSS", "ner": [ [ 37, 43, "CaMep2", "protein" ], [ 44, 49, "Tyr49", "residue_name_number" ], [ 53, 58, "plant", "taxonomy_domain" ], [ 59, 63, "AMTs", "protein_type" ], [ 83, 105, "Tyr\u2013His2 hydrogen bond", "site" ], [ 121, 125, "Mep2", "protein" ], [ 152, 158, "closed", "protein_state" ], [ 168, 173, "plant", "taxonomy_domain" ], [ 174, 186, "transporters", "protein_type" ] ] }, { "sid": 182, "sent": "We propose that intra-monomeric CTR-ICL1/ICL3 interactions lie at the basis of regulation of both fungal and plant ammonium transporters; close interactions generate open channels, whereas the lack of \u2018intra-' interactions leads to inactive states.", "section": "DISCUSS", "ner": [ [ 16, 58, "intra-monomeric CTR-ICL1/ICL3 interactions", "site" ], [ 98, 104, "fungal", "taxonomy_domain" ], [ 109, 114, "plant", "taxonomy_domain" ], [ 115, 136, "ammonium transporters", "protein_type" ], [ 166, 170, "open", "protein_state" ], [ 171, 179, "channels", "site" ], [ 193, 200, "lack of", "protein_state" ], [ 232, 240, "inactive", "protein_state" ] ] }, { "sid": 183, "sent": "The need to regulate in opposite ways may be the reason why the phosphorylation sites are in different parts of the CTR, that is, centrally located close to the ExxGxD motif in AMTs and peripherally in Mep2.", "section": "DISCUSS", "ner": [ [ 64, 85, "phosphorylation sites", "site" ], [ 116, 119, "CTR", "structure_element" ], [ 161, 173, "ExxGxD motif", "structure_element" ], [ 177, 181, "AMTs", "protein_type" ], [ 202, 206, "Mep2", "protein" ] ] }, { "sid": 184, "sent": "In this way, phosphorylation can either lead to channel closing (in the case of AMTs) or channel opening in the case of Mep2.", "section": "DISCUSS", "ner": [ [ 13, 28, "phosphorylation", "ptm" ], [ 48, 55, "channel", "site" ], [ 80, 84, "AMTs", "protein_type" ], [ 89, 96, "channel", "site" ], [ 120, 124, "Mep2", "protein" ] ] }, { "sid": 185, "sent": "Our model also provides an explanation for the observation that certain mutations within the CTR completely abolish transport activity.", "section": "DISCUSS", "ner": [ [ 64, 81, "certain mutations", "mutant" ], [ 93, 96, "CTR", "structure_element" ] ] }, { "sid": 186, "sent": "An example of an inactivating residue is the glycine of the ExxGxD motif of the CTR.", "section": "DISCUSS", "ner": [ [ 45, 52, "glycine", "residue_name" ], [ 60, 72, "ExxGxD motif", "structure_element" ], [ 80, 83, "CTR", "structure_element" ] ] }, { "sid": 187, "sent": "Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs).", "section": "DISCUSS", "ner": [ [ 0, 8, "Mutation", "experimental_method" ], [ 26, 30, "G393", "residue_name_number" ], [ 34, 40, "EcAmtB", "protein" ], [ 42, 46, "G456", "residue_name_number" ], [ 50, 59, "AtAmt-1;1", "protein" ], [ 73, 85, "transporters", "protein_type" ], [ 100, 116, "Escherichia coli", "species" ], [ 117, 121, "AmtB", "protein" ], [ 126, 137, "A. thaliana", "species" ], [ 138, 145, "Amt-1;1", "protein" ] ] }, { "sid": 188, "sent": "Such mutations likely cause structural changes in the CTR that prevent close contacts between the CTR and ICL1/ICL3, thereby stabilizing a closed state that may be similar to that observed in Mep2.", "section": "DISCUSS", "ner": [ [ 54, 57, "CTR", "structure_element" ], [ 98, 101, "CTR", "structure_element" ], [ 106, 110, "ICL1", "structure_element" ], [ 111, 115, "ICL3", "structure_element" ], [ 139, 145, "closed", "protein_state" ], [ 192, 196, "Mep2", "protein" ] ] }, { "sid": 189, "sent": "Regulation and modulation of membrane transport by phosphorylation is known to occur in, for example, aquaporins and urea transporters, and is likely to be a common theme for eukaryotic channels and transporters.", "section": "DISCUSS", "ner": [ [ 51, 66, "phosphorylation", "ptm" ], [ 102, 112, "aquaporins", "protein_type" ], [ 117, 134, "urea transporters", "protein_type" ], [ 175, 185, "eukaryotic", "taxonomy_domain" ], [ 186, 194, "channels", "protein_type" ], [ 199, 211, "transporters", "protein_type" ] ] }, { "sid": 190, "sent": "Recently, phosphorylation was also shown to modulate substrate affinity in nitrate transporters.", "section": "DISCUSS", "ner": [ [ 10, 25, "phosphorylation", "ptm" ], [ 75, 95, "nitrate transporters", "protein_type" ] ] }, { "sid": 191, "sent": "With respect to ammonium transport, phosphorylation has thus far only been shown for A. thaliana AMTs and for S. cerevisiae Mep2 (refs).", "section": "DISCUSS", "ner": [ [ 16, 24, "ammonium", "chemical" ], [ 36, 51, "phosphorylation", "ptm" ], [ 85, 96, "A. thaliana", "species" ], [ 97, 101, "AMTs", "protein_type" ], [ 110, 123, "S. cerevisiae", "species" ], [ 124, 128, "Mep2", "protein" ] ] }, { "sid": 192, "sent": "However, the absence of GlnK proteins in eukaryotes suggests that phosphorylation-based regulation of ammonium transport may be widespread.", "section": "DISCUSS", "ner": [ [ 13, 23, "absence of", "protein_state" ], [ 24, 37, "GlnK proteins", "protein_type" ], [ 41, 51, "eukaryotes", "taxonomy_domain" ], [ 66, 81, "phosphorylation", "ptm" ], [ 102, 110, "ammonium", "chemical" ] ] }, { "sid": 193, "sent": "With respect to Mep2-mediated signalling to induce pseudohyphal growth, two models have been put forward as to how this occurs and why it is specific to Mep2 proteins.", "section": "DISCUSS", "ner": [ [ 16, 20, "Mep2", "protein_type" ], [ 153, 166, "Mep2 proteins", "protein_type" ] ] }, { "sid": 194, "sent": "In one model, signalling is proposed to depend on the nature of the transported substrate, which might be different in certain subfamilies of ammonium transporters (for example, Mep1/Mep3 versus Mep2).", "section": "DISCUSS", "ner": [ [ 142, 163, "ammonium transporters", "protein_type" ], [ 178, 182, "Mep1", "protein" ], [ 183, 187, "Mep3", "protein" ], [ 195, 199, "Mep2", "protein" ] ] }, { "sid": 195, "sent": "For example, NH3 uniport or symport of NH3/H+ might result in changes in local pH, but NH4+ uniport might not, and this difference might determine signalling.", "section": "DISCUSS", "ner": [ [ 13, 16, "NH3", "chemical" ], [ 39, 42, "NH3", "chemical" ], [ 43, 45, "H+", "chemical" ], [ 87, 91, "NH4+", "chemical" ] ] }, { "sid": 196, "sent": "In the other model, signalling is thought to require a distinct conformation of the Mep2 transporter occurring during the transport cycle.", "section": "DISCUSS", "ner": [ [ 84, 88, "Mep2", "protein" ], [ 89, 100, "transporter", "protein_type" ] ] }, { "sid": 197, "sent": "While the current study does not specifically address the mechanism of signalling underlying pseudohyphal growth, our structures do show that Mep2 proteins can assume different conformations.", "section": "DISCUSS", "ner": [ [ 118, 128, "structures", "evidence" ], [ 142, 155, "Mep2 proteins", "protein_type" ] ] }, { "sid": 198, "sent": "It is clear that ammonium transport across biomembranes remains a fascinating and challenging field in large part due to the unique properties of the substrate.", "section": "DISCUSS", "ner": [ [ 17, 25, "ammonium", "chemical" ] ] }, { "sid": 199, "sent": "Our Mep2 structural work now provides a foundation for future studies to uncover the details of the structural changes that occur during eukaryotic ammonium transport and signaling, and to assess the possibility to utilize small molecules to shut down ammonium sensing and downstream signalling pathways in pathogenic fungi.", "section": "DISCUSS", "ner": [ [ 4, 8, "Mep2", "protein" ], [ 137, 147, "eukaryotic", "taxonomy_domain" ], [ 148, 156, "ammonium", "chemical" ], [ 252, 260, "ammonium", "chemical" ], [ 318, 323, "fungi", "taxonomy_domain" ] ] }, { "sid": 200, "sent": "X-ray crystal structures of Mep2 transceptors.", "section": "FIG", "ner": [ [ 0, 24, "X-ray crystal structures", "evidence" ], [ 28, 32, "Mep2", "protein" ], [ 33, 45, "transceptors", "protein_type" ] ] }, { "sid": 201, "sent": "(a) Monomer cartoon models viewed from the side for (left) A. fulgidus Amt-1 (PDB ID 2B2H), S. cerevisiae Mep2 (middle) and C. albicans Mep2 (right).", "section": "FIG", "ner": [ [ 4, 11, "Monomer", "oligomeric_state" ], [ 71, 76, "Amt-1", "protein" ], [ 92, 105, "S. cerevisiae", "species" ], [ 106, 110, "Mep2", "protein" ], [ 124, 135, "C. albicans", "species" ], [ 136, 140, "Mep2", "protein" ] ] }, { "sid": 202, "sent": "The region showing ICL1 (blue), ICL3 (green) and the CTR (red) is boxed for comparison.", "section": "FIG", "ner": [ [ 19, 23, "ICL1", "structure_element" ], [ 32, 36, "ICL3", "structure_element" ], [ 53, 56, "CTR", "structure_element" ] ] }, { "sid": 203, "sent": "(b) CaMep2 trimer viewed from the intracellular side (right).", "section": "FIG", "ner": [ [ 4, 10, "CaMep2", "protein" ], [ 11, 17, "trimer", "oligomeric_state" ] ] }, { "sid": 204, "sent": "One monomer is coloured as in a and one monomer is coloured by B-factor (blue, low; red; high).", "section": "FIG", "ner": [ [ 4, 11, "monomer", "oligomeric_state" ], [ 40, 47, "monomer", "oligomeric_state" ] ] }, { "sid": 205, "sent": "The CTR is boxed.", "section": "FIG", "ner": [ [ 4, 7, "CTR", "structure_element" ] ] }, { "sid": 206, "sent": " (c) Overlay of ScMep2 (grey) and CaMep2 (rainbow), illustrating the differences in the CTRs.", "section": "FIG", "ner": [ [ 5, 12, "Overlay", "experimental_method" ], [ 16, 22, "ScMep2", "protein" ], [ 34, 40, "CaMep2", "protein" ], [ 88, 92, "CTRs", "structure_element" ] ] }, { "sid": 207, "sent": "Sequence conservation in ammonium transporters.", "section": "FIG", "ner": [ [ 0, 21, "Sequence conservation", "evidence" ], [ 25, 46, "ammonium transporters", "protein_type" ] ] }, { "sid": 208, "sent": "ClustalW alignment of CaMep2, ScMep2, A. fulgidus Amt-1, E. coli AmtB and A. thaliana Amt-1;1.", "section": "FIG", "ner": [ [ 0, 18, "ClustalW alignment", "experimental_method" ], [ 22, 28, "CaMep2", "protein" ], [ 30, 36, "ScMep2", "protein" ], [ 38, 49, "A. fulgidus", "species" ], [ 50, 55, "Amt-1", "protein" ], [ 65, 69, "AmtB", "protein" ], [ 74, 85, "A. thaliana", "species" ], [ 86, 93, "Amt-1;1", "protein" ] ] }, { "sid": 209, "sent": "The secondary structure elements observed for CaMep2 are indicated, with the numbers corresponding to the centre of the TM segment.", "section": "FIG", "ner": [ [ 46, 52, "CaMep2", "protein" ], [ 120, 130, "TM segment", "structure_element" ] ] }, { "sid": 210, "sent": "The conserved RxK motif in ICL1 is boxed in blue, the ER motif in ICL2 in cyan, the conserved ExxGxD motif of the CTR in red and the AI region in yellow.", "section": "FIG", "ner": [ [ 4, 13, "conserved", "protein_state" ], [ 14, 23, "RxK motif", "structure_element" ], [ 27, 31, "ICL1", "structure_element" ], [ 54, 62, "ER motif", "structure_element" ], [ 66, 70, "ICL2", "structure_element" ], [ 84, 93, "conserved", "protein_state" ], [ 94, 106, "ExxGxD motif", "structure_element" ], [ 114, 117, "CTR", "structure_element" ], [ 133, 142, "AI region", "structure_element" ] ] }, { "sid": 211, "sent": " Coloured residues are functionally important and correspond to those of the Phe gate (blue), the binding site Trp residue (magenta) and the twin-His motif (red).", "section": "FIG", "ner": [ [ 77, 85, "Phe gate", "site" ], [ 98, 110, "binding site", "site" ], [ 111, 114, "Trp", "residue_name" ] ] }, { "sid": 212, "sent": "The Npr1 kinase site in the AI region is highlighted pink.", "section": "FIG", "ner": [ [ 4, 20, "Npr1 kinase site", "site" ], [ 28, 37, "AI region", "structure_element" ] ] }, { "sid": 213, "sent": "The grey sequences at the C termini of CaMep2 and ScMep2 are not visible in the structures and are likely disordered.", "section": "FIG", "ner": [ [ 39, 45, "CaMep2", "protein" ], [ 50, 56, "ScMep2", "protein" ], [ 80, 90, "structures", "evidence" ], [ 99, 116, "likely disordered", "protein_state" ] ] }, { "sid": 214, "sent": "Growth of ScMep2 variants on low ammonium medium.", "section": "FIG", "ner": [ [ 0, 6, "Growth", "experimental_method" ], [ 10, 25, "ScMep2 variants", "mutant" ] ] }, { "sid": 215, "sent": "(a) The triple mep\u0394 strain (black) and triple mep\u0394 npr1\u0394 strain (grey) containing plasmids expressing WT and variant ScMep2 were grown on minimal medium containing 1\u2009mM ammonium sulphate.", "section": "FIG", "ner": [ [ 8, 19, "triple mep\u0394", "mutant" ], [ 102, 104, "WT", "protein_state" ], [ 109, 123, "variant ScMep2", "mutant" ], [ 129, 152, "grown on minimal medium", "experimental_method" ], [ 169, 186, "ammonium sulphate", "chemical" ] ] }, { "sid": 216, "sent": "The quantified cell density reflects logarithmic growth after 24\u2009h. Error bars are the s.d. for three replicates of each strain (b) The strains used in a were also serially diluted and spotted onto minimal agar plates containing glutamate (0.1%) or ammonium sulphate (1\u2009mM), and grown for 3 days at 30\u2009\u00b0C.", "section": "FIG", "ner": [ [ 15, 27, "cell density", "evidence" ], [ 229, 238, "glutamate", "chemical" ], [ 249, 266, "ammonium sulphate", "chemical" ] ] }, { "sid": 217, "sent": "Structural differences between Mep2 and bacterial ammonium transporters.", "section": "FIG", "ner": [ [ 31, 35, "Mep2", "protein" ], [ 40, 49, "bacterial", "taxonomy_domain" ] ] }, { "sid": 218, "sent": "(a) ICL1 in AfAmt-1 (light blue) and CaMep2 (dark blue), showing unwinding and inward movement in the fungal protein. (b) Stereo diagram viewed from the cytosol of ICL1, ICL3 (green) and the CTR (red) in AfAmt-1 (light colours) and CaMep2 (dark colours).", "section": "FIG", "ner": [ [ 4, 8, "ICL1", "structure_element" ], [ 12, 19, "AfAmt-1", "protein" ], [ 37, 43, "CaMep2", "protein" ], [ 102, 108, "fungal", "taxonomy_domain" ], [ 164, 168, "ICL1", "structure_element" ], [ 170, 174, "ICL3", "structure_element" ], [ 191, 194, "CTR", "structure_element" ], [ 204, 211, "AfAmt-1", "protein" ], [ 232, 238, "CaMep2", "protein" ] ] }, { "sid": 219, "sent": "The side chains of residues in the RxK motif as well as those of Tyr49 and His342 are labelled.", "section": "FIG", "ner": [ [ 35, 44, "RxK motif", "structure_element" ], [ 65, 70, "Tyr49", "residue_name_number" ], [ 75, 81, "His342", "residue_name_number" ] ] }, { "sid": 220, "sent": " The numbering is for CaMep2.", "section": "FIG", "ner": [ [ 22, 28, "CaMep2", "protein" ] ] }, { "sid": 221, "sent": "(c) Conserved residues in ICL1-3 and the CTR.", "section": "FIG", "ner": [ [ 4, 13, "Conserved", "protein_state" ], [ 26, 32, "ICL1-3", "structure_element" ], [ 41, 44, "CTR", "structure_element" ] ] }, { "sid": 222, "sent": "Views from the cytosol for CaMep2 (left) and AfAmt-1, highlighting the large differences in conformation of the conserved residues in ICL1 (RxK motif; blue), ICL2 (ER motif; cyan), ICL3 (green) and the CTR (red).", "section": "FIG", "ner": [ [ 27, 33, "CaMep2", "protein" ], [ 45, 52, "AfAmt-1", "protein" ], [ 112, 121, "conserved", "protein_state" ], [ 134, 138, "ICL1", "structure_element" ], [ 158, 162, "ICL2", "structure_element" ], [ 164, 172, "ER motif", "structure_element" ], [ 181, 185, "ICL3", "structure_element" ], [ 202, 205, "CTR", "structure_element" ] ] }, { "sid": 223, "sent": "The labelled residues are analogous within both structures.", "section": "FIG", "ner": [ [ 48, 58, "structures", "evidence" ] ] }, { "sid": 224, "sent": "In b and c, the centre of the trimer is at top.", "section": "FIG", "ner": [ [ 30, 36, "trimer", "oligomeric_state" ] ] }, { "sid": 225, "sent": "Channel closures in Mep2.", "section": "FIG", "ner": [ [ 20, 24, "Mep2", "protein" ] ] }, { "sid": 226, "sent": "(a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2.", "section": "FIG", "ner": [ [ 11, 24, "superposition", "experimental_method" ], [ 28, 35, "AfAmt-1", "protein" ], [ 40, 46, "CaMep2", "protein" ], [ 75, 83, "Phe gate", "site" ], [ 85, 89, "His2", "residue_name_number" ], [ 97, 111, "twin-His motif", "structure_element" ], [ 120, 128, "tyrosine", "residue_name" ], [ 137, 140, "Y49", "residue_name_number" ], [ 144, 147, "TM1", "structure_element" ], [ 161, 174, "hydrogen bond", "bond_interaction" ], [ 180, 184, "His2", "residue_name_number" ], [ 188, 194, "CaMep2", "protein" ], [ 271, 284, "channel block", "structure_element" ], [ 314, 320, "CaMep2", "protein" ] ] }, { "sid": 227, "sent": "The Npr1 kinase target Ser453 is dephosphorylated and located in an electronegative pocket.", "section": "FIG", "ner": [ [ 4, 8, "Npr1", "protein" ], [ 9, 15, "kinase", "protein_type" ], [ 23, 29, "Ser453", "residue_name_number" ], [ 33, 49, "dephosphorylated", "protein_state" ], [ 68, 90, "electronegative pocket", "site" ] ] }, { "sid": 228, "sent": "(a) Stereoviews of CaMep2 showing 2Fo\u2013Fc electron density (contoured at 1.0\u2009\u03c3) for CTR residues Asp419-Met422 and for Tyr446-Thr455 of the AI region.", "section": "FIG", "ner": [ [ 19, 25, "CaMep2", "protein" ], [ 83, 86, "CTR", "structure_element" ], [ 96, 109, "Asp419-Met422", "residue_range" ], [ 118, 131, "Tyr446-Thr455", "residue_range" ], [ 139, 148, "AI region", "structure_element" ] ] }, { "sid": 229, "sent": "The phosphorylation target residue Ser453 is labelled in bold.", "section": "FIG", "ner": [ [ 4, 19, "phosphorylation", "ptm" ], [ 35, 41, "Ser453", "residue_name_number" ] ] }, { "sid": 230, "sent": "(b) Overlay of the CTRs of ScMep2 (grey) and CaMep2 (green), showing the similar electronegative environment surrounding the phosphorylation site (P).", "section": "FIG", "ner": [ [ 4, 11, "Overlay", "experimental_method" ], [ 19, 23, "CTRs", "structure_element" ], [ 27, 33, "ScMep2", "protein" ], [ 45, 51, "CaMep2", "protein" ], [ 125, 145, "phosphorylation site", "site" ] ] }, { "sid": 231, "sent": "The AI regions are coloured magenta.", "section": "FIG", "ner": [ [ 4, 14, "AI regions", "structure_element" ] ] }, { "sid": 232, "sent": "(c) Cytoplasmic view of the Mep2 trimer indicating the large distance between Ser453 and the channel exits (circles; Ile52 lining the channel exit is shown).", "section": "FIG", "ner": [ [ 28, 32, "Mep2", "protein" ], [ 33, 39, "trimer", "oligomeric_state" ], [ 78, 84, "Ser453", "residue_name_number" ], [ 93, 106, "channel exits", "site" ], [ 117, 122, "Ile52", "residue_name_number" ], [ 134, 146, "channel exit", "site" ] ] }, { "sid": 233, "sent": "Effect of removal of the AI region on Mep2 structure.", "section": "FIG", "ner": [ [ 10, 17, "removal", "experimental_method" ], [ 25, 34, "AI region", "structure_element" ], [ 38, 42, "Mep2", "protein" ], [ 43, 52, "structure", "evidence" ] ] }, { "sid": 234, "sent": "(a) Side views for WT CaMep2 (left) and the truncation mutant 442\u0394 (right).", "section": "FIG", "ner": [ [ 19, 21, "WT", "protein_state" ], [ 22, 28, "CaMep2", "protein" ], [ 44, 61, "truncation mutant", "protein_state" ], [ 62, 66, "442\u0394", "mutant" ] ] }, { "sid": 235, "sent": "The latter is shown as a putty model according to B-factors to illustrate the disorder in the protein on the cytoplasmic side.", "section": "FIG", "ner": [ [ 78, 86, "disorder", "protein_state" ] ] }, { "sid": 236, "sent": " Missing regions are labelled. (b) Stereo superpositions of WT CaMep2 and the truncation mutant.", "section": "FIG", "ner": [ [ 42, 56, "superpositions", "experimental_method" ], [ 60, 62, "WT", "protein_state" ], [ 63, 69, "CaMep2", "protein" ], [ 78, 95, "truncation mutant", "protein_state" ] ] }, { "sid": 237, "sent": "2Fo\u2013Fc electron density (contoured at 1.0\u2009\u03c3) for residues Tyr49 and His342 is shown for the truncation mutant.", "section": "FIG", "ner": [ [ 58, 63, "Tyr49", "residue_name_number" ], [ 68, 74, "His342", "residue_name_number" ], [ 92, 109, "truncation mutant", "protein_state" ] ] }, { "sid": 238, "sent": "Phosphorylation causes conformational changes in the CTR.", "section": "FIG", "ner": [ [ 0, 15, "Phosphorylation", "ptm" ], [ 53, 56, "CTR", "structure_element" ] ] }, { "sid": 239, "sent": "(a) Cytoplasmic view of the DD mutant trimer, with WT CaMep2 superposed in grey for one of the monomers.", "section": "FIG", "ner": [ [ 28, 37, "DD mutant", "mutant" ], [ 38, 44, "trimer", "oligomeric_state" ], [ 51, 53, "WT", "protein_state" ], [ 54, 60, "CaMep2", "protein" ], [ 61, 71, "superposed", "experimental_method" ], [ 95, 103, "monomers", "oligomeric_state" ] ] }, { "sid": 240, "sent": "The arrow indicates the phosphorylation site.", "section": "FIG", "ner": [ [ 24, 44, "phosphorylation site", "site" ] ] }, { "sid": 241, "sent": "The AI region is coloured magenta.", "section": "FIG", "ner": [ [ 4, 13, "AI region", "structure_element" ] ] }, { "sid": 242, "sent": "(b) Monomer side-view superposition of WT CaMep2 and the DD mutant, showing the conformational change and disorder around the ExxGxD motif.", "section": "FIG", "ner": [ [ 4, 11, "Monomer", "oligomeric_state" ], [ 22, 35, "superposition", "experimental_method" ], [ 39, 41, "WT", "protein_state" ], [ 42, 48, "CaMep2", "protein" ], [ 57, 66, "DD mutant", "mutant" ], [ 126, 138, "ExxGxD motif", "structure_element" ] ] }, { "sid": 243, "sent": "Side chains for residues 452 and 453 are shown as stick models.", "section": "FIG", "ner": [ [ 25, 28, "452", "residue_number" ], [ 33, 36, "453", "residue_number" ] ] }, { "sid": 244, "sent": "Schematic model for phosphorylation-based regulation of Mep2 ammonium transporters.", "section": "FIG", "ner": [ [ 56, 60, "Mep2", "protein" ] ] }, { "sid": 245, "sent": "(a) In the closed, non-phosphorylated state (i), the CTR (magenta) and ICL3 (green) are far apart with the latter blocking the intracellular channel exit (indicated with a hatched circle).", "section": "FIG", "ner": [ [ 11, 17, "closed", "protein_state" ], [ 19, 37, "non-phosphorylated", "protein_state" ], [ 53, 56, "CTR", "structure_element" ], [ 71, 75, "ICL3", "structure_element" ], [ 141, 153, "channel exit", "site" ] ] }, { "sid": 246, "sent": "Upon phosphorylation and mimicked by the CaMep2 S453D and DD mutants (ii), the region around the ExxGxD motif undergoes a conformational change that results in the CTR interacting with the inward-moving ICL3, opening the channel (full circle) (iii).", "section": "FIG", "ner": [ [ 5, 20, "phosphorylation", "ptm" ], [ 25, 33, "mimicked", "protein_state" ], [ 41, 47, "CaMep2", "protein" ], [ 48, 53, "S453D", "mutant" ], [ 58, 68, "DD mutants", "mutant" ], [ 97, 109, "ExxGxD motif", "structure_element" ], [ 164, 167, "CTR", "structure_element" ], [ 203, 207, "ICL3", "structure_element" ], [ 221, 228, "channel", "site" ] ] }, { "sid": 247, "sent": "The open-channel Mep2 structure is represented by archaebacterial Amt-1 and shown in lighter colours consistent with Fig. 4.", "section": "FIG", "ner": [ [ 4, 8, "open", "protein_state" ], [ 9, 16, "channel", "site" ], [ 17, 21, "Mep2", "protein" ], [ 22, 31, "structure", "evidence" ], [ 50, 65, "archaebacterial", "taxonomy_domain" ], [ 66, 71, "Amt-1", "protein" ] ] }, { "sid": 248, "sent": "As discussed in the text, similar structural arrangements may occur in plant AMTs.", "section": "FIG", "ner": [ [ 71, 76, "plant", "taxonomy_domain" ], [ 77, 81, "AMTs", "protein_type" ] ] }, { "sid": 249, "sent": "In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR\u2013ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow).", "section": "FIG", "ner": [ [ 26, 30, "open", "protein_state" ], [ 31, 38, "channel", "site" ], [ 58, 76, "non-phosphorylated", "protein_state" ], [ 84, 99, "phosphorylation", "ptm" ], [ 144, 151, "channel", "site" ], [ 216, 231, "phosphorylation", "ptm" ], [ 237, 241, "Mep2", "protein" ], [ 242, 249, "monomer", "oligomeric_state" ], [ 271, 275, "open", "protein_state" ], [ 276, 284, "channels", "site" ], [ 299, 305, "trimer", "oligomeric_state" ], [ 353, 356, "CTR", "structure_element" ], [ 361, 365, "ICL3", "structure_element" ], [ 384, 391, "monomer", "oligomeric_state" ] ] } ] }, "PMC4968113": { "annotations": [ { "sid": 0, "sent": "Structural diversity in a human antibody germline library", "section": "TITLE", "ner": [ [ 26, 31, "human", "species" ], [ 32, 40, "antibody", "protein_type" ] ] }, { "sid": 1, "sent": "To support antibody therapeutic development, the crystal structures of a set of 16 germline variants composed of 4 different kappa light chains paired with 4 different heavy chains have been determined.", "section": "ABSTRACT", "ner": [ [ 11, 19, "antibody", "protein_type" ], [ 49, 67, "crystal structures", "evidence" ], [ 125, 143, "kappa light chains", "structure_element" ], [ 168, 180, "heavy chains", "structure_element" ] ] }, { "sid": 2, "sent": "All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure.", "section": "ABSTRACT", "ner": [ [ 9, 21, "heavy chains", "structure_element" ], [ 29, 54, "antigen-binding fragments", "structure_element" ], [ 56, 60, "Fabs", "structure_element" ], [ 76, 110, "complementarity-determining region", "structure_element" ], [ 112, 115, "CDR", "structure_element" ], [ 117, 119, "H3", "structure_element" ], [ 152, 155, "Fab", "structure_element" ], [ 156, 165, "structure", "evidence" ] ] }, { "sid": 3, "sent": "The structure analyses include comparisons of the overall structures, canonical structures of the CDRs and the VH:VL packing interactions.", "section": "ABSTRACT", "ner": [ [ 4, 22, "structure analyses", "experimental_method" ], [ 58, 68, "structures", "evidence" ], [ 80, 90, "structures", "evidence" ], [ 98, 102, "CDRs", "structure_element" ], [ 111, 116, "VH:VL", "complex_assembly" ], [ 117, 137, "packing interactions", "bond_interaction" ] ] }, { "sid": 4, "sent": "The CDR conformations for the most part are tightly clustered, especially for the ones with shorter lengths.", "section": "ABSTRACT", "ner": [ [ 4, 7, "CDR", "structure_element" ] ] }, { "sid": 5, "sent": "The longer CDRs with tandem glycines or serines have more conformational diversity than the others.", "section": "ABSTRACT", "ner": [ [ 4, 10, "longer", "protein_state" ], [ 11, 15, "CDRs", "structure_element" ], [ 28, 36, "glycines", "residue_name" ], [ 40, 47, "serines", "residue_name" ] ] }, { "sid": 6, "sent": "CDR H3, despite having the same amino acid sequence, exhibits the largest conformational diversity.", "section": "ABSTRACT", "ner": [ [ 0, 3, "CDR", "structure_element" ], [ 4, 6, "H3", "structure_element" ] ] }, { "sid": 7, "sent": "About half of the structures have CDR H3 conformations similar to that of the parent; the others diverge significantly.", "section": "ABSTRACT", "ner": [ [ 18, 28, "structures", "evidence" ], [ 34, 37, "CDR", "structure_element" ], [ 38, 40, "H3", "structure_element" ] ] }, { "sid": 8, "sent": "One conclusion is that the CDR H3 conformations are influenced by both their amino acid sequence and their structural environment determined by the heavy and light chain pairing.", "section": "ABSTRACT", "ner": [ [ 27, 30, "CDR", "structure_element" ], [ 31, 33, "H3", "structure_element" ], [ 148, 153, "heavy", "structure_element" ], [ 158, 169, "light chain", "structure_element" ] ] }, { "sid": 9, "sent": "The stem regions of 14 of the variant pairs are in the \u2018kinked\u2019 conformation, and only 2 are in the extended conformation.", "section": "ABSTRACT", "ner": [ [ 4, 16, "stem regions", "structure_element" ], [ 56, 62, "kinked", "protein_state" ], [ 100, 108, "extended", "protein_state" ] ] }, { "sid": 10, "sent": "The packing of the VH and VL domains is consistent with our knowledge of antibody structure, and the tilt angles between these domains cover a range of 11 degrees.", "section": "ABSTRACT", "ner": [ [ 19, 21, "VH", "structure_element" ], [ 26, 28, "VL", "structure_element" ], [ 73, 81, "antibody", "protein_type" ], [ 82, 91, "structure", "evidence" ], [ 101, 112, "tilt angles", "evidence" ] ] }, { "sid": 11, "sent": "Two of 16 structures showed particularly large variations in the tilt angles when compared with the other pairings.", "section": "ABSTRACT", "ner": [ [ 10, 20, "structures", "evidence" ], [ 65, 76, "tilt angles", "evidence" ] ] }, { "sid": 12, "sent": "The structures and their analyses provide a rich foundation for future antibody modeling and engineering efforts.", "section": "ABSTRACT", "ner": [ [ 4, 14, "structures", "evidence" ], [ 71, 79, "antibody", "protein_type" ] ] }, { "sid": 13, "sent": "At present, therapeutic antibodies are the largest class of biotherapeutic proteins that are in clinical trials.", "section": "INTRO", "ner": [ [ 24, 34, "antibodies", "protein_type" ] ] }, { "sid": 14, "sent": "The use of monoclonal antibodies as therapeutics began in the early 1980s, and their composition has transitioned from murine antibodies to generally less immunogenic humanized and human antibodies.", "section": "INTRO", "ner": [ [ 22, 32, "antibodies", "protein_type" ], [ 119, 125, "murine", "taxonomy_domain" ], [ 126, 136, "antibodies", "protein_type" ], [ 181, 186, "human", "species" ], [ 187, 197, "antibodies", "protein_type" ] ] }, { "sid": 15, "sent": "The technologies currently used to obtain human antibodies include transgenic mice containing human antibody repertoires, cloning directly from human B cells, and in vitro selection from antibody libraries using various display technologies.", "section": "INTRO", "ner": [ [ 42, 47, "human", "species" ], [ 48, 58, "antibodies", "protein_type" ], [ 78, 82, "mice", "taxonomy_domain" ], [ 94, 99, "human", "species" ], [ 100, 108, "antibody", "protein_type" ], [ 144, 149, "human", "species" ], [ 163, 181, "in vitro selection", "experimental_method" ], [ 187, 205, "antibody libraries", "experimental_method" ] ] }, { "sid": 16, "sent": "Once a candidate antibody is identified, protein engineering is usually required to produce a molecule with the right biophysical and functional properties.", "section": "INTRO", "ner": [ [ 17, 25, "antibody", "protein_type" ], [ 41, 60, "protein engineering", "experimental_method" ] ] }, { "sid": 17, "sent": "All engineering efforts are guided by our understanding of the atomic structures of antibodies.", "section": "INTRO", "ner": [ [ 63, 80, "atomic structures", "evidence" ], [ 84, 94, "antibodies", "protein_type" ] ] }, { "sid": 18, "sent": "In such efforts, the crystal structure of the specific antibody may not be available, but modeling can be used to guide the engineering efforts.", "section": "INTRO", "ner": [ [ 21, 38, "crystal structure", "evidence" ], [ 55, 63, "antibody", "protein_type" ] ] }, { "sid": 19, "sent": "Today's antibody modeling approaches, which normally focus on the variable region, are being developed by the application of structural principles and insights that are evolving as our knowledge of antibody structures continues to expand.", "section": "INTRO", "ner": [ [ 8, 16, "antibody", "protein_type" ], [ 66, 81, "variable region", "structure_element" ], [ 198, 206, "antibody", "protein_type" ], [ 207, 217, "structures", "evidence" ] ] }, { "sid": 20, "sent": "Our current structural knowledge of antibodies is based on a multitude of studies that used many techniques to gain insight into the functional and structural properties of this class of macromolecule.", "section": "INTRO", "ner": [ [ 36, 46, "antibodies", "protein_type" ] ] }, { "sid": 21, "sent": "Five different antibody isotypes occur, IgG, IgD, IgE, IgA and IgM, and each isotype has a unique role in the adaptive immune system.", "section": "INTRO", "ner": [ [ 15, 23, "antibody", "protein_type" ], [ 40, 43, "IgG", "protein" ], [ 45, 48, "IgD", "protein" ], [ 50, 53, "IgE", "protein" ], [ 55, 58, "IgA", "protein" ], [ 63, 66, "IgM", "protein" ] ] }, { "sid": 22, "sent": "IgG, IgD and IgE isotypes are composed of 2 heavy chains (HCs) and 2 light chains (LCs) linked through disulfide bonds, while IgA and IgM are double and quintuple versions of antibodies, respectively.", "section": "INTRO", "ner": [ [ 0, 3, "IgG", "protein" ], [ 5, 8, "IgD", "protein" ], [ 13, 16, "IgE", "protein" ], [ 44, 56, "heavy chains", "structure_element" ], [ 58, 61, "HCs", "structure_element" ], [ 69, 81, "light chains", "structure_element" ], [ 83, 86, "LCs", "structure_element" ], [ 103, 118, "disulfide bonds", "ptm" ], [ 126, 129, "IgA", "protein" ], [ 134, 137, "IgM", "protein" ], [ 175, 185, "antibodies", "protein_type" ] ] }, { "sid": 23, "sent": "Isotypes IgG, IgD and IgA each have 4 domains, one variable (V) and 3 constant (C) domains, while IgE and IgM each have the same 4 domains along with an additional C domain.", "section": "INTRO", "ner": [ [ 9, 12, "IgG", "protein" ], [ 14, 17, "IgD", "protein" ], [ 22, 25, "IgA", "protein" ], [ 51, 59, "variable", "structure_element" ], [ 61, 62, "V", "structure_element" ], [ 70, 78, "constant", "structure_element" ], [ 80, 81, "C", "structure_element" ], [ 98, 101, "IgE", "protein" ], [ 106, 109, "IgM", "protein" ], [ 164, 172, "C domain", "structure_element" ] ] }, { "sid": 24, "sent": "These multimeric forms are linked with an additional J chain.", "section": "INTRO", "ner": [ [ 53, 54, "J", "structure_element" ] ] }, { "sid": 25, "sent": "The LCs that associate with the HCs are divided into 2 functionally indistinguishable classes, \u03ba and \u03bb.", "section": "INTRO", "ner": [ [ 4, 7, "LCs", "structure_element" ], [ 32, 35, "HCs", "structure_element" ], [ 95, 96, "\u03ba", "structure_element" ], [ 101, 102, "\u03bb", "structure_element" ] ] }, { "sid": 26, "sent": "Both \u03ba and \u03bb polypeptide chains are composed of a single V domain and a single C domain.", "section": "INTRO", "ner": [ [ 5, 6, "\u03ba", "structure_element" ], [ 11, 12, "\u03bb", "structure_element" ], [ 57, 65, "V domain", "structure_element" ], [ 79, 87, "C domain", "structure_element" ] ] }, { "sid": 27, "sent": "The heavy and light chains are composed of structural domains that have \u223c110 amino acid residues.", "section": "INTRO", "ner": [ [ 4, 9, "heavy", "structure_element" ], [ 14, 26, "light chains", "structure_element" ], [ 43, 61, "structural domains", "structure_element" ], [ 72, 96, "\u223c110 amino acid residues", "residue_range" ] ] }, { "sid": 28, "sent": "These domains have a common folding pattern often referred to as the \u201cimmunoglobulin fold,\u201d formed by the packing together of 2 anti-parallel \u03b2-sheets.", "section": "INTRO", "ner": [ [ 70, 89, "immunoglobulin fold", "structure_element" ], [ 128, 150, "anti-parallel \u03b2-sheets", "structure_element" ] ] }, { "sid": 29, "sent": "All immunoglobulin chains have an N-terminal V domain followed by 1 to 4 C domains, depending upon the chain type.", "section": "INTRO", "ner": [ [ 4, 25, "immunoglobulin chains", "protein_type" ], [ 45, 53, "V domain", "structure_element" ], [ 73, 82, "C domains", "structure_element" ] ] }, { "sid": 30, "sent": "In antibodies, the heavy and light chain V domains pack together forming the antigen combining site.", "section": "INTRO", "ner": [ [ 3, 13, "antibodies", "protein_type" ], [ 19, 40, "heavy and light chain", "structure_element" ], [ 41, 50, "V domains", "structure_element" ], [ 77, 99, "antigen combining site", "site" ] ] }, { "sid": 31, "sent": "This site, which interacts with the antigen (or target), is the focus of current antibody modeling efforts.", "section": "INTRO", "ner": [ [ 81, 89, "antibody", "protein_type" ] ] }, { "sid": 32, "sent": "This interaction site is composed of 6 complementarity-determining regions (CDRs) that were identified in early antibody amino acid sequence analyses to be hypervariable in nature, and thus are responsible for the sequence and structural diversity of our antibody repertoire.", "section": "INTRO", "ner": [ [ 5, 21, "interaction site", "site" ], [ 39, 74, "complementarity-determining regions", "structure_element" ], [ 76, 80, "CDRs", "structure_element" ], [ 112, 149, "antibody amino acid sequence analyses", "experimental_method" ], [ 156, 169, "hypervariable", "protein_state" ], [ 255, 263, "antibody", "protein_type" ] ] }, { "sid": 33, "sent": "The sequence diversity of the CDR regions presents a substantial challenge to antibody modeling.", "section": "INTRO", "ner": [ [ 30, 41, "CDR regions", "structure_element" ], [ 78, 86, "antibody", "protein_type" ] ] }, { "sid": 34, "sent": "However, an initial structural analysis of the combining sites of the small set of structures of immunoglobulin fragments available in the 1980s found that 5 of the 6 hypervariable loops or CDRs had canonical structures (a limited set of main-chain conformations).", "section": "INTRO", "ner": [ [ 20, 39, "structural analysis", "experimental_method" ], [ 47, 62, "combining sites", "site" ], [ 83, 93, "structures", "evidence" ], [ 167, 186, "hypervariable loops", "structure_element" ], [ 190, 194, "CDRs", "structure_element" ] ] }, { "sid": 35, "sent": "A CDR canonical structure is defined by its length and conserved residues located in the hypervariable loop and framework residues (V-region residues that are not part of the CDRs).", "section": "INTRO", "ner": [ [ 2, 5, "CDR", "structure_element" ], [ 89, 107, "hypervariable loop", "structure_element" ], [ 112, 130, "framework residues", "structure_element" ], [ 132, 140, "V-region", "structure_element" ], [ 175, 179, "CDRs", "structure_element" ] ] }, { "sid": 36, "sent": "Furthermore, studies of antibody sequences revealed that the total number of canonical structures are limited for each CDR, indicating possibly that antigen recognition may be affected by structural restrictions at the antigen-binding site.", "section": "INTRO", "ner": [ [ 24, 32, "antibody", "protein_type" ], [ 119, 122, "CDR", "structure_element" ], [ 219, 239, "antigen-binding site", "site" ] ] }, { "sid": 37, "sent": "Later studies found that the CDR loop length is the primary determining factor of antigen-binding site topography because it is the primary factor for determining a canonical structure.", "section": "INTRO", "ner": [ [ 29, 37, "CDR loop", "structure_element" ], [ 82, 102, "antigen-binding site", "site" ] ] }, { "sid": 38, "sent": "Additional efforts have led to our current understanding that the LC CDRs L1, L2, and L3 have preferred sets of canonical structures based on length and amino acid sequence composition.", "section": "INTRO", "ner": [ [ 66, 68, "LC", "structure_element" ], [ 69, 73, "CDRs", "structure_element" ], [ 74, 76, "L1", "structure_element" ], [ 78, 80, "L2", "structure_element" ], [ 86, 88, "L3", "structure_element" ] ] }, { "sid": 39, "sent": "This was also found to be the case for the H1 and H2 CDRs.", "section": "INTRO", "ner": [ [ 43, 45, "H1", "structure_element" ], [ 50, 52, "H2", "structure_element" ], [ 53, 57, "CDRs", "structure_element" ] ] }, { "sid": 40, "sent": "Classification schemes for the canonical structures of these 5 CDRs have emerged and evolved as the number of depositions in the Protein Data Bank of Fab fragments of antibodies grow.", "section": "INTRO", "ner": [ [ 63, 67, "CDRs", "structure_element" ], [ 150, 153, "Fab", "structure_element" ], [ 167, 177, "antibodies", "protein_type" ] ] }, { "sid": 41, "sent": "Recently, a comprehensive CDR classification scheme was reported identifying 72 clusters of conformations observed in antibody structures.", "section": "INTRO", "ner": [ [ 26, 29, "CDR", "structure_element" ], [ 118, 126, "antibody", "protein_type" ], [ 127, 137, "structures", "evidence" ] ] }, { "sid": 42, "sent": "The knowledge and predictability of these CDR canonical structures have greatly advanced antibody modeling efforts.", "section": "INTRO", "ner": [ [ 42, 45, "CDR", "structure_element" ], [ 56, 66, "structures", "evidence" ], [ 89, 97, "antibody", "protein_type" ] ] }, { "sid": 43, "sent": "In contrast to CDRs L1, L2, L3, H1 and H2, no canonical structures have been observed for CDR H3, which is the most variable in length and amino acid sequence.", "section": "INTRO", "ner": [ [ 15, 19, "CDRs", "structure_element" ], [ 20, 22, "L1", "structure_element" ], [ 24, 26, "L2", "structure_element" ], [ 28, 30, "L3", "structure_element" ], [ 32, 34, "H1", "structure_element" ], [ 39, 41, "H2", "structure_element" ], [ 56, 66, "structures", "evidence" ], [ 90, 93, "CDR", "structure_element" ], [ 94, 96, "H3", "structure_element" ] ] }, { "sid": 44, "sent": "Some clustering of conformations was observed for the shortest lengths; however, for the longer loops, only the portions nearest the framework (torso, stem or anchor region) were found to have defined conformations.", "section": "INTRO", "ner": [ [ 96, 101, "loops", "structure_element" ], [ 133, 142, "framework", "structure_element" ], [ 144, 149, "torso", "structure_element" ], [ 151, 155, "stem", "structure_element" ], [ 159, 172, "anchor region", "structure_element" ] ] }, { "sid": 45, "sent": "In the torso region, 2 primary groups could be identified, which led to sequence-based rules that can predict with some degree of reliability the conformation of the stem region.", "section": "INTRO", "ner": [ [ 7, 19, "torso region", "structure_element" ], [ 166, 177, "stem region", "structure_element" ] ] }, { "sid": 46, "sent": "The \u201ckinked\u201d or \u201cbulged\u201d conformation is the most prevalent, but an \u201cextended\u201d or \u201cnon-bulged\u201d conformation is also, but less frequently, observed.", "section": "INTRO", "ner": [ [ 5, 11, "kinked", "protein_state" ], [ 17, 23, "bulged", "protein_state" ], [ 69, 77, "extended", "protein_state" ], [ 83, 93, "non-bulged", "protein_state" ] ] }, { "sid": 47, "sent": "The cataloging and development of the rules for predicting the conformation of the anchor region of CDR H3 continue to be refined, producing new insight into the CDR H3 conformations and new tools for antibody engineering.", "section": "INTRO", "ner": [ [ 83, 96, "anchor region", "structure_element" ], [ 100, 103, "CDR", "structure_element" ], [ 104, 106, "H3", "structure_element" ], [ 162, 165, "CDR", "structure_element" ], [ 166, 168, "H3", "structure_element" ], [ 201, 209, "antibody", "protein_type" ] ] }, { "sid": 48, "sent": "Current antibody modeling approaches take advantage of the most recent advances in homology modeling, the evolving understanding of the CDR canonical structures, the emerging rules for CDR H3 modeling and the growing body of antibody structural data available from the PDB.", "section": "INTRO", "ner": [ [ 8, 16, "antibody", "protein_type" ], [ 83, 100, "homology modeling", "experimental_method" ], [ 136, 139, "CDR", "structure_element" ], [ 150, 160, "structures", "evidence" ], [ 185, 188, "CDR", "structure_element" ], [ 189, 191, "H3", "structure_element" ], [ 225, 233, "antibody", "protein_type" ] ] }, { "sid": 49, "sent": "Recent antibody modeling assessments show continued improvement in the quality of the models being generated by a variety of modeling methods.", "section": "INTRO", "ner": [ [ 7, 36, "antibody modeling assessments", "experimental_method" ] ] }, { "sid": 50, "sent": "Although antibody modeling is improving, the latest assessment revealed a number of challenges that need to be overcome to provide accurate 3-dimensional models of antibody V regions, including accuracies in the modeling of CDR H3.", "section": "INTRO", "ner": [ [ 9, 17, "antibody", "protein_type" ], [ 164, 172, "antibody", "protein_type" ], [ 173, 182, "V regions", "structure_element" ], [ 224, 227, "CDR", "structure_element" ], [ 228, 230, "H3", "structure_element" ] ] }, { "sid": 51, "sent": "The need for improvement in this area was also highlighted in a recent study reporting an approach and results that may influence future antibody modeling efforts.", "section": "INTRO", "ner": [ [ 137, 145, "antibody", "protein_type" ] ] }, { "sid": 52, "sent": "One important finding of the antibody modeling assessments was that errors in the structural templates that are used as the basis for homology models can propagate into the final models, producing inaccuracies that may negatively influence the predictive nature of the V region model.", "section": "INTRO", "ner": [ [ 29, 58, "antibody modeling assessments", "experimental_method" ], [ 134, 149, "homology models", "experimental_method" ], [ 269, 277, "V region", "structure_element" ] ] }, { "sid": 53, "sent": "To support antibody engineering and therapeutic development efforts, a phage library was designed and constructed based on a limited number of scaffolds built with frequently used human germ-line IGV and IGJ gene segments that encode antigen combining sites suitable for recognition of peptides and proteins.", "section": "INTRO", "ner": [ [ 11, 19, "antibody", "protein_type" ], [ 71, 84, "phage library", "experimental_method" ], [ 180, 185, "human", "species" ], [ 196, 199, "IGV", "structure_element" ], [ 204, 207, "IGJ", "structure_element" ], [ 234, 257, "antigen combining sites", "site" ] ] }, { "sid": 54, "sent": "This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all \u03ba), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1).", "section": "INTRO", "ner": [ [ 5, 8, "Fab", "structure_element" ], [ 34, 36, "HC", "structure_element" ], [ 48, 56, "IGHV1-69", "mutant" ], [ 58, 63, "H1-69", "mutant" ], [ 66, 74, "IGHV3-23", "mutant" ], [ 76, 81, "H3-23", "mutant" ], [ 87, 95, "IGHV5-51", "mutant" ], [ 96, 101, "H5-51", "mutant" ], [ 110, 112, "LC", "structure_element" ], [ 128, 129, "\u03ba", "structure_element" ], [ 132, 140, "IGKV1-39", "mutant" ], [ 142, 147, "L1-39", "mutant" ], [ 150, 158, "IGKV3-11", "mutant" ], [ 160, 165, "L3-11", "mutant" ], [ 168, 176, "IGKV3-20", "mutant" ], [ 178, 183, "L3-20", "mutant" ], [ 189, 196, "IGKV4-1", "mutant" ], [ 198, 202, "L4-1", "mutant" ] ] }, { "sid": 55, "sent": "Selection of these genes was based on the high frequency of their use and their cognate canonical structures that were found binding to peptides and proteins, as well as their ability to be expressed in bacteria and displayed on filamentous phage.", "section": "INTRO", "ner": [ [ 98, 108, "structures", "evidence" ], [ 190, 211, "expressed in bacteria", "experimental_method" ], [ 216, 246, "displayed on filamentous phage", "experimental_method" ] ] }, { "sid": 56, "sent": "The implementation of the library involves the diversification of the human germline genes to mimic that found in natural human libraries.", "section": "INTRO", "ner": [ [ 70, 75, "human", "species" ], [ 122, 127, "human", "species" ] ] }, { "sid": 57, "sent": "The crystal structure determinations and structural analyses of all germline Fabs in the library described above along with the structures of a fourth HC germline, IGHV3-53 (H3-53), paired with the 4 LCs of the library have been carried out to support antibody therapeutic development.", "section": "INTRO", "ner": [ [ 4, 36, "crystal structure determinations", "experimental_method" ], [ 41, 60, "structural analyses", "experimental_method" ], [ 77, 81, "Fabs", "structure_element" ], [ 128, 138, "structures", "evidence" ], [ 151, 153, "HC", "structure_element" ], [ 164, 172, "IGHV3-53", "mutant" ], [ 174, 179, "H3-53", "mutant" ], [ 200, 203, "LCs", "structure_element" ], [ 252, 260, "antibody", "protein_type" ] ] }, { "sid": 58, "sent": "All 16 HCs of the Fabs have the same CDR H3 that was reported in an earlier Fab structure.", "section": "INTRO", "ner": [ [ 7, 10, "HCs", "structure_element" ], [ 18, 22, "Fabs", "structure_element" ], [ 37, 40, "CDR", "structure_element" ], [ 41, 43, "H3", "structure_element" ], [ 76, 79, "Fab", "structure_element" ], [ 80, 89, "structure", "evidence" ] ] }, { "sid": 59, "sent": "This is the first systematic study of the same VH and VL structures in the context of different pairings.", "section": "INTRO", "ner": [ [ 47, 49, "VH", "structure_element" ], [ 54, 56, "VL", "structure_element" ], [ 57, 67, "structures", "evidence" ] ] }, { "sid": 60, "sent": "The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions.", "section": "INTRO", "ner": [ [ 58, 68, "structures", "evidence" ], [ 80, 90, "structures", "evidence" ], [ 98, 100, "L1", "structure_element" ], [ 102, 104, "L2", "structure_element" ], [ 106, 108, "L3", "structure_element" ], [ 110, 112, "H1", "structure_element" ], [ 117, 119, "H2", "structure_element" ], [ 120, 124, "CDRs", "structure_element" ], [ 130, 140, "structures", "evidence" ], [ 148, 151, "CDR", "structure_element" ], [ 152, 155, "H3s", "structure_element" ], [ 165, 170, "VH:VL", "complex_assembly" ], [ 171, 191, "packing interactions", "bond_interaction" ] ] }, { "sid": 61, "sent": "The structures and their analyses provide a foundation for future antibody engineering and structure determination efforts.", "section": "INTRO", "ner": [ [ 4, 14, "structures", "evidence" ], [ 66, 74, "antibody", "protein_type" ] ] }, { "sid": 62, "sent": "Crystal structures", "section": "RESULTS", "ner": [ [ 0, 18, "Crystal structures", "evidence" ] ] }, { "sid": 63, "sent": "Crystal data, X-ray data, and refinement statistics.", "section": "TABLE", "ner": [ [ 0, 12, "Crystal data", "evidence" ], [ 14, 24, "X-ray data", "evidence" ], [ 30, 51, "refinement statistics", "evidence" ] ] }, { "sid": 64, "sent": "(Continued) Crystal data, X-ray data, and refinement statistics.", "section": "TABLE", "ner": [ [ 12, 24, "Crystal data", "evidence" ], [ 26, 36, "X-ray data", "evidence" ], [ 42, 63, "refinement statistics", "evidence" ], [ 12, 24, "Crystal data", "evidence" ], [ 26, 36, "X-ray data", "evidence" ], [ 42, 63, "refinement statistics", "evidence" ], [ 12, 24, "Crystal data", "evidence" ], [ 26, 36, "X-ray data", "evidence" ], [ 42, 63, "refinement statistics", "evidence" ] ] }, { "sid": 65, "sent": "The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined.", "section": "RESULTS", "ner": [ [ 4, 22, "crystal structures", "evidence" ], [ 28, 44, "germline library", "experimental_method" ], [ 60, 64, "Fabs", "structure_element" ], [ 90, 93, "HCs", "structure_element" ], [ 95, 100, "H1-69", "mutant" ], [ 102, 107, "H3-23", "mutant" ], [ 109, 114, "H3-53", "mutant" ], [ 119, 124, "H5-51", "mutant" ], [ 132, 135, "LCs", "structure_element" ], [ 137, 142, "L1-39", "mutant" ], [ 144, 149, "L3-11", "mutant" ], [ 151, 156, "L3-20", "mutant" ], [ 161, 165, "L4-1", "mutant" ] ] }, { "sid": 66, "sent": "The Fab heavy and light chain sequences for the variants numbered according to Chothia are shown in Fig.\u00a0S1.", "section": "RESULTS", "ner": [ [ 4, 7, "Fab", "structure_element" ], [ 18, 29, "light chain", "structure_element" ] ] }, { "sid": 67, "sent": "The four different HCs all have the same CDR H3 sequence, ARYDGIYGELDF.", "section": "RESULTS", "ner": [ [ 19, 22, "HCs", "structure_element" ], [ 41, 44, "CDR", "structure_element" ], [ 45, 47, "H3", "structure_element" ], [ 58, 70, "ARYDGIYGELDF", "structure_element" ] ] }, { "sid": 68, "sent": "Crystallization of the 16 Fabs was previously reported.", "section": "RESULTS", "ner": [ [ 0, 15, "Crystallization", "experimental_method" ], [ 26, 30, "Fabs", "structure_element" ] ] }, { "sid": 69, "sent": "Three sets of the crystals were isomorphous with nearly identical unit cells (Table\u00a01).", "section": "RESULTS", "ner": [ [ 18, 26, "crystals", "evidence" ] ] }, { "sid": 70, "sent": "These include (1) H3-23:L3-11 and H3-23:L4-1 in P212121, (2) H3-53:L1-39, H3-53:L3-11 and H3-53:L3-20 in P6522, and (3) H5-51:L1-39, H5-51:L3-11 and H5-51:L3-20 in P212121.", "section": "RESULTS", "ner": [ [ 18, 29, "H3-23:L3-11", "complex_assembly" ], [ 34, 44, "H3-23:L4-1", "complex_assembly" ], [ 61, 72, "H3-53:L1-39", "complex_assembly" ], [ 74, 85, "H3-53:L3-11", "complex_assembly" ], [ 90, 101, "H3-53:L3-20", "complex_assembly" ], [ 120, 131, "H5-51:L1-39", "complex_assembly" ], [ 133, 144, "H5-51:L3-11", "complex_assembly" ], [ 149, 160, "H5-51:L3-20", "complex_assembly" ] ] }, { "sid": 71, "sent": "Variations occur in the pH (buffer) and the additives, and, in group 3, PEG 3350 is the precipitant for one variants while ammonium sulfate is the precipitant for the other two.", "section": "RESULTS", "ner": [ [ 72, 80, "PEG 3350", "chemical" ], [ 123, 139, "ammonium sulfate", "chemical" ] ] }, { "sid": 72, "sent": "The similarity in the crystal forms is attributed in part to cross-seeding using the microseed matrix screening for groups 2 and 3.", "section": "RESULTS", "ner": [ [ 22, 35, "crystal forms", "evidence" ], [ 85, 111, "microseed matrix screening", "experimental_method" ] ] }, { "sid": 73, "sent": "The crystal structures of the 16 Fabs have been determined at resolutions ranging from 3.3 \u212b to 1.65 \u212b (Table\u00a01).", "section": "RESULTS", "ner": [ [ 4, 22, "crystal structures", "evidence" ], [ 33, 37, "Fabs", "structure_element" ] ] }, { "sid": 74, "sent": "The number of Fab molecules in the crystallographic asymmetric unit varies from 1 (for 12 Fabs) to 2 (for 4 Fabs).", "section": "RESULTS", "ner": [ [ 14, 17, "Fab", "structure_element" ], [ 90, 94, "Fabs", "structure_element" ], [ 108, 112, "Fabs", "structure_element" ] ] }, { "sid": 75, "sent": "Overall the structures are fairly complete, and, as can be expected, the models for the higher resolution structures are more complete than those for the lower resolution structures (Table\u00a0S1).", "section": "RESULTS", "ner": [ [ 12, 22, "structures", "evidence" ], [ 106, 116, "structures", "evidence" ], [ 171, 181, "structures", "evidence" ] ] }, { "sid": 76, "sent": "Invariably, the HCs have more disorder than the LCs.", "section": "RESULTS", "ner": [ [ 16, 19, "HCs", "structure_element" ], [ 30, 38, "disorder", "protein_state" ], [ 48, 51, "LCs", "structure_element" ] ] }, { "sid": 77, "sent": "For the LC, the disorder is observed at 2 of the C-terminal residues with few exceptions.", "section": "RESULTS", "ner": [ [ 8, 10, "LC", "structure_element" ], [ 16, 24, "disorder", "protein_state" ] ] }, { "sid": 78, "sent": "Apart from the C-terminus, only a few surface residues in LC are disordered.", "section": "RESULTS", "ner": [ [ 58, 60, "LC", "structure_element" ], [ 65, 75, "disordered", "protein_state" ] ] }, { "sid": 79, "sent": "The HCs feature the largest number of disordered residues, with the lower resolution structures having the most.", "section": "RESULTS", "ner": [ [ 4, 7, "HCs", "structure_element" ], [ 38, 48, "disordered", "protein_state" ], [ 85, 95, "structures", "evidence" ] ] }, { "sid": 80, "sent": "The C-terminal residues including the 6xHis tags are disordered in all 16 structures.", "section": "RESULTS", "ner": [ [ 53, 63, "disordered", "protein_state" ], [ 74, 84, "structures", "evidence" ] ] }, { "sid": 81, "sent": "In addition to these, 2 primary disordered stretches of residues are observed in a number of structures (Table\u00a0S1).", "section": "RESULTS", "ner": [ [ 93, 103, "structures", "evidence" ] ] }, { "sid": 82, "sent": "One involves the loop connecting the first 2 \u03b2-strands of the constant domain (in all Fabs except H3-23:L1-39, H3-23:L3-11 and H3-53:L1-39).", "section": "RESULTS", "ner": [ [ 17, 21, "loop", "structure_element" ], [ 45, 54, "\u03b2-strands", "structure_element" ], [ 62, 77, "constant domain", "structure_element" ], [ 86, 90, "Fabs", "structure_element" ], [ 98, 109, "H3-23:L1-39", "complex_assembly" ], [ 111, 122, "H3-23:L3-11", "complex_assembly" ], [ 127, 138, "H3-53:L1-39", "complex_assembly" ] ] }, { "sid": 83, "sent": "The other is located in CDR H3 (in H5-51:L3-11, H5-51:L3-20 and in one of 2 copies of H3-23:L4-1).", "section": "RESULTS", "ner": [ [ 24, 27, "CDR", "structure_element" ], [ 28, 30, "H3", "structure_element" ], [ 35, 46, "H5-51:L3-11", "complex_assembly" ], [ 48, 59, "H5-51:L3-20", "complex_assembly" ], [ 86, 96, "H3-23:L4-1", "complex_assembly" ] ] }, { "sid": 84, "sent": "CDR H1 and CDR H2 also show some degree of disorder, but to a lesser extent.", "section": "RESULTS", "ner": [ [ 0, 3, "CDR", "structure_element" ], [ 4, 6, "H1", "structure_element" ], [ 11, 14, "CDR", "structure_element" ], [ 15, 17, "H2", "structure_element" ], [ 43, 51, "disorder", "protein_state" ] ] }, { "sid": 85, "sent": "CDR canonical structures", "section": "RESULTS", "ner": [ [ 0, 3, "CDR", "structure_element" ], [ 14, 24, "structures", "evidence" ] ] }, { "sid": 86, "sent": "Several CDR definitions have evolved over decades of antibody research.", "section": "RESULTS", "ner": [ [ 8, 11, "CDR", "structure_element" ], [ 53, 61, "antibody", "protein_type" ] ] }, { "sid": 87, "sent": "Depending on the focus of the study, the CDR boundaries differ slightly between various definitions.", "section": "RESULTS", "ner": [ [ 41, 44, "CDR", "structure_element" ] ] }, { "sid": 88, "sent": "In this work, we use the CDR definition of North et\u00a0al., which is similar to that of Martin with the following exceptions: 1) CDRs H1 and H3 begin immediately after the Cys; and 2) CDR L2 includes an additional residue at the N-terminal side, typically Tyr.", "section": "RESULTS", "ner": [ [ 25, 28, "CDR", "structure_element" ], [ 126, 130, "CDRs", "structure_element" ], [ 131, 133, "H1", "structure_element" ], [ 138, 140, "H3", "structure_element" ], [ 169, 172, "Cys", "residue_name" ], [ 181, 184, "CDR", "structure_element" ], [ 185, 187, "L2", "structure_element" ], [ 253, 256, "Tyr", "residue_name" ] ] }, { "sid": 89, "sent": "CDR H1", "section": "RESULTS", "ner": [ [ 0, 3, "CDR", "structure_element" ], [ 4, 6, "H1", "structure_element" ] ] }, { "sid": 90, "sent": "The superposition of CDR H1 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51.", "section": "FIG", "ner": [ [ 4, 17, "superposition", "experimental_method" ], [ 21, 24, "CDR", "structure_element" ], [ 25, 27, "H1", "structure_element" ], [ 46, 51, "HC:LC", "complex_assembly" ], [ 63, 75, "heavy chains", "structure_element" ], [ 81, 86, "H1-69", "mutant" ], [ 92, 97, "H3-23", "mutant" ], [ 103, 108, "H3-53", "mutant" ], [ 117, 122, "H5-51", "mutant" ] ] }, { "sid": 91, "sent": "CDRs are defined using the Dunbrack convention [12].", "section": "TABLE", "ner": [ [ 0, 4, "CDRs", "structure_element" ] ] }, { "sid": 92, "sent": "Assignments for 2 copies of the Fab in the asymmetric unit are given for 5 structures.", "section": "TABLE", "ner": [ [ 32, 35, "Fab", "structure_element" ], [ 75, 85, "structures", "evidence" ] ] }, { "sid": 93, "sent": "No assignment (NA) for CDRs with missing residues.", "section": "TABLE", "ner": [ [ 23, 27, "CDRs", "structure_element" ] ] }, { "sid": 94, "sent": "The four HCs feature CDR H1 of the same length, and their sequences are highly similar (Table\u00a02).", "section": "RESULTS", "ner": [ [ 9, 12, "HCs", "structure_element" ], [ 21, 24, "CDR", "structure_element" ], [ 25, 27, "H1", "structure_element" ] ] }, { "sid": 95, "sent": "The CDR H1 backbone conformations for all variants for each of the HCs are shown in Fig.\u00a01.", "section": "RESULTS", "ner": [ [ 4, 7, "CDR", "structure_element" ], [ 8, 10, "H1", "structure_element" ], [ 67, 70, "HCs", "structure_element" ] ] }, { "sid": 96, "sent": "Three of the HCs, H3-23, H3-53 and H5-51, have the same canonical structure, H1-13-1, and the backbone conformations are tightly clustered for each set of Fab structures as reflected in the rmsd values (Fig.\u00a01B-D).", "section": "RESULTS", "ner": [ [ 13, 16, "HCs", "structure_element" ], [ 18, 23, "H3-23", "mutant" ], [ 25, 30, "H3-53", "mutant" ], [ 35, 40, "H5-51", "mutant" ], [ 77, 84, "H1-13-1", "mutant" ], [ 155, 158, "Fab", "structure_element" ], [ 159, 169, "structures", "evidence" ], [ 190, 201, "rmsd values", "evidence" ] ] }, { "sid": 97, "sent": "Some deviation is observed for H3-53, mostly due to H3-53:L4-1, which exhibits a significant degree of disorder in CDR H1.", "section": "RESULTS", "ner": [ [ 31, 36, "H3-53", "mutant" ], [ 52, 62, "H3-53:L4-1", "complex_assembly" ], [ 115, 118, "CDR", "structure_element" ], [ 119, 121, "H1", "structure_element" ] ] }, { "sid": 98, "sent": "The electron density for the backbone is weak and discontinuous, and completely missing for several side chains.", "section": "RESULTS", "ner": [ [ 4, 20, "electron density", "evidence" ] ] }, { "sid": 99, "sent": "The CDR H1 structures with H1-69 shown in Fig.\u00a01A are quite variable, both for the structures with different LCs and for the copies of the same Fab in the asymmetric unit, H1-69:L3-11 and H1-69:L3-20.", "section": "RESULTS", "ner": [ [ 4, 7, "CDR", "structure_element" ], [ 8, 10, "H1", "structure_element" ], [ 11, 21, "structures", "evidence" ], [ 27, 32, "H1-69", "mutant" ], [ 83, 93, "structures", "evidence" ], [ 109, 112, "LCs", "structure_element" ], [ 144, 147, "Fab", "structure_element" ], [ 172, 183, "H1-69:L3-11", "complex_assembly" ], [ 188, 199, "H1-69:L3-20", "complex_assembly" ] ] }, { "sid": 100, "sent": "In total, 6 independent Fab structures produce 5 different canonical structures, namely H1-13-1, H1-13-3, H1-13-4, H1-13-6 and H1-13-10.", "section": "RESULTS", "ner": [ [ 24, 27, "Fab", "structure_element" ], [ 28, 38, "structures", "evidence" ], [ 69, 79, "structures", "evidence" ], [ 88, 95, "H1-13-1", "mutant" ], [ 97, 104, "H1-13-3", "mutant" ], [ 106, 113, "H1-13-4", "mutant" ], [ 115, 122, "H1-13-6", "mutant" ], [ 127, 135, "H1-13-10", "mutant" ] ] }, { "sid": 101, "sent": "A major difference of H1-69 from the other germlines in the experimental data set is the presence of Gly instead of Phe or Tyr at position 27 (residue 5 of 13 in CDR H1).", "section": "RESULTS", "ner": [ [ 22, 27, "H1-69", "mutant" ], [ 101, 104, "Gly", "residue_name" ], [ 116, 119, "Phe", "residue_name" ], [ 123, 126, "Tyr", "residue_name" ], [ 139, 141, "27", "residue_number" ], [ 162, 165, "CDR", "structure_element" ], [ 166, 168, "H1", "structure_element" ] ] }, { "sid": 102, "sent": "Glycine introduces the possibility of a higher degree of conformational flexibility that undoubtedly translates to the differences observed, and contributes to the elevated thermal parameters for the atoms in the amino acid residues in this region.", "section": "RESULTS", "ner": [ [ 0, 7, "Glycine", "residue_name" ] ] }, { "sid": 103, "sent": "CDR H2", "section": "RESULTS", "ner": [ [ 0, 3, "CDR", "structure_element" ], [ 4, 6, "H2", "structure_element" ] ] }, { "sid": 104, "sent": "The superposition of CDR H2 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51.", "section": "FIG", "ner": [ [ 4, 17, "superposition", "experimental_method" ], [ 21, 24, "CDR", "structure_element" ], [ 25, 27, "H2", "structure_element" ], [ 46, 51, "HC:LC", "complex_assembly" ], [ 63, 75, "heavy chains", "structure_element" ], [ 81, 86, "H1-69", "mutant" ], [ 92, 97, "H3-23", "mutant" ], [ 103, 108, "H3-53", "mutant" ], [ 117, 122, "H5-51", "mutant" ] ] }, { "sid": 105, "sent": "The canonical structures of CDR H2 have fairly consistent conformations (Table\u00a02, Fig.\u00a02).", "section": "RESULTS", "ner": [ [ 28, 31, "CDR", "structure_element" ], [ 32, 34, "H2", "structure_element" ] ] }, { "sid": 106, "sent": "Each of the 4 HCs adopts only one canonical structure regardless of the pairing LC.", "section": "RESULTS", "ner": [ [ 14, 17, "HCs", "structure_element" ], [ 80, 82, "LC", "structure_element" ] ] }, { "sid": 107, "sent": "Germlines H1-69 and H5-51 have the same canonical structure assignment H2-10-1, H3-23 has H2-10-2, and H3-53 has H2-9-3.", "section": "RESULTS", "ner": [ [ 10, 15, "H1-69", "mutant" ], [ 20, 25, "H5-51", "mutant" ], [ 71, 78, "H2-10-1", "mutant" ], [ 80, 85, "H3-23", "mutant" ], [ 90, 97, "H2-10-2", "mutant" ], [ 103, 108, "H3-53", "mutant" ], [ 113, 119, "H2-9-3", "mutant" ] ] }, { "sid": 108, "sent": "The conformations for all of these CDR H2s are tightly clustered (Fig.\u00a02).", "section": "RESULTS", "ner": [ [ 35, 38, "CDR", "structure_element" ], [ 39, 42, "H2s", "structure_element" ] ] }, { "sid": 109, "sent": "In one case, in the second Fab of H1-69:L3-20, CDR H2 is partially disordered (\u039455-60).", "section": "RESULTS", "ner": [ [ 27, 30, "Fab", "structure_element" ], [ 34, 45, "H1-69:L3-20", "complex_assembly" ], [ 47, 50, "CDR", "structure_element" ], [ 51, 53, "H2", "structure_element" ], [ 57, 77, "partially disordered", "protein_state" ], [ 79, 85, "\u039455-60", "mutant" ] ] }, { "sid": 110, "sent": "Although three of the germlines have CDR H2 of the same length, 10 residues, they adopt 2 distinctively different conformations depending mostly on the residue at position 71 from the so-called CDR H4.", "section": "RESULTS", "ner": [ [ 37, 40, "CDR", "structure_element" ], [ 41, 43, "H2", "structure_element" ], [ 64, 75, "10 residues", "residue_range" ], [ 172, 174, "71", "residue_number" ], [ 194, 197, "CDR", "structure_element" ], [ 198, 200, "H4", "structure_element" ] ] }, { "sid": 111, "sent": "Arg71 in H3-23 fills the space between CDRs H2 and H4, and defines the conformation of the tip of CDR H2 so that residue 54 points away from the antigen binding site.", "section": "RESULTS", "ner": [ [ 0, 5, "Arg71", "residue_name_number" ], [ 9, 14, "H3-23", "mutant" ], [ 39, 43, "CDRs", "structure_element" ], [ 44, 46, "H2", "structure_element" ], [ 51, 53, "H4", "structure_element" ], [ 98, 101, "CDR", "structure_element" ], [ 102, 104, "H2", "structure_element" ], [ 121, 123, "54", "residue_number" ], [ 145, 165, "antigen binding site", "site" ] ] }, { "sid": 112, "sent": "Germlines H1-69 and H5-51 are unique in the human repertoire in having an Ala at position 71 that leaves enough space for H-Pro52a to pack deeper against CDR H4 so that the following residues 53 and 54 point toward the putative antigen.", "section": "RESULTS", "ner": [ [ 10, 15, "H1-69", "mutant" ], [ 20, 25, "H5-51", "mutant" ], [ 44, 49, "human", "species" ], [ 74, 77, "Ala", "residue_name" ], [ 90, 92, "71", "residue_number" ], [ 122, 123, "H", "structure_element" ], [ 124, 130, "Pro52a", "residue_name_number" ], [ 154, 157, "CDR", "structure_element" ], [ 158, 160, "H4", "structure_element" ], [ 192, 194, "53", "residue_number" ], [ 199, 201, "54", "residue_number" ] ] }, { "sid": 113, "sent": "Conformations of CDR H2 in H1-69 and H5-51, both of which have canonical structure H2-10-1, show little deviation within each set of 4 structures.", "section": "RESULTS", "ner": [ [ 17, 20, "CDR", "structure_element" ], [ 21, 23, "H2", "structure_element" ], [ 27, 32, "H1-69", "mutant" ], [ 37, 42, "H5-51", "mutant" ], [ 83, 90, "H2-10-1", "mutant" ], [ 135, 145, "structures", "evidence" ] ] }, { "sid": 114, "sent": "However, there is a significant shift of the CDR as a rigid body when the 2 sets are superimposed.", "section": "RESULTS", "ner": [ [ 45, 48, "CDR", "structure_element" ], [ 85, 97, "superimposed", "experimental_method" ] ] }, { "sid": 115, "sent": "Most likely this is the result of interaction of CDR H2 with CDR H1, namely with the residue at position 33 (residue 11 of 13 in CDR H1).", "section": "RESULTS", "ner": [ [ 49, 52, "CDR", "structure_element" ], [ 53, 55, "H2", "structure_element" ], [ 61, 64, "CDR", "structure_element" ], [ 65, 67, "H1", "structure_element" ], [ 105, 107, "33", "residue_number" ], [ 129, 132, "CDR", "structure_element" ], [ 133, 135, "H1", "structure_element" ] ] }, { "sid": 116, "sent": "Germline H1-69 has Ala at position 33 whereas in H5-51 position 33 is occupied by a bulky Trp, which stacks against H-Tyr52 and drives CDR H2 away from the center.", "section": "RESULTS", "ner": [ [ 9, 14, "H1-69", "mutant" ], [ 19, 22, "Ala", "residue_name" ], [ 35, 37, "33", "residue_number" ], [ 49, 54, "H5-51", "mutant" ], [ 64, 66, "33", "residue_number" ], [ 90, 93, "Trp", "residue_name" ], [ 116, 117, "H", "structure_element" ], [ 118, 123, "Tyr52", "residue_name_number" ], [ 135, 138, "CDR", "structure_element" ], [ 139, 141, "H2", "structure_element" ] ] }, { "sid": 117, "sent": "CDR L1", "section": "RESULTS", "ner": [ [ 0, 3, "CDR", "structure_element" ], [ 4, 6, "L1", "structure_element" ] ] }, { "sid": 118, "sent": "The superposition of CDR L1 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1.", "section": "FIG", "ner": [ [ 4, 17, "superposition", "experimental_method" ], [ 21, 24, "CDR", "structure_element" ], [ 25, 27, "L1", "structure_element" ], [ 46, 51, "HC:LC", "complex_assembly" ], [ 63, 75, "light chains", "structure_element" ], [ 81, 86, "L1-39", "mutant" ], [ 92, 97, "L3-11", "mutant" ], [ 103, 108, "L3-20", "mutant" ], [ 117, 121, "L4-1", "mutant" ] ] }, { "sid": 119, "sent": "The four LC CDRs L1 feature 3 different lengths (11, 12 and 17 residues) having a total of 4 different canonical structure assignments.", "section": "RESULTS", "ner": [ [ 9, 11, "LC", "structure_element" ], [ 12, 16, "CDRs", "structure_element" ], [ 17, 19, "L1", "structure_element" ], [ 49, 51, "11", "residue_range" ], [ 53, 55, "12", "residue_range" ], [ 60, 62, "17", "residue_range" ] ] }, { "sid": 120, "sent": "Of these LCs, L1-39 and L3-11 have the same canonical structure, L1-11-1, and superimpose very well (Fig.\u00a03A, B).", "section": "RESULTS", "ner": [ [ 9, 12, "LCs", "structure_element" ], [ 14, 19, "L1-39", "mutant" ], [ 24, 29, "L3-11", "mutant" ], [ 65, 72, "L1-11-1", "mutant" ], [ 78, 89, "superimpose", "experimental_method" ] ] }, { "sid": 121, "sent": "For the remaining 2, L3-20 has 2 different assignments, L1-12-1 and L1-12-2, while L4-1 has a single assignment, L1-17-1.", "section": "RESULTS", "ner": [ [ 21, 26, "L3-20", "mutant" ], [ 56, 63, "L1-12-1", "mutant" ], [ 68, 75, "L1-12-2", "mutant" ], [ 83, 87, "L4-1", "mutant" ], [ 113, 120, "L1-17-1", "mutant" ] ] }, { "sid": 122, "sent": "L4-1 has the longest CDR L1, composed of 17 amino acid residues (Fig.\u00a03D).", "section": "RESULTS", "ner": [ [ 0, 4, "L4-1", "mutant" ], [ 21, 24, "CDR", "structure_element" ], [ 25, 27, "L1", "structure_element" ], [ 41, 63, "17 amino acid residues", "residue_range" ] ] }, { "sid": 123, "sent": "Despite this, the conformations are tightly clustered (rmsd is 0.20 \u212b).", "section": "RESULTS", "ner": [ [ 55, 59, "rmsd", "evidence" ] ] }, { "sid": 124, "sent": "The backbone conformations of the stem regions superimpose well.", "section": "RESULTS", "ner": [ [ 34, 46, "stem regions", "structure_element" ] ] }, { "sid": 125, "sent": "Some changes in conformation occur between residues 30a and 30f (residues 8 and 13 of 17 in CDR L1).", "section": "RESULTS", "ner": [ [ 52, 55, "30a", "residue_number" ], [ 60, 63, "30f", "residue_number" ], [ 74, 75, "8", "residue_number" ], [ 80, 82, "13", "residue_number" ], [ 86, 88, "17", "residue_number" ], [ 92, 95, "CDR", "structure_element" ], [ 96, 98, "L1", "structure_element" ] ] }, { "sid": 126, "sent": "This is the tip of the loop region, which appears to have similar conformations that fan out the structures because of the slight differences in torsion angles in the backbone near Tyr30a and Lys30f.", "section": "RESULTS", "ner": [ [ 23, 34, "loop region", "structure_element" ], [ 97, 107, "structures", "evidence" ], [ 181, 187, "Tyr30a", "residue_name_number" ], [ 192, 198, "Lys30f", "residue_name_number" ] ] }, { "sid": 127, "sent": "L3-20 is the most variable in CDR L1 among the 4 germlines as indicated by an rmsd of 0.54 \u212b (Fig.\u00a03C).", "section": "RESULTS", "ner": [ [ 0, 5, "L3-20", "mutant" ], [ 30, 33, "CDR", "structure_element" ], [ 34, 36, "L1", "structure_element" ], [ 78, 82, "rmsd", "evidence" ] ] }, { "sid": 128, "sent": "Two structures, H3-53:L3-20 and H5-51:L3-20 are assigned to canonical structure L1-12-1 with virtually identical backbone conformations.", "section": "RESULTS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 16, 27, "H3-53:L3-20", "complex_assembly" ], [ 32, 43, "H5-51:L3-20", "complex_assembly" ], [ 80, 87, "L1-12-1", "mutant" ] ] }, { "sid": 129, "sent": "The third structure, H3-23:L3-20, has CDR L1 as L1-12-2, which deviates from L1-12-1 at residues 29-32, i.e., at the site of insertion with respect to the 11-residue CDR.", "section": "RESULTS", "ner": [ [ 21, 32, "H3-23:L3-20", "complex_assembly" ], [ 38, 41, "CDR", "structure_element" ], [ 42, 44, "L1", "structure_element" ], [ 48, 55, "L1-12-2", "mutant" ], [ 77, 84, "L1-12-1", "mutant" ], [ 97, 102, "29-32", "residue_range" ], [ 155, 165, "11-residue", "residue_range" ], [ 166, 169, "CDR", "structure_element" ] ] }, { "sid": 130, "sent": "The fourth member of the set, H1-69:L3-20, was crystallized with 2 Fabs in the asymmetric unit.", "section": "RESULTS", "ner": [ [ 30, 41, "H1-69:L3-20", "complex_assembly" ], [ 47, 59, "crystallized", "experimental_method" ], [ 67, 71, "Fabs", "structure_element" ] ] }, { "sid": 131, "sent": "The conformation of CDR L1 in these 2 Fabs is slightly different, and both conformations fall somewhere between L1-12-1 and L1-12-2.", "section": "RESULTS", "ner": [ [ 20, 23, "CDR", "structure_element" ], [ 24, 26, "L1", "structure_element" ], [ 38, 42, "Fabs", "structure_element" ], [ 112, 119, "L1-12-1", "mutant" ], [ 124, 131, "L1-12-2", "mutant" ] ] }, { "sid": 132, "sent": "This reflects the lack of accuracy in the structure due to low resolution of the X-ray data (3.3 \u212b).", "section": "RESULTS", "ner": [ [ 42, 51, "structure", "evidence" ], [ 81, 91, "X-ray data", "evidence" ] ] }, { "sid": 133, "sent": "CDR L2", "section": "RESULTS", "ner": [ [ 0, 3, "CDR", "structure_element" ], [ 4, 6, "L2", "structure_element" ] ] }, { "sid": 134, "sent": "The superposition of CDR L2 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1.", "section": "FIG", "ner": [ [ 4, 17, "superposition", "experimental_method" ], [ 21, 24, "CDR", "structure_element" ], [ 25, 27, "L2", "structure_element" ], [ 46, 51, "HC:LC", "complex_assembly" ], [ 63, 75, "light chains", "structure_element" ], [ 81, 86, "L1-39", "mutant" ], [ 92, 97, "L3-11", "mutant" ], [ 103, 108, "L3-20", "mutant" ], [ 117, 121, "L4-1", "mutant" ] ] }, { "sid": 135, "sent": "All four LCs have CDR L2 of the same length and canonical structure, L2-8-1 (Table\u00a02).", "section": "RESULTS", "ner": [ [ 9, 12, "LCs", "structure_element" ], [ 18, 21, "CDR", "structure_element" ], [ 22, 24, "L2", "structure_element" ], [ 69, 75, "L2-8-1", "mutant" ] ] }, { "sid": 136, "sent": "The CDR L2 conformations for each of the LCs paired with the 4 HCs are clustered more tightly than any of the other CDRs (rmsd values are in the range 0.09-0.16 \u212b), and all 4 sets have virtually the same conformation despite the sequence diversity of the loop.", "section": "RESULTS", "ner": [ [ 4, 7, "CDR", "structure_element" ], [ 8, 10, "L2", "structure_element" ], [ 41, 44, "LCs", "structure_element" ], [ 63, 66, "HCs", "structure_element" ], [ 116, 120, "CDRs", "structure_element" ], [ 122, 126, "rmsd", "evidence" ], [ 255, 259, "loop", "structure_element" ] ] }, { "sid": 137, "sent": "CDR L3", "section": "RESULTS", "ner": [ [ 0, 3, "CDR", "structure_element" ], [ 4, 6, "L3", "structure_element" ] ] }, { "sid": 138, "sent": "The superposition of CDR L3 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1.", "section": "FIG", "ner": [ [ 4, 17, "superposition", "experimental_method" ], [ 21, 24, "CDR", "structure_element" ], [ 25, 27, "L3", "structure_element" ], [ 46, 51, "HC:LC", "complex_assembly" ], [ 63, 75, "light chains", "structure_element" ], [ 81, 86, "L1-39", "mutant" ], [ 92, 97, "L3-11", "mutant" ], [ 103, 108, "L3-20", "mutant" ], [ 117, 121, "L4-1", "mutant" ] ] }, { "sid": 139, "sent": "As with CDR L2, all 4 LCs have CDR L3 of the same length and canonical structure, L3-9-cis7-1 (Table\u00a02).", "section": "RESULTS", "ner": [ [ 8, 11, "CDR", "structure_element" ], [ 12, 14, "L2", "structure_element" ], [ 22, 25, "LCs", "structure_element" ], [ 31, 34, "CDR", "structure_element" ], [ 35, 37, "L3", "structure_element" ], [ 71, 80, "structure", "evidence" ], [ 82, 93, "L3-9-cis7-1", "mutant" ] ] }, { "sid": 140, "sent": "The conformations of CDR L3 for L1-39, L3-11, and particularly for L320, are not as tightly clustered as those of L4-1 (Fig.\u00a05).", "section": "RESULTS", "ner": [ [ 21, 24, "CDR", "structure_element" ], [ 25, 27, "L3", "structure_element" ], [ 32, 37, "L1-39", "mutant" ], [ 39, 44, "L3-11", "mutant" ], [ 114, 118, "L4-1", "mutant" ] ] }, { "sid": 141, "sent": "The slight conformational variability occurs in the region of amino acid residues 90-92, which is in contact with CDR H3.", "section": "RESULTS", "ner": [ [ 82, 87, "90-92", "residue_range" ], [ 114, 117, "CDR", "structure_element" ], [ 118, 120, "H3", "structure_element" ] ] }, { "sid": 142, "sent": "CDR H3 conformational diversity", "section": "RESULTS", "ner": [ [ 0, 3, "CDR", "structure_element" ], [ 4, 6, "H3", "structure_element" ] ] }, { "sid": 143, "sent": "As mentioned earlier, all 16 Fabs have the same CDR H3, for which the amino acid sequence is derived from the anti-CCL2 antibody CNTO 888.", "section": "RESULTS", "ner": [ [ 29, 33, "Fabs", "structure_element" ], [ 48, 51, "CDR", "structure_element" ], [ 52, 54, "H3", "structure_element" ], [ 120, 128, "antibody", "protein_type" ], [ 129, 137, "CNTO 888", "chemical" ] ] }, { "sid": 144, "sent": "The loop and the 2 \u03b2-strands of the CDR H3 in this \u2018parent\u2019 structure are stabilized by H-bonds between the carbonyl oxygen and peptide nitrogen atoms in the 2 strands.", "section": "RESULTS", "ner": [ [ 4, 8, "loop", "structure_element" ], [ 19, 28, "\u03b2-strands", "structure_element" ], [ 36, 39, "CDR", "structure_element" ], [ 40, 42, "H3", "structure_element" ], [ 60, 69, "structure", "evidence" ], [ 88, 95, "H-bonds", "bond_interaction" ] ] }, { "sid": 145, "sent": "An interesting feature of these CDR H3 structures is the presence of a water molecule that interacts with the peptide nitrogens and carbonyl oxygens near the bridging loop connecting the 2 \u03b2-strands.", "section": "RESULTS", "ner": [ [ 32, 35, "CDR", "structure_element" ], [ 36, 38, "H3", "structure_element" ], [ 39, 49, "structures", "evidence" ], [ 71, 76, "water", "chemical" ], [ 167, 171, "loop", "structure_element" ], [ 189, 198, "\u03b2-strands", "structure_element" ] ] }, { "sid": 146, "sent": "This water is present in both the bound (4DN4) and unbound (4DN3) forms of CNTO 888.", "section": "RESULTS", "ner": [ [ 5, 10, "water", "chemical" ], [ 34, 39, "bound", "protein_state" ], [ 51, 58, "unbound", "protein_state" ], [ 75, 83, "CNTO 888", "chemical" ] ] }, { "sid": 147, "sent": "The stem region of CDR H3 in the parental Fab is in a \u2018kinked\u2019 conformation, in which the indole nitrogen of Trp103 forms a hydrogen bond with the carbonyl oxygen of Leu100b.", "section": "RESULTS", "ner": [ [ 4, 15, "stem region", "structure_element" ], [ 19, 22, "CDR", "structure_element" ], [ 23, 25, "H3", "structure_element" ], [ 42, 45, "Fab", "structure_element" ], [ 55, 61, "kinked", "protein_state" ], [ 109, 115, "Trp103", "residue_name_number" ], [ 124, 137, "hydrogen bond", "bond_interaction" ], [ 166, 173, "Leu100b", "residue_name_number" ] ] }, { "sid": 148, "sent": "The carboxyl group of Asp101 forms a salt bridge with Arg94.", "section": "RESULTS", "ner": [ [ 22, 28, "Asp101", "residue_name_number" ], [ 37, 48, "salt bridge", "bond_interaction" ], [ 54, 59, "Arg94", "residue_name_number" ] ] }, { "sid": 149, "sent": "Ribbon representations of (A) the superposition of all CDR H3s of the structures with complete backbone traces. (B) The CDR H3s rotated 90\u00b0 about the y axis of the page.", "section": "FIG", "ner": [ [ 34, 47, "superposition", "experimental_method" ], [ 55, 58, "CDR", "structure_element" ], [ 59, 62, "H3s", "structure_element" ], [ 70, 80, "structures", "evidence" ], [ 120, 123, "CDR", "structure_element" ], [ 124, 127, "H3s", "structure_element" ] ] }, { "sid": 150, "sent": "The structure of each CDR H3 is represented with a different color.", "section": "FIG", "ner": [ [ 4, 13, "structure", "evidence" ], [ 22, 25, "CDR", "structure_element" ], [ 26, 28, "H3", "structure_element" ] ] }, { "sid": 151, "sent": "Despite having the same amino acid sequence in all variants, CDR H3 has the highest degree of structural diversity and disorder of all of the CDRs in the experimental set.", "section": "RESULTS", "ner": [ [ 61, 64, "CDR", "structure_element" ], [ 65, 67, "H3", "structure_element" ], [ 142, 146, "CDRs", "structure_element" ] ] }, { "sid": 152, "sent": "Three of the 21 Fab structures (including multiple copies in the asymmetric unit), H5-51:L3-11, H551:L3-20 and H3-23:L4-1 (one of the 2 Fabs), have missing (disordered) residues at the apex of the CDR loop.", "section": "RESULTS", "ner": [ [ 16, 19, "Fab", "structure_element" ], [ 20, 30, "structures", "evidence" ], [ 83, 94, "H5-51:L3-11", "complex_assembly" ], [ 96, 106, "H551:L3-20", "complex_assembly" ], [ 111, 121, "H3-23:L4-1", "complex_assembly" ], [ 136, 140, "Fabs", "structure_element" ], [ 148, 155, "missing", "protein_state" ], [ 157, 167, "disordered", "protein_state" ], [ 197, 205, "CDR loop", "structure_element" ] ] }, { "sid": 153, "sent": "Another four of the Fabs, H3-23:L1-39, H3-53:L1-39, H3-53:L3-11 and H3-53:L4-1 have missing side-chain atoms.", "section": "RESULTS", "ner": [ [ 20, 24, "Fabs", "structure_element" ], [ 26, 37, "H3-23:L1-39", "complex_assembly" ], [ 39, 50, "H3-53:L1-39", "complex_assembly" ], [ 52, 63, "H3-53:L3-11", "complex_assembly" ], [ 68, 78, "H3-53:L4-1", "complex_assembly" ] ] }, { "sid": 154, "sent": "The variations in CDR H3 conformation are illustrated in Fig.\u00a06 for the 18 Fab structures that have ordered backbone atoms.", "section": "RESULTS", "ner": [ [ 18, 21, "CDR", "structure_element" ], [ 22, 24, "H3", "structure_element" ], [ 75, 78, "Fab", "structure_element" ], [ 79, 89, "structures", "evidence" ] ] }, { "sid": 155, "sent": "A comparison of representatives of the \u201ckinked\u201d and \u201cextended\u201d structures.", "section": "FIG", "ner": [ [ 40, 46, "kinked", "protein_state" ], [ 53, 61, "extended", "protein_state" ], [ 63, 73, "structures", "evidence" ] ] }, { "sid": 156, "sent": "(A) The \u201ckinked\u201d CDR H3 of H1-69:L3-11 with purple carbon atoms and yellow dashed lines connecting the H-bond pairs for Leu100b O and Trp103 NE1, Arg94 NE and Asp101 OD1, and Arg94 NH2 and Asp101 OD2.", "section": "FIG", "ner": [ [ 9, 15, "kinked", "protein_state" ], [ 17, 20, "CDR", "structure_element" ], [ 21, 23, "H3", "structure_element" ], [ 27, 38, "H1-69:L3-11", "complex_assembly" ], [ 120, 127, "Leu100b", "residue_name_number" ], [ 134, 140, "Trp103", "residue_name_number" ], [ 146, 151, "Arg94", "residue_name_number" ], [ 159, 165, "Asp101", "residue_name_number" ], [ 175, 180, "Arg94", "residue_name_number" ], [ 189, 195, "Asp101", "residue_name_number" ] ] }, { "sid": 157, "sent": "(B) The \u201cextended\u201d CDR H3 of H1-69:L3-20 with green carbon atoms and yellow dashed lines connecting the H-bond pairs for Asp101 OD1 and OD2 and Trp103 NE1.", "section": "FIG", "ner": [ [ 9, 17, "extended", "protein_state" ], [ 19, 22, "CDR", "structure_element" ], [ 23, 25, "H3", "structure_element" ], [ 29, 40, "H1-69:L3-20", "complex_assembly" ], [ 121, 127, "Asp101", "residue_name_number" ], [ 144, 150, "Trp103", "residue_name_number" ] ] }, { "sid": 158, "sent": "In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3.", "section": "RESULTS", "ner": [ [ 16, 19, "Fab", "structure_element" ], [ 20, 30, "structures", "evidence" ], [ 32, 43, "H1-69:L1-39", "complex_assembly" ], [ 45, 56, "H1-69:L3-11", "complex_assembly" ], [ 60, 64, "Fabs", "structure_element" ], [ 67, 77, "H1-69:L4-1", "complex_assembly" ], [ 79, 90, "H3-23:L3-11", "complex_assembly" ], [ 94, 98, "Fabs", "structure_element" ], [ 101, 112, "H3-23:L3-20", "complex_assembly" ], [ 114, 125, "H3-53:L3-11", "complex_assembly" ], [ 127, 138, "H3-53:L3-20", "complex_assembly" ], [ 143, 154, "H5-51:L1-39", "complex_assembly" ], [ 160, 164, "CDRs", "structure_element" ] ] }, { "sid": 159, "sent": "The bases of these structures have the \u2018kinked\u2019 conformation with the H-bond between Trp103 and Leu100b.", "section": "RESULTS", "ner": [ [ 19, 29, "structures", "evidence" ], [ 40, 46, "kinked", "protein_state" ], [ 85, 91, "Trp103", "residue_name_number" ], [ 96, 103, "Leu100b", "residue_name_number" ] ] }, { "sid": 160, "sent": "A representative CDR H3 structure for H1-69:L1-39 illustrating this is shown in Fig.\u00a07A.", "section": "RESULTS", "ner": [ [ 17, 20, "CDR", "structure_element" ], [ 21, 23, "H3", "structure_element" ], [ 24, 33, "structure", "evidence" ], [ 38, 49, "H1-69:L1-39", "complex_assembly" ] ] }, { "sid": 161, "sent": "The largest backbone conformational deviation for the set is at Tyr99, where the C=O is rotated by 90\u00b0 relative to that observed in 4DN3.", "section": "RESULTS", "ner": [ [ 64, 69, "Tyr99", "residue_name_number" ] ] }, { "sid": 162, "sent": "Also, it is worth noting that only one of these structures, H1-69:L4-1, has the conserved water molecule in CDR H3 observed in the 4DN3 and 4DN4 structures.", "section": "RESULTS", "ner": [ [ 48, 58, "structures", "evidence" ], [ 60, 70, "H1-69:L4-1", "complex_assembly" ], [ 80, 89, "conserved", "protein_state" ], [ 90, 95, "water", "chemical" ], [ 108, 111, "CDR", "structure_element" ], [ 112, 114, "H3", "structure_element" ], [ 145, 155, "structures", "evidence" ] ] }, { "sid": 163, "sent": "In fact, it is the only Fab in the set that has a water molecule present at this site.", "section": "RESULTS", "ner": [ [ 24, 27, "Fab", "structure_element" ], [ 50, 55, "water", "chemical" ] ] }, { "sid": 164, "sent": "The CDR H3 for this structure is shown in Fig.\u00a0S3.", "section": "RESULTS", "ner": [ [ 4, 7, "CDR", "structure_element" ], [ 8, 10, "H3", "structure_element" ], [ 20, 29, "structure", "evidence" ] ] }, { "sid": 165, "sent": "The remaining 8 Fabs can be grouped into 5 different conformational classes.", "section": "RESULTS", "ner": [ [ 16, 20, "Fabs", "structure_element" ] ] }, { "sid": 166, "sent": "Three of the Fabs, H3-23:L1-39, H3-23:L4-1 and H3-53:L1-39, have distinctive conformations.", "section": "RESULTS", "ner": [ [ 13, 17, "Fabs", "structure_element" ], [ 19, 30, "H3-23:L1-39", "complex_assembly" ], [ 32, 42, "H3-23:L4-1", "complex_assembly" ], [ 47, 58, "H3-53:L1-39", "complex_assembly" ] ] }, { "sid": 167, "sent": "The stem regions in these 3 cases are in the \u2018kinked\u2019 conformation consistent with that observed for 4DN3.", "section": "RESULTS", "ner": [ [ 4, 16, "stem regions", "structure_element" ], [ 46, 52, "kinked", "protein_state" ] ] }, { "sid": 168, "sent": "The five remaining Fabs, H5-51:L4-1 (2 copies), H1-69:L3-20 (2 copies) and H3-53:L4-1, have 3 different CDR H3 conformations (Fig.\u00a0S4).", "section": "RESULTS", "ner": [ [ 19, 23, "Fabs", "structure_element" ], [ 25, 35, "H5-51:L4-1", "complex_assembly" ], [ 48, 59, "H1-69:L3-20", "complex_assembly" ], [ 75, 85, "H3-53:L4-1", "complex_assembly" ], [ 104, 107, "CDR", "structure_element" ], [ 108, 110, "H3", "structure_element" ] ] }, { "sid": 169, "sent": "The stem regions of CDR H3 for the H5-51:L4-1 Fabs are in the \u2018kinked\u2019 conformation while, surprisingly, those of the H1-69:L3-20 pair and H3-53:L4-1 are in the \u2018extended\u2019 conformation (Fig.\u00a07B).", "section": "RESULTS", "ner": [ [ 4, 16, "stem regions", "structure_element" ], [ 20, 23, "CDR", "structure_element" ], [ 24, 26, "H3", "structure_element" ], [ 35, 45, "H5-51:L4-1", "complex_assembly" ], [ 46, 50, "Fabs", "structure_element" ], [ 63, 69, "kinked", "protein_state" ], [ 118, 129, "H1-69:L3-20", "complex_assembly" ], [ 139, 149, "H3-53:L4-1", "complex_assembly" ], [ 162, 170, "extended", "protein_state" ] ] }, { "sid": 170, "sent": "VH:VL domain packing", "section": "RESULTS", "ner": [ [ 0, 5, "VH:VL", "complex_assembly" ] ] }, { "sid": 171, "sent": "The VH and VL domains have a \u03b2-sandwich structure (also often referred as a Greek key motif) and each is composed of a 4-stranded and a 5-stranded antiparallel \u03b2-sheets.", "section": "RESULTS", "ner": [ [ 4, 6, "VH", "structure_element" ], [ 11, 13, "VL", "structure_element" ], [ 29, 49, "\u03b2-sandwich structure", "structure_element" ], [ 76, 91, "Greek key motif", "structure_element" ], [ 119, 168, "4-stranded and a 5-stranded antiparallel \u03b2-sheets", "structure_element" ] ] }, { "sid": 172, "sent": "The two domains pack together such that the 5-stranded \u03b2-sheets, which have hydrophobic surfaces, interact with each other bringing the CDRs from both the VH and VL domains into close proximity.", "section": "RESULTS", "ner": [ [ 44, 63, "5-stranded \u03b2-sheets", "structure_element" ], [ 136, 140, "CDRs", "structure_element" ], [ 155, 157, "VH", "structure_element" ], [ 162, 164, "VL", "structure_element" ] ] }, { "sid": 173, "sent": "The domain packing of the variants was assessed by computing the domain interface interactions, the VH:VL tilt angles, the buried surface area and surface complementarity.", "section": "RESULTS", "ner": [ [ 65, 81, "domain interface", "site" ], [ 100, 105, "VH:VL", "complex_assembly" ], [ 106, 117, "tilt angles", "evidence" ] ] }, { "sid": 174, "sent": "VH:VL interface amino acid residue interactions", "section": "RESULTS", "ner": [ [ 0, 15, "VH:VL interface", "site" ] ] }, { "sid": 175, "sent": "The conserved VH:VL interactions as viewed along the VH/VL axis.", "section": "FIG", "ner": [ [ 4, 13, "conserved", "protein_state" ], [ 14, 19, "VH:VL", "complex_assembly" ], [ 53, 55, "VH", "structure_element" ], [ 56, 58, "VL", "structure_element" ] ] }, { "sid": 176, "sent": "The VH residues are in blue, the VL residues are in orange.", "section": "FIG", "ner": [ [ 4, 6, "VH", "structure_element" ], [ 33, 35, "VL", "structure_element" ] ] }, { "sid": 177, "sent": "The VH:VL interface is pseudosymmetric, and involves 2 stretches of the polypeptide chain from each domain, namely CDR3 and the framework region between CDRs 1 and 2.", "section": "RESULTS", "ner": [ [ 4, 19, "VH:VL interface", "site" ], [ 23, 38, "pseudosymmetric", "protein_state" ], [ 115, 119, "CDR3", "structure_element" ], [ 128, 144, "framework region", "structure_element" ], [ 153, 165, "CDRs 1 and 2", "structure_element" ] ] }, { "sid": 178, "sent": "These stretches form antiparallel \u03b2-hairpins within the internal 5-stranded \u03b2-sheet.", "section": "RESULTS", "ner": [ [ 21, 44, "antiparallel \u03b2-hairpins", "structure_element" ], [ 65, 83, "5-stranded \u03b2-sheet", "structure_element" ] ] }, { "sid": 179, "sent": "There are a few principal inter-domain interactions that are conserved not only in the experimental set of 16 Fabs, but in all human antibodies.", "section": "RESULTS", "ner": [ [ 110, 114, "Fabs", "structure_element" ], [ 127, 132, "human", "species" ], [ 133, 143, "antibodies", "protein_type" ] ] }, { "sid": 180, "sent": "They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig.\u00a08).", "section": "RESULTS", "ner": [ [ 29, 42, "hydrogen bond", "bond_interaction" ], [ 51, 52, "L", "structure_element" ], [ 53, 58, "Gln38", "residue_name_number" ], [ 63, 64, "H", "structure_element" ], [ 65, 70, "Gln39", "residue_name_number" ], [ 75, 76, "H", "structure_element" ], [ 77, 82, "Leu45", "residue_name_number" ], [ 88, 106, "hydrophobic pocket", "site" ], [ 115, 116, "L", "structure_element" ], [ 117, 122, "Phe98", "residue_name_number" ], [ 124, 125, "L", "structure_element" ], [ 126, 131, "Tyr87", "residue_name_number" ], [ 136, 137, "L", "structure_element" ], [ 138, 143, "Pro44", "residue_name_number" ], [ 148, 149, "L", "structure_element" ], [ 150, 155, "Pro44", "residue_name_number" ], [ 172, 173, "H", "structure_element" ], [ 174, 180, "Trp103", "residue_name_number" ], [ 189, 190, "L", "structure_element" ], [ 191, 196, "Ala43", "residue_name_number" ], [ 218, 219, "H", "structure_element" ], [ 220, 225, "Tyr91", "residue_name_number" ] ] }, { "sid": 181, "sent": "With the exception of L-Ala43, all other residues are conserved in human germlines.", "section": "RESULTS", "ner": [ [ 22, 23, "L", "structure_element" ], [ 24, 29, "Ala43", "residue_name_number" ], [ 67, 72, "human", "species" ] ] }, { "sid": 182, "sent": "Position 43 may be alternatively occupied by Ser, Val or Pro (as in L4-1), but the hydrophobic interaction with H-Tyr91 is preserved.", "section": "RESULTS", "ner": [ [ 9, 11, "43", "residue_number" ], [ 45, 48, "Ser", "residue_name" ], [ 50, 53, "Val", "residue_name" ], [ 57, 60, "Pro", "residue_name" ], [ 68, 72, "L4-1", "mutant" ], [ 83, 106, "hydrophobic interaction", "bond_interaction" ], [ 112, 113, "H", "structure_element" ], [ 114, 119, "Tyr91", "residue_name_number" ] ] }, { "sid": 183, "sent": "These core interactions provide enough stability to the VH:VL dimer so that additional VH-VL contacts can tolerate amino acid sequence variations in CDRs H3 and L3 that form part of the VH:VL interface.", "section": "RESULTS", "ner": [ [ 56, 61, "VH:VL", "complex_assembly" ], [ 62, 67, "dimer", "oligomeric_state" ], [ 87, 101, "VH-VL contacts", "site" ], [ 149, 153, "CDRs", "structure_element" ], [ 154, 156, "H3", "structure_element" ], [ 161, 163, "L3", "structure_element" ], [ 186, 201, "VH:VL interface", "site" ] ] }, { "sid": 184, "sent": "In total, about 20 residues are involved in the VH:VL interactions on each side (Fig.\u00a0S5).", "section": "RESULTS", "ner": [ [ 16, 27, "20 residues", "residue_range" ], [ 48, 53, "VH:VL", "complex_assembly" ] ] }, { "sid": 185, "sent": "Half of them are in the framework regions and those residues (except residue 61 in HC, which is actually in CDR2 in Kabat's definition) are conserved in the set of 16 Fabs.", "section": "RESULTS", "ner": [ [ 24, 41, "framework regions", "structure_element" ], [ 77, 79, "61", "residue_number" ], [ 83, 85, "HC", "structure_element" ], [ 108, 112, "CDR2", "structure_element" ], [ 167, 171, "Fabs", "structure_element" ] ] }, { "sid": 186, "sent": "One notable exception is H-Trp47, which exhibits 2 conformations of the indole ring.", "section": "RESULTS", "ner": [ [ 25, 26, "H", "structure_element" ], [ 27, 32, "Trp47", "residue_name_number" ] ] }, { "sid": 187, "sent": "In most of the structures, it has the \u03c72 angle of \u223c80\u00b0, while the ring is flipped over (\u03c72 = \u2212100\u00b0) in H5-51:L3:11 and H5-51:L3-20.", "section": "RESULTS", "ner": [ [ 15, 25, "structures", "evidence" ], [ 38, 40, "\u03c72", "evidence" ], [ 88, 90, "\u03c72", "evidence" ], [ 103, 114, "H5-51:L3:11", "complex_assembly" ], [ 119, 130, "H5-51:L3-20", "complex_assembly" ] ] }, { "sid": 188, "sent": "Interestingly, these are the only 2 structures with residues missing in CDR H3 because of disorder, although both structures are determined at high resolution and the rest of the structure is well defined.", "section": "RESULTS", "ner": [ [ 36, 46, "structures", "evidence" ], [ 61, 68, "missing", "protein_state" ], [ 72, 75, "CDR", "structure_element" ], [ 76, 78, "H3", "structure_element" ], [ 114, 124, "structures", "evidence" ], [ 179, 188, "structure", "evidence" ] ] }, { "sid": 189, "sent": "Apparently, residues flanking CDR H3 in the 2 VH:VL pairings are inconsistent with any stable conformation of CDR H3, which translates into a less restricted conformational space for some of them, including H-Trp47.", "section": "RESULTS", "ner": [ [ 30, 33, "CDR", "structure_element" ], [ 34, 36, "H3", "structure_element" ], [ 46, 51, "VH:VL", "complex_assembly" ], [ 87, 93, "stable", "protein_state" ], [ 110, 113, "CDR", "structure_element" ], [ 114, 116, "H3", "structure_element" ], [ 207, 208, "H", "structure_element" ], [ 209, 214, "Trp47", "residue_name_number" ] ] }, { "sid": 190, "sent": "VH:VL tilt angles", "section": "RESULTS", "ner": [ [ 0, 5, "VH:VL", "complex_assembly" ], [ 6, 17, "tilt angles", "evidence" ] ] }, { "sid": 191, "sent": "The relative orientation of VH and VL has been measured in a number of different ways.", "section": "RESULTS", "ner": [ [ 28, 30, "VH", "structure_element" ], [ 35, 37, "VL", "structure_element" ] ] }, { "sid": 192, "sent": "The first approach uses ABangles, the results of which are shown in Table\u00a0S2.", "section": "RESULTS", "ner": [ [ 24, 32, "ABangles", "experimental_method" ] ] }, { "sid": 193, "sent": "The four LCs all are classified as Type A because they have a proline at position 44, and the results for each orientation parameter are within the range of values of this type reported by Dunbar and co-workers.", "section": "RESULTS", "ner": [ [ 9, 12, "LCs", "structure_element" ], [ 62, 69, "proline", "residue_name" ], [ 82, 84, "44", "residue_number" ], [ 111, 132, "orientation parameter", "evidence" ] ] }, { "sid": 194, "sent": "In fact, the parameter values for the set of 16 Fabs are in the middle of the distribution observed for 351 non-redundant antibody structures determined at 3.0 \u212b resolution or better.", "section": "RESULTS", "ner": [ [ 48, 52, "Fabs", "structure_element" ], [ 122, 130, "antibody", "protein_type" ], [ 131, 141, "structures", "evidence" ] ] }, { "sid": 195, "sent": "The only exception is HC1, which is shifted toward smaller angles with the mean value of 70.8\u00b0 as compared to the distribution centered at 72\u00b0 for the entire PDB.", "section": "RESULTS", "ner": [ [ 22, 25, "HC1", "structure_element" ] ] }, { "sid": 196, "sent": "This probably reflects the invariance of CDR H3 in the current set as opposed to the CDR H3 diversity in the PDB.", "section": "RESULTS", "ner": [ [ 41, 44, "CDR", "structure_element" ], [ 45, 47, "H3", "structure_element" ], [ 85, 88, "CDR", "structure_element" ], [ 89, 91, "H3", "structure_element" ] ] }, { "sid": 197, "sent": "The second approach used for comparing tilt angles involved computing the difference in the tilt angles between all pairs of structures.", "section": "RESULTS", "ner": [ [ 39, 50, "tilt angles", "evidence" ], [ 74, 84, "difference", "evidence" ], [ 92, 103, "tilt angles", "evidence" ], [ 125, 135, "structures", "evidence" ] ] }, { "sid": 198, "sent": "For structures with 2 copies of the Fab in the asymmetric unit, only one structure was used.", "section": "RESULTS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 36, 39, "Fab", "structure_element" ], [ 73, 82, "structure", "evidence" ] ] }, { "sid": 199, "sent": "The differences between independent Fabs in the same structure are 4.9\u00b0 for H1-69:L3-20, 1.6\u00b0 for H1-69:L3-11, 1.4\u00b0 for H3-23:L4-1, 3.3\u00b0 for H3-23:L3-11, and 2.5\u00b0 for H5-51:L4-1.", "section": "RESULTS", "ner": [ [ 36, 40, "Fabs", "structure_element" ], [ 53, 62, "structure", "evidence" ], [ 76, 87, "H1-69:L3-20", "complex_assembly" ], [ 98, 109, "H1-69:L3-11", "complex_assembly" ], [ 120, 130, "H3-23:L4-1", "complex_assembly" ], [ 141, 152, "H3-23:L3-11", "complex_assembly" ], [ 167, 177, "H5-51:L4-1", "complex_assembly" ] ] }, { "sid": 200, "sent": "With the exception of H1-69:L3-20, the angles are within the range of 2-3\u00b0 as are observed in the identical structures in the PDB.", "section": "RESULTS", "ner": [ [ 22, 33, "H1-69:L3-20", "complex_assembly" ], [ 108, 118, "structures", "evidence" ] ] }, { "sid": 201, "sent": "In H1-69:L3-20, one of the Fabs is substantially disordered so that part of CDR H2 (the outer \u03b2-strand, residues 55-60) is completely missing.", "section": "RESULTS", "ner": [ [ 3, 14, "H1-69:L3-20", "complex_assembly" ], [ 27, 31, "Fabs", "structure_element" ], [ 49, 59, "disordered", "protein_state" ], [ 76, 79, "CDR", "structure_element" ], [ 80, 82, "H2", "structure_element" ], [ 94, 102, "\u03b2-strand", "structure_element" ], [ 113, 118, "55-60", "residue_range" ] ] }, { "sid": 202, "sent": "This kind of disorder may compromise the integrity of the VH domain and its interaction with the VL.", "section": "RESULTS", "ner": [ [ 58, 60, "VH", "structure_element" ], [ 97, 99, "VL", "structure_element" ] ] }, { "sid": 203, "sent": "Indeed, this Fab has the largest twist angle HC2 within the experimental set that exceeds the mean value by 2.5 standard deviations (Table\u00a0S2).", "section": "RESULTS", "ner": [ [ 13, 16, "Fab", "structure_element" ], [ 33, 44, "twist angle", "evidence" ], [ 45, 48, "HC2", "structure_element" ] ] }, { "sid": 204, "sent": "An illustration of the difference in tilt angle for 2 pairs of variants by the superposition of the VH domains of (A) H1-69:L3-20 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 10.5\u00b0) and (B) H1-69:L4-1 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 1.6\u00b0).", "section": "FIG", "ner": [ [ 79, 92, "superposition", "experimental_method" ], [ 100, 102, "VH", "structure_element" ], [ 118, 129, "H1-69:L3-20", "complex_assembly" ], [ 141, 152, "H5-51:L1-39", "complex_assembly" ], [ 158, 160, "VL", "structure_element" ], [ 219, 229, "H1-69:L4-1", "complex_assembly" ], [ 241, 252, "H5-51:L1-39", "complex_assembly" ], [ 258, 260, "VL", "structure_element" ] ] }, { "sid": 205, "sent": "Differences in VH:VL tilt angles.", "section": "TABLE", "ner": [ [ 15, 20, "VH:VL", "complex_assembly" ], [ 21, 32, "tilt angles", "evidence" ] ] }, { "sid": 206, "sent": "The differences in the tilt angle are shown for all pairs of V regions in Table\u00a03.", "section": "RESULTS", "ner": [ [ 4, 15, "differences", "evidence" ], [ 23, 33, "tilt angle", "evidence" ], [ 61, 70, "V regions", "structure_element" ] ] }, { "sid": 207, "sent": "The smallest differences in the tilt angle are between the Fabs in isomorphous crystal forms.", "section": "RESULTS", "ner": [ [ 32, 42, "tilt angle", "evidence" ], [ 59, 63, "Fabs", "structure_element" ], [ 79, 92, "crystal forms", "evidence" ] ] }, { "sid": 208, "sent": "The largest deviations in the tilt angle, up to 11.0\u00b0, are found for 2 structures, H1-69:L3-20 and H3-23:L3-20, that stand out from the other Fabs.", "section": "RESULTS", "ner": [ [ 30, 40, "tilt angle", "evidence" ], [ 71, 81, "structures", "evidence" ], [ 83, 94, "H1-69:L3-20", "complex_assembly" ], [ 99, 110, "H3-23:L3-20", "complex_assembly" ], [ 142, 146, "Fabs", "structure_element" ] ] }, { "sid": 209, "sent": "One of the 2 structures, H1-69:L3-20, has its CDR H3 in the \u2018extended\u2019 conformation; the other structure has it in the \u2018kinked\u2019 conformation.", "section": "RESULTS", "ner": [ [ 13, 23, "structures", "evidence" ], [ 25, 36, "H1-69:L3-20", "complex_assembly" ], [ 46, 49, "CDR", "structure_element" ], [ 50, 52, "H3", "structure_element" ], [ 61, 69, "extended", "protein_state" ], [ 120, 126, "kinked", "protein_state" ] ] }, { "sid": 210, "sent": "Two examples illustrating large (10.5\u00b0) and small (1.6\u00b0) differences in the tilt angles are shown in Fig.\u00a09.", "section": "RESULTS", "ner": [ [ 76, 87, "tilt angles", "evidence" ] ] }, { "sid": 211, "sent": "VH:VL buried surface area and complementarity", "section": "RESULTS", "ner": [ [ 0, 5, "VH:VL", "complex_assembly" ] ] }, { "sid": 212, "sent": "VH:VL surface areas and surface complementarity.", "section": "TABLE", "ner": [ [ 0, 5, "VH:VL", "complex_assembly" ] ] }, { "sid": 213, "sent": "Some side chain atoms in CDR H3 are missing.", "section": "TABLE", "ner": [ [ 25, 28, "CDR", "structure_element" ], [ 29, 31, "H3", "structure_element" ] ] }, { "sid": 214, "sent": "Residues in CDR H3 are missing: YGE in H5-51:L3-11, GIY in H5-51:L3-20.", "section": "TABLE", "ner": [ [ 12, 15, "CDR", "structure_element" ], [ 16, 18, "H3", "structure_element" ], [ 32, 35, "YGE", "structure_element" ], [ 39, 50, "H5-51:L3-11", "complex_assembly" ], [ 52, 55, "GIY", "structure_element" ], [ 59, 70, "H5-51:L3-20", "complex_assembly" ] ] }, { "sid": 215, "sent": "The results of the PISA contact surface calculation and surface complementarity calculation are shown in Table\u00a04.", "section": "RESULTS", "ner": [ [ 19, 23, "PISA", "experimental_method" ], [ 24, 51, "contact surface calculation", "experimental_method" ], [ 56, 91, "surface complementarity calculation", "experimental_method" ] ] }, { "sid": 216, "sent": "The interface areas are calculated as the average of the VH and VL contact surfaces.", "section": "RESULTS", "ner": [ [ 4, 13, "interface", "site" ], [ 57, 83, "VH and VL contact surfaces", "site" ] ] }, { "sid": 217, "sent": "Six of the 16 structures have CDR H3 side chains or complete residues missing, and therefore their interfaces are much smaller than in the other 10 structures with complete CDRs (the results are provided for all Fabs for completeness).", "section": "RESULTS", "ner": [ [ 14, 24, "structures", "evidence" ], [ 30, 33, "CDR", "structure_element" ], [ 34, 36, "H3", "structure_element" ], [ 70, 77, "missing", "protein_state" ], [ 99, 109, "interfaces", "site" ], [ 148, 158, "structures", "evidence" ], [ 164, 172, "complete", "protein_state" ], [ 173, 177, "CDRs", "structure_element" ], [ 212, 216, "Fabs", "structure_element" ] ] }, { "sid": 218, "sent": "Among the complete structures, the interface areas range from 684 to 836 \u212b2.", "section": "RESULTS", "ner": [ [ 10, 18, "complete", "protein_state" ], [ 19, 29, "structures", "evidence" ], [ 35, 44, "interface", "site" ] ] }, { "sid": 219, "sent": "Interestingly, the 2 structures that have the largest tilt angle differences with the other variants, H3-23:L3-20 and H1-69:L3-20, have the smallest VH:VL interfaces, 684 and 725 \u212b2, respectively.", "section": "RESULTS", "ner": [ [ 21, 31, "structures", "evidence" ], [ 54, 76, "tilt angle differences", "evidence" ], [ 102, 113, "H3-23:L3-20", "complex_assembly" ], [ 118, 129, "H1-69:L3-20", "complex_assembly" ], [ 149, 165, "VH:VL interfaces", "site" ] ] }, { "sid": 220, "sent": "H3-23:L3-20 is also unique in that it has the lowest value (0.676) of surface complementarity.", "section": "RESULTS", "ner": [ [ 0, 11, "H3-23:L3-20", "complex_assembly" ], [ 70, 93, "surface complementarity", "evidence" ] ] }, { "sid": 221, "sent": "Melting temperatures for the 16 Fabs.", "section": "TABLE", "ner": [ [ 0, 20, "Melting temperatures", "evidence" ], [ 32, 36, "Fabs", "structure_element" ] ] }, { "sid": 222, "sent": "Colors: blue (Tm < 70\u00b0C), green (70\u00b0C < Tm < 73\u00b0C), yellow (73\u00b0C < Tm < 78\u00b0C), orange (Tm > 78\u00b0C).", "section": "TABLE", "ner": [ [ 14, 16, "Tm", "evidence" ], [ 40, 42, "Tm", "evidence" ], [ 67, 69, "Tm", "evidence" ], [ 87, 89, "Tm", "evidence" ] ] }, { "sid": 223, "sent": "Melting temperatures (Tm) were measured for all Fabs using differential scanning calorimetry (Table\u00a05).", "section": "RESULTS", "ner": [ [ 0, 20, "Melting temperatures", "evidence" ], [ 22, 24, "Tm", "evidence" ], [ 48, 52, "Fabs", "structure_element" ], [ 59, 92, "differential scanning calorimetry", "experimental_method" ] ] }, { "sid": 224, "sent": "It appears that for each given LC, the Fabs with germlines H1-69 and H3-23 are substantially more stable than those with germlines H3-53 and H5-51.", "section": "RESULTS", "ner": [ [ 31, 33, "LC", "structure_element" ], [ 39, 43, "Fabs", "structure_element" ], [ 59, 64, "H1-69", "mutant" ], [ 69, 74, "H3-23", "mutant" ], [ 98, 104, "stable", "protein_state" ], [ 131, 136, "H3-53", "mutant" ], [ 141, 146, "H5-51", "mutant" ] ] }, { "sid": 225, "sent": "In addition, L1-39 provides a much higher degree of stabilization than the other 3 LC germlines when combined with any of the HCs.", "section": "RESULTS", "ner": [ [ 13, 18, "L1-39", "mutant" ], [ 83, 85, "LC", "structure_element" ], [ 126, 129, "HCs", "structure_element" ] ] }, { "sid": 226, "sent": "As a result, the Tm for pairs H1-69:L1-39 and H3-23:L1-39 is 12-13\u00b0 higher than for pairs H3-53:L3-20, H3-53:L4-1, H5-51:L3-20 and H5-51:L4-1.", "section": "RESULTS", "ner": [ [ 17, 19, "Tm", "evidence" ], [ 30, 41, "H1-69:L1-39", "complex_assembly" ], [ 46, 57, "H3-23:L1-39", "complex_assembly" ], [ 90, 101, "H3-53:L3-20", "complex_assembly" ], [ 103, 113, "H3-53:L4-1", "complex_assembly" ], [ 115, 126, "H5-51:L3-20", "complex_assembly" ], [ 131, 141, "H5-51:L4-1", "complex_assembly" ] ] }, { "sid": 227, "sent": "These findings correlate well with the degree of conformational disorder observed in the crystal structures.", "section": "RESULTS", "ner": [ [ 89, 107, "crystal structures", "evidence" ] ] }, { "sid": 228, "sent": "Parts of CDR H3 main chain are completely disordered, and were not modeled in Fabs H5-51:L3-20 and H5-51:L3-11 that have the lowest Tms in the set.", "section": "RESULTS", "ner": [ [ 9, 12, "CDR", "structure_element" ], [ 13, 15, "H3", "structure_element" ], [ 42, 52, "disordered", "protein_state" ], [ 78, 82, "Fabs", "structure_element" ], [ 83, 94, "H5-51:L3-20", "complex_assembly" ], [ 99, 110, "H5-51:L3-11", "complex_assembly" ], [ 132, 135, "Tms", "evidence" ] ] }, { "sid": 229, "sent": "No electron density is observed for a number of side chains in CDRs H3 and L3 in all Fabs with germline H3-53, which indicates loose packing of the variable domains.", "section": "RESULTS", "ner": [ [ 3, 19, "electron density", "evidence" ], [ 63, 67, "CDRs", "structure_element" ], [ 68, 70, "H3", "structure_element" ], [ 75, 77, "L3", "structure_element" ], [ 85, 89, "Fabs", "structure_element" ], [ 104, 109, "H3-53", "mutant" ], [ 148, 164, "variable domains", "structure_element" ] ] }, { "sid": 230, "sent": "All those molecules are relatively unstable, as is reflected in their low Tms.", "section": "RESULTS", "ner": [ [ 74, 77, "Tms", "evidence" ] ] }, { "sid": 231, "sent": "This is the first report of a systematic structural investigation of a phage germline library.", "section": "DISCUSS", "ner": [ [ 30, 65, "systematic structural investigation", "experimental_method" ], [ 71, 93, "phage germline library", "experimental_method" ] ] }, { "sid": 232, "sent": "The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3.", "section": "DISCUSS", "ner": [ [ 7, 10, "Fab", "structure_element" ], [ 11, 21, "structures", "evidence" ], [ 73, 76, "HCs", "structure_element" ], [ 78, 83, "H1-69", "mutant" ], [ 85, 90, "H3-23", "mutant" ], [ 92, 97, "H3-53", "mutant" ], [ 103, 108, "H5-51", "mutant" ], [ 126, 129, "LCs", "structure_element" ], [ 131, 136, "L1-39", "mutant" ], [ 138, 143, "L3-11", "mutant" ], [ 145, 150, "L3-20", "mutant" ], [ 155, 159, "L4-1", "mutant" ], [ 180, 183, "CDR", "structure_element" ], [ 184, 186, "H3", "structure_element" ] ] }, { "sid": 233, "sent": "The structural data set taken as a whole provides insight into how the backbone conformations of the CDRs of a specific heavy or light chain vary when it is paired with 4 different light or heavy chains, respectively.", "section": "DISCUSS", "ner": [ [ 4, 19, "structural data", "evidence" ], [ 101, 105, "CDRs", "structure_element" ], [ 129, 140, "light chain", "structure_element" ], [ 190, 202, "heavy chains", "structure_element" ] ] }, { "sid": 234, "sent": "A large variability in the CDR conformations for the sets of HCs and LCs is observed.", "section": "DISCUSS", "ner": [ [ 27, 30, "CDR", "structure_element" ], [ 61, 64, "HCs", "structure_element" ], [ 69, 72, "LCs", "structure_element" ] ] }, { "sid": 235, "sent": "In some cases the CDR conformations for all members of a set are virtually identical, for others subtle changes occur in a few members of a set, and in some cases larger deviations are observed within a set.", "section": "DISCUSS", "ner": [ [ 18, 21, "CDR", "structure_element" ] ] }, { "sid": 236, "sent": "The five variants that crystallized with 2 copies of the Fab in the asymmetric unit serve somewhat as controls for the influence of crystal packing on the conformations of the CDRs.", "section": "DISCUSS", "ner": [ [ 23, 35, "crystallized", "experimental_method" ], [ 57, 60, "Fab", "structure_element" ], [ 176, 180, "CDRs", "structure_element" ] ] }, { "sid": 237, "sent": "In four of the 5 structures the CDR conformations are consistent.", "section": "DISCUSS", "ner": [ [ 17, 27, "structures", "evidence" ], [ 32, 35, "CDR", "structure_element" ] ] }, { "sid": 238, "sent": "In only one case, that of H1-69:L3-20 (the lowest resolution structure), do we see differences in the conformations of the 2 copies of CDRs H1 and L1.", "section": "DISCUSS", "ner": [ [ 26, 37, "H1-69:L3-20", "complex_assembly" ], [ 61, 70, "structure", "evidence" ], [ 135, 139, "CDRs", "structure_element" ], [ 140, 142, "H1", "structure_element" ], [ 147, 149, "L1", "structure_element" ] ] }, { "sid": 239, "sent": "This variability is likely a result of 2 factors, crystal packing interactions and internal instability of the variable domain.", "section": "DISCUSS", "ner": [ [ 111, 126, "variable domain", "structure_element" ] ] }, { "sid": 240, "sent": "For the CDRs with canonical structures, the largest changes in conformation occur for CDR H1 of H1-69 and H3-53.", "section": "DISCUSS", "ner": [ [ 8, 12, "CDRs", "structure_element" ], [ 86, 89, "CDR", "structure_element" ], [ 90, 92, "H1", "structure_element" ], [ 96, 101, "H1-69", "mutant" ], [ 106, 111, "H3-53", "mutant" ] ] }, { "sid": 241, "sent": "The other 2 HCs, H3-23 and H5-51, have canonical structures that are remarkably well conserved (Fig.\u00a01).", "section": "DISCUSS", "ner": [ [ 12, 15, "HCs", "structure_element" ], [ 17, 22, "H3-23", "mutant" ], [ 27, 32, "H5-51", "mutant" ], [ 69, 94, "remarkably well conserved", "protein_state" ] ] }, { "sid": 242, "sent": "Of the 4 HCs, H1-69 has the greatest number of canonical structure assignments (Table\u00a02).", "section": "DISCUSS", "ner": [ [ 9, 12, "HCs", "structure_element" ], [ 14, 19, "H1-69", "mutant" ] ] }, { "sid": 243, "sent": "H1-69 is unique in having a pair of glycine residues at positions 26 and 27, which provide more conformational freedom in CDR H1.", "section": "DISCUSS", "ner": [ [ 0, 5, "H1-69", "mutant" ], [ 36, 43, "glycine", "residue_name" ], [ 66, 68, "26", "residue_number" ], [ 73, 75, "27", "residue_number" ], [ 96, 118, "conformational freedom", "protein_state" ], [ 122, 125, "CDR", "structure_element" ], [ 126, 128, "H1", "structure_element" ] ] }, { "sid": 244, "sent": "Besides IGHV1-69, only the germlines of the VH4 family possess double glycines in CDR H1, and it will be interesting to see if they are also conformationally unstable.", "section": "DISCUSS", "ner": [ [ 8, 16, "IGHV1-69", "mutant" ], [ 44, 47, "VH4", "structure_element" ], [ 70, 78, "glycines", "residue_name" ], [ 82, 85, "CDR", "structure_element" ], [ 86, 88, "H1", "structure_element" ], [ 141, 166, "conformationally unstable", "protein_state" ] ] }, { "sid": 245, "sent": "Having all 16 VH:VL pairs with the same CDR H3 provides some insights into why molecular modeling efforts of CDR H3 have proven so difficult.", "section": "DISCUSS", "ner": [ [ 14, 19, "VH:VL", "complex_assembly" ], [ 40, 43, "CDR", "structure_element" ], [ 44, 46, "H3", "structure_element" ], [ 109, 112, "CDR", "structure_element" ], [ 113, 115, "H3", "structure_element" ] ] }, { "sid": 246, "sent": "As mentioned in the Results section, this data set is composed of 21 Fabs, since 5 of the 16 variants have 2 Fab copies in the asymmetric unit.", "section": "DISCUSS", "ner": [ [ 69, 73, "Fabs", "structure_element" ], [ 109, 112, "Fab", "structure_element" ] ] }, { "sid": 247, "sent": "For the 18 Fabs with complete backbone atoms for CDR H3, 10 have conformations similar to that of the parent, while the others have significantly different conformations (Fig.\u00a06).", "section": "DISCUSS", "ner": [ [ 11, 15, "Fabs", "structure_element" ], [ 49, 52, "CDR", "structure_element" ], [ 53, 55, "H3", "structure_element" ] ] }, { "sid": 248, "sent": "Thus, it is likely that the CDR H3 conformation is dependent upon 2 dominating factors: 1) amino acid sequence; and 2) VH and VL context.", "section": "DISCUSS", "ner": [ [ 28, 31, "CDR", "structure_element" ], [ 32, 34, "H3", "structure_element" ], [ 119, 121, "VH", "structure_element" ], [ 126, 128, "VL", "structure_element" ] ] }, { "sid": 249, "sent": "More than half of the variants retain the conformation of the parent despite having differences in the VH:VL pairing.", "section": "DISCUSS", "ner": [ [ 103, 108, "VH:VL", "complex_assembly" ] ] }, { "sid": 250, "sent": "This subset includes 2 structures with 2 copies of the Fab in the asymmetric unit, all of which are nearly identical in conformation.", "section": "DISCUSS", "ner": [ [ 23, 33, "structures", "evidence" ], [ 55, 58, "Fab", "structure_element" ] ] }, { "sid": 251, "sent": "The remaining 8 structures exhibit \u201cnon-parental\u201d conformations, indicating that the VH and VL context can also be a dominating factor influencing CDR H3.", "section": "DISCUSS", "ner": [ [ 16, 26, "structures", "evidence" ], [ 85, 87, "VH", "structure_element" ], [ 92, 94, "VL", "structure_element" ], [ 147, 150, "CDR", "structure_element" ], [ 151, 153, "H3", "structure_element" ] ] }, { "sid": 252, "sent": "This subset also has 2 structures with 2 Fab copies in the asymmetric unit.", "section": "DISCUSS", "ner": [ [ 23, 33, "structures", "evidence" ], [ 41, 44, "Fab", "structure_element" ] ] }, { "sid": 253, "sent": "Interestingly, as described earlier, these 2 pairs differ in the stem regions with the H1-69:L3-20 pair in the \u2018extended\u2019 conformation and H5-51:L4-1 pair in the \u2018kinked\u2019 conformation.", "section": "DISCUSS", "ner": [ [ 65, 77, "stem regions", "structure_element" ], [ 87, 98, "H1-69:L3-20", "complex_assembly" ], [ 112, 120, "extended", "protein_state" ], [ 139, 149, "H5-51:L4-1", "complex_assembly" ], [ 163, 169, "kinked", "protein_state" ] ] }, { "sid": 254, "sent": "The CDR H3 conformational analysis shows that, for each set of variants of one HC paired with the 4 different LCs, both \u201cparental\u201d and \u201cnon-parental\u201d conformations are observed.", "section": "DISCUSS", "ner": [ [ 4, 7, "CDR", "structure_element" ], [ 8, 10, "H3", "structure_element" ], [ 11, 34, "conformational analysis", "experimental_method" ], [ 79, 81, "HC", "structure_element" ], [ 110, 113, "LCs", "structure_element" ] ] }, { "sid": 255, "sent": "The same variability is observed for the sets of variants composed of one LC paired with each of the 4 HCs.", "section": "DISCUSS", "ner": [ [ 74, 76, "LC", "structure_element" ], [ 103, 106, "HCs", "structure_element" ] ] }, { "sid": 256, "sent": "Thus, no patterns of conformational preference for a particular HC or LC emerge to shed any direct light on what drives the conformational differences.", "section": "DISCUSS", "ner": [ [ 64, 66, "HC", "structure_element" ], [ 70, 72, "LC", "structure_element" ] ] }, { "sid": 257, "sent": "This finding supports the hypothesis of Weitzner et\u00a0al. that the H3 conformation is controlled both by its sequence and its environment.", "section": "DISCUSS", "ner": [ [ 65, 67, "H3", "structure_element" ] ] }, { "sid": 258, "sent": "In looking at a possible correlation between the tilt angle and the conformation of CDR H3, no clear trends are observed.", "section": "DISCUSS", "ner": [ [ 49, 59, "tilt angle", "evidence" ], [ 84, 87, "CDR", "structure_element" ], [ 88, 90, "H3", "structure_element" ] ] }, { "sid": 259, "sent": "Two variants, H1-69:L3-20 and H3-23:L3-20, have the largest differences in the tilt angles compared to other variants as seen in Table\u00a03.", "section": "DISCUSS", "ner": [ [ 14, 25, "H1-69:L3-20", "complex_assembly" ], [ 30, 41, "H3-23:L3-20", "complex_assembly" ] ] }, { "sid": 260, "sent": "The absolute VH:VL orientation parameters for the 2 Fabs (Table\u00a0S2) show significant deviation in HL, LC1 and HC2 values (2-3 standard deviations from the mean).", "section": "DISCUSS", "ner": [ [ 13, 18, "VH:VL", "complex_assembly" ], [ 19, 41, "orientation parameters", "evidence" ], [ 52, 56, "Fabs", "structure_element" ], [ 85, 94, "deviation", "evidence" ], [ 98, 100, "HL", "structure_element" ], [ 102, 105, "LC1", "structure_element" ], [ 110, 113, "HC2", "structure_element" ] ] }, { "sid": 261, "sent": "One of the variants, H3-23:L3-20, has the CDR H3 conformation similar to the parent, but the other, H1-69:L3-20, is different.", "section": "DISCUSS", "ner": [ [ 21, 32, "H3-23:L3-20", "complex_assembly" ], [ 42, 45, "CDR", "structure_element" ], [ 46, 48, "H3", "structure_element" ], [ 100, 111, "H1-69:L3-20", "complex_assembly" ] ] }, { "sid": 262, "sent": "As noted in the Results section, the 2 variants, H1-69:L3-20 and H3-23:L3-20, are outliers in terms of the tilt angle; at the same time, both have the smallest VH:VL interface.", "section": "DISCUSS", "ner": [ [ 49, 60, "H1-69:L3-20", "complex_assembly" ], [ 65, 76, "H3-23:L3-20", "complex_assembly" ], [ 107, 117, "tilt angle", "evidence" ], [ 160, 175, "VH:VL interface", "site" ] ] }, { "sid": 263, "sent": "These smaller interfaces may perhaps translate to a significant deviation in how VH is oriented relative to VL than the other variants.", "section": "DISCUSS", "ner": [ [ 14, 24, "interfaces", "site" ], [ 81, 83, "VH", "structure_element" ], [ 108, 110, "VL", "structure_element" ] ] }, { "sid": 264, "sent": "These deviations from the other variants can also be seen to some extent in VH:VL orientation parameters in Table\u00a0S2, as well as in the smaller number of residues involved in the VH:VL interfaces of these 2 variants (Fig.\u00a0S5).", "section": "DISCUSS", "ner": [ [ 76, 81, "VH:VL", "complex_assembly" ], [ 179, 195, "VH:VL interfaces", "site" ] ] }, { "sid": 265, "sent": "These differences undoubtedly influence the conformation of the CDRs, in particular CDR H1 (Fig.\u00a01A) and CDR L1 (Fig.\u00a03C), especially with the tandem glycines and multiple serines present, respectively.", "section": "DISCUSS", "ner": [ [ 64, 68, "CDRs", "structure_element" ], [ 84, 87, "CDR", "structure_element" ], [ 88, 90, "H1", "structure_element" ], [ 105, 108, "CDR", "structure_element" ], [ 109, 111, "L1", "structure_element" ], [ 150, 158, "glycines", "residue_name" ], [ 172, 179, "serines", "residue_name" ] ] }, { "sid": 266, "sent": "Pairing of different germlines yields antibodies with various degrees of stability.", "section": "DISCUSS", "ner": [ [ 38, 48, "antibodies", "protein_type" ] ] }, { "sid": 267, "sent": "As indicated by the melting temperatures, germlines H1-69 and H3-23 for HC and germline L1-39 for LC produce more stable Fabs compared to the other germlines in the experimental set.", "section": "DISCUSS", "ner": [ [ 20, 40, "melting temperatures", "evidence" ], [ 52, 57, "H1-69", "mutant" ], [ 62, 67, "H3-23", "mutant" ], [ 72, 74, "HC", "structure_element" ], [ 88, 93, "L1-39", "mutant" ], [ 98, 100, "LC", "structure_element" ], [ 114, 120, "stable", "protein_state" ], [ 121, 125, "Fabs", "structure_element" ] ] }, { "sid": 268, "sent": "One possible explanation of the clear preference of LC germline L1-39 is that CDR L3 has smaller residues at positions 91 and 94, allowing for more room to accommodate CDR H3.", "section": "DISCUSS", "ner": [ [ 52, 54, "LC", "structure_element" ], [ 64, 69, "L1-39", "mutant" ], [ 78, 81, "CDR", "structure_element" ], [ 82, 84, "L3", "structure_element" ], [ 119, 121, "91", "residue_number" ], [ 126, 128, "94", "residue_number" ], [ 168, 171, "CDR", "structure_element" ], [ 172, 174, "H3", "structure_element" ] ] }, { "sid": 269, "sent": "Other germlines have bulky residues, Tyr, Arg and Trp, at these positions, whereas L1-39 has Ser and Thr.", "section": "DISCUSS", "ner": [ [ 37, 40, "Tyr", "residue_name" ], [ 42, 45, "Arg", "residue_name" ], [ 50, 53, "Trp", "residue_name" ], [ 83, 88, "L1-39", "mutant" ], [ 93, 96, "Ser", "residue_name" ], [ 101, 104, "Thr", "residue_name" ] ] }, { "sid": 270, "sent": "Various combinations of germline sequences for VL and VH impose certain constraints on CDR H3, which has to adapt to the environment.", "section": "DISCUSS", "ner": [ [ 47, 49, "VL", "structure_element" ], [ 54, 56, "VH", "structure_element" ], [ 87, 90, "CDR", "structure_element" ], [ 91, 93, "H3", "structure_element" ] ] }, { "sid": 271, "sent": "A more compact CDR L3 may be beneficial in this situation.", "section": "DISCUSS", "ner": [ [ 7, 14, "compact", "protein_state" ], [ 15, 18, "CDR", "structure_element" ], [ 19, 21, "L3", "structure_element" ] ] }, { "sid": 272, "sent": "At the other end of the stability range is LC germline L3-20, which yields antibodies with the lowest Tms.", "section": "DISCUSS", "ner": [ [ 43, 45, "LC", "structure_element" ], [ 55, 60, "L3-20", "mutant" ], [ 75, 85, "antibodies", "protein_type" ], [ 102, 105, "Tms", "evidence" ] ] }, { "sid": 273, "sent": "While pairings with H3-53 and H5-51 may be safely called a mismatch, those with H1-69 and H3-23 have Tms about 5-6\u00b0 higher.", "section": "DISCUSS", "ner": [ [ 20, 25, "H3-53", "mutant" ], [ 30, 35, "H5-51", "mutant" ], [ 80, 85, "H1-69", "mutant" ], [ 90, 95, "H3-23", "mutant" ], [ 101, 104, "Tms", "evidence" ] ] }, { "sid": 274, "sent": "Curiously, the 2 Fabs, H1-69:L3-20 and H3-23:L3-20, deviate markedly in their tilt angles from the rest of the panel.", "section": "DISCUSS", "ner": [ [ 17, 21, "Fabs", "structure_element" ], [ 23, 34, "H1-69:L3-20", "complex_assembly" ], [ 39, 50, "H3-23:L3-20", "complex_assembly" ], [ 78, 89, "tilt angles", "evidence" ] ] }, { "sid": 275, "sent": "It is possible that by adopting extreme tilt angles the structure modulates CDR H3 and its environment, which apparently cannot be achieved solely by conformational rearrangement of the CDR.", "section": "DISCUSS", "ner": [ [ 40, 51, "tilt angles", "evidence" ], [ 56, 65, "structure", "evidence" ], [ 76, 79, "CDR", "structure_element" ], [ 80, 82, "H3", "structure_element" ], [ 186, 189, "CDR", "structure_element" ] ] }, { "sid": 276, "sent": "Note that most of the VH:VL interface residues are invariant; therefore, significant change of the tilt angle must come with a penalty in free energy.", "section": "DISCUSS", "ner": [ [ 22, 37, "VH:VL interface", "site" ] ] }, { "sid": 277, "sent": "Yet, for the 2 antibodies, the total gain in stability merits the domain repacking.", "section": "DISCUSS", "ner": [ [ 15, 25, "antibodies", "protein_type" ] ] }, { "sid": 278, "sent": "Overall, the stability of the Fab, as measured by Tm, is a result of the mutual adjustment of the HC and LC variable domains and adjustment of CDR H3 to the VH:VL cleft.", "section": "DISCUSS", "ner": [ [ 30, 33, "Fab", "structure_element" ], [ 50, 52, "Tm", "evidence" ], [ 98, 100, "HC", "structure_element" ], [ 105, 107, "LC", "structure_element" ], [ 108, 124, "variable domains", "structure_element" ], [ 143, 146, "CDR", "structure_element" ], [ 147, 149, "H3", "structure_element" ], [ 157, 168, "VH:VL cleft", "site" ] ] }, { "sid": 279, "sent": "The final conformation represents an energetic minimum; however, in most cases it is very shallow, so that a single mutation can cause a dramatic rearrangement of the structure.", "section": "DISCUSS", "ner": [ [ 167, 176, "structure", "evidence" ] ] }, { "sid": 280, "sent": "In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs.", "section": "DISCUSS", "ner": [ [ 33, 51, "structural library", "experimental_method" ], [ 100, 103, "HCs", "structure_element" ], [ 109, 112, "LCs", "structure_element" ], [ 132, 135, "CDR", "structure_element" ], [ 136, 138, "H3", "structure_element" ], [ 173, 181, "antibody", "protein_type" ], [ 182, 191, "structure", "evidence" ], [ 276, 278, "L1", "structure_element" ], [ 280, 282, "L2", "structure_element" ], [ 284, 286, "L3", "structure_element" ], [ 288, 290, "H1", "structure_element" ], [ 295, 297, "H2", "structure_element" ], [ 298, 302, "CDRs", "structure_element" ] ] }, { "sid": 281, "sent": "Comparison of the CDR H3s reveals a large set of variants with conformations similar to the parent, while a second set has significant conformational variability, indicating that both the sequence and the structural context define the CDR H3 conformation.", "section": "DISCUSS", "ner": [ [ 18, 21, "CDR", "structure_element" ], [ 22, 25, "H3s", "structure_element" ], [ 235, 238, "CDR", "structure_element" ], [ 239, 241, "H3", "structure_element" ] ] }, { "sid": 282, "sent": "Quite unexpectedly, 2 of the variants, H1-69:L3-20 and H3-53:L4-1, have the \u2018extended\u2019 stem region differing from the other 14 that have a \u2018kinked\u2019 stem region.", "section": "DISCUSS", "ner": [ [ 39, 50, "H1-69:L3-20", "complex_assembly" ], [ 55, 65, "H3-53:L4-1", "complex_assembly" ], [ 77, 85, "extended", "protein_state" ], [ 87, 98, "stem region", "structure_element" ], [ 140, 146, "kinked", "protein_state" ], [ 148, 159, "stem region", "structure_element" ] ] }, { "sid": 283, "sent": "These data reveal the difficulty of modeling CDR H3 accurately, as shown again in Antibody Modeling Assessment II.", "section": "DISCUSS", "ner": [ [ 45, 48, "CDR", "structure_element" ], [ 49, 51, "H3", "structure_element" ] ] }, { "sid": 284, "sent": "Furthermore, antibody CDRs, H3 in particular, may go through conformational changes upon binding their targets, making structural prediction for docking purposes an even more difficult task.", "section": "DISCUSS", "ner": [ [ 13, 21, "antibody", "protein_type" ], [ 22, 26, "CDRs", "structure_element" ], [ 28, 30, "H3", "structure_element" ] ] }, { "sid": 285, "sent": "Fortunately, for most applications of antibody modeling, such as engineering affinity and biophysical properties, an accurate CDR H3 structure is not always necessary.", "section": "DISCUSS", "ner": [ [ 38, 46, "antibody", "protein_type" ], [ 126, 129, "CDR", "structure_element" ], [ 130, 132, "H3", "structure_element" ], [ 133, 142, "structure", "evidence" ] ] }, { "sid": 286, "sent": "For those applications where accurate CDR structures are essential, such as docking, the results in this work demonstrate the importance of experimental structures.", "section": "DISCUSS", "ner": [ [ 38, 41, "CDR", "structure_element" ], [ 42, 52, "structures", "evidence" ], [ 153, 163, "structures", "evidence" ] ] }, { "sid": 287, "sent": "With the recent advances in expression and crystallization methods, Fab structures can be obtained rapidly.", "section": "DISCUSS", "ner": [ [ 28, 66, "expression and crystallization methods", "experimental_method" ], [ 68, 71, "Fab", "structure_element" ], [ 72, 82, "structures", "evidence" ] ] }, { "sid": 288, "sent": "The set of 16 germline Fab structures offers a unique dataset to facilitate software development for antibody modeling.", "section": "DISCUSS", "ner": [ [ 23, 26, "Fab", "structure_element" ], [ 27, 37, "structures", "evidence" ], [ 101, 109, "antibody", "protein_type" ] ] }, { "sid": 289, "sent": "The results essentially support the underlying idea of canonical structures, indicating that most CDRs with germline sequences tend to adopt predefined conformations.", "section": "DISCUSS", "ner": [ [ 65, 75, "structures", "evidence" ], [ 98, 102, "CDRs", "structure_element" ] ] }, { "sid": 290, "sent": "From this point of view, a novel approach to design combinatorial antibody libraries would be to cover the range of CDR conformations that may not necessarily coincide with the germline usage in the human repertoire.", "section": "DISCUSS", "ner": [ [ 66, 74, "antibody", "protein_type" ], [ 116, 119, "CDR", "structure_element" ], [ 199, 204, "human", "species" ] ] }, { "sid": 291, "sent": "This would insure more structural diversity, leading to a more diverse panel of antibodies that would bind to a broad spectrum of targets.", "section": "DISCUSS", "ner": [ [ 80, 90, "antibodies", "protein_type" ] ] } ] }, "PMC4937829": { "annotations": [ { "sid": 0, "sent": "Visualizing chaperone-assisted protein folding", "section": "TITLE", "ner": [ [ 12, 21, "chaperone", "protein_type" ] ] }, { "sid": 1, "sent": "Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes.", "section": "ABSTRACT", "ner": [ [ 30, 40, "structures", "evidence" ] ] }, { "sid": 2, "sent": "One such process is chaperone-assisted protein folding, where obtaining structural ensembles of chaperone:substrate complexes would ultimately reveal how chaperones help proteins fold into their native state.", "section": "ABSTRACT", "ner": [ [ 20, 29, "chaperone", "protein_type" ], [ 96, 105, "chaperone", "protein_type" ], [ 154, 164, "chaperones", "protein_type" ] ] }, { "sid": 3, "sent": "To address this problem, we devised a novel structural biology approach based on X-ray crystallography, termed Residual Electron and Anomalous Density (READ).", "section": "ABSTRACT", "ner": [ [ 81, 102, "X-ray crystallography", "experimental_method" ], [ 111, 150, "Residual Electron and Anomalous Density", "experimental_method" ], [ 152, 156, "READ", "experimental_method" ] ] }, { "sid": 4, "sent": "READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy.", "section": "ABSTRACT", "ner": [ [ 0, 4, "READ", "experimental_method" ], [ 92, 110, "immunity protein 7", "protein" ], [ 112, 115, "Im7", "protein" ], [ 117, 132, "in complex with", "protein_state" ], [ 137, 144, "E. coli", "species" ], [ 145, 154, "chaperone", "protein_type" ], [ 155, 158, "Spy", "protein" ] ] }, { "sid": 5, "sent": "This study resulted in a series of snapshots depicting the various folding states of Im7 while bound to Spy.", "section": "ABSTRACT", "ner": [ [ 85, 88, "Im7", "protein" ], [ 95, 103, "bound to", "protein_state" ], [ 104, 107, "Spy", "protein" ] ] }, { "sid": 6, "sent": "The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone.", "section": "ABSTRACT", "ner": [ [ 24, 38, "Spy-associated", "protein_state" ], [ 39, 42, "Im7", "protein" ], [ 78, 86, "unfolded", "protein_state" ], [ 100, 106, "folded", "protein_state" ], [ 111, 117, "native", "protein_state" ], [ 199, 207, "bound to", "protein_state" ], [ 210, 219, "chaperone", "protein_type" ] ] }, { "sid": 7, "sent": "High-resolution structural models of protein-protein interactions are critical for obtaining mechanistic insights into biological processes.", "section": "INTRO", "ner": [ [ 16, 33, "structural models", "evidence" ] ] }, { "sid": 8, "sent": "However, many protein-protein interactions are highly dynamic, making it difficult to obtain high-resolution data.", "section": "INTRO", "ner": [ [ 47, 61, "highly dynamic", "protein_state" ] ] }, { "sid": 9, "sent": "Particularly challenging are interactions of intrinsically or conditionally disordered sections of proteins with their partner proteins.", "section": "INTRO", "ner": [ [ 45, 86, "intrinsically or conditionally disordered", "protein_state" ] ] }, { "sid": 10, "sent": "Recent advances in X-ray crystallography and NMR spectroscopy continue to improve our ability to analyze biomolecules that exist in multiple conformations.", "section": "INTRO", "ner": [ [ 19, 40, "X-ray crystallography", "experimental_method" ], [ 45, 61, "NMR spectroscopy", "experimental_method" ] ] }, { "sid": 11, "sent": "X-ray crystallography has historically provided valuable information on small-scale conformational changes, but observing large-amplitude heterogeneous conformational changes often falls beyond the reach of current crystallographic techniques.", "section": "INTRO", "ner": [ [ 0, 21, "X-ray crystallography", "experimental_method" ] ] }, { "sid": 12, "sent": "NMR can theoretically be used to determine heterogeneous ensembles, but in practice, this proves to be very challenging.", "section": "INTRO", "ner": [ [ 0, 3, "NMR", "experimental_method" ] ] }, { "sid": 13, "sent": "It is clear that molecular chaperones aid in protein folding.", "section": "INTRO", "ner": [ [ 27, 37, "chaperones", "protein_type" ] ] }, { "sid": 14, "sent": "Structural characterization of chaperone-assisted protein folding likely would help bring clarity to this question.", "section": "INTRO", "ner": [ [ 31, 40, "chaperone", "protein_type" ] ] }, { "sid": 15, "sent": "Structural models of chaperone-substrate complexes have recently begun to provide information as to how a chaperone can recognize its substrate.", "section": "INTRO", "ner": [ [ 0, 17, "Structural models", "evidence" ], [ 21, 30, "chaperone", "protein_type" ], [ 106, 115, "chaperone", "protein_type" ] ] }, { "sid": 16, "sent": "However, the impact that chaperones have on their substrates, and how these interactions affect the folding process remain largely unknown.", "section": "INTRO", "ner": [ [ 25, 35, "chaperones", "protein_type" ] ] }, { "sid": 17, "sent": "For most chaperones, it is still unclear whether the chaperone actively participates in and affects the folding of the substrate proteins, or merely provides a suitable microenvironment enabling the substrate to fold on its own.", "section": "INTRO", "ner": [ [ 9, 19, "chaperones", "protein_type" ], [ 53, 62, "chaperone", "protein_type" ] ] }, { "sid": 18, "sent": "This is a truly fundamental question in the chaperone field, and one that has eluded the community largely because of the highly dynamic nature of the chaperone-substrate complexes.", "section": "INTRO", "ner": [ [ 44, 53, "chaperone", "protein_type" ], [ 122, 136, "highly dynamic", "protein_state" ], [ 151, 160, "chaperone", "protein_type" ] ] }, { "sid": 19, "sent": "To address this question, we investigated the ATP-independent Escherichia coli periplasmic chaperone Spy.", "section": "INTRO", "ner": [ [ 46, 61, "ATP-independent", "protein_state" ], [ 62, 78, "Escherichia coli", "species" ], [ 91, 100, "chaperone", "protein_type" ], [ 101, 104, "Spy", "protein" ] ] }, { "sid": 20, "sent": "Spy prevents protein aggregation and aids in protein folding under various stress conditions, including treatment with tannin and butanol.", "section": "INTRO", "ner": [ [ 0, 3, "Spy", "protein" ], [ 119, 125, "tannin", "chemical" ], [ 130, 137, "butanol", "chemical" ] ] }, { "sid": 21, "sent": "We originally discovered Spy by its ability to stabilize the protein-folding model Im7 in vivo and recently demonstrated that Im7 folds while associated with Spy.", "section": "INTRO", "ner": [ [ 25, 28, "Spy", "protein" ], [ 83, 86, "Im7", "protein" ], [ 126, 129, "Im7", "protein" ], [ 158, 161, "Spy", "protein" ] ] }, { "sid": 22, "sent": "The crystal structure of Spy revealed that it forms a thin \u03b1-helical homodimeric cradle.", "section": "INTRO", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 25, 28, "Spy", "protein" ], [ 69, 80, "homodimeric", "oligomeric_state" ], [ 81, 87, "cradle", "site" ] ] }, { "sid": 23, "sent": "Crosslinking and genetic experiments suggested that Spy interacts with substrates somewhere on its concave side.", "section": "INTRO", "ner": [ [ 0, 36, "Crosslinking and genetic experiments", "experimental_method" ], [ 52, 55, "Spy", "protein" ] ] }, { "sid": 24, "sent": "By using a novel X-ray crystallography-based approach to model disorder in crystal structures, we have now determined the high-resolution ensemble of the dynamic Spy:Im7 complex.", "section": "INTRO", "ner": [ [ 17, 38, "X-ray crystallography", "experimental_method" ], [ 75, 93, "crystal structures", "evidence" ], [ 138, 146, "ensemble", "evidence" ], [ 154, 161, "dynamic", "protein_state" ], [ 162, 169, "Spy:Im7", "complex_assembly" ] ] }, { "sid": 25, "sent": "This work provides a detailed view of chaperone-mediated protein folding and shows how substrates like Im7 find their native fold while bound to their chaperones.", "section": "INTRO", "ner": [ [ 38, 47, "chaperone", "protein_type" ], [ 103, 106, "Im7", "protein" ], [ 136, 144, "bound to", "protein_state" ], [ 151, 161, "chaperones", "protein_type" ] ] }, { "sid": 26, "sent": "Crystallizing the Spy:Im7 complex", "section": "RESULTS", "ner": [ [ 0, 13, "Crystallizing", "experimental_method" ], [ 18, 25, "Spy:Im7", "complex_assembly" ] ] }, { "sid": 27, "sent": "We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence.", "section": "RESULTS", "ner": [ [ 27, 35, "crystals", "evidence" ], [ 57, 60, "Spy", "protein" ], [ 167, 193, "crystallization conditions", "experimental_method" ], [ 207, 210, "Spy", "protein" ], [ 211, 219, "crystals", "evidence" ], [ 227, 238, "presence of", "protein_state" ], [ 275, 282, "absence", "protein_state" ] ] }, { "sid": 28, "sent": "We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine \u03b1-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.", "section": "RESULTS", "ner": [ [ 13, 21, "screened", "experimental_method" ], [ 22, 48, "crystallization conditions", "experimental_method" ], [ 53, 56, "Spy", "protein" ], [ 123, 131, "unfolded", "protein_state" ], [ 132, 138, "bovine", "taxonomy_domain" ], [ 139, 147, "\u03b1-casein", "chemical" ], [ 157, 166, "wild-type", "protein_state" ], [ 168, 170, "WT", "protein_state" ], [ 172, 179, "E. coli", "species" ], [ 180, 183, "Im7", "protein" ], [ 188, 196, "unfolded", "protein_state" ], [ 208, 211, "Im7", "protein" ], [ 213, 217, "L18A", "mutant" ], [ 218, 222, "L19A", "mutant" ], [ 223, 227, "L37A", "mutant" ], [ 238, 253, "N-terminal half", "structure_element" ], [ 257, 260, "Im7", "protein" ], [ 262, 269, "Im76-45", "mutant" ], [ 301, 320, "Spy-binding portion", "structure_element" ], [ 324, 327, "Im7", "protein" ] ] }, { "sid": 29, "sent": "We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals.", "section": "RESULTS", "ner": [ [ 49, 64, "co-crystallized", "experimental_method" ], [ 65, 69, "with", "protein_state" ], [ 70, 73, "Spy", "protein" ], [ 88, 91, "Spy", "protein" ], [ 92, 97, "alone", "protein_state" ], [ 112, 120, "crystals", "evidence" ] ] }, { "sid": 30, "sent": "Subsequent crystal washing and dissolution experiments confirmed the presence of the substrates in the co-crystals (Supplementary Fig. 2).", "section": "RESULTS", "ner": [ [ 11, 42, "crystal washing and dissolution", "experimental_method" ], [ 103, 114, "co-crystals", "experimental_method" ] ] }, { "sid": 31, "sent": "The crystals diffracted to ~1.8 \u00c5 resolution.", "section": "RESULTS", "ner": [ [ 4, 12, "crystals", "evidence" ] ] }, { "sid": 32, "sent": "We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes.", "section": "RESULTS", "ner": [ [ 8, 19, "Spy:Im76-45", "complex_assembly" ], [ 20, 36, "selenomethionine", "chemical" ], [ 37, 45, "crystals", "evidence" ], [ 63, 102, "single-wavelength anomalous diffraction", "experimental_method" ], [ 104, 107, "SAD", "experimental_method" ], [ 171, 174, "Spy", "protein" ] ] }, { "sid": 33, "sent": "However, modeling of the substrate in the complex proved to be a substantial challenge, as the electron density of the substrate was discontinuous and fragmented.", "section": "RESULTS", "ner": [ [ 95, 111, "electron density", "evidence" ] ] }, { "sid": 34, "sent": "Even the minimal binding portion of Im7 (Im76-45) showed highly dispersed electron density (Fig. 1a).", "section": "RESULTS", "ner": [ [ 9, 32, "minimal binding portion", "structure_element" ], [ 36, 39, "Im7", "protein" ], [ 41, 48, "Im76-45", "mutant" ], [ 74, 90, "electron density", "evidence" ] ] }, { "sid": 35, "sent": "We hypothesized that the fragmented density was due to multiple, partially occupied conformations of the substrate bound within the crystal.", "section": "RESULTS", "ner": [ [ 36, 43, "density", "evidence" ], [ 132, 139, "crystal", "evidence" ] ] }, { "sid": 36, "sent": "Such residual density is typically not considered usable by traditional X-ray crystallography methods.", "section": "RESULTS", "ner": [ [ 72, 93, "X-ray crystallography", "experimental_method" ] ] }, { "sid": 37, "sent": "Thus, we developed a new approach to interpret the chaperone-bound substrate in multiple conformations.", "section": "RESULTS", "ner": [ [ 51, 66, "chaperone-bound", "protein_state" ] ] }, { "sid": 38, "sent": "READ: a strategy to visualize heterogeneous and dynamic biomolecules", "section": "RESULTS", "ner": [ [ 0, 4, "READ", "experimental_method" ] ] }, { "sid": 39, "sent": "To determine the structure of the substrate portion of these Spy:substrate complexes, we conceived of an approach that we term READ, for Residual Electron and Anomalous Density.", "section": "RESULTS", "ner": [ [ 17, 26, "structure", "evidence" ], [ 61, 64, "Spy", "protein" ], [ 127, 131, "READ", "experimental_method" ], [ 137, 176, "Residual Electron and Anomalous Density", "experimental_method" ] ] }, { "sid": 40, "sent": "We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate.", "section": "RESULTS", "ner": [ [ 72, 75, "Spy", "protein" ], [ 86, 96, "co-crystal", "evidence" ], [ 122, 131, "structure", "evidence" ], [ 139, 145, "folded", "protein_state" ], [ 146, 152, "domain", "structure_element" ], [ 156, 159, "Spy", "protein" ], [ 186, 211, "residual electron density", "evidence" ], [ 223, 230, "dynamic", "protein_state" ] ] }, { "sid": 41, "sent": "(2) We then labeled individual residues in the flexible regions of the substrate with the strong anomalous scatterer iodine, which serves to locate these residues in three-dimensional space using their anomalous density.", "section": "RESULTS", "ner": [ [ 47, 55, "flexible", "protein_state" ], [ 117, 123, "iodine", "chemical" ], [ 202, 219, "anomalous density", "evidence" ] ] }, { "sid": 42, "sent": "(3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density.", "section": "RESULTS", "ner": [ [ 17, 35, "molecular dynamics", "experimental_method" ], [ 37, 39, "MD", "experimental_method" ], [ 41, 52, "simulations", "experimental_method" ], [ 121, 128, "dynamic", "protein_state" ], [ 155, 182, "sample-and-select algorithm", "experimental_method" ], [ 257, 287, "residual and anomalous density", "evidence" ] ] }, { "sid": 43, "sent": "Importantly, even though we only labeled a subset of the residues in the flexible regions of the substrate with iodine, the residual electron density can provide spatial information on many of the other flexible residues.", "section": "RESULTS", "ner": [ [ 73, 81, "flexible", "protein_state" ], [ 112, 118, "iodine", "chemical" ], [ 124, 149, "residual electron density", "evidence" ], [ 203, 211, "flexible", "protein_state" ] ] }, { "sid": 44, "sent": "The electron density then allowed us to connect the labeled residues of the substrate by confining the protein chain within regions of detectable density.", "section": "RESULTS", "ner": [ [ 4, 20, "electron density", "evidence" ], [ 146, 153, "density", "evidence" ] ] }, { "sid": 45, "sent": "In this way, the two forms of data together were able to describe multiple conformations of the substrate within the crystal.", "section": "RESULTS", "ner": [ [ 117, 124, "crystal", "evidence" ] ] }, { "sid": 46, "sent": "As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy.", "section": "RESULTS", "ner": [ [ 47, 51, "READ", "experimental_method" ], [ 109, 127, "Spy-binding domain", "structure_element" ], [ 131, 134, "Im7", "protein" ], [ 142, 149, "Im76-45", "mutant" ], [ 164, 172, "bound to", "protein_state" ], [ 173, 176, "Spy", "protein" ] ] }, { "sid": 47, "sent": "However, we believe that READ will prove generally applicable to visualizing heterogeneous and dynamic complexes that have previously escaped detailed structural analysis.", "section": "RESULTS", "ner": [ [ 25, 29, "READ", "experimental_method" ] ] }, { "sid": 48, "sent": "Collecting READ data for the Spy:Im76-45 complex", "section": "RESULTS", "ner": [ [ 11, 15, "READ", "experimental_method" ], [ 29, 40, "Spy:Im76-45", "complex_assembly" ] ] }, { "sid": 49, "sent": "To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3).", "section": "RESULTS", "ner": [ [ 13, 27, "READ technique", "experimental_method" ], [ 69, 78, "chaperone", "protein_type" ], [ 79, 82, "Spy", "protein" ], [ 96, 103, "Im76-45", "mutant" ], [ 138, 141, "NMR", "experimental_method" ], [ 162, 169, "Im76-45", "mutant" ], [ 189, 197, "unfolded", "protein_state" ], [ 209, 215, "folded", "protein_state" ], [ 243, 246, "Im7", "protein" ] ] }, { "sid": 50, "sent": "Moreover, binding experiments indicated that Im76-45 comprises the entire Spy-binding region.", "section": "RESULTS", "ner": [ [ 10, 29, "binding experiments", "experimental_method" ], [ 45, 52, "Im76-45", "mutant" ], [ 74, 92, "Spy-binding region", "site" ] ] }, { "sid": 51, "sent": "To introduce the anomalous scatterer iodine, we replaced eight Im76-45 residues with the non-canonical amino acid 4-iodophenylalanine (pI-Phe).", "section": "RESULTS", "ner": [ [ 37, 43, "iodine", "chemical" ], [ 48, 56, "replaced", "experimental_method" ], [ 63, 70, "Im76-45", "mutant" ], [ 114, 133, "4-iodophenylalanine", "chemical" ], [ 135, 141, "pI-Phe", "chemical" ] ] }, { "sid": 52, "sent": "Its strong anomalous scattering allowed us to track the positions of these individual Im76-45 residues one at a time, potentially even if the residue was found in several locations in the same crystal.", "section": "RESULTS", "ner": [ [ 11, 31, "anomalous scattering", "evidence" ], [ 86, 93, "Im76-45", "mutant" ], [ 193, 200, "crystal", "evidence" ] ] }, { "sid": 53, "sent": "We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2).", "section": "RESULTS", "ner": [ [ 8, 23, "co-crystallized", "experimental_method" ], [ 24, 27, "Spy", "protein" ], [ 42, 49, "Im76-45", "mutant" ], [ 97, 103, "pI-Phe", "chemical" ], [ 104, 116, "substitution", "experimental_method" ], [ 147, 156, "collected", "experimental_method" ], [ 157, 171, "anomalous data", "evidence" ], [ 186, 197, "Spy:Im76-45", "complex_assembly" ] ] }, { "sid": 54, "sent": "Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site.", "section": "RESULTS", "ner": [ [ 20, 40, "electron density map", "evidence" ], [ 72, 89, "anomalous signals", "evidence" ], [ 105, 111, "cradle", "site" ], [ 115, 118, "Spy", "protein" ], [ 153, 156, "Im7", "protein" ], [ 157, 179, "substrate binding site", "site" ] ] }, { "sid": 55, "sent": "Consistent with the fragmented density, however, we observed multiple iodine positions for seven of the eight substituted residues.", "section": "RESULTS", "ner": [ [ 31, 38, "density", "evidence" ], [ 70, 76, "iodine", "chemical" ] ] }, { "sid": 56, "sent": "Together, these results indicated that the Im7 substrate binds Spy in multiple conformations.", "section": "RESULTS", "ner": [ [ 43, 46, "Im7", "protein" ], [ 63, 66, "Spy", "protein" ] ] }, { "sid": 57, "sent": "READ sample-and-select procedure", "section": "RESULTS", "ner": [ [ 0, 4, "READ", "experimental_method" ], [ 5, 22, "sample-and-select", "experimental_method" ] ] }, { "sid": 58, "sent": "To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes.", "section": "RESULTS", "ner": [ [ 42, 49, "Im76-45", "mutant" ], [ 63, 71, "bound to", "protein_state" ], [ 72, 75, "Spy", "protein" ], [ 93, 118, "residual electron density", "evidence" ], [ 127, 144, "anomalous signals", "evidence" ], [ 154, 160, "pI-Phe", "chemical" ], [ 173, 184, "Spy:Im76-45", "complex_assembly" ] ] }, { "sid": 59, "sent": "To generate an accurate depiction of the chaperone-substrate interactions, we devised a selection protocol based on a sample-and-select procedure employed in NMR spectroscopy.", "section": "RESULTS", "ner": [ [ 41, 50, "chaperone", "protein_type" ], [ 118, 135, "sample-and-select", "experimental_method" ], [ 158, 174, "NMR spectroscopy", "experimental_method" ] ] }, { "sid": 60, "sent": "During each round of the selection, a genetic algorithm alters the ensemble and its agreement to the experimental data is re-evaluated.", "section": "RESULTS", "ner": [ [ 38, 55, "genetic algorithm", "experimental_method" ] ] }, { "sid": 61, "sent": "If successful, the selection identifies the smallest group of specific conformations that best fits the residual electron density and anomalous signals.", "section": "RESULTS", "ner": [ [ 104, 129, "residual electron density", "evidence" ], [ 134, 151, "anomalous signals", "evidence" ] ] }, { "sid": 62, "sent": "The READ sample-and-select algorithm is diagrammed in Fig. 2.", "section": "RESULTS", "ner": [ [ 4, 8, "READ", "experimental_method" ], [ 9, 36, "sample-and-select algorithm", "experimental_method" ] ] }, { "sid": 63, "sent": "Prior to performing the selection, we generated a large and diverse pool of chaperone-substrate complexes using coarse-grained MD simulations in a pseudo-crystal environment (Fig. 2 and Supplementary Fig. 4).", "section": "RESULTS", "ner": [ [ 76, 85, "chaperone", "protein_type" ], [ 112, 141, "coarse-grained MD simulations", "experimental_method" ], [ 147, 173, "pseudo-crystal environment", "experimental_method" ] ] }, { "sid": 64, "sent": "The coarse-grained simulations are based on a single-residue resolution model for protein folding and were extended here to describe Spy-Im76-45 binding events (Online Methods).", "section": "RESULTS", "ner": [ [ 4, 30, "coarse-grained simulations", "experimental_method" ], [ 133, 144, "Spy-Im76-45", "complex_assembly" ] ] }, { "sid": 65, "sent": "The initial conditions of the binding simulations are not biased toward a particular conformation of the substrate or any specific chaperone-substrate interaction (Online Methods).", "section": "RESULTS", "ner": [ [ 30, 49, "binding simulations", "experimental_method" ], [ 131, 140, "chaperone", "protein_type" ] ] }, { "sid": 66, "sent": "Im76-45 binds and unbinds to Spy throughout the simulations.", "section": "RESULTS", "ner": [ [ 0, 7, "Im76-45", "mutant" ], [ 29, 32, "Spy", "protein" ], [ 48, 59, "simulations", "experimental_method" ] ] }, { "sid": 67, "sent": "This strategy allows a wide range of substrate conformations to interact with the chaperone.", "section": "RESULTS", "ner": [ [ 82, 91, "chaperone", "protein_type" ] ] }, { "sid": 68, "sent": "From the MD simulations, we extracted ~10,000 diverse Spy:Im76-45 complexes to be used by the ensuing selection.", "section": "RESULTS", "ner": [ [ 9, 11, "MD", "experimental_method" ], [ 12, 23, "simulations", "experimental_method" ], [ 54, 65, "Spy:Im76-45", "complex_assembly" ] ] }, { "sid": 69, "sent": "Each complex within this pool comprises one Spy dimer bound to a single Im76-45 substrate.", "section": "RESULTS", "ner": [ [ 44, 47, "Spy", "protein" ], [ 48, 53, "dimer", "oligomeric_state" ], [ 54, 62, "bound to", "protein_state" ], [ 72, 79, "Im76-45", "mutant" ] ] }, { "sid": 70, "sent": "This pool was then used by the selection algorithm to identify the minimal ensemble that best satisfies both the residual electron and anomalous crystallographic data.", "section": "RESULTS", "ner": [ [ 113, 166, "residual electron and anomalous crystallographic data", "evidence" ] ] }, { "sid": 71, "sent": "The anomalous scattering portion of the selection uses our basic knowledge of pI-Phe geometry: the iodine is separated from its respective C\u03b1 atom in each coarse-grained conformer by 6.5 \u00c5. The selection then picks ensembles that best reproduce the collection of iodine anomalous signals.", "section": "RESULTS", "ner": [ [ 4, 24, "anomalous scattering", "evidence" ], [ 78, 84, "pI-Phe", "chemical" ], [ 99, 105, "iodine", "chemical" ], [ 263, 269, "iodine", "chemical" ], [ 270, 287, "anomalous signals", "evidence" ] ] }, { "sid": 72, "sent": "Simultaneously, it uses the residual electron density to help choose ensembles.", "section": "RESULTS", "ner": [ [ 28, 53, "residual electron density", "evidence" ] ] }, { "sid": 73, "sent": "To make the electron density selection practical, we needed to develop a method to rapidly evaluate the agreement between the selected sub-ensembles and the experimental electron density on-the-fly during the selection procedure.", "section": "RESULTS", "ner": [ [ 12, 38, "electron density selection", "experimental_method" ], [ 170, 186, "electron density", "evidence" ] ] }, { "sid": 74, "sent": "To accomplish this task, we generated a compressed version of the experimental 2mFo\u2212DFc electron density map for use in the selection.", "section": "RESULTS", "ner": [ [ 79, 108, "2mFo\u2212DFc electron density map", "evidence" ] ] }, { "sid": 75, "sent": "This process provided us with a target map that the ensuing selection tried to recapitulate.", "section": "RESULTS", "ner": [ [ 39, 42, "map", "evidence" ] ] }, { "sid": 76, "sent": "To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations.", "section": "RESULTS", "ner": [ [ 65, 68, "map", "evidence" ], [ 95, 102, "density", "evidence" ], [ 150, 157, "Im76-45", "mutant" ], [ 165, 176, "Spy:Im76-45", "complex_assembly" ], [ 177, 179, "MD", "experimental_method" ], [ 180, 191, "simulations", "experimental_method" ] ] }, { "sid": 77, "sent": "For each of the ~10,000 complexes in the coarse-grained MD pool, the electron density at the C\u03b1 positions of Im76-45 was extracted and used to construct an electron density map (Online Methods).", "section": "RESULTS", "ner": [ [ 41, 55, "coarse-grained", "experimental_method" ], [ 56, 58, "MD", "experimental_method" ], [ 69, 85, "electron density", "evidence" ], [ 109, 116, "Im76-45", "mutant" ], [ 156, 176, "electron density map", "evidence" ] ] }, { "sid": 78, "sent": "These individual electron density maps from the separate conformers could then be combined (Fig. 2) and compared to the averaged experimental electron density map as part of the selection algorithm.", "section": "RESULTS", "ner": [ [ 17, 38, "electron density maps", "evidence" ], [ 142, 162, "electron density map", "evidence" ] ] }, { "sid": 79, "sent": "This approach allowed us to simultaneously use both the iodine anomalous signals and the residual electron density in the selection procedure.", "section": "RESULTS", "ner": [ [ 56, 62, "iodine", "chemical" ], [ 63, 80, "anomalous signals", "evidence" ], [ 89, 114, "residual electron density", "evidence" ] ] }, { "sid": 80, "sent": "The selection resulted in small ensembles from the MD pool that best fit the READ data (Fig. 1c,d).", "section": "RESULTS", "ner": [ [ 51, 53, "MD", "experimental_method" ], [ 77, 81, "READ", "experimental_method" ] ] }, { "sid": 81, "sent": "Before analyzing the details of the Spy:Im76-45 complex, we first engaged in a series of validation tests to verify the ensemble and selection procedure (Supplementary Note 1, Figures 1c,d, Supplemental Figures 5-7).", "section": "RESULTS", "ner": [ [ 36, 47, "Spy:Im76-45", "complex_assembly" ] ] }, { "sid": 82, "sent": "Of note, the final six-membered ensemble was the largest ensemble that could simultaneously decrease the RFree and pass the 10-fold cross-validation test.", "section": "RESULTS", "ner": [ [ 105, 110, "RFree", "evidence" ], [ 124, 153, "10-fold cross-validation test", "experimental_method" ] ] }, { "sid": 83, "sent": "This ensemble depicts the conformations that the substrate Im76-45 adopts while bound to the chaperone Spy (Fig. 3 Supplementary Movie 1, and Table 1).", "section": "RESULTS", "ner": [ [ 59, 66, "Im76-45", "mutant" ], [ 80, 88, "bound to", "protein_state" ], [ 93, 102, "chaperone", "protein_type" ], [ 103, 106, "Spy", "protein" ] ] }, { "sid": 84, "sent": "Folding and interactions of Im7 while bound to Spy", "section": "RESULTS", "ner": [ [ 28, 31, "Im7", "protein" ], [ 38, 46, "bound to", "protein_state" ], [ 47, 50, "Spy", "protein" ] ] }, { "sid": 85, "sent": "Our results showed that by using this novel READ approach, we were able to obtain structural information about the dynamic interaction of a chaperone with its substrate protein.", "section": "RESULTS", "ner": [ [ 44, 48, "READ", "experimental_method" ], [ 140, 149, "chaperone", "protein_type" ] ] }, { "sid": 86, "sent": "We were particularly interested in finding answers to one of the most fundamental questions in chaperone biology\u2014how does chaperone binding affect substrate structure and vice versa.", "section": "RESULTS", "ner": [ [ 95, 104, "chaperone", "protein_type" ], [ 122, 131, "chaperone", "protein_type" ] ] }, { "sid": 87, "sent": "By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound.", "section": "RESULTS", "ner": [ [ 28, 38, "structures", "evidence" ], [ 69, 76, "Im76-45", "mutant" ], [ 77, 85, "bound to", "protein_state" ], [ 86, 89, "Spy", "protein" ], [ 108, 115, "Im76-45", "mutant" ], [ 163, 168, "bound", "protein_state" ] ] }, { "sid": 88, "sent": "We found these conformations to be highly heterogeneous and to include unfolded, partially folded, and native-like states (Fig. 3).", "section": "RESULTS", "ner": [ [ 71, 79, "unfolded", "protein_state" ], [ 81, 97, "partially folded", "protein_state" ], [ 103, 114, "native-like", "protein_state" ] ] }, { "sid": 89, "sent": "The ensemble primarily encompasses Im76-45 laying diagonally within the Spy cradle in several different orientations, but some conformations traverse as far as the tips or even extend over the side of the cradle (Figs. 3,4a).", "section": "RESULTS", "ner": [ [ 35, 42, "Im76-45", "mutant" ], [ 72, 75, "Spy", "protein" ], [ 76, 82, "cradle", "site" ], [ 205, 211, "cradle", "site" ] ] }, { "sid": 90, "sent": "We constructed a contact map of the complex, which shows the frequency of interactions for chaperone-substrate residue pairs (Fig. 4).", "section": "RESULTS", "ner": [ [ 17, 28, "contact map", "evidence" ], [ 91, 100, "chaperone", "protein_type" ] ] }, { "sid": 91, "sent": "We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46).", "section": "RESULTS", "ner": [ [ 26, 43, "interaction sites", "site" ], [ 47, 50, "Spy", "protein" ], [ 82, 88, "Arg122", "residue_name_number" ], [ 90, 96, "Thr124", "residue_name_number" ], [ 102, 107, "Phe29", "residue_name_number" ], [ 147, 156, "chaperone", "protein_type" ], [ 158, 163, "Arg61", "residue_name_number" ], [ 165, 170, "Arg43", "residue_name_number" ], [ 172, 177, "Lys47", "residue_name_number" ], [ 179, 184, "His96", "residue_name_number" ], [ 190, 195, "Met46", "residue_name_number" ] ] }, { "sid": 92, "sent": "The Spy-contacting residues comprise a mixture of charged, polar, and hydrophobic residues.", "section": "RESULTS", "ner": [ [ 4, 27, "Spy-contacting residues", "site" ] ] }, { "sid": 93, "sent": "Surprisingly, we noted that in the ensemble, Im76-45 interacts with only 38% of the hydrophobic residues in the Spy cradle, but interacts with 61% of the hydrophilic residues in the cradle.", "section": "RESULTS", "ner": [ [ 45, 52, "Im76-45", "mutant" ], [ 112, 115, "Spy", "protein" ], [ 116, 122, "cradle", "site" ], [ 182, 188, "cradle", "site" ] ] }, { "sid": 94, "sent": "This mixture suggests the importance of both electrostatic and hydrophobic components in binding the Im76-45 ensemble.", "section": "RESULTS", "ner": [ [ 101, 108, "Im76-45", "mutant" ] ] }, { "sid": 95, "sent": "With respect to the substrate, we observed that nearly every residue in Im76-45 is in contact with Spy (Fig. 4a).", "section": "RESULTS", "ner": [ [ 72, 79, "Im76-45", "mutant" ], [ 99, 102, "Spy", "protein" ] ] }, { "sid": 96, "sent": "However, we did notice that despite this uniformity, regions of Im76-45 preferentially interact with different regions in Spy (Fig. 4b).", "section": "RESULTS", "ner": [ [ 64, 71, "Im76-45", "mutant" ], [ 122, 125, "Spy", "protein" ] ] }, { "sid": 97, "sent": "For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy\u2019s concave surface.", "section": "RESULTS", "ner": [ [ 17, 32, "N-terminal half", "structure_element" ], [ 36, 43, "Im76-45", "mutant" ], [ 75, 78, "Spy", "protein" ], [ 79, 85, "cradle", "site" ], [ 99, 114, "C-terminal half", "structure_element" ], [ 157, 160, "Spy", "protein" ], [ 163, 178, "concave surface", "site" ] ] }, { "sid": 98, "sent": "Not unexpectedly, we found that as Im76-45 progresses from the unfolded to the native state, its interactions with Spy shift accordingly.", "section": "RESULTS", "ner": [ [ 35, 42, "Im76-45", "mutant" ], [ 63, 71, "unfolded", "protein_state" ], [ 79, 85, "native", "protein_state" ], [ 115, 118, "Spy", "protein" ] ] }, { "sid": 99, "sent": "Whereas the least-folded Im76-45 pose in the ensemble forms the most hydrophobic contacts with Spy (Fig. 3), the two most-folded conformations form the fewest hydrophobic contacts (Fig. 3).", "section": "RESULTS", "ner": [ [ 12, 24, "least-folded", "protein_state" ], [ 25, 32, "Im76-45", "mutant" ], [ 95, 98, "Spy", "protein" ], [ 117, 128, "most-folded", "protein_state" ] ] }, { "sid": 100, "sent": "This shift in contacts is likely due to hydrophobic residues of Im76-45 preferentially forming intra-molecular contacts upon folding (i.e., hydrophobic collapse), effectively removing themselves from the interaction sites.", "section": "RESULTS", "ner": [ [ 64, 71, "Im76-45", "mutant" ], [ 204, 221, "interaction sites", "site" ] ] }, { "sid": 101, "sent": "The diversity of conformations and binding sites observed here emphasizes the dynamic and heterogeneous nature of the chaperone-substrate ensemble.", "section": "RESULTS", "ner": [ [ 35, 48, "binding sites", "site" ], [ 118, 127, "chaperone", "protein_type" ] ] }, { "sid": 102, "sent": "Although we do not yet have time resolution data of these various snapshots of Im76-45, this ensemble illustrates how a substrate samples its folding landscape while bound to a chaperone.", "section": "RESULTS", "ner": [ [ 79, 86, "Im76-45", "mutant" ], [ 166, 174, "bound to", "protein_state" ], [ 177, 186, "chaperone", "protein_type" ] ] }, { "sid": 103, "sent": "Spy changes conformation upon substrate binding", "section": "RESULTS", "ner": [ [ 0, 3, "Spy", "protein" ] ] }, { "sid": 104, "sent": "Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2).", "section": "RESULTS", "ner": [ [ 14, 23, "structure", "evidence" ], [ 27, 30, "Spy", "protein" ], [ 38, 53, "substrate-bound", "protein_state" ], [ 58, 61, "apo", "protein_state" ], [ 87, 90, "Spy", "protein" ], [ 91, 96, "dimer", "oligomeric_state" ] ] }, { "sid": 105, "sent": "Upon substrate binding, the Spy dimer twists 9\u00b0 about its center relative to its apo form.", "section": "RESULTS", "ner": [ [ 28, 31, "Spy", "protein" ], [ 32, 37, "dimer", "oligomeric_state" ], [ 81, 84, "apo", "protein_state" ] ] }, { "sid": 106, "sent": "This twist yields asymmetry and results in substantially different interaction patterns in the two Spy monomers (Fig. 4b).", "section": "RESULTS", "ner": [ [ 99, 102, "Spy", "protein" ], [ 103, 111, "monomers", "oligomeric_state" ] ] }, { "sid": 107, "sent": "It is possible that this twist serves to increase heterogeneity in Spy by providing more binding poses.", "section": "RESULTS", "ner": [ [ 67, 70, "Spy", "protein" ] ] }, { "sid": 108, "sent": "Additionally, we observed that the linker region (residues 47\u201357) of Spy, which participates in substrate interaction, becomes mostly disordered upon binding the substrate.", "section": "RESULTS", "ner": [ [ 35, 48, "linker region", "structure_element" ], [ 59, 64, "47\u201357", "residue_range" ], [ 69, 72, "Spy", "protein" ], [ 134, 144, "disordered", "protein_state" ] ] }, { "sid": 109, "sent": "This increased disorder might explain how Spy is able to recognize and bind different substrates and/or differing conformations of the same substrate.", "section": "RESULTS", "ner": [ [ 42, 45, "Spy", "protein" ] ] }, { "sid": 110, "sent": "Importantly, we observed the same structural changes in Spy regardless of which of the four substrates was bound (Fig. 5b, Table 1).", "section": "RESULTS", "ner": [ [ 56, 59, "Spy", "protein" ] ] }, { "sid": 111, "sent": "The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 \u00c5. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site.", "section": "RESULTS", "ner": [ [ 4, 8, "RMSD", "evidence" ], [ 21, 32, "well-folded", "protein_state" ], [ 45, 48, "Spy", "protein" ], [ 61, 70, "chaperone", "protein_type" ], [ 138, 161, "competition experiments", "experimental_method" ], [ 214, 217, "Spy", "protein" ], [ 328, 344, "Spy binding site", "site" ] ] }, { "sid": 112, "sent": "To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate.", "section": "DISCUSS", "ner": [ [ 21, 31, "chaperones", "protein_type" ], [ 112, 116, "READ", "experimental_method" ], [ 148, 171, "conformational ensemble", "evidence" ], [ 179, 188, "chaperone", "protein_type" ], [ 189, 192, "Spy", "protein" ], [ 193, 211, "bound to substrate", "protein_state" ] ] }, { "sid": 113, "sent": "As a substrate, we used Im76-45, the chaperone-interacting portion of the protein-folding model protein Im7.", "section": "DISCUSS", "ner": [ [ 24, 31, "Im76-45", "mutant" ], [ 37, 66, "chaperone-interacting portion", "structure_element" ], [ 104, 107, "Im7", "protein" ] ] }, { "sid": 114, "sent": "In the chaperone-bound ensemble, Im76-45 samples unfolded, partially folded, and native-like states.", "section": "DISCUSS", "ner": [ [ 7, 22, "chaperone-bound", "protein_state" ], [ 33, 40, "Im76-45", "mutant" ], [ 49, 57, "unfolded", "protein_state" ], [ 69, 75, "folded", "protein_state" ], [ 81, 87, "native", "protein_state" ] ] }, { "sid": 115, "sent": "The ensemble provides an unprecedented description of the conformations that a substrate assumes while exploring its chaperone-associated folding landscape.", "section": "DISCUSS", "ner": [ [ 117, 126, "chaperone", "protein_type" ] ] }, { "sid": 116, "sent": "This substrate-chaperone ensemble helps accomplish the longstanding goal of obtaining a detailed view of how a chaperone aids protein folding.", "section": "DISCUSS", "ner": [ [ 15, 24, "chaperone", "protein_type" ], [ 111, 120, "chaperone", "protein_type" ] ] }, { "sid": 117, "sent": "We recently showed that Im7 can fold while remaining continuously bound to Spy.", "section": "DISCUSS", "ner": [ [ 24, 27, "Im7", "protein" ], [ 53, 74, "continuously bound to", "protein_state" ], [ 75, 78, "Spy", "protein" ] ] }, { "sid": 118, "sent": "The high-resolution ensemble obtained here now provides insight into exactly how this occurs.", "section": "DISCUSS", "ner": [ [ 20, 28, "ensemble", "evidence" ] ] }, { "sid": 119, "sent": "The structures of our ensemble agree well with lower-resolution crosslinking data, which indicate that chaperone-substrate interactions primarily occur on the concave surface of Spy.", "section": "DISCUSS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 22, 30, "ensemble", "evidence" ], [ 103, 112, "chaperone", "protein_type" ], [ 159, 174, "concave surface", "site" ], [ 178, 181, "Spy", "protein" ] ] }, { "sid": 120, "sent": "The ensemble suggests a model in which Spy provides an amphipathic surface that allows substrate proteins to assume different conformations while bound to the chaperone.", "section": "DISCUSS", "ner": [ [ 4, 12, "ensemble", "evidence" ], [ 39, 42, "Spy", "protein" ], [ 55, 74, "amphipathic surface", "site" ], [ 146, 154, "bound to", "protein_state" ], [ 159, 168, "chaperone", "protein_type" ] ] }, { "sid": 121, "sent": "This model is consistent with previous studies postulating that the flexible binding of chaperones allows for substrate protein folding.", "section": "DISCUSS", "ner": [ [ 88, 98, "chaperones", "protein_type" ] ] }, { "sid": 122, "sent": "The amphipathic concave surface of Spy likely facilitates this flexible binding and may be a crucial feature for Spy and potentially other chaperones, allowing them to bind multiple conformations of many different substrates.", "section": "DISCUSS", "ner": [ [ 16, 31, "concave surface", "site" ], [ 35, 38, "Spy", "protein" ], [ 113, 116, "Spy", "protein" ], [ 139, 149, "chaperones", "protein_type" ] ] }, { "sid": 123, "sent": "In contrast to Spy\u2019s binding hotspots, Im76-45 displays substantially less specificity in its binding sites.", "section": "DISCUSS", "ner": [ [ 15, 18, "Spy", "protein" ], [ 21, 37, "binding hotspots", "site" ], [ 39, 46, "Im76-45", "mutant" ], [ 94, 107, "binding sites", "site" ] ] }, { "sid": 124, "sent": "Nearly all Im76-45 residues come in contact with Spy.", "section": "DISCUSS", "ner": [ [ 11, 18, "Im76-45", "mutant" ], [ 49, 52, "Spy", "protein" ] ] }, { "sid": 125, "sent": "Unfolded substrate conformers interact with Spy through both hydrophobic and hydrophilic interactions, whereas the binding of native-like states is mainly hydrophilic.", "section": "DISCUSS", "ner": [ [ 0, 8, "Unfolded", "protein_state" ], [ 44, 47, "Spy", "protein" ], [ 61, 101, "hydrophobic and hydrophilic interactions", "bond_interaction" ], [ 126, 137, "native-like", "protein_state" ] ] }, { "sid": 126, "sent": "This trend suggests that complex formation between an ATP-independent chaperone and its unfolded substrate may initially involve hydrophobic interactions, effectively shielding the exposed aggregation-sensitive hydrophobic regions in the substrate.", "section": "DISCUSS", "ner": [ [ 54, 69, "ATP-independent", "protein_state" ], [ 70, 79, "chaperone", "protein_type" ], [ 88, 96, "unfolded", "protein_state" ], [ 129, 153, "hydrophobic interactions", "bond_interaction" ], [ 211, 230, "hydrophobic regions", "site" ] ] }, { "sid": 127, "sent": "Once the substrate begins to fold within this protected environment, it progressively buries its own hydrophobic residues, and its interactions with the chaperone shift towards becoming more electrostatic.", "section": "DISCUSS", "ner": [ [ 153, 162, "chaperone", "protein_type" ] ] }, { "sid": 128, "sent": "Notably, the most frequent contacts between Spy and Im76-45 are charge-charge interactions.", "section": "DISCUSS", "ner": [ [ 44, 47, "Spy", "protein" ], [ 52, 59, "Im76-45", "mutant" ], [ 64, 90, "charge-charge interactions", "bond_interaction" ] ] }, { "sid": 129, "sent": "The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy\u2019s positively charged cradle in both the unfolded and native-like states.", "section": "DISCUSS", "ner": [ [ 23, 26, "Im7", "protein" ], [ 36, 41, "Glu21", "residue_name_number" ], [ 43, 48, "Asp32", "residue_name_number" ], [ 54, 59, "Asp35", "residue_name_number" ], [ 85, 88, "Im7", "protein" ], [ 116, 119, "Spy", "protein" ], [ 141, 147, "cradle", "site" ], [ 160, 168, "unfolded", "protein_state" ], [ 173, 184, "native-like", "protein_state" ] ] }, { "sid": 130, "sent": "Residues Asp32 and Asp35 are close to each other in the folded state of Im7.", "section": "DISCUSS", "ner": [ [ 9, 14, "Asp32", "residue_name_number" ], [ 19, 24, "Asp35", "residue_name_number" ], [ 56, 62, "folded", "protein_state" ], [ 72, 75, "Im7", "protein" ] ] }, { "sid": 131, "sent": "This proximity likely causes electrostatic repulsion that destabilizes Im7\u2019s native state.", "section": "DISCUSS", "ner": [ [ 71, 74, "Im7", "protein" ], [ 77, 83, "native", "protein_state" ] ] }, { "sid": 132, "sent": "Interaction with Spy\u2019s positively-charged residues likely relieves the charge repulsion between Asp32 and Asp35, promoting their compaction into a helical conformation.", "section": "DISCUSS", "ner": [ [ 17, 20, "Spy", "protein" ], [ 96, 101, "Asp32", "residue_name_number" ], [ 106, 111, "Asp35", "residue_name_number" ], [ 147, 167, "helical conformation", "protein_state" ] ] }, { "sid": 133, "sent": "As inter-molecular hydrophobic interactions between Spy and the substrate become progressively replaced by intra-molecular interactions within the substrate, the affinity between chaperone and substrates could decrease, eventually leading to release of the folded client protein.", "section": "DISCUSS", "ner": [ [ 19, 43, "hydrophobic interactions", "bond_interaction" ], [ 52, 55, "Spy", "protein" ], [ 179, 188, "chaperone", "protein_type" ], [ 257, 263, "folded", "protein_state" ] ] }, { "sid": 134, "sent": "Recently, we employed a genetic selection system to improve the chaperone activity of Spy.", "section": "DISCUSS", "ner": [ [ 24, 48, "genetic selection system", "experimental_method" ], [ 64, 73, "chaperone", "protein_type" ], [ 86, 89, "Spy", "protein" ] ] }, { "sid": 135, "sent": "This selection resulted in \u201cSuper Spy\u201d variants that were more effective at both preventing aggregation and promoting protein folding.", "section": "DISCUSS", "ner": [ [ 34, 37, "Spy", "protein" ], [ 39, 47, "variants", "protein_state" ] ] }, { "sid": 136, "sent": "In conjunction with our bound Im76-45 ensemble, these mutants now allowed us to investigate structural features important to chaperone function.", "section": "DISCUSS", "ner": [ [ 24, 29, "bound", "protein_state" ], [ 30, 37, "Im76-45", "mutant" ], [ 38, 46, "ensemble", "evidence" ], [ 125, 134, "chaperone", "protein_type" ] ] }, { "sid": 137, "sent": "Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both.", "section": "DISCUSS", "ner": [ [ 42, 45, "Spy", "protein" ], [ 46, 54, "variants", "protein_state" ], [ 62, 67, "bound", "protein_state" ], [ 68, 71, "Im7", "protein" ], [ 85, 87, "WT", "protein_state" ], [ 88, 91, "Spy", "protein" ], [ 103, 112, "chaperone", "protein_type" ], [ 141, 153, "H/D exchange", "experimental_method" ] ] }, { "sid": 138, "sent": "Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a).", "section": "DISCUSS", "ner": [ [ 4, 12, "ensemble", "evidence" ], [ 44, 47, "Spy", "protein" ], [ 48, 57, "mutations", "protein_state" ], [ 59, 63, "H96L", "mutant" ], [ 68, 73, "Q100L", "mutant" ], [ 92, 117, "chaperone contact surface", "site" ], [ 132, 139, "Im76-45", "mutant" ] ] }, { "sid": 139, "sent": "Moreover, our co-structure suggests that the L32P substitution, which increases Spy\u2019s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker.", "section": "DISCUSS", "ner": [ [ 14, 26, "co-structure", "evidence" ], [ 45, 49, "L32P", "mutant" ], [ 80, 83, "Spy", "protein" ], [ 130, 146, "N-terminal helix", "structure_element" ], [ 189, 199, "disordered", "protein_state" ], [ 200, 206, "linker", "structure_element" ] ] }, { "sid": 140, "sent": "This possibility is supported by the Spy:substrate structures, in which the linker region becomes more flexible compared to the apo state (Fig. 6a).", "section": "DISCUSS", "ner": [ [ 37, 40, "Spy", "protein" ], [ 51, 61, "structures", "evidence" ], [ 76, 89, "linker region", "structure_element" ], [ 128, 131, "apo", "protein_state" ] ] }, { "sid": 141, "sent": "By sampling multiple conformations, this linker region may allow diverse substrate conformations to be accommodated.", "section": "DISCUSS", "ner": [ [ 41, 54, "linker region", "structure_element" ] ] }, { "sid": 142, "sent": "Other Super Spy mutations (F115I and F115L) caused increased flexibility but not tighter substrate binding.", "section": "DISCUSS", "ner": [ [ 12, 15, "Spy", "protein" ], [ 16, 25, "mutations", "protein_state" ], [ 27, 32, "F115I", "mutant" ], [ 37, 42, "F115L", "mutant" ] ] }, { "sid": 143, "sent": "This residue does not directly contact Im76-45 in our READ-derived ensemble.", "section": "DISCUSS", "ner": [ [ 39, 46, "Im76-45", "mutant" ], [ 54, 58, "READ", "experimental_method" ], [ 67, 75, "ensemble", "evidence" ] ] }, { "sid": 144, "sent": "Instead, when Spy is bound to substrate, F115 engages in close CH\u22ef\u03c0 hydrogen bonds with Tyr104 (Fig. 6b).", "section": "DISCUSS", "ner": [ [ 14, 17, "Spy", "protein" ], [ 21, 29, "bound to", "protein_state" ], [ 41, 45, "F115", "residue_name_number" ], [ 68, 82, "hydrogen bonds", "bond_interaction" ], [ 88, 94, "Tyr104", "residue_name_number" ] ] }, { "sid": 145, "sent": "This interaction presumably reduces the mobility of the C-terminal helix.", "section": "DISCUSS", "ner": [ [ 56, 72, "C-terminal helix", "structure_element" ] ] }, { "sid": 146, "sent": "The F115I/L substitutions would replace these hydrogen bonds with hydrophobic interactions that have little angular dependence.", "section": "DISCUSS", "ner": [ [ 4, 9, "F115I", "mutant" ], [ 10, 11, "L", "mutant" ], [ 46, 60, "hydrogen bonds", "bond_interaction" ], [ 66, 90, "hydrophobic interactions", "bond_interaction" ] ] }, { "sid": 147, "sent": "As a result, the C-terminus, and possibly also the flexible linker, is likely to become more flexible and thus more accommodating of different conformations of substrates.", "section": "DISCUSS", "ner": [ [ 51, 59, "flexible", "protein_state" ], [ 60, 66, "linker", "structure_element" ] ] }, { "sid": 148, "sent": "Overall, comparison of our ensemble to the Super Spy variants provides specific examples to corroborate the importance of conformational flexibility in chaperone-substrate interactions.", "section": "DISCUSS", "ner": [ [ 27, 35, "ensemble", "evidence" ], [ 49, 52, "Spy", "protein" ], [ 53, 61, "variants", "protein_state" ], [ 152, 161, "chaperone", "protein_type" ] ] }, { "sid": 149, "sent": "Despite extensive studies, exactly how complex chaperone machines help proteins fold remains controversial.", "section": "DISCUSS", "ner": [ [ 47, 56, "chaperone", "protein_type" ] ] }, { "sid": 150, "sent": "Our study indicates that the chaperone Spy employs a simple surface binding approach that allows the substrate to explore various conformations and form transiently favorable interactions while being protected from aggregation.", "section": "DISCUSS", "ner": [ [ 29, 38, "chaperone", "protein_type" ], [ 39, 42, "Spy", "protein" ] ] }, { "sid": 151, "sent": "We speculate that many other chaperones could utilize a similar strategy.", "section": "DISCUSS", "ner": [ [ 29, 39, "chaperones", "protein_type" ] ] }, { "sid": 152, "sent": "ATP and co-chaperone dependencies may have emerged later through evolution to better modulate and control chaperone action.", "section": "DISCUSS", "ner": [ [ 0, 3, "ATP", "chemical" ], [ 11, 20, "chaperone", "protein_type" ], [ 106, 115, "chaperone", "protein_type" ] ] }, { "sid": 153, "sent": "In addition to insights into chaperone function, this work presents a new method for determining heterogeneous structural ensembles via a hybrid methodology of X-ray crystallography and computational modeling.", "section": "DISCUSS", "ner": [ [ 29, 38, "chaperone", "protein_type" ], [ 160, 181, "X-ray crystallography", "experimental_method" ], [ 186, 208, "computational modeling", "experimental_method" ] ] }, { "sid": 154, "sent": "Heterogeneous dynamic complexes or disordered regions of single proteins, once considered solely approachable by NMR spectroscopy, can now be visualized through X-ray crystallography.", "section": "DISCUSS", "ner": [ [ 35, 45, "disordered", "protein_state" ], [ 113, 129, "NMR spectroscopy", "experimental_method" ], [ 161, 182, "X-ray crystallography", "experimental_method" ] ] }, { "sid": 155, "sent": "Crystallographic data and ensemble selection. (a) 2mFo\u2212DFc omit map of residual Im76-45 and flexible linker electron density contoured at 0.5 \u03c3.", "section": "FIG", "ner": [ [ 50, 67, "2mFo\u2212DFc omit map", "evidence" ], [ 80, 87, "Im76-45", "mutant" ], [ 92, 107, "flexible linker", "structure_element" ], [ 108, 124, "electron density", "evidence" ] ] }, { "sid": 156, "sent": "This is the residual density that is used in the READ selection.", "section": "FIG", "ner": [ [ 21, 28, "density", "evidence" ], [ 49, 53, "READ", "experimental_method" ] ] }, { "sid": 157, "sent": "(b) Composites of iodine positions detected from anomalous signals using pI-Phe substitutions, colored and numbered by sequence.", "section": "FIG", "ner": [ [ 18, 24, "iodine", "chemical" ], [ 49, 66, "anomalous signals", "evidence" ], [ 73, 79, "pI-Phe", "chemical" ], [ 80, 93, "substitutions", "experimental_method" ] ] }, { "sid": 158, "sent": "Multiple iodine positions were detected for most residues.", "section": "FIG", "ner": [ [ 9, 15, "iodine", "chemical" ] ] }, { "sid": 159, "sent": "Agreement to the residual Im76-45 electron density (c) and anomalous iodine signals (d) for ensembles of varying size generated by randomly choosing from the MD pool (blue) and from the selection procedure (black).", "section": "FIG", "ner": [ [ 26, 33, "Im76-45", "mutant" ], [ 34, 50, "electron density", "evidence" ], [ 59, 83, "anomalous iodine signals", "evidence" ], [ 158, 160, "MD", "experimental_method" ] ] }, { "sid": 160, "sent": "The cost function, \u03c72, decreases as the agreement to the experimental data increases and is defined in the Online Methods.", "section": "FIG", "ner": [ [ 4, 17, "cost function", "evidence" ], [ 19, 21, "\u03c72", "evidence" ] ] }, { "sid": 161, "sent": "Flowchart of the READ sample-and-select process.", "section": "FIG", "ner": [ [ 17, 21, "READ", "experimental_method" ], [ 22, 39, "sample-and-select", "experimental_method" ] ] }, { "sid": 162, "sent": "Spy:Im76-45 ensemble, arranged by RMSD to native state of Im76-45. Although the six-membered ensemble from the READ selection should be considered only as an ensemble, for clarity, the individual conformers are shown separately here.", "section": "FIG", "ner": [ [ 0, 11, "Spy:Im76-45", "complex_assembly" ], [ 34, 38, "RMSD", "evidence" ], [ 42, 48, "native", "protein_state" ], [ 58, 65, "Im76-45", "mutant" ], [ 111, 115, "READ", "experimental_method" ] ] }, { "sid": 163, "sent": "Spy is depicted as a gray surface and the Im76-45 conformer is shown as orange balls.", "section": "FIG", "ner": [ [ 0, 3, "Spy", "protein" ], [ 42, 49, "Im76-45", "mutant" ] ] }, { "sid": 164, "sent": "Atoms that were either not directly selected in the READ procedure, or whose position could not be justified based on agreement with the residual electron density were removed, leading to non-contiguous sections.", "section": "FIG", "ner": [ [ 52, 56, "READ", "experimental_method" ], [ 137, 162, "residual electron density", "evidence" ] ] }, { "sid": 165, "sent": "Dashed lines connect non-contiguous segments of the Im76-45 substrate.", "section": "FIG", "ner": [ [ 52, 59, "Im76-45", "mutant" ] ] }, { "sid": 166, "sent": "Residues of the Spy flexible linker region that fit the residual electron density are shown as larger gray spheres.", "section": "FIG", "ner": [ [ 16, 19, "Spy", "protein" ], [ 29, 42, "linker region", "structure_element" ], [ 56, 81, "residual electron density", "evidence" ] ] }, { "sid": 167, "sent": "Shown below each ensemble member is the RMSD of each conformer to the native state of Im76-45, as well as the percentage of contacts between Im76-45 and Spy that are hydrophobic.", "section": "FIG", "ner": [ [ 40, 44, "RMSD", "evidence" ], [ 70, 76, "native", "protein_state" ], [ 86, 93, "Im76-45", "mutant" ], [ 141, 148, "Im76-45", "mutant" ], [ 153, 156, "Spy", "protein" ] ] }, { "sid": 168, "sent": "Contact maps of Spy:Im76-45 complex.", "section": "FIG", "ner": [ [ 0, 12, "Contact maps", "evidence" ], [ 16, 27, "Spy:Im76-45", "complex_assembly" ] ] }, { "sid": 169, "sent": "(a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures.", "section": "FIG", "ner": [ [ 4, 15, "Spy:Im76-45", "complex_assembly" ], [ 16, 27, "contact map", "evidence" ], [ 47, 52, "bound", "protein_state" ], [ 53, 56, "Spy", "protein" ], [ 57, 62, "dimer", "oligomeric_state" ], [ 75, 82, "Im76-45", "mutant" ], [ 91, 101, "structures", "evidence" ] ] }, { "sid": 170, "sent": "For clarity, Im76-45 is represented with a single conformation.", "section": "FIG", "ner": [ [ 13, 20, "Im76-45", "mutant" ] ] }, { "sid": 171, "sent": "The frequency plotted is calculated as the average contact frequency from Spy to every residue of Im76-45 and vice-versa.", "section": "FIG", "ner": [ [ 51, 68, "contact frequency", "evidence" ], [ 74, 77, "Spy", "protein" ], [ 98, 105, "Im76-45", "mutant" ] ] }, { "sid": 172, "sent": "As the residues involved in contacts are more evenly distributed in Im76-45 compared to Spy, its contact map was amplified. (b) Detailed contact maps of Spy:Im76-45.", "section": "FIG", "ner": [ [ 68, 75, "Im76-45", "mutant" ], [ 88, 91, "Spy", "protein" ], [ 97, 108, "contact map", "evidence" ], [ 137, 149, "contact maps", "evidence" ], [ 153, 164, "Spy:Im76-45", "complex_assembly" ] ] }, { "sid": 173, "sent": "Contacts to the two Spy monomers are depicted separately.", "section": "FIG", "ner": [ [ 20, 23, "Spy", "protein" ], [ 24, 32, "monomers", "oligomeric_state" ] ] }, { "sid": 174, "sent": "Note that the flexible linker region of Spy (residues 47\u201357) is not represented in the 2D contact maps.", "section": "FIG", "ner": [ [ 14, 22, "flexible", "protein_state" ], [ 23, 36, "linker region", "structure_element" ], [ 40, 43, "Spy", "protein" ], [ 54, 59, "47\u201357", "residue_range" ], [ 90, 102, "contact maps", "evidence" ] ] }, { "sid": 175, "sent": "Spy conformation changes upon substrate binding.", "section": "FIG", "ner": [ [ 0, 3, "Spy", "protein" ] ] }, { "sid": 176, "sent": "(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).", "section": "FIG", "ner": [ [ 4, 11, "Overlay", "experimental_method" ], [ 15, 18, "apo", "protein_state" ], [ 19, 22, "Spy", "protein" ], [ 48, 53, "bound", "protein_state" ], [ 54, 57, "Spy", "protein" ], [ 71, 78, "Overlay", "experimental_method" ], [ 82, 84, "WT", "protein_state" ], [ 85, 88, "Spy", "protein" ], [ 89, 97, "bound to", "protein_state" ], [ 98, 105, "Im76-45", "mutant" ], [ 115, 119, "H96L", "mutant" ], [ 120, 123, "Spy", "protein" ], [ 124, 132, "bound to", "protein_state" ], [ 133, 136, "Im7", "protein" ], [ 137, 141, "L18A", "mutant" ], [ 142, 147, "L19 A", "mutant" ], [ 147, 151, "L13A", "mutant" ], [ 160, 164, "H96L", "mutant" ], [ 165, 168, "Spy", "protein" ], [ 169, 177, "bound to", "protein_state" ], [ 178, 180, "WT", "protein_state" ], [ 181, 184, "Im7", "protein" ], [ 199, 201, "WT", "protein_state" ], [ 202, 205, "Spy", "protein" ], [ 206, 214, "bound to", "protein_state" ], [ 215, 221, "casein", "chemical" ], [ 236, 253, "Competition assay", "experimental_method" ], [ 262, 269, "Im76-45", "mutant" ], [ 284, 287, "Im7", "protein" ], [ 288, 292, "L18A", "mutant" ], [ 293, 297, "L19A", "mutant" ], [ 298, 302, "L37A", "mutant" ], [ 303, 307, "H40W", "mutant" ], [ 321, 333, "binding site", "site" ], [ 337, 340, "Spy", "protein" ], [ 350, 378, "substrate competition assays", "experimental_method" ] ] }, { "sid": 177, "sent": "Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green).", "section": "FIG", "ner": [ [ 15, 18, "Spy", "protein" ], [ 19, 32, "linker region", "structure_element" ], [ 53, 56, "Spy", "protein" ], [ 74, 77, "Spy", "protein" ], [ 78, 91, "linker region", "structure_element" ], [ 132, 135, "apo", "protein_state" ], [ 212, 217, "bound", "protein_state" ] ] }, { "sid": 178, "sent": "(b) F115 and L32 tether Spy\u2019s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility.", "section": "FIG", "ner": [ [ 4, 8, "F115", "residue_name_number" ], [ 13, 16, "L32", "residue_name_number" ], [ 24, 27, "Spy", "protein" ], [ 30, 43, "linker region", "structure_element" ], [ 51, 57, "cradle", "site" ], [ 70, 73, "Spy", "protein" ], [ 95, 108, "linker region", "structure_element" ] ] }, { "sid": 179, "sent": "The Super Spy mutants F115L, F115I, and L32P are proposed to gain activity by increasing the flexibility or size of this linker region.", "section": "FIG", "ner": [ [ 10, 13, "Spy", "protein" ], [ 22, 27, "F115L", "mutant" ], [ 29, 34, "F115I", "mutant" ], [ 40, 44, "L32P", "mutant" ], [ 121, 134, "linker region", "structure_element" ] ] }, { "sid": 180, "sent": "L32, F115, and Y104 are rendered in purple to illustrate residues that are most affected by Super Spy mutations; CH\u22ef\u03c0 hydrogen bonds are depicted by orange dashes.", "section": "FIG", "ner": [ [ 0, 3, "L32", "residue_name_number" ], [ 5, 9, "F115", "residue_name_number" ], [ 15, 19, "Y104", "residue_name_number" ], [ 98, 101, "Spy", "protein" ], [ 102, 111, "mutations", "protein_state" ], [ 118, 132, "hydrogen bonds", "bond_interaction" ] ] } ] }, "PMC4918766": { "annotations": [ { "sid": 0, "sent": "Mechanism of extracellular ion exchange and binding-site occlusion in the sodium-calcium exchanger", "section": "TITLE", "ner": [ [ 44, 56, "binding-site", "site" ], [ 74, 98, "sodium-calcium exchanger", "protein_type" ] ] }, { "sid": 1, "sent": "Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis.", "section": "ABSTRACT", "ner": [ [ 0, 19, "Na+/Ca2+ exchangers", "protein_type" ], [ 32, 35, "Na+", "chemical" ], [ 113, 117, "Ca2+", "chemical" ], [ 146, 150, "Ca2+", "chemical" ] ] }, { "sid": 2, "sent": "Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state.", "section": "ABSTRACT", "ner": [ [ 92, 111, "structural analysis", "experimental_method" ], [ 119, 128, "exchanger", "protein_type" ], [ 134, 158, "Methanococcus jannaschii", "species" ], [ 160, 166, "NCX_Mj", "protein" ], [ 168, 176, "bound to", "protein_state" ], [ 177, 180, "Na+", "chemical" ], [ 182, 186, "Ca2+", "chemical" ], [ 190, 194, "Sr2+", "chemical" ], [ 228, 231, "apo", "protein_state" ] ] }, { "sid": 3, "sent": "This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3Na+:1Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism.", "section": "ABSTRACT", "ner": [ [ 130, 133, "Na+", "chemical" ], [ 135, 139, "Ca2+", "chemical" ] ] }, { "sid": 4, "sent": "An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations.", "section": "ABSTRACT", "ner": [ [ 44, 80, "conformational free-energy landscape", "evidence" ], [ 84, 90, "NCX_Mj", "protein" ], [ 104, 117, "ion-occupancy", "protein_state" ], [ 135, 183, "enhanced-sampling molecular-dynamics simulations", "experimental_method" ], [ 207, 225, "crystal structures", "evidence" ] ] }, { "sid": 5, "sent": "These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport.", "section": "ABSTRACT", "ner": [ [ 6, 18, "calculations", "experimental_method" ], [ 58, 65, "outward", "protein_state" ], [ 69, 75, "inward", "protein_state" ], [ 182, 185, "Na+", "chemical" ], [ 186, 190, "Ca2+", "chemical" ] ] }, { "sid": 6, "sent": "Na+/Ca2+ exchangers (NCX) play physiologically essential roles in Ca2+ signaling and homeostasis.", "section": "INTRO", "ner": [ [ 0, 19, "Na+/Ca2+ exchangers", "protein_type" ], [ 21, 24, "NCX", "protein_type" ], [ 66, 70, "Ca2+", "chemical" ] ] }, { "sid": 7, "sent": "NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry.", "section": "INTRO", "ner": [ [ 0, 3, "NCX", "protein_type" ], [ 52, 56, "Ca2+", "chemical" ], [ 138, 141, "Na+", "chemical" ], [ 166, 169, "Na+", "chemical" ], [ 175, 179, "Ca2+", "chemical" ] ] }, { "sid": 8, "sent": "The mechanism of NCX proteins is therefore highly likely to be consistent with the alternating-access model of secondary-active transport.", "section": "INTRO", "ner": [ [ 17, 20, "NCX", "protein_type" ] ] }, { "sid": 9, "sent": "The basic functional unit for ion transport in NCX consists of ten membrane-spanning segments, comprising two homologous halves.", "section": "INTRO", "ner": [ [ 47, 50, "NCX", "protein_type" ], [ 67, 93, "membrane-spanning segments", "structure_element" ], [ 121, 127, "halves", "structure_element" ] ] }, { "sid": 10, "sent": "Each of these halves contains a highly conserved region, referred to as \u03b1-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1).", "section": "INTRO", "ner": [ [ 14, 20, "halves", "structure_element" ], [ 32, 48, "highly conserved", "protein_state" ], [ 72, 80, "\u03b1-repeat", "structure_element" ], [ 142, 152, "eukaryotic", "taxonomy_domain" ], [ 153, 156, "NCX", "protein_type" ], [ 166, 172, "halves", "structure_element" ], [ 198, 229, "intracellular regulatory domain", "structure_element" ], [ 240, 246, "absent", "protein_state" ], [ 250, 259, "microbial", "taxonomy_domain" ], [ 260, 263, "NCX", "protein_type" ] ] }, { "sid": 11, "sent": "Despite a long history of physiological and functional studies, the molecular mechanism of NCX has been elusive, owing to the lack of structural information.", "section": "INTRO", "ner": [ [ 91, 94, "NCX", "protein_type" ] ] }, { "sid": 12, "sent": "Our recent atomic-resolution structure of NCX_Mj from Methanococcus jannaschii provided the first view of the basic functional unit of an NCX protein.", "section": "INTRO", "ner": [ [ 29, 38, "structure", "evidence" ], [ 42, 48, "NCX_Mj", "protein" ], [ 54, 78, "Methanococcus jannaschii", "species" ], [ 138, 141, "NCX", "protein_type" ] ] }, { "sid": 13, "sent": "This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b).", "section": "INTRO", "ner": [ [ 5, 14, "structure", "evidence" ], [ 25, 34, "exchanger", "protein_type" ], [ 41, 55, "outward-facing", "protein_state" ], [ 95, 112, "ion-binding sites", "site" ], [ 126, 134, "internal", "site" ], [ 136, 140, "Sint", "site" ], [ 143, 151, "external", "site" ], [ 153, 157, "Sext", "site" ], [ 160, 172, "Ca2+-binding", "site" ], [ 174, 177, "SCa", "site" ], [ 183, 189, "middle", "site" ], [ 191, 195, "Smid", "site" ], [ 241, 254, "occluded from", "protein_state" ] ] }, { "sid": 14, "sent": "With similar ion exchange properties to those of its eukaryotic counterparts, NCX_Mj provides a compelling model system to investigate the structural basis for the specificity, stoichiometry and mechanism of the ion-exchange reaction catalyzed by NCX.", "section": "INTRO", "ner": [ [ 53, 63, "eukaryotic", "taxonomy_domain" ], [ 78, 84, "NCX_Mj", "protein" ], [ 247, 250, "NCX", "protein_type" ] ] }, { "sid": 15, "sent": "In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations.", "section": "INTRO", "ner": [ [ 43, 53, "structures", "evidence" ], [ 57, 71, "outward-facing", "protein_state" ], [ 72, 81, "wild-type", "protein_state" ], [ 82, 88, "NCX_Mj", "protein" ], [ 89, 104, "in complex with", "protein_state" ], [ 105, 108, "Na+", "chemical" ], [ 110, 114, "Ca2+", "chemical" ], [ 119, 123, "Sr2+", "chemical" ] ] }, { "sid": 16, "sent": "These structures reveal the mode of recognition of these ions, their relative affinities, and the mechanism of extracellular ion exchange, for a well-defined, functional conformation in a membrane-like environment.", "section": "INTRO", "ner": [ [ 6, 16, "structures", "evidence" ] ] }, { "sid": 17, "sent": "An independent analysis based on molecular-dynamics simulations demonstrates that the structures capture mechanistically relevant states.", "section": "INTRO", "ner": [ [ 33, 63, "molecular-dynamics simulations", "experimental_method" ], [ 86, 96, "structures", "evidence" ] ] }, { "sid": 18, "sent": "These calculations also reveal how the ion occupancy state of the outward-facing exchanger determines the feasibility of the transition to the inward-facing conformation, thereby addressing a key outstanding question in secondary-active transport, namely how the transported substrates control the alternating-access mechanism.", "section": "INTRO", "ner": [ [ 6, 18, "calculations", "experimental_method" ], [ 66, 80, "outward-facing", "protein_state" ], [ 81, 90, "exchanger", "protein_type" ], [ 143, 156, "inward-facing", "protein_state" ] ] }, { "sid": 19, "sent": "Extracellular Na+ binding", "section": "RESULTS", "ner": [ [ 14, 17, "Na+", "chemical" ] ] }, { "sid": 20, "sent": "The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals.", "section": "RESULTS", "ner": [ [ 27, 48, "central binding sites", "site" ], [ 87, 93, "NCX_Mj", "protein" ], [ 94, 103, "structure", "evidence" ], [ 141, 144, "Na+", "chemical" ], [ 149, 153, "Ca2+", "chemical" ], [ 169, 177, "crystals", "evidence" ] ] }, { "sid": 21, "sent": "To conclusively clarify this assignment, we first set out to examine the Na+ occupancy of these sites without Ca2+.", "section": "RESULTS", "ner": [ [ 73, 76, "Na+", "chemical" ], [ 110, 114, "Ca2+", "chemical" ] ] }, { "sid": 22, "sent": "Crystals were grown in 150 mM NaCl using the lipidic cubic phase (LCP) technique.", "section": "RESULTS", "ner": [ [ 0, 8, "Crystals", "evidence" ], [ 30, 34, "NaCl", "chemical" ], [ 45, 64, "lipidic cubic phase", "experimental_method" ], [ 66, 69, "LCP", "experimental_method" ] ] }, { "sid": 23, "sent": "The crystallization solutions around the LCP droplets were then slowly replaced by solutions containing different concentrations of NaCl and EGTA (Methods).", "section": "RESULTS", "ner": [ [ 41, 44, "LCP", "experimental_method" ], [ 132, 136, "NaCl", "chemical" ], [ 141, 145, "EGTA", "chemical" ] ] }, { "sid": 24, "sent": "X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c).", "section": "RESULTS", "ner": [ [ 0, 17, "X-ray diffraction", "experimental_method" ], [ 34, 42, "crystals", "evidence" ], [ 54, 57, "Na+", "chemical" ], [ 85, 114, "electron-density distribution", "evidence" ], [ 124, 128, "Sext", "site" ], [ 130, 133, "SCa", "site" ], [ 138, 142, "Sint", "site" ], [ 157, 160, "Na+", "chemical" ] ] }, { "sid": 25, "sent": "Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1).", "section": "RESULTS", "ner": [ [ 0, 20, "Occupancy refinement", "experimental_method" ], [ 35, 38, "Na+", "chemical" ], [ 52, 56, "Sint", "site" ], [ 61, 64, "SCa", "site" ], [ 72, 75, "Na+", "chemical" ], [ 131, 135, "Sint", "site" ] ] }, { "sid": 26, "sent": "Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c).", "section": "RESULTS", "ner": [ [ 19, 22, "Na+", "chemical" ], [ 26, 30, "Sext", "site" ], [ 70, 77, "density", "evidence" ], [ 106, 109, "Na+", "chemical" ], [ 130, 134, "Sext", "site" ], [ 174, 177, "Na+", "chemical" ] ] }, { "sid": 27, "sent": "The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated.", "section": "RESULTS", "ner": [ [ 4, 7, "Na+", "chemical" ], [ 22, 25, "SCa", "site" ], [ 57, 60, "Na+", "chemical" ], [ 62, 66, "Ca2+", "chemical" ], [ 121, 125, "Smid", "site" ] ] }, { "sid": 28, "sent": "Indeed, two observations indicate that a water molecule rather than a Na+ ion occupies Smid, as was predicted in a recent simulation study.", "section": "RESULTS", "ner": [ [ 41, 46, "water", "chemical" ], [ 70, 73, "Na+", "chemical" ], [ 87, 91, "Smid", "site" ], [ 122, 132, "simulation", "experimental_method" ] ] }, { "sid": 29, "sent": "First, the electron density at Smid does not depend significantly on the Na+ concentration.", "section": "RESULTS", "ner": [ [ 11, 27, "electron density", "evidence" ], [ 31, 35, "Smid", "site" ], [ 73, 76, "Na+", "chemical" ] ] }, { "sid": 30, "sent": "Second, the protein coordination geometry at Smid is clearly suboptimal for Na+ (Supplementary Fig. 1d).", "section": "RESULTS", "ner": [ [ 45, 49, "Smid", "site" ], [ 76, 79, "Na+", "chemical" ] ] }, { "sid": 31, "sent": "The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint.", "section": "RESULTS", "ner": [ [ 4, 9, "water", "chemical" ], [ 22, 26, "Smid", "site" ], [ 33, 47, "hydrogen-bonds", "bond_interaction" ], [ 57, 73, "highly conserved", "protein_state" ], [ 74, 79, "Glu54", "residue_name_number" ], [ 84, 90, "Glu213", "residue_name_number" ], [ 158, 168, "coordinate", "bond_interaction" ], [ 178, 181, "Na+", "chemical" ], [ 190, 194, "Sext", "site" ], [ 196, 199, "SCa", "site" ], [ 204, 208, "Sint", "site" ] ] }, { "sid": 32, "sent": "It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study.", "section": "RESULTS", "ner": [ [ 45, 50, "Glu54", "residue_name_number" ], [ 55, 61, "Glu213", "residue_name_number" ], [ 81, 87, "Asp240", "residue_name_number" ], [ 102, 106, "Smid", "site" ], [ 127, 130, "Asn", "residue_name" ], [ 134, 144, "eukaryotic", "taxonomy_domain" ], [ 145, 148, "NCX", "protein_type" ], [ 159, 169, "protonated", "protein_state" ], [ 206, 216, "simulation", "experimental_method" ] ] }, { "sid": 33, "sent": "Na+-dependent conformational change", "section": "RESULTS", "ner": [ [ 0, 3, "Na+", "chemical" ] ] }, { "sid": 34, "sent": "The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2).", "section": "RESULTS", "ner": [ [ 4, 10, "NCX_Mj", "protein" ], [ 11, 21, "structures", "evidence" ], [ 33, 36, "Na+", "chemical" ], [ 69, 72, "Na+", "chemical" ], [ 84, 88, "Sext", "site" ] ] }, { "sid": 35, "sent": "When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a).", "section": "RESULTS", "ner": [ [ 5, 8, "Na+", "chemical" ], [ 18, 22, "Sext", "site" ], [ 26, 30, "high", "protein_state" ], [ 51, 66, "N-terminal half", "structure_element" ], [ 70, 73, "TM7", "structure_element" ], [ 91, 104, "short helices", "structure_element" ], [ 106, 110, "TM7a", "structure_element" ], [ 115, 119, "TM7b", "structure_element" ] ] }, { "sid": 36, "sent": "TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d).", "section": "RESULTS", "ner": [ [ 0, 4, "TM7b", "structure_element" ], [ 23, 44, "central binding sites", "site" ], [ 103, 109, "Ala206", "residue_name_number" ], [ 110, 122, "coordinating", "bond_interaction" ], [ 127, 130, "Na+", "chemical" ] ] }, { "sid": 37, "sent": "However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d).", "section": "RESULTS", "ner": [ [ 14, 18, "Sext", "site" ], [ 27, 32, "empty", "protein_state" ], [ 36, 39, "low", "protein_state" ], [ 40, 43, "Na+", "chemical" ], [ 60, 64, "TM7a", "structure_element" ], [ 69, 73, "TM7b", "structure_element" ], [ 103, 108, "helix", "structure_element" ], [ 146, 152, "Ala206", "residue_name_number" ] ] }, { "sid": 38, "sent": "TM7a also forms hydrophobic contacts with the C-terminal half of TM6.", "section": "RESULTS", "ner": [ [ 0, 4, "TM7a", "structure_element" ], [ 16, 36, "hydrophobic contacts", "bond_interaction" ], [ 46, 61, "C-terminal half", "structure_element" ], [ 65, 68, "TM6", "structure_element" ] ] }, { "sid": 39, "sent": "These contacts are absent in the structure with Na+ at Sext, in which there is an open gap between the two helices (Fig. 2b).", "section": "RESULTS", "ner": [ [ 33, 42, "structure", "evidence" ], [ 48, 51, "Na+", "chemical" ], [ 55, 59, "Sext", "site" ], [ 107, 114, "helices", "structure_element" ] ] }, { "sid": 40, "sent": "This difference is noteworthy because TM6 and TM1 are believed to undergo a sliding motion, relative to the rest of the protein, when the transporter switches to the inward-facing conformation.", "section": "RESULTS", "ner": [ [ 38, 41, "TM6", "structure_element" ], [ 46, 49, "TM1", "structure_element" ], [ 138, 149, "transporter", "protein_type" ], [ 166, 179, "inward-facing", "protein_state" ] ] }, { "sid": 41, "sent": "The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d).", "section": "RESULTS", "ner": [ [ 21, 26, "TM7ab", "structure_element" ], [ 84, 88, "Sext", "site" ], [ 93, 97, "Smid", "site" ], [ 105, 108, "SCa", "site" ], [ 113, 117, "Sint", "site" ], [ 125, 133, "occluded", "protein_state" ] ] }, { "sid": 42, "sent": "Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively.", "section": "RESULTS", "ner": [ [ 10, 20, "structures", "evidence" ], [ 24, 28, "high", "protein_state" ], [ 33, 36, "low", "protein_state" ], [ 37, 40, "Na+", "chemical" ], [ 70, 84, "outward-facing", "protein_state" ], [ 85, 93, "occluded", "protein_state" ], [ 98, 112, "partially open", "protein_state" ] ] }, { "sid": 43, "sent": "This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa.", "section": "RESULTS", "ner": [ [ 47, 50, "Na+", "chemical" ], [ 64, 68, "Sext", "site" ], [ 85, 88, "Na+", "chemical" ], [ 106, 110, "Sint", "site" ], [ 115, 118, "SCa", "site" ] ] }, { "sid": 44, "sent": "Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations.", "section": "RESULTS", "ner": [ [ 4, 41, "crystallographic titration experiment", "experimental_method" ], [ 61, 65, "K1/2", "evidence" ], [ 74, 77, "Na+", "chemical" ], [ 145, 149, "Sext", "site" ], [ 153, 171, "partially occupied", "protein_state" ], [ 180, 186, "NCX_Mj", "protein" ], [ 187, 194, "crystal", "evidence" ], [ 220, 228, "occluded", "protein_state" ], [ 233, 247, "partially open", "protein_state" ] ] }, { "sid": 45, "sent": "This structurally-derived Na+ affinity agrees well with the external Na+ concentration required for NCX activation in eukaryotes.", "section": "RESULTS", "ner": [ [ 26, 38, "Na+ affinity", "evidence" ], [ 69, 72, "Na+", "chemical" ], [ 100, 103, "NCX", "protein_type" ], [ 118, 128, "eukaryotes", "taxonomy_domain" ] ] }, { "sid": 46, "sent": "The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX.", "section": "RESULTS", "ner": [ [ 21, 24, "Na+", "chemical" ], [ 105, 116, "transporter", "protein_type" ], [ 150, 166, "Hill coefficient", "evidence" ], [ 174, 177, "Na+", "chemical" ], [ 210, 220, "eukaryotic", "taxonomy_domain" ], [ 221, 224, "NCX", "protein_type" ] ] }, { "sid": 47, "sent": "Extracellular Ca2+ and Sr2+ binding and their competition with Na+", "section": "RESULTS", "ner": [ [ 14, 18, "Ca2+", "chemical" ], [ 23, 27, "Sr2+", "chemical" ], [ 63, 66, "Na+", "chemical" ] ] }, { "sid": 48, "sent": "To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods).", "section": "RESULTS", "ner": [ [ 17, 21, "Ca2+", "chemical" ], [ 31, 37, "NCX_Mj", "protein" ], [ 56, 59, "Na+", "chemical" ], [ 70, 91, "titrated the crystals", "experimental_method" ], [ 97, 101, "Sr2+", "chemical" ] ] }, { "sid": 49, "sent": "Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity.", "section": "RESULTS", "ner": [ [ 0, 4, "Sr2+", "chemical" ], [ 23, 26, "NCX", "protein_type" ], [ 40, 44, "Ca2+", "chemical" ], [ 75, 78, "Na+", "chemical" ], [ 94, 120, "electron-density intensity", "evidence" ] ] }, { "sid": 50, "sent": "Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a).", "section": "RESULTS", "ner": [ [ 0, 23, "Protein crystals soaked", "experimental_method" ], [ 35, 39, "Sr2+", "chemical" ], [ 51, 54, "Na+", "chemical" ], [ 73, 94, "electron-density peak", "evidence" ], [ 103, 106, "SCa", "site" ], [ 139, 143, "Sr2+", "chemical" ] ] }, { "sid": 51, "sent": "The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations.", "section": "RESULTS", "ner": [ [ 4, 15, "Sr2+-loaded", "protein_state" ], [ 16, 22, "NCX_Mj", "protein" ], [ 23, 32, "structure", "evidence" ], [ 44, 58, "partially open", "protein_state" ], [ 88, 91, "Na+", "chemical" ] ] }, { "sid": 52, "sent": "Binding of Sr2+, however, excludes Na+ entirely.", "section": "RESULTS", "ner": [ [ 11, 15, "Sr2+", "chemical" ], [ 35, 38, "Na+", "chemical" ] ] }, { "sid": 53, "sent": "Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b).", "section": "RESULTS", "ner": [ [ 0, 18, "Crystal titrations", "experimental_method" ], [ 24, 34, "decreasing", "experimental_method" ], [ 35, 39, "Sr2+", "chemical" ], [ 43, 53, "increasing", "experimental_method" ], [ 54, 57, "Na+", "chemical" ], [ 76, 80, "Sr2+", "chemical" ], [ 94, 108, "outward-facing", "protein_state" ], [ 109, 115, "NCX_Mj", "protein" ], [ 170, 173, "Na+", "chemical" ] ] }, { "sid": 54, "sent": "Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state.", "section": "RESULTS", "ner": [ [ 16, 19, "Na+", "chemical" ], [ 30, 34, "Sr2+", "chemical" ], [ 36, 39, "Na+", "chemical" ], [ 60, 64, "Sr2+", "chemical" ], [ 88, 94, "NCX_Mj", "protein" ], [ 102, 112, "Na+-loaded", "protein_state" ], [ 114, 128, "fully occluded", "protein_state" ] ] }, { "sid": 55, "sent": "Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b).", "section": "RESULTS", "ner": [ [ 8, 29, "titration experiments", "experimental_method" ], [ 42, 46, "Ca2+", "chemical" ], [ 51, 55, "Sr2+", "chemical" ], [ 67, 73, "NCX_Mj", "protein" ], [ 100, 129, "electron density distribution", "evidence" ], [ 135, 153, "crystals soaked in", "experimental_method" ], [ 154, 158, "high", "protein_state" ], [ 159, 163, "Ca2+", "chemical" ], [ 168, 171, "low", "protein_state" ], [ 172, 175, "Na+", "chemical" ], [ 192, 196, "Ca2+", "chemical" ], [ 209, 213, "Smid", "site" ], [ 225, 228, "SCa", "site" ], [ 252, 255, "SCa", "site" ] ] }, { "sid": 56, "sent": "Binding of Ca2+ to both sites simultaneously is highly improbable due to their close proximity, and at least one water molecule can be discerned coordinating the ion (Fig. 3b).", "section": "RESULTS", "ner": [ [ 11, 15, "Ca2+", "chemical" ], [ 113, 118, "water", "chemical" ], [ 145, 157, "coordinating", "bond_interaction" ] ] }, { "sid": 57, "sent": "The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+.", "section": "RESULTS", "ner": [ [ 4, 11, "partial", "protein_state" ], [ 12, 16, "Ca2+", "chemical" ], [ 17, 26, "occupancy", "protein_state" ], [ 30, 34, "Smid", "site" ], [ 55, 61, "Asp240", "residue_name_number" ], [ 107, 117, "coordinate", "bond_interaction" ], [ 118, 122, "Ca2+", "chemical" ] ] }, { "sid": 58, "sent": "Previous functional and computational studies, however, indicate Asp240 becomes protonated during transport.", "section": "RESULTS", "ner": [ [ 9, 45, "functional and computational studies", "experimental_method" ], [ 65, 71, "Asp240", "residue_name_number" ], [ 80, 90, "protonated", "protein_state" ] ] }, { "sid": 59, "sent": "Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid.", "section": "RESULTS", "ner": [ [ 16, 19, "NCX", "protein_type" ], [ 29, 35, "Asp240", "residue_name_number" ], [ 39, 50, "substituted", "experimental_method" ], [ 54, 57, "Asn", "residue_name" ], [ 97, 101, "Ca2+", "chemical" ], [ 113, 117, "Smid", "site" ] ] }, { "sid": 60, "sent": "SCa is therefore the functional Ca2+ site.", "section": "RESULTS", "ner": [ [ 0, 3, "SCa", "site" ], [ 32, 41, "Ca2+ site", "site" ] ] }, { "sid": 61, "sent": "Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c).", "section": "RESULTS", "ner": [ [ 13, 17, "Sr2+", "chemical" ], [ 19, 23, "Ca2+", "chemical" ], [ 39, 47, "affinity", "evidence" ], [ 51, 65, "outward-facing", "protein_state" ], [ 66, 72, "NCX_Mj", "protein" ], [ 105, 108, "Na+", "chemical" ] ] }, { "sid": 62, "sent": "This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side.", "section": "RESULTS", "ner": [ [ 32, 66, "physiological and biochemical data", "evidence" ], [ 76, 86, "eukaryotic", "taxonomy_domain" ], [ 87, 90, "NCX", "protein_type" ], [ 95, 101, "NCX_Mj", "protein" ], [ 131, 144, "Ca2+ affinity", "evidence" ] ] }, { "sid": 63, "sent": "Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM).", "section": "RESULTS", "ner": [ [ 18, 50, "crystallographic titration assay", "experimental_method" ], [ 61, 65, "Ca2+", "chemical" ], [ 92, 100, "affinity", "evidence" ], [ 147, 162, "Ca2+ affinities", "evidence" ], [ 196, 199, "NCX", "protein_type" ], [ 201, 203, "Km", "evidence" ], [ 219, 225, "NCX_Mj", "protein" ], [ 227, 229, "Km", "evidence" ] ] }, { "sid": 64, "sent": "Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.", "section": "RESULTS", "ner": [ [ 22, 51, "crystal titration experiments", "experimental_method" ], [ 78, 91, "binding sites", "site" ], [ 95, 109, "outward-facing", "protein_state" ], [ 110, 116, "NCX_Mj", "protein" ], [ 148, 152, "Sint", "site" ], [ 157, 161, "Sext", "site" ], [ 166, 169, "Na+", "chemical" ], [ 187, 190, "SCa", "site" ], [ 222, 226, "Ca2+", "chemical" ], [ 251, 254, "Na+", "chemical" ], [ 279, 289, "simulation", "experimental_method" ], [ 308, 312, "Sr2+", "chemical" ], [ 314, 318, "Smid", "site" ], [ 352, 356, "Ca2+", "chemical" ], [ 378, 382, "Smid", "site" ], [ 410, 415, "water", "chemical" ] ] }, { "sid": 65, "sent": "The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers.", "section": "RESULTS", "ner": [ [ 4, 21, "ion-binding sites", "site" ], [ 25, 31, "NCX_Mj", "protein" ], [ 70, 73, "Na+", "chemical" ], [ 122, 125, "Na+", "chemical" ], [ 130, 134, "Ca2+", "chemical" ], [ 139, 143, "Sr2+", "chemical" ], [ 188, 198, "eukaryotic", "taxonomy_domain" ], [ 199, 209, "exchangers", "protein_type" ] ] }, { "sid": 66, "sent": "A structure of NCX_Mj without Na+ or Ca2+ bound", "section": "RESULTS", "ner": [ [ 2, 11, "structure", "evidence" ], [ 15, 21, "NCX_Mj", "protein" ], [ 22, 29, "without", "protein_state" ], [ 30, 33, "Na+", "chemical" ], [ 37, 41, "Ca2+", "chemical" ], [ 42, 47, "bound", "protein_state" ] ] }, { "sid": 67, "sent": "An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM).", "section": "RESULTS", "ner": [ [ 3, 6, "apo", "protein_state" ], [ 16, 30, "outward-facing", "protein_state" ], [ 31, 37, "NCX_Mj", "protein" ], [ 140, 143, "Na+", "chemical" ], [ 158, 162, "Ca2+", "chemical" ] ] }, { "sid": 68, "sent": "We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods).", "section": "RESULTS", "ner": [ [ 29, 32, "apo", "protein_state" ], [ 39, 48, "structure", "evidence" ], [ 52, 58, "NCX_Mj", "protein" ], [ 63, 76, "crystallizing", "experimental_method" ], [ 92, 100, "lower pH", "protein_state" ], [ 112, 122, "absence of", "protein_state" ], [ 123, 126, "Na+", "chemical" ] ] }, { "sid": 69, "sent": "This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences.", "section": "RESULTS", "ner": [ [ 5, 14, "structure", "evidence" ], [ 33, 47, "partially open", "protein_state" ], [ 48, 57, "structure", "evidence" ], [ 67, 70, "Na+", "chemical" ], [ 85, 89, "Ca2+", "chemical" ], [ 97, 101, "Sr2+", "chemical" ] ] }, { "sid": 70, "sent": "First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites.", "section": "RESULTS", "ner": [ [ 7, 12, "TM7ab", "structure_element" ], [ 28, 46, "extracellular half", "structure_element" ], [ 54, 57, "TM6", "structure_element" ], [ 62, 65, "TM1", "structure_element" ], [ 168, 181, "binding sites", "site" ] ] }, { "sid": 71, "sent": "Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d).", "section": "RESULTS", "ner": [ [ 8, 13, "Glu54", "residue_name_number" ], [ 18, 24, "Glu213", "residue_name_number" ], [ 58, 71, "binding sites", "site" ], [ 91, 105, "hydrogen-bonds", "bond_interaction" ], [ 132, 148, "ion coordination", "bond_interaction" ], [ 156, 172, "fully Na+-loaded", "protein_state" ], [ 173, 182, "structure", "evidence" ] ] }, { "sid": 72, "sent": "Although the binding sites are thus fully accessible to the external solution (Fig. 3e), the lack of electron density therein indicates no ions or ordered solvent molecules.", "section": "RESULTS", "ner": [ [ 13, 26, "binding sites", "site" ], [ 36, 52, "fully accessible", "protein_state" ], [ 101, 117, "electron density", "evidence" ] ] }, { "sid": 73, "sent": "This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj.", "section": "RESULTS", "ner": [ [ 5, 8, "apo", "protein_state" ], [ 9, 18, "structure", "evidence" ], [ 49, 57, "unloaded", "protein_state" ], [ 59, 63, "open", "protein_state" ], [ 73, 87, "outward-facing", "protein_state" ], [ 88, 94, "NCX_Mj", "protein" ] ] }, { "sid": 74, "sent": "Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind.", "section": "RESULTS", "ner": [ [ 20, 29, "structure", "evidence" ], [ 46, 62, "fully protonated", "protein_state" ], [ 76, 87, "transporter", "protein_type" ], [ 98, 101, "Na+", "chemical" ], [ 106, 110, "Ca2+", "chemical" ] ] }, { "sid": 75, "sent": "Such interpretation would be consistent with the computer simulations reported below.", "section": "RESULTS", "ner": [ [ 49, 69, "computer simulations", "experimental_method" ] ] }, { "sid": 76, "sent": "Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle.", "section": "RESULTS", "ner": [ [ 8, 24, "transport assays", "experimental_method" ], [ 28, 34, "NCX_Mj", "protein" ], [ 63, 74, "presence of", "protein_state" ], [ 75, 78, "Na+", "chemical" ], [ 82, 86, "Ca2+", "chemical" ], [ 88, 94, "low pH", "protein_state" ], [ 95, 106, "inactivates", "protein_state" ] ] }, { "sid": 77, "sent": "Ion occupancy determines the free-energy landscape of NCX_Mj", "section": "RESULTS", "ner": [ [ 54, 60, "NCX_Mj", "protein" ] ] }, { "sid": 78, "sent": "That secondary-active transporters are able to harness an electrochemical gradient of one substrate to power the uphill transport of another relies on a seemingly simple principle: they must not transition between outward- and inward-open conformations unless in two precise substrate occupancy states.", "section": "RESULTS", "ner": [ [ 5, 34, "secondary-active transporters", "protein_type" ], [ 214, 222, "outward-", "protein_state" ], [ 227, 238, "inward-open", "protein_state" ] ] }, { "sid": 79, "sent": "NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state.", "section": "RESULTS", "ner": [ [ 0, 3, "NCX", "protein_type" ], [ 33, 36, "Na+", "chemical" ], [ 42, 46, "Ca2+", "chemical" ], [ 78, 88, "antiporter", "protein_type" ], [ 90, 100, "symporters", "protein_type" ], [ 217, 222, "bound", "protein_state" ], [ 234, 237, "apo", "protein_state" ] ] }, { "sid": 80, "sent": "To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations.", "section": "RESULTS", "ner": [ [ 64, 100, "conformational free-energy landscape", "evidence" ], [ 104, 110, "NCX_Mj", "protein" ], [ 175, 193, "molecular dynamics", "experimental_method" ], [ 195, 197, "MD", "experimental_method" ], [ 199, 210, "simulations", "experimental_method" ] ] }, { "sid": 81, "sent": "This computational analysis was based solely on the published structure of NCX_Mj, independently of the crystallographic studies described above.", "section": "RESULTS", "ner": [ [ 62, 71, "structure", "evidence" ], [ 75, 81, "NCX_Mj", "protein" ], [ 104, 128, "crystallographic studies", "experimental_method" ] ] }, { "sid": 82, "sent": "As it happens, the results confirm that the structures now available are representing interconverting states of the functional cycle of NCX_Mj, while revealing how the alternating-access mechanism is controlled by the ion-occupancy state.", "section": "RESULTS", "ner": [ [ 44, 54, "structures", "evidence" ], [ 136, 142, "NCX_Mj", "protein" ] ] }, { "sid": 83, "sent": "A series of exploratory MD simulations was initially carried out to examine what features of the NCX_Mj structure might depend on the ion-binding sites occupancy.", "section": "RESULTS", "ner": [ [ 24, 38, "MD simulations", "experimental_method" ], [ 97, 103, "NCX_Mj", "protein" ], [ 104, 113, "structure", "evidence" ], [ 134, 151, "ion-binding sites", "site" ] ] }, { "sid": 84, "sent": "Specifically, we first simulated the outward-occluded form, in the ion configuration we previously predicted, now confirmed by the high-Na+ crystal structure described above (Fig. 1b).", "section": "RESULTS", "ner": [ [ 23, 32, "simulated", "experimental_method" ], [ 37, 53, "outward-occluded", "protein_state" ], [ 131, 139, "high-Na+", "protein_state" ], [ 140, 157, "crystal structure", "evidence" ] ] }, { "sid": 85, "sent": "That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid.", "section": "RESULTS", "ner": [ [ 9, 12, "Na+", "chemical" ], [ 25, 29, "Sext", "site" ], [ 31, 34, "SCa", "site" ], [ 40, 44, "Sint", "site" ], [ 52, 56, "D240", "residue_name_number" ], [ 60, 70, "protonated", "protein_state" ], [ 77, 82, "water", "chemical" ], [ 101, 105, "Smid", "site" ] ] }, { "sid": 86, "sent": "The Na+ ion at Sext was then relocated from the site to the bulk solution (Methods), and this system was then allowed to evolve freely in time.", "section": "RESULTS", "ner": [ [ 4, 7, "Na+", "chemical" ], [ 15, 19, "Sext", "site" ] ] }, { "sid": 87, "sent": "The Na+ ions at SCa and Sint were displaced subsequently, and an analogous simulation was then carried out.", "section": "RESULTS", "ner": [ [ 4, 7, "Na+", "chemical" ], [ 16, 19, "SCa", "site" ], [ 24, 28, "Sint", "site" ], [ 75, 85, "simulation", "experimental_method" ] ] }, { "sid": 88, "sent": "These initial simulations revealed noticeable changes in the transporter, consistent with those observed in the new crystal structures.", "section": "RESULTS", "ner": [ [ 14, 25, "simulations", "experimental_method" ], [ 61, 72, "transporter", "protein_type" ], [ 116, 134, "crystal structures", "evidence" ] ] }, { "sid": 89, "sent": "The most notable change upon displacement of Na+ from Sext was the straightening of TM7ab (Fig. 4a).", "section": "RESULTS", "ner": [ [ 45, 48, "Na+", "chemical" ], [ 54, 58, "Sext", "site" ], [ 84, 89, "TM7ab", "structure_element" ] ] }, { "sid": 90, "sent": "When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear \u03b1-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b).", "section": "RESULTS", "ner": [ [ 7, 10, "Na+", "chemical" ], [ 20, 25, "bound", "protein_state" ], [ 27, 32, "TM7ab", "structure_element" ], [ 80, 99, "\u03b1-helical fragments", "structure_element" ], [ 150, 164, "hydrogen-bonds", "bond_interaction" ], [ 173, 177, "F202", "residue_name_number" ], [ 182, 186, "A206", "residue_name_number" ], [ 192, 196, "T203", "residue_name_number" ], [ 201, 205, "F207", "residue_name_number" ] ] }, { "sid": 91, "sent": "This distortion occludes Sext from the exterior (Fig. 4d, 4h-i) and appears to be induced by the Na+ ion itself, which pulls the carbonyl group of A206 into its coordination sphere (Fig. 4g).", "section": "RESULTS", "ner": [ [ 25, 29, "Sext", "site" ], [ 97, 100, "Na+", "chemical" ], [ 147, 151, "A206", "residue_name_number" ] ] }, { "sid": 92, "sent": "With Sext empty, however, TM7ab forms a canonical \u03b1-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded.", "section": "RESULTS", "ner": [ [ 5, 9, "Sext", "site" ], [ 10, 15, "empty", "protein_state" ], [ 26, 31, "TM7ab", "structure_element" ], [ 50, 57, "\u03b1-helix", "structure_element" ], [ 111, 114, "TM3", "structure_element" ], [ 119, 122, "TM7", "structure_element" ], [ 145, 150, "water", "chemical" ], [ 202, 206, "Sext", "site" ], [ 233, 244, "transporter", "protein_type" ], [ 248, 266, "no longer occluded", "protein_state" ] ] }, { "sid": 93, "sent": "Displacement of Na+ from SCa and Sint induces further changes (Fig. 4c).", "section": "RESULTS", "ner": [ [ 16, 19, "Na+", "chemical" ], [ 25, 28, "SCa", "site" ], [ 33, 37, "Sint", "site" ] ] }, { "sid": 94, "sent": "The most noticeable is an increased separation between TM7 and TM2 (Fig. 4f), previously brought together by concurrent backbone interactions with the Na+ ion at SCa (Fig. 4d-e).", "section": "RESULTS", "ner": [ [ 55, 58, "TM7", "structure_element" ], [ 63, 66, "TM2", "structure_element" ], [ 151, 154, "Na+", "chemical" ], [ 162, 165, "SCa", "site" ] ] }, { "sid": 95, "sent": "TM1 and TM6 also slide further towards the membrane center, relative to the outward-occluded state (Fig. 4c).", "section": "RESULTS", "ner": [ [ 0, 3, "TM1", "structure_element" ], [ 8, 11, "TM6", "structure_element" ], [ 76, 92, "outward-occluded", "protein_state" ] ] }, { "sid": 96, "sent": "Together, these changes open a second aqueous channel leading directly into SCa and Sint (Fig. 4f, Fig. 4h-i).", "section": "RESULTS", "ner": [ [ 38, 53, "aqueous channel", "site" ], [ 76, 79, "SCa", "site" ], [ 84, 88, "Sint", "site" ] ] }, { "sid": 97, "sent": "The transporter thus becomes fully outward-open.", "section": "RESULTS", "ner": [ [ 4, 15, "transporter", "protein_type" ], [ 29, 47, "fully outward-open", "protein_state" ] ] }, { "sid": 98, "sent": "To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods).", "section": "RESULTS", "ner": [ [ 111, 120, "exchanger", "protein_type" ], [ 167, 182, "MD calculations", "experimental_method" ], [ 242, 258, "outward-occluded", "protein_state" ], [ 263, 281, "fully outward-open", "protein_state" ], [ 312, 333, "free-energy landscape", "evidence" ] ] }, { "sid": 99, "sent": "As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+.", "section": "RESULTS", "ner": [ [ 68, 71, "Na+", "chemical" ], [ 75, 79, "Sext", "site" ], [ 81, 84, "SCa", "site" ], [ 89, 93, "Sint", "site" ], [ 100, 103, "Na+", "chemical" ], [ 112, 115, "SCa", "site" ], [ 120, 124, "Sint", "site" ], [ 130, 137, "without", "protein_state" ], [ 138, 141, "Na+", "chemical" ] ] }, { "sid": 100, "sent": "These calculations demonstrate that the Na+ occupancy state of the transporter has a profound effect on its conformational free-energy landscape.", "section": "RESULTS", "ner": [ [ 6, 18, "calculations", "experimental_method" ], [ 40, 43, "Na+", "chemical" ], [ 67, 78, "transporter", "protein_type" ], [ 108, 144, "conformational free-energy landscape", "evidence" ] ] }, { "sid": 101, "sent": "When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b).", "section": "RESULTS", "ner": [ [ 9, 18, "Na+ sites", "site" ], [ 44, 63, "free-energy minimum", "evidence" ], [ 168, 173, "TM7ab", "structure_element" ], [ 205, 208, "TM2", "structure_element" ], [ 213, 216, "TM3", "structure_element" ], [ 229, 242, "binding sites", "site" ] ] }, { "sid": 102, "sent": "At a small energetic cost, however, the transporter can adopt a metastable \u2018half-open\u2019 conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b).", "section": "RESULTS", "ner": [ [ 40, 51, "transporter", "protein_type" ], [ 64, 74, "metastable", "protein_state" ], [ 76, 85, "half-open", "protein_state" ], [ 109, 114, "TM7ab", "structure_element" ], [ 142, 146, "Sext", "site" ], [ 150, 154, "open", "protein_state" ] ] }, { "sid": 103, "sent": "The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O\u2013A206:N hydrogen-bond (Fig. 5c).", "section": "RESULTS", "ner": [ [ 4, 7, "Na+", "chemical" ], [ 15, 19, "Sext", "site" ], [ 28, 45, "fully coordinated", "protein_state" ], [ 62, 67, "water", "chemical" ], [ 111, 115, "A206", "residue_name_number" ], [ 147, 151, "F202", "residue_name_number" ], [ 154, 158, "A206", "residue_name_number" ], [ 161, 174, "hydrogen-bond", "bond_interaction" ] ] }, { "sid": 104, "sent": "This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 \u00d7 Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation.", "section": "RESULTS", "ner": [ [ 5, 14, "semi-open", "protein_state" ], [ 91, 94, "Na+", "chemical" ], [ 109, 112, "SCa", "site" ], [ 117, 121, "Sint", "site" ], [ 127, 130, "Na+", "chemical" ], [ 185, 188, "Na+", "chemical" ], [ 192, 196, "Sext", "site" ], [ 212, 222, "metastable", "protein_state" ] ] }, { "sid": 105, "sent": "Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c).", "section": "RESULTS", "ner": [ [ 92, 107, "aqueous channel", "site" ], [ 141, 144, "SCa", "site" ], [ 153, 156, "TM7", "structure_element" ], [ 161, 164, "TM2", "structure_element" ], [ 178, 192, "gating helices", "structure_element" ], [ 193, 196, "TM1", "structure_element" ], [ 201, 204, "TM6", "structure_element" ], [ 219, 223, "open", "protein_state" ] ] }, { "sid": 106, "sent": "Crucially, though, the free-energy landscape for this partially occupied state demonstrates that the occluded conformation is no longer energetically feasible (Fig. 5a).", "section": "RESULTS", "ner": [ [ 23, 44, "free-energy landscape", "evidence" ], [ 54, 72, "partially occupied", "protein_state" ], [ 101, 109, "occluded", "protein_state" ] ] }, { "sid": 107, "sent": "Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c).", "section": "RESULTS", "ner": [ [ 34, 37, "Na+", "chemical" ], [ 48, 51, "SCa", "site" ], [ 56, 60, "Sint", "site" ], [ 82, 103, "free-energy landscape", "evidence" ], [ 111, 122, "transporter", "protein_type" ], [ 170, 180, "fully open", "protein_state" ], [ 205, 221, "aqueous channels", "site" ] ] }, { "sid": 108, "sent": "The transition to the occluded state in this apo state is again energetically unfeasible.", "section": "RESULTS", "ner": [ [ 22, 30, "occluded", "protein_state" ], [ 45, 48, "apo", "protein_state" ] ] }, { "sid": 109, "sent": "From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete.", "section": "RESULTS", "ner": [ [ 67, 71, "open", "protein_state" ], [ 76, 85, "semi-open", "protein_state" ], [ 132, 135, "Na+", "chemical" ], [ 175, 178, "Na+", "chemical" ], [ 287, 308, "free-energy landscape", "evidence" ], [ 316, 327, "transporter", "protein_type" ], [ 346, 354, "occluded", "protein_state" ], [ 392, 417, "Na+ occupancy is complete", "protein_state" ] ] }, { "sid": 110, "sent": "This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state.", "section": "RESULTS", "ner": [ [ 41, 44, "Na+", "chemical" ], [ 93, 103, "Ca2+-bound", "protein_state" ], [ 144, 155, "transporter", "protein_type" ], [ 223, 235, "outward-open", "protein_state" ], [ 276, 280, "Ca2+", "chemical" ], [ 295, 303, "occluded", "protein_state" ], [ 331, 340, "semi-open", "protein_state" ], [ 385, 396, "inward-open", "protein_state" ] ] }, { "sid": 111, "sent": "By contrast, occupancy by H+, which as mentioned are not transported, might be compatible with a semi-open state as well as with the fully open conformation, but should not be conducive to occlusion.", "section": "RESULTS", "ner": [ [ 26, 28, "H+", "chemical" ], [ 97, 106, "semi-open", "protein_state" ], [ 133, 143, "fully open", "protein_state" ] ] }, { "sid": 112, "sent": "To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted).", "section": "RESULTS", "ner": [ [ 42, 71, "enhanced-sampling simulations", "experimental_method" ], [ 80, 84, "Ca2+", "protein_state" ], [ 89, 97, "H+-bound", "protein_state" ], [ 108, 122, "outward-facing", "protein_state" ], [ 123, 129, "NCX_Mj", "protein" ], [ 169, 172, "Na+", "chemical" ], [ 249, 259, "structures", "evidence" ], [ 267, 277, "Ca2+-bound", "protein_state" ] ] }, { "sid": 113, "sent": "The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 \u00d7 Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).", "section": "RESULTS", "ner": [ [ 4, 14, "calculated", "experimental_method" ], [ 15, 36, "free-energy landscape", "evidence" ], [ 41, 51, "Ca2+-bound", "protein_state" ], [ 52, 58, "NCX_Mj", "protein" ], [ 103, 107, "Ca2+", "chemical" ], [ 149, 155, "NCX_Mj", "protein" ], [ 176, 180, "Ca2+", "chemical" ], [ 186, 194, "occluded", "protein_state" ], [ 196, 206, "dehydrated", "protein_state" ], [ 274, 283, "exchanger", "protein_type" ], [ 303, 312, "semi-open", "protein_state" ], [ 317, 321, "open", "protein_state" ], [ 356, 360, "Ca2+", "chemical" ], [ 373, 376, "Na+", "chemical" ], [ 402, 425, "aqueous access channels", "site" ], [ 439, 443, "Sext", "site" ], [ 448, 451, "SCa", "site" ] ] }, { "sid": 114, "sent": "By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 \u00d7 H+, Fig. 6a-c).", "section": "RESULTS", "ner": [ [ 13, 24, "protonation", "protein_state" ], [ 28, 33, "Glu54", "residue_name_number" ], [ 38, 44, "Glu213", "residue_name_number" ], [ 55, 63, "occluded", "protein_state" ], [ 133, 139, "NCX_Mj", "protein" ], [ 159, 166, "protons", "chemical" ], [ 176, 184, "H+-bound", "protein_state" ], [ 204, 213, "exchanger", "protein_type" ], [ 228, 237, "semi-open", "protein_state" ], [ 267, 273, "low pH", "protein_state" ], [ 275, 278, "apo", "protein_state" ], [ 279, 296, "crystal structure", "evidence" ], [ 302, 304, "H+", "chemical" ] ] }, { "sid": 115, "sent": "Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible.", "section": "RESULTS", "ner": [ [ 21, 54, "systematic computational analysis", "experimental_method" ], [ 58, 72, "outward-facing", "protein_state" ], [ 73, 79, "NCX_Mj", "protein" ], [ 168, 186, "Na+/Ca2+ exchanger", "protein_type" ], [ 253, 274, "free-energy landscape", "evidence" ], [ 335, 343, "occluded", "protein_state" ] ] }, { "sid": 116, "sent": "This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites.", "section": "RESULTS", "ner": [ [ 5, 13, "occluded", "protein_state" ], [ 74, 81, "outward", "protein_state" ], [ 86, 97, "inward-open", "protein_state" ], [ 137, 148, "dehydration", "protein_state" ], [ 189, 207, "complete occupancy", "protein_state" ], [ 215, 228, "binding sites", "site" ] ] }, { "sid": 117, "sent": "The alternating-access hypothesis implicitly dictates that the switch between outward- and inward-open conformations of a given secondary-active transporter must not occur unless the appropriate type and number of substrates are recognized.", "section": "DISCUSS", "ner": [ [ 78, 85, "outward", "protein_state" ], [ 91, 102, "inward-open", "protein_state" ], [ 128, 144, "secondary-active", "protein_state" ], [ 145, 156, "transporter", "protein_type" ] ] }, { "sid": 118, "sent": "It is however also non-trivial: antiporters, for example, do not undergo the alternating-access transition without a cargo, but this is precisely how membrane symporters reset their transport cycles.", "section": "DISCUSS", "ner": [ [ 32, 43, "antiporters", "protein_type" ], [ 150, 169, "membrane symporters", "protein_type" ] ] }, { "sid": 119, "sent": "Similarly puzzling is that a given antiporter will undergo this transition upon recognition of substrates of different charge, size and number.", "section": "DISCUSS", "ner": [ [ 35, 45, "antiporter", "protein_type" ] ] }, { "sid": 120, "sent": "Yet, when multiple species are to be co-translocated, by either an antiporter or a symporter, partial occupancies must not be conducive to the alternating-access switch.", "section": "DISCUSS", "ner": [ [ 67, 77, "antiporter", "protein_type" ], [ 83, 92, "symporter", "protein_type" ], [ 143, 168, "alternating-access switch", "site" ] ] }, { "sid": 121, "sent": "Here, we have provided novel insights into this intriguing mechanism of conformational control through structural studies and quantitative molecular simulations of a Na+/Ca2+ exchanger.", "section": "DISCUSS", "ner": [ [ 103, 121, "structural studies", "experimental_method" ], [ 126, 160, "quantitative molecular simulations", "experimental_method" ], [ 166, 184, "Na+/Ca2+ exchanger", "protein_type" ] ] }, { "sid": 122, "sent": "Specifically, our studies of NCX_Mj reveal the mechanism of forward ion exchange (Fig. 7).", "section": "DISCUSS", "ner": [ [ 29, 35, "NCX_Mj", "protein" ] ] }, { "sid": 123, "sent": "The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter.", "section": "DISCUSS", "ner": [ [ 25, 39, "outward-facing", "protein_state" ], [ 40, 46, "NCX_Mj", "protein" ], [ 55, 68, "inward-facing", "protein_state" ], [ 69, 87, "crystal structures", "evidence" ], [ 99, 117, "Ca2+/H+ exchangers", "protein_type" ], [ 168, 171, "NCX", "protein_type" ], [ 209, 212, "TM1", "structure_element" ], [ 217, 220, "TM6", "structure_element" ], [ 249, 260, "transporter", "protein_type" ] ] }, { "sid": 124, "sent": "Here, we demonstrate that conformational changes in the extracellular region of the TM2-TM3 and TM7-TM8 bundle precede and are necessary for the transition, and are associated with ion recognition and/or release.", "section": "DISCUSS", "ner": [ [ 56, 76, "extracellular region", "structure_element" ], [ 84, 91, "TM2-TM3", "structure_element" ], [ 96, 110, "TM7-TM8 bundle", "structure_element" ] ] }, { "sid": 125, "sent": "The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution.", "section": "DISCUSS", "ner": [ [ 48, 63, "N-terminal half", "structure_element" ], [ 67, 70, "TM7", "structure_element" ], [ 72, 77, "TM7ab", "structure_element" ], [ 123, 126, "TM2", "structure_element" ], [ 131, 134, "TM3", "structure_element" ], [ 151, 156, "TM7ab", "structure_element" ], [ 213, 229, "aqueous channels", "site" ], [ 247, 264, "ion-binding sites", "site" ] ] }, { "sid": 126, "sent": "Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation.", "section": "DISCUSS", "ner": [ [ 30, 33, "TM7", "structure_element" ], [ 71, 88, "ion-binding sites", "site" ], [ 123, 126, "TM6", "structure_element" ], [ 145, 148, "TM6", "structure_element" ], [ 153, 156, "TM1", "structure_element" ], [ 180, 193, "inward-facing", "protein_state" ] ] }, { "sid": 127, "sent": "The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states.", "section": "DISCUSS", "ner": [ [ 4, 22, "crystal structures", "evidence" ], [ 26, 32, "NCX_Mj", "protein" ], [ 60, 64, "Na+,", "chemical" ], [ 65, 70, "Ca2+,", "chemical" ], [ 71, 75, "Sr2+", "chemical" ], [ 79, 81, "H+", "chemical" ], [ 82, 87, "bound", "protein_state" ], [ 101, 110, "exchanger", "protein_type" ] ] }, { "sid": 128, "sent": "These states can only represent a subset among all possible, but they ought to reflect inherent preferences of the transporter, modulated by the experimental conditions.", "section": "DISCUSS", "ner": [ [ 115, 126, "transporter", "protein_type" ] ] }, { "sid": 129, "sent": "For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure).", "section": "DISCUSS", "ner": [ [ 20, 27, "crystal", "evidence" ], [ 31, 37, "NCX_Mj", "protein" ], [ 41, 44, "LCP", "experimental_method" ], [ 50, 68, "extracellular half", "structure_element" ], [ 76, 90, "gating helices", "structure_element" ], [ 92, 95, "TM6", "structure_element" ], [ 100, 103, "TM1", "structure_element" ], [ 190, 207, "ion-binding sites", "site" ], [ 235, 238, "apo", "protein_state" ], [ 240, 246, "low pH", "protein_state" ], [ 247, 256, "structure", "evidence" ] ] }, { "sid": 130, "sent": "Nonetheless, the calculated free-energy landscapes, derived without knowledge of the experimental data, reassuringly confirm that the crystallized structures correspond to mechanistically relevant, interconverting states.", "section": "DISCUSS", "ner": [ [ 17, 50, "calculated free-energy landscapes", "evidence" ], [ 134, 157, "crystallized structures", "evidence" ] ] }, { "sid": 131, "sent": "The simulations also demonstrate how this landscape is drastically re-shaped upon each ion-binding event.", "section": "DISCUSS", "ner": [ [ 4, 15, "simulations", "experimental_method" ] ] }, { "sid": 132, "sent": "Indeed, we show that it is the presence or absence of the occluded state in this landscape that explains the antiport function of NCX_Mj and its 3Na+:1Ca2+ stoichiometry.", "section": "DISCUSS", "ner": [ [ 58, 66, "occluded", "protein_state" ], [ 130, 136, "NCX_Mj", "protein" ], [ 146, 149, "Na+", "chemical" ], [ 151, 155, "Ca2+", "chemical" ] ] }, { "sid": 133, "sent": "We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms.", "section": "DISCUSS", "ner": [ [ 89, 101, "transporters", "protein_type" ], [ 140, 150, "symporters", "protein_type" ], [ 155, 166, "antiporters", "protein_type" ], [ 197, 205, "occluded", "protein_state" ], [ 228, 272, "protein conformational free-energy landscape", "evidence" ] ] }, { "sid": 134, "sent": "In multiple ways, our findings provide an explanation for, existing functional, biochemical and biophysical data for both NCX_Mj and its eukaryotic homologues.", "section": "DISCUSS", "ner": [ [ 122, 128, "NCX_Mj", "protein" ], [ 137, 147, "eukaryotic", "taxonomy_domain" ] ] }, { "sid": 135, "sent": "The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above.", "section": "DISCUSS", "ner": [ [ 48, 70, "ion-binding affinities", "evidence" ], [ 89, 116, "crystallographic titrations", "experimental_method" ], [ 125, 127, "Km", "evidence" ], [ 132, 143, "K1/2 values", "evidence" ], [ 168, 185, "functional assays", "experimental_method" ] ] }, { "sid": 136, "sent": "Consistent with that finding, mutations that have been shown to inactivate or diminish the transport activity of NCX_Mj and cardiac NCX perfectly map to the first ion-coordination shell in our NCX_Mj structures (Supplementary Fig. 4c-d).", "section": "DISCUSS", "ner": [ [ 113, 119, "NCX_Mj", "protein" ], [ 132, 135, "NCX", "protein_type" ], [ 193, 199, "NCX_Mj", "protein" ], [ 200, 210, "structures", "evidence" ] ] }, { "sid": 137, "sent": "The crystallographic data also provides the long-sought structural basis for the \u2018two-site\u2019 model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger.", "section": "DISCUSS", "ner": [ [ 4, 25, "crystallographic data", "evidence" ], [ 149, 159, "eukaryotic", "taxonomy_domain" ], [ 160, 163, "NCX", "protein_type" ], [ 212, 218, "NCX_Mj", "protein" ], [ 237, 255, "Na+/Ca2+ exchanger", "protein_type" ] ] }, { "sid": 138, "sent": "Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.", "section": "DISCUSS", "ner": [ [ 18, 36, "crystal titrations", "experimental_method" ], [ 66, 69, "Na+", "chemical" ], [ 70, 74, "Ca2+", "chemical" ], [ 91, 95, "Sint", "site" ], [ 100, 103, "SCa", "site" ], [ 111, 115, "Ca2+", "chemical" ], [ 120, 123, "Na+", "chemical" ], [ 162, 165, "Na+", "chemical" ], [ 211, 214, "Na+", "chemical" ], [ 239, 242, "Na+", "chemical" ], [ 261, 266, "water", "chemical" ], [ 280, 284, "Smid", "site" ], [ 313, 317, "Ca2+", "chemical" ], [ 334, 350, "Hill coefficient", "evidence" ], [ 361, 364, "Na+", "chemical" ], [ 389, 393, "Ca2+", "chemical" ] ] }, { "sid": 139, "sent": "The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state.", "section": "DISCUSS", "ner": [ [ 4, 8, "Sext", "site" ], [ 51, 66, "activation site", "site" ], [ 78, 81, "Na+", "chemical" ], [ 127, 130, "Na+", "chemical" ], [ 149, 152, "Na+", "chemical" ], [ 199, 207, "occluded", "protein_state" ] ] }, { "sid": 140, "sent": "Interestingly, binding of Ca2+ to Smid appears to be also possible, but available evidence indicates that this event transiently blocks the exchange cycle.", "section": "DISCUSS", "ner": [ [ 26, 30, "Ca2+", "chemical" ], [ 34, 38, "Smid", "site" ] ] }, { "sid": 141, "sent": "Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium.", "section": "DISCUSS", "ner": [ [ 8, 18, "structures", "evidence" ], [ 22, 28, "NCX_Mj", "protein" ], [ 29, 37, "bound to", "protein_state" ], [ 38, 42, "Cd2+", "chemical" ], [ 46, 50, "Mn2+", "chemical" ], [ 104, 108, "Smid", "site" ], [ 123, 127, "Sr2+", "chemical" ], [ 142, 145, "SCa", "site" ], [ 182, 185, "NCX", "protein_type" ], [ 199, 206, "calcium", "chemical" ] ] }, { "sid": 142, "sent": "Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations.", "section": "DISCUSS", "ner": [ [ 37, 43, "NCX_Mj", "protein" ], [ 72, 76, "Ca2+", "chemical" ], [ 80, 83, "Na+", "chemical" ], [ 147, 174, "hydrogen-deuterium exchange", "experimental_method" ], [ 176, 179, "HDX", "experimental_method" ], [ 184, 190, "NCX_Mj", "protein" ], [ 199, 207, "presence", "protein_state" ], [ 211, 221, "absence of", "protein_state" ], [ 259, 273, "outward-facing", "protein_state" ] ] }, { "sid": 143, "sent": "Specifically, saturating amounts of Ca2+ or Na+ resulted in a noticeable slowdown in the HDX rate for extracellular portions of the \u03b1-repeat helices.", "section": "DISCUSS", "ner": [ [ 36, 40, "Ca2+", "chemical" ], [ 44, 47, "Na+", "chemical" ], [ 89, 97, "HDX rate", "evidence" ], [ 132, 148, "\u03b1-repeat helices", "structure_element" ] ] }, { "sid": 144, "sent": "We interpret these observations as reflecting that the solvent accessibility of the protein interior is diminished upon ion recognition, consistent with our finding that opening and closing of extracellular aqueous pathways to the ion-binding sites depend on ion occupancy state.", "section": "DISCUSS", "ner": [ [ 231, 248, "ion-binding sites", "site" ] ] }, { "sid": 145, "sent": "In addition, the increased compactness of the protein tertiary structure in the occluded state would also slow down the dynamics of the secondary-structure elements, and thus further reduce the HDX rate.", "section": "DISCUSS", "ner": [ [ 80, 88, "occluded", "protein_state" ], [ 194, 202, "HDX rate", "evidence" ] ] }, { "sid": 146, "sent": "Our data would also explain the observation that the reduction in the HDX rate is comparable for Na+ and Ca2+, as well as the finding that the degree of deuterium incorporation remains non-negligible even under saturating ion concentrations.", "section": "DISCUSS", "ner": [ [ 70, 78, "HDX rate", "evidence" ], [ 97, 100, "Na+", "chemical" ], [ 105, 109, "Ca2+", "chemical" ] ] }, { "sid": 147, "sent": "As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates.", "section": "DISCUSS", "ner": [ [ 7, 40, "calculated free-energy landscapes", "evidence" ], [ 47, 50, "Na+", "chemical" ], [ 55, 59, "Ca2+", "chemical" ], [ 88, 99, "transporter", "protein_type" ], [ 136, 145, "ion-bound", "protein_state" ], [ 219, 229, "fully open", "protein_state" ] ] }, { "sid": 148, "sent": "Na+ binding to outward-facing NCX_Mj.", "section": "FIG", "ner": [ [ 0, 3, "Na+", "chemical" ], [ 15, 29, "outward-facing", "protein_state" ], [ 30, 36, "NCX_Mj", "protein" ] ] }, { "sid": 149, "sent": "(a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+.", "section": "FIG", "ner": [ [ 12, 21, "structure", "evidence" ], [ 25, 31, "native", "protein_state" ], [ 32, 46, "outward-facing", "protein_state" ], [ 47, 53, "NCX_Mj", "protein" ], [ 59, 73, "crystals grown", "experimental_method" ], [ 84, 87, "Na+", "chemical" ] ] }, { "sid": 150, "sent": "Colored spheres represent the bound Na+ (green) and water (red).", "section": "FIG", "ner": [ [ 36, 39, "Na+", "chemical" ], [ 52, 57, "water", "chemical" ] ] }, { "sid": 151, "sent": "(b) Structural details and definition of the four central binding sites.", "section": "FIG", "ner": [ [ 50, 71, "central binding sites", "site" ] ] }, { "sid": 152, "sent": "The electron density (grey mesh, 1.9 \u00c5 Fo-Fc ion omit map contoured at 4\u03c3) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres).", "section": "FIG", "ner": [ [ 4, 20, "electron density", "evidence" ], [ 39, 57, "Fo-Fc ion omit map", "evidence" ], [ 78, 82, "Smid", "site" ], [ 98, 103, "water", "chemical" ], [ 130, 134, "Sext", "site" ], [ 136, 139, "SCa", "site" ], [ 144, 148, "Sint", "site" ], [ 152, 155, "Na+", "chemical" ] ] }, { "sid": 153, "sent": "Further details are shown in Supplementary Fig. 1. (c) Concentration-dependent change in Na+ occupancy (see also Table 1).", "section": "FIG", "ner": [ [ 89, 92, "Na+", "chemical" ] ] }, { "sid": 154, "sent": "All Fo \u2013 Fc ion-omit maps are calculated to 2.4 \u00c5 and contoured at 3\u03c3 for comparison.", "section": "FIG", "ner": [ [ 4, 25, "Fo \u2013 Fc ion-omit maps", "evidence" ] ] }, { "sid": 155, "sent": "The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively).", "section": "FIG", "ner": [ [ 20, 24, "A206", "residue_name_number" ], [ 39, 42, "Na+", "chemical" ], [ 85, 99, "partially open", "protein_state" ], [ 107, 115, "occluded", "protein_state" ], [ 148, 151, "Na+", "chemical" ] ] }, { "sid": 156, "sent": "At 20 mM Na+, both conformations co-exist.", "section": "FIG", "ner": [ [ 9, 12, "Na+", "chemical" ] ] }, { "sid": 157, "sent": "No significant changes were observed in the side-chains involved in ion or water coordination at the SCa, Sint and Smid sites.", "section": "FIG", "ner": [ [ 75, 80, "water", "chemical" ], [ 101, 104, "SCa", "site" ], [ 106, 110, "Sint", "site" ], [ 115, 119, "Smid", "site" ] ] }, { "sid": 158, "sent": "Na+-occupancy dependent conformational change in NCX_Mj.", "section": "FIG", "ner": [ [ 0, 3, "Na+", "chemical" ], [ 49, 55, "NCX_Mj", "protein" ] ] }, { "sid": 159, "sent": "(a) Superimposition of the NCX_Mj crystal structures obtained in high (100 mM, cyan cylinders) and low (10 mM, brown cylinders) Na+ concentrations.", "section": "FIG", "ner": [ [ 4, 19, "Superimposition", "experimental_method" ], [ 27, 33, "NCX_Mj", "protein" ], [ 34, 52, "crystal structures", "evidence" ], [ 128, 131, "Na+", "chemical" ] ] }, { "sid": 160, "sent": "(b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively).", "section": "FIG", "ner": [ [ 25, 34, "interface", "site" ], [ 43, 46, "TM6", "structure_element" ], [ 51, 56, "TM7ab", "structure_element" ], [ 64, 70, "NCX_Mj", "protein" ], [ 71, 81, "structures", "evidence" ], [ 107, 110, "Na+", "chemical" ] ] }, { "sid": 161, "sent": "Residues forming van-der-Waals contacts in the structure at low Na+ concentration are shown in detail.", "section": "FIG", "ner": [ [ 47, 56, "structure", "evidence" ], [ 60, 63, "low", "protein_state" ], [ 64, 67, "Na+", "chemical" ] ] }, { "sid": 162, "sent": "(c) Close-up view of the Na+-binding sites.", "section": "FIG", "ner": [ [ 25, 42, "Na+-binding sites", "site" ] ] }, { "sid": 163, "sent": "The vacant Sext site in the structure at low Na+ concentration is indicated with a white sphere.", "section": "FIG", "ner": [ [ 11, 15, "Sext", "site" ], [ 28, 37, "structure", "evidence" ], [ 41, 44, "low", "protein_state" ], [ 45, 48, "Na+", "chemical" ] ] }, { "sid": 164, "sent": "Residues surrounding this site are also indicated; note A206 (labeled in red) coordinates Na+ at Sext via its backbone carbonyl oxygen.", "section": "FIG", "ner": [ [ 56, 60, "A206", "residue_name_number" ], [ 78, 89, "coordinates", "bond_interaction" ], [ 90, 93, "Na+", "chemical" ], [ 97, 101, "Sext", "site" ] ] }, { "sid": 165, "sent": "(d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+].", "section": "FIG", "ner": [ [ 47, 64, "ion binding sites", "site" ], [ 72, 82, "structures", "evidence" ], [ 86, 90, "high", "protein_state" ], [ 95, 98, "low", "protein_state" ], [ 100, 103, "Na+", "chemical" ] ] }, { "sid": 166, "sent": "Putative solvent channels are represented as light-purple surfaces.", "section": "FIG", "ner": [ [ 9, 25, "solvent channels", "site" ] ] }, { "sid": 167, "sent": "Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).", "section": "FIG", "ner": [ [ 28, 31, "apo", "protein_state" ], [ 32, 41, "structure", "evidence" ], [ 45, 51, "NCX_Mj", "protein" ], [ 66, 70, "Sr2+", "chemical" ], [ 99, 102, "SCa", "site" ], [ 106, 123, "crystals titrated", "experimental_method" ], [ 135, 139, "Sr2+", "chemical" ], [ 151, 154, "Na+", "chemical" ] ] }, { "sid": 168, "sent": "Residues involved in Sr2+ coordination are labeled.", "section": "FIG", "ner": [ [ 21, 25, "Sr2+", "chemical" ] ] }, { "sid": 169, "sent": "There are no significant changes in the side-chains involved in ion coordination, relative to the Na+-bound state.", "section": "FIG", "ner": [ [ 98, 107, "Na+-bound", "protein_state" ] ] }, { "sid": 170, "sent": "T50 and T209 (labeled in red) coordinate Sr2+ through their backbone carbonyl-oxygen atoms.", "section": "FIG", "ner": [ [ 0, 3, "T50", "residue_name_number" ], [ 8, 12, "T209", "residue_name_number" ], [ 30, 40, "coordinate", "bond_interaction" ], [ 41, 45, "Sr2+", "chemical" ] ] }, { "sid": 171, "sent": "High Na+ concentration (100 mM) completely displaces Sr2+ and reverts NCX_Mj to the occluded state (right panel).", "section": "FIG", "ner": [ [ 5, 8, "Na+", "chemical" ], [ 53, 57, "Sr2+", "chemical" ], [ 70, 76, "NCX_Mj", "protein" ], [ 84, 92, "occluded", "protein_state" ] ] }, { "sid": 172, "sent": "The contour level of the Fo \u2013 Fc omit map of the structure at high Na+ concentration was lowered (to 4\u03c3) so as to visualize the density from Na+ ions and H2O.", "section": "FIG", "ner": [ [ 25, 41, "Fo \u2013 Fc omit map", "evidence" ], [ 49, 58, "structure", "evidence" ], [ 67, 70, "Na+", "chemical" ], [ 128, 135, "density", "evidence" ], [ 141, 144, "Na+", "chemical" ], [ 154, 157, "H2O", "chemical" ] ] }, { "sid": 173, "sent": "(b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).", "section": "FIG", "ner": [ [ 4, 8, "Ca2+", "chemical" ], [ 42, 45, "SCa", "site" ], [ 49, 53, "Smid", "site" ], [ 57, 74, "crystals titrated", "experimental_method" ], [ 86, 90, "Ca2+", "chemical" ], [ 102, 105, "Na+", "chemical" ] ] }, { "sid": 174, "sent": "The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state.", "section": "FIG", "ner": [ [ 60, 75, "Superimposition", "experimental_method" ], [ 79, 85, "NCX_Mj", "protein" ], [ 86, 96, "structures", "evidence" ], [ 113, 116, "Na+", "chemical" ], [ 169, 179, "absence of", "protein_state" ], [ 180, 183, "Na+", "chemical" ], [ 188, 192, "pH 4", "protein_state" ], [ 223, 226, "apo", "protein_state" ], [ 259, 276, "ion-binding sites", "site" ], [ 284, 287, "apo", "protein_state" ], [ 292, 299, "high H+", "protein_state" ] ] }, { "sid": 175, "sent": "The side chains of E54 and E213 from the low Na+ structure are also shown (light brown) for comparison.", "section": "FIG", "ner": [ [ 19, 22, "E54", "residue_name_number" ], [ 27, 31, "E213", "residue_name_number" ], [ 41, 48, "low Na+", "protein_state" ], [ 49, 58, "structure", "evidence" ] ] }, { "sid": 176, "sent": "White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj.", "section": "FIG", "ner": [ [ 36, 40, "Sint", "site" ], [ 42, 46, "Smid", "site" ], [ 47, 50, "SCa", "site" ], [ 99, 116, "ion-binding sites", "site" ], [ 120, 123, "apo", "protein_state" ], [ 124, 130, "NCX_Mj", "protein" ] ] }, { "sid": 177, "sent": "Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+.", "section": "FIG", "ner": [ [ 27, 36, "structure", "evidence" ], [ 40, 56, "outward-occluded", "protein_state" ], [ 58, 76, "fully Na+-occupied", "protein_state" ], [ 77, 83, "NCX_Mj", "protein" ], [ 132, 135, "Na+", "chemical" ] ] }, { "sid": 178, "sent": "(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).", "section": "FIG", "ner": [ [ 19, 29, "simulation", "experimental_method" ], [ 43, 49, "NCX_Mj", "protein" ], [ 65, 68, "Na+", "chemical" ], [ 69, 77, "bound at", "protein_state" ], [ 78, 82, "Sext", "site" ], [ 84, 87, "SCa", "site" ], [ 92, 96, "Sint", "site" ], [ 139, 142, "Na+", "chemical" ], [ 143, 156, "bound only at", "protein_state" ], [ 157, 160, "SCa", "site" ], [ 165, 169, "Sint", "site" ], [ 240, 255, "N-terminal half", "structure_element" ], [ 259, 262, "TM7", "structure_element" ], [ 264, 269, "TM7ab", "structure_element" ], [ 284, 287, "Na+", "chemical" ] ] }, { "sid": 179, "sent": "(c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons).", "section": "FIG", "ner": [ [ 19, 39, "simulation snapshots", "evidence" ], [ 61, 64, "Na+", "chemical" ], [ 65, 73, "bound at", "protein_state" ], [ 74, 77, "SCa", "site" ], [ 82, 86, "Sint", "site" ], [ 125, 132, "without", "protein_state" ], [ 137, 140, "Na+", "chemical" ], [ 141, 146, "bound", "protein_state" ] ] }, { "sid": 180, "sent": "(d) Close-up of the ion-binding region in the fully Na+-occupied state.", "section": "FIG", "ner": [ [ 20, 38, "ion-binding region", "site" ], [ 46, 64, "fully Na+-occupied", "protein_state" ] ] }, { "sid": 181, "sent": "Approximate distances between TM2, TM3 and TM7 are indicated in \u00c5. (e) Close-up of the ion-binding region in the partially Na+-occupied state.", "section": "FIG", "ner": [ [ 30, 33, "TM2", "structure_element" ], [ 35, 38, "TM3", "structure_element" ], [ 43, 46, "TM7", "structure_element" ], [ 87, 105, "ion-binding region", "site" ], [ 113, 135, "partially Na+-occupied", "protein_state" ] ] }, { "sid": 182, "sent": "(f) Close-up of the ion-binding region in the Na+-free state. (g-i) Analytical descriptors of the changes just described, calculated from the simulations of each Na+-occupancy state depicted in panels (a-f).", "section": "FIG", "ner": [ [ 20, 38, "ion-binding region", "site" ], [ 46, 54, "Na+-free", "protein_state" ], [ 142, 153, "simulations", "experimental_method" ], [ 162, 175, "Na+-occupancy", "protein_state" ] ] }, { "sid": 183, "sent": "These descriptors were employed as collective variables in the Bias-Exchange Metadynamics simulations (Methods).", "section": "FIG", "ner": [ [ 63, 101, "Bias-Exchange Metadynamics simulations", "experimental_method" ] ] }, { "sid": 184, "sent": "(g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1).", "section": "FIG", "ner": [ [ 4, 29, "Probability distributions", "evidence" ], [ 74, 90, "hydrogen-bonding", "bond_interaction" ], [ 102, 107, "TM7ab", "structure_element" ], [ 223, 227, "Sext", "site" ], [ 348, 351, "SCa", "site" ] ] }, { "sid": 185, "sent": "Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound.", "section": "FIG", "ner": [ [ 108, 111, "NCX", "protein_type" ], [ 117, 165, "Calculated conformational free-energy landscapes", "evidence" ], [ 170, 184, "outward-facing", "protein_state" ], [ 185, 191, "NCX_Mj", "protein" ], [ 211, 214, "Na+", "chemical" ], [ 254, 267, "no ions bound", "protein_state" ] ] }, { "sid": 186, "sent": "The free energy is plotted as a function of two coordinates, each describing the degree of opening of the aqueous channels leading to the Sext and SCa sites, respectively (see Methods).", "section": "FIG", "ner": [ [ 4, 15, "free energy", "evidence" ], [ 106, 122, "aqueous channels", "site" ], [ 138, 142, "Sext", "site" ], [ 147, 150, "SCa", "site" ] ] }, { "sid": 187, "sent": "Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study.", "section": "FIG", "ner": [ [ 22, 38, "X-ray structures", "evidence" ], [ 42, 48, "NCX_Mj", "protein" ], [ 61, 65, "high", "protein_state" ], [ 70, 73, "low", "protein_state" ], [ 74, 77, "Na+", "chemical" ], [ 112, 118, "low pH", "protein_state" ] ] }, { "sid": 188, "sent": "(b) Density isosurfaces for water molecules within 12 \u00c5 of the ion-binding region (grey volumes), for each of the major conformational free-energy minima in each ion-occupancy state.", "section": "FIG", "ner": [ [ 4, 23, "Density isosurfaces", "evidence" ], [ 28, 33, "water", "chemical" ], [ 63, 81, "ion-binding region", "site" ], [ 120, 153, "conformational free-energy minima", "evidence" ] ] }, { "sid": 189, "sent": "Na+ ions are shown as green spheres.", "section": "FIG", "ner": [ [ 0, 3, "Na+", "chemical" ] ] }, { "sid": 190, "sent": "The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine).", "section": "FIG", "ner": [ [ 8, 33, "inverted-topology repeats", "structure_element" ], [ 41, 52, "transporter", "protein_type" ], [ 53, 62, "structure", "evidence" ], [ 111, 116, "TM1-5", "structure_element" ], [ 126, 132, "TM6-10", "structure_element" ] ] }, { "sid": 191, "sent": "(c) Close-up views of the ion-binding region in the same conformational free-energy minima.", "section": "FIG", "ner": [ [ 26, 44, "ion-binding region", "site" ], [ 57, 90, "conformational free-energy minima", "evidence" ] ] }, { "sid": 192, "sent": "Key residues involved in Na+ and water coordination (W) are highlighted (sticks, black lines).", "section": "FIG", "ner": [ [ 25, 28, "Na+", "chemical" ], [ 33, 38, "water", "chemical" ] ] }, { "sid": 193, "sent": "The water-density maps in (b) is shown here as a grey mesh.", "section": "FIG", "ner": [ [ 4, 22, "water-density maps", "evidence" ] ] }, { "sid": 194, "sent": "Note D240 is protonated, while E54 and E213 are ionized.", "section": "FIG", "ner": [ [ 5, 9, "D240", "residue_name_number" ], [ 31, 34, "E54", "residue_name_number" ], [ 39, 43, "E213", "residue_name_number" ] ] }, { "sid": 195, "sent": "Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state.", "section": "FIG", "ner": [ [ 108, 111, "NCX", "protein_type" ], [ 117, 150, "Calculated free-energy landscapes", "evidence" ], [ 155, 169, "outward-facing", "protein_state" ], [ 170, 176, "NCX_Mj", "protein" ], [ 186, 190, "Ca2+", "chemical" ], [ 199, 215, "fully protonated", "protein_state" ] ] }, { "sid": 196, "sent": "The free energy is plotted as in Fig. 5.", "section": "FIG", "ner": [ [ 4, 15, "free energy", "evidence" ] ] }, { "sid": 197, "sent": "For Ca2+, a map is shown in which a correction for the charge-transfer between the ion and the protein is introduced, alongside the uncorrected map (see Supplementary Notes 3-4 and Supplementary Fig. 5-6).", "section": "FIG", "ner": [ [ 4, 8, "Ca2+", "chemical" ], [ 12, 15, "map", "evidence" ], [ 144, 147, "map", "evidence" ] ] }, { "sid": 198, "sent": "The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+).", "section": "FIG", "ner": [ [ 16, 19, "map", "evidence" ], [ 39, 43, "open", "protein_state" ], [ 66, 75, "semi-open", "protein_state" ], [ 80, 88, "occluded", "protein_state" ], [ 166, 174, "bound to", "protein_state" ], [ 218, 221, "Na+", "chemical" ] ] }, { "sid": 199, "sent": "Black circles map the crystal structures obtained at high Ca2+ concentration and at low pH (or high H+) reported in this study.", "section": "FIG", "ner": [ [ 22, 40, "crystal structures", "evidence" ], [ 58, 62, "Ca2+", "chemical" ], [ 84, 90, "low pH", "protein_state" ], [ 95, 102, "high H+", "protein_state" ] ] }, { "sid": 200, "sent": "(b) Water-density isosurfaces analogous to those in Fig. 5 are shown for each of the major conformational free-energy minima in the free-energy maps.", "section": "FIG", "ner": [ [ 4, 29, "Water-density isosurfaces", "evidence" ], [ 106, 124, "free-energy minima", "evidence" ], [ 132, 148, "free-energy maps", "evidence" ] ] }, { "sid": 201, "sent": "The Ca2+ ion is shown as a red sphere; the protein is shown as in Fig. 5. (c) Close-up views of the ion-binding region in the same conformational free-energy minima.", "section": "FIG", "ner": [ [ 4, 8, "Ca2+", "chemical" ], [ 100, 118, "ion-binding region", "site" ], [ 131, 164, "conformational free-energy minima", "evidence" ] ] }, { "sid": 202, "sent": "Key residues involved in Ca2+ and water coordination (W) are highlighted (sticks, black lines).", "section": "FIG", "ner": [ [ 25, 29, "Ca2+", "chemical" ], [ 34, 39, "water", "chemical" ] ] }, { "sid": 203, "sent": "The water-density maps in (b) are shown here as a grey mesh.", "section": "FIG", "ner": [ [ 4, 22, "water-density maps", "evidence" ] ] }, { "sid": 204, "sent": "In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210.", "section": "FIG", "ner": [ [ 7, 15, "occluded", "protein_state" ], [ 27, 31, "Ca2+", "chemical" ], [ 32, 37, "bound", "protein_state" ], [ 39, 44, "helix", "structure_element" ], [ 45, 50, "TM7ab", "structure_element" ], [ 83, 97, "fully occupied", "protein_state" ], [ 98, 101, "Na+", "chemical" ], [ 128, 134, "Ala206", "residue_name_number" ], [ 143, 171, "hydrogen-bonding interaction", "bond_interaction" ], [ 177, 183, "Ser210", "residue_name_number" ] ] }, { "sid": 205, "sent": "Structural mechanism of extracellular forward ion exchange in NCX.", "section": "FIG", "ner": [ [ 62, 65, "NCX", "protein_type" ] ] }, { "sid": 206, "sent": "The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes.", "section": "FIG", "ner": [ [ 23, 28, "Ala47", "residue_name_number" ], [ 33, 37, "TM2b", "structure_element" ], [ 43, 49, "Ala206", "residue_name_number" ], [ 54, 58, "TM7b", "structure_element" ], [ 84, 89, "Glu54", "residue_name_number" ], [ 94, 98, "TM2c", "structure_element" ], [ 104, 110, "Glu213", "residue_name_number" ], [ 115, 119, "TM7c", "structure_element" ] ] }, { "sid": 207, "sent": "The green open cylinders represent the gating helices TM1 and TM6.", "section": "FIG", "ner": [ [ 39, 53, "gating helices", "structure_element" ], [ 54, 57, "TM1", "structure_element" ], [ 62, 65, "TM6", "structure_element" ] ] }, { "sid": 208, "sent": "Asterisks mark the states whose crystal structures have been determined in this study.", "section": "FIG", "ner": [ [ 32, 50, "crystal structures", "evidence" ] ] }, { "sid": 209, "sent": "These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state.", "section": "FIG", "ner": [ [ 65, 98, "calculated free-energy landscapes", "evidence" ], [ 120, 131, "Ca2+-loaded", "protein_state" ], [ 132, 146, "outward-facing", "protein_state" ], [ 147, 155, "occluded", "protein_state" ], [ 170, 178, "unloaded", "protein_state" ], [ 180, 190, "fully open", "protein_state" ] ] } ] }, "PMC4786784": { "annotations": [ { "sid": 0, "sent": "An extended U2AF65\u2013RNA-binding domain recognizes the 3\u2032 splice site signal", "section": "TITLE", "ner": [ [ 3, 11, "extended", "protein_state" ], [ 12, 37, "U2AF65\u2013RNA-binding domain", "structure_element" ], [ 53, 67, "3\u2032 splice site", "site" ] ] }, { "sid": 1, "sent": "How the essential pre-mRNA splicing factor U2AF65 recognizes the polypyrimidine (Py) signals of the major class of 3\u2032 splice sites in human gene transcripts remains incompletely understood.", "section": "ABSTRACT", "ner": [ [ 18, 42, "pre-mRNA splicing factor", "protein_type" ], [ 43, 49, "U2AF65", "protein" ], [ 65, 79, "polypyrimidine", "chemical" ], [ 81, 83, "Py", "chemical" ], [ 115, 130, "3\u2032 splice sites", "site" ], [ 134, 139, "human", "species" ] ] }, { "sid": 2, "sent": "We determined four structures of an extended U2AF65\u2013RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5\u2009\u00c5. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract.", "section": "ABSTRACT", "ner": [ [ 3, 29, "determined four structures", "experimental_method" ], [ 36, 44, "extended", "protein_state" ], [ 45, 70, "U2AF65\u2013RNA-binding domain", "structure_element" ], [ 71, 79, "bound to", "protein_state" ], [ 80, 105, "Py-tract oligonucleotides", "chemical" ], [ 150, 160, "structures", "evidence" ], [ 175, 206, "RNA binding and splicing assays", "experimental_method" ], [ 235, 241, "U2AF65", "protein" ], [ 242, 263, "inter-domain residues", "site" ], [ 281, 291, "contiguous", "structure_element" ], [ 298, 308, "nucleotide", "chemical" ], [ 309, 317, "Py tract", "chemical" ] ] }, { "sid": 3, "sent": "The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3\u2032 terminus and third nucleotide.", "section": "ABSTRACT", "ner": [ [ 4, 10, "U2AF65", "protein" ], [ 11, 17, "linker", "structure_element" ], [ 44, 66, "RNA recognition motifs", "structure_element" ], [ 68, 72, "RRMs", "structure_element" ], [ 96, 106, "nucleotide", "chemical" ], [ 138, 152, "RRM extensions", "structure_element" ], [ 167, 178, "3\u2032 terminus", "site" ], [ 183, 188, "third", "residue_number" ], [ 189, 199, "nucleotide", "chemical" ] ] }, { "sid": 4, "sent": "Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF65 RRMs are complementary mechanisms for Py-tract association.", "section": "ABSTRACT", "ner": [ [ 0, 20, "Single-molecule FRET", "experimental_method" ], [ 94, 100, "U2AF65", "protein" ], [ 101, 105, "RRMs", "structure_element" ], [ 139, 147, "Py-tract", "chemical" ] ] }, { "sid": 5, "sent": "Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals.", "section": "ABSTRACT", "ner": [ [ 110, 121, "splice site", "site" ] ] }, { "sid": 6, "sent": " The pre-mRNA splicing factor U2AF65 recognizes 3\u2032 splice sites in human gene transcripts, but the details are not fully understood.", "section": "ABSTRACT", "ner": [ [ 5, 29, "pre-mRNA splicing factor", "protein_type" ], [ 30, 36, "U2AF65", "protein" ], [ 48, 63, "3\u2032 splice sites", "site" ], [ 67, 72, "human", "species" ] ] }, { "sid": 7, "sent": "Here, the authors report U2AF65 structures and single molecule FRET that reveal mechanistic insights into splice site recognition.", "section": "ABSTRACT", "ner": [ [ 25, 31, "U2AF65", "protein" ], [ 32, 42, "structures", "evidence" ], [ 47, 67, "single molecule FRET", "experimental_method" ], [ 106, 117, "splice site", "site" ] ] }, { "sid": 8, "sent": "The differential skipping or inclusion of alternatively spliced pre-mRNA regions is a major source of diversity for nearly all human gene transcripts.", "section": "INTRO", "ner": [ [ 64, 80, "pre-mRNA regions", "structure_element" ], [ 127, 132, "human", "species" ] ] }, { "sid": 9, "sent": "The splice sites are marked by relatively short consensus sequences and are regulated by additional pre-mRNA motifs (reviewed in ref.).", "section": "INTRO", "ner": [ [ 4, 16, "splice sites", "site" ], [ 42, 67, "short consensus sequences", "structure_element" ], [ 100, 115, "pre-mRNA motifs", "structure_element" ] ] }, { "sid": 10, "sent": "At the 3\u2032 splice site of the major intron class, these include a polypyrimidine (Py) tract comprising primarily Us or Cs, which is preceded by a branch point sequence (BPS) that ultimately serves as the nucleophile in the splicing reaction and an AG-dinucleotide at the 3\u2032 splice site junction.", "section": "INTRO", "ner": [ [ 7, 21, "3\u2032 splice site", "site" ], [ 65, 90, "polypyrimidine (Py) tract", "chemical" ], [ 112, 113, "U", "residue_name" ], [ 118, 119, "C", "residue_name" ], [ 145, 166, "branch point sequence", "site" ], [ 168, 171, "BPS", "site" ], [ 247, 262, "AG-dinucleotide", "chemical" ], [ 270, 284, "3\u2032 splice site", "site" ] ] }, { "sid": 11, "sent": "Disease-causing mutations often compromise pre-mRNA splicing (reviewed in refs), yet a priori predictions of splice sites and the consequences of their mutations are challenged by the brevity and degeneracy of known splice site sequences.", "section": "INTRO", "ner": [ [ 43, 51, "pre-mRNA", "chemical" ], [ 109, 121, "splice sites", "site" ], [ 216, 227, "splice site", "site" ] ] }, { "sid": 12, "sent": "High-resolution structures of intact splicing factor\u2013RNA complexes would offer key insights regarding the juxtaposition of the distinct splice site consensus sequences and their relationship to disease-causing point mutations.", "section": "INTRO", "ner": [ [ 16, 26, "structures", "evidence" ], [ 30, 36, "intact", "protein_state" ], [ 37, 56, "splicing factor\u2013RNA", "complex_assembly" ], [ 136, 147, "splice site", "site" ] ] }, { "sid": 13, "sent": "The early-stage pre-mRNA splicing factor U2AF65 is essential for viability in vertebrates and other model organisms (for example, ref.).", "section": "INTRO", "ner": [ [ 16, 40, "pre-mRNA splicing factor", "protein_type" ], [ 41, 47, "U2AF65", "protein" ], [ 78, 89, "vertebrates", "taxonomy_domain" ] ] }, { "sid": 14, "sent": "A tightly controlled assembly among U2AF65, the pre-mRNA, and partner proteins sequentially identifies the 3\u2032 splice site and promotes association of the spliceosome, which ultimately accomplishes the task of splicing.", "section": "INTRO", "ner": [ [ 21, 29, "assembly", "complex_assembly" ], [ 36, 42, "U2AF65", "protein" ], [ 48, 56, "pre-mRNA", "chemical" ], [ 107, 121, "3\u2032 splice site", "site" ], [ 154, 165, "spliceosome", "complex_assembly" ] ] }, { "sid": 15, "sent": "Initially U2AF65 recognizes the Py-tract splice site signal.", "section": "INTRO", "ner": [ [ 10, 16, "U2AF65", "protein" ], [ 32, 40, "Py-tract", "chemical" ], [ 41, 52, "splice site", "site" ] ] }, { "sid": 16, "sent": "In turn, the ternary complex of U2AF65 with SF1 and U2AF35 identifies the surrounding BPS and 3\u2032 splice site junctions.", "section": "INTRO", "ner": [ [ 13, 28, "ternary complex", "complex_assembly" ], [ 32, 38, "U2AF65", "protein" ], [ 44, 47, "SF1", "protein" ], [ 52, 58, "U2AF35", "protein" ], [ 86, 89, "BPS", "site" ], [ 94, 108, "3\u2032 splice site", "site" ] ] }, { "sid": 17, "sent": "Subsequently U2AF65 recruits the U2 small nuclear ribonucleoprotein particle (snRNP) and ultimately dissociates from the active spliceosome.", "section": "INTRO", "ner": [ [ 13, 19, "U2AF65", "protein" ], [ 33, 76, "U2 small nuclear ribonucleoprotein particle", "complex_assembly" ], [ 78, 83, "snRNP", "complex_assembly" ], [ 121, 127, "active", "protein_state" ], [ 128, 139, "spliceosome", "complex_assembly" ] ] }, { "sid": 18, "sent": "Biochemical characterizations of U2AF65 demonstrated that tandem RNA recognition motifs (RRM1 and RRM2) recognize the Py tract (Fig. 1a).", "section": "INTRO", "ner": [ [ 0, 29, "Biochemical characterizations", "experimental_method" ], [ 33, 39, "U2AF65", "protein" ], [ 65, 87, "RNA recognition motifs", "structure_element" ], [ 89, 93, "RRM1", "structure_element" ], [ 98, 102, "RRM2", "structure_element" ], [ 118, 126, "Py tract", "chemical" ] ] }, { "sid": 19, "sent": "Milestone crystal structures of the core U2AF65 RRM1 and RRM2 connected by a shortened inter-RRM linker (dU2AF651,2) detailed a subset of nucleotide interactions with the individual U2AF65 RRMs.", "section": "INTRO", "ner": [ [ 10, 28, "crystal structures", "evidence" ], [ 36, 40, "core", "protein_state" ], [ 41, 47, "U2AF65", "protein" ], [ 48, 52, "RRM1", "structure_element" ], [ 57, 61, "RRM2", "structure_element" ], [ 77, 86, "shortened", "protein_state" ], [ 87, 103, "inter-RRM linker", "structure_element" ], [ 105, 115, "dU2AF651,2", "mutant" ], [ 182, 188, "U2AF65", "protein" ], [ 189, 193, "RRMs", "structure_element" ] ] }, { "sid": 20, "sent": "A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation.", "section": "INTRO", "ner": [ [ 13, 16, "NMR", "experimental_method" ], [ 17, 26, "structure", "evidence" ], [ 45, 57, "side-by-side", "protein_state" ], [ 77, 84, "minimal", "protein_state" ], [ 85, 91, "U2AF65", "protein" ], [ 92, 96, "RRM1", "structure_element" ], [ 101, 105, "RRM2", "structure_element" ], [ 121, 127, "linker", "structure_element" ], [ 131, 145, "natural length", "protein_state" ], [ 147, 156, "U2AF651,2", "mutant" ], [ 179, 189, "dU2AF651,2", "mutant" ], [ 190, 208, "crystal structures", "evidence" ], [ 213, 216, "RNA", "chemical" ], [ 261, 277, "inter-RRM linker", "structure_element" ] ] }, { "sid": 21, "sent": "As such, the molecular mechanisms for Py-tract recognition by the intact U2AF65\u2013RNA-binding domain remained unknown.", "section": "INTRO", "ner": [ [ 38, 46, "Py-tract", "chemical" ], [ 66, 72, "intact", "protein_state" ], [ 73, 98, "U2AF65\u2013RNA-binding domain", "structure_element" ] ] }, { "sid": 22, "sent": "Here, we use X-ray crystallography and biochemical studies to reveal new roles in Py-tract recognition for the inter-RRM linker and key residues surrounding the core U2AF65 RRMs.", "section": "INTRO", "ner": [ [ 13, 34, "X-ray crystallography", "experimental_method" ], [ 39, 58, "biochemical studies", "experimental_method" ], [ 82, 90, "Py-tract", "chemical" ], [ 111, 127, "inter-RRM linker", "structure_element" ], [ 161, 165, "core", "protein_state" ], [ 166, 172, "U2AF65", "protein" ], [ 173, 177, "RRMs", "structure_element" ] ] }, { "sid": 23, "sent": "We use single-molecule F\u00f6rster resonance energy transfer (smFRET) to characterize the conformational dynamics of this extended U2AF65\u2013RNA-binding domain during Py-tract recognition.", "section": "INTRO", "ner": [ [ 7, 56, "single-molecule F\u00f6rster resonance energy transfer", "experimental_method" ], [ 58, 64, "smFRET", "experimental_method" ], [ 86, 109, "conformational dynamics", "evidence" ], [ 118, 126, "extended", "protein_state" ], [ 127, 152, "U2AF65\u2013RNA-binding domain", "structure_element" ], [ 160, 168, "Py-tract", "chemical" ] ] }, { "sid": 24, "sent": "Cognate U2AF65\u2013Py-tract recognition requires RRM extensions", "section": "RESULTS", "ner": [ [ 8, 14, "U2AF65", "protein" ], [ 15, 23, "Py-tract", "chemical" ], [ 45, 59, "RRM extensions", "structure_element" ] ] }, { "sid": 25, "sent": "The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1\u2013RRM2 folds (U2AF651,2, residues 148\u2013336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1).", "section": "RESULTS", "ner": [ [ 4, 16, "RNA affinity", "evidence" ], [ 24, 31, "minimal", "protein_state" ], [ 32, 41, "U2AF651,2", "mutant" ], [ 64, 68, "core", "protein_state" ], [ 69, 73, "RRM1", "structure_element" ], [ 74, 78, "RRM2", "structure_element" ], [ 79, 84, "folds", "structure_element" ], [ 86, 95, "U2AF651,2", "mutant" ], [ 106, 113, "148\u2013336", "residue_range" ], [ 148, 159, "full-length", "protein_state" ], [ 160, 166, "U2AF65", "protein" ] ] }, { "sid": 26, "sent": "Historically, this difference was attributed to the U2AF65 arginine\u2013serine rich domain, which contacts pre-mRNA\u2013U2 snRNA duplexes outside of the Py tract.", "section": "RESULTS", "ner": [ [ 52, 58, "U2AF65", "protein" ], [ 59, 86, "arginine\u2013serine rich domain", "structure_element" ], [ 103, 129, "pre-mRNA\u2013U2 snRNA duplexes", "complex_assembly" ], [ 145, 153, "Py tract", "chemical" ] ] }, { "sid": 27, "sent": "We noticed that the RNA-binding affinity of the U2AF651,2 domain was greatly enhanced by the addition of seven and six residues at the respective N and C termini of the minimal RRM1 and RRM2 (U2AF651,2L, residues 141\u2013342; Fig. 1a).", "section": "RESULTS", "ner": [ [ 20, 40, "RNA-binding affinity", "evidence" ], [ 48, 57, "U2AF651,2", "mutant" ], [ 93, 127, "addition of seven and six residues", "experimental_method" ], [ 169, 176, "minimal", "protein_state" ], [ 177, 181, "RRM1", "structure_element" ], [ 186, 190, "RRM2", "structure_element" ], [ 192, 202, "U2AF651,2L", "mutant" ], [ 213, 220, "141\u2013342", "residue_range" ] ] }, { "sid": 28, "sent": "In a fluorescence anisotropy assay for binding a representative Py tract derived from the well-characterized splice site of the adenovirus major late promoter (AdML), the RNA affinity of U2AF651,2L increased by 100-fold relative to U2AF651,2 to comparable levels as full-length U2AF65 (Fig. 1b; Supplementary Fig. 1a\u2013d).", "section": "RESULTS", "ner": [ [ 5, 34, "fluorescence anisotropy assay", "experimental_method" ], [ 64, 72, "Py tract", "chemical" ], [ 109, 120, "splice site", "site" ], [ 128, 158, "adenovirus major late promoter", "gene" ], [ 160, 164, "AdML", "gene" ], [ 171, 183, "RNA affinity", "evidence" ], [ 187, 197, "U2AF651,2L", "mutant" ], [ 232, 241, "U2AF651,2", "mutant" ], [ 266, 277, "full-length", "protein_state" ], [ 278, 284, "U2AF65", "protein" ] ] }, { "sid": 29, "sent": "Likewise, both U2AF651,2L and full-length U2AF65 showed similar sequence specificity for U-rich stretches in the 5\u2032-region of the Py tract and promiscuity for C-rich regions in the 3\u2032-region (Fig. 1c, Supplementary Fig. 1e\u2013h).", "section": "RESULTS", "ner": [ [ 15, 25, "U2AF651,2L", "mutant" ], [ 30, 41, "full-length", "protein_state" ], [ 42, 48, "U2AF65", "protein" ], [ 64, 84, "sequence specificity", "evidence" ], [ 89, 105, "U-rich stretches", "structure_element" ], [ 113, 122, "5\u2032-region", "site" ], [ 130, 138, "Py tract", "chemical" ], [ 159, 173, "C-rich regions", "structure_element" ], [ 181, 190, "3\u2032-region", "site" ] ] }, { "sid": 30, "sent": "U2AF65-bound Py tract comprises nine contiguous nucleotides", "section": "RESULTS", "ner": [ [ 0, 12, "U2AF65-bound", "protein_state" ], [ 13, 21, "Py tract", "chemical" ], [ 37, 47, "contiguous", "structure_element" ], [ 48, 59, "nucleotides", "chemical" ] ] }, { "sid": 31, "sent": "To investigate the structural basis for cognate U2AF65 recognition of a contiguous Py tract, we determined four crystal structures of U2AF651,2L bound to Py-tract oligonucleotides (Fig. 2a; Table 1).", "section": "RESULTS", "ner": [ [ 48, 54, "U2AF65", "protein" ], [ 72, 82, "contiguous", "structure_element" ], [ 83, 91, "Py tract", "chemical" ], [ 96, 106, "determined", "experimental_method" ], [ 112, 130, "crystal structures", "evidence" ], [ 134, 144, "U2AF651,2L", "mutant" ], [ 145, 153, "bound to", "protein_state" ], [ 154, 179, "Py-tract oligonucleotides", "chemical" ] ] }, { "sid": 32, "sent": "By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5\u2009\u00c5 resolution.", "section": "RESULTS", "ner": [ [ 3, 28, "sequential boot strapping", "experimental_method" ], [ 57, 72, "oligonucleotide", "chemical" ], [ 99, 104, "Br-dU", "chemical" ], [ 139, 149, "nucleotide", "chemical" ], [ 151, 153, "rU", "residue_name" ], [ 155, 157, "dU", "residue_name" ], [ 162, 164, "rC", "residue_name" ], [ 191, 201, "U2AF651,2L", "mutant" ], [ 202, 210, "bound to", "protein_state" ], [ 211, 221, "contiguous", "structure_element" ], [ 222, 231, "Py tracts", "chemical" ] ] }, { "sid": 33, "sent": "The protein and oligonucleotide conformations are nearly identical among the four new U2AF651,2L structures (Supplementary Fig. 2a).", "section": "RESULTS", "ner": [ [ 16, 31, "oligonucleotide", "chemical" ], [ 86, 96, "U2AF651,2L", "mutant" ], [ 97, 107, "structures", "evidence" ] ] }, { "sid": 34, "sent": "The U2AF651,2L RRM1 and RRM2 associate with the Py tract in a parallel, side-by-side arrangement (shown for representative structure iv in Fig. 2b,c; Supplementary Movie 1).", "section": "RESULTS", "ner": [ [ 4, 14, "U2AF651,2L", "mutant" ], [ 15, 19, "RRM1", "structure_element" ], [ 24, 28, "RRM2", "structure_element" ], [ 48, 56, "Py tract", "chemical" ], [ 62, 70, "parallel", "protein_state" ], [ 72, 84, "side-by-side", "protein_state" ] ] }, { "sid": 35, "sent": "An extended conformation of the U2AF65 inter-RRM linker traverses across the \u03b1-helical surface of RRM1 and the central \u03b2-strands of RRM2 and is well defined in the electron density (Fig. 2b).", "section": "RESULTS", "ner": [ [ 3, 24, "extended conformation", "protein_state" ], [ 32, 38, "U2AF65", "protein" ], [ 39, 55, "inter-RRM linker", "structure_element" ], [ 77, 94, "\u03b1-helical surface", "structure_element" ], [ 98, 102, "RRM1", "structure_element" ], [ 119, 128, "\u03b2-strands", "structure_element" ], [ 132, 136, "RRM2", "structure_element" ], [ 164, 180, "electron density", "evidence" ] ] }, { "sid": 36, "sent": "The extensions at the N terminus of RRM1 and C terminus of RRM2 adopt well-ordered \u03b1-helices.", "section": "RESULTS", "ner": [ [ 4, 14, "extensions", "structure_element" ], [ 36, 40, "RRM1", "structure_element" ], [ 59, 63, "RRM2", "structure_element" ], [ 83, 92, "\u03b1-helices", "structure_element" ] ] }, { "sid": 37, "sent": "Both RRM1/RRM2 extensions and the inter-RRM linker of U2AF651,2L directly recognize the bound oligonucleotide.", "section": "RESULTS", "ner": [ [ 5, 9, "RRM1", "structure_element" ], [ 10, 14, "RRM2", "structure_element" ], [ 15, 25, "extensions", "structure_element" ], [ 34, 50, "inter-RRM linker", "structure_element" ], [ 54, 64, "U2AF651,2L", "mutant" ], [ 88, 93, "bound", "protein_state" ], [ 94, 109, "oligonucleotide", "chemical" ] ] }, { "sid": 38, "sent": "We compare the global conformation of the U2AF651,2L structures with the prior dU2AF651,2 crystal structure and U2AF651,2 NMR structure in the Supplementary Discussion and Supplementary Fig. 2.", "section": "RESULTS", "ner": [ [ 42, 52, "U2AF651,2L", "mutant" ], [ 53, 63, "structures", "evidence" ], [ 79, 89, "dU2AF651,2", "mutant" ], [ 90, 107, "crystal structure", "evidence" ], [ 112, 121, "U2AF651,2", "mutant" ], [ 122, 125, "NMR", "experimental_method" ], [ 126, 135, "structure", "evidence" ] ] }, { "sid": 39, "sent": "The discovery of nine U2AF65-binding sites for contiguous Py-tract nucleotides was unexpected.", "section": "RESULTS", "ner": [ [ 22, 42, "U2AF65-binding sites", "site" ], [ 47, 57, "contiguous", "structure_element" ], [ 58, 78, "Py-tract nucleotides", "chemical" ] ] }, { "sid": 40, "sent": "Based on dU2AF651,2 structures, we originally hypothesized that the U2AF65 RRMs would bind the minimal seven nucleotides observed in these structures.", "section": "RESULTS", "ner": [ [ 9, 19, "dU2AF651,2", "mutant" ], [ 20, 30, "structures", "evidence" ], [ 68, 74, "U2AF65", "protein" ], [ 75, 79, "RRMs", "structure_element" ], [ 95, 102, "minimal", "protein_state" ], [ 109, 120, "nucleotides", "chemical" ], [ 139, 149, "structures", "evidence" ] ] }, { "sid": 41, "sent": "Surprisingly, the RRM2 extension/inter-RRM linker contribute new central nucleotide-binding sites near the RRM1/RRM2 junction and the RRM1 extension recognizes the 3\u2032-terminal nucleotide (Fig. 2c; Supplementary Movie 1).", "section": "RESULTS", "ner": [ [ 18, 32, "RRM2 extension", "structure_element" ], [ 33, 49, "inter-RRM linker", "structure_element" ], [ 73, 97, "nucleotide-binding sites", "site" ], [ 107, 125, "RRM1/RRM2 junction", "site" ], [ 134, 148, "RRM1 extension", "structure_element" ], [ 176, 186, "nucleotide", "chemical" ] ] }, { "sid": 42, "sent": "The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2\u2032-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure.", "section": "RESULTS", "ner": [ [ 4, 14, "U2AF651,2L", "mutant" ], [ 15, 25, "structures", "evidence" ], [ 39, 45, "ribose", "chemical" ], [ 47, 48, "r", "chemical" ], [ 50, 61, "nucleotides", "chemical" ], [ 76, 89, "binding sites", "site" ], [ 101, 108, "seventh", "residue_number" ], [ 113, 119, "eighth", "residue_number" ], [ 120, 130, "deoxy-(d)U", "chemical" ], [ 191, 200, "RNA-bound", "protein_state" ], [ 201, 211, "dU2AF651,2", "mutant" ], [ 212, 221, "structure", "evidence" ] ] }, { "sid": 43, "sent": "Qualitatively, a subset of the U2AF651,2L-nucleotide-binding sites (sites 1\u20133 and 7\u20139) share similar locations to those of the dU2AF651,2 structures (Supplementary Figs 2c,d and 3).", "section": "RESULTS", "ner": [ [ 31, 66, "U2AF651,2L-nucleotide-binding sites", "site" ], [ 68, 77, "sites 1\u20133", "site" ], [ 82, 85, "7\u20139", "site" ], [ 127, 137, "dU2AF651,2", "mutant" ], [ 138, 148, "structures", "evidence" ] ] }, { "sid": 44, "sent": "Yet, only the U2AF651,2L interactions at sites 1 and 7 are nearly identical to those of the dU2AF651,2 structures (Supplementary Fig. 3a,f).", "section": "RESULTS", "ner": [ [ 14, 24, "U2AF651,2L", "mutant" ], [ 41, 54, "sites 1 and 7", "site" ], [ 92, 102, "dU2AF651,2", "mutant" ], [ 103, 113, "structures", "evidence" ] ] }, { "sid": 45, "sent": "In striking departures from prior partial views, the U2AF651,2L structures reveal three unanticipated nucleotide-binding sites at the centre of the Py tract, as well as numerous new interactions that underlie cognate recognition of the Py tract (Fig. 3a\u2013h).", "section": "RESULTS", "ner": [ [ 53, 63, "U2AF651,2L", "mutant" ], [ 64, 74, "structures", "evidence" ], [ 102, 126, "nucleotide-binding sites", "site" ], [ 148, 156, "Py tract", "chemical" ], [ 236, 244, "Py tract", "chemical" ] ] }, { "sid": 46, "sent": "U2AF65 inter-RRM linker interacts with the Py tract", "section": "RESULTS", "ner": [ [ 0, 6, "U2AF65", "protein" ], [ 7, 23, "inter-RRM linker", "structure_element" ], [ 43, 51, "Py tract", "chemical" ] ] }, { "sid": 47, "sent": "The U2AF651,2L RRM2, the inter-RRM linker and RRM1 concomitantly recognize the three central nucleotides of the Py tract, which are likely to coordinate the conformational arrangement of these disparate portions of the protein.", "section": "RESULTS", "ner": [ [ 4, 14, "U2AF651,2L", "mutant" ], [ 15, 19, "RRM2", "structure_element" ], [ 25, 41, "inter-RRM linker", "structure_element" ], [ 46, 50, "RRM1", "structure_element" ], [ 93, 104, "nucleotides", "chemical" ], [ 112, 120, "Py tract", "chemical" ] ] }, { "sid": 48, "sent": "Residues in the C-terminal region of the U2AF65 inter-RRM linker comprise a centrally located binding site for the fifth nucleotide on the RRM2 surface and abutting the RRM1/RRM2 interface (Fig. 3d).", "section": "RESULTS", "ner": [ [ 16, 33, "C-terminal region", "structure_element" ], [ 41, 47, "U2AF65", "protein" ], [ 48, 64, "inter-RRM linker", "structure_element" ], [ 94, 106, "binding site", "site" ], [ 115, 120, "fifth", "residue_number" ], [ 121, 131, "nucleotide", "chemical" ], [ 139, 151, "RRM2 surface", "site" ], [ 169, 188, "RRM1/RRM2 interface", "site" ] ] }, { "sid": 49, "sent": "The backbone amide of the linker V254 and the carbonyl of T252 engage in hydrogen bonds with the rU5-O4 and -N3H atoms.", "section": "RESULTS", "ner": [ [ 26, 32, "linker", "structure_element" ], [ 33, 37, "V254", "residue_name_number" ], [ 58, 62, "T252", "residue_name_number" ], [ 73, 87, "hydrogen bonds", "bond_interaction" ], [ 97, 100, "rU5", "residue_name_number" ] ] }, { "sid": 50, "sent": "In the C-terminal \u03b2-strand of RRM1, the side chains of K225 and R227 donate additional hydrogen bonds to the rU5-O2 lone pair electrons.", "section": "RESULTS", "ner": [ [ 18, 26, "\u03b2-strand", "structure_element" ], [ 30, 34, "RRM1", "structure_element" ], [ 55, 59, "K225", "residue_name_number" ], [ 64, 68, "R227", "residue_name_number" ], [ 87, 101, "hydrogen bonds", "bond_interaction" ], [ 109, 112, "rU5", "residue_name_number" ] ] }, { "sid": 51, "sent": "The C-terminal region of the inter-RRM linker also participates in the preceding rU4-binding site, where the V254 backbone carbonyl and D256 carboxylate position the K260 side chain to hydrogen bond with the rU4-O4 (Fig. 3c).", "section": "RESULTS", "ner": [ [ 4, 21, "C-terminal region", "structure_element" ], [ 29, 45, "inter-RRM linker", "structure_element" ], [ 81, 97, "rU4-binding site", "site" ], [ 109, 113, "V254", "residue_name_number" ], [ 136, 140, "D256", "residue_name_number" ], [ 166, 170, "K260", "residue_name_number" ], [ 185, 198, "hydrogen bond", "bond_interaction" ], [ 208, 211, "rU4", "residue_name_number" ] ] }, { "sid": 52, "sent": "Otherwise, the rU4 nucleotide packs against F304 in the signature ribonucleoprotein consensus motif (RNP)-2 of RRM2.", "section": "RESULTS", "ner": [ [ 15, 18, "rU4", "residue_name_number" ], [ 19, 29, "nucleotide", "chemical" ], [ 44, 48, "F304", "residue_name_number" ], [ 66, 107, "ribonucleoprotein consensus motif (RNP)-2", "structure_element" ], [ 111, 115, "RRM2", "structure_element" ] ] }, { "sid": 53, "sent": "At the opposite side of the central fifth nucleotide, the sixth rU6 nucleotide is located at the inter-RRM1/RRM2 interface (Fig. 3e; Supplementary Movie 1).", "section": "RESULTS", "ner": [ [ 36, 41, "fifth", "residue_number" ], [ 42, 52, "nucleotide", "chemical" ], [ 58, 63, "sixth", "residue_number" ], [ 64, 67, "rU6", "residue_name_number" ], [ 68, 78, "nucleotide", "chemical" ], [ 97, 122, "inter-RRM1/RRM2 interface", "site" ] ] }, { "sid": 54, "sent": "This nucleotide twists to face away from the U2AF65 linker and instead inserts the rU6-uracil into a sandwich between the \u03b22/\u03b23 loops of RRM1 and RRM2.", "section": "RESULTS", "ner": [ [ 5, 15, "nucleotide", "chemical" ], [ 45, 51, "U2AF65", "protein" ], [ 52, 58, "linker", "structure_element" ], [ 83, 86, "rU6", "residue_name_number" ], [ 87, 93, "uracil", "residue_name" ], [ 122, 133, "\u03b22/\u03b23 loops", "structure_element" ], [ 137, 141, "RRM1", "structure_element" ], [ 146, 150, "RRM2", "structure_element" ] ] }, { "sid": 55, "sent": "The rU6 base edge is relatively solvent exposed; accordingly, the rU6 hydrogen bonds with U2AF65 are water mediated apart from a single direct interaction by the RRM1-N196 side chain.", "section": "RESULTS", "ner": [ [ 4, 7, "rU6", "residue_name_number" ], [ 32, 47, "solvent exposed", "protein_state" ], [ 66, 69, "rU6", "residue_name_number" ], [ 70, 84, "hydrogen bonds", "bond_interaction" ], [ 90, 96, "U2AF65", "protein" ], [ 101, 106, "water", "chemical" ], [ 162, 166, "RRM1", "structure_element" ], [ 167, 171, "N196", "residue_name_number" ] ] }, { "sid": 56, "sent": "We tested the contribution of the U2AF651,2L interactions with the new central nucleotide to Py-tract affinity (Fig. 3i; Supplementary Fig. 4a,b).", "section": "RESULTS", "ner": [ [ 3, 26, "tested the contribution", "experimental_method" ], [ 34, 44, "U2AF651,2L", "mutant" ], [ 79, 89, "nucleotide", "chemical" ], [ 93, 110, "Py-tract affinity", "evidence" ] ] }, { "sid": 57, "sent": "Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1\u2013R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively.", "section": "RESULTS", "ner": [ [ 0, 11, "Mutagenesis", "experimental_method" ], [ 22, 26, "V254", "residue_name_number" ], [ 34, 40, "U2AF65", "protein" ], [ 41, 57, "inter-RRM linker", "structure_element" ], [ 61, 68, "proline", "residue_name" ], [ 72, 76, "RRM1", "structure_element" ], [ 77, 81, "R227", "residue_name_number" ], [ 85, 92, "alanine", "residue_name" ], [ 111, 124, "hydrogen bond", "bond_interaction" ], [ 134, 139, "fifth", "residue_number" ], [ 140, 146, "uracil", "residue_name" ], [ 170, 180, "affinities", "evidence" ], [ 184, 194, "U2AF651,2L", "mutant" ], [ 218, 222, "AdML", "gene" ], [ 223, 231, "Py tract", "chemical" ] ] }, { "sid": 58, "sent": "The energetic penalties due to these mutations (\u0394\u0394G 0.8\u20130.9\u2009kcal\u2009mol\u22121) are consistent with the loss of each hydrogen bond with the rU5 base and support the relevance of the central nucleotide interactions observed in the U2AF651,2L structures.", "section": "RESULTS", "ner": [ [ 48, 51, "\u0394\u0394G", "evidence" ], [ 109, 122, "hydrogen bond", "bond_interaction" ], [ 132, 135, "rU5", "residue_name_number" ], [ 222, 232, "U2AF651,2L", "mutant" ], [ 233, 243, "structures", "evidence" ] ] }, { "sid": 59, "sent": "U2AF65 RRM extensions interact with the Py tract", "section": "RESULTS", "ner": [ [ 0, 6, "U2AF65", "protein" ], [ 7, 21, "RRM extensions", "structure_element" ], [ 40, 48, "Py tract", "chemical" ] ] }, { "sid": 60, "sent": "The N- and C-terminal extensions of the U2AF65 RRM1 and RRM2 directly contact the bound Py tract.", "section": "RESULTS", "ner": [ [ 4, 32, "N- and C-terminal extensions", "structure_element" ], [ 40, 46, "U2AF65", "protein" ], [ 47, 51, "RRM1", "structure_element" ], [ 56, 60, "RRM2", "structure_element" ], [ 82, 87, "bound", "protein_state" ], [ 88, 96, "Py tract", "chemical" ] ] }, { "sid": 61, "sent": "Rather than interacting with a new 5\u2032-terminal nucleotide as we had hypothesized, the C-terminal \u03b1-helix of RRM2 instead folds across one surface of rU3 in the third binding site (Fig. 3b).", "section": "RESULTS", "ner": [ [ 47, 57, "nucleotide", "chemical" ], [ 97, 104, "\u03b1-helix", "structure_element" ], [ 108, 112, "RRM2", "structure_element" ], [ 149, 152, "rU3", "residue_name_number" ], [ 160, 178, "third binding site", "site" ] ] }, { "sid": 62, "sent": "There, a salt bridge between the K340 side chain and nucleotide phosphate, as well as G338-base stacking and a hydrogen bond between the backbone amide of G338 and the rU3-O4, secure the RRM2 extension.", "section": "RESULTS", "ner": [ [ 9, 20, "salt bridge", "bond_interaction" ], [ 33, 37, "K340", "residue_name_number" ], [ 53, 63, "nucleotide", "chemical" ], [ 86, 90, "G338", "residue_name_number" ], [ 96, 104, "stacking", "bond_interaction" ], [ 111, 124, "hydrogen bond", "bond_interaction" ], [ 155, 159, "G338", "residue_name_number" ], [ 168, 171, "rU3", "residue_name_number" ], [ 187, 201, "RRM2 extension", "structure_element" ] ] }, { "sid": 63, "sent": "Indirectly, the additional contacts with the third nucleotide shift the rU2 nucleotide in the second binding site closer to the C-terminal \u03b2-strand of RRM2.", "section": "RESULTS", "ner": [ [ 45, 50, "third", "residue_number" ], [ 51, 61, "nucleotide", "chemical" ], [ 72, 75, "rU2", "residue_name_number" ], [ 76, 86, "nucleotide", "chemical" ], [ 94, 113, "second binding site", "site" ], [ 139, 147, "\u03b2-strand", "structure_element" ], [ 151, 155, "RRM2", "structure_element" ] ] }, { "sid": 64, "sent": "Consequently, the U2AF651,2L-bound rU2-O4 and -N3H form dual hydrogen bonds with the K329 backbone atoms (Fig. 3a), rather than a single hydrogen bond with the K329 side chain as in the prior dU2AF651,2 structure (Supplementary Fig. 3b).", "section": "RESULTS", "ner": [ [ 18, 34, "U2AF651,2L-bound", "protein_state" ], [ 35, 38, "rU2", "residue_name_number" ], [ 61, 75, "hydrogen bonds", "bond_interaction" ], [ 85, 89, "K329", "residue_name_number" ], [ 137, 150, "hydrogen bond", "bond_interaction" ], [ 160, 164, "K329", "residue_name_number" ], [ 192, 202, "dU2AF651,2", "mutant" ], [ 203, 212, "structure", "evidence" ] ] }, { "sid": 65, "sent": "At the N terminus, the \u03b1-helical extension of U2AF65 RRM1 positions the Q147 side chain to bridge the eighth and ninth nucleotides at the 3\u2032 terminus of the Py tract (Fig. 3f\u2013h).", "section": "RESULTS", "ner": [ [ 23, 42, "\u03b1-helical extension", "structure_element" ], [ 46, 52, "U2AF65", "protein" ], [ 53, 57, "RRM1", "structure_element" ], [ 72, 76, "Q147", "residue_name_number" ], [ 102, 108, "eighth", "residue_number" ], [ 113, 118, "ninth", "residue_number" ], [ 119, 130, "nucleotides", "chemical" ], [ 138, 149, "3\u2032 terminus", "site" ], [ 157, 165, "Py tract", "chemical" ] ] }, { "sid": 66, "sent": "The Q147 residue participates in hydrogen bonds with the -N3H of the eighth uracil and -O2 of the ninth pyrimidine.", "section": "RESULTS", "ner": [ [ 4, 8, "Q147", "residue_name_number" ], [ 33, 47, "hydrogen bonds", "bond_interaction" ], [ 69, 75, "eighth", "residue_number" ], [ 76, 82, "uracil", "residue_name" ], [ 98, 103, "ninth", "residue_number" ], [ 104, 114, "pyrimidine", "chemical" ] ] }, { "sid": 67, "sent": "The adjacent R146 guanidinium group donates hydrogen bonds to the 3\u2032-terminal ribose-O2\u2032 and O3\u2032 atoms, where it could form a salt bridge with a phospho-diester group in the context of a longer pre-mRNA.", "section": "RESULTS", "ner": [ [ 13, 17, "R146", "residue_name_number" ], [ 44, 58, "hydrogen bonds", "bond_interaction" ], [ 78, 84, "ribose", "chemical" ], [ 126, 137, "salt bridge", "bond_interaction" ], [ 194, 202, "pre-mRNA", "chemical" ] ] }, { "sid": 68, "sent": "Consistent with loss of a hydrogen bond with the ninth pyrimidine-O2 (\u0394\u0394G 1.0\u2009kcal\u2009mol\u22121), mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold (Fig. 3i; Supplementary Fig. 4c).", "section": "RESULTS", "ner": [ [ 26, 39, "hydrogen bond", "bond_interaction" ], [ 49, 54, "ninth", "residue_number" ], [ 55, 65, "pyrimidine", "chemical" ], [ 70, 73, "\u0394\u0394G", "evidence" ], [ 91, 99, "mutation", "experimental_method" ], [ 107, 111, "Q147", "residue_name_number" ], [ 118, 125, "alanine", "residue_name" ], [ 134, 153, "U2AF651,2L affinity", "evidence" ], [ 162, 166, "AdML", "gene" ], [ 167, 175, "Py tract", "chemical" ] ] }, { "sid": 69, "sent": "We compare U2AF65 interactions with uracil relative to cytosine pyrimidines at the ninth binding site in Fig. 3g,h and the Supplementary Discussion.", "section": "RESULTS", "ner": [ [ 3, 10, "compare", "experimental_method" ], [ 11, 17, "U2AF65", "protein" ], [ 36, 42, "uracil", "residue_name" ], [ 55, 63, "cytosine", "residue_name" ], [ 64, 75, "pyrimidines", "chemical" ], [ 83, 101, "ninth binding site", "site" ] ] }, { "sid": 70, "sent": "Versatile primary sequence of the U2AF65 inter-RRM linker", "section": "RESULTS", "ner": [ [ 34, 40, "U2AF65", "protein" ], [ 41, 57, "inter-RRM linker", "structure_element" ] ] }, { "sid": 71, "sent": "The U2AF651,2L structures reveal that the inter-RRM linker mediates an extensive interface with the second \u03b1-helix of RRM1, the \u03b22/\u03b23 strands of RRM2 and the N-terminal \u03b1-helical extension of RRM1.", "section": "RESULTS", "ner": [ [ 4, 14, "U2AF651,2L", "mutant" ], [ 15, 25, "structures", "evidence" ], [ 42, 58, "inter-RRM linker", "structure_element" ], [ 71, 90, "extensive interface", "site" ], [ 107, 114, "\u03b1-helix", "structure_element" ], [ 118, 122, "RRM1", "structure_element" ], [ 128, 141, "\u03b22/\u03b23 strands", "structure_element" ], [ 145, 149, "RRM2", "structure_element" ], [ 169, 188, "\u03b1-helical extension", "structure_element" ], [ 192, 196, "RRM1", "structure_element" ] ] }, { "sid": 72, "sent": "Altogether, the U2AF65 inter-RRM linker residues (R228\u2013K260) bury 2,800\u2009\u00c52 of surface area in the U2AF651,2L holo-protein, suggestive of a cognate interface compared with 1,900\u2009\u00c52 for a typical protein\u2013protein complex.", "section": "RESULTS", "ner": [ [ 16, 22, "U2AF65", "protein" ], [ 23, 39, "inter-RRM linker", "structure_element" ], [ 50, 59, "R228\u2013K260", "residue_range" ], [ 98, 108, "U2AF651,2L", "mutant" ], [ 109, 121, "holo-protein", "protein_state" ], [ 139, 156, "cognate interface", "site" ] ] }, { "sid": 73, "sent": "The path of the linker initiates at P229 following the core RRM1 \u03b2-strand, in a kink that is positioned by intra-molecular stacking among the consecutive R228, Y232 and P234 side chains (Fig. 4a, lower right).", "section": "RESULTS", "ner": [ [ 16, 22, "linker", "structure_element" ], [ 36, 40, "P229", "residue_name_number" ], [ 55, 59, "core", "protein_state" ], [ 60, 64, "RRM1", "structure_element" ], [ 65, 73, "\u03b2-strand", "structure_element" ], [ 80, 84, "kink", "structure_element" ], [ 107, 131, "intra-molecular stacking", "bond_interaction" ], [ 154, 158, "R228", "residue_name_number" ], [ 160, 164, "Y232", "residue_name_number" ], [ 169, 173, "P234", "residue_name_number" ] ] }, { "sid": 74, "sent": "A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal \u03b1-helical RRM1 extension and the core RRM1 \u03b12-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site.", "section": "RESULTS", "ner": [ [ 2, 13, "second kink", "structure_element" ], [ 17, 21, "P236", "residue_name_number" ], [ 62, 66, "L235", "residue_name_number" ], [ 71, 75, "M238", "residue_name_number" ], [ 106, 130, "\u03b1-helical RRM1 extension", "structure_element" ], [ 139, 143, "core", "protein_state" ], [ 144, 148, "RRM1", "structure_element" ], [ 149, 157, "\u03b12-helix", "structure_element" ], [ 189, 205, "inter-RRM linker", "structure_element" ], [ 218, 237, "RRM1/RRM2 interface", "site" ], [ 256, 272, "RNA-binding site", "site" ] ] }, { "sid": 75, "sent": "In the neighbouring apical region of the linker, the V244 and V246 side chains pack in a hydrophobic pocket between two \u03b1-helices of the core RRM1.", "section": "RESULTS", "ner": [ [ 41, 47, "linker", "structure_element" ], [ 53, 57, "V244", "residue_name_number" ], [ 62, 66, "V246", "residue_name_number" ], [ 89, 107, "hydrophobic pocket", "site" ], [ 120, 129, "\u03b1-helices", "structure_element" ], [ 137, 141, "core", "protein_state" ], [ 142, 146, "RRM1", "structure_element" ] ] }, { "sid": 76, "sent": "The adjacent V249 and V250 are notable for their respective interactions that connect RRM1 and RRM2 at this distal interface from the RNA-binding site (Fig. 4a, top).", "section": "RESULTS", "ner": [ [ 13, 17, "V249", "residue_name_number" ], [ 22, 26, "V250", "residue_name_number" ], [ 86, 90, "RRM1", "structure_element" ], [ 95, 99, "RRM2", "structure_element" ], [ 115, 124, "interface", "site" ], [ 134, 150, "RNA-binding site", "site" ] ] }, { "sid": 77, "sent": "A third kink stacks P247 and G248 with Y245 and re-orients the C-terminal region of the linker towards the RRM2 and bound RNA.", "section": "RESULTS", "ner": [ [ 2, 12, "third kink", "structure_element" ], [ 13, 19, "stacks", "bond_interaction" ], [ 20, 24, "P247", "residue_name_number" ], [ 29, 33, "G248", "residue_name_number" ], [ 39, 43, "Y245", "residue_name_number" ], [ 63, 80, "C-terminal region", "structure_element" ], [ 88, 94, "linker", "structure_element" ], [ 107, 111, "RRM2", "structure_element" ], [ 116, 121, "bound", "protein_state" ], [ 122, 125, "RNA", "chemical" ] ] }, { "sid": 78, "sent": "At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the \u03b2-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left).", "section": "RESULTS", "ner": [ [ 7, 10, "RNA", "chemical" ], [ 28, 32, "V254", "residue_name_number" ], [ 53, 58, "fifth", "residue_number" ], [ 59, 65, "uracil", "residue_name" ], [ 81, 101, "hydrophobic contacts", "bond_interaction" ], [ 133, 148, "\u03b2-sheet surface", "structure_element" ], [ 152, 156, "RRM2", "structure_element" ], [ 180, 184, "RNP1", "structure_element" ], [ 185, 189, "F304", "residue_name_number" ], [ 203, 209, "stacks", "bond_interaction" ], [ 219, 225, "fourth", "residue_number" ], [ 226, 232, "uracil", "residue_name" ] ] }, { "sid": 79, "sent": "Few direct contacts are made between the remaining residues of the linker and the U2AF65 RRM2; instead, the C-terminal conformation of the linker appears primarily RNA mediated (Fig. 3c,d).", "section": "RESULTS", "ner": [ [ 67, 73, "linker", "structure_element" ], [ 82, 88, "U2AF65", "protein" ], [ 89, 93, "RRM2", "structure_element" ], [ 139, 145, "linker", "structure_element" ], [ 164, 167, "RNA", "chemical" ] ] }, { "sid": 80, "sent": "We investigated whether the observed contacts between the RRMs and linker were critical for RNA binding by structure-guided mutagenesis (Fig. 4b).", "section": "RESULTS", "ner": [ [ 58, 62, "RRMs", "structure_element" ], [ 67, 73, "linker", "structure_element" ], [ 107, 135, "structure-guided mutagenesis", "experimental_method" ] ] }, { "sid": 81, "sent": "We titrated these mutant U2AF651,2L proteins into fluorescein-labelled AdML Py-tract RNA and fit the fluorescence anisotropy changes to obtain the apparent equilibrium affinities (Supplementary Fig. 4d\u2013h).", "section": "RESULTS", "ner": [ [ 3, 11, "titrated", "experimental_method" ], [ 18, 24, "mutant", "protein_state" ], [ 25, 35, "U2AF651,2L", "mutant" ], [ 50, 61, "fluorescein", "chemical" ], [ 71, 75, "AdML", "gene" ], [ 76, 88, "Py-tract RNA", "chemical" ], [ 101, 132, "fluorescence anisotropy changes", "evidence" ], [ 156, 178, "equilibrium affinities", "evidence" ] ] }, { "sid": 82, "sent": "We introduced glycine substitutions to maximally reduce the buried surface area without directly interfering with its hydrogen bonds between backbone atoms and the base.", "section": "RESULTS", "ner": [ [ 14, 21, "glycine", "residue_name" ], [ 22, 35, "substitutions", "experimental_method" ], [ 118, 132, "hydrogen bonds", "bond_interaction" ] ] }, { "sid": 83, "sent": "First, we replaced V249 and V250 at the RRM1/RRM2 interface and V254 at the bound RNA site with glycine (3Gly).", "section": "RESULTS", "ner": [ [ 10, 18, "replaced", "experimental_method" ], [ 19, 23, "V249", "residue_name_number" ], [ 28, 32, "V250", "residue_name_number" ], [ 40, 59, "RRM1/RRM2 interface", "site" ], [ 64, 68, "V254", "residue_name_number" ], [ 76, 81, "bound", "protein_state" ], [ 82, 85, "RNA", "chemical" ], [ 96, 103, "glycine", "residue_name" ], [ 105, 109, "3Gly", "mutant" ] ] }, { "sid": 84, "sent": "However, the resulting decrease in the AdML RNA affinity of the U2AF651,2L-3Gly mutant relative to wild-type protein was not significant (Fig. 4b).", "section": "RESULTS", "ner": [ [ 39, 43, "AdML", "gene" ], [ 44, 56, "RNA affinity", "evidence" ], [ 64, 79, "U2AF651,2L-3Gly", "mutant" ], [ 80, 86, "mutant", "protein_state" ], [ 99, 108, "wild-type", "protein_state" ], [ 109, 116, "protein", "protein" ] ] }, { "sid": 85, "sent": "In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein.", "section": "RESULTS", "ner": [ [ 16, 24, "replaced", "experimental_method" ], [ 30, 45, "linker residues", "structure_element" ], [ 47, 51, "S251", "residue_name_number" ], [ 53, 57, "T252", "residue_name_number" ], [ 59, 63, "V253", "residue_name_number" ], [ 65, 69, "V254", "residue_name_number" ], [ 74, 78, "P255", "residue_name_number" ], [ 87, 116, "fifth nucleotide-binding site", "site" ], [ 122, 130, "glycines", "residue_name" ], [ 132, 136, "5Gly", "mutant" ], [ 162, 174, "RNA affinity", "evidence" ], [ 182, 197, "U2AF651,2L-5Gly", "mutant" ], [ 198, 204, "mutant", "protein_state" ], [ 250, 259, "wild-type", "protein_state" ], [ 260, 267, "protein", "protein" ] ] }, { "sid": 86, "sent": "A more conservative substitution of these five residues (251\u2013255) with an unrelated sequence capable of backbone-mediated hydrogen bonds (STVVP>NLALA) confirmed the subtle impact of this versatile inter-RRM sequence on affinity for the AdML Py tract.", "section": "RESULTS", "ner": [ [ 7, 32, "conservative substitution", "experimental_method" ], [ 57, 64, "251\u2013255", "residue_range" ], [ 122, 136, "hydrogen bonds", "bond_interaction" ], [ 138, 149, "STVVP>NLALA", "mutant" ], [ 197, 215, "inter-RRM sequence", "structure_element" ], [ 219, 227, "affinity", "evidence" ], [ 236, 240, "AdML", "gene" ], [ 241, 249, "Py tract", "chemical" ] ] }, { "sid": 87, "sent": "Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly).", "section": "RESULTS", "ner": [ [ 81, 87, "linker", "structure_element" ], [ 88, 91, "RRM", "structure_element" ], [ 105, 116, "substituted", "experimental_method" ], [ 117, 124, "glycine", "residue_name" ], [ 180, 196, "inter-RRM linker", "structure_element" ], [ 208, 212, "M144", "residue_name_number" ], [ 214, 218, "L235", "residue_name_number" ], [ 220, 224, "M238", "residue_name_number" ], [ 226, 230, "V244", "residue_name_number" ], [ 232, 236, "V246", "residue_name_number" ], [ 238, 242, "V249", "residue_name_number" ], [ 244, 248, "V250", "residue_name_number" ], [ 250, 254, "S251", "residue_name_number" ], [ 256, 260, "T252", "residue_name_number" ], [ 262, 266, "V253", "residue_name_number" ], [ 268, 272, "V254", "residue_name_number" ], [ 274, 278, "P255", "residue_name_number" ], [ 287, 292, "12Gly", "mutant" ] ] }, { "sid": 88, "sent": "Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein (Fig. 4b), which is less than the penalty of the V254P mutation that disrupts the rU5 hydrogen bond (Fig. 3d,i).", "section": "RESULTS", "ner": [ [ 8, 31, "12 concurrent mutations", "experimental_method" ], [ 37, 41, "AdML", "gene" ], [ 42, 54, "RNA affinity", "evidence" ], [ 62, 78, "U2AF651,2L-12Gly", "mutant" ], [ 79, 86, "variant", "protein_state" ], [ 134, 144, "unmodified", "protein_state" ], [ 145, 152, "protein", "protein" ], [ 202, 207, "V254P", "mutant" ], [ 235, 238, "rU5", "residue_name_number" ], [ 239, 252, "hydrogen bond", "bond_interaction" ] ] }, { "sid": 89, "sent": "To test the interplay of the U2AF65 inter-RRM linker with its N- and C-terminal RRM extensions, we constructed an internal linker deletion of 20-residues within the extended RNA-binding domain (dU2AF651,2L).", "section": "RESULTS", "ner": [ [ 29, 35, "U2AF65", "protein" ], [ 36, 52, "inter-RRM linker", "structure_element" ], [ 80, 94, "RRM extensions", "structure_element" ], [ 99, 110, "constructed", "experimental_method" ], [ 123, 138, "linker deletion", "experimental_method" ], [ 142, 153, "20-residues", "residue_range" ], [ 165, 173, "extended", "protein_state" ], [ 174, 192, "RNA-binding domain", "structure_element" ], [ 194, 205, "dU2AF651,2L", "mutant" ] ] }, { "sid": 90, "sent": "We found that the affinity of dU2AF651,2L for the AdML RNA was significantly reduced relative to U2AF651,2L (four-fold, Figs 1b and 4b; Supplementary Fig. 4i).", "section": "RESULTS", "ner": [ [ 18, 26, "affinity", "evidence" ], [ 30, 41, "dU2AF651,2L", "mutant" ], [ 50, 54, "AdML", "gene" ], [ 55, 58, "RNA", "chemical" ], [ 97, 107, "U2AF651,2L", "mutant" ] ] }, { "sid": 91, "sent": "Yet, it is well known that the linker deletion in the context of the minimal RRM1\u2013RRM2 boundaries has no detectable effect on the RNA affinities of dU2AF651,2 compared with U2AF651,2 (refs; Figs 1b and 4b; Supplementary Fig. 4j).", "section": "RESULTS", "ner": [ [ 31, 46, "linker deletion", "experimental_method" ], [ 69, 76, "minimal", "protein_state" ], [ 77, 81, "RRM1", "structure_element" ], [ 82, 86, "RRM2", "structure_element" ], [ 130, 144, "RNA affinities", "evidence" ], [ 148, 158, "dU2AF651,2", "mutant" ], [ 173, 182, "U2AF651,2", "mutant" ] ] }, { "sid": 92, "sent": "The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24\u2009\u00c5 distance between U2AF651,2L R227-C\u03b1\u2013H259-C\u03b1 atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs.", "section": "RESULTS", "ner": [ [ 4, 14, "U2AF651,2L", "mutant" ], [ 15, 25, "structures", "evidence" ], [ 42, 63, "extended conformation", "protein_state" ], [ 71, 80, "truncated", "protein_state" ], [ 81, 91, "dU2AF651,2", "mutant" ], [ 92, 108, "inter-RRM linker", "structure_element" ], [ 138, 148, "U2AF651,2L", "mutant" ], [ 149, 153, "RRM1", "structure_element" ], [ 186, 190, "RRM2", "structure_element" ], [ 214, 224, "U2AF651,2L", "mutant" ], [ 225, 229, "R227", "residue_name_number" ], [ 233, 237, "H259", "residue_name_number" ], [ 279, 293, "RNA affinities", "evidence" ], [ 297, 307, "dU2AF651,2", "mutant" ], [ 312, 321, "U2AF651,2", "mutant" ], [ 322, 326, "dual", "protein_state" ], [ 327, 331, "RRMs", "structure_element" ], [ 350, 360, "individual", "protein_state" ], [ 361, 367, "U2AF65", "protein" ], [ 368, 372, "RRMs", "structure_element" ] ] }, { "sid": 93, "sent": "However, stretching of the truncated dU2AF651,2L linker to connect the RRM termini is expected to disrupt its nucleotide interactions.", "section": "RESULTS", "ner": [ [ 27, 36, "truncated", "protein_state" ], [ 37, 48, "dU2AF651,2L", "mutant" ], [ 49, 55, "linker", "structure_element" ], [ 71, 82, "RRM termini", "structure_element" ] ] }, { "sid": 94, "sent": "Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L.", "section": "RESULTS", "ner": [ [ 10, 18, "deletion", "experimental_method" ], [ 37, 51, "RRM1 extension", "structure_element" ], [ 59, 68, "shortened", "protein_state" ], [ 132, 138, "linker", "structure_element" ], [ 144, 155, "kinked turn", "structure_element" ], [ 166, 170, "P229", "residue_name_number" ], [ 208, 222, "RNA affinities", "evidence" ], [ 226, 237, "dU2AF651,2L", "mutant" ], [ 239, 249, "dU2AF651,2", "mutant" ], [ 254, 263, "U2AF651,2", "mutant" ], [ 278, 288, "U2AF651,2L", "mutant" ] ] }, { "sid": 95, "sent": "To further test cooperation among the U2AF65 RRM extensions and inter-RRM linker for RNA recognition, we tested the impact of a triple Q147A/V254P/R227A mutation (U2AF651,2L-3Mut) for RNA binding (Fig. 4b; Supplementary Fig. 4d).", "section": "RESULTS", "ner": [ [ 38, 44, "U2AF65", "protein" ], [ 45, 59, "RRM extensions", "structure_element" ], [ 64, 80, "inter-RRM linker", "structure_element" ], [ 135, 140, "Q147A", "mutant" ], [ 141, 146, "V254P", "mutant" ], [ 147, 152, "R227A", "mutant" ], [ 153, 161, "mutation", "experimental_method" ], [ 163, 178, "U2AF651,2L-3Mut", "mutant" ] ] }, { "sid": 96, "sent": "Notably, the Q147A/V254P/R227A mutation reduced the RNA affinity of the U2AF651,2L-3Mut protein by 30-fold more than would be expected based on simple addition of the \u0394\u0394G's for the single mutations.", "section": "RESULTS", "ner": [ [ 13, 18, "Q147A", "mutant" ], [ 19, 24, "V254P", "mutant" ], [ 25, 30, "R227A", "mutant" ], [ 31, 39, "mutation", "experimental_method" ], [ 52, 64, "RNA affinity", "evidence" ], [ 72, 87, "U2AF651,2L-3Mut", "mutant" ], [ 167, 170, "\u0394\u0394G", "evidence" ] ] }, { "sid": 97, "sent": "This difference indicates that the linearly distant regions of the U2AF65 primary sequence, including Q147 in the N-terminal RRM1 extension and R227/V254 in the N-/C-terminal linker regions at the fifth nucleotide site, cooperatively recognize the Py tract.", "section": "RESULTS", "ner": [ [ 35, 51, "linearly distant", "protein_state" ], [ 52, 59, "regions", "structure_element" ], [ 67, 73, "U2AF65", "protein" ], [ 102, 106, "Q147", "residue_name_number" ], [ 125, 139, "RRM1 extension", "structure_element" ], [ 144, 148, "R227", "residue_name_number" ], [ 149, 153, "V254", "residue_name_number" ], [ 175, 189, "linker regions", "structure_element" ], [ 197, 218, "fifth nucleotide site", "site" ], [ 248, 256, "Py tract", "chemical" ] ] }, { "sid": 98, "sent": "Altogether, we conclude that the conformation of the U2AF65 inter-RRM linker is key for recognizing RNA and is positioned by the RRM extension but otherwise relatively independent of the side chain composition.", "section": "RESULTS", "ner": [ [ 53, 59, "U2AF65", "protein" ], [ 60, 76, "inter-RRM linker", "structure_element" ], [ 100, 103, "RNA", "chemical" ], [ 129, 142, "RRM extension", "structure_element" ] ] }, { "sid": 99, "sent": "The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs.", "section": "RESULTS", "ner": [ [ 32, 37, "Q147A", "mutant" ], [ 38, 43, "V254P", "mutant" ], [ 44, 49, "R227A", "mutant" ], [ 50, 65, "triple mutation", "experimental_method" ], [ 127, 133, "U2AF65", "protein" ], [ 134, 149, "linker deletion", "experimental_method" ], [ 215, 221, "U2AF65", "protein" ], [ 222, 228, "linker", "structure_element" ], [ 237, 265, "N- and C-terminal extensions", "structure_element" ], [ 279, 283, "core", "protein_state" ], [ 284, 288, "RRMs", "structure_element" ] ] }, { "sid": 100, "sent": "Importance of U2AF65\u2013RNA contacts for pre-mRNA splicing", "section": "RESULTS", "ner": [ [ 14, 24, "U2AF65\u2013RNA", "complex_assembly" ], [ 38, 46, "pre-mRNA", "chemical" ] ] }, { "sid": 101, "sent": "We proceeded to test the importance of new U2AF65\u2013Py-tract interactions for splicing of a model pre-mRNA substrate in a human cell line (Fig. 5; Supplementary Fig. 5).", "section": "RESULTS", "ner": [ [ 43, 58, "U2AF65\u2013Py-tract", "complex_assembly" ], [ 96, 104, "pre-mRNA", "chemical" ], [ 120, 125, "human", "species" ] ] }, { "sid": 102, "sent": "As a representative splicing substrate, we utilized a well-characterized minigene splicing reporter (called pyPY) comprising a weak (that is, degenerate, py) and strong (that is, U-rich, PY) polypyrimidine tracts preceding two alternative splice sites (Fig. 5a).", "section": "RESULTS", "ner": [ [ 73, 99, "minigene splicing reporter", "chemical" ], [ 108, 112, "pyPY", "chemical" ], [ 154, 156, "py", "chemical" ], [ 179, 185, "U-rich", "structure_element" ], [ 187, 189, "PY", "chemical" ], [ 191, 212, "polypyrimidine tracts", "chemical" ], [ 239, 251, "splice sites", "site" ] ] }, { "sid": 103, "sent": "When transfected into HEK293T cells containing only endogenous U2AF65, the PY splice site is used and the remaining transcript remains unspliced.", "section": "RESULTS", "ner": [ [ 5, 16, "transfected", "experimental_method" ], [ 52, 62, "endogenous", "protein_state" ], [ 63, 69, "U2AF65", "protein" ], [ 75, 89, "PY splice site", "site" ] ] }, { "sid": 104, "sent": "When co-transfected with an expression plasmid for wild-type U2AF65, use of the py splice site significantly increases (by more than five-fold) and as documented converts a fraction of the unspliced to spliced transcript.", "section": "RESULTS", "ner": [ [ 5, 19, "co-transfected", "experimental_method" ], [ 28, 46, "expression plasmid", "experimental_method" ], [ 51, 60, "wild-type", "protein_state" ], [ 61, 67, "U2AF65", "protein" ], [ 80, 94, "py splice site", "site" ] ] }, { "sid": 105, "sent": "The strong PY splice site is insensitive to added U2AF65, suggesting that endogenous U2AF65 levels are sufficient to saturate this site (Supplementary Fig. 5b).", "section": "RESULTS", "ner": [ [ 11, 25, "PY splice site", "site" ], [ 50, 56, "U2AF65", "protein" ], [ 74, 84, "endogenous", "protein_state" ], [ 85, 91, "U2AF65", "protein" ] ] }, { "sid": 106, "sent": "We introduced the triple mutation (V254P/R227A/Q147A) that significantly reduced U2AF651,2L association with the Py tract (Fig. 4b) in the context of full-length U2AF65 (U2AF65-3Mut).", "section": "RESULTS", "ner": [ [ 18, 33, "triple mutation", "experimental_method" ], [ 35, 40, "V254P", "mutant" ], [ 41, 46, "R227A", "mutant" ], [ 47, 52, "Q147A", "mutant" ], [ 81, 91, "U2AF651,2L", "mutant" ], [ 113, 121, "Py tract", "chemical" ], [ 150, 161, "full-length", "protein_state" ], [ 162, 168, "U2AF65", "protein" ], [ 170, 181, "U2AF65-3Mut", "mutant" ] ] }, { "sid": 107, "sent": "Co-transfection of the U2AF65-3Mut with the pyPY splicing substrate significantly reduced splicing of the weak \u2018py' splice site relative to wild-type U2AF65 (Fig. 5b,c).", "section": "RESULTS", "ner": [ [ 0, 15, "Co-transfection", "experimental_method" ], [ 23, 34, "U2AF65-3Mut", "mutant" ], [ 44, 48, "pyPY", "chemical" ], [ 111, 127, "\u2018py' splice site", "site" ], [ 140, 149, "wild-type", "protein_state" ], [ 150, 156, "U2AF65", "protein" ] ] }, { "sid": 108, "sent": "We conclude that the Py-tract interactions with these residues of the U2AF65 inter-RRM linker and RRM extensions are important for splicing as well as for binding a representative of the major U2-class of splice sites.", "section": "RESULTS", "ner": [ [ 21, 29, "Py-tract", "chemical" ], [ 70, 76, "U2AF65", "protein" ], [ 77, 93, "inter-RRM linker", "structure_element" ], [ 98, 112, "RRM extensions", "structure_element" ], [ 187, 217, "major U2-class of splice sites", "structure_element" ] ] }, { "sid": 109, "sent": "Sparse inter-RRM contacts underlie apo-U2AF65 dynamics", "section": "RESULTS", "ner": [ [ 7, 16, "inter-RRM", "structure_element" ], [ 35, 38, "apo", "protein_state" ], [ 39, 45, "U2AF65", "protein" ] ] }, { "sid": 110, "sent": "The direct interface between U2AF651,2L RRM1 and RRM2 is minor, burying 265\u2009\u00c52 of solvent accessible surface area compared with 570\u2009\u00c52 on average for a crystal packing interface.", "section": "RESULTS", "ner": [ [ 11, 20, "interface", "site" ], [ 29, 39, "U2AF651,2L", "mutant" ], [ 40, 44, "RRM1", "structure_element" ], [ 49, 53, "RRM2", "structure_element" ] ] }, { "sid": 111, "sent": "A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c).", "section": "RESULTS", "ner": [ [ 13, 22, "inter-RRM", "structure_element" ], [ 23, 37, "hydrogen bonds", "bond_interaction" ], [ 78, 82, "RRM1", "structure_element" ], [ 83, 87, "N155", "residue_name_number" ], [ 92, 96, "RRM2", "structure_element" ], [ 97, 101, "K292", "residue_name_number" ], [ 103, 107, "RRM1", "structure_element" ], [ 108, 112, "N155", "residue_name_number" ], [ 117, 121, "RRM2", "structure_element" ], [ 122, 126, "D272", "residue_name_number" ], [ 160, 164, "RRM1", "structure_element" ], [ 165, 169, "G221", "residue_name_number" ], [ 174, 178, "RRM2", "structure_element" ], [ 179, 183, "D273", "residue_name_number" ] ] }, { "sid": 112, "sent": "This minor U2AF65 RRM1/RRM2 interface, coupled with the versatile sequence of the inter-RRM linker, highlighted the potential role for inter-RRM conformational dynamics in U2AF65-splice site recognition.", "section": "RESULTS", "ner": [ [ 11, 17, "U2AF65", "protein" ], [ 18, 37, "RRM1/RRM2 interface", "site" ], [ 82, 98, "inter-RRM linker", "structure_element" ], [ 135, 144, "inter-RRM", "structure_element" ], [ 172, 178, "U2AF65", "protein" ] ] }, { "sid": 113, "sent": "Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or \u2018closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor \u2018open' conformation resembling the RNA-bound inter-RRM arrangement.", "section": "RESULTS", "ner": [ [ 0, 34, "Paramagnetic resonance enhancement", "experimental_method" ], [ 36, 39, "PRE", "experimental_method" ], [ 93, 105, "back-to-back", "protein_state" ], [ 111, 117, "closed", "protein_state" ], [ 139, 142, "apo", "protein_state" ], [ 143, 152, "U2AF651,2", "mutant" ], [ 153, 157, "RRM1", "structure_element" ], [ 162, 166, "RRM2", "structure_element" ], [ 196, 200, "open", "protein_state" ], [ 230, 239, "RNA-bound", "protein_state" ], [ 240, 249, "inter-RRM", "structure_element" ] ] }, { "sid": 114, "sent": "Yet, small-angle X-ray scattering (SAXS) data indicated that both the minimal U2AF651,2 and longer constructs comprise a highly diverse continuum of conformations in the absence of RNA that includes the \u2018closed' and \u2018open' conformations.", "section": "RESULTS", "ner": [ [ 5, 33, "small-angle X-ray scattering", "experimental_method" ], [ 35, 39, "SAXS", "experimental_method" ], [ 70, 77, "minimal", "protein_state" ], [ 78, 87, "U2AF651,2", "mutant" ], [ 121, 162, "highly diverse continuum of conformations", "protein_state" ], [ 170, 180, "absence of", "protein_state" ], [ 181, 184, "RNA", "chemical" ], [ 204, 210, "closed", "protein_state" ], [ 217, 221, "open", "protein_state" ] ] }, { "sid": 115, "sent": "To complement the static portraits of U2AF651,2L structure that we had determined by X-ray crystallography, we used smFRET to characterize the probability distribution functions and time dependence of U2AF65 inter-RRM conformational dynamics in solution.", "section": "RESULTS", "ner": [ [ 38, 48, "U2AF651,2L", "mutant" ], [ 49, 58, "structure", "evidence" ], [ 85, 106, "X-ray crystallography", "experimental_method" ], [ 116, 122, "smFRET", "experimental_method" ], [ 143, 177, "probability distribution functions", "evidence" ], [ 201, 207, "U2AF65", "protein" ], [ 208, 217, "inter-RRM", "structure_element" ] ] }, { "sid": 116, "sent": "The inter-RRM dynamics of U2AF65 were followed using FRET between fluorophores attached to RRM1 and RRM2 (Fig. 6a,b, Methods).", "section": "RESULTS", "ner": [ [ 4, 13, "inter-RRM", "structure_element" ], [ 26, 32, "U2AF65", "protein" ], [ 53, 57, "FRET", "experimental_method" ], [ 66, 78, "fluorophores", "chemical" ], [ 91, 95, "RRM1", "structure_element" ], [ 100, 104, "RRM2", "structure_element" ] ] }, { "sid": 117, "sent": "The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the \u2018closed' model of apo-U2AF651,2.", "section": "RESULTS", "ner": [ [ 24, 32, "cysteine", "residue_name" ], [ 33, 42, "mutations", "experimental_method" ], [ 47, 58, "fluorophore", "chemical" ], [ 71, 76, "A181C", "mutant" ], [ 80, 84, "RRM1", "structure_element" ], [ 89, 94, "Q324C", "mutant" ], [ 98, 102, "RRM2", "structure_element" ], [ 143, 153, "U2AF651,2L", "mutant" ], [ 154, 164, "structures", "evidence" ], [ 174, 180, "closed", "protein_state" ], [ 191, 194, "apo", "protein_state" ], [ 195, 204, "U2AF651,2", "mutant" ] ] }, { "sid": 118, "sent": "Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50\u2009\u00c5 for U2AF651,2L\u2013Py tract and 30\u2009\u00c5 for the closed apo-model) that are expected to be near the F\u00f6rster radius (Ro) for the Cy3/Cy5 pair (56\u2009\u00c5), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the \u2018closed' apo-model as opposed to the \u2018open' RNA-bound structures (by \u223c30%).", "section": "RESULTS", "ner": [ [ 107, 114, "RRM/RNA", "complex_assembly" ], [ 118, 138, "inter-RRM interfaces", "site" ], [ 175, 194, "U2AF651,2L\u2013Py tract", "complex_assembly" ], [ 212, 218, "closed", "protein_state" ], [ 219, 222, "apo", "protein_state" ], [ 263, 282, "F\u00f6rster radius (Ro)", "experimental_method" ], [ 291, 294, "Cy3", "chemical" ], [ 295, 298, "Cy5", "chemical" ], [ 405, 422, "FRET efficiencies", "evidence" ], [ 480, 486, "closed", "protein_state" ], [ 488, 491, "apo", "protein_state" ], [ 517, 521, "open", "protein_state" ], [ 523, 532, "RNA-bound", "protein_state" ], [ 533, 543, "structures", "evidence" ] ] }, { "sid": 119, "sent": "The FRET efficiencies of either of these structurally characterized conformations also are expected to be significantly greater than elongated U2AF65 conformations that lack inter-RRM contacts.", "section": "RESULTS", "ner": [ [ 4, 21, "FRET efficiencies", "evidence" ], [ 133, 142, "elongated", "protein_state" ], [ 143, 149, "U2AF65", "protein" ], [ 169, 173, "lack", "protein_state" ], [ 180, 183, "RRM", "structure_element" ] ] }, { "sid": 120, "sent": "Double-cysteine variant of U2AF651,2 was modified with equimolar amount of Cy3 and Cy5.", "section": "RESULTS", "ner": [ [ 7, 15, "cysteine", "residue_name" ], [ 16, 23, "variant", "protein_state" ], [ 27, 36, "U2AF651,2", "mutant" ], [ 41, 49, "modified", "experimental_method" ], [ 75, 78, "Cy3", "chemical" ], [ 83, 86, "Cy5", "chemical" ] ] }, { "sid": 121, "sent": "Only traces that showed single photobleaching events for both donor and acceptor dyes and anti-correlated changes in acceptor and donor fluorescence were included in smFRET data analysis.", "section": "RESULTS", "ner": [ [ 5, 11, "traces", "evidence" ], [ 166, 172, "smFRET", "experimental_method" ] ] }, { "sid": 122, "sent": "We first characterized the conformational dynamics spectrum of U2AF65 in the absence of RNA (Fig. 6c,d; Supplementary Fig. 7a,b).", "section": "RESULTS", "ner": [ [ 63, 69, "U2AF65", "protein" ], [ 77, 87, "absence of", "protein_state" ], [ 88, 91, "RNA", "chemical" ] ] }, { "sid": 123, "sent": "The double-labelled U2AF651,2LFRET(Cy3/Cy5) protein was tethered to a slide via biotin-NTA/Ni+2 resin.", "section": "RESULTS", "ner": [ [ 20, 34, "U2AF651,2LFRET", "mutant" ], [ 35, 38, "Cy3", "chemical" ], [ 39, 42, "Cy5", "chemical" ], [ 56, 64, "tethered", "protein_state" ], [ 80, 101, "biotin-NTA/Ni+2 resin", "chemical" ] ] }, { "sid": 124, "sent": "Virtually no fluorescent molecules were detected in the absence of biotin-NTA/Ni+2, which demonstrates the absence of detectable non-specific binding of U2AF651,2LFRET to the slide.", "section": "RESULTS", "ner": [ [ 56, 66, "absence of", "protein_state" ], [ 67, 82, "biotin-NTA/Ni+2", "chemical" ], [ 107, 117, "absence of", "protein_state" ], [ 153, 167, "U2AF651,2LFRET", "mutant" ] ] }, { "sid": 125, "sent": "The FRET distribution histogram built from more than a thousand traces of U2AF651,2LFRET(Cy3/Cy5) in the absence of ligand showed an extremely broad distribution centred at a FRET efficiency of \u223c0.4 (Fig. 6d).", "section": "RESULTS", "ner": [ [ 4, 31, "FRET distribution histogram", "evidence" ], [ 64, 70, "traces", "evidence" ], [ 74, 88, "U2AF651,2LFRET", "mutant" ], [ 89, 92, "Cy3", "chemical" ], [ 93, 96, "Cy5", "chemical" ], [ 105, 115, "absence of", "protein_state" ], [ 116, 122, "ligand", "chemical" ], [ 175, 190, "FRET efficiency", "evidence" ] ] }, { "sid": 126, "sent": "Approximately 40% of the smFRET traces showed apparent transitions between multiple FRET values (for example, Fig. 6c).", "section": "RESULTS", "ner": [ [ 25, 31, "smFRET", "experimental_method" ], [ 32, 38, "traces", "evidence" ], [ 84, 95, "FRET values", "evidence" ] ] }, { "sid": 127, "sent": "Despite the large width of the FRET-distribution histogram, the majority (80%) of traces that showed fluctuations sampled only two distinct FRET states (for example, Supplementary Fig. 7a).", "section": "RESULTS", "ner": [ [ 31, 58, "FRET-distribution histogram", "evidence" ], [ 82, 88, "traces", "evidence" ], [ 140, 151, "FRET states", "evidence" ] ] }, { "sid": 128, "sent": "Approximately 70% of observed fluctuations were interchanges between the \u223c0.65 and \u223c0.45 FRET values (Supplementary Fig. 7b).", "section": "RESULTS", "ner": [ [ 89, 100, "FRET values", "evidence" ] ] }, { "sid": 129, "sent": "We cannot exclude a possibility that tethering of U2AF651,2LFRET(Cy3/Cy5) to the microscope slide introduces structural heterogeneity into the protein and, thus, contributes to the breadth of the FRET distribution histogram.", "section": "RESULTS", "ner": [ [ 50, 64, "U2AF651,2LFRET", "mutant" ], [ 65, 68, "Cy3", "chemical" ], [ 69, 72, "Cy5", "chemical" ], [ 196, 223, "FRET distribution histogram", "evidence" ] ] }, { "sid": 130, "sent": "However, the presence of repetitive fluctuations between particular FRET values supports the hypothesis that RNA-free U2AF65 samples several distinct conformations.", "section": "RESULTS", "ner": [ [ 68, 79, "FRET values", "evidence" ], [ 109, 117, "RNA-free", "protein_state" ], [ 118, 124, "U2AF65", "protein" ] ] }, { "sid": 131, "sent": "This result is consistent with the broad ensembles of extended solution conformations that best fit the SAXS data collected for U2AF651,2 as well as for a longer construct (residues 136\u2013347).", "section": "RESULTS", "ner": [ [ 54, 62, "extended", "protein_state" ], [ 104, 108, "SAXS", "experimental_method" ], [ 128, 137, "U2AF651,2", "mutant" ], [ 182, 189, "136\u2013347", "residue_range" ] ] }, { "sid": 132, "sent": "We conclude that weak contacts between the U2AF65 RRM1 and RRM2 permit dissociation of these RRMs in the absence of RNA.", "section": "RESULTS", "ner": [ [ 43, 49, "U2AF65", "protein" ], [ 50, 54, "RRM1", "structure_element" ], [ 59, 63, "RRM2", "structure_element" ], [ 93, 97, "RRMs", "structure_element" ], [ 105, 115, "absence of", "protein_state" ], [ 116, 119, "RNA", "chemical" ] ] }, { "sid": 133, "sent": "U2AF65 conformational selection and induced fit by bound RNA", "section": "RESULTS", "ner": [ [ 0, 6, "U2AF65", "protein" ], [ 51, 56, "bound", "protein_state" ], [ 57, 60, "RNA", "chemical" ] ] }, { "sid": 134, "sent": "We next used smFRET to probe the conformational selection of distinct inter-RRM arrangements following association of U2AF65 with the AdML Py-tract prototype.", "section": "RESULTS", "ner": [ [ 13, 19, "smFRET", "experimental_method" ], [ 70, 79, "inter-RRM", "structure_element" ], [ 118, 124, "U2AF65", "protein" ], [ 134, 138, "AdML", "gene" ], [ 139, 147, "Py-tract", "chemical" ] ] }, { "sid": 135, "sent": "Addition of the AdML RNA to tethered U2AF651,2LFRET(Cy3/Cy5) selectively increases a fraction of molecules showing an \u223c0.45 apparent FRET efficiency, suggesting that RNA binding stabilizes a single conformation, which corresponds to the 0.45 FRET state (Fig. 6e,f).", "section": "RESULTS", "ner": [ [ 16, 20, "AdML", "gene" ], [ 21, 24, "RNA", "chemical" ], [ 28, 36, "tethered", "protein_state" ], [ 37, 51, "U2AF651,2LFRET", "mutant" ], [ 52, 55, "Cy3", "chemical" ], [ 56, 59, "Cy5", "chemical" ], [ 133, 148, "FRET efficiency", "evidence" ], [ 242, 252, "FRET state", "evidence" ] ] }, { "sid": 136, "sent": "To assess the possible contributions of RNA-free conformations of U2AF65 and/or structural heterogeneity introduced by tethering of U2AF651,2LFRET(Cy3/Cy5) to the slide to the observed distribution of FRET values, we reversed the immobilization scheme.", "section": "RESULTS", "ner": [ [ 40, 48, "RNA-free", "protein_state" ], [ 66, 72, "U2AF65", "protein" ], [ 119, 128, "tethering", "experimental_method" ], [ 132, 146, "U2AF651,2LFRET", "mutant" ], [ 147, 150, "Cy3", "chemical" ], [ 151, 154, "Cy5", "chemical" ], [ 185, 212, "distribution of FRET values", "evidence" ], [ 217, 251, "reversed the immobilization scheme", "experimental_method" ] ] }, { "sid": 137, "sent": "We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin (Fig. 6g,h; Supplementary Fig. 7c\u2013g).", "section": "RESULTS", "ner": [ [ 3, 11, "tethered", "protein_state" ], [ 16, 20, "AdML", "gene" ], [ 21, 24, "RNA", "chemical" ], [ 44, 76, "biotinylated oligonucleotide DNA", "chemical" ], [ 88, 93, "added", "experimental_method" ], [ 94, 108, "U2AF651,2LFRET", "mutant" ], [ 109, 112, "Cy3", "chemical" ], [ 113, 116, "Cy5", "chemical" ], [ 125, 135, "absence of", "protein_state" ], [ 136, 152, "biotin-NTA resin", "chemical" ] ] }, { "sid": 138, "sent": "A 0.45 FRET value was again predominant, indicating a similar RNA-bound conformation and structural dynamics for the untethered and tethered U2AF651,2LFRET(Cy3/Cy5).", "section": "RESULTS", "ner": [ [ 7, 17, "FRET value", "evidence" ], [ 62, 71, "RNA-bound", "protein_state" ], [ 117, 127, "untethered", "protein_state" ], [ 132, 140, "tethered", "protein_state" ], [ 141, 155, "U2AF651,2LFRET", "mutant" ], [ 156, 159, "Cy3", "chemical" ], [ 160, 163, "Cy5", "chemical" ] ] }, { "sid": 139, "sent": "We examined the effect on U2AF651,2L conformations of purine interruptions that often occur in relatively degenerate human Py tracts.", "section": "RESULTS", "ner": [ [ 26, 36, "U2AF651,2L", "mutant" ], [ 54, 74, "purine interruptions", "experimental_method" ], [ 117, 122, "human", "species" ], [ 123, 132, "Py tracts", "chemical" ] ] }, { "sid": 140, "sent": "We introduced an rArA purine dinucleotide within a variant of the AdML Py tract (detailed in Methods).", "section": "RESULTS", "ner": [ [ 3, 13, "introduced", "experimental_method" ], [ 17, 21, "rArA", "chemical" ], [ 22, 41, "purine dinucleotide", "chemical" ], [ 66, 70, "AdML", "gene" ], [ 71, 79, "Py tract", "chemical" ] ] }, { "sid": 141, "sent": "Insertion of adenine nucleotides decreased binding affinity of U2AF65 to RNA by approximately five-fold.", "section": "RESULTS", "ner": [ [ 0, 9, "Insertion", "experimental_method" ], [ 13, 32, "adenine nucleotides", "chemical" ], [ 43, 59, "binding affinity", "evidence" ], [ 63, 69, "U2AF65", "protein" ], [ 73, 76, "RNA", "chemical" ] ] }, { "sid": 142, "sent": "Nevertheless, in the presence of saturating concentrations of rArA-interrupted RNA slide-tethered U2AF651,2LFRET(Cy3/Cy5) showed a prevalent \u223c0.45 apparent FRET value (Fig. 6i,j), which was also predominant in the presence of continuous Py tract.", "section": "RESULTS", "ner": [ [ 62, 66, "rArA", "chemical" ], [ 79, 82, "RNA", "chemical" ], [ 83, 97, "slide-tethered", "protein_state" ], [ 98, 112, "U2AF651,2LFRET", "mutant" ], [ 113, 116, "Cy3", "chemical" ], [ 117, 120, "Cy5", "chemical" ], [ 156, 166, "FRET value", "evidence" ], [ 237, 245, "Py tract", "chemical" ] ] }, { "sid": 143, "sent": "Therefore, RRM1-to-RRM2 distance remains similar regardless of whether U2AF65 is bound to interrupted or continuous Py tract.", "section": "RESULTS", "ner": [ [ 11, 15, "RRM1", "structure_element" ], [ 19, 23, "RRM2", "structure_element" ], [ 71, 77, "U2AF65", "protein" ], [ 81, 89, "bound to", "protein_state" ], [ 116, 124, "Py tract", "chemical" ] ] }, { "sid": 144, "sent": "The inter-fluorophore distances derived from the observed 0.45 FRET state agree with the distances between the \u03b1-carbon atoms of the respective residues in the crystal structures of U2AF651,2L bound to Py-tract oligonucleotides.", "section": "RESULTS", "ner": [ [ 4, 31, "inter-fluorophore distances", "evidence" ], [ 63, 73, "FRET state", "evidence" ], [ 160, 178, "crystal structures", "evidence" ], [ 182, 192, "U2AF651,2L", "mutant" ], [ 193, 201, "bound to", "protein_state" ], [ 202, 227, "Py-tract oligonucleotides", "chemical" ] ] }, { "sid": 145, "sent": "It should be noted that inferring distances from FRET values is prone to significant error because of uncertainties in the determination of fluorophore orientation factor \u03ba2 and F\u00f6rster radius R0, the parameters used in distance calculations.", "section": "RESULTS", "ner": [ [ 49, 60, "FRET values", "evidence" ] ] }, { "sid": 146, "sent": "Nevertheless, the predominant 0.45 FRET state in the presence of RNA agrees with the Py-tract-bound crystal structure of U2AF651,2L.", "section": "RESULTS", "ner": [ [ 35, 45, "FRET state", "evidence" ], [ 65, 68, "RNA", "chemical" ], [ 85, 99, "Py-tract-bound", "protein_state" ], [ 100, 117, "crystal structure", "evidence" ], [ 121, 131, "U2AF651,2L", "mutant" ] ] }, { "sid": 147, "sent": "Importantly, the majority of traces (\u223c70%) of U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA lacked FRET fluctuations and predominately exhibited a \u223c0.45 FRET value (for example, Fig. 6g).", "section": "RESULTS", "ner": [ [ 29, 35, "traces", "evidence" ], [ 46, 60, "U2AF651,2LFRET", "mutant" ], [ 61, 64, "Cy3", "chemical" ], [ 65, 68, "Cy5", "chemical" ], [ 70, 78, "bound to", "protein_state" ], [ 98, 101, "RNA", "chemical" ], [ 163, 173, "FRET value", "evidence" ] ] }, { "sid": 148, "sent": "The remaining \u223c30% of traces for U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA showed fluctuations between distinct FRET values.", "section": "RESULTS", "ner": [ [ 22, 28, "traces", "evidence" ], [ 33, 47, "U2AF651,2LFRET", "mutant" ], [ 48, 51, "Cy3", "chemical" ], [ 52, 55, "Cy5", "chemical" ], [ 57, 65, "bound to", "protein_state" ], [ 85, 88, "RNA", "chemical" ], [ 126, 137, "FRET values", "evidence" ] ] }, { "sid": 149, "sent": "The majority of traces that show fluctuations began at high (0.65\u20130.8) FRET value and transitioned to a \u223c0.45 FRET value (Supplementary Fig. 7c\u2013g).", "section": "RESULTS", "ner": [ [ 16, 22, "traces", "evidence" ], [ 71, 81, "FRET value", "evidence" ], [ 110, 120, "FRET value", "evidence" ] ] }, { "sid": 150, "sent": "Hidden Markov modelling analysis of smFRET traces suggests that RNA-bound U2AF651,2L can sample at least two other conformations corresponding to \u223c0.7\u20130.8 and \u223c0.3 FRET values in addition to the predominant conformation corresponding to the 0.45 FRET state.", "section": "RESULTS", "ner": [ [ 0, 32, "Hidden Markov modelling analysis", "experimental_method" ], [ 36, 42, "smFRET", "experimental_method" ], [ 43, 49, "traces", "evidence" ], [ 64, 73, "RNA-bound", "protein_state" ], [ 74, 84, "U2AF651,2L", "mutant" ], [ 164, 175, "FRET values", "evidence" ], [ 246, 256, "FRET state", "evidence" ] ] }, { "sid": 151, "sent": "Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to \u223c0.7\u20130.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to \u223c0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures.", "section": "RESULTS", "ner": [ [ 63, 73, "U2AF651,2L", "mutant" ], [ 100, 111, "FRET values", "evidence" ], [ 121, 124, "RNA", "chemical" ], [ 129, 132, "RNA", "chemical" ], [ 148, 155, "compact", "protein_state" ], [ 173, 183, "U2AF651,2L", "mutant" ], [ 257, 267, "FRET value", "evidence" ], [ 297, 309, "side-by-side", "protein_state" ], [ 310, 319, "inter-RRM", "structure_element" ], [ 339, 349, "U2AF651,2L", "mutant" ], [ 350, 368, "crystal structures", "evidence" ] ] }, { "sid": 152, "sent": "Thus, the sequence of structural rearrangements of U2AF65 observed in smFRET traces (Supplementary Fig. 7c\u2013g) suggests that a \u2018conformational selection' mechanism of Py-tract recognition (that is, RNA ligand stabilization of a pre-configured U2AF65 conformation) is complemented by \u2018induced fit' (that is, RNA-induced rearrangement of the U2AF65 RRMs to achieve the final \u2018side-by-side' conformation), as discussed below.", "section": "RESULTS", "ner": [ [ 51, 57, "U2AF65", "protein" ], [ 70, 76, "smFRET", "experimental_method" ], [ 77, 83, "traces", "evidence" ], [ 166, 174, "Py-tract", "chemical" ], [ 227, 241, "pre-configured", "protein_state" ], [ 242, 248, "U2AF65", "protein" ], [ 339, 345, "U2AF65", "protein" ], [ 346, 350, "RRMs", "structure_element" ], [ 373, 385, "side-by-side", "protein_state" ] ] }, { "sid": 153, "sent": "The U2AF65 structures and analyses presented here represent a successful step towards defining a molecular map of the 3\u2032 splice site.", "section": "DISCUSS", "ner": [ [ 4, 10, "U2AF65", "protein" ], [ 11, 21, "structures", "evidence" ], [ 26, 34, "analyses", "evidence" ], [ 118, 132, "3\u2032 splice site", "site" ] ] }, { "sid": 154, "sent": "Several observations indicate that the numerous intramolecular contacts, here revealed among the inter-RRM linker and RRM1, RRM2, and the N-terminal RRM1 extension, synergistically coordinate U2AF65\u2013Py-tract recognition.", "section": "DISCUSS", "ner": [ [ 97, 113, "inter-RRM linker", "structure_element" ], [ 118, 122, "RRM1", "structure_element" ], [ 124, 128, "RRM2", "structure_element" ], [ 149, 163, "RRM1 extension", "structure_element" ], [ 192, 198, "U2AF65", "protein" ], [ 199, 207, "Py-tract", "chemical" ] ] }, { "sid": 155, "sent": "Truncation of U2AF65 to the core RRM1\u2013RRM2 region reduces its RNA affinity by 100-fold.", "section": "DISCUSS", "ner": [ [ 0, 10, "Truncation", "experimental_method" ], [ 14, 20, "U2AF65", "protein" ], [ 28, 32, "core", "protein_state" ], [ 33, 49, "RRM1\u2013RRM2 region", "structure_element" ], [ 62, 74, "RNA affinity", "evidence" ] ] }, { "sid": 156, "sent": "Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65\u2013RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions.", "section": "DISCUSS", "ner": [ [ 10, 18, "deletion", "experimental_method" ], [ 22, 24, "20", "residue_range" ], [ 25, 50, "inter-RRM linker residues", "structure_element" ], [ 73, 79, "U2AF65", "protein" ], [ 80, 83, "RNA", "chemical" ], [ 135, 141, "longer", "protein_state" ], [ 142, 152, "U2AF651,2L", "mutant" ], [ 178, 192, "RRM extensions", "structure_element" ], [ 221, 227, "linker", "structure_element" ], [ 232, 235, "RNA", "chemical" ] ] }, { "sid": 157, "sent": "Notably, a triple mutation of three residues (V254P, Q147A and R227A) in the respective inter-RRM linker, N- and C-terminal extensions non-additively reduce RNA binding by 150-fold.", "section": "DISCUSS", "ner": [ [ 11, 26, "triple mutation", "protein_state" ], [ 46, 51, "V254P", "mutant" ], [ 53, 58, "Q147A", "mutant" ], [ 63, 68, "R227A", "mutant" ], [ 88, 104, "inter-RRM linker", "structure_element" ], [ 106, 134, "N- and C-terminal extensions", "structure_element" ], [ 157, 168, "RNA binding", "evidence" ] ] }, { "sid": 158, "sent": "Altogether, these data indicate that interactions among the U2AF65 RRM1/RRM2, inter-RRM linker, N-and C-terminal extensions are mutually inter-dependent for cognate Py-tract recognition.", "section": "DISCUSS", "ner": [ [ 60, 66, "U2AF65", "protein" ], [ 67, 71, "RRM1", "structure_element" ], [ 72, 76, "RRM2", "structure_element" ], [ 78, 94, "inter-RRM linker", "structure_element" ], [ 96, 123, "N-and C-terminal extensions", "structure_element" ], [ 165, 173, "Py-tract", "chemical" ] ] }, { "sid": 159, "sent": "The implications of this finding for U2AF65 conservation and Py-tract recognition are detailed in the Supplementary Discussion.", "section": "DISCUSS", "ner": [ [ 37, 43, "U2AF65", "protein" ], [ 61, 69, "Py-tract", "chemical" ] ] }, { "sid": 160, "sent": "Recently, high-throughput sequencing studies have shown that somatic mutations in pre-mRNA splicing factors occur in the majority of patients with myelodysplastic syndrome (MDS).", "section": "DISCUSS", "ner": [ [ 10, 44, "high-throughput sequencing studies", "experimental_method" ], [ 82, 107, "pre-mRNA splicing factors", "protein_type" ] ] }, { "sid": 161, "sent": "MDS-relevant mutations are common in the small U2AF subunit (U2AF35, or U2AF1), yet such mutations are rare in the large U2AF65 subunit (also called U2AF2)\u2014possibly due to the selective versus nearly universal requirements of these factors for splicing.", "section": "DISCUSS", "ner": [ [ 41, 46, "small", "protein_state" ], [ 47, 59, "U2AF subunit", "protein_type" ], [ 61, 67, "U2AF35", "protein" ], [ 72, 77, "U2AF1", "protein" ], [ 115, 120, "large", "protein_state" ], [ 121, 127, "U2AF65", "protein" ], [ 149, 154, "U2AF2", "protein" ] ] }, { "sid": 162, "sent": "A confirmed somatic mutation of U2AF65 in patients with MDS, L187V, is located on a solvent-exposed surface of RRM1 that is distinct from the RNA interface (Fig. 7a).", "section": "DISCUSS", "ner": [ [ 32, 38, "U2AF65", "protein" ], [ 61, 66, "L187V", "mutant" ], [ 84, 107, "solvent-exposed surface", "site" ], [ 111, 115, "RRM1", "structure_element" ], [ 142, 155, "RNA interface", "site" ] ] }, { "sid": 163, "sent": "This L187 surface is oriented towards the N terminus of the U2AF651,2L construct, where it is expected to abut the U2AF35-binding site in the context of the full-length U2AF heterodimer.", "section": "DISCUSS", "ner": [ [ 5, 9, "L187", "residue_name_number" ], [ 60, 70, "U2AF651,2L", "mutant" ], [ 115, 134, "U2AF35-binding site", "site" ], [ 157, 168, "full-length", "protein_state" ], [ 169, 173, "U2AF", "protein" ], [ 174, 185, "heterodimer", "oligomeric_state" ] ] }, { "sid": 164, "sent": "Likewise, an unconfirmed M144I mutation reported by the same group corresponds to the N-terminal residue of U2AF651,2L, which is separated by only \u223c20 residues from the U2AF35-binding site.", "section": "DISCUSS", "ner": [ [ 25, 30, "M144I", "mutant" ], [ 108, 118, "U2AF651,2L", "mutant" ], [ 169, 188, "U2AF35-binding site", "site" ] ] }, { "sid": 165, "sent": "As such, we suggest that the MDS-relevant U2AF65 mutations contribute to MDS progression indirectly, by destabilizing a relevant conformation of the conjoined U2AF35 subunit rather than affecting U2AF65 functions in RNA binding or spliceosome recruitment per se.", "section": "DISCUSS", "ner": [ [ 42, 48, "U2AF65", "protein" ], [ 159, 165, "U2AF35", "protein" ], [ 196, 202, "U2AF65", "protein" ], [ 216, 219, "RNA", "chemical" ], [ 231, 242, "spliceosome", "complex_assembly" ] ] }, { "sid": 166, "sent": "Our smFRET results agree with prior NMR/PRE evidence for multi-domain conformational selection as one mechanistic basis for U2AF65\u2013RNA association (Fig. 7b).", "section": "DISCUSS", "ner": [ [ 4, 10, "smFRET", "experimental_method" ], [ 36, 39, "NMR", "experimental_method" ], [ 40, 43, "PRE", "experimental_method" ], [ 124, 130, "U2AF65", "protein" ], [ 131, 134, "RNA", "chemical" ] ] }, { "sid": 167, "sent": "An \u223c0.45 FRET value is likely to correspond to the U2AF65 conformation visualized in our U2AF651,2L crystal structures, in which the RRM1 and RRM2 bind side-by-side to the Py-tract oligonucleotide.", "section": "DISCUSS", "ner": [ [ 9, 19, "FRET value", "evidence" ], [ 51, 57, "U2AF65", "protein" ], [ 89, 99, "U2AF651,2L", "mutant" ], [ 100, 118, "crystal structures", "evidence" ], [ 133, 137, "RRM1", "structure_element" ], [ 142, 146, "RRM2", "structure_element" ], [ 152, 164, "side-by-side", "protein_state" ], [ 172, 196, "Py-tract oligonucleotide", "chemical" ] ] }, { "sid": 168, "sent": "The lesser 0.65\u20130.8 and 0.2\u20130.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the \u2018closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs.", "section": "DISCUSS", "ner": [ [ 32, 43, "FRET values", "evidence" ], [ 51, 61, "untethered", "protein_state" ], [ 62, 76, "U2AF651,2LFRET", "mutant" ], [ 77, 80, "Cy3", "chemical" ], [ 81, 84, "Cy5", "chemical" ], [ 145, 151, "closed", "protein_state" ], [ 154, 166, "back-to-back", "protein_state" ], [ 167, 173, "U2AF65", "protein" ], [ 205, 208, "NMR", "experimental_method" ], [ 209, 212, "PRE", "experimental_method" ], [ 225, 233, "extended", "protein_state" ], [ 234, 240, "U2AF65", "protein" ], [ 284, 288, "RRM1", "structure_element" ], [ 289, 293, "RRM2", "structure_element" ], [ 328, 335, "protein", "protein" ], [ 339, 347, "bound to", "protein_state" ], [ 348, 351, "RNA", "chemical" ], [ 356, 362, "single", "protein_state" ], [ 363, 367, "RRMs", "structure_element" ] ] }, { "sid": 169, "sent": "An increased prevalence of the \u223c0.45 FRET value following U2AF65\u2013RNA binding, coupled with the apparent absence of transitions in many \u223c0.45-value single molecule traces (for example, Fig. 6e), suggests a population shift in which RNA binds to (and draws the equilibrium towards) a pre-configured inter-RRM proximity that most often corresponds to the \u223c0.45 FRET value.", "section": "DISCUSS", "ner": [ [ 37, 47, "FRET value", "evidence" ], [ 58, 64, "U2AF65", "protein" ], [ 65, 68, "RNA", "chemical" ], [ 104, 114, "absence of", "protein_state" ], [ 163, 169, "traces", "evidence" ], [ 231, 234, "RNA", "chemical" ], [ 282, 296, "pre-configured", "protein_state" ], [ 297, 306, "inter-RRM", "structure_element" ], [ 358, 368, "FRET value", "evidence" ] ] }, { "sid": 170, "sent": "Notably, our smFRET results reveal that U2AF65\u2013Py-tract recognition can be characterized by an \u2018extended conformational selection' model (Fig. 7b).", "section": "DISCUSS", "ner": [ [ 13, 19, "smFRET", "experimental_method" ], [ 40, 46, "U2AF65", "protein" ], [ 47, 55, "Py-tract", "chemical" ] ] }, { "sid": 171, "sent": "Examples of \u2018extended conformational selection' during ligand binding have been characterized for a growing number of macromolecules (for example, adenylate kinase, LAO-binding protein, poly-ubiquitin, maltose-binding protein and the preQ1 riboswitch, among others).", "section": "DISCUSS", "ner": [ [ 13, 21, "extended", "protein_state" ], [ 147, 163, "adenylate kinase", "protein_type" ], [ 165, 184, "LAO-binding protein", "protein_type" ], [ 186, 200, "poly-ubiquitin", "protein_type" ], [ 202, 225, "maltose-binding protein", "protein_type" ], [ 234, 250, "preQ1 riboswitch", "protein_type" ] ] }, { "sid": 172, "sent": "Here, the majority of changes in smFRET traces for U2AF651,2LFRET(Cy3/Cy5) bound to slide-tethered RNA began at high (0.65\u20130.8) FRET value and transition to the predominant 0.45 FRET value (Supplementary Fig. 7c\u2013g).", "section": "DISCUSS", "ner": [ [ 33, 39, "smFRET", "experimental_method" ], [ 40, 46, "traces", "evidence" ], [ 51, 65, "U2AF651,2LFRET", "mutant" ], [ 66, 69, "Cy3", "chemical" ], [ 70, 73, "Cy5", "chemical" ], [ 75, 83, "bound to", "protein_state" ], [ 99, 102, "RNA", "chemical" ], [ 128, 138, "FRET value", "evidence" ], [ 178, 188, "FRET value", "evidence" ] ] }, { "sid": 173, "sent": "These transitions could correspond to rearrangement from the \u2018closed' NMR/PRE-based U2AF65 conformation in which the RNA-binding surface of only a single RRM is exposed and available for RNA binding, to the structural state seen in the side-by-side, RNA-bound crystal structure.", "section": "DISCUSS", "ner": [ [ 62, 68, "closed", "protein_state" ], [ 70, 73, "NMR", "experimental_method" ], [ 74, 77, "PRE", "experimental_method" ], [ 84, 90, "U2AF65", "protein" ], [ 117, 136, "RNA-binding surface", "site" ], [ 147, 153, "single", "protein_state" ], [ 154, 157, "RRM", "structure_element" ], [ 236, 248, "side-by-side", "protein_state" ], [ 250, 259, "RNA-bound", "protein_state" ], [ 260, 277, "crystal structure", "evidence" ] ] }, { "sid": 174, "sent": "As such, the smFRET approach reconciles prior inconsistencies between two major conformations that were detected by NMR/PRE experiments and a broad ensemble of diverse inter-RRM arrangements that fit the SAXS data for the apo-protein.", "section": "DISCUSS", "ner": [ [ 13, 19, "smFRET", "experimental_method" ], [ 116, 119, "NMR", "experimental_method" ], [ 120, 123, "PRE", "experimental_method" ], [ 168, 177, "inter-RRM", "structure_element" ], [ 204, 208, "SAXS", "experimental_method" ], [ 222, 225, "apo", "protein_state" ], [ 226, 233, "protein", "protein" ] ] }, { "sid": 175, "sent": "Similar interdisciplinary structural approaches are likely to illuminate whether similar mechanistic bases for RNA binding are widespread among other members of the vast multi-RRM family.", "section": "DISCUSS", "ner": [ [ 176, 179, "RRM", "structure_element" ] ] }, { "sid": 176, "sent": "The finding that U2AF65 recognizes a nine base pair Py tract contributes to an elusive \u2018code' for predicting splicing patterns from primary sequences in the post-genomic era (reviewed in ref.).", "section": "DISCUSS", "ner": [ [ 17, 23, "U2AF65", "protein" ], [ 52, 60, "Py tract", "chemical" ] ] }, { "sid": 177, "sent": "Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein.", "section": "DISCUSS", "ner": [ [ 21, 35, "RNA affinities", "evidence" ], [ 39, 45, "U2AF65", "protein" ], [ 50, 60, "U2AF651,2L", "mutant" ], [ 110, 120, "U2AF651,2L", "mutant" ], [ 121, 131, "structures", "evidence" ], [ 188, 197, "penalties", "evidence" ], [ 201, 227, "structure-guided mutations", "experimental_method" ], [ 231, 262, "RNA binding and splicing assays", "experimental_method" ], [ 284, 292, "extended", "protein_state" ], [ 293, 310, "inter-RRM regions", "structure_element" ], [ 318, 328, "U2AF651,2L", "mutant" ], [ 329, 339, "structures", "evidence" ], [ 357, 365, "Py-tract", "chemical" ], [ 385, 396, "full-length", "protein_state" ], [ 397, 403, "U2AF65", "protein" ] ] }, { "sid": 178, "sent": "Further research will be needed to understand the roles of SF1 and U2AF35 subunits in the conformational equilibria underlying U2AF65 association with Py tracts.", "section": "DISCUSS", "ner": [ [ 59, 62, "SF1", "protein" ], [ 67, 73, "U2AF35", "protein" ], [ 127, 133, "U2AF65", "protein" ], [ 151, 160, "Py tracts", "chemical" ] ] }, { "sid": 179, "sent": "Moreover, structural differences among U2AF65 homologues and paralogues may regulate splice site selection.", "section": "DISCUSS", "ner": [ [ 39, 45, "U2AF65", "protein" ], [ 85, 96, "splice site", "site" ] ] }, { "sid": 180, "sent": "Ultimately, these guidelines will assist the identification of 3\u2032 splice sites and the relationship of disease-causing mutations to penalties for U2AF65 association.", "section": "DISCUSS", "ner": [ [ 63, 78, "3\u2032 splice sites", "site" ], [ 146, 152, "U2AF65", "protein" ] ] }, { "sid": 181, "sent": "The intact U2AF65 RRM1/RRM2-containing domain and flanking residues are required for binding contiguous Py tracts.", "section": "FIG", "ner": [ [ 4, 10, "intact", "protein_state" ], [ 11, 17, "U2AF65", "protein" ], [ 18, 22, "RRM1", "structure_element" ], [ 23, 27, "RRM2", "structure_element" ], [ 93, 103, "contiguous", "structure_element" ], [ 104, 113, "Py tracts", "chemical" ] ] }, { "sid": 182, "sent": "(a) Domain organization of full-length (fl) U2AF65 and constructs used for RNA binding and structural experiments.", "section": "FIG", "ner": [ [ 27, 38, "full-length", "protein_state" ], [ 40, 42, "fl", "protein_state" ], [ 44, 50, "U2AF65", "protein" ], [ 75, 78, "RNA", "chemical" ] ] }, { "sid": 183, "sent": "An internal deletion (d, \u0394) of residues 238\u2013257 removes a portion of the inter-RRM linker from the dU2AF651,2 and dU2AF651,2L constructs.", "section": "FIG", "ner": [ [ 22, 23, "d", "mutant" ], [ 25, 26, "\u0394", "mutant" ], [ 40, 47, "238\u2013257", "residue_range" ], [ 73, 89, "inter-RRM linker", "structure_element" ], [ 99, 109, "dU2AF651,2", "mutant" ], [ 114, 125, "dU2AF651,2L", "mutant" ] ] }, { "sid": 184, "sent": "(b) Comparison of the apparent equilibrium affinities of various U2AF65 constructs for binding the prototypical AdML Py tract (5\u2032-CCCUUUUUUUUCC-3\u2032).", "section": "FIG", "ner": [ [ 31, 53, "equilibrium affinities", "evidence" ], [ 65, 71, "U2AF65", "protein" ], [ 112, 116, "AdML", "gene" ], [ 117, 125, "Py tract", "chemical" ], [ 127, 146, "5\u2032-CCCUUUUUUUUCC-3\u2032", "chemical" ] ] }, { "sid": 185, "sent": "The flU2AF65 protein includes a heterodimerization domain of the U2AF35 subunit to promote solubility and folding.", "section": "FIG", "ner": [ [ 4, 12, "flU2AF65", "protein" ], [ 32, 57, "heterodimerization domain", "structure_element" ], [ 65, 71, "U2AF35", "protein" ] ] }, { "sid": 186, "sent": "The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30\u00b13\u2009nM; U2AF651,2L, 35\u00b16\u2009nM; U2AF651,2, 3,600\u00b1300\u2009nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5\u2032- (light grey fill) or 3\u2032- (dark grey fill) regions.", "section": "FIG", "ner": [ [ 13, 47, "equilibrium dissociation constants", "evidence" ], [ 49, 51, "KD", "evidence" ], [ 69, 73, "AdML", "gene" ], [ 96, 104, "flU2AF65", "protein" ], [ 115, 125, "U2AF651,2L", "mutant" ], [ 136, 145, "U2AF651,2", "mutant" ], [ 183, 209, "RNA sequence specificities", "evidence" ], [ 213, 221, "flU2AF65", "protein" ], [ 226, 236, "U2AF651,2L", "mutant" ], [ 256, 262, "C-rich", "structure_element" ], [ 263, 272, "Py tracts", "chemical" ] ] }, { "sid": 187, "sent": "The KD's for binding 5\u2032-CCUUUUCCCCCCC-3\u2032 are: flU2AF65, 41\u00b12\u2009nM; U2AF651,2L, 31\u00b13\u2009nM. The KD's for binding 5\u2032-CCCCCCCUUUUCC-3\u2032 are: flU2AF65, 414\u00b112\u2009nM; U2AF651,2L, 417\u00b110\u2009nM. Bar graphs are hatched to match the constructs shown in a. The average apparent equilibrium affinity (KA) and s.e.m. for three independent titrations are plotted.", "section": "FIG", "ner": [ [ 4, 6, "KD", "evidence" ], [ 21, 40, "5\u2032-CCUUUUCCCCCCC-3\u2032", "chemical" ], [ 46, 54, "flU2AF65", "protein" ], [ 65, 75, "U2AF651,2L", "mutant" ], [ 90, 92, "KD", "evidence" ], [ 107, 126, "5\u2032-CCCCCCCUUUUCC-3\u2032", "chemical" ], [ 132, 140, "flU2AF65", "protein" ], [ 153, 163, "U2AF651,2L", "mutant" ], [ 239, 276, "average apparent equilibrium affinity", "evidence" ], [ 278, 280, "KA", "evidence" ] ] }, { "sid": 188, "sent": "The purified protein and average fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 1.", "section": "FIG", "ner": [ [ 25, 82, "average fitted fluorescence anisotropy RNA-binding curves", "evidence" ] ] }, { "sid": 189, "sent": "RRM, RNA recognition motif; RS, arginine-serine rich; UHM, U2AF homology motif; ULM, U2AF ligand motif.", "section": "FIG", "ner": [ [ 0, 3, "RRM", "structure_element" ], [ 5, 26, "RNA recognition motif", "structure_element" ], [ 28, 30, "RS", "structure_element" ], [ 32, 52, "arginine-serine rich", "structure_element" ], [ 54, 57, "UHM", "structure_element" ], [ 59, 78, "U2AF homology motif", "structure_element" ], [ 80, 83, "ULM", "structure_element" ], [ 85, 102, "U2AF ligand motif", "structure_element" ] ] }, { "sid": 190, "sent": "Structures of U2AF651,2L recognizing a contiguous Py tract.", "section": "FIG", "ner": [ [ 0, 10, "Structures", "evidence" ], [ 14, 24, "U2AF651,2L", "mutant" ], [ 39, 49, "contiguous", "structure_element" ], [ 50, 58, "Py tract", "chemical" ] ] }, { "sid": 191, "sent": "(a) Alignment of oligonucleotide sequences that were co-crystallized in the indicated U2AF651,2L structures.", "section": "FIG", "ner": [ [ 4, 13, "Alignment", "experimental_method" ], [ 17, 32, "oligonucleotide", "chemical" ], [ 53, 68, "co-crystallized", "experimental_method" ], [ 86, 96, "U2AF651,2L", "mutant" ], [ 97, 107, "structures", "evidence" ] ] }, { "sid": 192, "sent": "The regions of RRM1, RRM2 and linker contacts are indicated above by respective black and blue arrows from N- to C-terminus.", "section": "FIG", "ner": [ [ 15, 19, "RRM1", "structure_element" ], [ 21, 25, "RRM2", "structure_element" ], [ 30, 36, "linker", "structure_element" ] ] }, { "sid": 193, "sent": "For clarity, we consistently number the U2AF651,2L nucleotide-binding sites from one to nine, although in some cases the co-crystallized oligonucleotide comprises eight nucleotides and as such leaves the first binding site empty.", "section": "FIG", "ner": [ [ 40, 50, "U2AF651,2L", "mutant" ], [ 51, 75, "nucleotide-binding sites", "site" ], [ 121, 136, "co-crystallized", "experimental_method" ], [ 137, 152, "oligonucleotide", "chemical" ], [ 169, 180, "nucleotides", "chemical" ], [ 204, 222, "first binding site", "site" ] ] }, { "sid": 194, "sent": "The prior dU2AF651,2 nucleotide-binding sites are given in parentheses (site 4' interacts with dU2AF65 RRM1 and RRM2 by crystallographic symmetry).", "section": "FIG", "ner": [ [ 10, 20, "dU2AF651,2", "mutant" ], [ 21, 45, "nucleotide-binding sites", "site" ], [ 95, 102, "dU2AF65", "mutant" ], [ 103, 107, "RRM1", "structure_element" ], [ 112, 116, "RRM2", "structure_element" ] ] }, { "sid": 195, "sent": "(b) Stereo views of a \u2018kicked' 2|Fo|\u2212|Fc| electron density map contoured at 1\u03c3 for the inter-RRM linker, N- and C-terminal residues (blue) or bound oligonucleotide of a representative U2AF651,2L structure (structure iv, bound to 5\u2032-(P)rUrUrUdUrUrU(BrdU)dUrC) (magenta).", "section": "FIG", "ner": [ [ 31, 62, "2|Fo|\u2212|Fc| electron density map", "evidence" ], [ 87, 103, "inter-RRM linker", "structure_element" ], [ 148, 163, "oligonucleotide", "chemical" ], [ 184, 194, "U2AF651,2L", "mutant" ], [ 220, 228, "bound to", "protein_state" ] ] }, { "sid": 196, "sent": "Crystallographic statistics are given in Table 1 and the overall conformations of U2AF651,2L and prior dU2AF651,2/U2AF651,2 structures are compared in Supplementary Fig. 2.", "section": "FIG", "ner": [ [ 82, 92, "U2AF651,2L", "mutant" ], [ 103, 113, "dU2AF651,2", "mutant" ], [ 114, 123, "U2AF651,2", "mutant" ], [ 124, 134, "structures", "evidence" ] ] }, { "sid": 197, "sent": "BrdU, 5-bromo-deoxy-uridine; d, deoxy-ribose; P-, 5\u2032-phosphorylation; r, ribose.", "section": "FIG", "ner": [ [ 0, 4, "BrdU", "chemical" ], [ 6, 27, "5-bromo-deoxy-uridine", "chemical" ], [ 29, 30, "d", "chemical" ], [ 32, 44, "deoxy-ribose", "chemical" ], [ 46, 48, "P-", "chemical" ], [ 50, 68, "5\u2032-phosphorylation", "chemical" ], [ 70, 71, "r", "chemical" ], [ 73, 79, "ribose", "chemical" ] ] }, { "sid": 198, "sent": "Representative views of the U2AF651,2L interactions with each new nucleotide of the bound Py tract.", "section": "FIG", "ner": [ [ 28, 38, "U2AF651,2L", "mutant" ], [ 66, 76, "nucleotide", "chemical" ], [ 84, 89, "bound", "protein_state" ], [ 90, 98, "Py tract", "chemical" ] ] }, { "sid": 199, "sent": "New residues of the U2AF651,2L structures are coloured a darker shade of blue, apart from residues that were tested by site-directed mutagenesis, which are coloured yellow.", "section": "FIG", "ner": [ [ 20, 30, "U2AF651,2L", "mutant" ], [ 31, 41, "structures", "evidence" ], [ 119, 144, "site-directed mutagenesis", "experimental_method" ] ] }, { "sid": 200, "sent": "The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a\u2013h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2\u2013RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv.", "section": "FIG", "ner": [ [ 4, 28, "nucleotide-binding sites", "site" ], [ 36, 46, "U2AF651,2L", "mutant" ], [ 57, 67, "dU2AF651,2", "mutant" ], [ 68, 77, "structure", "evidence" ], [ 123, 165, "first and seventh U2AF651,2L-binding sites", "site" ], [ 195, 209, "dU2AF651,2\u2013RNA", "complex_assembly" ], [ 210, 219, "structure", "evidence" ], [ 275, 285, "U2AF651,2L", "mutant" ], [ 286, 296, "structures", "evidence" ], [ 362, 372, "ninth site", "site" ], [ 438, 448, "U2AF651,2L", "mutant" ], [ 449, 458, "structure", "evidence" ], [ 499, 516, "ribose nucleotide", "chemical" ], [ 540, 543, "rU2", "residue_name_number" ], [ 565, 568, "rU3", "residue_name_number" ], [ 591, 594, "rU4", "residue_name_number" ], [ 615, 618, "rU5", "residue_name_number" ], [ 641, 644, "rU6", "residue_name_number" ], [ 666, 669, "dU8", "residue_name_number" ], [ 692, 695, "dU9", "residue_name_number" ], [ 718, 721, "rC9", "residue_name_number" ] ] }, { "sid": 201, "sent": "(i) Bar graph of apparent equilibrium affinities (KA) of the wild type (blue) and the indicated mutant (yellow) U2AF651,2L proteins binding the AdML Py tract (5\u2032-CCCUUUUUUUUCC-3\u2032).", "section": "FIG", "ner": [ [ 26, 48, "equilibrium affinities", "evidence" ], [ 50, 52, "KA", "evidence" ], [ 61, 70, "wild type", "protein_state" ], [ 96, 102, "mutant", "protein_state" ], [ 112, 122, "U2AF651,2L", "mutant" ], [ 144, 148, "AdML", "gene" ], [ 149, 157, "Py tract", "chemical" ], [ 159, 178, "5\u2032-CCCUUUUUUUUCC-3\u2032", "chemical" ] ] }, { "sid": 202, "sent": "The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35\u00b16\u2009nM; R227A, 166\u00b12\u2009nM; V254P, 137\u00b110\u2009nM; Q147A, 171\u00b121\u2009nM. The average KA and s.e.m. for three independent titrations are plotted.", "section": "FIG", "ner": [ [ 13, 47, "equilibrium dissociation constants", "evidence" ], [ 49, 51, "KD", "evidence" ], [ 60, 70, "U2AF651,2L", "mutant" ], [ 71, 77, "mutant", "protein_state" ], [ 92, 101, "wild type", "protein_state" ], [ 103, 105, "WT", "protein_state" ], [ 117, 122, "R227A", "mutant" ], [ 134, 139, "V254P", "mutant" ], [ 152, 157, "Q147A", "mutant" ], [ 182, 184, "KA", "evidence" ] ] }, { "sid": 203, "sent": "The average fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 4a\u2013c.", "section": "FIG", "ner": [ [ 43, 61, "RNA-binding curves", "evidence" ] ] }, { "sid": 204, "sent": "The U2AF65 linker/RRM and inter-RRM interactions.", "section": "FIG", "ner": [ [ 4, 10, "U2AF65", "protein" ], [ 11, 17, "linker", "structure_element" ], [ 18, 21, "RRM", "structure_element" ], [ 32, 35, "RRM", "structure_element" ] ] }, { "sid": 205, "sent": "(a) Contacts of the U2AF65 inter-RRM linker with the RRMs.", "section": "FIG", "ner": [ [ 20, 26, "U2AF65", "protein" ], [ 27, 43, "inter-RRM linker", "structure_element" ], [ 53, 57, "RRMs", "structure_element" ] ] }, { "sid": 206, "sent": "A semi-transparent space-filling surface is shown for the RRM1 (green) and RRM2 (light blue).", "section": "FIG", "ner": [ [ 58, 62, "RRM1", "structure_element" ], [ 75, 79, "RRM2", "structure_element" ] ] }, { "sid": 207, "sent": "Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant.", "section": "FIG", "ner": [ [ 9, 13, "V249", "residue_name_number" ], [ 15, 19, "V250", "residue_name_number" ], [ 21, 25, "V254", "residue_name_number" ], [ 39, 46, "mutated", "experimental_method" ], [ 50, 55, "V249G", "mutant" ], [ 56, 61, "V250G", "mutant" ], [ 62, 67, "V254G", "mutant" ], [ 75, 86, "3Gly mutant", "mutant" ], [ 97, 101, "S251", "residue_name_number" ], [ 103, 107, "T252", "residue_name_number" ], [ 109, 113, "V253", "residue_name_number" ], [ 115, 119, "P255", "residue_name_number" ], [ 137, 141, "V254", "residue_name_number" ], [ 146, 153, "mutated", "experimental_method" ], [ 157, 162, "S251G", "mutant" ], [ 163, 168, "T252G", "mutant" ], [ 169, 174, "V253G", "mutant" ], [ 175, 180, "V254G", "mutant" ], [ 181, 186, "P255G", "mutant" ], [ 194, 205, "5Gly mutant", "mutant" ], [ 212, 217, "S251N", "mutant" ], [ 218, 223, "T252L", "mutant" ], [ 224, 229, "V253A", "mutant" ], [ 230, 235, "V254L", "mutant" ], [ 236, 241, "P255A", "mutant" ], [ 249, 261, "NLALA mutant", "mutant" ], [ 272, 276, "M144", "residue_name_number" ], [ 278, 282, "L235", "residue_name_number" ], [ 284, 288, "M238", "residue_name_number" ], [ 290, 294, "V244", "residue_name_number" ], [ 296, 300, "V246", "residue_name_number" ], [ 321, 325, "V249", "residue_name_number" ], [ 327, 331, "V250", "residue_name_number" ], [ 333, 337, "S251", "residue_name_number" ], [ 339, 343, "T252", "residue_name_number" ], [ 345, 349, "V253", "residue_name_number" ], [ 351, 355, "V254", "residue_name_number" ], [ 357, 361, "P255", "residue_name_number" ], [ 366, 373, "mutated", "experimental_method" ], [ 377, 382, "M144G", "mutant" ], [ 383, 388, "L235G", "mutant" ], [ 389, 394, "M238G", "mutant" ], [ 395, 400, "V244G", "mutant" ], [ 401, 406, "V246G", "mutant" ], [ 407, 412, "V249G", "mutant" ], [ 414, 419, "V250G", "mutant" ], [ 420, 425, "S251G", "mutant" ], [ 426, 431, "T252G", "mutant" ], [ 432, 437, "V253G", "mutant" ], [ 438, 443, "V254G", "mutant" ], [ 444, 449, "P255G", "mutant" ], [ 457, 469, "12Gly mutant", "mutant" ] ] }, { "sid": 208, "sent": "Other linker residues are coloured either dark blue for new residues in the U2AF651,2L structure or light blue for the remaining inter-RRM residues.", "section": "FIG", "ner": [ [ 6, 12, "linker", "structure_element" ], [ 76, 86, "U2AF651,2L", "mutant" ], [ 129, 138, "inter-RRM", "structure_element" ] ] }, { "sid": 209, "sent": "The central panel shows an overall view with stick diagrams for mutated residues; boxed regions are expanded to show the C-terminal (bottom left) and central linker regions (top) at the inter-RRM interfaces, and N-terminal linker region contacts with RRM1 (bottom right).", "section": "FIG", "ner": [ [ 150, 172, "central linker regions", "structure_element" ], [ 186, 206, "inter-RRM interfaces", "structure_element" ], [ 251, 255, "RRM1", "structure_element" ] ] }, { "sid": 210, "sent": "(b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5\u2032-CCCUUUUUUUUCC-3\u2032) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1\u2013RRM2 region (residues 148\u2013237, 258\u2013336) or dU2AF651,2L (residues 141\u2013237, 258\u2013342).", "section": "FIG", "ner": [ [ 26, 48, "equilibrium affinities", "evidence" ], [ 50, 52, "KA", "evidence" ], [ 62, 66, "AdML", "gene" ], [ 67, 75, "Py tract", "chemical" ], [ 77, 96, "5\u2032-CCCUUUUUUUUCC-3\u2032", "chemical" ], [ 105, 114, "wild-type", "protein_state" ], [ 122, 132, "U2AF651,2L", "mutant" ], [ 193, 197, "3Gly", "mutant" ], [ 208, 212, "5Gly", "mutant" ], [ 220, 225, "NLALA", "mutant" ], [ 241, 246, "12Gly", "mutant" ], [ 264, 280, "linker deletions", "experimental_method" ], [ 281, 291, "dU2AF651,2", "mutant" ], [ 299, 306, "minimal", "protein_state" ], [ 307, 323, "RRM1\u2013RRM2 region", "structure_element" ], [ 334, 341, "148\u2013237", "residue_range" ], [ 343, 350, "258\u2013336", "residue_range" ], [ 355, 366, "dU2AF651,2L", "mutant" ], [ 377, 384, "141\u2013237", "residue_range" ], [ 386, 393, "258\u2013342", "residue_range" ] ] }, { "sid": 211, "sent": "The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35\u00b16\u2009nM; 3Gly, 47\u00b14\u2009nM; 5Gly, 61\u00b13\u2009nM; 12Gly, 88\u00b121\u2009nM; NLALA, 45\u00b13\u2009nM; dU2AF651,2L, 123\u00b15\u2009nM; dU2AF651,2, 5000\u00b1100\u2009nM; 3Mut, 5630\u00b170\u2009nM. The average KA and s.e.m. for three independent titrations are plotted.", "section": "FIG", "ner": [ [ 13, 47, "equilibrium dissociation constants", "evidence" ], [ 49, 51, "KD", "evidence" ], [ 60, 70, "U2AF651,2L", "mutant" ], [ 71, 77, "mutant", "protein_state" ], [ 92, 101, "wild type", "protein_state" ], [ 103, 105, "WT", "protein_state" ], [ 117, 121, "3Gly", "mutant" ], [ 132, 136, "5Gly", "mutant" ], [ 147, 152, "12Gly", "mutant" ], [ 164, 169, "NLALA", "mutant" ], [ 180, 191, "dU2AF651,2L", "mutant" ], [ 203, 213, "dU2AF651,2", "mutant" ], [ 228, 232, "3Mut", "mutant" ], [ 258, 260, "KA", "evidence" ] ] }, { "sid": 212, "sent": "The fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 4d\u2013j. (c) Close view of the U2AF65 RRM1/RRM2 interface following a two-fold rotation about the x-axis relative to a.", "section": "FIG", "ner": [ [ 35, 53, "RNA-binding curves", "evidence" ], [ 114, 120, "U2AF65", "protein" ], [ 121, 140, "RRM1/RRM2 interface", "site" ] ] }, { "sid": 213, "sent": "U2AF65 inter-domain residues are important for splicing a representative pre-mRNA substrate in human cells.", "section": "FIG", "ner": [ [ 0, 6, "U2AF65", "protein" ], [ 73, 81, "pre-mRNA", "chemical" ], [ 95, 100, "human", "species" ] ] }, { "sid": 214, "sent": "(a) Schematic diagram of the pyPY reporter minigene construct comprising two alternative splice sites preceded by either the weak IgM Py tract (py) or the strong AdML Py tract (PY) (sequences inset).", "section": "FIG", "ner": [ [ 29, 33, "pyPY", "chemical" ], [ 89, 101, "splice sites", "site" ], [ 134, 142, "Py tract", "chemical" ], [ 144, 146, "py", "chemical" ], [ 162, 166, "AdML", "gene" ], [ 167, 175, "Py tract", "chemical" ], [ 177, 179, "PY", "chemical" ] ] }, { "sid": 215, "sent": "(b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65).", "section": "FIG", "ner": [ [ 19, 25, "RT-PCR", "experimental_method" ], [ 29, 33, "pyPY", "chemical" ], [ 65, 79, "co-transfected", "experimental_method" ], [ 109, 113, "pyPY", "chemical" ], [ 134, 143, "wild-type", "protein_state" ], [ 145, 147, "WT", "protein_state" ], [ 149, 155, "U2AF65", "protein" ], [ 168, 174, "U2AF65", "protein" ], [ 175, 181, "mutant", "protein_state" ], [ 183, 187, "3Mut", "mutant" ], [ 192, 197, "Q147A", "mutant" ], [ 199, 204, "R227A", "mutant" ], [ 209, 214, "V254P", "mutant" ], [ 274, 276, "py", "chemical" ], [ 285, 289, "mRNA", "chemical" ], [ 317, 321, "pyPY", "chemical" ], [ 401, 407, "U2AF65", "protein" ], [ 422, 424, "WT", "protein_state" ], [ 425, 431, "U2AF65", "protein" ], [ 439, 443, "3Mut", "mutant" ], [ 444, 450, "U2AF65", "protein" ] ] }, { "sid": 216, "sent": "Protein overexpression and qRT-PCR results are shown in Supplementary Fig. 5.", "section": "FIG", "ner": [ [ 0, 22, "Protein overexpression", "experimental_method" ], [ 27, 34, "qRT-PCR", "experimental_method" ] ] }, { "sid": 217, "sent": "RNA binding stabilizes the side-by-side conformation of U2AF65 RRMs.", "section": "FIG", "ner": [ [ 27, 39, "side-by-side", "protein_state" ], [ 56, 62, "U2AF65", "protein" ], [ 63, 67, "RRMs", "structure_element" ] ] }, { "sid": 218, "sent": "(a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of \u2018side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or \u2018closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2.", "section": "FIG", "ner": [ [ 15, 19, "FRET", "experimental_method" ], [ 68, 72, "RRM1", "structure_element" ], [ 77, 81, "RRM2", "structure_element" ], [ 89, 106, "crystal structure", "evidence" ], [ 111, 123, "side-by-side", "protein_state" ], [ 125, 135, "U2AF651,2L", "mutant" ], [ 136, 140, "RRMs", "structure_element" ], [ 141, 149, "bound to", "protein_state" ], [ 152, 176, "Py-tract oligonucleotide", "chemical" ], [ 214, 220, "closed", "protein_state" ], [ 222, 225, "NMR", "experimental_method" ], [ 226, 229, "PRE", "experimental_method" ], [ 245, 254, "U2AF651,2", "mutant" ], [ 301, 305, "RRM2", "structure_element" ] ] }, { "sid": 219, "sent": "The U2AF651,2LFRET proteins were doubly labelled at A181C/Q324C such that a mixture of Cy3/Cy5 fluorophores are expected to be present at each site.", "section": "FIG", "ner": [ [ 4, 18, "U2AF651,2LFRET", "mutant" ], [ 52, 57, "A181C", "mutant" ], [ 58, 63, "Q324C", "mutant" ], [ 87, 90, "Cy3", "chemical" ], [ 91, 94, "Cy5", "chemical" ], [ 95, 107, "fluorophores", "chemical" ] ] }, { "sid": 220, "sent": "(c\u2013f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5\u2009\u03bcM AdML Py-tract RNA (5\u2032-CCUUUUUUUUCC-3\u2032) (e,f), or in the presence of 10\u2009\u03bcM adenosine-interrupted variant RNA (5\u2032-CUUUUUAAUUUCCA-3\u2032) (i,j).", "section": "FIG", "ner": [ [ 14, 28, "U2AF651,2LFRET", "mutant" ], [ 29, 32, "Cy3", "chemical" ], [ 33, 36, "Cy5", "chemical" ], [ 90, 105, "biotin-NTA/Ni+2", "chemical" ], [ 217, 227, "absence of", "protein_state" ], [ 228, 235, "ligands", "chemical" ], [ 267, 271, "AdML", "gene" ], [ 272, 284, "Py-tract RNA", "chemical" ], [ 286, 304, "5\u2032-CCUUUUUUUUCC-3\u2032", "chemical" ], [ 341, 350, "adenosine", "residue_name" ], [ 371, 374, "RNA", "chemical" ], [ 376, 396, "5\u2032-CUUUUUAAUUUCCA-3\u2032", "chemical" ] ] }, { "sid": 221, "sent": "The untethered U2AF651,2LFRET(Cy3/Cy5) protein (1\u2009nM) was added to AdML RNA\u2013polyethylene-glycol-linker\u2013DNA oligonucleotide (10\u2009nM), which was immobilized on the microscope slide by annealing with a complementary biotinyl-DNA oligonucleotide (black vertical line).", "section": "FIG", "ner": [ [ 4, 14, "untethered", "protein_state" ], [ 15, 29, "U2AF651,2LFRET", "mutant" ], [ 30, 33, "Cy3", "chemical" ], [ 34, 37, "Cy5", "chemical" ], [ 67, 71, "AdML", "gene" ], [ 72, 122, "RNA\u2013polyethylene-glycol-linker\u2013DNA oligonucleotide", "chemical" ], [ 212, 240, "biotinyl-DNA oligonucleotide", "chemical" ] ] }, { "sid": 222, "sent": "Typical single-molecule FRET traces (c,e,g,i) show fluorescence intensities from Cy3 (green) and Cy5 (red) and the calculated apparent FRET efficiency (blue).", "section": "FIG", "ner": [ [ 8, 28, "single-molecule FRET", "experimental_method" ], [ 29, 35, "traces", "evidence" ], [ 81, 84, "Cy3", "chemical" ], [ 97, 100, "Cy5", "chemical" ], [ 115, 150, "calculated apparent FRET efficiency", "evidence" ] ] }, { "sid": 223, "sent": "Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j).", "section": "FIG", "ner": [ [ 11, 17, "traces", "evidence" ], [ 22, 32, "untethered", "protein_state" ], [ 34, 43, "RNA-bound", "protein_state" ], [ 44, 58, "U2AF651,2LFRET", "mutant" ], [ 59, 62, "Cy3", "chemical" ], [ 63, 66, "Cy5", "chemical" ], [ 106, 116, "Histograms", "evidence" ], [ 136, 163, "distribution of FRET values", "evidence" ], [ 167, 175, "RNA-free", "protein_state" ], [ 177, 191, "slide-tethered", "protein_state" ], [ 192, 206, "U2AF651,2LFRET", "mutant" ], [ 207, 210, "Cy3", "chemical" ], [ 211, 214, "Cy5", "chemical" ], [ 221, 225, "AdML", "gene" ], [ 226, 235, "RNA-bound", "protein_state" ], [ 237, 251, "slide-tethered", "protein_state" ], [ 252, 266, "U2AF651,2LFRET", "mutant" ], [ 267, 270, "Cy3", "chemical" ], [ 271, 274, "Cy5", "chemical" ], [ 281, 285, "AdML", "gene" ], [ 286, 295, "RNA-bound", "protein_state" ], [ 297, 307, "untethered", "protein_state" ], [ 308, 322, "U2AF651,2LFRET", "mutant" ], [ 323, 326, "Cy3", "chemical" ], [ 327, 330, "Cy5", "chemical" ], [ 362, 371, "RNA-bound", "protein_state" ], [ 373, 387, "slide-tethered", "protein_state" ], [ 388, 402, "U2AF651,2LFRET", "mutant" ], [ 403, 406, "Cy3", "chemical" ], [ 407, 410, "Cy5", "chemical" ] ] }, { "sid": 224, "sent": "N is the number of single-molecule traces compiled for each histogram.", "section": "FIG", "ner": [ [ 35, 41, "traces", "evidence" ], [ 60, 69, "histogram", "evidence" ] ] }, { "sid": 225, "sent": "Schematic models of U2AF65 recognizing the Py tract.", "section": "FIG", "ner": [ [ 20, 26, "U2AF65", "protein" ], [ 43, 51, "Py tract", "chemical" ] ] }, { "sid": 226, "sent": "(a) Diagram of the U2AF65, SF1 and U2AF35 splicing factors bound to the consensus elements of the 3\u2032 splice site.", "section": "FIG", "ner": [ [ 19, 25, "U2AF65", "protein" ], [ 27, 30, "SF1", "protein" ], [ 35, 41, "U2AF35", "protein" ], [ 59, 67, "bound to", "protein_state" ], [ 98, 112, "3\u2032 splice site", "site" ] ] }, { "sid": 227, "sent": "A surface representation of U2AF651,2L is shown bound to nine nucleotides (nt); the relative distances and juxtaposition of the branch point sequence (BPS) and consensus AG dinucleotide at the 3\u2032 splice site are unknown.", "section": "FIG", "ner": [ [ 28, 38, "U2AF651,2L", "mutant" ], [ 48, 56, "bound to", "protein_state" ], [ 62, 73, "nucleotides", "chemical" ], [ 128, 149, "branch point sequence", "site" ], [ 151, 154, "BPS", "site" ], [ 170, 185, "AG dinucleotide", "chemical" ], [ 193, 207, "3\u2032 splice site", "site" ] ] }, { "sid": 228, "sent": "MDS-relevant mutated residues of U2AF65 are shown as yellow spheres (L187 and M144).", "section": "FIG", "ner": [ [ 33, 39, "U2AF65", "protein" ], [ 69, 73, "L187", "residue_name_number" ], [ 78, 82, "M144", "residue_name_number" ] ] }, { "sid": 229, "sent": "(b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the \u2018closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to \u223c0.45 FRET value, which is consistent with \u2018open', side-by-side RRMs such as the U2AF651,2L crystal structures.", "section": "FIG", "ner": [ [ 29, 41, "Py-tract RNA", "chemical" ], [ 75, 84, "high FRET", "evidence" ], [ 110, 116, "closed", "protein_state" ], [ 119, 131, "back-to-back", "protein_state" ], [ 132, 135, "apo", "protein_state" ], [ 136, 142, "U2AF65", "protein" ], [ 164, 167, "PRE", "experimental_method" ], [ 168, 171, "NMR", "experimental_method" ], [ 262, 272, "FRET value", "evidence" ], [ 300, 304, "open", "protein_state" ], [ 307, 319, "side-by-side", "protein_state" ], [ 320, 324, "RRMs", "structure_element" ], [ 337, 347, "U2AF651,2L", "mutant" ], [ 348, 366, "crystal structures", "evidence" ] ] }, { "sid": 230, "sent": "Alternatively, a conformation of U2AF65 corresponding to \u223c0.45 FRET value can directly bind to RNA; RNA binding stabilizes the \u2018open', side-by-side conformation and thus shifts the U2AF65 population towards the \u223c0.45 FRET value.", "section": "FIG", "ner": [ [ 33, 39, "U2AF65", "protein" ], [ 63, 73, "FRET value", "evidence" ], [ 95, 98, "RNA", "chemical" ], [ 100, 103, "RNA", "chemical" ], [ 128, 132, "open", "protein_state" ], [ 135, 147, "side-by-side", "protein_state" ], [ 181, 187, "U2AF65", "protein" ], [ 217, 227, "FRET value", "evidence" ] ] }, { "sid": 231, "sent": "RRM1, green; RRM2, pale blue; RRM extensions/linker, blue.", "section": "FIG", "ner": [ [ 0, 4, "RRM1", "structure_element" ], [ 13, 17, "RRM2", "structure_element" ], [ 30, 44, "RRM extensions", "structure_element" ], [ 45, 51, "linker", "structure_element" ] ] } ] }, "PMC4854314": { "annotations": [ { "sid": 0, "sent": "RNA protects a nucleoprotein complex against radiation damage", "section": "TITLE", "ner": [ [ 0, 3, "RNA", "chemical" ], [ 15, 28, "nucleoprotein", "complex_assembly" ] ] }, { "sid": 1, "sent": "Systematic analysis of radiation damage within a protein\u2013RNA complex over a large dose range (1.3\u201325\u2005MGy) reveals significant differential susceptibility of RNA and protein.", "section": "ABSTRACT", "ner": [ [ 49, 60, "protein\u2013RNA", "complex_assembly" ], [ 157, 160, "RNA", "chemical" ] ] }, { "sid": 2, "sent": "A new method of difference electron-density quantification is presented.", "section": "ABSTRACT", "ner": [ [ 16, 58, "difference electron-density quantification", "experimental_method" ] ] }, { "sid": 3, "sent": "Radiation damage during macromolecular X-ray crystallographic data collection is still the main impediment for many macromolecular structure determinations.", "section": "ABSTRACT", "ner": [ [ 24, 77, "macromolecular X-ray crystallographic data collection", "experimental_method" ], [ 116, 155, "macromolecular structure determinations", "experimental_method" ] ] }, { "sid": 4, "sent": "Although this has been well characterized within protein crystals, far less is known about specific damage effects within the larger class of nucleoprotein complexes.", "section": "ABSTRACT", "ner": [ [ 57, 65, "crystals", "evidence" ] ] }, { "sid": 5, "sent": "Here, a methodology has been developed whereby per-atom density changes could be quantified with increasing dose over a wide (1.3\u201325.0\u2005MGy) range and at higher resolution (1.98\u2005\u00c5) than the previous systematic specific damage study on a protein\u2013DNA complex.", "section": "ABSTRACT", "ner": [ [ 47, 71, "per-atom density changes", "evidence" ], [ 244, 247, "DNA", "chemical" ] ] }, { "sid": 6, "sent": "Specific damage manifestations were determined within the large trp RNA-binding attenuation protein (TRAP) bound to a single-stranded RNA that forms a belt around the protein.", "section": "ABSTRACT", "ner": [ [ 64, 99, "trp RNA-binding attenuation protein", "protein_type" ], [ 101, 105, "TRAP", "complex_assembly" ], [ 107, 115, "bound to", "protein_state" ], [ 134, 137, "RNA", "chemical" ] ] }, { "sid": 7, "sent": "Over a large dose range, the RNA was found to be far less susceptible to radiation-induced chemical changes than the protein.", "section": "ABSTRACT", "ner": [ [ 29, 32, "RNA", "chemical" ] ] }, { "sid": 8, "sent": "The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility.", "section": "ABSTRACT", "ner": [ [ 24, 28, "TRAP", "complex_assembly" ], [ 91, 96, "bound", "protein_state" ], [ 97, 100, "RNA", "chemical" ], [ 160, 163, "RNA", "chemical" ] ] }, { "sid": 9, "sent": "The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule.", "section": "ABSTRACT", "ner": [ [ 33, 37, "TRAP", "complex_assembly" ], [ 38, 42, "ring", "structure_element" ], [ 95, 98, "Glu", "residue_name" ], [ 103, 106, "Asp", "residue_name" ], [ 129, 132, "RNA", "chemical" ], [ 236, 255, "RNA-binding pockets", "site" ] ] }, { "sid": 10, "sent": "Additionally, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density.", "section": "ABSTRACT", "ner": [ [ 88, 91, "Lys", "residue_name" ], [ 96, 99, "Phe", "residue_name" ], [ 117, 120, "RNA", "chemical" ], [ 147, 163, "electron density", "evidence" ] ] }, { "sid": 11, "sent": "With the wide use of high-flux third-generation synchrotron sources, radiation damage (RD) has once again become a dominant reason for the failure of structure determination using macromolecular crystallography (MX) in experiments conducted both at room temperature and under cryocooled conditions (100\u2005K).", "section": "INTRO", "ner": [ [ 150, 173, "structure determination", "experimental_method" ], [ 180, 210, "macromolecular crystallography", "experimental_method" ], [ 212, 214, "MX", "experimental_method" ] ] }, { "sid": 12, "sent": "Significant progress has been made in recent years in understanding the inevitable manifestations of X-ray-induced RD within protein crystals, and there is now a body of literature on possible strategies to mitigate the effects of RD (e.g. Zeldin, Brockhauser et al., 2013; Bourenkov & Popov, 2010).", "section": "INTRO", "ner": [ [ 133, 141, "crystals", "evidence" ] ] }, { "sid": 13, "sent": "However, there is still no general consensus within the field on how to minimize RD during MX data collection, and debates on the dependence of RD progression on incident X-ray energy (Shimizu et al., 2007; Liebschner et al., 2015) and the efficacy of radical scavengers (Allan et al., 2013) have yet to be resolved.", "section": "INTRO", "ner": [ [ 91, 93, "MX", "experimental_method" ] ] }, { "sid": 14, "sent": "Global radiation damage is observed within reciprocal space as the overall decay of the summed intensity of reflections detected within the diffraction pattern as dose increases (Garman, 2010; Murray & Garman, 2002).", "section": "INTRO", "ner": [ [ 140, 159, "diffraction pattern", "evidence" ] ] }, { "sid": 15, "sent": "Dose is defined as the absorbed energy per unit mass of crystal in grays (Gy; 1\u2005Gy = 1\u2005J\u2005kg\u22121), and is the metric against which damage progression should be monitored during MX data collection, as opposed to time.", "section": "INTRO", "ner": [ [ 174, 176, "MX", "experimental_method" ] ] }, { "sid": 16, "sent": "At 100\u2005K, an experimental dose limit of 30\u2005MGy has been recommended as an upper limit beyond which the biological information derived from any macromolecular crystal may be compromised (Owen et al., 2006).", "section": "INTRO", "ner": [ [ 158, 165, "crystal", "evidence" ] ] }, { "sid": 17, "sent": " Specific radiation damage (SRD) is observed in the real-space electron density, and has been detected at much lower doses than any observable decay in the intensity of reflections.", "section": "INTRO", "ner": [ [ 1, 26, "Specific radiation damage", "experimental_method" ], [ 28, 31, "SRD", "experimental_method" ], [ 52, 79, "real-space electron density", "evidence" ] ] }, { "sid": 18, "sent": "Indeed, the C\u2014Se bond in selenomethionine, the stability of which is key for the success of experimental phasing methods, can be cleaved at a dose as low as 2\u2005MGy for a crystal maintained at 100\u2005K (Holton, 2007).", "section": "INTRO", "ner": [ [ 14, 16, "Se", "chemical" ], [ 25, 41, "selenomethionine", "chemical" ], [ 169, 176, "crystal", "evidence" ] ] }, { "sid": 19, "sent": "SRD has been well characterized in a large range of proteins, and is seen to follow a reproducible order: metallo-centre reduction, disulfide-bond cleavage, acidic residue decarboxylation and methionine methylthio cleavage (Ravelli & McSweeney, 2000; Burmeister, 2000; Weik et al., 2000; Yano et al., 2005).", "section": "INTRO", "ner": [ [ 132, 146, "disulfide-bond", "ptm" ] ] }, { "sid": 20, "sent": "There are a number of cases where SRD manifestations have compromised the biological information extracted from MX-determined structures at much lower doses than the recommended 30\u2005MGy limit, leading to false structural interpretations of protein mechanisms.", "section": "INTRO", "ner": [ [ 112, 125, "MX-determined", "experimental_method" ], [ 126, 136, "structures", "evidence" ] ] }, { "sid": 21, "sent": "Active-site residues appear to be particularly susceptible, particularly for photosensitive proteins and in instances where chemical strain is an intrinsic feature of the reaction mechanism.", "section": "INTRO", "ner": [ [ 0, 20, "Active-site residues", "site" ] ] }, { "sid": 22, "sent": "For instance, structure determination of the purple membrane protein bacterio\u00adrhodopsin required careful corrections for radiation-induced structural changes before the correct photosensitive intermediate states could be isolated (Matsui et al., 2002).", "section": "INTRO", "ner": [ [ 14, 37, "structure determination", "experimental_method" ], [ 69, 87, "bacterio\u00adrhodopsin", "protein_type" ] ] }, { "sid": 23, "sent": "The significant chemical strain required for catalysis within the active site of phosphoserine aminotransferase has been observed to diminish during X-ray exposure (Dubnovitsky et al., 2005).", "section": "INTRO", "ner": [ [ 66, 77, "active site", "site" ], [ 81, 111, "phosphoserine aminotransferase", "protein_type" ] ] }, { "sid": 24, "sent": "Since the majority of SRD studies to date have focused on proteins, much less is known about the effects of X-ray irradiation on the wider class of crystalline nucleoprotein complexes or how to correct for such radiation-induced structural changes.", "section": "INTRO", "ner": [ [ 22, 33, "SRD studies", "experimental_method" ], [ 160, 173, "nucleoprotein", "complex_assembly" ] ] }, { "sid": 25, "sent": "Understanding RD to such complexes is crucial, since DNA is rarely naked within a cell, instead dynamically interacting with proteins, facilitating replication, transcription, modification and DNA repair.", "section": "INTRO", "ner": [ [ 53, 56, "DNA", "chemical" ], [ 193, 196, "DNA", "chemical" ] ] }, { "sid": 26, "sent": "As of early 2016, >5400 nucleoprotein complex structures have been deposited within the PDB, with 91% solved by MX.", "section": "INTRO", "ner": [ [ 24, 37, "nucleoprotein", "complex_assembly" ], [ 46, 56, "structures", "evidence" ], [ 112, 114, "MX", "experimental_method" ] ] }, { "sid": 27, "sent": "It is essential to understand how these increasingly complex macromolecular structures are affected by the radiation used to solve them.", "section": "INTRO", "ner": [ [ 76, 86, "structures", "evidence" ] ] }, { "sid": 28, "sent": "Nucleoproteins also represent one of the main targets of radiotherapy, and an insight into the damage mechanisms induced by X-ray irradiation could inform innovative treatments.", "section": "INTRO", "ner": [ [ 0, 14, "Nucleoproteins", "complex_assembly" ] ] }, { "sid": 29, "sent": "Investigations on sub-ionization-level LEEs (0\u201315\u2005eV) interacting with both dried and aqueous oligonucleotides (Alizadeh & Sanche, 2014; Simons, 2006) concluded that resonant electron attachment to DNA bases and the sugar-phosphate backbone could lead to the preferential cleavage of strong (\u223c4\u2005eV, 385\u2005kJ\u2005mol\u22121) sugar-phosphate C\u2014O covalent bonds within the DNA backbone and then base-sugar N1\u2014C bonds, eventually leading to single-strand breakages (SSBs; Ptasi\u0144ska & Sanche, 2007).", "section": "INTRO", "ner": [ [ 198, 201, "DNA", "chemical" ], [ 359, 362, "DNA", "chemical" ] ] }, { "sid": 30, "sent": "Electrons have been shown to be mobile at 77\u2005K by electron spin resonance spectroscopy studies (Symons, 1997; Jones et al., 1987), with rapid electron quantum tunnelling and positive hole migration along the protein backbone and through stacked DNA bases indicated as a dominant mechanism by which oxidative and reductive damage localizes at distances from initial ionization sites at 100\u2005K (O\u2019Neill et al., 2002).", "section": "INTRO", "ner": [ [ 50, 86, "electron spin resonance spectroscopy", "experimental_method" ], [ 245, 248, "DNA", "chemical" ], [ 365, 381, "ionization sites", "site" ] ] }, { "sid": 31, "sent": "The investigation of naturally forming nucleoprotein complexes circumvents the inherent challenges in making controlled comparisons of damage mechanisms between protein and nucleic acids crystallized separately. Recently, for a well characterized bacterial protein\u2013DNA complex (C.Esp1396I; PDB entry 3clc; resolution 2.8\u2005\u00c5; McGeehan et al., 2008) it was concluded that over a wide dose range (2.1\u201344.6\u2005MGy) the protein was far more susceptible to SRD than the DNA within the crystal (Bury et al., 2015).", "section": "INTRO", "ner": [ [ 187, 199, "crystallized", "experimental_method" ], [ 247, 256, "bacterial", "taxonomy_domain" ], [ 265, 268, "DNA", "chemical" ], [ 278, 288, "C.Esp1396I", "complex_assembly" ], [ 460, 463, "DNA", "chemical" ], [ 475, 482, "crystal", "evidence" ] ] }, { "sid": 32, "sent": "Only at doses above 20\u2005MGy were precursors of phosphodiester-bond cleavage observed within AT-rich regions of the 35-mer DNA.", "section": "INTRO", "ner": [ [ 91, 106, "AT-rich regions", "structure_element" ], [ 121, 124, "DNA", "chemical" ] ] }, { "sid": 33, "sent": "For crystalline complexes such as C.Esp1396I, whether the protein is intrinsically more susceptible to X-ray-induced damage or whether the protein scavenges electrons to protect the DNA remains unclear in the absence of a non-nucleic acid-bound protein control obtained under exactly the same crystallization and data-collection conditions.", "section": "INTRO", "ner": [ [ 34, 44, "C.Esp1396I", "complex_assembly" ], [ 182, 185, "DNA", "chemical" ] ] }, { "sid": 34, "sent": "To monitor the effects of nucleic acid binding on protein damage susceptibility, a crystal containing two protein molecules per asymmetric unit, only one of which was bound to RNA, is reported here (Fig. 1 \u25b8).", "section": "INTRO", "ner": [ [ 83, 90, "crystal", "evidence" ], [ 167, 175, "bound to", "protein_state" ], [ 176, 179, "RNA", "chemical" ] ] }, { "sid": 35, "sent": "Using newly developed methodology, we present a controlled SRD investigation at 1.98\u2005\u00c5 resolution using a large (\u223c91\u2005kDa) crystalline protein\u2013RNA complex: trp RNA-binding attenuation protein (TRAP) bound to a 53\u2005bp RNA sequence (GAGUU)10GAG (PDB entry 1gtf; Hopcroft et al., 2002).", "section": "INTRO", "ner": [ [ 48, 62, "controlled SRD", "experimental_method" ], [ 134, 145, "protein\u2013RNA", "complex_assembly" ], [ 155, 190, "trp RNA-binding attenuation protein", "protein_type" ], [ 192, 196, "TRAP", "complex_assembly" ], [ 198, 206, "bound to", "protein_state" ], [ 215, 218, "RNA", "chemical" ], [ 228, 240, "(GAGUU)10GAG", "chemical" ] ] }, { "sid": 36, "sent": "TRAP consists of 11 identical subunits assembled into a ring with 11-fold rotational symmetry.", "section": "INTRO", "ner": [ [ 0, 4, "TRAP", "complex_assembly" ], [ 30, 38, "subunits", "structure_element" ], [ 56, 60, "ring", "structure_element" ] ] }, { "sid": 37, "sent": "It binds with high affinity (K d \u2243 1.0\u2005nM) to RNA segments containing 11 GAG/UAG triplets separated by two or three spacer nucleotides (Elliott et al., 2001) to regulate the transcription of tryptophan biosynthetic genes in Bacillus subtilis (Antson et al., 1999).", "section": "INTRO", "ner": [ [ 29, 32, "K d", "evidence" ], [ 46, 49, "RNA", "chemical" ], [ 73, 89, "GAG/UAG triplets", "structure_element" ], [ 116, 134, "spacer nucleotides", "structure_element" ], [ 191, 201, "tryptophan", "chemical" ], [ 224, 241, "Bacillus subtilis", "species" ] ] }, { "sid": 38, "sent": "In this structure, the bases of the G1-A2-G3 nucleotides form direct hydrogen bonds to the protein, unlike the U4-U5 nucleotides, which appear to be more flexible.", "section": "INTRO", "ner": [ [ 8, 17, "structure", "evidence" ], [ 36, 44, "G1-A2-G3", "chemical" ], [ 69, 83, "hydrogen bonds", "bond_interaction" ], [ 111, 116, "U4-U5", "chemical" ] ] }, { "sid": 39, "sent": "Ten successive 1.98\u2005\u00c5 resolution MX data sets were collected from the same TRAP\u2013RNA crystal to analyse X-ray-induced structural changes over a large dose range (d 1 = 1.3\u2005MGy to d 10 = 25.0\u2005MGy).", "section": "INTRO", "ner": [ [ 33, 35, "MX", "experimental_method" ], [ 75, 83, "TRAP\u2013RNA", "complex_assembly" ], [ 84, 91, "crystal", "evidence" ] ] }, { "sid": 40, "sent": "To avoid the previous necessity for visual inspection of electron-density maps to detect SRD sites, a computational approach was designed to quantify the electron-density change for each refined atom with increasing dose, thus providing a rapid systematic method for SRD study on such large multimeric complexes.", "section": "INTRO", "ner": [ [ 57, 78, "electron-density maps", "evidence" ], [ 89, 98, "SRD sites", "site" ], [ 154, 177, "electron-density change", "evidence" ] ] }, { "sid": 41, "sent": "By employing the high 11-fold structural symmetry within each TRAP macromolecule, this approach permitted a thorough statistical quantification of the RD effects of RNA binding to TRAP.", "section": "INTRO", "ner": [ [ 62, 66, "TRAP", "complex_assembly" ], [ 165, 168, "RNA", "chemical" ], [ 180, 184, "TRAP", "complex_assembly" ] ] }, { "sid": 42, "sent": "Per-atom quantification of electron density \u00a0 ", "section": "RESULTS", "ner": [ [ 0, 43, "Per-atom quantification of electron density", "experimental_method" ] ] }, { "sid": 43, "sent": "To quantify the exact effects of nucleic acid binding to a protein on SRD susceptibility, a high-throughput and automated pipeline was created to systematically calculate the electron-density change for every refined atom within the TRAP\u2013RNA structure as a function of dose.", "section": "RESULTS", "ner": [ [ 175, 198, "electron-density change", "evidence" ], [ 233, 241, "TRAP\u2013RNA", "complex_assembly" ], [ 242, 251, "structure", "evidence" ] ] }, { "sid": 44, "sent": "This provides an atom-specific quantification of density\u2013dose dynamics, which was previously lacking within the field.", "section": "RESULTS", "ner": [ [ 49, 70, "density\u2013dose dynamics", "evidence" ] ] }, { "sid": 45, "sent": "Previous studies have characterized SRD sites by reporting magnitudes of F obs(d n) \u2212 F obs(d 1) Fourier difference map peaks in terms of the sigma (\u03c3) contour level (the number of standard deviations from the mean map electron-density value) at which peaks become visible.", "section": "RESULTS", "ner": [ [ 36, 45, "SRD sites", "site" ], [ 73, 125, "F obs(d n) \u2212 F obs(d 1) Fourier difference map peaks", "evidence" ], [ 142, 147, "sigma", "evidence" ], [ 149, 150, "\u03c3", "evidence" ], [ 181, 200, "standard deviations", "evidence" ], [ 210, 241, "mean map electron-density value", "evidence" ] ] }, { "sid": 46, "sent": "However, these \u03c3 levels depend on the standard deviation values of the map, which can deviate between data sets, and are thus unsuitable for quantitative comparison of density between different dose data sets.", "section": "RESULTS", "ner": [ [ 15, 16, "\u03c3", "evidence" ], [ 38, 56, "standard deviation", "evidence" ], [ 71, 74, "map", "evidence" ], [ 168, 175, "density", "evidence" ] ] }, { "sid": 47, "sent": "Instead, we use here a maximum density-loss metric (D loss), which is the per-atom equivalent of the magnitude of these negative Fourier difference map peaks in units of e\u2005\u00c5\u22123.", "section": "RESULTS", "ner": [ [ 23, 50, "maximum density-loss metric", "evidence" ], [ 52, 58, "D loss", "evidence" ], [ 120, 157, "negative Fourier difference map peaks", "evidence" ] ] }, { "sid": 48, "sent": "Large positive D loss values indicate radiation-induced atomic disordering reproducibly throughout the unit cells with respect to the initial low-dose data set.", "section": "RESULTS", "ner": [ [ 15, 21, "D loss", "evidence" ] ] }, { "sid": 49, "sent": "For each TRAP\u2013RNA data set, the D loss metric successfully identified the recognized forms of protein SRD (Fig. 2 \u25b8 a), with clear Glu and Asp side-chain decarboxylation even in the first difference map calculated (3.9\u2005MGy; Fig. 3 \u25b8 a).", "section": "RESULTS", "ner": [ [ 9, 17, "TRAP\u2013RNA", "complex_assembly" ], [ 32, 45, "D loss metric", "evidence" ], [ 102, 105, "SRD", "experimental_method" ], [ 131, 134, "Glu", "residue_name" ], [ 139, 142, "Asp", "residue_name" ], [ 188, 202, "difference map", "evidence" ] ] }, { "sid": 50, "sent": "The main sequence of TRAP does not contain any Trp and Cys residues (and thus contains no disulfide bonds).", "section": "RESULTS", "ner": [ [ 21, 25, "TRAP", "complex_assembly" ], [ 47, 50, "Trp", "residue_name" ], [ 55, 58, "Cys", "residue_name" ] ] }, { "sid": 51, "sent": "The substrate Trp amino-acid ligands also exhibited disordering of the free terminal carboxyl groups at higher doses (Fig. 2 \u25b8 a); however, no clear Fourier difference peaks could be observed visually.", "section": "RESULTS", "ner": [ [ 14, 17, "Trp", "chemical" ], [ 149, 173, "Fourier difference peaks", "evidence" ] ] }, { "sid": 52, "sent": "Even for radiation-insensitive residues (e.g. Gly) the average D loss increases with dose: this is the effect of global radiation damage, since as dose increases the electron density associated with each refined atom becomes weaker as the atomic occupancy decreases (Fig. 2 \u25b8 b).", "section": "RESULTS", "ner": [ [ 46, 49, "Gly", "residue_name" ], [ 63, 69, "D loss", "evidence" ], [ 166, 182, "electron density", "evidence" ] ] }, { "sid": 53, "sent": "Only Glu and Asp residues exhibit a rate of D loss increase that consistently exceeds the average decay (Fig. 2 \u25b8 b, dashed line) at each dose.", "section": "RESULTS", "ner": [ [ 5, 8, "Glu", "residue_name" ], [ 13, 16, "Asp", "residue_name" ], [ 44, 50, "D loss", "evidence" ] ] }, { "sid": 54, "sent": "The rate of D loss (attributed to side-chain decarboxylation) was consistently larger for Glu compared with Asp residues over the large dose range (Fig. 2 \u25b8 b and Supplementary Fig. S3); this observation is consistent with our calculations on model systems (see above) that suggest that, without considering differential hydrogen-bonding environments, CO2 loss is more exothermic by around 8\u2005kJ\u2005mol\u22121 from oxidized Glu residues than from their Asp counterparts.", "section": "RESULTS", "ner": [ [ 12, 18, "D loss", "evidence" ], [ 90, 93, "Glu", "residue_name" ], [ 108, 111, "Asp", "residue_name" ], [ 321, 337, "hydrogen-bonding", "bond_interaction" ], [ 352, 355, "CO2", "chemical" ], [ 406, 414, "oxidized", "protein_state" ], [ 415, 418, "Glu", "residue_name" ], [ 444, 447, "Asp", "residue_name" ] ] }, { "sid": 55, "sent": "RNA is less susceptible to electron-density loss than protein within the TRAP\u2013RNA complex \u00a0 ", "section": "RESULTS", "ner": [ [ 0, 3, "RNA", "chemical" ], [ 27, 43, "electron-density", "evidence" ], [ 73, 81, "TRAP\u2013RNA", "complex_assembly" ] ] }, { "sid": 56, "sent": "Visual inspection of Fourier difference maps illustrated the clear lack of RNA electron-density degradation with increasing dose compared with the obvious protein damage manifestations (Figs. 3 \u25b8 b and 3 \u25b8 c).", "section": "RESULTS", "ner": [ [ 0, 20, "Visual inspection of", "experimental_method" ], [ 21, 44, "Fourier difference maps", "evidence" ], [ 75, 78, "RNA", "chemical" ], [ 79, 107, "electron-density degradation", "evidence" ] ] }, { "sid": 57, "sent": "Only at the highest doses investigated (>20\u2005MGy) was density loss observed at the RNA phosphate and C\u2014O bonds of the phosphodiester backbone.", "section": "RESULTS", "ner": [ [ 82, 85, "RNA", "chemical" ] ] }, { "sid": 58, "sent": "However, the median D loss was lower by a factor of >2 for RNA P atoms than for Glu and Asp side-chain groups at 25.0\u2005MGy (Supplementary Fig. S4), and furthermore could not be numerically distinguished from Gly C\u03b1 atoms within TRAP, which are not radiation-sensitive at the doses tested here (Supplementary Fig. S3).", "section": "RESULTS", "ner": [ [ 20, 26, "D loss", "evidence" ], [ 59, 62, "RNA", "chemical" ], [ 80, 83, "Glu", "residue_name" ], [ 88, 91, "Asp", "residue_name" ], [ 207, 210, "Gly", "residue_name" ], [ 227, 231, "TRAP", "complex_assembly" ] ] }, { "sid": 59, "sent": "RNA binding protects radiation-sensitive residues \u00a0 ", "section": "RESULTS", "ner": [ [ 0, 3, "RNA", "chemical" ] ] }, { "sid": 60, "sent": "For the large number of acidic residues per TRAP ring (four Asp and six Glu residues per protein monomer), a strong dependence of decarboxylation susceptibility on local environment was observed (Fig. 4 \u25b8).", "section": "RESULTS", "ner": [ [ 44, 48, "TRAP", "complex_assembly" ], [ 49, 53, "ring", "structure_element" ], [ 60, 63, "Asp", "residue_name" ], [ 72, 75, "Glu", "residue_name" ], [ 97, 104, "monomer", "oligomeric_state" ] ] }, { "sid": 61, "sent": "For each Glu\u2005C\u03b4 or Asp\u2005C\u03b3 atom, D loss provided a direct measure of the rate of side-chain carboxyl-group disordering and subsequent decarboxylation.", "section": "RESULTS", "ner": [ [ 9, 12, "Glu", "residue_name" ], [ 19, 22, "Asp", "residue_name" ], [ 32, 38, "D loss", "evidence" ] ] }, { "sid": 62, "sent": "For acidic residues with no differing interactions between nonbound and bound TRAP (Fig. 4 \u25b8 a), similar damage was apparent between the two rings within the asymmetric unit, as expected.", "section": "RESULTS", "ner": [ [ 59, 67, "nonbound", "protein_state" ], [ 72, 77, "bound", "protein_state" ], [ 78, 82, "TRAP", "complex_assembly" ] ] }, { "sid": 63, "sent": "However, TRAP residues directly on the RNA-binding interfaces exhibited greater damage accumulation in nonbound TRAP (Fig. 4 \u25b8 b), and for residues at the ring\u2013ring interfaces (where crystal contacts were detected) bound TRAP exhibited enhanced SRD accumulation (Fig. 4 \u25b8 c).", "section": "RESULTS", "ner": [ [ 9, 13, "TRAP", "complex_assembly" ], [ 39, 61, "RNA-binding interfaces", "site" ], [ 103, 111, "nonbound", "protein_state" ], [ 112, 116, "TRAP", "complex_assembly" ], [ 155, 175, "ring\u2013ring interfaces", "site" ], [ 215, 220, "bound", "protein_state" ], [ 221, 225, "TRAP", "complex_assembly" ] ] }, { "sid": 64, "sent": "Three acidic residues (Glu36, Asp39 and Glu42) are involved in RNA interactions within each of the 11 TRAP ring subunits, and Fig. 5 \u25b8 shows their density changes with increasing dose.", "section": "RESULTS", "ner": [ [ 23, 28, "Glu36", "residue_name_number" ], [ 30, 35, "Asp39", "residue_name_number" ], [ 40, 45, "Glu42", "residue_name_number" ], [ 63, 66, "RNA", "chemical" ], [ 102, 106, "TRAP", "complex_assembly" ], [ 107, 111, "ring", "structure_element" ], [ 112, 120, "subunits", "structure_element" ], [ 147, 162, "density changes", "evidence" ] ] }, { "sid": 65, "sent": "Hotelling\u2019s T-squared test (the multivariate counterpart of Student\u2019s t-test) was used to reject the null hypothesis that the means of the D loss metric were equal for the bound and nonbound groups in Fig. 5 \u25b8.", "section": "RESULTS", "ner": [ [ 0, 26, "Hotelling\u2019s T-squared test", "experimental_method" ], [ 60, 76, "Student\u2019s t-test", "experimental_method" ], [ 139, 152, "D loss metric", "evidence" ], [ 172, 177, "bound", "protein_state" ], [ 182, 190, "nonbound", "protein_state" ] ] }, { "sid": 66, "sent": "A significant reduction in D loss is seen for Glu36 in RNA-bound compared with nonbound TRAP, indicative of a lower rate of side-chain decarboxylation (Fig. 5 \u25b8 a; p = 6.06 \u00d7 10\u22125).", "section": "RESULTS", "ner": [ [ 27, 33, "D loss", "evidence" ], [ 46, 51, "Glu36", "residue_name_number" ], [ 55, 64, "RNA-bound", "protein_state" ], [ 79, 87, "nonbound", "protein_state" ], [ 88, 92, "TRAP", "complex_assembly" ] ] }, { "sid": 67, "sent": "For each TRAP ring subunit, the Glu36 side-chain carboxyl group accepts a pair of hydrogen bonds from the two N atoms of the G3 RNA base.", "section": "RESULTS", "ner": [ [ 9, 13, "TRAP", "complex_assembly" ], [ 14, 18, "ring", "structure_element" ], [ 19, 26, "subunit", "structure_element" ], [ 32, 37, "Glu36", "residue_name_number" ], [ 82, 96, "hydrogen bonds", "bond_interaction" ], [ 125, 127, "G3", "residue_name_number" ], [ 128, 131, "RNA", "chemical" ] ] }, { "sid": 68, "sent": "In our analysis, Asp39 in the TRAP\u2013(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP\u2013RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 \u25b8 b, p = 0.0925).", "section": "RESULTS", "ner": [ [ 17, 22, "Asp39", "residue_name_number" ], [ 30, 47, "TRAP\u2013(GAGUU)10GAG", "complex_assembly" ], [ 48, 57, "structure", "evidence" ], [ 90, 104, "hydrogen bonds", "bond_interaction" ], [ 112, 114, "G1", "residue_name_number" ], [ 142, 161, "TRAP\u2013RNA interfaces", "site" ], [ 171, 176, "Glu36", "residue_name_number" ], [ 180, 182, "G3", "residue_name_number" ], [ 210, 217, "density", "evidence" ], [ 235, 238, "RNA", "chemical" ], [ 275, 280, "Asp39", "residue_name_number" ], [ 290, 295, "Glu36", "residue_name_number" ] ] }, { "sid": 69, "sent": "RNA binding reduces radiation-induced disorder on the atomic scale \u00a0 ", "section": "RESULTS", "ner": [ [ 0, 3, "RNA", "chemical" ] ] }, { "sid": 70, "sent": "One oxygen (O\u220a1) of Glu42 appears to form a hydrogen bond to a nearby water within each TRAP RNA-binding pocket, with the other (O\u220a2) being involved in a salt-bridge interaction with Arg58 (Hopcroft et al., 2002; Antson et al., 1999).", "section": "RESULTS", "ner": [ [ 20, 25, "Glu42", "residue_name_number" ], [ 44, 57, "hydrogen bond", "bond_interaction" ], [ 70, 75, "water", "chemical" ], [ 88, 111, "TRAP RNA-binding pocket", "site" ], [ 154, 165, "salt-bridge", "bond_interaction" ], [ 183, 188, "Arg58", "residue_name_number" ] ] }, { "sid": 71, "sent": "Salt-bridge interactions have previously been suggested to reduce the glutamate decarboxylation rate within the large (\u223c62.4\u2005kDa) myrosinase protein structure (Burmeister, 2000).", "section": "RESULTS", "ner": [ [ 0, 11, "Salt-bridge", "bond_interaction" ], [ 70, 79, "glutamate", "residue_name" ], [ 130, 140, "myrosinase", "protein_type" ], [ 149, 158, "structure", "evidence" ] ] }, { "sid": 72, "sent": "A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42\u2005O\u220a1 atoms (Fig. 5 \u25b8 c; p\u00a0=\u00a00.007) but not for the Glu42\u2005O\u220a2 atoms (Fig. 5 \u25b8 d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA.", "section": "RESULTS", "ner": [ [ 50, 65, "D loss dynamics", "evidence" ], [ 74, 82, "nonbound", "protein_state" ], [ 83, 88, "bound", "protein_state" ], [ 89, 94, "Glu42", "residue_name_number" ], [ 145, 150, "Glu42", "residue_name_number" ], [ 235, 246, "salt-bridge", "bond_interaction" ], [ 278, 281, "RNA", "chemical" ], [ 303, 308, "water", "chemical" ], [ 318, 331, "hydrogen bond", "bond_interaction" ], [ 399, 409, "absence of", "protein_state" ], [ 410, 413, "RNA", "chemical" ] ] }, { "sid": 73, "sent": "The density-change dynamics were statistically indistinguishable between bound and nonbound TRAP for each Glu42 carboxyl group C\u03b4 atom (p = 0.435), indicating that upon RNA binding the conserved salt-bridge interaction ultimately dictated the overall Glu42 decarboxylation rate.", "section": "RESULTS", "ner": [ [ 4, 27, "density-change dynamics", "evidence" ], [ 73, 78, "bound", "protein_state" ], [ 83, 91, "nonbound", "protein_state" ], [ 92, 96, "TRAP", "complex_assembly" ], [ 106, 111, "Glu42", "residue_name_number" ], [ 169, 172, "RNA", "chemical" ], [ 195, 206, "salt-bridge", "bond_interaction" ], [ 251, 256, "Glu42", "residue_name_number" ] ] }, { "sid": 74, "sent": "The RNA-stabilizing effect was not restricted to radiation-sensitive acidic residues.", "section": "RESULTS", "ner": [ [ 4, 7, "RNA", "chemical" ] ] }, { "sid": 75, "sent": "The side chain of Phe32 stacks against the G3 base within the 11 TRAP RNA-binding interfaces (Antson et al., 1999).", "section": "RESULTS", "ner": [ [ 18, 23, "Phe32", "residue_name_number" ], [ 43, 45, "G3", "residue_name_number" ], [ 65, 92, "TRAP RNA-binding interfaces", "site" ] ] }, { "sid": 76, "sent": "With increasing dose, the D loss associated with the Phe32 side chain was significantly reduced upon RNA binding (Fig. 5 \u25b8 e; Phe32\u2005C\u03b6; p = 0.0014), an indication that radiation-induced conformation disordering of Phe32 had been reduced.", "section": "RESULTS", "ner": [ [ 26, 32, "D loss", "evidence" ], [ 53, 58, "Phe32", "residue_name_number" ], [ 101, 104, "RNA", "chemical" ], [ 126, 131, "Phe32", "residue_name_number" ], [ 214, 219, "Phe32", "residue_name_number" ] ] }, { "sid": 77, "sent": "The extended aliphatic Lys37 side chain stacks against the nearby G1 base, making a series of nonpolar contacts within each RNA-binding interface.", "section": "RESULTS", "ner": [ [ 23, 28, "Lys37", "residue_name_number" ], [ 66, 68, "G1", "residue_name_number" ], [ 94, 111, "nonpolar contacts", "bond_interaction" ], [ 124, 145, "RNA-binding interface", "site" ] ] }, { "sid": 78, "sent": "The D loss for Lys37 side-chain atoms was also reduced when stacked against the G1 base (Fig. 5 \u25b8 f; p = 0.0243 for Lys37\u2005C\u220a atoms).", "section": "RESULTS", "ner": [ [ 4, 10, "D loss", "evidence" ], [ 15, 20, "Lys37", "residue_name_number" ], [ 60, 67, "stacked", "bond_interaction" ], [ 80, 82, "G1", "residue_name_number" ], [ 116, 121, "Lys37", "residue_name_number" ] ] }, { "sid": 79, "sent": "Representative Phe32 and Lys37 atoms were selected to illustrate these trends.", "section": "RESULTS", "ner": [ [ 15, 20, "Phe32", "residue_name_number" ], [ 25, 30, "Lys37", "residue_name_number" ] ] }, { "sid": 80, "sent": "Here, MX radiation-induced specific structural changes within the large TRAP\u2013RNA assembly over a large dose range (1.3\u201325.0\u2005MGy) have been analysed using a high-throughput quantitative approach, providing a measure of the electron-density distribution for each refined atom with increasing dose, D loss.", "section": "DISCUSS", "ner": [ [ 6, 8, "MX", "experimental_method" ], [ 72, 80, "TRAP\u2013RNA", "complex_assembly" ], [ 222, 251, "electron-density distribution", "evidence" ], [ 296, 302, "D loss", "evidence" ] ] }, { "sid": 81, "sent": "Compared with previous studies, the results provide a further step in the detailed characterization of SRD effects in MX.", "section": "DISCUSS", "ner": [ [ 118, 120, "MX", "experimental_method" ] ] }, { "sid": 82, "sent": "Our method\u00adology, which eliminated tedious and error-prone visual inspection, permitted the determination on a per-atom basis of the most damaged sites, as characterized by F obs(d n) \u2212 F obs(d 1) Fourier difference map peaks between successive data sets collected from the same crystal.", "section": "DISCUSS", "ner": [ [ 173, 225, "F obs(d n) \u2212 F obs(d 1) Fourier difference map peaks", "evidence" ], [ 279, 286, "crystal", "evidence" ] ] }, { "sid": 83, "sent": "Here, it provided the precision required to quantify the role of RNA in the damage susceptibilities of equivalent atoms between RNA-bound and nonbound TRAP, but it is applicable to any MX SRD study.", "section": "DISCUSS", "ner": [ [ 65, 68, "RNA", "chemical" ], [ 128, 137, "RNA-bound", "protein_state" ], [ 142, 150, "nonbound", "protein_state" ], [ 151, 155, "TRAP", "complex_assembly" ], [ 185, 187, "MX", "experimental_method" ] ] }, { "sid": 84, "sent": "The RNA was found to be substantially more radiation-resistant than the protein, even at the highest doses investigated (\u223c25.0\u2005MGy), which is in strong concurrence with our previous SRD investigation of the C.Esp1396I protein\u2013DNA complex (Bury et al., 2015).", "section": "DISCUSS", "ner": [ [ 4, 7, "RNA", "chemical" ], [ 43, 62, "radiation-resistant", "protein_state" ], [ 182, 199, "SRD investigation", "experimental_method" ], [ 207, 217, "C.Esp1396I", "complex_assembly" ], [ 226, 229, "DNA", "chemical" ] ] }, { "sid": 85, "sent": "Consistent with that study, at high doses of above \u223c20\u2005MGy, F obs(d n) \u2212 F obs(d 1) map density was detected around P, O3\u2032 and O5\u2032 atoms of the RNA backbone, with no significant difference density localized to RNA ribose and basic subunits.", "section": "DISCUSS", "ner": [ [ 60, 95, "F obs(d n) \u2212 F obs(d 1) map density", "evidence" ], [ 144, 147, "RNA", "chemical" ], [ 178, 196, "difference density", "evidence" ], [ 210, 213, "RNA", "chemical" ], [ 231, 239, "subunits", "structure_element" ] ] }, { "sid": 86, "sent": "RNA backbone disordering thus appears to be the main radiation-induced effect in RNA, with the protein\u2013base interactions maintained even at high doses (>20\u2005MGy).", "section": "DISCUSS", "ner": [ [ 0, 3, "RNA", "chemical" ], [ 81, 84, "RNA", "chemical" ] ] }, { "sid": 87, "sent": "The U4 phosphate exhibited marginally larger D loss values above 20\u2005MGy than G1, A2 and G3 (Supplementary Fig. S4).", "section": "DISCUSS", "ner": [ [ 4, 6, "U4", "residue_name_number" ], [ 7, 16, "phosphate", "chemical" ], [ 45, 51, "D loss", "evidence" ], [ 77, 79, "G1", "residue_name_number" ], [ 81, 83, "A2", "residue_name_number" ], [ 88, 90, "G3", "residue_name_number" ] ] }, { "sid": 88, "sent": "Since U4 is the only refined nucleotide not to exhibit significant base\u2013protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility.", "section": "DISCUSS", "ner": [ [ 6, 8, "U4", "residue_name_number" ], [ 100, 104, "TRAP", "complex_assembly" ], [ 113, 118, "water", "chemical" ], [ 128, 141, "hydrogen bond", "bond_interaction" ], [ 175, 183, "subunits", "structure_element" ], [ 197, 202, "Arg58", "residue_name_number" ], [ 203, 216, "hydrogen bond", "bond_interaction" ], [ 245, 253, "subunits", "structure_element" ], [ 271, 273, "U4", "residue_name_number" ], [ 274, 280, "D loss", "evidence" ] ] }, { "sid": 89, "sent": "At 25.0\u2005MGy, the magnitude of the RNA backbone D loss was of the same order as for the radiation-insensitive Gly\u2005C\u03b1 atoms and on average less than half that of the acidic residues of the protein (Supplementary Fig. S3).", "section": "DISCUSS", "ner": [ [ 34, 37, "RNA", "chemical" ], [ 47, 53, "D loss", "evidence" ], [ 109, 112, "Gly", "residue_name" ] ] }, { "sid": 90, "sent": "Consequently, no clear single-strand breaks could be located, and since RNA-binding within the current TRAP\u2013(GAGUU)10GAG complex is mediated predominantly through base\u2013protein interactions, the biological integrity of the RNA complex was dictated by the rate at which protein decarboxylation occurred.", "section": "DISCUSS", "ner": [ [ 72, 75, "RNA", "chemical" ], [ 103, 120, "TRAP\u2013(GAGUU)10GAG", "complex_assembly" ], [ 222, 225, "RNA", "chemical" ] ] }, { "sid": 91, "sent": "RNA interacting with TRAP was shown to offer significant protection against radiation-induced structural changes.", "section": "DISCUSS", "ner": [ [ 0, 3, "RNA", "chemical" ], [ 21, 25, "TRAP", "complex_assembly" ] ] }, { "sid": 92, "sent": "Both Glu36 and Asp39 bind directly to RNA, each through two hydrogen bonds to guanine bases (G3 and G1, respectively).", "section": "DISCUSS", "ner": [ [ 5, 10, "Glu36", "residue_name_number" ], [ 15, 20, "Asp39", "residue_name_number" ], [ 38, 41, "RNA", "chemical" ], [ 60, 74, "hydrogen bonds", "bond_interaction" ], [ 78, 85, "guanine", "chemical" ], [ 93, 95, "G3", "residue_name_number" ], [ 100, 102, "G1", "residue_name_number" ] ] }, { "sid": 93, "sent": "However, compared with Asp39, Glu36 is strikingly less decarboxylated when bound to RNA (Fig. 4 \u25b8).", "section": "DISCUSS", "ner": [ [ 23, 28, "Asp39", "residue_name_number" ], [ 30, 35, "Glu36", "residue_name_number" ], [ 75, 83, "bound to", "protein_state" ], [ 84, 87, "RNA", "chemical" ] ] }, { "sid": 94, "sent": "This is in good agreement with previous mutagenesis and nucleoside analogue studies (Elliott et al., 2001), which indicated that the G1 nucleotide does not bind to TRAP as strongly as do A2 and G3, and plays little role in the high RNA-binding affinity of TRAP (K d \u2243 1.1 \u00b1 0.4\u2005nM).", "section": "DISCUSS", "ner": [ [ 40, 83, "mutagenesis and nucleoside analogue studies", "experimental_method" ], [ 133, 135, "G1", "residue_name_number" ], [ 164, 168, "TRAP", "complex_assembly" ], [ 187, 189, "A2", "residue_name_number" ], [ 194, 196, "G3", "residue_name_number" ], [ 232, 252, "RNA-binding affinity", "evidence" ], [ 256, 260, "TRAP", "complex_assembly" ], [ 262, 265, "K d", "evidence" ] ] }, { "sid": 95, "sent": "For Glu36 and Asp39, no direct quantitative correlation could be established between hydrogen-bond length and D loss (linear R 2 of <0.23 for all doses; Supplementary Fig. S5).", "section": "DISCUSS", "ner": [ [ 4, 9, "Glu36", "residue_name_number" ], [ 14, 19, "Asp39", "residue_name_number" ], [ 85, 98, "hydrogen-bond", "bond_interaction" ], [ 110, 116, "D loss", "evidence" ], [ 118, 128, "linear R 2", "evidence" ] ] }, { "sid": 96, "sent": "Thus, another factor must be responsible for this clear reduction in Glu36 CO2 decarboxyl\u00adation in RNA-bound TRAP.", "section": "DISCUSS", "ner": [ [ 69, 74, "Glu36", "residue_name_number" ], [ 99, 108, "RNA-bound", "protein_state" ], [ 109, 113, "TRAP", "complex_assembly" ] ] }, { "sid": 97, "sent": "The Glu36 carboxyl side chain also potentially forms hydrogen bonds to His34 and Lys56, but since these interactions are conserved irrespective of G3 nucleotide binding, this cannot directly account for the stabilization effect on Glu36 in RNA-bound TRAP.", "section": "DISCUSS", "ner": [ [ 4, 9, "Glu36", "residue_name_number" ], [ 53, 67, "hydrogen bonds", "bond_interaction" ], [ 71, 76, "His34", "residue_name_number" ], [ 81, 86, "Lys56", "residue_name_number" ], [ 121, 130, "conserved", "protein_state" ], [ 147, 149, "G3", "residue_name_number" ], [ 231, 236, "Glu36", "residue_name_number" ], [ 240, 249, "RNA-bound", "protein_state" ], [ 250, 254, "TRAP", "complex_assembly" ] ] }, { "sid": 98, "sent": "When bound to RNA, the average solvent-accessible area of the Glu36 side-chain O atoms is reduced from \u223c15 to 0\u2005\u00c52.", "section": "DISCUSS", "ner": [ [ 5, 13, "bound to", "protein_state" ], [ 14, 17, "RNA", "chemical" ], [ 62, 67, "Glu36", "residue_name_number" ] ] }, { "sid": 99, "sent": "We propose that with no solvent accessibility Glu36 decarboxylation is inhibited, since the CO2-formation rate K 2 is greatly reduced, and suggest that steric hindrance prevents each radicalized Glu36 CO2 group from achieving the planar conformation required for complete dissociation from TRAP.", "section": "DISCUSS", "ner": [ [ 46, 51, "Glu36", "residue_name_number" ], [ 92, 114, "CO2-formation rate K 2", "evidence" ], [ 195, 200, "Glu36", "residue_name_number" ], [ 290, 294, "TRAP", "complex_assembly" ] ] }, { "sid": 100, "sent": "The electron-recombination rate K \u22121 remains high, however, owing to rapid electron migration through the protein\u2013RNA complex to refill the Glu36 positive hole (the precursor for Glu decarboxylation).", "section": "DISCUSS", "ner": [ [ 4, 36, "electron-recombination rate K \u22121", "evidence" ], [ 106, 117, "protein\u2013RNA", "complex_assembly" ], [ 140, 145, "Glu36", "residue_name_number" ], [ 146, 159, "positive hole", "site" ], [ 179, 182, "Glu", "residue_name" ] ] }, { "sid": 101, "sent": "Upon RNA binding, the Asp39 side-chain carboxyl group solvent-accessible area changes from \u223c75 to 35\u2005\u00c52, still allowing a high CO2-formation rate K 2.", "section": "DISCUSS", "ner": [ [ 5, 8, "RNA", "chemical" ], [ 22, 27, "Asp39", "residue_name_number" ], [ 127, 130, "CO2", "chemical" ], [ 141, 149, "rate K 2", "evidence" ] ] }, { "sid": 102, "sent": "The prevalence of radical attack from solvent channels surrounding the protein in the crystal is a questionable cause, considering previous observations indicating that the strongly oxidizing hydroxyl radical is immobile at 100\u2005K (Allan et al., 2013; Owen et al., 2012).", "section": "DISCUSS", "ner": [ [ 86, 93, "crystal", "evidence" ] ] }, { "sid": 103, "sent": "By comparing equivalent acidic residues with and without RNA, we have now deconvoluted the role of solvent accessibility from other local protein environment factors, and thus propose a suitable mechanism by which exceptionally low solvent accessibility can reduce the rate of decarboxylation.", "section": "DISCUSS", "ner": [ [ 40, 44, "with", "protein_state" ], [ 49, 56, "without", "protein_state" ], [ 57, 60, "RNA", "chemical" ] ] }, { "sid": 104, "sent": "Apart from these RNA-binding interfaces, RNA binding was seen to enhance decarboxylation for residues Glu50, Glu71 and Glu73, all of which are involved in crystal contacts between TRAP rings (Fig. 4 \u25b8 c).", "section": "DISCUSS", "ner": [ [ 17, 39, "RNA-binding interfaces", "site" ], [ 41, 44, "RNA", "chemical" ], [ 102, 107, "Glu50", "residue_name_number" ], [ 109, 114, "Glu71", "residue_name_number" ], [ 119, 124, "Glu73", "residue_name_number" ], [ 180, 184, "TRAP", "complex_assembly" ], [ 185, 190, "rings", "structure_element" ] ] }, { "sid": 105, "sent": "However, for each of these residues the exact crystal contacts are not preserved between bound and nonbound TRAP or even between monomers within one TRAP ring.", "section": "DISCUSS", "ner": [ [ 89, 94, "bound", "protein_state" ], [ 99, 107, "nonbound", "protein_state" ], [ 108, 112, "TRAP", "complex_assembly" ], [ 149, 153, "TRAP", "complex_assembly" ], [ 154, 158, "ring", "structure_element" ] ] }, { "sid": 106, "sent": "For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit.", "section": "DISCUSS", "ner": [ [ 16, 21, "bound", "protein_state" ], [ 22, 26, "TRAP", "complex_assembly" ], [ 28, 33, "Glu73", "residue_name_number" ], [ 61, 67, "lysine", "residue_name" ], [ 86, 94, "subunits", "structure_element" ], [ 107, 115, "nonbound", "protein_state" ], [ 116, 120, "TRAP", "complex_assembly" ], [ 152, 157, "Glu73", "residue_name_number" ], [ 202, 208, "waters", "chemical" ], [ 217, 224, "subunit", "structure_element" ] ] }, { "sid": 107, "sent": "Radiation-induced side-chain conformational changes have been poorly characterized in previous SRD investigations owing to their strong dependence on packing density and geometric strain.", "section": "DISCUSS", "ner": [ [ 95, 113, "SRD investigations", "experimental_method" ] ] }, { "sid": 108, "sent": "Such structural changes are known to have significant roles within enzymatic pathways, and experimenters must be aware of these possible confounding factors when assigning true functional mechanisms using MX.", "section": "DISCUSS", "ner": [ [ 205, 207, "MX", "experimental_method" ] ] }, { "sid": 109, "sent": "Our results show that RNA binding to TRAP physically stabilizes non-acidic residues within the TRAP macromolecule, most notably Lys37 and Phe32, which stack against the G1 and G3 bases, respectively.", "section": "DISCUSS", "ner": [ [ 22, 25, "RNA", "chemical" ], [ 37, 41, "TRAP", "complex_assembly" ], [ 95, 99, "TRAP", "complex_assembly" ], [ 128, 133, "Lys37", "residue_name_number" ], [ 138, 143, "Phe32", "residue_name_number" ], [ 169, 171, "G1", "residue_name_number" ], [ 176, 178, "G3", "residue_name_number" ] ] }, { "sid": 110, "sent": "It has been suggested (Burmeister, 2000) that Tyr residues can lose their aromatic \u2013OH group owing to radiation-induced effects; however, no energetically favourable pathway for \u2013OH cleavage exists and this has not been detected in aqueous radiation-chemistry studies.", "section": "DISCUSS", "ner": [ [ 46, 49, "Tyr", "residue_name" ] ] }, { "sid": 111, "sent": "In TRAP, D loss increased at a similar rate for both the Tyr O atoms and aromatic ring atoms, suggesting that full ring conformational disordering is more likely.", "section": "DISCUSS", "ner": [ [ 3, 7, "TRAP", "complex_assembly" ], [ 9, 15, "D loss", "evidence" ], [ 57, 60, "Tyr", "residue_name" ], [ 82, 86, "ring", "structure_element" ], [ 115, 119, "ring", "structure_element" ] ] }, { "sid": 112, "sent": "Indeed, no convincing reproducible Fourier difference peaks above the background map noise were observed around any Tyr terminal \u2013OH groups.", "section": "DISCUSS", "ner": [ [ 35, 59, "Fourier difference peaks", "evidence" ], [ 81, 84, "map", "evidence" ], [ 116, 119, "Tyr", "residue_name" ] ] }, { "sid": 113, "sent": "The RNA-stabilization effects on protein are observed at short ranges and are restricted to within the RNA-binding interfaces around the TRAP ring.", "section": "DISCUSS", "ner": [ [ 4, 7, "RNA", "chemical" ], [ 103, 125, "RNA-binding interfaces", "site" ], [ 137, 141, "TRAP", "complex_assembly" ], [ 142, 146, "ring", "structure_element" ] ] }, { "sid": 114, "sent": "For example, Asp17 is located \u223c6.8\u2005\u00c5 from the G1 base, outside the RNA-binding interfaces, and has indistinguishable C\u03b3 atom D loss dose-dynamics between RNA-bound and nonbound TRAP (p > 0.9).", "section": "DISCUSS", "ner": [ [ 13, 18, "Asp17", "residue_name_number" ], [ 46, 48, "G1", "residue_name_number" ], [ 67, 89, "RNA-binding interfaces", "site" ], [ 127, 145, "loss dose-dynamics", "evidence" ], [ 154, 163, "RNA-bound", "protein_state" ], [ 168, 176, "nonbound", "protein_state" ], [ 177, 181, "TRAP", "complex_assembly" ] ] }, { "sid": 115, "sent": "An increase in the dose at which functionally important residues remain intact has biological ramifications for understanding the mechanisms at which ionizing radiation damage is mitigated within naturally forming DNA\u2013protein and RNA\u2013protein complexes.", "section": "DISCUSS", "ner": [ [ 214, 225, "DNA\u2013protein", "complex_assembly" ], [ 230, 241, "RNA\u2013protein", "complex_assembly" ] ] }, { "sid": 116, "sent": "Observations of lower protein radiation-sensitivity in DNA-bound forms have been recorded in solution at RT at much lower doses (\u223c1\u2005kGy) than those used for typical MX experiments [e.g. an oestrogen response element\u2013receptor complex (St\u00edsov\u00e1 et al., 2006) and a DNA glycosylase and its abasic DNA target site (Gillard et al., 2004)].", "section": "DISCUSS", "ner": [ [ 55, 64, "DNA-bound", "protein_state" ], [ 165, 167, "MX", "experimental_method" ], [ 262, 277, "DNA glycosylase", "protein_type" ], [ 286, 308, "abasic DNA target site", "site" ] ] }, { "sid": 117, "sent": "In these studies, the main damaging species is predicted to be the oxidizing hydroxyl radical produced through solvent irradiation, which is known to add to double covalent bonds within both DNA and RNA bases to induce strand breaks and base modification (Spotheim-Maurizot & Dav\u00eddkov\u00e1, 2011; Chance et al., 1997).", "section": "DISCUSS", "ner": [ [ 191, 194, "DNA", "chemical" ], [ 199, 202, "RNA", "chemical" ] ] }, { "sid": 118, "sent": "It was suggested that physical screening of DNA by protein shielded the DNA\u2013protein interaction sites from radical damage, yielding an extended life-dose for the nucleoprotein complex compared with separate protein and DNA constituents at RT.", "section": "DISCUSS", "ner": [ [ 44, 47, "DNA", "chemical" ], [ 72, 101, "DNA\u2013protein interaction sites", "site" ], [ 219, 222, "DNA", "chemical" ] ] }, { "sid": 119, "sent": "However, in the current MX study at 100\u2005K, the main damaging species are believed to be migrating LEEs and holes produced directly within the protein\u2013RNA components or in closely associated solvent.", "section": "DISCUSS", "ner": [ [ 24, 26, "MX", "experimental_method" ], [ 142, 153, "protein\u2013RNA", "complex_assembly" ] ] }, { "sid": 120, "sent": "The results presented here suggest that biologically relevant nucleoprotein complexes also exhibit prolonged life-doses under the effect of LEE-induced structural changes, involving direct physical protection of key RNA-binding residues.", "section": "DISCUSS", "ner": [ [ 62, 75, "nucleoprotein", "complex_assembly" ], [ 216, 236, "RNA-binding residues", "site" ] ] }, { "sid": 121, "sent": "Such reduced radiation-sensitivity in this case ensures that the interacting protein remains bound long enough to the RNA to complete its function, even whilst exposed to ionizing radiation.", "section": "DISCUSS", "ner": [ [ 93, 98, "bound", "protein_state" ], [ 118, 121, "RNA", "chemical" ] ] }, { "sid": 122, "sent": "Within the nonbound TRAP macromolecule, the acidic residues within the unoccupied RNA-binding interfaces (Asp39, Glu36, Glu42) are notably amongst the most susceptible residues within the asymmetric unit (Fig. 4 \u25b8).", "section": "DISCUSS", "ner": [ [ 11, 19, "nonbound", "protein_state" ], [ 20, 24, "TRAP", "complex_assembly" ], [ 82, 104, "RNA-binding interfaces", "site" ], [ 106, 111, "Asp39", "residue_name_number" ], [ 113, 118, "Glu36", "residue_name_number" ], [ 120, 125, "Glu42", "residue_name_number" ] ] }, { "sid": 123, "sent": "When exposed to X-rays, these residues will be preferentially damaged by X-rays and subsequently reduce the affinity with which TRAP binds to RNA.", "section": "DISCUSS", "ner": [ [ 128, 132, "TRAP", "complex_assembly" ], [ 142, 145, "RNA", "chemical" ] ] }, { "sid": 124, "sent": "Within the cellular environment, this mechanism could reduce the risk that radiation-damaged proteins might bind to RNA, thus avoiding the detrimental introduction of incorrect DNA-repair, transcriptional and base-modification pathways.", "section": "DISCUSS", "ner": [ [ 116, 119, "RNA", "chemical" ], [ 177, 180, "DNA", "chemical" ] ] }, { "sid": 125, "sent": "The TRAP\u2013(GAGUU)10GAG complex asymmetric unit (PDB entry 1gtf; Hopcroft et al., 2002).", "section": "FIG", "ner": [ [ 4, 21, "TRAP\u2013(GAGUU)10GAG", "complex_assembly" ] ] }, { "sid": 126, "sent": "Bound tryptophan ligands are represented as coloured spheres.", "section": "FIG", "ner": [ [ 0, 5, "Bound", "protein_state" ], [ 6, 16, "tryptophan", "chemical" ] ] }, { "sid": 127, "sent": "RNA is shown is yellow.", "section": "FIG", "ner": [ [ 0, 3, "RNA", "chemical" ] ] }, { "sid": 128, "sent": "(a) Electron-density loss sites as indicated by D loss in the TRAP\u2013RNA complex crystal by residue/nucleotide type for five doses [sites determined above the 4\u00d7 average D loss threshold, calculated over the TRAP\u2013RNA structure for the first difference map: F obs(d 2) \u2212 F obs(d 1)].", "section": "FIG", "ner": [ [ 62, 70, "TRAP\u2013RNA", "complex_assembly" ], [ 79, 86, "crystal", "evidence" ], [ 206, 214, "TRAP\u2013RNA", "complex_assembly" ], [ 215, 224, "structure", "evidence" ], [ 239, 253, "difference map", "evidence" ] ] }, { "sid": 129, "sent": "(b) Average D loss for each residue/nucleotide type with respect to the DWD (diffraction-weighted dose; Zeldin, Brock\u00adhauser et al., 2013).", "section": "FIG", "ner": [ [ 72, 75, "DWD", "evidence" ], [ 77, 102, "diffraction-weighted dose", "evidence" ] ] }, { "sid": 130, "sent": "Only a subset of key TRAP residue types are included.", "section": "FIG", "ner": [ [ 21, 25, "TRAP", "complex_assembly" ] ] }, { "sid": 131, "sent": "The average D loss (calculated over the whole TRAP asymmetric unit) is shown at each dose (dashed line).", "section": "FIG", "ner": [ [ 46, 50, "TRAP", "complex_assembly" ] ] }, { "sid": 132, "sent": "In (a) clear difference density is observed around the Glu42 carboxyl side chain in chain H, within the lowest dose difference map at d 2 = 3.9\u2005MGy.", "section": "FIG", "ner": [ [ 13, 31, "difference density", "evidence" ], [ 55, 60, "Glu42", "residue_name_number" ], [ 104, 130, "lowest dose difference map", "evidence" ] ] }, { "sid": 133, "sent": "Radiation-induced protein disordering is evident across the large dose range (b, c); in comparison, no clear deterioration of the RNA density was observed.", "section": "FIG", "ner": [ [ 130, 133, "RNA", "chemical" ], [ 134, 141, "density", "evidence" ] ] }, { "sid": 134, "sent": " D loss calculated for all side-chain carboxyl group Glu\u2005C\u03b4 and Asp\u2005C\u03b3 atoms within the TRAP\u2013RNA complex for a dose of 19.3\u2005MGy (d 8).", "section": "FIG", "ner": [ [ 53, 56, "Glu", "residue_name" ], [ 64, 67, "Asp", "residue_name" ], [ 88, 96, "TRAP\u2013RNA", "complex_assembly" ] ] }, { "sid": 135, "sent": "Residues have been grouped by amino-acid number, and split into bound and nonbound groupings, with each bar representing the mean calculated over 11 equivalent atoms around a TRAP ring.", "section": "FIG", "ner": [ [ 64, 69, "bound", "protein_state" ], [ 74, 82, "nonbound", "protein_state" ], [ 175, 179, "TRAP", "complex_assembly" ], [ 180, 184, "ring", "structure_element" ] ] }, { "sid": 136, "sent": " D loss against dose for (a) Glu36\u2005C\u03b4, (b) Asp39\u2005C\u03b3, (c) Glu42\u2005O\u220a1, (d) Glu42\u2005O\u220a2, (e) Phe32\u2005C\u03b6 and (f) Lys37\u2005C\u220a atoms.", "section": "FIG", "ner": [ [ 29, 34, "Glu36", "residue_name_number" ], [ 43, 48, "Asp39", "residue_name_number" ], [ 57, 62, "Glu42", "residue_name_number" ], [ 72, 77, "Glu42", "residue_name_number" ], [ 87, 92, "Phe32", "residue_name_number" ], [ 104, 109, "Lys37", "residue_name_number" ] ] }, { "sid": 137, "sent": "95% CI are included for each set of 11 equivalent atoms grouped as bound/nonbound.", "section": "FIG", "ner": [ [ 67, 72, "bound", "protein_state" ], [ 73, 81, "nonbound", "protein_state" ] ] }, { "sid": 138, "sent": "RNA-binding interface interactions are shown for TRAP chain N, with the F obs(d 7) \u2212 F obs(d 1) Fourier difference map (dose 16.7\u2005MGy) overlaid and contoured at a \u00b14\u03c3 level.", "section": "FIG", "ner": [ [ 0, 21, "RNA-binding interface", "site" ], [ 49, 53, "TRAP", "complex_assembly" ] ] } ] }, "PMC4802042": { "annotations": [ { "sid": 0, "sent": "A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation", "section": "TITLE", "ner": [ [ 21, 55, "JNK/p38-specific MAPK phosphatases", "protein_type" ], [ 77, 81, "JNK1", "protein" ] ] }, { "sid": 1, "sent": "Mitogen-activated protein kinases (MAPKs), important in a large array of signalling pathways, are tightly controlled by a cascade of protein kinases and by MAPK phosphatases (MKPs).", "section": "ABSTRACT", "ner": [ [ 0, 33, "Mitogen-activated protein kinases", "protein_type" ], [ 35, 40, "MAPKs", "protein_type" ], [ 133, 148, "protein kinases", "protein_type" ], [ 156, 173, "MAPK phosphatases", "protein_type" ], [ 175, 179, "MKPs", "protein_type" ] ] }, { "sid": 2, "sent": "MAPK signalling efficiency and specificity is modulated by protein\u2013protein interactions between individual MAPKs and the docking motifs in cognate binding partners.", "section": "ABSTRACT", "ner": [ [ 0, 4, "MAPK", "protein_type" ], [ 107, 112, "MAPKs", "protein_type" ], [ 121, 135, "docking motifs", "structure_element" ] ] }, { "sid": 3, "sent": "Two types of docking interactions have been identified: D-motif-mediated interaction and FXF-docking interaction.", "section": "ABSTRACT", "ner": [ [ 56, 63, "D-motif", "structure_element" ], [ 89, 112, "FXF-docking interaction", "site" ] ] }, { "sid": 4, "sent": "Here we report the crystal structure of JNK1 bound to the catalytic domain of MKP7 at 2.4-\u00c5 resolution, providing high-resolution structural insight into the FXF-docking interaction.", "section": "ABSTRACT", "ner": [ [ 19, 36, "crystal structure", "evidence" ], [ 40, 44, "JNK1", "protein" ], [ 45, 53, "bound to", "protein_state" ], [ 58, 74, "catalytic domain", "structure_element" ], [ 78, 82, "MKP7", "protein" ], [ 158, 181, "FXF-docking interaction", "site" ] ] }, { "sid": 5, "sent": "The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix \u03b1G. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function.", "section": "ABSTRACT", "ner": [ [ 4, 22, "285FNFL288 segment", "structure_element" ], [ 26, 30, "MKP7", "protein" ], [ 51, 67, "hydrophobic site", "site" ], [ 71, 75, "JNK1", "protein" ], [ 93, 97, "MAPK", "protein_type" ], [ 112, 117, "helix", "structure_element" ], [ 118, 120, "\u03b1G", "structure_element" ], [ 122, 141, "Biochemical studies", "experimental_method" ], [ 167, 183, "highly conserved", "protein_state" ], [ 184, 200, "structural motif", "structure_element" ], [ 234, 244, "MKP family", "protein_type" ], [ 305, 308, "MKP", "protein_type" ], [ 309, 313, "MAPK", "protein_type" ] ] }, { "sid": 6, "sent": " The important MAPK family of signalling proteins is controlled by MAPK phosphatases (MKPs).", "section": "ABSTRACT", "ner": [ [ 15, 26, "MAPK family", "protein_type" ], [ 67, 84, "MAPK phosphatases", "protein_type" ], [ 86, 90, "MKPs", "protein_type" ] ] }, { "sid": 7, "sent": "Here, the authors report the structure of MKP7 bound to JNK1 and characterise the conserved MKP-MAPK interaction.", "section": "ABSTRACT", "ner": [ [ 29, 38, "structure", "evidence" ], [ 42, 46, "MKP7", "protein" ], [ 47, 55, "bound to", "protein_state" ], [ 56, 60, "JNK1", "protein" ], [ 82, 91, "conserved", "protein_state" ], [ 92, 95, "MKP", "protein_type" ], [ 96, 100, "MAPK", "protein_type" ] ] }, { "sid": 8, "sent": "The mitogen-activated protein kinases (MAPKs) are central components of the signal-transduction pathways, which mediate the cellular response to a variety of extracellular stimuli, ranging from growth factors to environmental stresses.", "section": "INTRO", "ner": [ [ 4, 37, "mitogen-activated protein kinases", "protein_type" ], [ 39, 44, "MAPKs", "protein_type" ] ] }, { "sid": 9, "sent": "The MAPK signalling pathways are evolutionally highly conserved.", "section": "INTRO", "ner": [ [ 4, 8, "MAPK", "protein_type" ] ] }, { "sid": 10, "sent": "The basic assembly of MAPK pathways is a three-tier kinase module that establishes a sequential activation cascade: a MAPK kinase kinase activates a MAPK kinase, which in turn activates a MAPK.", "section": "INTRO", "ner": [ [ 22, 26, "MAPK", "protein_type" ], [ 52, 58, "kinase", "protein_type" ], [ 118, 136, "MAPK kinase kinase", "protein_type" ], [ 149, 160, "MAPK kinase", "protein_type" ], [ 188, 192, "MAPK", "protein_type" ] ] }, { "sid": 11, "sent": "The three best-characterized MAPK signalling pathways are mediated by the kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38.", "section": "INTRO", "ner": [ [ 29, 33, "MAPK", "protein_type" ], [ 74, 81, "kinases", "protein_type" ], [ 82, 119, "extracellular signal-regulated kinase", "protein_type" ], [ 121, 124, "ERK", "protein_type" ], [ 127, 150, "c-Jun N-terminal kinase", "protein_type" ], [ 152, 155, "JNK", "protein_type" ], [ 161, 164, "p38", "protein_type" ] ] }, { "sid": 12, "sent": "The ERK pathway is activated by various mitogens and phorbol esters, whereas the JNK and p38 pathways are stimulated mainly by environmental stress and inflammatory cytokines.", "section": "INTRO", "ner": [ [ 4, 7, "ERK", "protein_type" ], [ 81, 84, "JNK", "protein_type" ], [ 89, 92, "p38", "protein_type" ], [ 165, 174, "cytokines", "protein_type" ] ] }, { "sid": 13, "sent": "The MAPKs are activated by MAPK kinases that phosphorylate the MAPKs at conserved threonine and tyrosine residues within their activation loop.", "section": "INTRO", "ner": [ [ 4, 9, "MAPKs", "protein_type" ], [ 27, 39, "MAPK kinases", "protein_type" ], [ 63, 68, "MAPKs", "protein_type" ], [ 72, 81, "conserved", "protein_state" ], [ 82, 91, "threonine", "residue_name" ], [ 96, 104, "tyrosine", "residue_name" ], [ 127, 142, "activation loop", "structure_element" ] ] }, { "sid": 14, "sent": "After activation, each MAPK phosphorylates a distinct set of protein substrates, which act as the critical effectors that enable cells to mount the appropriate responses to varied stimuli.", "section": "INTRO", "ner": [ [ 23, 27, "MAPK", "protein_type" ] ] }, { "sid": 15, "sent": "MAPKs lie at the bottom of conserved three-component phosphorylation cascades and utilize docking interactions to link module components and bind substrates.", "section": "INTRO", "ner": [ [ 0, 5, "MAPKs", "protein_type" ] ] }, { "sid": 16, "sent": "Two types of docking motifs have been identified in MAPK substrates and cognate proteins: kinase-interacting motif (D-motif) and FXF-motif (also called DEF motif, docking site for ERK FXF).", "section": "INTRO", "ner": [ [ 13, 27, "docking motifs", "structure_element" ], [ 52, 56, "MAPK", "protein_type" ], [ 90, 114, "kinase-interacting motif", "structure_element" ], [ 116, 123, "D-motif", "structure_element" ], [ 129, 138, "FXF-motif", "structure_element" ], [ 152, 161, "DEF motif", "structure_element" ], [ 163, 175, "docking site", "site" ], [ 180, 183, "ERK", "protein_type" ], [ 184, 187, "FXF", "structure_element" ] ] }, { "sid": 17, "sent": "The best-studied docking interactions are those between MAP kinases and \u2018D-motifs', which consists of two or more basic residues followed by a short linker and a cluster of hydrophobic residues.", "section": "INTRO", "ner": [ [ 56, 67, "MAP kinases", "protein_type" ], [ 73, 81, "D-motifs", "structure_element" ], [ 143, 155, "short linker", "structure_element" ] ] }, { "sid": 18, "sent": "The D-motif-docking site (D-site) in MAPKs is situated in a noncatalytic region opposite of the kinase catalytic pocket and is comprised of a highly acidic patch and a hydrophobic groove.", "section": "INTRO", "ner": [ [ 4, 24, "D-motif-docking site", "site" ], [ 26, 32, "D-site", "site" ], [ 37, 42, "MAPKs", "protein_type" ], [ 60, 79, "noncatalytic region", "site" ], [ 96, 102, "kinase", "protein_type" ], [ 103, 119, "catalytic pocket", "site" ], [ 142, 161, "highly acidic patch", "site" ], [ 168, 186, "hydrophobic groove", "site" ] ] }, { "sid": 19, "sent": "D-motifs are found in many MAPK-interacting proteins, including substrates, activating kinases and inactivating phosphatases, as well as scaffolding proteins.", "section": "INTRO", "ner": [ [ 0, 8, "D-motifs", "structure_element" ], [ 27, 52, "MAPK-interacting proteins", "protein_type" ], [ 87, 94, "kinases", "protein_type" ], [ 112, 124, "phosphatases", "protein_type" ] ] }, { "sid": 20, "sent": "A second docking motif for MAPKs consists of two Phe residues separated by one residue (FXF-motif).", "section": "INTRO", "ner": [ [ 2, 22, "second docking motif", "structure_element" ], [ 27, 32, "MAPKs", "protein_type" ], [ 49, 52, "Phe", "residue_name" ], [ 88, 97, "FXF-motif", "structure_element" ] ] }, { "sid": 21, "sent": "This motif has been observed in several MAPK substrates.", "section": "INTRO", "ner": [ [ 40, 44, "MAPK", "protein_type" ] ] }, { "sid": 22, "sent": "The FXF-motif-binding site of ERK2 has been mapped to a hydrophobic pocket formed between the P+1 site, \u03b1G helix and the MAPK insert.", "section": "INTRO", "ner": [ [ 4, 26, "FXF-motif-binding site", "site" ], [ 30, 34, "ERK2", "protein" ], [ 56, 74, "hydrophobic pocket", "site" ], [ 94, 102, "P+1 site", "site" ], [ 104, 112, "\u03b1G helix", "structure_element" ], [ 121, 132, "MAPK insert", "structure_element" ] ] }, { "sid": 23, "sent": "However, the generality and mechanism of the FXF-mediated interaction is unclear.", "section": "INTRO", "ner": [ [ 45, 48, "FXF", "structure_element" ] ] }, { "sid": 24, "sent": "The physiological outcome of MAPK signalling depends on both the magnitude and the duration of kinase activation.", "section": "INTRO", "ner": [ [ 29, 33, "MAPK", "protein_type" ] ] }, { "sid": 25, "sent": "Downregulation of MAPK activity can be achieved through direct dephosphorylation of the phospho-threonine and/or tyrosine residues by various serine/threonine phosphatases, tyrosine phosphatases and dual-specificity phosphatases (DUSPs) termed MKPs.", "section": "INTRO", "ner": [ [ 18, 22, "MAPK", "protein_type" ], [ 88, 121, "phospho-threonine and/or tyrosine", "residue_name" ], [ 142, 171, "serine/threonine phosphatases", "protein_type" ], [ 173, 194, "tyrosine phosphatases", "protein_type" ], [ 199, 228, "dual-specificity phosphatases", "protein_type" ], [ 230, 235, "DUSPs", "protein_type" ], [ 244, 248, "MKPs", "protein_type" ] ] }, { "sid": 26, "sent": "MKPs constitute a group of DUSPs that are characterized by their ability to dephosphorylate both phosphotyrosine and phosphoserine/phospho-threonine residues within a substrate.", "section": "INTRO", "ner": [ [ 0, 4, "MKPs", "protein_type" ], [ 27, 32, "DUSPs", "protein_type" ], [ 97, 112, "phosphotyrosine", "residue_name" ], [ 117, 130, "phosphoserine", "residue_name" ], [ 131, 148, "phospho-threonine", "residue_name" ] ] }, { "sid": 27, "sent": "Dysregulated expression of MKPs has been associated with pathogenesis of various diseases, and understanding their precise recognition mechanism presents an important challenge and opportunity for drug development.", "section": "INTRO", "ner": [ [ 27, 31, "MKPs", "protein_type" ] ] }, { "sid": 28, "sent": "Here, we present the crystal structure of JNK1 in complex with the catalytic domain of MKP7.", "section": "INTRO", "ner": [ [ 21, 38, "crystal structure", "evidence" ], [ 42, 46, "JNK1", "protein" ], [ 47, 62, "in complex with", "protein_state" ], [ 67, 83, "catalytic domain", "structure_element" ], [ 87, 91, "MKP7", "protein" ] ] }, { "sid": 29, "sent": "This structure reveals the molecular mechanism underlying the docking interaction between MKP7 and JNK1.", "section": "INTRO", "ner": [ [ 5, 14, "structure", "evidence" ], [ 90, 94, "MKP7", "protein" ], [ 99, 103, "JNK1", "protein" ] ] }, { "sid": 30, "sent": "In the JNK1\u2013MKP7 complex, a hydrophobic motif (285FNFL288) that initiates the helix \u03b15 in the MKP7 catalytic domain directly binds to the FXF-motif-binding site on JNK1, providing the structural insight into the classic FXF-type docking interaction.", "section": "INTRO", "ner": [ [ 7, 16, "JNK1\u2013MKP7", "complex_assembly" ], [ 28, 45, "hydrophobic motif", "structure_element" ], [ 47, 57, "285FNFL288", "structure_element" ], [ 78, 83, "helix", "structure_element" ], [ 84, 86, "\u03b15", "structure_element" ], [ 94, 98, "MKP7", "protein" ], [ 99, 115, "catalytic domain", "structure_element" ], [ 138, 160, "FXF-motif-binding site", "site" ], [ 164, 168, "JNK1", "protein" ], [ 220, 248, "FXF-type docking interaction", "site" ] ] }, { "sid": 31, "sent": "Biochemical and modelling studies further demonstrate that the molecular interactions mediate this key element for substrate recognition are highly conserved among all MKP-family members.", "section": "INTRO", "ner": [ [ 0, 33, "Biochemical and modelling studies", "experimental_method" ], [ 168, 186, "MKP-family members", "protein_type" ] ] }, { "sid": 32, "sent": "Thus, our study reveals a hitherto unrecognized interaction mode for encoding complex target specificity among MAPK isoforms.", "section": "INTRO", "ner": [ [ 111, 124, "MAPK isoforms", "protein_type" ] ] }, { "sid": 33, "sent": "Interaction of JNK1 with the MKP7 catalytic domain", "section": "RESULTS", "ner": [ [ 15, 19, "JNK1", "protein" ], [ 29, 33, "MKP7", "protein" ], [ 34, 50, "catalytic domain", "structure_element" ] ] }, { "sid": 34, "sent": "DUSPs belong to the protein-tyrosine phosphatases (PTPase) superfamily, which is defined by the PTPase-signature motif CXXGXXR.", "section": "RESULTS", "ner": [ [ 0, 5, "DUSPs", "protein_type" ], [ 20, 49, "protein-tyrosine phosphatases", "protein_type" ], [ 51, 57, "PTPase", "protein_type" ], [ 96, 102, "PTPase", "protein_type" ], [ 119, 126, "CXXGXXR", "structure_element" ] ] }, { "sid": 35, "sent": "MKPs represent a distinct subfamily within a larger group of DUSPs.", "section": "RESULTS", "ner": [ [ 0, 4, "MKPs", "protein_type" ], [ 61, 66, "DUSPs", "protein_type" ] ] }, { "sid": 36, "sent": "In mammalian cells, the MKP subfamily includes 10 distinct catalytically active MKPs.", "section": "RESULTS", "ner": [ [ 3, 12, "mammalian", "taxonomy_domain" ], [ 24, 37, "MKP subfamily", "protein_type" ], [ 59, 79, "catalytically active", "protein_state" ], [ 80, 84, "MKPs", "protein_type" ] ] }, { "sid": 37, "sent": "All MKPs contain a highly conserved C-terminal catalytic domain (CD) and an N-terminal kinase-binding domain (KBD).", "section": "RESULTS", "ner": [ [ 4, 8, "MKPs", "protein_type" ], [ 19, 35, "highly conserved", "protein_state" ], [ 47, 63, "catalytic domain", "structure_element" ], [ 65, 67, "CD", "structure_element" ], [ 87, 108, "kinase-binding domain", "structure_element" ], [ 110, 113, "KBD", "structure_element" ] ] }, { "sid": 38, "sent": "The KBD is homologous to the rhodanese family and contains an intervening cluster of basic amino acids, which has been suggested to be important for interacting with the target MAPKs.", "section": "RESULTS", "ner": [ [ 4, 7, "KBD", "structure_element" ], [ 29, 45, "rhodanese family", "protein_type" ], [ 177, 182, "MAPKs", "protein_type" ] ] }, { "sid": 39, "sent": "On the basis of sequence similarity, substrate specificity and predominant subcellular localization, the MKP family can be further divided into three groups (Fig. 1).", "section": "RESULTS", "ner": [ [ 105, 115, "MKP family", "protein_type" ] ] }, { "sid": 40, "sent": "Biochemical and structural studies have revealed that the KBD of MKPs is critical for MKP3 docking to ERK2, and MKP5 binding to p38\u03b1, although their binding mechanisms are completely different.", "section": "RESULTS", "ner": [ [ 0, 34, "Biochemical and structural studies", "experimental_method" ], [ 58, 61, "KBD", "structure_element" ], [ 65, 69, "MKPs", "protein_type" ], [ 86, 90, "MKP3", "protein" ], [ 102, 106, "ERK2", "protein" ], [ 112, 116, "MKP5", "protein" ], [ 128, 132, "p38\u03b1", "protein" ] ] }, { "sid": 41, "sent": "However, it is unknown if other MAPKs can interact with the KBD of their cognate phosphatases in the same manner as observed for recognition of ERK2 and p38\u03b1 by their MKPs, or whether they recognize distinct docking motifs of MKPs.", "section": "RESULTS", "ner": [ [ 32, 37, "MAPKs", "protein_type" ], [ 60, 63, "KBD", "structure_element" ], [ 81, 93, "phosphatases", "protein_type" ], [ 144, 148, "ERK2", "protein" ], [ 153, 157, "p38\u03b1", "protein" ], [ 167, 171, "MKPs", "protein_type" ], [ 208, 222, "docking motifs", "structure_element" ], [ 226, 230, "MKPs", "protein_type" ] ] }, { "sid": 42, "sent": "MKP7, the biggest molecule in the MKP family, selectively inactivates JNK and p38 following stress activation.", "section": "RESULTS", "ner": [ [ 0, 4, "MKP7", "protein" ], [ 34, 44, "MKP family", "protein_type" ], [ 70, 73, "JNK", "protein_type" ], [ 78, 81, "p38", "protein_type" ] ] }, { "sid": 43, "sent": "In addition to the CD and KBD, MKP7 has a long C-terminal region that contains both nuclear localization and export sequences by which MKP7 shuttles between the nucleus and the cytoplasm (Fig. 2a).", "section": "RESULTS", "ner": [ [ 19, 21, "CD", "structure_element" ], [ 26, 29, "KBD", "structure_element" ], [ 31, 35, "MKP7", "protein" ], [ 47, 64, "C-terminal region", "structure_element" ], [ 135, 139, "MKP7", "protein" ] ] }, { "sid": 44, "sent": "To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7\u0394C304, residues 5\u2013303) and MKP7-CD (residues 156\u2013301) towards phosphorylated JNK1 (pJNK1).", "section": "RESULTS", "ner": [ [ 49, 66, "N-terminal domain", "structure_element" ], [ 74, 78, "MKP7", "protein" ], [ 89, 93, "JNK1", "protein" ], [ 94, 111, "dephosphorylation", "ptm" ], [ 135, 142, "kinetic", "evidence" ], [ 172, 182, "truncation", "experimental_method" ], [ 186, 190, "MKP7", "protein" ], [ 192, 201, "MKP7\u0394C304", "mutant" ], [ 212, 217, "5\u2013303", "residue_range" ], [ 223, 227, "MKP7", "protein" ], [ 228, 230, "CD", "structure_element" ], [ 241, 248, "156\u2013301", "residue_range" ], [ 258, 272, "phosphorylated", "protein_state" ], [ 273, 277, "JNK1", "protein" ], [ 279, 280, "p", "protein_state" ], [ 280, 284, "JNK1", "protein" ] ] }, { "sid": 45, "sent": "Figure 2b shows the variation of initial rates of the MKP7\u0394C304 and MKP7-CD-catalysed reaction with the concentration of phospho-JNK1. Because the concentrations of MKP7 and pJNK1 were comparable in the reaction, the assumption that the free-substrate concentration is equal to the total substrate concentration is not valid.", "section": "RESULTS", "ner": [ [ 20, 46, "variation of initial rates", "evidence" ], [ 54, 63, "MKP7\u0394C304", "mutant" ], [ 68, 72, "MKP7", "protein" ], [ 73, 75, "CD", "structure_element" ], [ 121, 128, "phospho", "protein_state" ], [ 129, 133, "JNK1", "protein" ], [ 165, 169, "MKP7", "protein" ], [ 174, 175, "p", "protein_state" ], [ 175, 179, "JNK1", "protein" ] ] }, { "sid": 46, "sent": "Thus, the kinetic data were analysed using the general initial velocity equation, taking substrate depletion into account:", "section": "RESULTS", "ner": [ [ 10, 22, "kinetic data", "evidence" ], [ 55, 80, "initial velocity equation", "evidence" ] ] }, { "sid": 47, "sent": "The kcat and Km of the MKP7-CD (0.028\u2009s\u22121 and 0.26\u2009\u03bcM) so determined were nearly identical to those of MKP7\u0394C304 (0.029\u2009s\u22121 and 0.27\u2009\u03bcM), indicating that the MKP7-KBD has no effect on enzyme catalysis.", "section": "RESULTS", "ner": [ [ 4, 8, "kcat", "evidence" ], [ 13, 15, "Km", "evidence" ], [ 23, 27, "MKP7", "protein" ], [ 28, 30, "CD", "structure_element" ], [ 103, 112, "MKP7\u0394C304", "mutant" ], [ 158, 162, "MKP7", "protein" ], [ 163, 166, "KBD", "structure_element" ] ] }, { "sid": 48, "sent": "We next examined the interaction of JNK1 with the CD and KBD of MKP7 by gel filtration analysis.", "section": "RESULTS", "ner": [ [ 36, 40, "JNK1", "protein" ], [ 50, 52, "CD", "structure_element" ], [ 57, 60, "KBD", "structure_element" ], [ 64, 68, "MKP7", "protein" ], [ 72, 95, "gel filtration analysis", "experimental_method" ] ] }, { "sid": 49, "sent": "When 3\u2009molar equivalents of CD were mixed with 1\u2009molar equivalent of JNK1, a significant amount of CD co-migrated with JNK1 to earlier fractions, and the excess amount of CD was eluted from the size exclusion column as a monomer, indicating stable complex formation (Fig. 2c).", "section": "RESULTS", "ner": [ [ 28, 30, "CD", "structure_element" ], [ 69, 73, "JNK1", "protein" ], [ 99, 101, "CD", "structure_element" ], [ 119, 123, "JNK1", "protein" ], [ 171, 173, "CD", "structure_element" ], [ 221, 228, "monomer", "oligomeric_state" ] ] }, { "sid": 50, "sent": "In contrast, no KBD\u2013JNK1 complex was detected when 3\u2009molar equivalents of KBD were mixed with 1\u2009molar equivalent of JNK1.", "section": "RESULTS", "ner": [ [ 16, 24, "KBD\u2013JNK1", "complex_assembly" ], [ 74, 77, "KBD", "structure_element" ], [ 116, 120, "JNK1", "protein" ] ] }, { "sid": 51, "sent": "To further confirm the JNK1\u2013MKP7-CD interaction, we performed a pull-down assay using the purified proteins.", "section": "RESULTS", "ner": [ [ 23, 35, "JNK1\u2013MKP7-CD", "complex_assembly" ], [ 64, 79, "pull-down assay", "experimental_method" ] ] }, { "sid": 52, "sent": "As shown in Fig. 2d, the CD of MKP7 can be pulled down by JNK1, while the KBD failed to bind to the counterpart protein.", "section": "RESULTS", "ner": [ [ 25, 27, "CD", "structure_element" ], [ 31, 35, "MKP7", "protein" ], [ 58, 62, "JNK1", "protein" ], [ 74, 77, "KBD", "structure_element" ] ] }, { "sid": 53, "sent": "Taken together, our data indicate that the CD of MKP7, but not the KBD domain, is responsible for JNK substrate-binding and enzymatic specificity.", "section": "RESULTS", "ner": [ [ 43, 45, "CD", "structure_element" ], [ 49, 53, "MKP7", "protein" ], [ 67, 70, "KBD", "structure_element" ], [ 98, 101, "JNK", "protein_type" ] ] }, { "sid": 54, "sent": "Crystal structure of JNK1 in complex with the MKP7-CD", "section": "RESULTS", "ner": [ [ 0, 17, "Crystal structure", "evidence" ], [ 21, 25, "JNK1", "protein" ], [ 26, 41, "in complex with", "protein_state" ], [ 46, 50, "MKP7", "protein" ], [ 51, 53, "CD", "structure_element" ] ] }, { "sid": 55, "sent": "To understand the molecular basis of JNK1 recognition by MKP7, we determined the crystal structure of unphosphorylated JNK1 in complex with the MKP7-CD (Fig. 3a, Supplementary Fig. 1a and Table 1).", "section": "RESULTS", "ner": [ [ 37, 41, "JNK1", "protein" ], [ 57, 61, "MKP7", "protein" ], [ 81, 98, "crystal structure", "evidence" ], [ 102, 118, "unphosphorylated", "protein_state" ], [ 119, 123, "JNK1", "protein" ], [ 124, 139, "in complex with", "protein_state" ], [ 144, 148, "MKP7", "protein" ], [ 149, 151, "CD", "structure_element" ] ] }, { "sid": 56, "sent": "In the complex, JNK1 has its characteristic bilobal structure comprising an N-terminal lobe rich in \u03b2-sheet and a C-terminal lobe that is mostly \u03b1-helical.", "section": "RESULTS", "ner": [ [ 16, 20, "JNK1", "protein" ], [ 76, 91, "N-terminal lobe", "structure_element" ], [ 100, 107, "\u03b2-sheet", "structure_element" ], [ 114, 129, "C-terminal lobe", "structure_element" ], [ 145, 154, "\u03b1-helical", "structure_element" ] ] }, { "sid": 57, "sent": "The overall folding of MKP7-CD is typical of DUSPs, with a central twisted five-stranded \u03b2-sheet surrounded by six \u03b1-helices.", "section": "RESULTS", "ner": [ [ 23, 27, "MKP7", "protein" ], [ 28, 30, "CD", "structure_element" ], [ 45, 50, "DUSPs", "protein_type" ], [ 67, 96, "twisted five-stranded \u03b2-sheet", "structure_element" ], [ 115, 124, "\u03b1-helices", "structure_element" ] ] }, { "sid": 58, "sent": "One side of the \u03b2-sheet is covered with two \u03b1-helices and the other is covered with four \u03b1-helices (Fig. 3b).", "section": "RESULTS", "ner": [ [ 16, 23, "\u03b2-sheet", "structure_element" ], [ 44, 53, "\u03b1-helices", "structure_element" ], [ 89, 98, "\u03b1-helices", "structure_element" ] ] }, { "sid": 59, "sent": "The catalytic domain of MKP7 interacts with JNK1 through a contiguous surface area that is remote from the active site.", "section": "RESULTS", "ner": [ [ 4, 20, "catalytic domain", "structure_element" ], [ 24, 28, "MKP7", "protein" ], [ 44, 48, "JNK1", "protein" ], [ 107, 118, "active site", "site" ] ] }, { "sid": 60, "sent": "MKP7-CD is positioned onto the JNK1 molecule so that the active site of the phosphatase faces towards the activation segment.", "section": "RESULTS", "ner": [ [ 0, 4, "MKP7", "protein" ], [ 5, 7, "CD", "structure_element" ], [ 31, 35, "JNK1", "protein" ], [ 57, 68, "active site", "site" ], [ 76, 87, "phosphatase", "protein_type" ], [ 106, 124, "activation segment", "structure_element" ] ] }, { "sid": 61, "sent": "In an alignment of the structure of MKP7-CD with that of VHR, an atypical \u2018MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (root mean squared deviation) of 1.05\u2009\u00c5 (Fig. 3c).", "section": "RESULTS", "ner": [ [ 6, 15, "alignment", "experimental_method" ], [ 23, 32, "structure", "evidence" ], [ 36, 40, "MKP7", "protein" ], [ 41, 43, "CD", "structure_element" ], [ 57, 60, "VHR", "protein" ], [ 75, 78, "MKP", "protein_type" ], [ 101, 117, "catalytic domain", "structure_element" ], [ 130, 134, "MKP7", "protein" ], [ 135, 137, "CD", "structure_element" ], [ 156, 168, "superimposed", "experimental_method" ], [ 176, 184, "r.m.s.d.", "evidence" ], [ 186, 213, "root mean squared deviation", "evidence" ] ] }, { "sid": 62, "sent": "The most striking difference is that helix \u03b10 and loop \u03b10\u2013\u03b21 of VHR are absent in MKP7-CD.", "section": "RESULTS", "ner": [ [ 37, 42, "helix", "structure_element" ], [ 43, 45, "\u03b10", "structure_element" ], [ 50, 54, "loop", "structure_element" ], [ 55, 60, "\u03b10\u2013\u03b21", "structure_element" ], [ 64, 67, "VHR", "protein" ], [ 82, 86, "MKP7", "protein" ], [ 87, 89, "CD", "structure_element" ] ] }, { "sid": 63, "sent": "Another region that cannot be aligned with VHR is found in loop \u03b23\u2013\u03b24.", "section": "RESULTS", "ner": [ [ 43, 46, "VHR", "protein" ], [ 59, 63, "loop", "structure_element" ], [ 64, 69, "\u03b23\u2013\u03b24", "structure_element" ] ] }, { "sid": 64, "sent": "This loop is shortened by nine residues in MKP7-CD compared with that in VHR.", "section": "RESULTS", "ner": [ [ 5, 9, "loop", "structure_element" ], [ 43, 47, "MKP7", "protein" ], [ 48, 50, "CD", "structure_element" ], [ 73, 76, "VHR", "protein" ] ] }, { "sid": 65, "sent": "Since helix \u03b10 and the following loop \u03b10\u2013\u03b21 are known for a substrate-recognition motif of VHR and other phosphatases, the absence of these moieties implicates a different substrate-binding mode of MKP7.", "section": "RESULTS", "ner": [ [ 6, 11, "helix", "structure_element" ], [ 12, 14, "\u03b10", "structure_element" ], [ 33, 37, "loop", "structure_element" ], [ 38, 43, "\u03b10\u2013\u03b21", "structure_element" ], [ 60, 87, "substrate-recognition motif", "site" ], [ 91, 94, "VHR", "protein" ], [ 105, 117, "phosphatases", "protein_type" ], [ 198, 202, "MKP7", "protein" ] ] }, { "sid": 66, "sent": "The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b).", "section": "RESULTS", "ner": [ [ 4, 15, "active site", "site" ], [ 19, 23, "MKP7", "protein" ], [ 40, 62, "phosphate-binding loop", "structure_element" ], [ 64, 70, "P-loop", "structure_element" ], [ 72, 78, "Cys244", "residue_name_number" ], [ 79, 85, "Leu245", "residue_name_number" ], [ 86, 92, "Ala246", "residue_name_number" ], [ 93, 99, "Gly247", "residue_name_number" ], [ 100, 106, "Ile248", "residue_name_number" ], [ 107, 113, "Ser249", "residue_name_number" ], [ 114, 120, "Arg250", "residue_name_number" ], [ 127, 133, "Asp213", "residue_name_number" ], [ 141, 158, "general acid loop", "structure_element" ] ] }, { "sid": 67, "sent": "The MKP7-CD structure near the active site exhibits a typical active conformation as found in VHR and other PTPs.", "section": "RESULTS", "ner": [ [ 4, 8, "MKP7", "protein" ], [ 9, 11, "CD", "structure_element" ], [ 12, 21, "structure", "evidence" ], [ 31, 42, "active site", "site" ], [ 62, 81, "active conformation", "protein_state" ], [ 94, 97, "VHR", "protein" ], [ 108, 112, "PTPs", "protein_type" ] ] }, { "sid": 68, "sent": "The catalytic residue, Cys244, is located just after strand \u03b25 and optimally positioned for nucleophilic attack.", "section": "RESULTS", "ner": [ [ 4, 21, "catalytic residue", "site" ], [ 23, 29, "Cys244", "residue_name_number" ], [ 53, 59, "strand", "structure_element" ], [ 60, 62, "\u03b25", "structure_element" ] ] }, { "sid": 69, "sent": "Asp213 in MKP7 also adopts a position similar to that of Asp92 in VHR (Supplementary Fig. 1c), indicating that Asp213 is likely to function as the general acid in MKP7.", "section": "RESULTS", "ner": [ [ 0, 6, "Asp213", "residue_name_number" ], [ 10, 14, "MKP7", "protein" ], [ 57, 62, "Asp92", "residue_name_number" ], [ 66, 69, "VHR", "protein" ], [ 111, 117, "Asp213", "residue_name_number" ], [ 163, 167, "MKP7", "protein" ] ] }, { "sid": 70, "sent": "We also observed the binding of a chloride ion in the active site of MKP7-CD.", "section": "RESULTS", "ner": [ [ 34, 42, "chloride", "chemical" ], [ 54, 65, "active site", "site" ], [ 69, 73, "MKP7", "protein" ], [ 74, 76, "CD", "structure_element" ] ] }, { "sid": 71, "sent": "It is located 3.36\u2009\u00c5 from the Cys244 side chain and makes electrostatic interactions with the dipole moment of helix \u03b13 and with several main-chain amide groups.", "section": "RESULTS", "ner": [ [ 30, 36, "Cys244", "residue_name_number" ], [ 58, 84, "electrostatic interactions", "bond_interaction" ], [ 111, 116, "helix", "structure_element" ], [ 117, 119, "\u03b13", "structure_element" ] ] }, { "sid": 72, "sent": "The side chain of strictly conserved Arg250 is oriented towards the negatively charged chloride, similar to the canonical phosphate-coordinating conformation.", "section": "RESULTS", "ner": [ [ 18, 36, "strictly conserved", "protein_state" ], [ 37, 43, "Arg250", "residue_name_number" ], [ 87, 95, "chloride", "chemical" ], [ 122, 157, "phosphate-coordinating conformation", "structure_element" ] ] }, { "sid": 73, "sent": "Thus this chloride ion is a mimic for the phosphate group of the substrate, as revealed by a comparison with the structure of PTP1B in complex with phosphotyrosine (Supplementary Fig. 1d).", "section": "RESULTS", "ner": [ [ 10, 18, "chloride", "chemical" ], [ 42, 51, "phosphate", "chemical" ], [ 113, 122, "structure", "evidence" ], [ 126, 131, "PTP1B", "protein" ], [ 132, 147, "in complex with", "protein_state" ], [ 148, 163, "phosphotyrosine", "residue_name" ] ] }, { "sid": 74, "sent": "Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c).", "section": "RESULTS", "ner": [ [ 49, 53, "MKP7", "protein" ], [ 54, 56, "CD", "structure_element" ], [ 88, 91, "VHR", "protein" ], [ 113, 119, "P-loop", "structure_element" ], [ 123, 127, "MKP7", "protein" ], [ 192, 195, "VHR", "protein" ], [ 197, 203, "Cys124", "residue_name_number" ], [ 204, 210, "Arg125", "residue_name_number" ], [ 211, 217, "Glu126", "residue_name_number" ], [ 218, 224, "Gly127", "residue_name_number" ], [ 225, 231, "Tyr128", "residue_name_number" ], [ 232, 238, "Gly129", "residue_name_number" ], [ 239, 245, "Arg130", "residue_name_number" ] ] }, { "sid": 75, "sent": "The difference in the polarity/hydrophobicity of the surface may also point to the origin of the differences in the substrate-recognition mechanism for these two phosphatases (Supplementary Fig. 1e,f).", "section": "RESULTS", "ner": [ [ 162, 174, "phosphatases", "protein_type" ] ] }, { "sid": 76, "sent": "In the complex, MKP7-CD and JNK1 form extensive protein\u2013protein interactions involving the C-terminal helices of MKP7-CD and C-lobe of JNK1 (Fig. 3d,e).", "section": "RESULTS", "ner": [ [ 16, 20, "MKP7", "protein" ], [ 21, 23, "CD", "structure_element" ], [ 28, 32, "JNK1", "protein" ], [ 91, 109, "C-terminal helices", "structure_element" ], [ 113, 117, "MKP7", "protein" ], [ 118, 120, "CD", "structure_element" ], [ 125, 131, "C-lobe", "structure_element" ], [ 135, 139, "JNK1", "protein" ] ] }, { "sid": 77, "sent": "As a result, the buried solvent-accessible surface area is \u223c1,315\u2009\u00c5. In the C-terminal domain, JNK1 has an insertion after the helix \u03b1G. This insertion consists of two helices (\u03b11L14 and \u03b12L14) that are common to all members of the MAPK family.", "section": "RESULTS", "ner": [ [ 76, 93, "C-terminal domain", "structure_element" ], [ 95, 99, "JNK1", "protein" ], [ 127, 132, "helix", "structure_element" ], [ 133, 135, "\u03b1G", "structure_element" ], [ 168, 175, "helices", "structure_element" ], [ 177, 182, "\u03b11L14", "structure_element" ], [ 187, 192, "\u03b12L14", "structure_element" ], [ 232, 243, "MAPK family", "protein_type" ] ] }, { "sid": 78, "sent": "The interactive surface in JNK1, formed by the helices \u03b1G and \u03b12L14, displays a hydrophobic region, centred at Trp234 (Fig. 3d).", "section": "RESULTS", "ner": [ [ 4, 23, "interactive surface", "site" ], [ 27, 31, "JNK1", "protein" ], [ 47, 54, "helices", "structure_element" ], [ 55, 57, "\u03b1G", "structure_element" ], [ 62, 67, "\u03b12L14", "structure_element" ], [ 80, 98, "hydrophobic region", "site" ], [ 111, 117, "Trp234", "residue_name_number" ] ] }, { "sid": 79, "sent": "The MKP7-docking region includes two helices, \u03b14 and \u03b15, and the general acid loop.", "section": "RESULTS", "ner": [ [ 4, 23, "MKP7-docking region", "site" ], [ 37, 44, "helices", "structure_element" ], [ 46, 48, "\u03b14", "structure_element" ], [ 53, 55, "\u03b15", "structure_element" ], [ 65, 82, "general acid loop", "structure_element" ] ] }, { "sid": 80, "sent": "The aromatic ring of Phe285 on MKP7 \u03b15-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g).", "section": "RESULTS", "ner": [ [ 21, 27, "Phe285", "residue_name_number" ], [ 31, 35, "MKP7", "protein" ], [ 36, 44, "\u03b15-helix", "structure_element" ], [ 61, 79, "hydrophobic pocket", "site" ], [ 83, 87, "JNK1", "protein" ], [ 114, 120, "Ile197", "residue_name_number" ], [ 122, 128, "Leu198", "residue_name_number" ], [ 130, 136, "Ile231", "residue_name_number" ], [ 138, 144, "Trp234", "residue_name_number" ], [ 146, 152, "Val256", "residue_name_number" ], [ 154, 160, "Tyr259", "residue_name_number" ], [ 162, 168, "Val260", "residue_name_number" ], [ 198, 204, "His230", "residue_name_number" ] ] }, { "sid": 81, "sent": "In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1.", "section": "RESULTS", "ner": [ [ 23, 37, "hydrogen bonds", "bond_interaction" ], [ 46, 52, "Ser282", "residue_name_number" ], [ 57, 63, "Asn286", "residue_name_number" ], [ 67, 71, "MKP7", "protein" ], [ 76, 82, "His230", "residue_name_number" ], [ 87, 93, "Thr255", "residue_name_number" ], [ 97, 101, "JNK1", "protein" ], [ 125, 131, "Phe215", "residue_name_number" ], [ 139, 156, "general acid loop", "structure_element" ], [ 160, 164, "MKP7", "protein" ], [ 168, 183, "hydrogen-bonded", "bond_interaction" ], [ 205, 211, "Gln253", "residue_name_number" ], [ 215, 219, "JNK1", "protein" ] ] }, { "sid": 82, "sent": "The second interactive area involves the \u03b14 helix of MKP7 and charged/polar residues of JNK1 (Fig. 3e).", "section": "RESULTS", "ner": [ [ 4, 27, "second interactive area", "site" ], [ 41, 49, "\u03b14 helix", "structure_element" ], [ 53, 57, "MKP7", "protein" ], [ 88, 92, "JNK1", "protein" ] ] }, { "sid": 83, "sent": "The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively.", "section": "RESULTS", "ner": [ [ 19, 25, "Asp268", "residue_name_number" ], [ 29, 33, "MKP7", "protein" ], [ 42, 53, "salt bridge", "bond_interaction" ], [ 73, 79, "Arg263", "residue_name_number" ], [ 83, 87, "JNK1", "protein" ], [ 93, 99, "Lys275", "residue_name_number" ], [ 103, 107, "MKP7", "protein" ], [ 116, 129, "hydrogen bond", "bond_interaction" ], [ 136, 147, "salt bridge", "bond_interaction" ], [ 153, 159, "Thr228", "residue_name_number" ], [ 164, 170, "Asp229", "residue_name_number" ], [ 174, 178, "JNK1", "protein" ] ] }, { "sid": 84, "sent": "Mutational analysis of the JNK1\u2013MKP7 docking interface", "section": "RESULTS", "ner": [ [ 0, 19, "Mutational analysis", "experimental_method" ], [ 27, 54, "JNK1\u2013MKP7 docking interface", "site" ] ] }, { "sid": 85, "sent": "To assess the importance of the aforementioned interactions, we generated a series of point mutations on the MKP7-CD and examined their effect on the MKP7-catalysed JNK1 dephosphorylation (Fig. 4a).", "section": "RESULTS", "ner": [ [ 86, 101, "point mutations", "experimental_method" ], [ 109, 113, "MKP7", "protein" ], [ 114, 116, "CD", "structure_element" ], [ 150, 154, "MKP7", "protein" ], [ 165, 169, "JNK1", "protein" ], [ 170, 187, "dephosphorylation", "ptm" ] ] }, { "sid": 86, "sent": "When the hydrophobic residues Phe285 and Phe287 on the \u03b15 of MKP7-CD were replaced by Asp or Ala, their phosphatase activities for JNK1 dephosphorylation decreased \u223c10-fold.", "section": "RESULTS", "ner": [ [ 30, 36, "Phe285", "residue_name_number" ], [ 41, 47, "Phe287", "residue_name_number" ], [ 55, 57, "\u03b15", "structure_element" ], [ 61, 65, "MKP7", "protein" ], [ 66, 68, "CD", "structure_element" ], [ 74, 82, "replaced", "experimental_method" ], [ 86, 89, "Asp", "residue_name" ], [ 93, 96, "Ala", "residue_name" ], [ 131, 135, "JNK1", "protein" ], [ 136, 153, "dephosphorylation", "ptm" ] ] }, { "sid": 87, "sent": "In comparison, replacement of the other residues (Phe215, Asp268, Lys275, Ser282, Asn286 and Leu292) with an Ala or Asp individually led to a modest decrease in catalytic efficiencies, suggesting that this position may only affect some selectivity of MKP.", "section": "RESULTS", "ner": [ [ 15, 26, "replacement", "experimental_method" ], [ 50, 56, "Phe215", "residue_name_number" ], [ 58, 64, "Asp268", "residue_name_number" ], [ 66, 72, "Lys275", "residue_name_number" ], [ 74, 80, "Ser282", "residue_name_number" ], [ 82, 88, "Asn286", "residue_name_number" ], [ 93, 99, "Leu292", "residue_name_number" ], [ 109, 112, "Ala", "residue_name" ], [ 116, 119, "Asp", "residue_name" ], [ 251, 254, "MKP", "protein_type" ] ] }, { "sid": 88, "sent": "Mutation of Leu288 markedly reduced its solubility when expressed in Escherichia coli, resulting in the insoluble aggregation of the mutant protein.", "section": "RESULTS", "ner": [ [ 0, 8, "Mutation", "experimental_method" ], [ 12, 18, "Leu288", "residue_name_number" ], [ 69, 85, "Escherichia coli", "species" ], [ 133, 139, "mutant", "protein_state" ] ] }, { "sid": 89, "sent": "Gel filtration analysis further confirmed the key role of Phe285 in the MKP7\u2013JNK1 interaction: no F285D\u2013JNK1 complex was detected when 3\u2009molar equivalents of MKP7-CD (F285D) were mixed with 1\u2009molar equivalent of JNK1 (Fig. 4b).", "section": "RESULTS", "ner": [ [ 0, 23, "Gel filtration analysis", "experimental_method" ], [ 58, 64, "Phe285", "residue_name_number" ], [ 72, 76, "MKP7", "protein" ], [ 77, 81, "JNK1", "protein" ], [ 98, 108, "F285D\u2013JNK1", "complex_assembly" ], [ 158, 162, "MKP7", "protein" ], [ 163, 165, "CD", "structure_element" ], [ 167, 172, "F285D", "mutant" ], [ 212, 216, "JNK1", "protein" ] ] }, { "sid": 90, "sent": "Interestingly, mutation of Phe287 results in a considerable loss of activity against pJNK1 without altering the affinity of MKP7-CD for JNK1 (Supplementary Fig. 2a).", "section": "RESULTS", "ner": [ [ 15, 23, "mutation", "experimental_method" ], [ 27, 33, "Phe287", "residue_name_number" ], [ 85, 86, "p", "protein_state" ], [ 86, 90, "JNK1", "protein" ], [ 112, 120, "affinity", "evidence" ], [ 124, 128, "MKP7", "protein" ], [ 129, 131, "CD", "structure_element" ], [ 136, 140, "JNK1", "protein" ] ] }, { "sid": 91, "sent": "We also generated a series of point mutations in the JNK1 and assessed the effect on JNK1 binding using the GST pull-down assay (Fig. 4c).", "section": "RESULTS", "ner": [ [ 30, 45, "point mutations", "experimental_method" ], [ 53, 57, "JNK1", "protein" ], [ 85, 89, "JNK1", "protein" ], [ 108, 127, "GST pull-down assay", "experimental_method" ] ] }, { "sid": 92, "sent": "Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK.", "section": "RESULTS", "ner": [ [ 0, 12, "Substitution", "experimental_method" ], [ 16, 22, "Asp229", "residue_name_number" ], [ 24, 30, "Trp234", "residue_name_number" ], [ 32, 38, "Thr255", "residue_name_number" ], [ 40, 46, "Val256", "residue_name_number" ], [ 48, 54, "Tyr259", "residue_name_number" ], [ 59, 65, "Val260", "residue_name_number" ], [ 92, 108, "binding affinity", "evidence" ], [ 112, 116, "MKP7", "protein" ], [ 117, 119, "CD", "structure_element" ], [ 124, 127, "JNK", "protein_type" ] ] }, { "sid": 93, "sent": "To determine whether the deficiencies in their abilities to bind partner proteins or carry out catalytic function are owing to misfolding of the purified mutant proteins, we also examined the folding properties of the JNK1 and MKP7 mutants with circular dichroism.", "section": "RESULTS", "ner": [ [ 154, 160, "mutant", "protein_state" ], [ 218, 222, "JNK1", "protein" ], [ 227, 231, "MKP7", "protein" ], [ 232, 239, "mutants", "protein_state" ], [ 245, 263, "circular dichroism", "experimental_method" ] ] }, { "sid": 94, "sent": "The spectra of these mutants are similar to the wild-type proteins, indicating that these mutants fold as well as the wild-type proteins (Fig. 4d,e).", "section": "RESULTS", "ner": [ [ 4, 11, "spectra", "evidence" ], [ 21, 28, "mutants", "protein_state" ], [ 48, 57, "wild-type", "protein_state" ], [ 90, 97, "mutants", "protein_state" ], [ 118, 127, "wild-type", "protein_state" ] ] }, { "sid": 95, "sent": "Taken together, these results are consistent with the present crystallographic model, which reveal the hydrophobic contacts between the MKP7 catalytic domain and JNK1 have a predominant role in the enzyme\u2013substrate interaction, and hydrophobic residue Phe285 in the MKP7-CD is a key residue for its high-affinity binding to JNK1.", "section": "RESULTS", "ner": [ [ 62, 84, "crystallographic model", "evidence" ], [ 103, 123, "hydrophobic contacts", "bond_interaction" ], [ 136, 140, "MKP7", "protein" ], [ 141, 157, "catalytic domain", "structure_element" ], [ 162, 166, "JNK1", "protein" ], [ 252, 258, "Phe285", "residue_name_number" ], [ 266, 270, "MKP7", "protein" ], [ 271, 273, "CD", "structure_element" ], [ 324, 328, "JNK1", "protein" ] ] }, { "sid": 96, "sent": "It has previously been reported that several cytosolic and inducible nuclear MKPs undergo catalytic activation upon interaction with the MAPK substrates.", "section": "RESULTS", "ner": [ [ 77, 81, "MKPs", "protein_type" ], [ 137, 141, "MAPK", "protein_type" ] ] }, { "sid": 97, "sent": "This allosteric activation of MKP3 has been well-documented in vitro using pNPP, a small-molecule phosphotyrosine analogue of its normal substrate.", "section": "RESULTS", "ner": [ [ 30, 34, "MKP3", "protein" ], [ 75, 79, "pNPP", "chemical" ], [ 98, 113, "phosphotyrosine", "residue_name" ] ] }, { "sid": 98, "sent": "We then assayed pNPPase activities of MKP7\u0394C304 and MKP7-CD in the presence of JNK1.", "section": "RESULTS", "ner": [ [ 16, 23, "pNPPase", "protein_type" ], [ 38, 47, "MKP7\u0394C304", "mutant" ], [ 52, 56, "MKP7", "protein" ], [ 57, 59, "CD", "structure_element" ], [ 67, 78, "presence of", "protein_state" ], [ 79, 83, "JNK1", "protein" ] ] }, { "sid": 99, "sent": "Incubation of MKP7 with JNK1 did not markedly stimulate the phosphatase activity, which is consistent with previous results that MKP7 solely possesses the intrinsic activity (Supplementary Fig. 2b).", "section": "RESULTS", "ner": [ [ 0, 10, "Incubation", "experimental_method" ], [ 14, 18, "MKP7", "protein" ], [ 24, 28, "JNK1", "protein" ], [ 60, 71, "phosphatase", "protein_type" ], [ 129, 133, "MKP7", "protein" ] ] }, { "sid": 100, "sent": "The small pNPP molecule binds directly at the enzyme active site and can be used to probe the reaction mechanism of protein phosphatases.", "section": "RESULTS", "ner": [ [ 10, 14, "pNPP", "chemical" ], [ 53, 64, "active site", "site" ], [ 116, 136, "protein phosphatases", "protein_type" ] ] }, { "sid": 101, "sent": "We therefore examined the effects of the MKP7-CD mutants on their pNPPase activities.", "section": "RESULTS", "ner": [ [ 41, 45, "MKP7", "protein" ], [ 46, 48, "CD", "structure_element" ], [ 49, 56, "mutants", "protein_state" ], [ 66, 73, "pNPPase", "protein_type" ] ] }, { "sid": 102, "sent": "As shown in Fig. 4f, all the mutants, except F287D/A, showed little or no activity change compared with the wild-type MKP7-CD.", "section": "RESULTS", "ner": [ [ 29, 36, "mutants", "protein_state" ], [ 45, 52, "F287D/A", "mutant" ], [ 108, 117, "wild-type", "protein_state" ], [ 118, 122, "MKP7", "protein" ], [ 123, 125, "CD", "structure_element" ] ] }, { "sid": 103, "sent": "In the JNK1/MKP7-CD complex structure, Phe287 of MKP7 does not make contacts with JNK1 substrate.", "section": "RESULTS", "ner": [ [ 7, 19, "JNK1/MKP7-CD", "complex_assembly" ], [ 28, 37, "structure", "evidence" ], [ 39, 45, "Phe287", "residue_name_number" ], [ 49, 53, "MKP7", "protein" ], [ 82, 86, "JNK1", "protein" ] ] }, { "sid": 104, "sent": "It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c).", "section": "RESULTS", "ner": [ [ 21, 27, "pocket", "site" ], [ 56, 62, "P-loop", "structure_element" ], [ 67, 84, "general acid loop", "structure_element" ], [ 95, 115, "hydrophobic contacts", "bond_interaction" ], [ 162, 168, "Arg250", "residue_name_number" ], [ 170, 176, "Glu217", "residue_name_number" ], [ 181, 187, "Ile219", "residue_name_number" ], [ 205, 211, "Phe287", "residue_name_number" ], [ 215, 219, "MKP7", "protein" ], [ 291, 295, "PTPs", "protein_type" ], [ 297, 303, "Gln266", "residue_name_number" ], [ 307, 312, "PTP1B", "protein" ], [ 318, 321, "VHR", "protein" ], [ 323, 329, "Phe166", "residue_name_number" ], [ 333, 336, "VHR", "protein" ], [ 366, 386, "active-site residues", "site" ], [ 390, 394, "MKP7", "protein" ] ] }, { "sid": 105, "sent": "Kinase-associated phosphatase (KAP), a member of the DUSP family, plays a crucial role in cell cycle regulation by dephosphorylating the pThr160 residue of CDK2 (cyclin-dependent kinase 2).", "section": "RESULTS", "ner": [ [ 0, 29, "Kinase-associated phosphatase", "protein" ], [ 31, 34, "KAP", "protein" ], [ 53, 64, "DUSP family", "protein_type" ], [ 137, 144, "pThr160", "ptm" ], [ 156, 160, "CDK2", "protein" ], [ 162, 187, "cyclin-dependent kinase 2", "protein" ] ] }, { "sid": 106, "sent": "The crystal structure of the CDK2/KAP complex has been determined at 3.0\u2009\u00c5 (Fig. 5a).", "section": "RESULTS", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 29, 37, "CDK2/KAP", "complex_assembly" ] ] }, { "sid": 107, "sent": "The interface between these two proteins consists of three discontinuous contact regions.", "section": "RESULTS", "ner": [ [ 4, 13, "interface", "site" ] ] }, { "sid": 108, "sent": "Biochemical results suggested that the affinity and specificity between KAP and CDK2 results from the recognition site comprising CDK2 residues from the \u03b1G helix and L14 loop and the N-terminal helical region of KAP (Fig. 5b).", "section": "RESULTS", "ner": [ [ 72, 75, "KAP", "protein" ], [ 80, 84, "CDK2", "protein" ], [ 102, 118, "recognition site", "site" ], [ 130, 134, "CDK2", "protein" ], [ 153, 161, "\u03b1G helix", "structure_element" ], [ 166, 174, "L14 loop", "structure_element" ], [ 183, 208, "N-terminal helical region", "structure_element" ], [ 212, 215, "KAP", "protein" ] ] }, { "sid": 109, "sent": "There is a hydrogen bond between the main-chain nitrogen of Ile183 (KAP) and side chain oxygen of Glu208 (CDK2), and salt bridges between Lys184 of KAP and Asp235 of CDK2.", "section": "RESULTS", "ner": [ [ 11, 24, "hydrogen bond", "bond_interaction" ], [ 60, 66, "Ile183", "residue_name_number" ], [ 68, 71, "KAP", "protein" ], [ 98, 104, "Glu208", "residue_name_number" ], [ 106, 110, "CDK2", "protein" ], [ 138, 144, "Lys184", "residue_name_number" ], [ 148, 151, "KAP", "protein" ], [ 156, 162, "Asp235", "residue_name_number" ], [ 166, 170, "CDK2", "protein" ] ] }, { "sid": 110, "sent": "Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c).", "section": "RESULTS", "ner": [ [ 0, 19, "Structural analysis", "experimental_method" ], [ 24, 42, "sequence alignment", "experimental_method" ], [ 90, 94, "MKP7", "protein" ], [ 95, 97, "CD", "structure_element" ], [ 102, 105, "KAP", "protein" ], [ 113, 137, "substrate-binding region", "site" ], [ 167, 171, "FNFL", "structure_element" ], [ 175, 179, "MKP7", "protein" ], [ 180, 182, "CD", "structure_element" ], [ 205, 209, "IKQY", "structure_element" ], [ 213, 216, "KAP", "protein" ] ] }, { "sid": 111, "sent": "The substitution of the two hydrophobic residues with charged/polar residues (F285I/N286K) seriously disrupts the hydrophobic interaction required for MKP7 binding on JNK1 (Fig. 4a).", "section": "RESULTS", "ner": [ [ 4, 16, "substitution", "experimental_method" ], [ 78, 83, "F285I", "mutant" ], [ 84, 89, "N286K", "mutant" ], [ 114, 137, "hydrophobic interaction", "bond_interaction" ], [ 151, 155, "MKP7", "protein" ], [ 167, 171, "JNK1", "protein" ] ] }, { "sid": 112, "sent": "In addition, His230 and Val256 in JNK1 are replaced by the negatively charged residues Glu208 and Asp235 in CDK2 (Fig. 5d), and the charge distribution on the CDK2 interactive surface is quite different from that of JNK.", "section": "RESULTS", "ner": [ [ 13, 19, "His230", "residue_name_number" ], [ 24, 30, "Val256", "residue_name_number" ], [ 34, 38, "JNK1", "protein" ], [ 87, 93, "Glu208", "residue_name_number" ], [ 98, 104, "Asp235", "residue_name_number" ], [ 108, 112, "CDK2", "protein" ], [ 159, 163, "CDK2", "protein" ], [ 164, 183, "interactive surface", "site" ], [ 216, 219, "JNK", "protein_type" ] ] }, { "sid": 113, "sent": "These data indicated that a unique hydrophobic pocket formed between the MAPK insert and \u03b1G helix plays a major role in the substrate recognition by MKPs.", "section": "RESULTS", "ner": [ [ 35, 53, "hydrophobic pocket", "site" ], [ 73, 84, "MAPK insert", "structure_element" ], [ 89, 97, "\u03b1G helix", "structure_element" ], [ 149, 153, "MKPs", "protein_type" ] ] }, { "sid": 114, "sent": "F-site interaction is crucial for JNK1 inactivation in vivo", "section": "RESULTS", "ner": [ [ 0, 6, "F-site", "site" ], [ 34, 38, "JNK1", "protein" ] ] }, { "sid": 115, "sent": "JNK is activated following cellular exposure to a number of acute stimuli such as anisomycin, H2O2, ultraviolet light, sorbitol, DNA-damaging agents and several strong apoptosis inducers (etoposide, cisplatin and taxol).", "section": "RESULTS", "ner": [ [ 0, 3, "JNK", "protein_type" ], [ 82, 92, "anisomycin", "chemical" ], [ 94, 98, "H2O2", "chemical" ], [ 119, 127, "sorbitol", "chemical" ], [ 188, 197, "etoposide", "chemical" ], [ 199, 208, "cisplatin", "chemical" ], [ 213, 218, "taxol", "chemical" ] ] }, { "sid": 116, "sent": "To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7\u0394C304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment.", "section": "RESULTS", "ner": [ [ 25, 29, "MKP7", "protein" ], [ 38, 45, "mutants", "protein_state" ], [ 78, 81, "JNK", "protein_type" ], [ 148, 157, "HA-tagged", "protein_state" ], [ 173, 184, "full-length", "protein_state" ], [ 185, 189, "MKP7", "protein" ], [ 191, 200, "MKP7\u0394C304", "mutant" ], [ 205, 209, "MKP7", "protein" ], [ 210, 212, "CD", "structure_element" ], [ 216, 220, "MKP7", "protein" ], [ 221, 228, "mutants", "protein_state" ], [ 265, 274, "etoposide", "chemical" ] ] }, { "sid": 117, "sent": "As shown in Fig. 6a\u2013c, immunobloting showed similar expression levels for the different MKP7 constructs in all the cells.", "section": "RESULTS", "ner": [ [ 23, 36, "immunobloting", "experimental_method" ], [ 88, 92, "MKP7", "protein" ] ] }, { "sid": 118, "sent": "Overexpressed full-length MKP7, MKP7\u0394C304 and MKP7-CD significantly reduced the endogenous level of phosphorylated JNK compared with vector-transfected cells.", "section": "RESULTS", "ner": [ [ 0, 13, "Overexpressed", "experimental_method" ], [ 14, 25, "full-length", "protein_state" ], [ 26, 30, "MKP7", "protein" ], [ 32, 41, "MKP7\u0394C304", "mutant" ], [ 46, 50, "MKP7", "protein" ], [ 51, 53, "CD", "structure_element" ], [ 100, 114, "phosphorylated", "protein_state" ], [ 115, 118, "JNK", "protein_type" ] ] }, { "sid": 119, "sent": "Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo.", "section": "RESULTS", "ner": [ [ 45, 52, "D-motif", "structure_element" ], [ 53, 60, "mutants", "protein_state" ], [ 62, 66, "R56A", "mutant" ], [ 67, 71, "R57A", "mutant" ], [ 76, 80, "V63A", "mutant" ], [ 81, 85, "I65A", "mutant" ], [ 87, 103, "dephosphorylated", "protein_state" ], [ 104, 107, "JNK", "protein_type" ], [ 119, 128, "wild type", "protein_state" ], [ 184, 188, "MKP7", "protein" ], [ 189, 192, "KBD", "structure_element" ], [ 217, 220, "JNK", "protein_type" ] ] }, { "sid": 120, "sent": "Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK.", "section": "RESULTS", "ner": [ [ 48, 62, "phosphorylated", "protein_state" ], [ 63, 66, "JNK", "protein_type" ], [ 96, 100, "MKP7", "protein" ], [ 101, 110, "FXF-motif", "structure_element" ], [ 111, 118, "mutants", "protein_state" ], [ 120, 125, "F285D", "mutant" ], [ 127, 132, "F287D", "mutant" ], [ 137, 142, "L288D", "mutant" ], [ 171, 175, "MKP7", "protein" ], [ 176, 181, "D268A", "mutant" ], [ 186, 191, "N286A", "mutant" ], [ 192, 199, "mutants", "protein_state" ], [ 261, 264, "JNK", "protein_type" ] ] }, { "sid": 121, "sent": "We next tested in vivo interactions between JNK1 mutants and full-length MKP7 by coimmunoprecipitation experiments under unstimulated conditions.", "section": "RESULTS", "ner": [ [ 44, 48, "JNK1", "protein" ], [ 49, 56, "mutants", "protein_state" ], [ 61, 72, "full-length", "protein_state" ], [ 73, 77, "MKP7", "protein" ], [ 81, 114, "coimmunoprecipitation experiments", "experimental_method" ] ] }, { "sid": 122, "sent": "When co-expressed in HEK293T cells, wild-type (HA)-JNK1 was readily precipitated with (Myc)-MKP7 (Fig. 6d), indicating that MKP7 binds dephosphorylated JNK1 protein in vivo.", "section": "RESULTS", "ner": [ [ 5, 17, "co-expressed", "experimental_method" ], [ 36, 45, "wild-type", "protein_state" ], [ 51, 55, "JNK1", "protein" ], [ 92, 96, "MKP7", "protein" ], [ 124, 128, "MKP7", "protein" ], [ 135, 151, "dephosphorylated", "protein_state" ], [ 152, 156, "JNK1", "protein" ] ] }, { "sid": 123, "sent": "In agreement with the in vitro pull-down results, the mutants D229A, W234D and Y259D were not co-precipitated with MKP7, and the I231D mutant had only little effect on the JNK1\u2013MKP7 interaction (Fig. 6d and Supplementary Fig. 3a).", "section": "RESULTS", "ner": [ [ 22, 40, "in vitro pull-down", "experimental_method" ], [ 54, 61, "mutants", "protein_state" ], [ 62, 67, "D229A", "mutant" ], [ 69, 74, "W234D", "mutant" ], [ 79, 84, "Y259D", "mutant" ], [ 115, 119, "MKP7", "protein" ], [ 129, 134, "I231D", "mutant" ], [ 135, 141, "mutant", "protein_state" ], [ 172, 181, "JNK1\u2013MKP7", "complex_assembly" ] ] }, { "sid": 124, "sent": "Activation of the JNK signalling pathway is frequently associated with apoptotic cell death, and inhibition of JNK can prevent apoptotic death of multiple cells.", "section": "RESULTS", "ner": [ [ 18, 21, "JNK", "protein_type" ], [ 111, 114, "JNK", "protein_type" ] ] }, { "sid": 125, "sent": "To examine whether the inhibition of JNK activity by MKP7 would provide protections against the apoptosis, we analysed the rate of apoptosis in ultraviolet-irradiated cells transfected with MKP7 (wild type or mutants) by flow cytometry.", "section": "RESULTS", "ner": [ [ 37, 40, "JNK", "protein_type" ], [ 53, 57, "MKP7", "protein" ], [ 190, 194, "MKP7", "protein" ], [ 196, 205, "wild type", "protein_state" ], [ 209, 216, "mutants", "protein_state" ], [ 221, 235, "flow cytometry", "experimental_method" ] ] }, { "sid": 126, "sent": "The results showed similar apoptotic rates between cells transfected with blank vector or with MKP7 (wild type or mutants) under unstimulated conditions (Supplementary Fig. 3b), while ultraviolet-irradiation significantly increased apoptotic rate in cells transfected with blank vector (Fig. 6e).", "section": "RESULTS", "ner": [ [ 95, 99, "MKP7", "protein" ], [ 101, 110, "wild type", "protein_state" ], [ 114, 121, "mutants", "protein_state" ] ] }, { "sid": 127, "sent": "Expressions of wild-type MKP7, MKP7\u0394C304 and MKP7-CD significantly decreased the proportion of apoptotic cells after ultraviolet treatment.", "section": "RESULTS", "ner": [ [ 0, 11, "Expressions", "experimental_method" ], [ 15, 24, "wild-type", "protein_state" ], [ 25, 29, "MKP7", "protein" ], [ 31, 40, "MKP7\u0394C304", "mutant" ], [ 45, 49, "MKP7", "protein" ], [ 50, 52, "CD", "structure_element" ] ] }, { "sid": 128, "sent": "Moreover, treatment of cells expressing MKP7-KBD mutants (R56A/R57A and V63A/I65A) decreased the apoptosis rates to a similar extent as MKP7 wild type did.", "section": "RESULTS", "ner": [ [ 40, 44, "MKP7", "protein" ], [ 45, 48, "KBD", "structure_element" ], [ 49, 56, "mutants", "protein_state" ], [ 58, 62, "R56A", "mutant" ], [ 63, 67, "R57A", "mutant" ], [ 72, 76, "V63A", "mutant" ], [ 77, 81, "I65A", "mutant" ], [ 136, 140, "MKP7", "protein" ], [ 141, 150, "wild type", "protein_state" ] ] }, { "sid": 129, "sent": "In contrast, cells transfected with the MKP7 FXF-motif mutants (F285D, F287D and L288D) showed little protective effect after ultraviolet treatment and similar levels of apoptosis rates were detected to cells transfected with control vectors (Fig. 6e,f).", "section": "RESULTS", "ner": [ [ 40, 44, "MKP7", "protein" ], [ 45, 54, "FXF-motif", "structure_element" ], [ 55, 62, "mutants", "protein_state" ], [ 64, 69, "F285D", "mutant" ], [ 71, 76, "F287D", "mutant" ], [ 81, 86, "L288D", "mutant" ] ] }, { "sid": 130, "sent": "Taken together, our results suggested that FXF-motif-mediated, rather than KBD-mediated, interaction is essential for MKP7 to block ultraviolet-induced apoptosis.", "section": "RESULTS", "ner": [ [ 43, 52, "FXF-motif", "structure_element" ], [ 75, 78, "KBD", "structure_element" ], [ 118, 122, "MKP7", "protein" ] ] }, { "sid": 131, "sent": "A similar docking mechanism for JNK1 recognition by MKP5", "section": "RESULTS", "ner": [ [ 32, 36, "JNK1", "protein" ], [ 52, 56, "MKP5", "protein" ] ] }, { "sid": 132, "sent": "MKP5 belongs to the same subfamily as MKP7.", "section": "RESULTS", "ner": [ [ 0, 4, "MKP5", "protein" ], [ 38, 42, "MKP7", "protein" ] ] }, { "sid": 133, "sent": "MKP5 is unique among the MKPs in possessing an additional domain of unknown function at the N-terminus (Fig. 7a).", "section": "RESULTS", "ner": [ [ 0, 4, "MKP5", "protein" ], [ 25, 29, "MKPs", "protein_type" ] ] }, { "sid": 134, "sent": "The KBD of MKP5 interacts with the D-site of p38\u03b1 to mediate the enzyme\u2013substrate interaction.", "section": "RESULTS", "ner": [ [ 4, 7, "KBD", "structure_element" ], [ 11, 15, "MKP5", "protein" ], [ 35, 41, "D-site", "site" ], [ 45, 49, "p38\u03b1", "protein" ] ] }, { "sid": 135, "sent": "Deletion of the KBD in MKP5 leads to a 280-fold increase in Km for p38\u03b1 substrate.", "section": "RESULTS", "ner": [ [ 0, 11, "Deletion of", "experimental_method" ], [ 16, 19, "KBD", "structure_element" ], [ 23, 27, "MKP5", "protein" ], [ 60, 62, "Km", "evidence" ], [ 67, 71, "p38\u03b1", "protein" ] ] }, { "sid": 136, "sent": "In contrast to p38\u03b1 substrate, deletion of the MKP5-KBD had little effects on the kinetic parameters for the JNK1 dephosphorylation, indicating that the KBD of MKP5 is not required for the JNK1 dephosphorylation (Fig. 7b).", "section": "RESULTS", "ner": [ [ 15, 19, "p38\u03b1", "protein" ], [ 31, 42, "deletion of", "experimental_method" ], [ 47, 51, "MKP5", "protein" ], [ 52, 55, "KBD", "structure_element" ], [ 109, 113, "JNK1", "protein" ], [ 153, 156, "KBD", "structure_element" ], [ 160, 164, "MKP5", "protein" ], [ 189, 193, "JNK1", "protein" ] ] }, { "sid": 137, "sent": "The substrate specificity constant kcat /Km value for MKP5-CD was calculated as 1.0 \u00d7 105\u2009M\u22121\u2009s\u22121, which is very close to that of MKP7-CD (1.07 \u00d7 105\u2009M\u22121\u2009s\u22121).", "section": "RESULTS", "ner": [ [ 4, 34, "substrate specificity constant", "evidence" ], [ 35, 43, "kcat /Km", "evidence" ], [ 54, 58, "MKP5", "protein" ], [ 59, 61, "CD", "structure_element" ], [ 130, 134, "MKP7", "protein" ], [ 135, 137, "CD", "structure_element" ] ] }, { "sid": 138, "sent": "The crystal structure of human MKP5-CD has been determined.", "section": "RESULTS", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 25, 30, "human", "species" ], [ 31, 35, "MKP5", "protein" ], [ 36, 38, "CD", "structure_element" ] ] }, { "sid": 139, "sent": "Comparisons between catalytic domains structures of MKP5 and MKP7 reveal that the overall folds of the two proteins are highly similar, with only a few regions exhibiting small deviations (r.m.s.d. of 0.79\u2009\u00c5; Fig. 7c).", "section": "RESULTS", "ner": [ [ 20, 37, "catalytic domains", "structure_element" ], [ 38, 48, "structures", "evidence" ], [ 52, 56, "MKP5", "protein" ], [ 61, 65, "MKP7", "protein" ], [ 189, 197, "r.m.s.d.", "evidence" ] ] }, { "sid": 140, "sent": "Given the distinct interaction mode revealed by the crystal structure of JNK1\u2013MKP7-CD, one obvious question is whether this is a general mechanism used by all members of the JNK-specific MKPs.", "section": "RESULTS", "ner": [ [ 52, 69, "crystal structure", "evidence" ], [ 73, 85, "JNK1\u2013MKP7-CD", "complex_assembly" ], [ 174, 191, "JNK-specific MKPs", "protein_type" ] ] }, { "sid": 141, "sent": "To address this issue, we first examined the docking ability of JNK1 to the KBD and CD of MKP5 using gel filtration analysis and pull-down assays.", "section": "RESULTS", "ner": [ [ 64, 68, "JNK1", "protein" ], [ 76, 79, "KBD", "structure_element" ], [ 84, 86, "CD", "structure_element" ], [ 90, 94, "MKP5", "protein" ], [ 101, 124, "gel filtration analysis", "experimental_method" ], [ 129, 145, "pull-down assays", "experimental_method" ] ] }, { "sid": 142, "sent": "It can be seen from gel filtration experiments that JNK1 can forms a stable heterodimer with MKP5-CD in solution, but no detectable interaction was found with the KBD domain (Fig. 7d).", "section": "RESULTS", "ner": [ [ 20, 46, "gel filtration experiments", "experimental_method" ], [ 52, 56, "JNK1", "protein" ], [ 69, 75, "stable", "protein_state" ], [ 76, 87, "heterodimer", "oligomeric_state" ], [ 93, 97, "MKP5", "protein" ], [ 98, 100, "CD", "structure_element" ], [ 163, 166, "KBD", "structure_element" ] ] }, { "sid": 143, "sent": "Pull-down assays also confirmed the protein\u2013protein interactions observed above.", "section": "RESULTS", "ner": [ [ 0, 16, "Pull-down assays", "experimental_method" ] ] }, { "sid": 144, "sent": "The catalytic domain of MKP5, but not its KBD, was able to pull-down a detectable amount of JNK1 (Fig. 7e), implicating a different substrate-recognition mechanisms for p38 and JNK MAPKs.", "section": "RESULTS", "ner": [ [ 4, 20, "catalytic domain", "structure_element" ], [ 24, 28, "MKP5", "protein" ], [ 42, 45, "KBD", "structure_element" ], [ 92, 96, "JNK1", "protein" ], [ 169, 172, "p38", "protein_type" ], [ 177, 180, "JNK", "protein_type" ], [ 181, 186, "MAPKs", "protein_type" ] ] }, { "sid": 145, "sent": "To further test our hypothesis, we generated forms of MKP5-CD bearing mutations corresponding to the changes we made on MKP7-CD on the basis of sequence and structural alignment and examined their effects on the phosphatase activity.", "section": "RESULTS", "ner": [ [ 54, 58, "MKP5", "protein" ], [ 59, 61, "CD", "structure_element" ], [ 70, 79, "mutations", "experimental_method" ], [ 120, 124, "MKP7", "protein" ], [ 125, 127, "CD", "structure_element" ], [ 144, 177, "sequence and structural alignment", "experimental_method" ], [ 212, 223, "phosphatase", "protein_type" ] ] }, { "sid": 146, "sent": "As shown in Fig. 7f, the T432A and L449F MKP5 mutant showed little or no difference in phosphatase activity, whereas the other mutants showed reduced specific activities of MKP5.", "section": "RESULTS", "ner": [ [ 25, 30, "T432A", "mutant" ], [ 35, 40, "L449F", "mutant" ], [ 41, 45, "MKP5", "protein" ], [ 46, 52, "mutant", "protein_state" ], [ 127, 134, "mutants", "protein_state" ], [ 173, 177, "MKP5", "protein" ] ] }, { "sid": 147, "sent": "As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h).", "section": "RESULTS", "ner": [ [ 18, 22, "MKP7", "protein" ], [ 32, 39, "mutants", "protein_state" ], [ 48, 55, "F451D/A", "mutant" ], [ 67, 74, "pNPPase", "protein_type" ], [ 110, 119, "wild-type", "protein_state" ], [ 120, 124, "MKP5", "protein" ], [ 125, 127, "CD", "structure_element" ], [ 147, 162, "point mutations", "experimental_method" ], [ 166, 170, "JNK1", "protein" ], [ 188, 204, "binding affinity", "evidence" ], [ 208, 212, "MKP5", "protein" ], [ 213, 215, "CD", "structure_element" ], [ 220, 224, "JNK1", "protein" ] ] }, { "sid": 148, "sent": "In addition, there were no significant differences in the CD spectra between wild-type and mutant proteins, indicating that the overall structures of these mutants did not change significantly from that of wild-type MKP5 protein (Supplementary Fig. 4a).", "section": "RESULTS", "ner": [ [ 58, 68, "CD spectra", "evidence" ], [ 77, 86, "wild-type", "protein_state" ], [ 91, 97, "mutant", "protein_state" ], [ 136, 146, "structures", "evidence" ], [ 156, 163, "mutants", "protein_state" ], [ 206, 215, "wild-type", "protein_state" ], [ 216, 220, "MKP5", "protein" ] ] }, { "sid": 149, "sent": "Taken together, our results suggest that MKP5 binds JNK1 in a docking mode similar to that in the JNK1\u2013MKP7 complex, and the detailed interaction model can be generated using molecular dynamics simulation based on the structure of JNK1\u2013MKP7-CD complex (Supplementary Fig. 4b,c).", "section": "RESULTS", "ner": [ [ 41, 45, "MKP5", "protein" ], [ 52, 56, "JNK1", "protein" ], [ 98, 107, "JNK1\u2013MKP7", "complex_assembly" ], [ 175, 204, "molecular dynamics simulation", "experimental_method" ], [ 218, 227, "structure", "evidence" ], [ 231, 243, "JNK1\u2013MKP7-CD", "complex_assembly" ] ] }, { "sid": 150, "sent": "In this model, the MKP5-CD adopts a conformation nearly identical to that in its unbound form, suggesting that the conformation of the catalytic domain undergoes little change, if any at all, upon JNK1 binding.", "section": "RESULTS", "ner": [ [ 19, 23, "MKP5", "protein" ], [ 24, 26, "CD", "structure_element" ], [ 81, 88, "unbound", "protein_state" ], [ 135, 151, "catalytic domain", "structure_element" ], [ 197, 201, "JNK1", "protein" ] ] }, { "sid": 151, "sent": "In particular, Leu449 of MKP5, which is equivalent to the key residue Phe285 of MKP7, buried deeply within the hydrophobic pocket of JNK1 in the same way as Phe285 in the JNK1\u2013MKP7-CD complex (Supplementary Fig. 4d).", "section": "RESULTS", "ner": [ [ 15, 21, "Leu449", "residue_name_number" ], [ 25, 29, "MKP5", "protein" ], [ 70, 76, "Phe285", "residue_name_number" ], [ 80, 84, "MKP7", "protein" ], [ 111, 129, "hydrophobic pocket", "site" ], [ 133, 137, "JNK1", "protein" ], [ 157, 163, "Phe285", "residue_name_number" ], [ 171, 183, "JNK1\u2013MKP7-CD", "complex_assembly" ] ] }, { "sid": 152, "sent": "Despite the strong similarities between JNK1\u2013MKP5-CD and JNK1\u2013MKP7-CD, however, there are differences.", "section": "RESULTS", "ner": [ [ 40, 44, "JNK1", "protein" ], [ 45, 49, "MKP5", "protein" ], [ 50, 52, "CD", "structure_element" ], [ 57, 69, "JNK1\u2013MKP7-CD", "complex_assembly" ] ] }, { "sid": 153, "sent": "The JNK1\u2013MKP7-CD interaction is better and more extensive.", "section": "RESULTS", "ner": [ [ 4, 16, "JNK1\u2013MKP7-CD", "complex_assembly" ] ] }, { "sid": 154, "sent": "Asp268 of MKP7-CD forms salt bridge with JNK1 Arg263, whereas the corresponding residue Thr432 in MKP5-CD may not interact with JNK1.", "section": "RESULTS", "ner": [ [ 0, 6, "Asp268", "residue_name_number" ], [ 10, 14, "MKP7", "protein" ], [ 15, 17, "CD", "structure_element" ], [ 24, 35, "salt bridge", "bond_interaction" ], [ 41, 45, "JNK1", "protein" ], [ 46, 52, "Arg263", "residue_name_number" ], [ 88, 94, "Thr432", "residue_name_number" ], [ 98, 102, "MKP5", "protein" ], [ 103, 105, "CD", "structure_element" ], [ 128, 132, "JNK1", "protein" ] ] }, { "sid": 155, "sent": "In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1.", "section": "RESULTS", "ner": [ [ 45, 49, "MKP7", "protein" ], [ 50, 52, "CD", "structure_element" ], [ 54, 60, "Phe215", "residue_name_number" ], [ 62, 68, "Leu267", "residue_name_number" ], [ 73, 79, "Leu288", "residue_name_number" ], [ 124, 130, "Asn379", "residue_name_number" ], [ 132, 138, "Met431", "residue_name_number" ], [ 143, 149, "Met452", "residue_name_number" ], [ 153, 157, "MKP5", "protein" ], [ 158, 160, "CD", "structure_element" ], [ 213, 237, "hydrophobic interactions", "bond_interaction" ], [ 246, 250, "MKP5", "protein" ], [ 251, 253, "CD", "structure_element" ], [ 258, 262, "JNK1", "protein" ] ] }, { "sid": 156, "sent": "This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is \u223c20-fold higher than that of MKP7-CD).", "section": "RESULTS", "ner": [ [ 66, 70, "JNK1", "protein" ], [ 80, 84, "MKP7", "protein" ], [ 85, 87, "CD", "structure_element" ], [ 111, 115, "MKP5", "protein" ], [ 116, 118, "CD", "structure_element" ], [ 132, 136, "MKP5", "protein" ], [ 137, 139, "CD", "structure_element" ], [ 144, 145, "p", "protein_state" ], [ 145, 149, "JNK1", "protein" ], [ 192, 196, "MKP7", "protein" ], [ 197, 199, "CD", "structure_element" ] ] }, { "sid": 157, "sent": "The MAPKs p38, ERK and JNK, are central to evolutionarily conserved signalling pathways that are present in all eukaryotic cells.", "section": "DISCUSS", "ner": [ [ 4, 9, "MAPKs", "protein_type" ], [ 10, 13, "p38", "protein_type" ], [ 15, 18, "ERK", "protein_type" ], [ 23, 26, "JNK", "protein_type" ], [ 112, 122, "eukaryotic", "taxonomy_domain" ] ] }, { "sid": 158, "sent": "Each MAPK cascade is activated in response to a diverse array of extracellular signals and culminates in the dual-phosphorylation of a threonine and a tyrosine residue in the MAPK-activation loop.", "section": "DISCUSS", "ner": [ [ 5, 9, "MAPK", "protein_type" ], [ 109, 129, "dual-phosphorylation", "ptm" ], [ 135, 144, "threonine", "residue_name" ], [ 151, 159, "tyrosine", "residue_name" ], [ 175, 195, "MAPK-activation loop", "structure_element" ] ] }, { "sid": 159, "sent": "The propagation of MAPK signals is attenuated through the actions of the MKPs.", "section": "DISCUSS", "ner": [ [ 19, 23, "MAPK", "protein_type" ], [ 73, 77, "MKPs", "protein_type" ] ] }, { "sid": 160, "sent": "Most studies have focused on the dephosphorylation of MAPKs by phosphatases containing the \u2018kinase-interaction motif ' (D-motif), including a group of DUSPs (MKPs) and a distinct subfamily of tyrosine phosphatases (HePTP, STEP and PTP-SL).", "section": "DISCUSS", "ner": [ [ 54, 59, "MAPKs", "protein_type" ], [ 63, 75, "phosphatases", "protein_type" ], [ 92, 116, "kinase-interaction motif", "structure_element" ], [ 120, 127, "D-motif", "structure_element" ], [ 151, 156, "DUSPs", "protein_type" ], [ 158, 162, "MKPs", "protein_type" ], [ 192, 213, "tyrosine phosphatases", "protein_type" ], [ 215, 220, "HePTP", "protein" ], [ 222, 226, "STEP", "protein" ], [ 231, 237, "PTP-SL", "protein" ] ] }, { "sid": 161, "sent": "Crystal structures of ERK2 bound with the D-motif sequences derived from MKP3 and HePTP have been reported.", "section": "DISCUSS", "ner": [ [ 0, 18, "Crystal structures", "evidence" ], [ 22, 26, "ERK2", "protein" ], [ 27, 37, "bound with", "protein_state" ], [ 42, 49, "D-motif", "structure_element" ], [ 73, 77, "MKP3", "protein" ], [ 82, 87, "HePTP", "protein" ] ] }, { "sid": 162, "sent": "These structures revealed that linear docking motifs in interacting proteins bind to a common docking site on MAPKs outside the kinase active site.", "section": "DISCUSS", "ner": [ [ 6, 16, "structures", "evidence" ], [ 31, 52, "linear docking motifs", "structure_element" ], [ 94, 106, "docking site", "site" ], [ 110, 115, "MAPKs", "protein_type" ], [ 128, 134, "kinase", "protein_type" ], [ 135, 146, "active site", "site" ] ] }, { "sid": 163, "sent": "The particular amino acids and their spacing within D-motif sequences and amino acid composition of the docking sites on MAPKs appear to determine the specificity of D-motifs for individual MAPKs.", "section": "DISCUSS", "ner": [ [ 52, 59, "D-motif", "structure_element" ], [ 104, 117, "docking sites", "site" ], [ 121, 126, "MAPKs", "protein_type" ], [ 166, 174, "D-motifs", "structure_element" ], [ 190, 195, "MAPKs", "protein_type" ] ] }, { "sid": 164, "sent": "Recently, the crystal structure of a complex between the KBD of MKP5 and p38\u03b1 has been obtained.", "section": "DISCUSS", "ner": [ [ 14, 31, "crystal structure", "evidence" ], [ 57, 60, "KBD", "structure_element" ], [ 64, 68, "MKP5", "protein" ], [ 73, 77, "p38\u03b1", "protein" ] ] }, { "sid": 165, "sent": "This complex has revealed a distinct interaction mode for MKP5.", "section": "DISCUSS", "ner": [ [ 58, 62, "MKP5", "protein" ] ] }, { "sid": 166, "sent": "The KBD of MKP5 binds to p38\u03b1 in the opposite polypeptide direction compared with how the D-motif of MKP3 binds to ERK2.", "section": "DISCUSS", "ner": [ [ 4, 7, "KBD", "structure_element" ], [ 11, 15, "MKP5", "protein" ], [ 25, 29, "p38\u03b1", "protein" ], [ 90, 97, "D-motif", "structure_element" ], [ 101, 105, "MKP3", "protein" ], [ 115, 119, "ERK2", "protein" ] ] }, { "sid": 167, "sent": "In contrast to the canonical D-motif-binding mode, separate helices, \u03b12 and \u03b13\u2032, in the KBD of MKP5 engage the p38\u03b1-docking site.", "section": "DISCUSS", "ner": [ [ 29, 49, "D-motif-binding mode", "site" ], [ 60, 67, "helices", "structure_element" ], [ 69, 71, "\u03b12", "structure_element" ], [ 76, 79, "\u03b13\u2032", "structure_element" ], [ 88, 91, "KBD", "structure_element" ], [ 95, 99, "MKP5", "protein" ], [ 111, 128, "p38\u03b1-docking site", "site" ] ] }, { "sid": 168, "sent": "Further structural and biochemical studies indicate that KBD of MKP7 may interact with p38\u03b1 in a similar manner to that of MKP5.", "section": "DISCUSS", "ner": [ [ 8, 42, "structural and biochemical studies", "experimental_method" ], [ 57, 60, "KBD", "structure_element" ], [ 64, 68, "MKP7", "protein" ], [ 87, 91, "p38\u03b1", "protein" ], [ 123, 127, "MKP5", "protein" ] ] }, { "sid": 169, "sent": "In contrast to MKP5, removal of the KBD domain from MKP7 does not drastically affect enzyme catalysis, and the kinetic parameters of MKP7-CD for p38\u03b1 substrate are very similar to those for JNK1 substrate.", "section": "DISCUSS", "ner": [ [ 15, 19, "MKP5", "protein" ], [ 21, 31, "removal of", "experimental_method" ], [ 36, 39, "KBD", "structure_element" ], [ 52, 56, "MKP7", "protein" ], [ 133, 137, "MKP7", "protein" ], [ 138, 140, "CD", "structure_element" ], [ 145, 149, "p38\u03b1", "protein" ], [ 190, 194, "JNK1", "protein" ] ] }, { "sid": 170, "sent": "Taken together, these results suggest that MKP7 utilizes a bipartite recognition mechanism to achieve the efficiency and fidelity of p38\u03b1 signalling.", "section": "DISCUSS", "ner": [ [ 43, 47, "MKP7", "protein" ], [ 133, 137, "p38\u03b1", "protein" ] ] }, { "sid": 171, "sent": "The MKP7-KBD docks to the D-site located on the back side of the p38\u03b1 catalytic pocket for high-affinity association, whereas the interaction of the MKP7-CD with another p38\u03b1 structural region, which is close to the activation loop, may not only stabilize binding but also provide contacts crucial for organizing the MKP7 active site with respect to the phosphoreceptor in the p38\u03b1 substrate for efficient dephosphorylation.", "section": "DISCUSS", "ner": [ [ 4, 8, "MKP7", "protein" ], [ 9, 12, "KBD", "structure_element" ], [ 26, 32, "D-site", "site" ], [ 65, 69, "p38\u03b1", "protein" ], [ 70, 86, "catalytic pocket", "site" ], [ 149, 153, "MKP7", "protein" ], [ 154, 156, "CD", "structure_element" ], [ 170, 174, "p38\u03b1", "protein" ], [ 216, 231, "activation loop", "structure_element" ], [ 317, 321, "MKP7", "protein" ], [ 322, 333, "active site", "site" ], [ 377, 381, "p38\u03b1", "protein" ] ] }, { "sid": 172, "sent": "In addition to the canonical D-site, the MAPK ERK2 contains a second binding site utilized by transcription factor substrates and phosphatases, the FXF-motif-binding site (also called F-site), that is exposed in active ERK2 and the D-motif peptide-induced conformation of MAPKs.", "section": "DISCUSS", "ner": [ [ 29, 35, "D-site", "site" ], [ 41, 45, "MAPK", "protein_type" ], [ 46, 50, "ERK2", "protein" ], [ 62, 81, "second binding site", "site" ], [ 130, 142, "phosphatases", "protein_type" ], [ 148, 170, "FXF-motif-binding site", "site" ], [ 184, 190, "F-site", "site" ], [ 212, 218, "active", "protein_state" ], [ 219, 223, "ERK2", "protein" ], [ 232, 239, "D-motif", "structure_element" ], [ 272, 277, "MAPKs", "protein_type" ] ] }, { "sid": 173, "sent": "This hydrophobic site was first identified by changes in deuterium exchange profiles, and is near the MAPK insertion and helix \u03b1G. Interestingly, many of the equivalent residues in JNK1, important for MKP7-CD recognition, are also used for substrate binding by ERK2 (ref.), indicating that this site is overlapped with the DEF-site previously identified in ERK2 (Fig. 5d).", "section": "DISCUSS", "ner": [ [ 5, 21, "hydrophobic site", "site" ], [ 46, 84, "changes in deuterium exchange profiles", "evidence" ], [ 102, 116, "MAPK insertion", "structure_element" ], [ 121, 126, "helix", "structure_element" ], [ 127, 129, "\u03b1G", "structure_element" ], [ 181, 185, "JNK1", "protein" ], [ 201, 205, "MKP7", "protein" ], [ 206, 208, "CD", "structure_element" ], [ 261, 265, "ERK2", "protein" ], [ 323, 331, "DEF-site", "site" ], [ 357, 361, "ERK2", "protein" ] ] }, { "sid": 174, "sent": "MKP3 is highly specific in dephosphorylating and inactivating ERK2, and the phosphatase activity of the MKP3-catalysed pNPP reaction can be markedly increased in the presence of ERK2 (refs).", "section": "DISCUSS", "ner": [ [ 0, 4, "MKP3", "protein" ], [ 62, 66, "ERK2", "protein" ], [ 104, 108, "MKP3", "protein" ], [ 119, 123, "pNPP", "chemical" ], [ 166, 177, "presence of", "protein_state" ], [ 178, 182, "ERK2", "protein" ] ] }, { "sid": 175, "sent": "Sequence alignment of all MKPs reveals a high degree of conservation of residues surrounding the interacting region observed in JNK1\u2013MKP7-CD complex (Supplementary Fig. 5).", "section": "DISCUSS", "ner": [ [ 0, 18, "Sequence alignment", "experimental_method" ], [ 26, 30, "MKPs", "protein_type" ], [ 97, 115, "interacting region", "site" ], [ 128, 140, "JNK1\u2013MKP7-CD", "complex_assembly" ] ] }, { "sid": 176, "sent": "Therefore, it is tempting to speculate that the catalytic domain of MKP3 may bind to ERK2 in a manner analogous to the way by which MKP7-CD binds to JNK1.", "section": "DISCUSS", "ner": [ [ 48, 64, "catalytic domain", "structure_element" ], [ 68, 72, "MKP3", "protein" ], [ 85, 89, "ERK2", "protein" ], [ 132, 136, "MKP7", "protein" ], [ 137, 139, "CD", "structure_element" ], [ 149, 153, "JNK1", "protein" ] ] }, { "sid": 177, "sent": "A comprehensive examination of the molecular basis of the specific ERK2 recognition by MKP3 is underway.", "section": "DISCUSS", "ner": [ [ 67, 71, "ERK2", "protein" ], [ 87, 91, "MKP3", "protein" ] ] }, { "sid": 178, "sent": "The ongoing work demonstrates that although the overall interaction modes are similar between the JNK1\u2013MKP7-CD and ERK2\u2013MKP3-CD complexes, the ERK2\u2013MKP3-CD interaction is less extensive and helix \u03b14 from MKP3-CD does not interact directly with ERK2.", "section": "DISCUSS", "ner": [ [ 98, 110, "JNK1\u2013MKP7-CD", "complex_assembly" ], [ 115, 127, "ERK2\u2013MKP3-CD", "complex_assembly" ], [ 143, 155, "ERK2\u2013MKP3-CD", "complex_assembly" ], [ 190, 195, "helix", "structure_element" ], [ 196, 198, "\u03b14", "structure_element" ], [ 204, 208, "MKP3", "protein" ], [ 209, 211, "CD", "structure_element" ], [ 244, 248, "ERK2", "protein" ] ] }, { "sid": 179, "sent": "The FXF-motif-mediated interaction is critical for both pERK2 inactivation and ERK2-induced MKP3 activation (manuscript in preparation).", "section": "DISCUSS", "ner": [ [ 4, 13, "FXF-motif", "structure_element" ], [ 56, 57, "p", "protein_state" ], [ 57, 61, "ERK2", "protein" ], [ 79, 83, "ERK2", "protein" ], [ 92, 96, "MKP3", "protein" ] ] }, { "sid": 180, "sent": "In summary, we have resolved the structure of JNK1 in complex with the catalytic domain of MKP7.", "section": "DISCUSS", "ner": [ [ 33, 42, "structure", "evidence" ], [ 46, 50, "JNK1", "protein" ], [ 51, 66, "in complex with", "protein_state" ], [ 71, 87, "catalytic domain", "structure_element" ], [ 91, 95, "MKP7", "protein" ] ] }, { "sid": 181, "sent": "This structure reveals an FXF-docking interaction mode between MAPK and MKP.", "section": "DISCUSS", "ner": [ [ 5, 14, "structure", "evidence" ], [ 26, 54, "FXF-docking interaction mode", "site" ], [ 63, 67, "MAPK", "protein_type" ], [ 72, 75, "MKP", "protein_type" ] ] }, { "sid": 182, "sent": "Results from biochemical characterization of the Phe285 and Phe287 MKP7 mutants combined with structural information support the conclusion that the two Phe residues serve different roles in the catalytic reaction.", "section": "DISCUSS", "ner": [ [ 13, 41, "biochemical characterization", "experimental_method" ], [ 49, 55, "Phe285", "residue_name_number" ], [ 60, 66, "Phe287", "residue_name_number" ], [ 67, 71, "MKP7", "protein" ], [ 72, 79, "mutants", "protein_state" ], [ 94, 116, "structural information", "evidence" ], [ 153, 156, "Phe", "residue_name" ] ] }, { "sid": 183, "sent": "Phe285 is essential for JNK1 substrate binding, whereas Phe287 plays a role for the precise alignment of active-site residues, which are important for transition-state stabilization.", "section": "DISCUSS", "ner": [ [ 0, 6, "Phe285", "residue_name_number" ], [ 24, 28, "JNK1", "protein" ], [ 56, 62, "Phe287", "residue_name_number" ], [ 105, 125, "active-site residues", "site" ] ] }, { "sid": 184, "sent": "This newly identified FXF-type motif is present in all MKPs, except that the residue at the first position in MKP5 is an equivalent hydrophobic leucine residue (see also Fig. 7f,g), suggesting that these two Phe residues would play a similar role in facilitating substrate recognition and catalysis, respectively.", "section": "DISCUSS", "ner": [ [ 22, 36, "FXF-type motif", "structure_element" ], [ 55, 59, "MKPs", "protein_type" ], [ 110, 114, "MKP5", "protein" ], [ 144, 151, "leucine", "residue_name" ], [ 208, 211, "Phe", "residue_name" ] ] }, { "sid": 185, "sent": "An important feature of MKP\u2013JNK1 interactions is that MKP7 or MKP5 only interact with the F-site of JNK1.", "section": "DISCUSS", "ner": [ [ 24, 27, "MKP", "protein_type" ], [ 28, 32, "JNK1", "protein" ], [ 54, 58, "MKP7", "protein" ], [ 62, 66, "MKP5", "protein" ], [ 90, 96, "F-site", "site" ], [ 100, 104, "JNK1", "protein" ] ] }, { "sid": 186, "sent": "One possible explanation is that JNK1 needs to use the D-site to interact with JIP-1, a scaffold protein for JNK signalling.", "section": "DISCUSS", "ner": [ [ 33, 37, "JNK1", "protein" ], [ 55, 61, "D-site", "site" ], [ 79, 84, "JIP-1", "protein" ], [ 109, 112, "JNK", "protein_type" ] ] }, { "sid": 187, "sent": "The N-terminal JNK-binding domain of JIP-1 interacts with the D-site on JNK and this interaction is required for JIP-1-mediated enhancement of JNK activation.", "section": "DISCUSS", "ner": [ [ 15, 33, "JNK-binding domain", "structure_element" ], [ 37, 42, "JIP-1", "protein" ], [ 62, 68, "D-site", "site" ], [ 72, 75, "JNK", "protein_type" ], [ 113, 118, "JIP-1", "protein" ], [ 143, 146, "JNK", "protein_type" ] ] }, { "sid": 188, "sent": "In addition, JIP-1 can also associate with MKP7 via the C-terminal region of MKP7 (ref.).", "section": "DISCUSS", "ner": [ [ 13, 18, "JIP-1", "protein" ], [ 43, 47, "MKP7", "protein" ], [ 56, 73, "C-terminal region", "structure_element" ], [ 77, 81, "MKP7", "protein" ] ] }, { "sid": 189, "sent": "When MKP7 is bound to JIP-1, it reduces JNK activation, leading to reduced phosphorylation of the JNK target c-Jun.", "section": "DISCUSS", "ner": [ [ 5, 9, "MKP7", "protein" ], [ 13, 21, "bound to", "protein_state" ], [ 22, 27, "JIP-1", "protein" ], [ 40, 43, "JNK", "protein_type" ], [ 98, 101, "JNK", "protein_type" ], [ 109, 114, "c-Jun", "protein_type" ] ] }, { "sid": 190, "sent": "Thus, our biochemical and structural data allow us to present a model for the JNK1\u2013JIP-1\u2013MKP7 ternary complex and provide an important insight into the assembly and function of JNK signalling modules (Supplementary Fig. 6).", "section": "DISCUSS", "ner": [ [ 10, 41, "biochemical and structural data", "evidence" ], [ 78, 93, "JNK1\u2013JIP-1\u2013MKP7", "complex_assembly" ], [ 177, 180, "JNK", "protein_type" ] ] }, { "sid": 191, "sent": "The cDNAs of human MKP7 and MKP5 were kindly provided by Dr Mathijs Baens (University of Leuven) and Dr Eisuke Nishida (Kyoto University), respectively.", "section": "METHODS", "ner": [ [ 13, 18, "human", "species" ] ] }, { "sid": 192, "sent": "The cDNAs of human ASK1, MKK4, MKK7 and JNK1 were kindly provided by Dr Zhenguo Wu (Hong Kong University of Science and Technology).", "section": "METHODS", "ner": [ [ 13, 18, "human", "species" ] ] }, { "sid": 193, "sent": "The human full-length JNK1, MKK4, MKK7 and the kinase domain of ASK1 (659\u2013951) were cloned into pGEX4T-2, pET15b and/or pET21b vectors to produce a GST- or His-tagged protein.", "section": "METHODS", "ner": [ [ 4, 9, "human", "species" ] ] }, { "sid": 194, "sent": "The crystal structure of unphosphorylated JNK1 in complex with the catalytic domain of MKP7 was refined to 2.4\u2009\u00c5 resolution.", "section": "METHODS", "ner": [ [ 67, 83, "catalytic domain", "structure_element" ] ] }, { "sid": 195, "sent": "Domain structures of ten human MKPs and the atypical VHR.", "section": "FIG", "ner": [ [ 7, 17, "structures", "evidence" ], [ 25, 30, "human", "species" ], [ 31, 35, "MKPs", "protein_type" ], [ 53, 56, "VHR", "protein" ] ] }, { "sid": 196, "sent": "On the basis of sequence similarity, protein structure, substrate specificity and subcellular localization, the ten members of MKP family can be divided into three groups.", "section": "FIG", "ner": [ [ 45, 54, "structure", "evidence" ], [ 127, 137, "MKP family", "protein_type" ] ] }, { "sid": 197, "sent": "The first subfamily comprises MKP1, MKP2, PAC1 and hVH3, which are inducible nuclear phosphatases and can dephosphorylate ERK (and JNK, p38) MAPKs.", "section": "FIG", "ner": [ [ 30, 34, "MKP1", "protein" ], [ 36, 40, "MKP2", "protein" ], [ 42, 46, "PAC1", "protein" ], [ 51, 55, "hVH3", "protein" ], [ 67, 76, "inducible", "protein_state" ], [ 77, 97, "nuclear phosphatases", "protein_type" ], [ 122, 125, "ERK", "protein_type" ], [ 131, 134, "JNK", "protein_type" ], [ 136, 139, "p38", "protein_type" ], [ 141, 146, "MAPKs", "protein_type" ] ] }, { "sid": 198, "sent": "The second subfamily contains MKP3, MKP4 and MKPX, which are cytoplasmic ERK-specific MKPs.", "section": "FIG", "ner": [ [ 30, 34, "MKP3", "protein" ], [ 36, 40, "MKP4", "protein" ], [ 45, 49, "MKPX", "protein" ], [ 73, 90, "ERK-specific MKPs", "protein_type" ] ] }, { "sid": 199, "sent": "The third subfamily comprises MKP5, MKP7 and hVH5, which were located in both nucleus and cytoplasm, and selectively inactivate JNK and p38.", "section": "FIG", "ner": [ [ 30, 34, "MKP5", "protein" ], [ 36, 40, "MKP7", "protein" ], [ 45, 49, "hVH5", "protein" ], [ 128, 131, "JNK", "protein_type" ], [ 136, 139, "p38", "protein_type" ] ] }, { "sid": 200, "sent": "All MKPs contain both the CD and KBD domains, whereas VHR, an atypical MKP, only contains a highly conserved catalytic domain.", "section": "FIG", "ner": [ [ 4, 8, "MKPs", "protein_type" ], [ 26, 28, "CD", "structure_element" ], [ 33, 36, "KBD", "structure_element" ], [ 54, 57, "VHR", "protein" ], [ 71, 74, "MKP", "protein_type" ], [ 92, 108, "highly conserved", "protein_state" ], [ 109, 125, "catalytic domain", "structure_element" ] ] }, { "sid": 201, "sent": "In addition to the CD and KBD, MKP7 contains a unique long C-terminal region that contains NES, NLS and PEST motifs, which has no effect on the binding ability and phosphatase activity of MKP7 toward MAPKs.", "section": "FIG", "ner": [ [ 19, 21, "CD", "structure_element" ], [ 26, 29, "KBD", "structure_element" ], [ 31, 35, "MKP7", "protein" ], [ 59, 76, "C-terminal region", "structure_element" ], [ 91, 94, "NES", "structure_element" ], [ 96, 99, "NLS", "structure_element" ], [ 104, 115, "PEST motifs", "structure_element" ], [ 188, 192, "MKP7", "protein" ], [ 200, 205, "MAPKs", "protein_type" ] ] }, { "sid": 202, "sent": "NES, nuclear export signal; NLS, nuclear localization signal; PEST, C-terminal sequence rich in prolines, glutamates, serines and threonines.", "section": "FIG", "ner": [ [ 0, 3, "NES", "structure_element" ], [ 5, 26, "nuclear export signal", "structure_element" ], [ 28, 31, "NLS", "structure_element" ], [ 33, 60, "nuclear localization signal", "structure_element" ], [ 62, 66, "PEST", "structure_element" ], [ 68, 92, "C-terminal sequence rich", "structure_element" ], [ 96, 104, "prolines", "residue_name" ], [ 106, 116, "glutamates", "residue_name" ], [ 118, 125, "serines", "residue_name" ], [ 130, 140, "threonines", "residue_name" ] ] }, { "sid": 203, "sent": "MKP7-CD is crucial for JNK1 binding and enzyme catalysis.", "section": "FIG", "ner": [ [ 0, 4, "MKP7", "protein" ], [ 5, 7, "CD", "structure_element" ], [ 23, 27, "JNK1", "protein" ] ] }, { "sid": 204, "sent": "(a) Domain organization of human MKP7 and JNK1.", "section": "FIG", "ner": [ [ 27, 32, "human", "species" ], [ 33, 37, "MKP7", "protein" ], [ 42, 46, "JNK1", "protein" ] ] }, { "sid": 205, "sent": "The KBD and CD of MKP7 are shown in green and blue, and the N-lobe and C-lobe of JNK1 are coloured in lemon and yellow, respectively.", "section": "FIG", "ner": [ [ 4, 7, "KBD", "structure_element" ], [ 12, 14, "CD", "structure_element" ], [ 18, 22, "MKP7", "protein" ], [ 60, 66, "N-lobe", "structure_element" ], [ 71, 77, "C-lobe", "structure_element" ], [ 81, 85, "JNK1", "protein" ] ] }, { "sid": 206, "sent": "The colour scheme is the same in the following figures unless indicated otherwise. (b) Plots of initial velocity of the MKP7-catalysed reaction versus phospho-JNK1 concentration.", "section": "FIG", "ner": [ [ 87, 112, "Plots of initial velocity", "evidence" ], [ 120, 124, "MKP7", "protein" ], [ 151, 158, "phospho", "ptm" ], [ 159, 163, "JNK1", "protein" ] ] }, { "sid": 207, "sent": "The error bars represent s.e.m. (c) Gel filtration analysis for interaction of JNK1 with MKP7-CD and MKP7-KBD.", "section": "FIG", "ner": [ [ 36, 59, "Gel filtration analysis", "experimental_method" ], [ 79, 83, "JNK1", "protein" ], [ 89, 93, "MKP7", "protein" ], [ 94, 96, "CD", "structure_element" ], [ 101, 105, "MKP7", "protein" ], [ 106, 109, "KBD", "structure_element" ] ] }, { "sid": 208, "sent": "(d) GST-mediated pull-down assay for interaction of JNK1 with MKP7-CD and MKP7-KBD.", "section": "FIG", "ner": [ [ 4, 32, "GST-mediated pull-down assay", "experimental_method" ], [ 52, 56, "JNK1", "protein" ], [ 62, 66, "MKP7", "protein" ], [ 67, 69, "CD", "structure_element" ], [ 74, 78, "MKP7", "protein" ], [ 79, 82, "KBD", "structure_element" ] ] }, { "sid": 209, "sent": "The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays.", "section": "FIG", "ner": [ [ 33, 43, "affinities", "evidence" ], [ 47, 51, "MKP7", "protein" ], [ 52, 54, "CD", "structure_element" ], [ 59, 63, "MKP7", "protein" ], [ 64, 67, "KBD", "structure_element" ], [ 71, 75, "JNK1", "protein" ], [ 86, 94, "affinity", "evidence" ], [ 98, 102, "MKP7", "protein" ], [ 103, 105, "CD", "structure_element" ], [ 174, 178, "MKP7", "protein" ], [ 183, 187, "JNK1", "protein" ], [ 194, 214, "GST pull-down assays", "experimental_method" ] ] }, { "sid": 210, "sent": "The protein amounts of MKP7 used are shown at the bottom.", "section": "FIG", "ner": [ [ 23, 27, "MKP7", "protein" ] ] }, { "sid": 211, "sent": "Structure of JNK1 in complex with MKP7-CD.", "section": "FIG", "ner": [ [ 0, 9, "Structure", "evidence" ], [ 13, 17, "JNK1", "protein" ], [ 18, 33, "in complex with", "protein_state" ], [ 34, 38, "MKP7", "protein" ], [ 39, 41, "CD", "structure_element" ] ] }, { "sid": 212, "sent": "(a) Ribbon diagram of JNK1\u2013MKP7-CD complex in two views related by a 45\u00b0 rotation around a vertical axis. (b) Structure of MKP7-CD with its active site highlight in cyan.", "section": "FIG", "ner": [ [ 22, 34, "JNK1\u2013MKP7-CD", "complex_assembly" ], [ 110, 119, "Structure", "evidence" ], [ 123, 127, "MKP7", "protein" ], [ 128, 130, "CD", "structure_element" ], [ 140, 151, "active site", "site" ] ] }, { "sid": 213, "sent": "The 2Fo\u2212Fc omit map (contoured at 1.5\u03c3) for the P-loop of MKP7-CD is shown at inset of b. (c) Structure of VHR with its active site highlighted in marine blue. (d) Close-up view of the JNK1\u2013MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan).", "section": "FIG", "ner": [ [ 4, 19, "2Fo\u2212Fc omit map", "evidence" ], [ 48, 54, "P-loop", "structure_element" ], [ 58, 62, "MKP7", "protein" ], [ 63, 65, "CD", "structure_element" ], [ 94, 103, "Structure", "evidence" ], [ 107, 110, "VHR", "protein" ], [ 120, 131, "active site", "site" ], [ 185, 204, "JNK1\u2013MKP7 interface", "site" ], [ 240, 244, "JNK1", "protein" ], [ 258, 262, "MKP7", "protein" ], [ 263, 265, "CD", "structure_element" ] ] }, { "sid": 214, "sent": "The JNK1 is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red).", "section": "FIG", "ner": [ [ 4, 8, "JNK1", "protein" ] ] }, { "sid": 215, "sent": "(e) Interaction networks mainly involving helices \u03b14 and \u03b15 from MKP7-CD, and \u03b1G and \u03b12L14 of JNK1.", "section": "FIG", "ner": [ [ 4, 24, "Interaction networks", "site" ], [ 42, 49, "helices", "structure_element" ], [ 50, 52, "\u03b14", "structure_element" ], [ 57, 59, "\u03b15", "structure_element" ], [ 65, 69, "MKP7", "protein" ], [ 70, 72, "CD", "structure_element" ], [ 78, 80, "\u03b1G", "structure_element" ], [ 85, 90, "\u03b12L14", "structure_element" ], [ 94, 98, "JNK1", "protein" ] ] }, { "sid": 216, "sent": "MKP7-CD is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red).", "section": "FIG", "ner": [ [ 0, 4, "MKP7", "protein" ], [ 5, 7, "CD", "structure_element" ] ] }, { "sid": 217, "sent": "Blue dashed lines represent polar interactions.", "section": "FIG", "ner": [ [ 28, 46, "polar interactions", "bond_interaction" ], [ 28, 46, "polar interactions", "bond_interaction" ] ] }, { "sid": 218, "sent": "(f) The 2Fo\u2212Fc omit map (contoured at 1.5\u03c3) clearly shows electron density for the 285FNFL288 segment of MKP7-CD.", "section": "FIG", "ner": [ [ 8, 23, "2Fo\u2212Fc omit map", "evidence" ], [ 58, 74, "electron density", "evidence" ], [ 83, 101, "285FNFL288 segment", "structure_element" ], [ 105, 109, "MKP7", "protein" ], [ 110, 112, "CD", "structure_element" ] ] }, { "sid": 219, "sent": "Mutational analysis on interactions between MKP7-CD and JNK1.", "section": "FIG", "ner": [ [ 0, 19, "Mutational analysis", "experimental_method" ], [ 44, 48, "MKP7", "protein" ], [ 49, 51, "CD", "structure_element" ], [ 56, 60, "JNK1", "protein" ] ] }, { "sid": 220, "sent": "(a) Effects of mutations in MKP7-CD on the JNK1 dephosphorylation (mean\u00b1s.e.m., n=3).", "section": "FIG", "ner": [ [ 28, 32, "MKP7", "protein" ], [ 33, 35, "CD", "structure_element" ], [ 43, 47, "JNK1", "protein" ], [ 48, 65, "dephosphorylation", "ptm" ] ] }, { "sid": 221, "sent": "Residues involved in hydrophobic and hydrophilic contacts are coloured in red and blue, respectively. (b) Gel filtration analysis for interaction of JNK1 with MKP7-CD mutant F285D.", "section": "FIG", "ner": [ [ 21, 57, "hydrophobic and hydrophilic contacts", "bond_interaction" ], [ 106, 129, "Gel filtration analysis", "experimental_method" ], [ 149, 153, "JNK1", "protein" ], [ 159, 163, "MKP7", "protein" ], [ 164, 166, "CD", "structure_element" ], [ 167, 173, "mutant", "protein_state" ], [ 174, 179, "F285D", "mutant" ] ] }, { "sid": 222, "sent": "Mutant F285D and JNK1 were eluted as monomers, with the molecular masses of \u223c17 and 44\u2009kDa, respectively.", "section": "FIG", "ner": [ [ 0, 6, "Mutant", "protein_state" ], [ 7, 12, "F285D", "mutant" ], [ 17, 21, "JNK1", "protein" ], [ 37, 45, "monomers", "oligomeric_state" ] ] }, { "sid": 223, "sent": "However, in contrast to the wild-type MKP7-CD, mutant F285D did not co-migrate with JNK1.", "section": "FIG", "ner": [ [ 28, 37, "wild-type", "protein_state" ], [ 38, 42, "MKP7", "protein" ], [ 43, 45, "CD", "structure_element" ], [ 47, 53, "mutant", "protein_state" ], [ 54, 59, "F285D", "mutant" ], [ 84, 88, "JNK1", "protein" ] ] }, { "sid": 224, "sent": "(c) Pull-down assays of MKP7-CD by GST-tagged JNK1 mutants.", "section": "FIG", "ner": [ [ 4, 20, "Pull-down assays", "experimental_method" ], [ 24, 28, "MKP7", "protein" ], [ 29, 31, "CD", "structure_element" ], [ 35, 45, "GST-tagged", "protein_state" ], [ 46, 50, "JNK1", "protein" ], [ 51, 58, "mutants", "protein_state" ] ] }, { "sid": 225, "sent": "The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays.", "section": "FIG", "ner": [ [ 33, 43, "affinities", "evidence" ], [ 47, 51, "MKP7", "protein" ], [ 52, 54, "CD", "structure_element" ], [ 58, 62, "JNK1", "protein" ], [ 63, 70, "mutants", "protein_state" ], [ 81, 89, "affinity", "evidence" ], [ 93, 102, "wild-type", "protein_state" ], [ 103, 107, "JNK1", "protein" ], [ 176, 180, "MKP7", "protein" ], [ 181, 183, "CD", "structure_element" ], [ 188, 192, "JNK1", "protein" ], [ 193, 200, "mutants", "protein_state" ], [ 207, 227, "GST pull-down assays", "experimental_method" ] ] }, { "sid": 226, "sent": "The protein amounts of MKP7-CD used are shown at the bottom. (d) Circular dichroism spectra for MKP7-CD wild type and mutants.", "section": "FIG", "ner": [ [ 23, 27, "MKP7", "protein" ], [ 28, 30, "CD", "structure_element" ], [ 65, 83, "Circular dichroism", "experimental_method" ], [ 84, 91, "spectra", "evidence" ], [ 96, 100, "MKP7", "protein" ], [ 101, 103, "CD", "structure_element" ], [ 104, 113, "wild type", "protein_state" ], [ 118, 125, "mutants", "protein_state" ] ] }, { "sid": 227, "sent": "Measurements were averaged for three scans. (e) Circular dichroism spectra for JNK1 wild type and mutants.", "section": "FIG", "ner": [ [ 48, 66, "Circular dichroism", "experimental_method" ], [ 67, 74, "spectra", "evidence" ], [ 79, 83, "JNK1", "protein" ], [ 84, 93, "wild type", "protein_state" ], [ 98, 105, "mutants", "protein_state" ] ] }, { "sid": 228, "sent": "(f) Effects of mutations in MKP7-CD on the pNPP hydrolysis reaction (mean\u00b1s.e.m., n=3).", "section": "FIG", "ner": [ [ 15, 24, "mutations", "experimental_method" ], [ 28, 32, "MKP7", "protein" ], [ 33, 35, "CD", "structure_element" ], [ 43, 47, "pNPP", "chemical" ] ] }, { "sid": 229, "sent": "Comparison of CDK2-KAP and JNK1\u2013MKP7-CD.", "section": "FIG", "ner": [ [ 14, 22, "CDK2-KAP", "complex_assembly" ], [ 27, 39, "JNK1\u2013MKP7-CD", "complex_assembly" ] ] }, { "sid": 230, "sent": "(a) Superposition of the complex structures of CDK2-KAP (PDB 1FQ1) and JNK1\u2013MKP7-CD.", "section": "FIG", "ner": [ [ 4, 17, "Superposition", "experimental_method" ], [ 33, 43, "structures", "evidence" ], [ 47, 55, "CDK2-KAP", "complex_assembly" ], [ 71, 83, "JNK1\u2013MKP7-CD", "complex_assembly" ] ] }, { "sid": 231, "sent": "The N-lobe and C-lobe of CDK2 are coloured in grey and pink, respectively, and KAP is coloured in green.", "section": "FIG", "ner": [ [ 4, 10, "N-lobe", "structure_element" ], [ 15, 21, "C-lobe", "structure_element" ], [ 25, 29, "CDK2", "protein" ], [ 79, 82, "KAP", "protein" ] ] }, { "sid": 232, "sent": "The interactions between these two proteins consist of three discontinuous contact regions, centred at the multiple hydrogen bonds between the pThr160 of CDK2 and the active site of KAP (region I).", "section": "FIG", "ner": [ [ 75, 90, "contact regions", "site" ], [ 116, 130, "hydrogen bonds", "bond_interaction" ], [ 143, 150, "pThr160", "ptm" ], [ 154, 158, "CDK2", "protein" ], [ 167, 178, "active site", "site" ], [ 182, 185, "KAP", "protein" ], [ 187, 195, "region I", "structure_element" ] ] }, { "sid": 233, "sent": "Interestingly, the recognition of CDK2 by KAP is augmented by a similar interface as that observed in the complex of JNK1 and MKP7-CD (region II).", "section": "FIG", "ner": [ [ 34, 38, "CDK2", "protein" ], [ 42, 45, "KAP", "protein" ], [ 72, 81, "interface", "site" ], [ 117, 121, "JNK1", "protein" ], [ 126, 130, "MKP7", "protein" ], [ 131, 133, "CD", "structure_element" ], [ 135, 144, "region II", "structure_element" ] ] }, { "sid": 234, "sent": "(b) Interactions networks at the auxiliary region II mainly involving helix \u03b17 from KAP and the \u03b1G helix and following L14 loop of CDK2.", "section": "FIG", "ner": [ [ 33, 52, "auxiliary region II", "structure_element" ], [ 70, 75, "helix", "structure_element" ], [ 76, 78, "\u03b17", "structure_element" ], [ 84, 87, "KAP", "protein" ], [ 96, 104, "\u03b1G helix", "structure_element" ], [ 119, 127, "L14 loop", "structure_element" ], [ 131, 135, "CDK2", "protein" ] ] }, { "sid": 235, "sent": "The CDK2 is shown in surface representation coloured according to the electrostatic potential (positive, blue; negative, red).", "section": "FIG", "ner": [ [ 4, 8, "CDK2", "protein" ] ] }, { "sid": 236, "sent": "Residues of KAP and CDK2 are highlighted as green and red sticks, respectively.", "section": "FIG", "ner": [ [ 12, 15, "KAP", "protein" ], [ 20, 24, "CDK2", "protein" ] ] }, { "sid": 237, "sent": "One remarkable difference between these two kinase-phosphatase complexes is that helix \u03b16 of KAP (corresponding to helix \u03b14 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs.", "section": "FIG", "ner": [ [ 81, 86, "helix", "structure_element" ], [ 87, 89, "\u03b16", "structure_element" ], [ 93, 96, "KAP", "protein" ], [ 115, 120, "helix", "structure_element" ], [ 121, 123, "\u03b14", "structure_element" ], [ 127, 131, "MKP7", "protein" ], [ 132, 134, "CD", "structure_element" ], [ 185, 191, "stable", "protein_state" ], [ 192, 203, "heterodimer", "oligomeric_state" ], [ 207, 211, "CDK2", "protein" ], [ 216, 219, "KAP", "protein" ], [ 225, 243, "Sequence alignment", "experimental_method" ], [ 251, 274, "JNK-interacting regions", "site" ], [ 278, 282, "MKPs", "protein_type" ] ] }, { "sid": 238, "sent": "Residues of MKP7-CD involved in JNK1 recognition are indicated by cyan asterisks, and the conserved FXF-motif is highlighted in cyan.", "section": "FIG", "ner": [ [ 12, 16, "MKP7", "protein" ], [ 17, 19, "CD", "structure_element" ], [ 32, 36, "JNK1", "protein" ], [ 90, 99, "conserved", "protein_state" ], [ 100, 109, "FXF-motif", "structure_element" ] ] }, { "sid": 239, "sent": "The secondary structure assignments of MKP7-CD and KAP are shown above and below each sequence.", "section": "FIG", "ner": [ [ 39, 43, "MKP7", "protein" ], [ 44, 46, "CD", "structure_element" ], [ 51, 54, "KAP", "protein" ] ] }, { "sid": 240, "sent": "(d) Sequence alignment of the F-site regions on MAPKs.", "section": "FIG", "ner": [ [ 4, 22, "Sequence alignment", "experimental_method" ], [ 30, 44, "F-site regions", "structure_element" ], [ 48, 53, "MAPKs", "protein_type" ] ] }, { "sid": 241, "sent": "Residues of JNK1 involved in recognition of MKP7 are indicated by orange asterisks, and those forming the F-site are highlighted in yellow.", "section": "FIG", "ner": [ [ 12, 16, "JNK1", "protein" ], [ 44, 48, "MKP7", "protein" ], [ 106, 112, "F-site", "site" ] ] }, { "sid": 242, "sent": "FXF-motif is critical for controlling the phosphorylation of JNK and ultraviolet-induced apoptosis.", "section": "FIG", "ner": [ [ 0, 9, "FXF-motif", "structure_element" ], [ 42, 57, "phosphorylation", "ptm" ], [ 61, 64, "JNK", "protein_type" ] ] }, { "sid": 243, "sent": "(a\u2013c) FXF-motif is essential for the dephosphorylation of JNK by MKP7.", "section": "FIG", "ner": [ [ 6, 15, "FXF-motif", "structure_element" ], [ 58, 61, "JNK", "protein_type" ], [ 65, 69, "MKP7", "protein" ] ] }, { "sid": 244, "sent": "HEK293T cells were infected with lentiviruses expressing MKP7 and its mutants (1.0\u2009\u03bcg).", "section": "FIG", "ner": [ [ 33, 45, "lentiviruses", "taxonomy_domain" ], [ 57, 61, "MKP7", "protein" ], [ 70, 77, "mutants", "protein_state" ] ] }, { "sid": 245, "sent": "After 36\u2009h infection, cells were untreated in a, stimulated with 30\u2009\u03bcM etoposide for 3\u2009h in b or irradiated with 25\u2009J\u2009m\u22122 ultraviolet light at 30\u2009min before lysis in c. Whole-cell extracts were then immunoblotted with antibody indicated.", "section": "FIG", "ner": [ [ 71, 80, "etoposide", "chemical" ] ] }, { "sid": 246, "sent": "Shown is a typical immunoblot for phosphorylated JNK from three independent experiments.", "section": "FIG", "ner": [ [ 34, 48, "phosphorylated", "protein_state" ], [ 49, 52, "JNK", "protein_type" ] ] }, { "sid": 247, "sent": "(d) F-site is required for JNK1 to interact with MKP7.", "section": "FIG", "ner": [ [ 4, 10, "F-site", "site" ], [ 27, 31, "JNK1", "protein" ], [ 49, 53, "MKP7", "protein" ] ] }, { "sid": 248, "sent": "HEK293T cells were co-transfected with MKP7 full-length (1.0\u2009\u03bcg) and JNK1 (wild type or mutants as indicated, 1.0\u2009\u03bcg).", "section": "FIG", "ner": [ [ 19, 33, "co-transfected", "experimental_method" ], [ 39, 43, "MKP7", "protein" ], [ 44, 55, "full-length", "protein_state" ], [ 69, 73, "JNK1", "protein" ], [ 75, 84, "wild type", "protein_state" ], [ 88, 95, "mutants", "protein_state" ] ] }, { "sid": 249, "sent": "Whole-cell extracts were then immunoprecipitated with antibody against Myc for MKP7; immunobloting was carried out with antibodies indicated.", "section": "FIG", "ner": [ [ 30, 48, "immunoprecipitated", "experimental_method" ], [ 79, 83, "MKP7", "protein" ] ] }, { "sid": 250, "sent": "IP, immunoprecipitation; TCL, total cell lysate.", "section": "FIG", "ner": [ [ 0, 2, "IP", "experimental_method" ], [ 4, 23, "immunoprecipitation", "experimental_method" ] ] }, { "sid": 251, "sent": "(e) Effect of MKP7 (wild type or mutants) expression on ultraviolet-induced apoptosis.", "section": "FIG", "ner": [ [ 14, 18, "MKP7", "protein" ], [ 20, 29, "wild type", "protein_state" ], [ 33, 40, "mutants", "protein_state" ] ] }, { "sid": 252, "sent": "HeLa cells were infected with lentiviruses expressing MKP7 full-length and its mutants.", "section": "FIG", "ner": [ [ 30, 42, "lentiviruses", "taxonomy_domain" ], [ 54, 58, "MKP7", "protein" ], [ 59, 70, "full-length", "protein_state" ], [ 79, 86, "mutants", "protein_state" ] ] }, { "sid": 253, "sent": "Cells were then subjected to flow cytometry analysis.", "section": "FIG", "ner": [ [ 29, 43, "flow cytometry", "experimental_method" ] ] }, { "sid": 254, "sent": "Apoptotic cells were determined by Annexin-V-APC/PI staining.", "section": "FIG", "ner": [ [ 35, 48, "Annexin-V-APC", "chemical" ], [ 49, 51, "PI", "chemical" ] ] }, { "sid": 255, "sent": "The results using Annexin-V stain for membrane phosphatidylserine eversion, combined with propidium iodide (PI) uptake to evaluate cells whose membranes had been compromised.", "section": "FIG", "ner": [ [ 18, 27, "Annexin-V", "chemical" ], [ 90, 106, "propidium iodide", "chemical" ], [ 108, 110, "PI", "chemical" ] ] }, { "sid": 256, "sent": "Staining with both Annexin-V and PI indicate apoptosis (upper right quadrant).", "section": "FIG", "ner": [ [ 19, 28, "Annexin-V", "chemical" ], [ 33, 35, "PI", "chemical" ] ] }, { "sid": 257, "sent": "(f) Statistical analysis of apoptotic cells (mean\u00b1s.e.m., n=3), *P<0.05, ***P<0.001 (ANOVA followed by Tukey's test).", "section": "FIG", "ner": [ [ 64, 66, "*P", "evidence" ], [ 73, 77, "***P", "evidence" ], [ 85, 90, "ANOVA", "experimental_method" ], [ 103, 115, "Tukey's test", "experimental_method" ] ] }, { "sid": 258, "sent": "MKP5-CD is crucial for JNK1 binding and enzyme catalysis.", "section": "FIG", "ner": [ [ 0, 4, "MKP5", "protein" ], [ 5, 7, "CD", "structure_element" ], [ 23, 27, "JNK1", "protein" ] ] }, { "sid": 259, "sent": "(a) Domain organization of human MKP5.", "section": "FIG", "ner": [ [ 27, 32, "human", "species" ], [ 33, 37, "MKP5", "protein" ] ] }, { "sid": 260, "sent": "The KBD and CD of MKP5 are shown in brown and grey, respectively. (b) Plots of initial velocity of the MKP5-catalysed reaction versus phospho-JNK1 concentration.", "section": "FIG", "ner": [ [ 4, 7, "KBD", "structure_element" ], [ 12, 14, "CD", "structure_element" ], [ 18, 22, "MKP5", "protein" ], [ 70, 95, "Plots of initial velocity", "evidence" ], [ 103, 107, "MKP5", "protein" ], [ 134, 141, "phospho", "protein_state" ], [ 142, 146, "JNK1", "protein" ] ] }, { "sid": 261, "sent": "The solid lines are best-fitting results according to the Michaelis\u2013Menten equation with Km and kcat values indicated.", "section": "FIG", "ner": [ [ 89, 91, "Km", "evidence" ], [ 96, 100, "kcat", "evidence" ] ] }, { "sid": 262, "sent": "The error bars represent s.e.m. (c) Structural comparison of the JNK-interacting residues on MKP5-CD (PDB 1ZZW) and MKP7-CD.", "section": "FIG", "ner": [ [ 36, 57, "Structural comparison", "experimental_method" ], [ 65, 89, "JNK-interacting residues", "site" ], [ 93, 97, "MKP5", "protein" ], [ 98, 100, "CD", "structure_element" ], [ 116, 120, "MKP7", "protein" ], [ 121, 123, "CD", "structure_element" ] ] }, { "sid": 263, "sent": "The corresponding residues on MKP5 are depicted as orange sticks, and MKP5 residues numbers are in parentheses.", "section": "FIG", "ner": [ [ 30, 34, "MKP5", "protein" ], [ 70, 74, "MKP5", "protein" ] ] }, { "sid": 264, "sent": "(d) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. (e) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD.", "section": "FIG", "ner": [ [ 4, 27, "Gel filtration analysis", "experimental_method" ], [ 47, 51, "JNK1", "protein" ], [ 57, 61, "MKP5", "protein" ], [ 62, 64, "CD", "structure_element" ], [ 69, 73, "MKP5", "protein" ], [ 74, 77, "KBD", "structure_element" ], [ 83, 112, "GST-mediated pull-down assays", "experimental_method" ], [ 132, 136, "JNK1", "protein" ], [ 142, 146, "MKP5", "protein" ], [ 147, 149, "CD", "structure_element" ], [ 154, 158, "MKP5", "protein" ], [ 159, 162, "KBD", "structure_element" ] ] }, { "sid": 265, "sent": "The panels are arranged the same as in Fig. 2d. (f) Effects of mutations in MKP5-CD on the JNK1 dephosphorylation (mean\u00b1s.e.m., n=3).", "section": "FIG", "ner": [ [ 63, 72, "mutations", "experimental_method" ], [ 76, 80, "MKP5", "protein" ], [ 81, 83, "CD", "structure_element" ], [ 91, 95, "JNK1", "protein" ] ] }, { "sid": 266, "sent": "(g) Effects of mutations in MKP5-CD on the pNPP hydrolysis reaction (mean\u00b1s.e.m., n=3).", "section": "FIG", "ner": [ [ 15, 24, "mutations", "experimental_method" ], [ 28, 32, "MKP5", "protein" ], [ 33, 35, "CD", "structure_element" ], [ 43, 47, "pNPP", "chemical" ] ] }, { "sid": 267, "sent": "(h) Pull-down assays of MKP5-CD by GST-tagged JNK1 mutants.", "section": "FIG", "ner": [ [ 4, 20, "Pull-down assays", "experimental_method" ], [ 24, 28, "MKP5", "protein" ], [ 29, 31, "CD", "structure_element" ], [ 35, 45, "GST-tagged", "protein_state" ], [ 46, 50, "JNK1", "protein" ], [ 51, 58, "mutants", "protein_state" ] ] } ] }, "PMC4848090": { "annotations": [ { "sid": 0, "sent": "Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission", "section": "TITLE", "ner": [ [ 27, 42, "peptide hormone", "protein_type" ] ] }, { "sid": 1, "sent": "Plants constantly renew during their life cycle and thus require to shed senescent and damaged organs.", "section": "ABSTRACT", "ner": [ [ 0, 6, "Plants", "taxonomy_domain" ] ] }, { "sid": 2, "sent": "Floral abscission is controlled by the leucine-rich repeat receptor kinase (LRR-RK) HAESA and the peptide hormone IDA.", "section": "ABSTRACT", "ner": [ [ 39, 74, "leucine-rich repeat receptor kinase", "protein_type" ], [ 76, 82, "LRR-RK", "protein_type" ], [ 84, 89, "HAESA", "protein" ], [ 98, 113, "peptide hormone", "protein_type" ], [ 114, 117, "IDA", "protein" ] ] }, { "sid": 3, "sent": "It is unknown how expression of IDA in the abscission zone leads to HAESA activation.", "section": "ABSTRACT", "ner": [ [ 32, 35, "IDA", "protein" ], [ 68, 73, "HAESA", "protein" ] ] }, { "sid": 4, "sent": "Here we show that IDA is sensed directly by the HAESA ectodomain.", "section": "ABSTRACT", "ner": [ [ 18, 21, "IDA", "protein" ], [ 48, 53, "HAESA", "protein" ], [ 54, 64, "ectodomain", "structure_element" ] ] }, { "sid": 5, "sent": "Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide.", "section": "ABSTRACT", "ner": [ [ 0, 18, "Crystal structures", "evidence" ], [ 22, 27, "HAESA", "protein" ], [ 28, 43, "in complex with", "protein_state" ], [ 44, 47, "IDA", "protein" ], [ 57, 79, "hormone binding pocket", "site" ], [ 101, 107, "active", "protein_state" ], [ 108, 117, "dodecamer", "structure_element" ], [ 118, 125, "peptide", "chemical" ] ] }, { "sid": 6, "sent": "A central hydroxyproline residue anchors IDA to the receptor.", "section": "ABSTRACT", "ner": [ [ 10, 24, "hydroxyproline", "residue_name" ], [ 41, 44, "IDA", "protein" ] ] }, { "sid": 7, "sent": "The HAESA co-receptor SERK1, a positive regulator of the floral abscission pathway, allows for high-affinity sensing of the peptide hormone by binding to an Arg-His-Asn motif in IDA.", "section": "ABSTRACT", "ner": [ [ 4, 9, "HAESA", "protein" ], [ 10, 21, "co-receptor", "protein_type" ], [ 22, 27, "SERK1", "protein" ], [ 124, 139, "peptide hormone", "protein_type" ], [ 157, 174, "Arg-His-Asn motif", "structure_element" ], [ 178, 181, "IDA", "protein" ] ] }, { "sid": 8, "sent": "This sequence pattern is conserved among diverse plant peptides, suggesting that plant peptide hormone receptors may share a common ligand binding mode and activation mechanism.", "section": "ABSTRACT", "ner": [ [ 25, 34, "conserved", "protein_state" ], [ 49, 54, "plant", "taxonomy_domain" ], [ 55, 63, "peptides", "chemical" ], [ 81, 86, "plant", "taxonomy_domain" ], [ 87, 112, "peptide hormone receptors", "protein_type" ] ] }, { "sid": 9, "sent": "Plants can shed their leaves, flowers or other organs when they no longer need them. But how does a leaf or a flower know when to let go? A receptor protein called HAESA is found on the surface of the cells that surround a future break point on the plant. When its time to shed an organ, a hormone called IDA instructs HAESA to trigger the shedding process.", "section": "ABSTRACT", "ner": [ [ 0, 6, "Plants", "taxonomy_domain" ], [ 140, 156, "receptor protein", "protein_type" ], [ 164, 169, "HAESA", "protein" ], [ 290, 297, "hormone", "chemical" ], [ 305, 308, "IDA", "protein" ], [ 319, 324, "HAESA", "protein" ] ] }, { "sid": 10, "sent": "However, the molecular details of how IDA triggers organ shedding are not clear.", "section": "ABSTRACT", "ner": [ [ 38, 41, "IDA", "protein" ] ] }, { "sid": 11, "sent": "The shedding of floral organs (or leaves) can be easily studied in a model plant called Arabidopsis.", "section": "ABSTRACT", "ner": [ [ 75, 80, "plant", "taxonomy_domain" ], [ 88, 99, "Arabidopsis", "taxonomy_domain" ] ] }, { "sid": 12, "sent": "Santiago et al. used protein biochemistry, structural biology and genetics to uncover how the IDA hormone activates HAESA.", "section": "ABSTRACT", "ner": [ [ 21, 41, "protein biochemistry", "experimental_method" ], [ 43, 61, "structural biology", "experimental_method" ], [ 66, 74, "genetics", "experimental_method" ], [ 94, 97, "IDA", "protein" ], [ 98, 105, "hormone", "chemical" ], [ 116, 121, "HAESA", "protein" ] ] }, { "sid": 13, "sent": "The experiments show that IDA binds directly to a canyon shaped pocket in HAESA that extends out from the surface of the cell.", "section": "ABSTRACT", "ner": [ [ 26, 29, "IDA", "protein" ], [ 30, 47, "binds directly to", "protein_state" ], [ 50, 63, "canyon shaped", "protein_state" ], [ 64, 70, "pocket", "site" ], [ 74, 79, "HAESA", "protein" ] ] }, { "sid": 14, "sent": "IDA binding to HAESA allows another receptor protein called SERK1 to bind to HAESA, which results in the release of signals inside the cell that trigger the shedding of organs.", "section": "ABSTRACT", "ner": [ [ 0, 3, "IDA", "protein" ], [ 15, 20, "HAESA", "protein" ], [ 36, 52, "receptor protein", "protein_type" ], [ 60, 65, "SERK1", "protein" ], [ 66, 76, "to bind to", "protein_state" ], [ 77, 82, "HAESA", "protein" ] ] }, { "sid": 15, "sent": "The next step following on from this work is to understand what signals are produced when IDA activates HAESA.", "section": "ABSTRACT", "ner": [ [ 90, 93, "IDA", "protein" ], [ 104, 109, "HAESA", "protein" ] ] }, { "sid": 16, "sent": "Another challenge will be to find out where IDA is produced in the plant and what causes it to accumulate in specific places in preparation for organ shedding.", "section": "ABSTRACT", "ner": [ [ 44, 47, "IDA", "protein" ], [ 67, 72, "plant", "taxonomy_domain" ] ] }, { "sid": 17, "sent": "The HAESA ectodomain folds into a superhelical assembly of 21 leucine-rich repeats.", "section": "FIG", "ner": [ [ 4, 9, "HAESA", "protein" ], [ 10, 20, "ectodomain", "structure_element" ], [ 34, 55, "superhelical assembly", "structure_element" ], [ 62, 82, "leucine-rich repeats", "structure_element" ] ] }, { "sid": 18, "sent": "(A) SDS PAGE analysis of the purified Arabidopsis thaliana HAESA ectodomain (residues 20\u2013620) obtained by secreted expression in insect cells.", "section": "FIG", "ner": [ [ 4, 12, "SDS PAGE", "experimental_method" ], [ 38, 58, "Arabidopsis thaliana", "species" ], [ 59, 64, "HAESA", "protein" ], [ 65, 75, "ectodomain", "structure_element" ], [ 86, 92, "20\u2013620", "residue_range" ], [ 106, 141, "secreted expression in insect cells", "experimental_method" ] ] }, { "sid": 19, "sent": "The calculated molecular mass is 65.7 kDa, the actual molecular mass obtained by mass spectrometry is 74,896 Da, accounting for the N-glycans. (B) Ribbon diagrams showing front (left panel) and side views (right panel) of the isolated HAESA LRR domain.", "section": "FIG", "ner": [ [ 81, 98, "mass spectrometry", "experimental_method" ], [ 132, 141, "N-glycans", "chemical" ], [ 235, 240, "HAESA", "protein" ], [ 241, 251, "LRR domain", "structure_element" ] ] }, { "sid": 20, "sent": "The N- (residues 20\u201388) and C-terminal (residues 593\u2013615) capping domains are shown in yellow, the central 21 LRR motifs are in blue and disulphide bonds are highlighted in green (in bonds representation). (C) Structure based sequence alignment of the 21 leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison.", "section": "FIG", "ner": [ [ 17, 22, "20\u201388", "residue_range" ], [ 49, 56, "593\u2013615", "residue_range" ], [ 58, 73, "capping domains", "structure_element" ], [ 110, 120, "LRR motifs", "structure_element" ], [ 137, 153, "disulphide bonds", "ptm" ], [ 210, 244, "Structure based sequence alignment", "experimental_method" ], [ 255, 275, "leucine-rich repeats", "structure_element" ], [ 279, 284, "HAESA", "protein" ], [ 294, 299, "plant", "taxonomy_domain" ], [ 300, 303, "LRR", "structure_element" ] ] }, { "sid": 21, "sent": "Conserved hydrophobic residues are shaded in gray, N-glycosylation sites visible in our structures are highlighted in blue, cysteine residues involved in disulphide bridge formation in green. (D) Asn-linked glycans mask the N-terminal portion of the HAESA ectodomain.", "section": "FIG", "ner": [ [ 0, 9, "Conserved", "protein_state" ], [ 10, 21, "hydrophobic", "protein_state" ], [ 22, 30, "residues", "structure_element" ], [ 51, 72, "N-glycosylation sites", "site" ], [ 88, 98, "structures", "evidence" ], [ 124, 132, "cysteine", "residue_name" ], [ 154, 171, "disulphide bridge", "ptm" ], [ 196, 214, "Asn-linked glycans", "ptm" ], [ 250, 255, "HAESA", "protein" ], [ 256, 266, "ectodomain", "structure_element" ] ] }, { "sid": 22, "sent": "Oligomannose core structures (containing two N-actylglucosamines and three terminal mannose units) as found in Trichoplusia ni cells and in plants were modeled onto the seven glycosylation sites observed in our HAESA structures, to visualize the surface areas potentially not masked by carbohydrate.", "section": "FIG", "ner": [ [ 0, 12, "Oligomannose", "chemical" ], [ 45, 64, "N-actylglucosamines", "chemical" ], [ 84, 91, "mannose", "chemical" ], [ 111, 126, "Trichoplusia ni", "species" ], [ 140, 146, "plants", "taxonomy_domain" ], [ 175, 194, "glycosylation sites", "site" ], [ 211, 216, "HAESA", "protein" ], [ 217, 227, "structures", "evidence" ], [ 286, 298, "carbohydrate", "chemical" ] ] }, { "sid": 23, "sent": "The HAESA ectodomain is shown in blue (in surface representation), the glycan structures are shown in yellow.", "section": "FIG", "ner": [ [ 4, 9, "HAESA", "protein" ], [ 10, 20, "ectodomain", "structure_element" ], [ 71, 77, "glycan", "chemical" ] ] }, { "sid": 24, "sent": "Hydrophobic contacts and a hydrogen-bond network mediate the interaction between HAESA and the peptide hormone IDA.", "section": "FIG", "ner": [ [ 0, 20, "Hydrophobic contacts", "bond_interaction" ], [ 27, 48, "hydrogen-bond network", "site" ], [ 81, 86, "HAESA", "protein" ], [ 95, 110, "peptide hormone", "protein_type" ], [ 111, 114, "IDA", "protein" ] ] }, { "sid": 25, "sent": "(A) Details of the IDA binding pocket.", "section": "FIG", "ner": [ [ 19, 37, "IDA binding pocket", "site" ] ] }, { "sid": 26, "sent": "HAESA is shown in blue (ribbon diagram), the C-terminal Arg-His-Asn motif (left panel), the central Hyp anchor (center) and the N-terminal Pro-rich motif in IDA (right panel) are shown in yellow (in bonds representation).", "section": "FIG", "ner": [ [ 0, 5, "HAESA", "protein" ], [ 56, 73, "Arg-His-Asn motif", "structure_element" ], [ 100, 110, "Hyp anchor", "structure_element" ], [ 139, 153, "Pro-rich motif", "structure_element" ], [ 157, 160, "IDA", "protein" ] ] }, { "sid": 27, "sent": "HAESA interface residues are shown as sticks, selected hydrogen bond interactions are denoted as dotted lines (in magenta). (B) View of the complete IDA (in bonds representation, in yellow) binding pocket in HAESA (surface view, in blue).", "section": "FIG", "ner": [ [ 0, 24, "HAESA interface residues", "site" ], [ 55, 81, "hydrogen bond interactions", "bond_interaction" ], [ 149, 152, "IDA", "protein" ], [ 190, 204, "binding pocket", "site" ], [ 208, 213, "HAESA", "protein" ] ] }, { "sid": 28, "sent": "Orientation as in (A). (C) Structure based sequence alignment of leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison.", "section": "FIG", "ner": [ [ 27, 61, "Structure based sequence alignment", "experimental_method" ], [ 65, 85, "leucine-rich repeats", "structure_element" ], [ 89, 94, "HAESA", "protein" ], [ 104, 109, "plant", "taxonomy_domain" ], [ 110, 113, "LRR", "structure_element" ], [ 114, 132, "consensus sequence", "evidence" ] ] }, { "sid": 29, "sent": "Residues mediating hydrophobic interactions with the IDA peptide are highlighted in blue, residues contributing to hydrogen bond interactions and/or salt bridges are shown in red.", "section": "FIG", "ner": [ [ 19, 43, "hydrophobic interactions", "bond_interaction" ], [ 53, 64, "IDA peptide", "chemical" ], [ 115, 141, "hydrogen bond interactions", "bond_interaction" ], [ 149, 161, "salt bridges", "bond_interaction" ] ] }, { "sid": 30, "sent": "The IDA binding pocket covers LRRs 2\u201314 and all residues originate from the inner surface of the HAESA superhelix.", "section": "FIG", "ner": [ [ 4, 22, "IDA binding pocket", "site" ], [ 30, 39, "LRRs 2\u201314", "structure_element" ], [ 97, 102, "HAESA", "protein" ], [ 103, 113, "superhelix", "structure_element" ] ] }, { "sid": 31, "sent": "The IDA-HAESA and SERK1-HAESA complex interfaces are conserved among HAESA and HAESA-like\u00a0proteins from different plant species.", "section": "FIG", "ner": [ [ 4, 13, "IDA-HAESA", "complex_assembly" ], [ 18, 29, "SERK1-HAESA", "complex_assembly" ], [ 38, 48, "interfaces", "site" ], [ 53, 62, "conserved", "protein_state" ], [ 69, 74, "HAESA", "protein" ], [ 79, 98, "HAESA-like\u00a0proteins", "protein_type" ], [ 114, 119, "plant", "taxonomy_domain" ] ] }, { "sid": 32, "sent": "Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39).", "section": "FIG", "ner": [ [ 0, 34, "Structure-based sequence alignment", "experimental_method" ], [ 42, 62, "HAESA family members", "protein_type" ], [ 64, 84, "Arabidopsis thaliana", "species" ], [ 85, 90, "HAESA", "protein" ], [ 137, 157, "Arabidopsis thaliana", "species" ], [ 158, 162, "HSL2", "protein" ], [ 184, 200, "Capsella rubella", "species" ], [ 201, 206, "HAESA", "protein" ], [ 228, 245, "Citrus clementina", "species" ], [ 246, 250, "HSL2", "protein" ], [ 272, 286, "Vitis vinifera", "species" ], [ 287, 292, "HAESA", "protein" ] ] }, { "sid": 33, "sent": "The alignment includes a secondary structure assignment calculated with the program DSSP and colored according to Figure 1, with the N- and C-terminal caps and the 21 LRR motifs indicated in orange and blue, respectively.", "section": "FIG", "ner": [ [ 151, 155, "caps", "structure_element" ], [ 167, 177, "LRR motifs", "structure_element" ] ] }, { "sid": 34, "sent": "Cysteine residues engaged in disulphide bonds are depicted in green.", "section": "FIG", "ner": [ [ 0, 8, "Cysteine", "residue_name" ], [ 29, 45, "disulphide bonds", "ptm" ] ] }, { "sid": 35, "sent": "HAESA residues interacting with the IDA peptide and/or the SERK1 co-receptor kinase ectodomain are highlighted in blue and orange, respectively.", "section": "FIG", "ner": [ [ 0, 5, "HAESA", "protein" ], [ 36, 47, "IDA peptide", "chemical" ], [ 59, 64, "SERK1", "protein" ], [ 65, 83, "co-receptor kinase", "protein_type" ], [ 84, 94, "ectodomain", "structure_element" ] ] }, { "sid": 36, "sent": "The peptide hormone IDA binds to the HAESA LRR ectodomain.", "section": "FIG", "ner": [ [ 4, 19, "peptide hormone", "protein_type" ], [ 20, 23, "IDA", "protein" ], [ 37, 42, "HAESA", "protein" ], [ 43, 57, "LRR ectodomain", "structure_element" ] ] }, { "sid": 37, "sent": "(A) Multiple sequence alignment of selected IDA family members.", "section": "FIG", "ner": [ [ 4, 31, "Multiple sequence alignment", "experimental_method" ], [ 44, 62, "IDA family members", "protein_type" ] ] }, { "sid": 38, "sent": "The conserved PIP motif is highlighted in yellow, the central Hyp in blue.", "section": "FIG", "ner": [ [ 4, 13, "conserved", "protein_state" ], [ 14, 23, "PIP motif", "structure_element" ], [ 62, 65, "Hyp", "residue_name" ] ] }, { "sid": 39, "sent": "The PKGV motif present in our N-terminally extended IDA peptide is highlighted in red. (B) Isothermal titration calorimetry of the HAESA ectodomain vs. IDA and including the synthetic peptide sequence.", "section": "FIG", "ner": [ [ 4, 14, "PKGV motif", "structure_element" ], [ 30, 51, "N-terminally extended", "protein_state" ], [ 52, 63, "IDA peptide", "chemical" ], [ 91, 123, "Isothermal titration calorimetry", "experimental_method" ], [ 131, 136, "HAESA", "protein" ], [ 137, 147, "ectodomain", "structure_element" ], [ 152, 155, "IDA", "protein" ], [ 174, 183, "synthetic", "protein_state" ], [ 184, 191, "peptide", "chemical" ] ] }, { "sid": 40, "sent": "(C) Structure of the HAESA \u2013 IDA complex with HAESA shown in blue (ribbon diagram).", "section": "FIG", "ner": [ [ 21, 32, "HAESA \u2013 IDA", "complex_assembly" ], [ 46, 51, "HAESA", "protein" ] ] }, { "sid": 41, "sent": "IDA (in bonds representation, surface view included) is depicted in yellow.", "section": "FIG", "ner": [ [ 0, 3, "IDA", "protein" ] ] }, { "sid": 42, "sent": "The peptide binding pocket covers HAESA LRRs 2\u201314. (D) Close-up view of the entire IDA (in yellow) peptide binding site in HAESA (in blue).", "section": "FIG", "ner": [ [ 4, 26, "peptide binding pocket", "site" ], [ 34, 39, "HAESA", "protein" ], [ 40, 49, "LRRs 2\u201314", "structure_element" ], [ 83, 86, "IDA", "protein" ], [ 99, 119, "peptide binding site", "site" ], [ 123, 128, "HAESA", "protein" ] ] }, { "sid": 43, "sent": "Details of the interactions between the central Hyp anchor in IDA and the C-terminal Arg-His-Asn motif with HAESA\u00a0are highlighted in (E) and (F), respectively.", "section": "FIG", "ner": [ [ 48, 58, "Hyp anchor", "structure_element" ], [ 62, 65, "IDA", "protein" ], [ 85, 102, "Arg-His-Asn motif", "structure_element" ], [ 108, 113, "HAESA", "protein" ] ] }, { "sid": 44, "sent": "Hydrogren bonds are depicted as dotted lines (in magenta), a water molecule is shown as a red sphere.", "section": "FIG", "ner": [ [ 61, 66, "water", "chemical" ] ] }, { "sid": 45, "sent": "During their growth, development and reproduction plants use cell separation processes to detach no-longer required, damaged or senescent organs.", "section": "INTRO", "ner": [ [ 50, 56, "plants", "taxonomy_domain" ] ] }, { "sid": 46, "sent": "Abscission of floral organs in Arabidopsis is a model system to study these cell separation processes in molecular detail.", "section": "INTRO", "ner": [ [ 31, 42, "Arabidopsis", "taxonomy_domain" ] ] }, { "sid": 47, "sent": "The LRR-RKs HAESA (greek: to adhere to) and HAESA-LIKE 2 (HSL2) redundantly control floral abscission.", "section": "INTRO", "ner": [ [ 4, 11, "LRR-RKs", "structure_element" ], [ 12, 17, "HAESA", "protein" ], [ 44, 56, "HAESA-LIKE 2", "protein" ], [ 58, 62, "HSL2", "protein" ] ] }, { "sid": 48, "sent": "Loss-of-function of the secreted small protein INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) causes floral organs to remain attached while its over-expression leads to premature shedding.", "section": "INTRO", "ner": [ [ 47, 84, "INFLORESCENCE DEFICIENT IN ABSCISSION", "protein" ], [ 86, 89, "IDA", "protein" ] ] }, { "sid": 49, "sent": "Full-length IDA is proteolytically processed and a conserved stretch of 20 amino-acids (termed EPIP) can rescue the IDA loss-of-function phenotype (Figure 1A).", "section": "INTRO", "ner": [ [ 0, 11, "Full-length", "protein_state" ], [ 12, 15, "IDA", "protein" ], [ 19, 44, "proteolytically processed", "ptm" ], [ 51, 60, "conserved", "protein_state" ], [ 61, 86, "stretch of 20 amino-acids", "residue_range" ], [ 95, 99, "EPIP", "structure_element" ], [ 116, 119, "IDA", "protein" ] ] }, { "sid": 50, "sent": "It has been demonstrated that a dodecamer peptide within EPIP is able to activate HAESA and\u00a0HSL2 in transient assays in tobacco cells.", "section": "INTRO", "ner": [ [ 32, 41, "dodecamer", "structure_element" ], [ 42, 49, "peptide", "chemical" ], [ 57, 61, "EPIP", "structure_element" ], [ 82, 87, "HAESA", "protein" ], [ 92, 96, "HSL2", "protein" ], [ 100, 116, "transient assays", "experimental_method" ], [ 120, 127, "tobacco", "taxonomy_domain" ] ] }, { "sid": 51, "sent": "This sequence motif is highly conserved among IDA family members (IDA-LIKE PROTEINS, IDLs) and contains a central Pro residue, presumed to be post-translationally modified to hydroxyproline (Hyp; Figure 1A).", "section": "INTRO", "ner": [ [ 0, 19, "This sequence motif", "structure_element" ], [ 23, 39, "highly conserved", "protein_state" ], [ 46, 64, "IDA family members", "protein_type" ], [ 66, 83, "IDA-LIKE PROTEINS", "protein_type" ], [ 85, 89, "IDLs", "protein_type" ], [ 114, 117, "Pro", "residue_name" ], [ 142, 171, "post-translationally modified", "protein_state" ], [ 175, 189, "hydroxyproline", "residue_name" ], [ 191, 194, "Hyp", "residue_name" ] ] }, { "sid": 52, "sent": "The available genetic and biochemical evidence suggests that IDA and HAESA together control floral abscission, but it is poorly understood if IDA is directly sensed by the receptor kinase HAESA and how IDA binding at the cell surface would activate the receptor.", "section": "INTRO", "ner": [ [ 61, 64, "IDA", "protein" ], [ 69, 74, "HAESA", "protein" ], [ 142, 145, "IDA", "protein" ], [ 172, 187, "receptor kinase", "protein_type" ], [ 188, 193, "HAESA", "protein" ], [ 202, 205, "IDA", "protein" ] ] }, { "sid": 53, "sent": "IDA directly binds to the LRR domain of HAESA", "section": "RESULTS", "ner": [ [ 0, 3, "IDA", "protein" ], [ 26, 36, "LRR domain", "structure_element" ], [ 40, 45, "HAESA", "protein" ] ] }, { "sid": 54, "sent": "Active IDA-family peptide hormones are hydroxyprolinated dodecamers.", "section": "FIG", "ner": [ [ 0, 6, "Active", "protein_state" ], [ 7, 34, "IDA-family peptide hormones", "protein_type" ], [ 39, 56, "hydroxyprolinated", "protein_state" ], [ 57, 67, "dodecamers", "structure_element" ] ] }, { "sid": 55, "sent": "Close-up views of (A) IDA, (B) the N-terminally extended PKGV-IDA and (C) IDL1 bound to the HAESA hormone binding pocket (in bonds representation, in yellow) and including simulated annealing 2Fo\u2013Fc omit electron density maps contoured at 1.0 \u03c3.", "section": "FIG", "ner": [ [ 22, 25, "IDA", "protein" ], [ 35, 56, "N-terminally extended", "protein_state" ], [ 57, 65, "PKGV-IDA", "mutant" ], [ 74, 78, "IDL1", "protein" ], [ 79, 87, "bound to", "protein_state" ], [ 92, 97, "HAESA", "protein" ], [ 98, 120, "hormone binding pocket", "site" ], [ 172, 191, "simulated annealing", "experimental_method" ], [ 192, 225, "2Fo\u2013Fc omit electron density maps", "evidence" ] ] }, { "sid": 56, "sent": "Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (\u0394H), binding entropies (\u0394S) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( \u00b1 fitting errors; n.d.", "section": "FIG", "ner": [ [ 10, 15, "Pro58", "residue_name_number" ], [ 15, 18, "IDA", "protein" ], [ 23, 28, "Leu67", "residue_name_number" ], [ 28, 31, "IDA", "protein" ], [ 66, 82, "electron density", "evidence" ], [ 88, 96, "bound to", "protein_state" ], [ 101, 106, "HAESA", "protein" ], [ 107, 117, "ectodomain", "structure_element" ], [ 143, 177, "equilibrium dissociation constants", "evidence" ], [ 179, 181, "Kd", "evidence" ], [ 184, 202, "binding enthalpies", "evidence" ], [ 204, 206, "\u0394H", "evidence" ], [ 209, 226, "binding entropies", "evidence" ], [ 228, 230, "\u0394S", "evidence" ], [ 270, 282, "IDA peptides", "chemical" ], [ 298, 303, "HAESA", "protein" ], [ 304, 314, "ectodomain", "structure_element" ] ] }, { "sid": 57, "sent": "no detectable binding). (E) Structural superposition of the active IDA (in bonds representation, in gray) and IDL1 peptide (in yellow) hormones bound to the HAESA ectodomain.", "section": "FIG", "ner": [ [ 28, 52, "Structural superposition", "experimental_method" ], [ 60, 66, "active", "protein_state" ], [ 67, 70, "IDA", "protein" ], [ 110, 122, "IDL1 peptide", "chemical" ], [ 144, 152, "bound to", "protein_state" ], [ 157, 162, "HAESA", "protein" ], [ 163, 173, "ectodomain", "structure_element" ] ] }, { "sid": 58, "sent": "Root mean square deviation (r.m.s.d.) is 1.0 \u00c5 comparing 100 corresponding atoms.", "section": "FIG", "ner": [ [ 0, 26, "Root mean square deviation", "evidence" ], [ 28, 36, "r.m.s.d.", "evidence" ] ] }, { "sid": 59, "sent": "The receptor kinase SERK1 acts as a HAESA co-receptor and promotes high-affinity IDA sensing.", "section": "FIG", "ner": [ [ 4, 19, "receptor kinase", "protein_type" ], [ 20, 25, "SERK1", "protein" ], [ 36, 53, "HAESA co-receptor", "protein_type" ], [ 81, 84, "IDA", "protein" ] ] }, { "sid": 60, "sent": "(A) Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers.", "section": "FIG", "ner": [ [ 4, 31, "Petal break-strength assays", "experimental_method" ], [ 131, 135, "serk", "gene" ], [ 136, 142, "mutant", "protein_state" ], [ 143, 149, "plants", "taxonomy_domain" ], [ 162, 167, "haesa", "gene" ], [ 168, 172, "hsl2", "gene" ], [ 173, 179, "mutant", "protein_state" ], [ 190, 199, "wild-type", "protein_state" ] ] }, { "sid": 61, "sent": "Petal break-strength was found significantly increased in almost all positions (indicated with a *) for haesa/hsl2 and serk1-1 mutant plants with respect to the Col-0 control.", "section": "FIG", "ner": [ [ 104, 109, "haesa", "gene" ], [ 110, 114, "hsl2", "gene" ], [ 119, 126, "serk1-1", "gene" ], [ 127, 133, "mutant", "protein_state" ], [ 134, 140, "plants", "taxonomy_domain" ] ] }, { "sid": 62, "sent": "(B) Analytical size-exclusion chromatography.", "section": "FIG", "ner": [ [ 4, 44, "Analytical size-exclusion chromatography", "experimental_method" ] ] }, { "sid": 63, "sent": "The HAESA LRR domain elutes as a monomer (black dotted line), as does the isolated SERK1 ectodomain (blue dotted line).", "section": "FIG", "ner": [ [ 4, 9, "HAESA", "protein" ], [ 10, 20, "LRR domain", "structure_element" ], [ 33, 40, "monomer", "oligomeric_state" ], [ 83, 88, "SERK1", "protein" ], [ 89, 99, "ectodomain", "structure_element" ] ] }, { "sid": 64, "sent": "A HAESA \u2013 IDA \u2013 SERK1 complex elutes as an apparent heterodimer (red line), while a mixture of HAESA and SERK1 yields two isolated peaks that correspond to monomeric HAESA and SERK1, respectively (black line).", "section": "FIG", "ner": [ [ 2, 21, "HAESA \u2013 IDA \u2013 SERK1", "complex_assembly" ], [ 52, 63, "heterodimer", "oligomeric_state" ], [ 95, 100, "HAESA", "protein" ], [ 105, 110, "SERK1", "protein" ], [ 156, 165, "monomeric", "oligomeric_state" ], [ 166, 171, "HAESA", "protein" ], [ 176, 181, "SERK1", "protein" ] ] }, { "sid": 65, "sent": "Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da).", "section": "FIG", "ner": [ [ 113, 126, "Thyroglobulin", "protein" ], [ 143, 151, "Ferritin", "protein" ], [ 167, 175, "Aldolase", "protein" ], [ 192, 202, "Conalbumin", "protein" ], [ 218, 227, "Ovalbumin", "protein" ], [ 243, 261, "Carbonic anhydrase", "protein" ], [ 113, 126, "Thyroglobulin", "protein" ], [ 143, 151, "Ferritin", "protein" ], [ 167, 175, "Aldolase", "protein" ], [ 192, 202, "Conalbumin", "protein" ], [ 218, 227, "Ovalbumin", "protein" ], [ 243, 261, "Carbonic anhydrase", "protein" ] ] }, { "sid": 66, "sent": "A SDS PAGE of the peak fractions is shown alongside.", "section": "FIG", "ner": [ [ 2, 10, "SDS PAGE", "experimental_method" ], [ 2, 10, "SDS PAGE", "experimental_method" ] ] }, { "sid": 67, "sent": "Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. (C) Isothermal titration calorimetry of wild-type and Hyp64\u2192Pro IDA versus the HAESA and SERK1 ectodomains.", "section": "FIG", "ner": [ [ 9, 14, "HAESA", "protein" ], [ 19, 24, "SERK1", "protein" ], [ 64, 96, "Isothermal titration calorimetry", "experimental_method" ], [ 100, 109, "wild-type", "protein_state" ], [ 114, 123, "Hyp64\u2192Pro", "ptm" ], [ 114, 119, "Hyp64", "ptm" ], [ 124, 127, "IDA", "protein" ], [ 139, 144, "HAESA", "protein" ], [ 149, 154, "SERK1", "protein" ], [ 155, 166, "ectodomains", "structure_element" ] ] }, { "sid": 68, "sent": "The titration of IDA wild-type versus the isolated HAESA ectodomain from Figure 1B is shown for comparison (red line; n.d.", "section": "FIG", "ner": [ [ 4, 13, "titration", "experimental_method" ], [ 17, 20, "IDA", "protein" ], [ 21, 30, "wild-type", "protein_state" ], [ 51, 56, "HAESA", "protein" ], [ 57, 67, "ectodomain", "structure_element" ] ] }, { "sid": 69, "sent": "no detectable binding) (D) Analytical size-exclusion chromatography in the presence of the IDA Hyp64\u2192Pro mutant peptide reveals no complex formation between HAESA and SERK1 ectodomains.", "section": "FIG", "ner": [ [ 27, 67, "Analytical size-exclusion chromatography", "experimental_method" ], [ 75, 86, "presence of", "protein_state" ], [ 91, 94, "IDA", "protein" ], [ 95, 104, "Hyp64\u2192Pro", "ptm" ], [ 95, 100, "Hyp64", "ptm" ], [ 105, 111, "mutant", "protein_state" ], [ 112, 119, "peptide", "chemical" ], [ 157, 162, "HAESA", "protein" ], [ 167, 172, "SERK1", "protein" ], [ 173, 184, "ectodomains", "structure_element" ] ] }, { "sid": 70, "sent": "(E) In vitro kinase assays of the HAESA and SERK1 kinase domains.", "section": "FIG", "ner": [ [ 4, 26, "In vitro kinase assays", "experimental_method" ], [ 34, 39, "HAESA", "protein" ], [ 44, 49, "SERK1", "protein" ], [ 50, 64, "kinase domains", "structure_element" ] ] }, { "sid": 71, "sent": "Wild-type HAESA and SERK1 kinase domains (KDs) exhibit auto-phosphorylation activities (lanes 1 + 3).", "section": "FIG", "ner": [ [ 0, 9, "Wild-type", "protein_state" ], [ 10, 15, "HAESA", "protein" ], [ 20, 25, "SERK1", "protein" ], [ 26, 40, "kinase domains", "structure_element" ], [ 42, 45, "KDs", "structure_element" ] ] }, { "sid": 72, "sent": "Mutant (m)\u00a0versions, which carry point mutations in their active sites (Asp837HAESA\u2192Asn, Asp447SERK1\u2192Asn) possess no autophosphorylation activity (lanes 2+4).", "section": "FIG", "ner": [ [ 0, 6, "Mutant", "protein_state" ], [ 33, 48, "point mutations", "experimental_method" ], [ 58, 70, "active sites", "site" ], [ 72, 87, "Asp837HAESA\u2192Asn", "mutant" ], [ 89, 104, "Asp447SERK1\u2192Asn", "mutant" ] ] }, { "sid": 73, "sent": "Transphosphorylation activity from the active kinase to the mutated form can be observed in both directions (lanes 5+6).", "section": "FIG", "ner": [ [ 39, 45, "active", "protein_state" ], [ 60, 67, "mutated", "protein_state" ] ] }, { "sid": 74, "sent": "We purified the HAESA ectodomain (residues 20\u2013620) from baculovirus-infected insect cells (Figure 1\u2014figure supplement 1A, see Materials\u00a0and\u00a0methods) and quantified the interaction of the ~75\u2009kDa glycoprotein with synthetic IDA peptides using isothermal titration calorimetry (ITC).", "section": "RESULTS", "ner": [ [ 3, 11, "purified", "experimental_method" ], [ 16, 21, "HAESA", "protein" ], [ 22, 32, "ectodomain", "structure_element" ], [ 43, 49, "20\u2013620", "residue_range" ], [ 56, 89, "baculovirus-infected insect cells", "experimental_method" ], [ 195, 207, "glycoprotein", "protein_type" ], [ 213, 222, "synthetic", "protein_state" ], [ 223, 235, "IDA peptides", "chemical" ], [ 242, 274, "isothermal titration calorimetry", "experimental_method" ], [ 276, 279, "ITC", "experimental_method" ] ] }, { "sid": 75, "sent": "A Hyp-modified dodecamer comprising the highly conserved PIP motif in IDA (Figure 1A) interacts with HAESA with 1:1 stoichiometry (N) and with a dissociation constant (Kd) of ~20\u2009\u03bcM (Figure 1B).", "section": "RESULTS", "ner": [ [ 2, 14, "Hyp-modified", "protein_state" ], [ 15, 24, "dodecamer", "structure_element" ], [ 40, 56, "highly conserved", "protein_state" ], [ 57, 66, "PIP motif", "structure_element" ], [ 70, 73, "IDA", "protein" ], [ 101, 106, "HAESA", "protein" ], [ 145, 166, "dissociation constant", "evidence" ], [ 168, 170, "Kd", "evidence" ] ] }, { "sid": 76, "sent": "We next determined crystal structures of the apo HAESA ectodomain and of a HAESA-IDA complex, at 1.74 and 1.86\u2009\u00c5 resolution, respectively (Figure 1C; Figure 1\u2014figure supplement 1B\u2013D; Tables 1,2).", "section": "RESULTS", "ner": [ [ 19, 37, "crystal structures", "evidence" ], [ 45, 48, "apo", "protein_state" ], [ 49, 54, "HAESA", "protein" ], [ 55, 65, "ectodomain", "structure_element" ], [ 75, 84, "HAESA-IDA", "complex_assembly" ] ] }, { "sid": 77, "sent": "IDA binds in a completely extended conformation along the inner surface of the HAESA ectodomain, covering LRRs 2\u201314 (Figure 1C,D, Figure 1\u2014figure supplement 2).", "section": "RESULTS", "ner": [ [ 0, 3, "IDA", "protein" ], [ 15, 47, "completely extended conformation", "protein_state" ], [ 79, 84, "HAESA", "protein" ], [ 85, 95, "ectodomain", "structure_element" ], [ 106, 115, "LRRs 2\u201314", "structure_element" ] ] }, { "sid": 78, "sent": "The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8\u201310, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1\u2014figure supplement 3).", "section": "RESULTS", "ner": [ [ 12, 17, "Hyp64", "ptm" ], [ 17, 20, "IDA", "protein" ], [ 45, 51, "pocket", "site" ], [ 62, 67, "HAESA", "protein" ], [ 68, 77, "LRRs 8\u201310", "structure_element" ], [ 116, 130, "hydrogen bonds", "bond_interaction" ], [ 140, 158, "strictly conserved", "protein_state" ], [ 159, 165, "Glu266", "residue_name_number" ], [ 165, 170, "HAESA", "protein" ], [ 182, 187, "water", "chemical" ], [ 256, 262, "Phe289", "residue_name_number" ], [ 262, 267, "HAESA", "protein" ], [ 272, 278, "Ser311", "residue_name_number" ], [ 278, 283, "HAESA", "protein" ] ] }, { "sid": 79, "sent": "The restricted size of the Hyp pocket suggests that IDA does not require arabinosylation of Hyp64IDA for activity in vivo, a modification that has been reported for Hyp residues in plant CLE peptide hormones.", "section": "RESULTS", "ner": [ [ 27, 37, "Hyp pocket", "site" ], [ 52, 55, "IDA", "protein" ], [ 73, 88, "arabinosylation", "ptm" ], [ 92, 97, "Hyp64", "ptm" ], [ 97, 100, "IDA", "protein" ], [ 165, 168, "Hyp", "residue_name" ], [ 181, 186, "plant", "taxonomy_domain" ], [ 187, 207, "CLE peptide hormones", "protein_type" ] ] }, { "sid": 80, "sent": "The C-terminal Arg-His-Asn motif in IDA maps to a cavity formed by HAESA LRRs 11\u201314 (Figure 1D,F).", "section": "RESULTS", "ner": [ [ 15, 32, "Arg-His-Asn motif", "structure_element" ], [ 36, 39, "IDA", "protein" ], [ 50, 56, "cavity", "site" ], [ 67, 72, "HAESA", "protein" ], [ 73, 83, "LRRs 11\u201314", "structure_element" ] ] }, { "sid": 81, "sent": "The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D,F, 2D).", "section": "RESULTS", "ner": [ [ 18, 23, "Asn69", "residue_name_number" ], [ 23, 26, "IDA", "protein" ], [ 53, 59, "Arg407", "residue_name_number" ], [ 59, 64, "HAESA", "protein" ], [ 69, 75, "Arg409", "residue_name_number" ], [ 75, 80, "HAESA", "protein" ], [ 85, 90, "HAESA", "protein" ], [ 105, 126, "C-terminally extended", "protein_state" ], [ 127, 135, "IDA-SFVN", "mutant" ] ] }, { "sid": 82, "sent": "This suggests that the conserved Asn69IDA may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein.", "section": "RESULTS", "ner": [ [ 23, 32, "conserved", "protein_state" ], [ 33, 38, "Asn69", "residue_name_number" ], [ 38, 41, "IDA", "protein" ], [ 84, 90, "mature", "protein_state" ], [ 91, 102, "IDA peptide", "chemical" ], [ 106, 112, "planta", "taxonomy_domain" ], [ 122, 128, "active", "protein_state" ], [ 129, 132, "IDA", "protein" ] ] }, { "sid": 83, "sent": "Mutation of Arg417HSL2 (which corresponds to Arg409HAESA) causes a loss-of-function phenotype in HSL2, which indicates that the peptide binding pockets in different HAESA receptors have common structural and sequence features.", "section": "RESULTS", "ner": [ [ 0, 8, "Mutation", "experimental_method" ], [ 12, 18, "Arg417", "residue_name_number" ], [ 18, 22, "HSL2", "protein" ], [ 45, 51, "Arg409", "residue_name_number" ], [ 51, 56, "HAESA", "protein" ], [ 97, 101, "HSL2", "protein" ], [ 128, 151, "peptide binding pockets", "site" ], [ 165, 180, "HAESA receptors", "protein_type" ] ] }, { "sid": 84, "sent": "Indeed, we find many of the residues contributing to the formation of the IDA binding surface in HAESA to be conserved in HSL2 and in\u00a0other HAESA-type receptors in different plant species (Figure 1\u2014figure supplement 3).", "section": "RESULTS", "ner": [ [ 74, 93, "IDA binding surface", "site" ], [ 97, 102, "HAESA", "protein" ], [ 109, 118, "conserved", "protein_state" ], [ 122, 126, "HSL2", "protein" ], [ 140, 160, "HAESA-type receptors", "protein_type" ], [ 174, 179, "plant", "taxonomy_domain" ] ] }, { "sid": 85, "sent": "A N-terminal Pro-rich motif in IDA makes contacts with\u00a0LRRs 2\u20136 of the receptor (Figure 1D, Figure 1\u2014figure supplement 2A\u2013C).", "section": "RESULTS", "ner": [ [ 13, 27, "Pro-rich motif", "structure_element" ], [ 31, 34, "IDA", "protein" ], [ 55, 63, "LRRs 2\u20136", "structure_element" ] ] }, { "sid": 86, "sent": "Other hydrophobic and polar interactions are mediated by Ser62IDA, Ser65IDA and by backbone atoms along the IDA peptide (Figure 1D, Figure 1\u2014figure supplement 2A\u2013C).", "section": "RESULTS", "ner": [ [ 6, 40, "hydrophobic and polar interactions", "bond_interaction" ], [ 57, 62, "Ser62", "residue_name_number" ], [ 62, 65, "IDA", "protein" ], [ 67, 72, "Ser65", "residue_name_number" ], [ 72, 75, "IDA", "protein" ], [ 108, 119, "IDA peptide", "chemical" ] ] }, { "sid": 87, "sent": "HAESA specifically senses IDA-family dodecamer peptides", "section": "RESULTS", "ner": [ [ 0, 5, "HAESA", "protein" ], [ 26, 36, "IDA-family", "protein_type" ], [ 37, 46, "dodecamer", "structure_element" ], [ 47, 55, "peptides", "chemical" ] ] }, { "sid": 88, "sent": "We next investigated whether HAESA binds N-terminally extended versions of IDA.", "section": "RESULTS", "ner": [ [ 29, 34, "HAESA", "protein" ], [ 41, 62, "N-terminally extended", "protein_state" ], [ 75, 78, "IDA", "protein" ] ] }, { "sid": 89, "sent": "We obtained a structure of HAESA in complex with a PKGV-IDA peptide at 1.94\u2009\u00c5 resolution (Table 2).", "section": "RESULTS", "ner": [ [ 14, 23, "structure", "evidence" ], [ 27, 32, "HAESA", "protein" ], [ 33, 48, "in complex with", "protein_state" ], [ 51, 59, "PKGV-IDA", "mutant" ], [ 60, 67, "peptide", "chemical" ] ] }, { "sid": 90, "sent": "In this structure, no additional electron density accounts for the PKGV motif at the IDA N-terminus (Figure 2A,B).", "section": "RESULTS", "ner": [ [ 8, 17, "structure", "evidence" ], [ 33, 49, "electron density", "evidence" ], [ 67, 77, "PKGV motif", "structure_element" ], [ 85, 88, "IDA", "protein" ] ] }, { "sid": 91, "sent": "Consistently, PKGV-IDA and IDA have similar binding affinities in our ITC assays, further indicating that HAESA senses a dodecamer peptide comprising residues 58-69IDA (Figure 2D).", "section": "RESULTS", "ner": [ [ 14, 22, "PKGV-IDA", "mutant" ], [ 27, 30, "IDA", "protein" ], [ 44, 62, "binding affinities", "evidence" ], [ 70, 80, "ITC assays", "experimental_method" ], [ 106, 111, "HAESA", "protein" ], [ 121, 130, "dodecamer", "structure_element" ], [ 131, 138, "peptide", "chemical" ], [ 159, 164, "58-69", "residue_range" ], [ 164, 167, "IDA", "protein" ] ] }, { "sid": 92, "sent": "We next tested if HAESA binds other IDA peptide family members.", "section": "RESULTS", "ner": [ [ 18, 23, "HAESA", "protein" ], [ 36, 62, "IDA peptide family members", "chemical" ] ] }, { "sid": 93, "sent": "IDL1, which can rescue IDA loss-of-function mutants when introduced in abscission zone cells, can also be sensed by HAESA, albeit with lower affinity (Figure 2D).", "section": "RESULTS", "ner": [ [ 0, 4, "IDL1", "protein" ], [ 23, 26, "IDA", "protein" ], [ 116, 121, "HAESA", "protein" ], [ 141, 149, "affinity", "evidence" ] ] }, { "sid": 94, "sent": "A 2.56\u2009\u00c5 co-crystal structure with IDL1 reveals that different IDA family members use a common binding mode to interact with HAESA-type receptors (Figure 2A\u2013C,E, Table 2).", "section": "RESULTS", "ner": [ [ 9, 29, "co-crystal structure", "evidence" ], [ 35, 39, "IDL1", "protein" ], [ 63, 81, "IDA family members", "protein_type" ], [ 125, 145, "HAESA-type receptors", "protein_type" ] ] }, { "sid": 95, "sent": "We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (\u0394N69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D).", "section": "RESULTS", "ner": [ [ 37, 42, "HAESA", "protein" ], [ 49, 58, "synthetic", "protein_state" ], [ 59, 66, "peptide", "chemical" ], [ 67, 89, "missing the C-terminal", "protein_state" ], [ 90, 95, "Asn69", "residue_name_number" ], [ 95, 98, "IDA", "protein" ], [ 100, 104, "\u0394N69", "mutant" ], [ 142, 160, "polar interactions", "bond_interaction" ], [ 173, 176, "IDA", "protein" ], [ 198, 204, "Arg407", "residue_name_number" ], [ 204, 209, "HAESA", "protein" ], [ 210, 216, "Arg409", "residue_name_number" ], [ 216, 221, "HAESA", "protein" ] ] }, { "sid": 96, "sent": "Replacing Hyp64IDA, which is common to all IDLs, with proline impairs the interaction with the receptor, as does the Lys66IDA/Arg67IDA \u2192 Ala double-mutant discussed below (Figure 1A, 2D).", "section": "RESULTS", "ner": [ [ 0, 9, "Replacing", "experimental_method" ], [ 10, 15, "Hyp64", "ptm" ], [ 15, 18, "IDA", "protein" ], [ 43, 47, "IDLs", "protein_type" ], [ 54, 61, "proline", "residue_name" ], [ 117, 140, "Lys66IDA/Arg67IDA \u2192 Ala", "mutant" ], [ 141, 154, "double-mutant", "protein_state" ] ] }, { "sid": 97, "sent": "Notably, HAESA can discriminate between IDLs and functionally unrelated dodecamer peptides with Hyp modifications, such as CLV3 (Figures 2D, 7).", "section": "RESULTS", "ner": [ [ 9, 14, "HAESA", "protein" ], [ 40, 44, "IDLs", "protein_type" ], [ 49, 71, "functionally unrelated", "protein_state" ], [ 72, 81, "dodecamer", "structure_element" ], [ 82, 90, "peptides", "chemical" ], [ 96, 113, "Hyp modifications", "ptm" ], [ 123, 127, "CLV3", "protein" ] ] }, { "sid": 98, "sent": "The co-receptor kinase SERK1 allows for high-affinity IDA sensing", "section": "RESULTS", "ner": [ [ 4, 22, "co-receptor kinase", "protein_type" ], [ 23, 28, "SERK1", "protein" ] ] }, { "sid": 99, "sent": "Our binding assays reveal that IDA family\u00a0peptides are sensed by the isolated HAESA ectodomain with relatively weak binding affinities (Figures 1B, 2A\u2013D).", "section": "RESULTS", "ner": [ [ 4, 18, "binding assays", "experimental_method" ], [ 31, 50, "IDA family\u00a0peptides", "chemical" ], [ 69, 77, "isolated", "protein_state" ], [ 78, 83, "HAESA", "protein" ], [ 84, 94, "ectodomain", "structure_element" ], [ 116, 134, "binding affinities", "evidence" ] ] }, { "sid": 100, "sent": "It has been recently reported that SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) are positive regulators of floral abscission and can interact with HAESA and HSL2 in an IDA-dependent manner.", "section": "RESULTS", "ner": [ [ 35, 73, "SOMATIC EMBRYOGENESIS RECEPTOR KINASES", "protein_type" ], [ 75, 80, "SERKs", "protein_type" ], [ 149, 154, "HAESA", "protein" ], [ 159, 163, "HSL2", "protein" ] ] }, { "sid": 101, "sent": "As all five SERK family members appear to be expressed in the Arabidopsis abscission zone, we quantified their relative contribution to floral abscission in Arabidopsis using a petal break-strength assay.", "section": "RESULTS", "ner": [ [ 12, 31, "SERK family members", "protein_type" ], [ 62, 73, "Arabidopsis", "taxonomy_domain" ], [ 157, 168, "Arabidopsis", "taxonomy_domain" ], [ 177, 203, "petal break-strength assay", "experimental_method" ] ] }, { "sid": 102, "sent": "Our experiments suggest that among the SERK family members, SERK1 is a positive regulator of floral abscission.", "section": "RESULTS", "ner": [ [ 39, 58, "SERK family members", "protein_type" ], [ 60, 65, "SERK1", "protein" ] ] }, { "sid": 103, "sent": "We found that the force required to remove the petals of serk1-1 mutants is significantly higher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants, and that floral abscission is delayed in serk1-1 (Figure 3A).", "section": "RESULTS", "ner": [ [ 57, 64, "serk1-1", "gene" ], [ 65, 72, "mutants", "protein_state" ], [ 118, 127, "wild-type", "protein_state" ], [ 128, 134, "plants", "taxonomy_domain" ], [ 163, 168, "haesa", "gene" ], [ 169, 173, "hsl2", "gene" ], [ 174, 181, "mutants", "protein_state" ], [ 224, 231, "serk1-1", "gene" ] ] }, { "sid": 104, "sent": "The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines showed a petal break-strength profile not significantly different from wild-type plants.", "section": "RESULTS", "ner": [ [ 4, 11, "serk2-2", "gene" ], [ 13, 20, "serk3-1", "gene" ], [ 22, 29, "serk4-1", "gene" ], [ 34, 41, "serk5-1", "gene" ], [ 42, 48, "mutant", "protein_state" ], [ 126, 135, "wild-type", "protein_state" ], [ 136, 142, "plants", "taxonomy_domain" ] ] }, { "sid": 105, "sent": "Possibly because SERKs have additional roles in plant development such as in pollen formation and brassinosteroid signaling, we found that higher-order SERK mutants exhibit pleiotropic phenotypes in the flower, rendering their analysis and comparison by quantitative petal break-strength assays difficult.", "section": "RESULTS", "ner": [ [ 17, 22, "SERKs", "protein_type" ], [ 254, 294, "quantitative petal break-strength assays", "experimental_method" ] ] }, { "sid": 106, "sent": "We thus focused on analyzing the contribution of SERK1 to HAESA ligand sensing and receptor activation.", "section": "RESULTS", "ner": [ [ 49, 54, "SERK1", "protein" ], [ 58, 63, "HAESA", "protein" ] ] }, { "sid": 107, "sent": "In vitro, the LRR ectodomain of SERK1 (residues 24\u2013213) forms stable, IDA-dependent heterodimeric complexes with HAESA in size exclusion chromatography experiments (Figure 3B).", "section": "RESULTS", "ner": [ [ 14, 28, "LRR ectodomain", "structure_element" ], [ 32, 37, "SERK1", "protein" ], [ 48, 54, "24\u2013213", "residue_range" ], [ 62, 68, "stable", "protein_state" ], [ 70, 83, "IDA-dependent", "protein_state" ], [ 84, 97, "heterodimeric", "oligomeric_state" ], [ 98, 112, "complexes with", "protein_state" ], [ 113, 118, "HAESA", "protein" ], [ 122, 151, "size exclusion chromatography", "experimental_method" ] ] }, { "sid": 108, "sent": "We next quantified the contribution of SERK1 to IDA recognition by HAESA.", "section": "RESULTS", "ner": [ [ 39, 44, "SERK1", "protein" ], [ 48, 51, "IDA", "protein" ], [ 67, 72, "HAESA", "protein" ] ] }, { "sid": 109, "sent": "We found that HAESA senses IDA with a ~60\u00a0fold higher binding affinity in the presence of SERK1, suggesting that SERK1 is involved in the specific recognition of the peptide hormone (Figure 3C).", "section": "RESULTS", "ner": [ [ 14, 19, "HAESA", "protein" ], [ 27, 30, "IDA", "protein" ], [ 54, 70, "binding affinity", "evidence" ], [ 78, 89, "presence of", "protein_state" ], [ 90, 95, "SERK1", "protein" ], [ 113, 118, "SERK1", "protein" ], [ 166, 181, "peptide hormone", "protein_type" ] ] }, { "sid": 110, "sent": "We next titrated SERK1 into a solution containing only the HAESA ectodomain.", "section": "RESULTS", "ner": [ [ 8, 16, "titrated", "experimental_method" ], [ 17, 22, "SERK1", "protein" ], [ 59, 64, "HAESA", "protein" ], [ 65, 75, "ectodomain", "structure_element" ] ] }, { "sid": 111, "sent": "In this case, there was no detectable interaction between receptor and co-receptor, while in the presence of IDA, SERK1 strongly binds HAESA with a dissociation constant in the mid-nanomolar range (Figure 3C).", "section": "RESULTS", "ner": [ [ 97, 108, "presence of", "protein_state" ], [ 109, 112, "IDA", "protein" ], [ 114, 119, "SERK1", "protein" ], [ 135, 140, "HAESA", "protein" ], [ 148, 169, "dissociation constant", "evidence" ] ] }, { "sid": 112, "sent": "This suggests that IDA itself promotes receptor \u2013 co-receptor association, as previously described for the steroid hormone brassinolide and for other LRR-RK complexes.", "section": "RESULTS", "ner": [ [ 19, 22, "IDA", "protein" ], [ 107, 122, "steroid hormone", "chemical" ], [ 123, 135, "brassinolide", "chemical" ], [ 150, 156, "LRR-RK", "complex_assembly" ] ] }, { "sid": 113, "sent": "Importantly, hydroxyprolination of IDA is critical for HAESA-IDA-SERK1 complex formation (Figure 3C,D).", "section": "RESULTS", "ner": [ [ 13, 31, "hydroxyprolination", "ptm" ], [ 35, 38, "IDA", "protein" ], [ 55, 70, "HAESA-IDA-SERK1", "complex_assembly" ] ] }, { "sid": 114, "sent": "Our calorimetry experiments now reveal that SERKs may render HAESA, and potentially other receptor kinases, competent for high-affinity sensing of their cognate ligands.", "section": "RESULTS", "ner": [ [ 4, 15, "calorimetry", "experimental_method" ], [ 44, 49, "SERKs", "protein_type" ], [ 61, 66, "HAESA", "protein" ], [ 90, 106, "receptor kinases", "protein_type" ] ] }, { "sid": 115, "sent": "Upon IDA binding at the cell surface, the kinase domains of HAESA and SERK1, which have been shown to be active protein kinases, may interact in the cytoplasm to activate each other.", "section": "RESULTS", "ner": [ [ 5, 8, "IDA", "protein" ], [ 42, 56, "kinase domains", "structure_element" ], [ 60, 65, "HAESA", "protein" ], [ 70, 75, "SERK1", "protein" ], [ 105, 111, "active", "protein_state" ], [ 112, 127, "protein kinases", "protein_type" ] ] }, { "sid": 116, "sent": "Consistently, the HAESA kinase domain can transphosphorylate SERK1 and vice versa in in vitro transphosphorylation assays (Figure 3E).", "section": "RESULTS", "ner": [ [ 18, 23, "HAESA", "protein" ], [ 24, 37, "kinase domain", "structure_element" ], [ 61, 66, "SERK1", "protein" ], [ 94, 121, "transphosphorylation assays", "experimental_method" ] ] }, { "sid": 117, "sent": "Together, our genetic and biochemical experiments implicate SERK1 as a HAESA co-receptor in the Arabidopsis abscission zone.", "section": "RESULTS", "ner": [ [ 14, 49, "genetic and biochemical experiments", "experimental_method" ], [ 60, 65, "SERK1", "protein" ], [ 71, 88, "HAESA co-receptor", "protein_type" ], [ 96, 107, "Arabidopsis", "taxonomy_domain" ] ] }, { "sid": 118, "sent": "SERK1 senses a conserved motif in IDA family peptides", "section": "RESULTS", "ner": [ [ 0, 5, "SERK1", "protein" ], [ 15, 24, "conserved", "protein_state" ], [ 25, 30, "motif", "structure_element" ], [ 34, 53, "IDA family peptides", "chemical" ] ] }, { "sid": 119, "sent": "Crystal structure of a HAESA \u2013 IDA \u2013 SERK1 signaling complex.", "section": "FIG", "ner": [ [ 0, 17, "Crystal structure", "evidence" ], [ 23, 42, "HAESA \u2013 IDA \u2013 SERK1", "complex_assembly" ] ] }, { "sid": 120, "sent": "(A) Overview of the ternary complex with HAESA in blue (surface representation), IDA in yellow (bonds representation) and SERK1 in orange (surface view). (B) The HAESA ectodomain undergoes a conformational change upon SERK1 co-receptor binding.", "section": "FIG", "ner": [ [ 41, 46, "HAESA", "protein" ], [ 81, 84, "IDA", "protein" ], [ 122, 127, "SERK1", "protein" ], [ 162, 167, "HAESA", "protein" ], [ 168, 178, "ectodomain", "structure_element" ], [ 218, 223, "SERK1", "protein" ] ] }, { "sid": 121, "sent": "Shown are C\u03b1 traces of a structural superposition of the unbound (yellow) and SERK1-bound (blue) HAESA ectodomains (r.m.s.d. is 1.5 \u00c5 between 572 corresponding C\u03b1 atoms).", "section": "FIG", "ner": [ [ 25, 49, "structural superposition", "experimental_method" ], [ 57, 64, "unbound", "protein_state" ], [ 78, 89, "SERK1-bound", "protein_state" ], [ 97, 102, "HAESA", "protein" ], [ 103, 114, "ectodomains", "structure_element" ], [ 116, 124, "r.m.s.d.", "evidence" ] ] }, { "sid": 122, "sent": "SERK1 (in orange) and IDA (in red) are shown alongside.", "section": "FIG", "ner": [ [ 0, 5, "SERK1", "protein" ], [ 22, 25, "IDA", "protein" ] ] }, { "sid": 123, "sent": "The conformational change in the C-terminal LRRs and capping domain is indicated by an arrow. (C) SERK1 forms an integral part of the receptor's peptide binding pocket.", "section": "FIG", "ner": [ [ 44, 48, "LRRs", "structure_element" ], [ 53, 67, "capping domain", "structure_element" ], [ 98, 103, "SERK1", "protein" ], [ 145, 167, "peptide binding pocket", "site" ] ] }, { "sid": 124, "sent": "The N-terminal capping domain of SERK1 (in orange) directly contacts the C-terminal part of IDA (in yellow, in bonds representation) and the receptor HAESA (in blue).", "section": "FIG", "ner": [ [ 15, 29, "capping domain", "structure_element" ], [ 33, 38, "SERK1", "protein" ], [ 92, 95, "IDA", "protein" ], [ 141, 149, "receptor", "protein_type" ], [ 150, 155, "HAESA", "protein" ] ] }, { "sid": 125, "sent": "Polar contacts of SERK1 with IDA are shown in magenta, with the HAESA LRR domain in gray. (D) Details of the zipper-like SERK1-HAESA interface.", "section": "FIG", "ner": [ [ 0, 14, "Polar contacts", "bond_interaction" ], [ 18, 23, "SERK1", "protein" ], [ 29, 32, "IDA", "protein" ], [ 64, 69, "HAESA", "protein" ], [ 70, 80, "LRR domain", "structure_element" ], [ 109, 120, "zipper-like", "structure_element" ], [ 121, 142, "SERK1-HAESA interface", "site" ] ] }, { "sid": 126, "sent": "Ribbon diagrams of HAESA (in blue) and SERK1 (in orange) are shown with selected interface residues (in bonds representation).", "section": "FIG", "ner": [ [ 19, 24, "HAESA", "protein" ], [ 39, 44, "SERK1", "protein" ], [ 81, 99, "interface residues", "site" ] ] }, { "sid": 127, "sent": "Polar interactions are highlighted as dotted lines (in magenta).", "section": "FIG", "ner": [ [ 0, 18, "Polar interactions", "bond_interaction" ] ] }, { "sid": 128, "sent": "To understand in molecular terms how SERK1 contributes to high-affinity IDA recognition, we solved a 2.43\u2009\u00c5 crystal structure of the ternary HAESA \u2013 IDA \u2013 SERK1 complex (Figure 4A, Table 2).", "section": "RESULTS", "ner": [ [ 37, 42, "SERK1", "protein" ], [ 72, 75, "IDA", "protein" ], [ 108, 125, "crystal structure", "evidence" ], [ 141, 160, "HAESA \u2013 IDA \u2013 SERK1", "complex_assembly" ] ] }, { "sid": 129, "sent": "HAESA LRRs 16\u201321 and its C-terminal capping domain undergo a conformational change upon SERK1 binding (Figure 4B).", "section": "RESULTS", "ner": [ [ 0, 5, "HAESA", "protein" ], [ 6, 16, "LRRs 16\u201321", "structure_element" ], [ 36, 50, "capping domain", "structure_element" ], [ 88, 93, "SERK1", "protein" ] ] }, { "sid": 130, "sent": "The SERK1 ectodomain interacts with the IDA peptide binding site using a loop region (residues 51-59SERK1) from its N-terminal cap (Figure 4A,C).", "section": "RESULTS", "ner": [ [ 4, 9, "SERK1", "protein" ], [ 10, 20, "ectodomain", "structure_element" ], [ 40, 64, "IDA peptide binding site", "site" ], [ 73, 84, "loop region", "structure_element" ], [ 95, 100, "51-59", "residue_range" ], [ 100, 105, "SERK1", "protein" ], [ 127, 130, "cap", "structure_element" ] ] }, { "sid": 131, "sent": "SERK1 loop residues establish multiple hydrophobic and polar contacts with Lys66IDA and the C-terminal Arg-His-Asn motif in IDA (Figure 4C).", "section": "RESULTS", "ner": [ [ 0, 5, "SERK1", "protein" ], [ 6, 10, "loop", "structure_element" ], [ 39, 69, "hydrophobic and polar contacts", "bond_interaction" ], [ 75, 80, "Lys66", "residue_name_number" ], [ 80, 83, "IDA", "protein" ], [ 103, 120, "Arg-His-Asn motif", "structure_element" ], [ 124, 127, "IDA", "protein" ] ] }, { "sid": 132, "sent": "SERK1 LRRs 1\u20135 and its C-terminal capping domain form an additional zipper-like interface with residues originating from HAESA LRRs 15\u201321 and from the HAESA C-terminal cap (Figure 4D).", "section": "RESULTS", "ner": [ [ 0, 5, "SERK1", "protein" ], [ 6, 14, "LRRs 1\u20135", "structure_element" ], [ 34, 48, "capping domain", "structure_element" ], [ 68, 79, "zipper-like", "structure_element" ], [ 80, 89, "interface", "site" ], [ 121, 126, "HAESA", "protein" ], [ 127, 137, "LRRs 15\u201321", "structure_element" ], [ 151, 156, "HAESA", "protein" ], [ 168, 171, "cap", "structure_element" ] ] }, { "sid": 133, "sent": "SERK1 binds HAESA using these two distinct interaction surfaces (Figure 1\u2014figure supplement 3), with the N-cap of the SERK1 LRR domain partially covering the IDA peptide binding cleft.", "section": "RESULTS", "ner": [ [ 0, 5, "SERK1", "protein" ], [ 12, 17, "HAESA", "protein" ], [ 43, 63, "interaction surfaces", "site" ], [ 105, 110, "N-cap", "structure_element" ], [ 118, 123, "SERK1", "protein" ], [ 124, 134, "LRR domain", "structure_element" ], [ 158, 183, "IDA peptide binding cleft", "site" ] ] }, { "sid": 134, "sent": "The IDA C-terminal motif is required for HAESA-SERK1 complex formation and for IDA bioactivity.", "section": "FIG", "ner": [ [ 4, 7, "IDA", "protein" ], [ 8, 24, "C-terminal motif", "structure_element" ], [ 41, 52, "HAESA-SERK1", "complex_assembly" ] ] }, { "sid": 135, "sent": "(A) Size exclusion chromatography experiments similar to Figure 3B,D reveal that IDA mutant peptides targeting the C-terminal motif do not form biochemically stable HAESA-IDA-SERK1 complexes.", "section": "FIG", "ner": [ [ 4, 33, "Size exclusion chromatography", "experimental_method" ], [ 81, 84, "IDA", "protein" ], [ 85, 91, "mutant", "protein_state" ], [ 92, 100, "peptides", "chemical" ], [ 115, 131, "C-terminal motif", "structure_element" ], [ 144, 164, "biochemically stable", "protein_state" ], [ 165, 180, "HAESA-IDA-SERK1", "complex_assembly" ] ] }, { "sid": 136, "sent": "Deletion of the C-terminal Asn69IDA completely inhibits complex formation.", "section": "FIG", "ner": [ [ 0, 8, "Deletion", "experimental_method" ], [ 27, 32, "Asn69", "residue_name_number" ], [ 32, 35, "IDA", "protein" ], [ 47, 55, "inhibits", "protein_state" ] ] }, { "sid": 137, "sent": "Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively.", "section": "FIG", "ner": [ [ 0, 8, "Purified", "experimental_method" ], [ 9, 14, "HAESA", "protein" ], [ 19, 24, "SERK1", "protein" ] ] }, { "sid": 138, "sent": "Left panel: IDA K66A/R67A; center: IDA\u00a0\u0394N69, right panel: SDS-PAGE of peak fractions.", "section": "FIG", "ner": [ [ 12, 25, "IDA K66A/R67A", "mutant" ], [ 35, 43, "IDA\u00a0\u0394N69", "mutant" ], [ 58, 66, "SDS-PAGE", "experimental_method" ] ] }, { "sid": 139, "sent": "Note that the HAESA and SERK1 input lanes have already been shown in Figure 3D. (B) Isothermal titration thermographs of wild-type and mutant IDA peptides titrated into a HAESA - SERK1 mixture in the cell.", "section": "FIG", "ner": [ [ 14, 19, "HAESA", "protein" ], [ 24, 29, "SERK1", "protein" ], [ 84, 117, "Isothermal titration thermographs", "evidence" ], [ 121, 130, "wild-type", "protein_state" ], [ 135, 141, "mutant", "protein_state" ], [ 142, 154, "IDA peptides", "chemical" ], [ 155, 163, "titrated", "experimental_method" ], [ 171, 176, "HAESA", "protein" ], [ 179, 184, "SERK1", "protein" ] ] }, { "sid": 140, "sent": "Table summaries for calorimetric binding constants and stoichoimetries for different IDA peptides binding to the HAESA \u2013 SERK1 ectodomain mixture ( \u00b1 fitting errors; n.d.", "section": "FIG", "ner": [ [ 20, 50, "calorimetric binding constants", "evidence" ], [ 85, 97, "IDA peptides", "chemical" ], [ 113, 118, "HAESA", "protein" ], [ 121, 126, "SERK1", "protein" ], [ 127, 137, "ectodomain", "structure_element" ] ] }, { "sid": 141, "sent": "(C) Quantitative petal break-strength assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers.", "section": "FIG", "ner": [ [ 17, 43, "petal break-strength assay", "experimental_method" ], [ 54, 63, "wild-type", "protein_state" ], [ 76, 79, "35S", "gene" ], [ 81, 84, "IDA", "protein" ], [ 85, 94, "wild-type", "protein_state" ], [ 99, 102, "35S", "gene" ], [ 104, 117, "IDA K66A/R67A", "mutant" ], [ 118, 124, "mutant", "protein_state" ] ] }, { "sid": 142, "sent": "35S::IDA plants showed significantly increased abscission compared to Col-0 controls in inflorescence positions 2 and 3 (a).", "section": "FIG", "ner": [ [ 0, 3, "35S", "gene" ], [ 5, 8, "IDA", "protein" ], [ 9, 15, "plants", "taxonomy_domain" ] ] }, { "sid": 143, "sent": "Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression \u00b1 standard error; ida: -0.02 \u00b1 0.001; Col-0: 1 \u00b1 0.11; 35S::IDA 124 \u00b1 0.75; 35S::IDA K66A/R67A: 159 \u00b1 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines.", "section": "FIG", "ner": [ [ 47, 50, "35S", "gene" ], [ 52, 65, "IDA K66A/R67A", "mutant" ], [ 66, 72, "mutant", "protein_state" ], [ 73, 79, "plants", "taxonomy_domain" ], [ 139, 145, "plants", "taxonomy_domain" ], [ 154, 157, "35S", "gene" ], [ 159, 162, "IDA", "protein" ], [ 163, 169, "plants", "taxonomy_domain" ], [ 283, 286, "35S", "gene" ], [ 288, 291, "IDA", "protein" ], [ 304, 307, "35S", "gene" ], [ 309, 322, "IDA K66A/R67A", "mutant" ], [ 339, 342, "IDA", "protein" ], [ 343, 352, "wild-type", "protein_state" ], [ 357, 363, "mutant", "protein_state" ], [ 383, 417, "35S promoter over-expression lines", "experimental_method" ], [ 494, 497, "35S", "gene" ], [ 499, 502, "IDA", "protein" ], [ 510, 519, "wild-type", "protein_state" ], [ 524, 527, "35S", "gene" ], [ 529, 542, "IDA K66A/R67A", "mutant" ], [ 543, 556, "double-mutant", "protein_state" ], [ 557, 576, "T3 transgenic lines", "experimental_method" ] ] }, { "sid": 144, "sent": "15 out of 15 35S::IDA plants, 0 out of 15 Col-0 plants and 0 out of 15 35S::IDA K66A/R67A double-mutant plants, showed an enlarged abscission zone, respectively (3 independent lines were analyzed).", "section": "FIG", "ner": [ [ 13, 16, "35S", "gene" ], [ 18, 21, "IDA", "protein" ], [ 22, 28, "plants", "taxonomy_domain" ], [ 48, 54, "plants", "taxonomy_domain" ], [ 71, 74, "35S", "gene" ], [ 76, 89, "IDA K66A/R67A", "mutant" ], [ 90, 103, "double-mutant", "protein_state" ], [ 104, 110, "plants", "taxonomy_domain" ] ] }, { "sid": 145, "sent": "The four C-terminal residues in IDA (Lys66IDA-Asn69IDA) are conserved among IDA family members and are in direct contact with SERK1 (Figures 1A, 4C).", "section": "RESULTS", "ner": [ [ 32, 35, "IDA", "protein" ], [ 37, 54, "Lys66IDA-Asn69IDA", "residue_range" ], [ 60, 69, "conserved", "protein_state" ], [ 76, 94, "IDA family members", "protein_type" ], [ 126, 131, "SERK1", "protein" ] ] }, { "sid": 146, "sent": "We thus assessed their contribution to HAESA \u2013 SERK1 complex formation.", "section": "RESULTS", "ner": [ [ 39, 52, "HAESA \u2013 SERK1", "complex_assembly" ] ] }, { "sid": 147, "sent": "Deletion of the buried Asn69IDA completely inhibits receptor \u2013 co-receptor complex formation and HSL2 activation (Figure 5A,B).", "section": "RESULTS", "ner": [ [ 0, 8, "Deletion", "experimental_method" ], [ 23, 28, "Asn69", "residue_name_number" ], [ 28, 31, "IDA", "protein" ], [ 32, 51, "completely inhibits", "protein_state" ] ] }, { "sid": 148, "sent": "A synthetic Lys66IDA/Arg67IDA \u2192 Ala mutant peptide (IDA K66A/R66A) showed a 10 fold reduced binding affinity when titrated in a HAESA/SERK1 protein solution (Figures 5A,B, 2D).", "section": "RESULTS", "ner": [ [ 2, 11, "synthetic", "protein_state" ], [ 12, 35, "Lys66IDA/Arg67IDA \u2192 Ala", "mutant" ], [ 36, 42, "mutant", "protein_state" ], [ 43, 50, "peptide", "chemical" ], [ 52, 65, "IDA K66A/R66A", "mutant" ], [ 92, 108, "binding affinity", "evidence" ], [ 114, 122, "titrated", "experimental_method" ], [ 128, 133, "HAESA", "protein" ], [ 134, 139, "SERK1", "protein" ] ] }, { "sid": 149, "sent": "We over-expressed full-length wild-type IDA or this Lys66IDA/Arg67IDA \u2192 Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D).", "section": "RESULTS", "ner": [ [ 3, 17, "over-expressed", "experimental_method" ], [ 18, 29, "full-length", "protein_state" ], [ 30, 39, "wild-type", "protein_state" ], [ 40, 43, "IDA", "protein" ], [ 52, 75, "Lys66IDA/Arg67IDA \u2192 Ala", "mutant" ], [ 76, 89, "double-mutant", "protein_state" ], [ 117, 128, "Arabidopsis", "taxonomy_domain" ], [ 129, 135, "plants", "taxonomy_domain" ] ] }, { "sid": 150, "sent": "We found that over-expression of wild-type IDA leads to early floral abscission and an enlargement of the abscission zone (Figure 5C\u2013E).", "section": "RESULTS", "ner": [ [ 14, 29, "over-expression", "experimental_method" ], [ 33, 42, "wild-type", "protein_state" ], [ 43, 46, "IDA", "protein" ] ] }, { "sid": 151, "sent": "In contrast, over-expression of the IDA Lys66IDA/Arg67IDA \u2192 Ala double mutant significantly delays floral abscission when compared to wild-type control plants, suggesting that the mutant IDA peptide has reduced activity in planta (Figure 5C\u2013E).", "section": "RESULTS", "ner": [ [ 13, 28, "over-expression", "experimental_method" ], [ 36, 63, "IDA Lys66IDA/Arg67IDA \u2192 Ala", "mutant" ], [ 64, 77, "double mutant", "protein_state" ], [ 134, 143, "wild-type", "protein_state" ], [ 152, 158, "plants", "taxonomy_domain" ], [ 180, 186, "mutant", "protein_state" ], [ 187, 198, "IDA peptide", "chemical" ], [ 223, 229, "planta", "taxonomy_domain" ] ] }, { "sid": 152, "sent": "Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA \u2192 Ala may cause a weak dominant negative effect (Figure 5C\u2013E).", "section": "RESULTS", "ner": [ [ 14, 17, "35S", "gene" ], [ 19, 22, "IDA", "protein" ], [ 23, 32, "wild-type", "protein_state" ], [ 37, 43, "mutant", "protein_state" ], [ 44, 50, "plants", "taxonomy_domain" ], [ 74, 82, "mutation", "experimental_method" ], [ 86, 109, "Lys66IDA/Arg67IDA \u2192 Ala", "mutant" ] ] }, { "sid": 153, "sent": "In agreement with our structures and biochemical assays, this experiment suggests a role of the conserved IDA C-terminus in the control of floral abscission.", "section": "RESULTS", "ner": [ [ 22, 32, "structures", "evidence" ], [ 37, 55, "biochemical assays", "experimental_method" ], [ 96, 105, "conserved", "protein_state" ], [ 106, 109, "IDA", "protein" ] ] }, { "sid": 154, "sent": "In contrast to animal LRR receptors, plant LRR-RKs harbor spiral-shaped ectodomains and thus they require shape-complementary co-receptor proteins for receptor activation.", "section": "DISCUSS", "ner": [ [ 15, 21, "animal", "taxonomy_domain" ], [ 22, 35, "LRR receptors", "protein_type" ], [ 37, 42, "plant", "taxonomy_domain" ], [ 43, 50, "LRR-RKs", "structure_element" ], [ 58, 71, "spiral-shaped", "protein_state" ], [ 72, 83, "ectodomains", "structure_element" ], [ 106, 125, "shape-complementary", "protein_state" ], [ 126, 146, "co-receptor proteins", "protein_type" ] ] }, { "sid": 155, "sent": "For a rapidly growing number of plant signaling pathways, SERK proteins act as these essential co-receptors (;\u00a0).", "section": "DISCUSS", "ner": [ [ 32, 37, "plant", "taxonomy_domain" ], [ 58, 71, "SERK proteins", "protein_type" ], [ 95, 107, "co-receptors", "protein_type" ] ] }, { "sid": 156, "sent": "\u00a0SERK1 has been previously reported as a positive regulator in plant embryogenesis, male sporogenesis, brassinosteroid signaling and in phytosulfokine perception.", "section": "DISCUSS", "ner": [ [ 63, 68, "plant", "taxonomy_domain" ] ] }, { "sid": 157, "sent": "Recent findings by and our mechanistic studies now also support a positive role for SERK1 in floral abscission.", "section": "DISCUSS", "ner": [ [ 84, 89, "SERK1", "protein" ] ] }, { "sid": 158, "sent": "As serk1-1 mutant plants show intermediate abscission phenotypes when compared to haesa/hsl2 mutants, SERK1 likely acts redundantly with other SERKs in the abscission zone (Figure 3A).", "section": "DISCUSS", "ner": [ [ 3, 10, "serk1-1", "gene" ], [ 11, 17, "mutant", "protein_state" ], [ 18, 24, "plants", "taxonomy_domain" ], [ 82, 87, "haesa", "gene" ], [ 93, 100, "mutants", "protein_state" ], [ 102, 107, "SERK1", "protein" ], [ 143, 148, "SERKs", "protein_type" ] ] }, { "sid": 159, "sent": "It has been previously suggested that SERK1 can inhibit cell separation.", "section": "DISCUSS", "ner": [ [ 38, 43, "SERK1", "protein" ] ] }, { "sid": 160, "sent": "However our results show that SERK1 also can activate this process upon IDA sensing, indicating that SERKs may fulfill several different functions in the course of the abscission process.", "section": "DISCUSS", "ner": [ [ 30, 35, "SERK1", "protein" ], [ 72, 75, "IDA", "protein" ], [ 101, 106, "SERKs", "protein_type" ] ] }, { "sid": 161, "sent": "While the sequence of the mature IDA peptide has not been experimentally determined in planta, our HAESA-IDA complex structures and calorimetry assays suggest that active IDLs are hydroxyprolinated dodecamers.", "section": "DISCUSS", "ner": [ [ 26, 32, "mature", "protein_state" ], [ 33, 44, "IDA peptide", "chemical" ], [ 87, 93, "planta", "taxonomy_domain" ], [ 99, 108, "HAESA-IDA", "complex_assembly" ], [ 117, 127, "structures", "evidence" ], [ 132, 150, "calorimetry assays", "evidence" ], [ 164, 170, "active", "protein_state" ], [ 171, 175, "IDLs", "protein_type" ], [ 180, 197, "hydroxyprolinated", "protein_state" ], [ 198, 208, "dodecamers", "structure_element" ] ] }, { "sid": 162, "sent": "It will be thus interesting to see if proteolytic processing of full-length IDA in vivo is regulated in a cell-type or tissue-specific manner.", "section": "DISCUSS", "ner": [ [ 64, 75, "full-length", "protein_state" ], [ 76, 79, "IDA", "protein" ] ] }, { "sid": 163, "sent": "The central Hyp residue in IDA is found buried in the HAESA peptide binding surface and thus this post-translational modification may regulate IDA bioactivity.", "section": "DISCUSS", "ner": [ [ 12, 15, "Hyp", "residue_name" ], [ 27, 30, "IDA", "protein" ], [ 54, 59, "HAESA", "protein" ], [ 60, 83, "peptide binding surface", "site" ], [ 143, 146, "IDA", "protein" ] ] }, { "sid": 164, "sent": "Our comparative structural and biochemical analysis further suggests that IDLs share a common receptor binding mode, but may preferably bind to HAESA, HSL1 or HSL2 in different plant tissues and organs.", "section": "DISCUSS", "ner": [ [ 4, 51, "comparative structural and biochemical analysis", "experimental_method" ], [ 74, 78, "IDLs", "protein_type" ], [ 144, 149, "HAESA", "protein" ], [ 151, 155, "HSL1", "protein" ], [ 159, 163, "HSL2", "protein" ], [ 177, 182, "plant", "taxonomy_domain" ] ] }, { "sid": 165, "sent": "In our quantitative biochemical assays, the presence of SERK1 dramatically increases the HAESA binding specificity and affinity for IDA.", "section": "DISCUSS", "ner": [ [ 7, 38, "quantitative biochemical assays", "experimental_method" ], [ 44, 55, "presence of", "protein_state" ], [ 56, 61, "SERK1", "protein" ], [ 89, 94, "HAESA", "protein" ], [ 132, 135, "IDA", "protein" ] ] }, { "sid": 166, "sent": "This observation is consistent with our complex structure in which receptor and co-receptor together form the IDA binding pocket.", "section": "DISCUSS", "ner": [ [ 48, 57, "structure", "evidence" ], [ 110, 128, "IDA binding pocket", "site" ] ] }, { "sid": 167, "sent": "The fact that SERK1 specifically interacts with the very C-terminus of IDLs may allow for the rational design of peptide hormone antagonists, as previously demonstrated for the brassinosteroid pathway.", "section": "DISCUSS", "ner": [ [ 14, 19, "SERK1", "protein" ], [ 71, 75, "IDLs", "protein_type" ], [ 113, 140, "peptide hormone antagonists", "chemical" ] ] }, { "sid": 168, "sent": "Importantly, our calorimetry assays reveal that the SERK1 ectodomain binds HAESA with nanomolar affinity, but only in the presence of IDA (Figure 3C).", "section": "DISCUSS", "ner": [ [ 17, 35, "calorimetry assays", "experimental_method" ], [ 52, 57, "SERK1", "protein" ], [ 58, 68, "ectodomain", "structure_element" ], [ 69, 74, "binds", "protein_state" ], [ 75, 80, "HAESA", "protein" ], [ 122, 133, "presence of", "protein_state" ], [ 134, 137, "IDA", "protein" ] ] }, { "sid": 169, "sent": "This ligand-induced formation of a receptor \u2013 co-receptor complex may allow the HAESA and SERK1 kinase domains to efficiently trans-phosphorylate and activate each other in the cytoplasm.", "section": "DISCUSS", "ner": [ [ 80, 85, "HAESA", "protein" ], [ 90, 95, "SERK1", "protein" ], [ 96, 110, "kinase domains", "structure_element" ] ] }, { "sid": 170, "sent": "It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus\u00a0might differ in the context of the full-length, membrane-embedded signaling complex.", "section": "DISCUSS", "ner": [ [ 32, 50, "binding affinities", "evidence" ], [ 55, 58, "IDA", "protein" ], [ 63, 68, "SERK1", "protein" ], [ 94, 103, "synthetic", "protein_state" ], [ 104, 112, "peptides", "chemical" ], [ 121, 129, "isolated", "experimental_method" ], [ 130, 135, "HAESA", "protein" ], [ 140, 145, "SERK1", "protein" ], [ 146, 157, "ectodomains", "structure_element" ], [ 203, 214, "full-length", "protein_state" ], [ 216, 233, "membrane-embedded", "protein_state" ] ] }, { "sid": 171, "sent": "SERK1 uses partially overlapping surface areas to activate different plant signaling receptors.", "section": "FIG", "ner": [ [ 0, 5, "SERK1", "protein" ], [ 69, 74, "plant", "taxonomy_domain" ], [ 75, 94, "signaling receptors", "protein_type" ] ] }, { "sid": 172, "sent": "(A) Structural comparison of plant steroid and peptide hormone membrane signaling complexes.", "section": "FIG", "ner": [ [ 4, 25, "Structural comparison", "experimental_method" ], [ 29, 34, "plant", "taxonomy_domain" ], [ 35, 42, "steroid", "chemical" ], [ 47, 62, "peptide hormone", "protein_type" ], [ 63, 91, "membrane signaling complexes", "protein_type" ] ] }, { "sid": 173, "sent": "Left panel: Ribbon diagram of HAESA (in blue), SERK1 (in orange) and IDA (in bonds and surface represention).", "section": "FIG", "ner": [ [ 30, 35, "HAESA", "protein" ], [ 47, 52, "SERK1", "protein" ], [ 69, 72, "IDA", "protein" ] ] }, { "sid": 174, "sent": "Right panel: Ribbon diagram of the plant steroid receptor BRI1 (in blue) bound to brassinolide (in gray, in bonds representation) and to SERK1, shown in the same orientation (PDB-ID. 4lsx).", "section": "FIG", "ner": [ [ 35, 40, "plant", "taxonomy_domain" ], [ 41, 57, "steroid receptor", "protein_type" ], [ 58, 62, "BRI1", "protein" ], [ 73, 81, "bound to", "protein_state" ], [ 82, 94, "brassinolide", "chemical" ], [ 137, 142, "SERK1", "protein" ] ] }, { "sid": 175, "sent": "(B) View of the inner surface of the SERK1 LRR domain (PDB-ID 4lsc, surface representation, in gray).", "section": "FIG", "ner": [ [ 37, 42, "SERK1", "protein" ], [ 43, 53, "LRR domain", "structure_element" ] ] }, { "sid": 176, "sent": "A ribbon diagram of SERK1 in the same orientation is shown alongside.", "section": "FIG", "ner": [ [ 20, 25, "SERK1", "protein" ] ] }, { "sid": 177, "sent": "Residues interacting with the HAESA or BRI1 LRR domains are shown in orange or magenta, respectively.", "section": "FIG", "ner": [ [ 30, 35, "HAESA", "protein" ], [ 39, 43, "BRI1", "protein" ], [ 44, 55, "LRR domains", "structure_element" ] ] }, { "sid": 178, "sent": "Comparison of our HAESA \u2013 IDA \u2013 SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 \u2013 14; HAESA; LRRs 21 \u2013 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A).", "section": "DISCUSS", "ner": [ [ 0, 10, "Comparison", "experimental_method" ], [ 18, 37, "HAESA \u2013 IDA \u2013 SERK1", "complex_assembly" ], [ 38, 47, "structure", "evidence" ], [ 107, 112, "SERK1", "protein" ], [ 126, 137, "co-receptor", "protein_type" ], [ 158, 167, "conserved", "protein_state" ], [ 176, 181, "SERK1", "protein" ], [ 201, 223, "ligand binding pockets", "site" ], [ 284, 295, "LRRs 2 \u2013 14", "structure_element" ], [ 297, 302, "HAESA", "protein" ], [ 304, 316, "LRRs 21 \u2013 25", "structure_element" ], [ 318, 322, "BRI1", "protein" ], [ 358, 362, "BRI1", "protein" ], [ 372, 377, "HAESA", "protein" ] ] }, { "sid": 179, "sent": "Several residues in the SERK1\u2009N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the\u00a0LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B).", "section": "DISCUSS", "ner": [ [ 24, 29, "SERK1", "protein" ], [ 41, 55, "capping domain", "structure_element" ], [ 57, 62, "Thr59", "residue_name_number" ], [ 62, 67, "SERK1", "protein" ], [ 69, 74, "Phe61", "residue_name_number" ], [ 74, 79, "SERK1", "protein" ], [ 89, 106, "LRR inner surface", "site" ], [ 108, 113, "Asp75", "residue_name_number" ], [ 113, 118, "SERK1", "protein" ], [ 120, 126, "Tyr101", "residue_name_number" ], [ 126, 131, "SERK1", "protein" ], [ 133, 139, "SER121", "residue_name_number" ], [ 139, 144, "SERK1", "protein" ], [ 146, 152, "Phe145", "residue_name_number" ], [ 152, 157, "SERK1", "protein" ] ] }, { "sid": 180, "sent": "In addition, residues 53-55SERK1 from the SERK1\u2009N-terminal cap mediate specific interactions with the IDA peptide (Figures 4C, 6B).", "section": "DISCUSS", "ner": [ [ 22, 27, "53-55", "residue_range" ], [ 27, 32, "SERK1", "protein" ], [ 42, 47, "SERK1", "protein" ], [ 59, 62, "cap", "structure_element" ], [ 102, 113, "IDA peptide", "chemical" ] ] }, { "sid": 181, "sent": "These residues are not involved in the sensing of the steroid hormone brassinolide.", "section": "DISCUSS", "ner": [ [ 54, 69, "steroid hormone", "chemical" ], [ 70, 82, "brassinolide", "chemical" ] ] }, { "sid": 182, "sent": "In both cases however, the co-receptor completes the hormone binding pocket.", "section": "DISCUSS", "ner": [ [ 53, 75, "hormone binding pocket", "site" ] ] }, { "sid": 183, "sent": "This fact together with the largely overlapping SERK1 binding surfaces in HAESA and BRI1 allows us to speculate that SERK1 may promote high-affinity peptide hormone and brassinosteroid sensing by simply slowing down dissociation of the ligand from its cognate receptor.", "section": "DISCUSS", "ner": [ [ 48, 70, "SERK1 binding surfaces", "site" ], [ 74, 79, "HAESA", "protein" ], [ 84, 88, "BRI1", "protein" ], [ 117, 122, "SERK1", "protein" ], [ 149, 164, "peptide hormone", "protein_type" ] ] }, { "sid": 184, "sent": "Different plant peptide hormone families contain a C-terminal (Arg)-His-Asn motif, which in IDA represents the co-receptor recognition site.", "section": "FIG", "ner": [ [ 10, 15, "plant", "taxonomy_domain" ], [ 16, 40, "peptide hormone families", "protein_type" ], [ 62, 81, "(Arg)-His-Asn motif", "structure_element" ], [ 92, 95, "IDA", "protein" ], [ 111, 139, "co-receptor recognition site", "site" ] ] }, { "sid": 185, "sent": "Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 \u2013 EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR \u2013 GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana.", "section": "FIG", "ner": [ [ 0, 44, "Structure-guided multiple sequence alignment", "experimental_method" ], [ 48, 51, "IDA", "protein" ], [ 56, 73, "IDA-like peptides", "chemical" ], [ 85, 90, "plant", "taxonomy_domain" ], [ 91, 115, "peptide hormone families", "protein_type" ], [ 127, 171, "CLAVATA3 \u2013 EMBRYO SURROUNDING REGION-RELATED", "protein_type" ], [ 173, 181, "CLV3/CLE", "protein_type" ], [ 184, 211, "ROOT GROWTH FACTOR \u2013 GOLVEN", "protein_type" ], [ 213, 220, "RGF/GLV", "protein_type" ], [ 223, 245, "PRECURSOR GENE PROPEP1", "protein_type" ], [ 247, 251, "PEP1", "protein_type" ], [ 258, 278, "Arabidopsis thaliana", "species" ] ] }, { "sid": 186, "sent": "The conserved (Arg)-His-Asn motif is highlighted in red, the central Hyp residue in IDLs and CLEs is marked in blue.", "section": "FIG", "ner": [ [ 4, 13, "conserved", "protein_state" ], [ 14, 33, "(Arg)-His-Asn motif", "structure_element" ], [ 69, 72, "Hyp", "residue_name" ], [ 84, 88, "IDLs", "protein_type" ], [ 93, 97, "CLEs", "protein_type" ] ] }, { "sid": 187, "sent": "Our experiments reveal that SERK1 recognizes a C-terminal Arg-His-Asn motif in IDA.", "section": "DISCUSS", "ner": [ [ 28, 33, "SERK1", "protein" ], [ 58, 75, "Arg-His-Asn motif", "structure_element" ], [ 79, 82, "IDA", "protein" ] ] }, { "sid": 188, "sent": "Importantly, this motif can also be found in other peptide hormone families (Figure 7).", "section": "DISCUSS", "ner": [ [ 13, 23, "this motif", "structure_element" ], [ 51, 75, "peptide hormone families", "protein_type" ] ] }, { "sid": 189, "sent": "Among these are the CLE peptides regulating stem cell maintenance in the shoot and the root.", "section": "DISCUSS", "ner": [ [ 20, 32, "CLE peptides", "chemical" ] ] }, { "sid": 190, "sent": "It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA.", "section": "DISCUSS", "ner": [ [ 32, 36, "CLEs", "protein_type" ], [ 46, 57, "mature form", "protein_state" ], [ 67, 84, "hydroxyprolinated", "protein_state" ], [ 85, 95, "dodecamers", "structure_element" ], [ 113, 125, "surface area", "site" ], [ 133, 163, "BARELY ANY MERISTEM 1 receptor", "protein_type" ], [ 201, 218, "IDA binding cleft", "site" ], [ 222, 227, "HAESA", "protein" ] ] }, { "sid": 191, "sent": "Diverse plant peptide hormones may thus also bind their LRR-RK receptors in an extended conformation along the inner surface of the LRR domain and may also use small, shape-complementary co-receptors for high-affinity ligand binding and receptor activation.", "section": "DISCUSS", "ner": [ [ 8, 13, "plant", "taxonomy_domain" ], [ 14, 30, "peptide hormones", "protein_type" ], [ 56, 72, "LRR-RK receptors", "protein_type" ], [ 79, 100, "extended conformation", "protein_state" ], [ 132, 142, "LRR domain", "structure_element" ], [ 160, 165, "small", "protein_state" ], [ 167, 186, "shape-complementary", "protein_state" ], [ 187, 199, "co-receptors", "protein_type" ] ] } ] }, "PMC4896748": { "annotations": [ { "sid": 0, "sent": "Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome", "section": "TITLE", "ner": [ [ 9, 16, "cryo-EM", "experimental_method" ], [ 26, 34, "inchworm", "protein_state" ], [ 59, 64, "viral", "taxonomy_domain" ], [ 65, 69, "IRES", "site" ], [ 82, 90, "ribosome", "complex_assembly" ] ] }, { "sid": 1, "sent": "Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs.", "section": "ABSTRACT", "ner": [ [ 0, 29, "Internal ribosome entry sites", "site" ], [ 31, 36, "IRESs", "site" ], [ 77, 82, "viral", "taxonomy_domain" ], [ 83, 88, "mRNAs", "chemical" ] ] }, { "sid": 2, "sent": "Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2\u2022GTP bound with sordarin.", "section": "ABSTRACT", "ner": [ [ 6, 30, "electron cryo-microscopy", "experimental_method" ], [ 69, 77, "ribosome", "complex_assembly" ], [ 78, 88, "structures", "evidence" ], [ 105, 125, "Taura syndrome virus", "species" ], [ 126, 130, "IRES", "site" ], [ 135, 146, "translocase", "protein_type" ], [ 147, 155, "eEF2\u2022GTP", "complex_assembly" ], [ 156, 166, "bound with", "protein_state" ], [ 167, 175, "sordarin", "chemical" ] ] }, { "sid": 3, "sent": "The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics.", "section": "ABSTRACT", "ner": [ [ 4, 14, "structures", "evidence" ], [ 39, 43, "IRES", "site" ], [ 84, 94, "initiation", "protein_state" ], [ 133, 137, "eEF2", "protein" ] ] }, { "sid": 4, "sent": "The IRES rearranges from extended to bent to extended conformations.", "section": "ABSTRACT", "ner": [ [ 4, 8, "IRES", "site" ], [ 25, 33, "extended", "protein_state" ], [ 37, 41, "bent", "protein_state" ], [ 45, 53, "extended", "protein_state" ] ] }, { "sid": 5, "sent": "This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel.", "section": "ABSTRACT", "ner": [ [ 5, 13, "inchworm", "protein_state" ], [ 81, 84, "40S", "complex_assembly" ], [ 85, 89, "head", "structure_element" ] ] }, { "sid": 6, "sent": "eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site.", "section": "ABSTRACT", "ner": [ [ 0, 4, "eEF2", "protein" ], [ 22, 25, "60S", "complex_assembly" ], [ 26, 33, "subunit", "structure_element" ], [ 61, 64, "40S", "complex_assembly" ], [ 65, 72, "subunit", "structure_element" ], [ 65, 72, "subunit", "structure_element" ], [ 86, 92, "A site", "site" ] ] }, { "sid": 7, "sent": "Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center.", "section": "ABSTRACT", "ner": [ [ 4, 15, "diphthamide", "ptm" ], [ 38, 40, "IV", "structure_element" ], [ 55, 82, "tRNA-mRNA-like pseudoknot I", "structure_element" ], [ 84, 87, "PKI", "structure_element" ], [ 96, 100, "IRES", "site" ], [ 110, 125, "decoding center", "site" ] ] }, { "sid": 8, "sent": "This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites.", "section": "ABSTRACT", "ner": [ [ 13, 16, "40S", "complex_assembly" ], [ 39, 43, "head", "structure_element" ], [ 63, 67, "IRES", "site" ], [ 122, 125, "PKI", "structure_element" ], [ 147, 160, "A and P sites", "site" ] ] }, { "sid": 9, "sent": "The structures suggest missing links in our understanding of tRNA translocation.", "section": "ABSTRACT", "ner": [ [ 4, 14, "structures", "evidence" ], [ 61, 65, "tRNA", "chemical" ] ] }, { "sid": 10, "sent": "Virus propagation relies on the host translational apparatus.", "section": "INTRO", "ner": [ [ 0, 5, "Virus", "taxonomy_domain" ] ] }, { "sid": 11, "sent": "To efficiently compete with host mRNAs and engage in translation under stress, some viral mRNAs undergo cap-independent translation.", "section": "INTRO", "ner": [ [ 33, 38, "mRNAs", "chemical" ], [ 84, 89, "viral", "taxonomy_domain" ], [ 90, 95, "mRNAs", "chemical" ] ] }, { "sid": 12, "sent": "To this end, internal ribosome entry site (IRES) RNAs are employed (reviewed in.", "section": "INTRO", "ner": [ [ 13, 41, "internal ribosome entry site", "site" ], [ 43, 47, "IRES", "site" ], [ 49, 53, "RNAs", "chemical" ] ] }, { "sid": 13, "sent": "An IRES is located at the 5\u2019 untranslated region of the viral mRNA, preceding an open reading frame (ORF).", "section": "INTRO", "ner": [ [ 3, 7, "IRES", "site" ], [ 26, 48, "5\u2019 untranslated region", "structure_element" ], [ 56, 61, "viral", "taxonomy_domain" ], [ 62, 66, "mRNA", "chemical" ], [ 81, 99, "open reading frame", "structure_element" ], [ 101, 104, "ORF", "structure_element" ] ] }, { "sid": 14, "sent": "To initiate translation, a structured IRES RNA interacts with the 40S subunit or the 80S ribosome, resulting in precise positioning of the downstream start codon in the small 40S subunit.", "section": "INTRO", "ner": [ [ 27, 37, "structured", "protein_state" ], [ 38, 42, "IRES", "site" ], [ 43, 46, "RNA", "chemical" ], [ 66, 69, "40S", "complex_assembly" ], [ 70, 77, "subunit", "structure_element" ], [ 85, 97, "80S ribosome", "complex_assembly" ], [ 169, 174, "small", "protein_state" ], [ 175, 178, "40S", "complex_assembly" ], [ 179, 186, "subunit", "structure_element" ] ] }, { "sid": 15, "sent": "The canonical scenario of cap-dependent and IRES-dependent initiation involves positioning of the AUG start codon and the initiator tRNAMet in the ribosomal peptidyl-tRNA (P) site, facilitated by interaction with initiation factors.", "section": "INTRO", "ner": [ [ 44, 48, "IRES", "site" ], [ 132, 139, "tRNAMet", "chemical" ], [ 157, 179, "peptidyl-tRNA (P) site", "site" ], [ 213, 231, "initiation factors", "protein_type" ] ] }, { "sid": 16, "sent": "Subsequent binding of an elongator aminoacyl-tRNA to the ribosomal A site transitions the initiation complex into the elongation cycle of translation.", "section": "INTRO", "ner": [ [ 35, 49, "aminoacyl-tRNA", "chemical" ], [ 67, 73, "A site", "site" ], [ 90, 108, "initiation complex", "complex_assembly" ] ] }, { "sid": 17, "sent": "Upon peptide bond formation, the two tRNAs and their respective mRNA codons translocate from the A and P to P and E (exit) sites, freeing the A site for the next elongator tRNA.", "section": "INTRO", "ner": [ [ 37, 42, "tRNAs", "chemical" ], [ 64, 68, "mRNA", "chemical" ], [ 97, 104, "A and P", "site" ], [ 108, 128, "P and E (exit) sites", "site" ], [ 142, 148, "A site", "site" ], [ 172, 176, "tRNA", "chemical" ] ] }, { "sid": 18, "sent": "An unusual strategy of initiation is used by intergenic-region (IGR) IRESs found in Dicistroviridae arthropod-infecting viruses.", "section": "INTRO", "ner": [ [ 23, 33, "initiation", "protein_state" ], [ 45, 62, "intergenic-region", "structure_element" ], [ 64, 67, "IGR", "structure_element" ], [ 69, 74, "IRESs", "site" ], [ 84, 109, "Dicistroviridae arthropod", "species" ], [ 120, 127, "viruses", "taxonomy_domain" ] ] }, { "sid": 19, "sent": "These include shrimp-infecting Taura syndrome virus (TSV), and insect viruses Plautia stali intestine virus (PSIV) and Cricket paralysis virus (CrPV).", "section": "INTRO", "ner": [ [ 14, 20, "shrimp", "taxonomy_domain" ], [ 31, 51, "Taura syndrome virus", "species" ], [ 53, 56, "TSV", "species" ], [ 63, 69, "insect", "taxonomy_domain" ], [ 78, 107, "Plautia stali intestine virus", "species" ], [ 109, 113, "PSIV", "species" ], [ 119, 142, "Cricket paralysis virus", "species" ], [ 144, 148, "CrPV", "species" ] ] }, { "sid": 20, "sent": "The IGR IRES mRNAs do not contain an AUG start codon.", "section": "INTRO", "ner": [ [ 4, 7, "IGR", "structure_element" ], [ 8, 12, "IRES", "site" ], [ 13, 18, "mRNAs", "chemical" ] ] }, { "sid": 21, "sent": "The IGR-IRES-driven initiation does not involve initiator tRNAMet and initiation factors.", "section": "INTRO", "ner": [ [ 4, 7, "IGR", "structure_element" ], [ 8, 12, "IRES", "site" ], [ 20, 30, "initiation", "protein_state" ], [ 58, 65, "tRNAMet", "chemical" ], [ 70, 80, "initiation", "protein_state" ] ] }, { "sid": 22, "sent": "As such, this group of IRESs represents the most streamlined mechanism of eukaryotic translation initiation.", "section": "INTRO", "ner": [ [ 23, 28, "IRESs", "site" ], [ 74, 84, "eukaryotic", "taxonomy_domain" ], [ 97, 107, "initiation", "protein_state" ] ] }, { "sid": 23, "sent": "A recent demonstration of bacterial translation initiation by an IGR IRES indicates that the IRESs take advantage of conserved structural and dynamic properties of the ribosome.", "section": "INTRO", "ner": [ [ 26, 35, "bacterial", "taxonomy_domain" ], [ 48, 58, "initiation", "protein_state" ], [ 65, 68, "IGR", "structure_element" ], [ 69, 73, "IRES", "site" ], [ 93, 98, "IRESs", "site" ], [ 168, 176, "ribosome", "complex_assembly" ] ] }, { "sid": 24, "sent": "Early electron cryo-microscopy (cryo-EM) studies have found that the CrPV IRES packs in the ribosome intersubunit space.", "section": "INTRO", "ner": [ [ 6, 30, "electron cryo-microscopy", "experimental_method" ], [ 32, 39, "cryo-EM", "experimental_method" ], [ 69, 73, "CrPV", "species" ], [ 74, 78, "IRES", "site" ], [ 92, 100, "ribosome", "complex_assembly" ], [ 101, 119, "intersubunit space", "site" ] ] }, { "sid": 25, "sent": "Recent cryo-EM structures of ribosome-bound TSV IRES and CrPV IRES revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA.", "section": "INTRO", "ner": [ [ 7, 14, "cryo-EM", "experimental_method" ], [ 15, 25, "structures", "evidence" ], [ 29, 43, "ribosome-bound", "protein_state" ], [ 44, 47, "TSV", "species" ], [ 48, 52, "IRES", "site" ], [ 57, 61, "CrPV", "species" ], [ 62, 66, "IRES", "site" ], [ 81, 84, "IGR", "structure_element" ], [ 85, 90, "IRESs", "site" ], [ 104, 107, "ORF", "structure_element" ], [ 135, 143, "ribosome", "complex_assembly" ], [ 144, 154, "bound with", "protein_state" ], [ 155, 159, "tRNA", "chemical" ], [ 164, 168, "mRNA", "chemical" ] ] }, { "sid": 26, "sent": "The ~200-nt IRES RNAs span from the A site beyond the E site.", "section": "INTRO", "ner": [ [ 12, 16, "IRES", "site" ], [ 17, 21, "RNAs", "chemical" ], [ 36, 42, "A site", "site" ], [ 54, 60, "E site", "site" ] ] }, { "sid": 27, "sent": "A conserved tRNA-mRNA\u2013like structural element of pseudoknot I (PKI) interacts with the decoding center in the A site of the 40S subunit.", "section": "INTRO", "ner": [ [ 2, 11, "conserved", "protein_state" ], [ 12, 45, "tRNA-mRNA\u2013like structural element", "structure_element" ], [ 49, 61, "pseudoknot I", "structure_element" ], [ 63, 66, "PKI", "structure_element" ], [ 87, 102, "decoding center", "site" ], [ 110, 116, "A site", "site" ], [ 124, 127, "40S", "complex_assembly" ], [ 128, 135, "subunit", "structure_element" ] ] }, { "sid": 28, "sent": "The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA).", "section": "INTRO", "ner": [ [ 4, 30, "codon-anticodon-like helix", "structure_element" ], [ 34, 37, "PKI", "structure_element" ], [ 77, 98, "universally conserved", "protein_state" ], [ 99, 114, "decoding-center", "site" ], [ 127, 131, "G577", "residue_name_number" ], [ 133, 138, "A1755", "residue_name_number" ], [ 143, 148, "A1756", "residue_name_number" ], [ 150, 154, "G530", "residue_name_number" ], [ 156, 161, "A1492", "residue_name_number" ], [ 166, 171, "A1493", "residue_name_number" ], [ 175, 182, "E. coli", "species" ], [ 197, 200, "RNA", "chemical" ], [ 205, 209, "rRNA", "chemical" ] ] }, { "sid": 29, "sent": "The downstream initiation codon\u2014coding for alanine\u2014is placed in the mRNA tunnel, preceding the decoding center.", "section": "INTRO", "ner": [ [ 43, 50, "alanine", "residue_name" ], [ 68, 79, "mRNA tunnel", "site" ], [ 95, 110, "decoding center", "site" ] ] }, { "sid": 30, "sent": "PKI of IGR IRESs therefore mimics an A-site elongator\u00a0tRNA interacting with an mRNA sense codon, but not a P-site initiator\u00a0tRNAMet and the AUG start codon.", "section": "INTRO", "ner": [ [ 0, 3, "PKI", "structure_element" ], [ 7, 10, "IGR", "structure_element" ], [ 11, 16, "IRESs", "site" ], [ 37, 43, "A-site", "site" ], [ 54, 58, "tRNA", "chemical" ], [ 79, 83, "mRNA", "chemical" ], [ 107, 113, "P-site", "site" ], [ 124, 131, "tRNAMet", "chemical" ] ] }, { "sid": 31, "sent": "How this non-canonical initiation complex transitions to the elongation step is not fully understood.", "section": "INTRO", "ner": [ [ 23, 33, "initiation", "protein_state" ] ] }, { "sid": 32, "sent": "For a cognate aminoacyl-tRNA to bind the first viral mRNA codon, PKI has to be translocated from the A site, so that the first codon can be presented in the A site.", "section": "INTRO", "ner": [ [ 14, 28, "aminoacyl-tRNA", "chemical" ], [ 47, 52, "viral", "taxonomy_domain" ], [ 53, 57, "mRNA", "chemical" ], [ 65, 68, "PKI", "structure_element" ], [ 101, 107, "A site", "site" ], [ 157, 163, "A site", "site" ] ] }, { "sid": 33, "sent": "A cryo-EM structure of the ribosome bound with a CrPV IRES and release factor eRF1 occupying the A site provided insight into the post-translocation state.", "section": "INTRO", "ner": [ [ 2, 9, "cryo-EM", "experimental_method" ], [ 10, 19, "structure", "evidence" ], [ 27, 35, "ribosome", "complex_assembly" ], [ 36, 46, "bound with", "protein_state" ], [ 49, 53, "CrPV", "species" ], [ 54, 58, "IRES", "site" ], [ 63, 77, "release factor", "protein_type" ], [ 78, 82, "eRF1", "protein" ], [ 97, 103, "A site", "site" ], [ 130, 148, "post-translocation", "protein_state" ] ] }, { "sid": 34, "sent": "In this structure, PKI is positioned in the P site and the first mRNA codon is located in the A site.", "section": "INTRO", "ner": [ [ 8, 17, "structure", "evidence" ], [ 19, 22, "PKI", "structure_element" ], [ 44, 50, "P site", "site" ], [ 65, 69, "mRNA", "chemical" ], [ 94, 100, "A site", "site" ] ] }, { "sid": 35, "sent": "How the large IRES RNA translocates within the ribosome, allowing PKI translocation from the A to P site is not known.", "section": "INTRO", "ner": [ [ 8, 13, "large", "protein_state" ], [ 14, 18, "IRES", "site" ], [ 19, 22, "RNA", "chemical" ], [ 47, 55, "ribosome", "complex_assembly" ], [ 66, 69, "PKI", "structure_element" ], [ 93, 104, "A to P site", "site" ] ] }, { "sid": 36, "sent": "The structural similarity of PKI and the tRNA anticodon stem loop (ASL) bound to a codon suggests that their mechanisms of translocation are similar to some extent.", "section": "INTRO", "ner": [ [ 29, 32, "PKI", "structure_element" ], [ 41, 45, "tRNA", "chemical" ], [ 46, 65, "anticodon stem loop", "structure_element" ], [ 67, 70, "ASL", "structure_element" ], [ 72, 80, "bound to", "protein_state" ] ] }, { "sid": 37, "sent": "Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2), a structural and functional homolog of the well-studied bacterial EF-G. Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA\u2022mRNA complex).", "section": "INTRO", "ner": [ [ 21, 25, "IRES", "site" ], [ 29, 38, "tRNA-mRNA", "complex_assembly" ], [ 48, 58, "eukaryotic", "taxonomy_domain" ], [ 59, 78, "elongation factor 2", "protein" ], [ 80, 84, "eEF2", "protein" ], [ 143, 152, "bacterial", "taxonomy_domain" ], [ 153, 157, "EF-G", "protein" ], [ 159, 176, "Pre-translocation", "protein_state" ], [ 177, 187, "tRNA-bound", "protein_state" ], [ 188, 197, "ribosomes", "complex_assembly" ], [ 208, 233, "peptidyl- and deacyl-tRNA", "chemical" ], [ 255, 259, "mRNA", "chemical" ], [ 274, 287, "A and P sites", "site" ], [ 296, 306, "2tRNA\u2022mRNA", "complex_assembly" ] ] }, { "sid": 38, "sent": "Translocation of 2tRNA\u2022mRNA involves two major large-scale ribosome rearrangements (Figure 1\u2014figure supplement 1) (reviewed in).", "section": "INTRO", "ner": [ [ 17, 27, "2tRNA\u2022mRNA", "complex_assembly" ], [ 59, 67, "ribosome", "complex_assembly" ] ] }, { "sid": 39, "sent": "First, studies of bacterial ribosomes showed that a ~10\u00b0 rotation of the small subunit relative to the large subunit, known as intersubunit rotation, or ratcheting, is required for translocation.", "section": "INTRO", "ner": [ [ 18, 27, "bacterial", "taxonomy_domain" ], [ 28, 37, "ribosomes", "complex_assembly" ], [ 73, 86, "small subunit", "structure_element" ], [ 103, 116, "large subunit", "structure_element" ] ] }, { "sid": 40, "sent": "Intersubunit rotation occurs spontaneously upon peptidyl transfer, and is coupled with formation of hybrid tRNA states.", "section": "INTRO", "ner": [ [ 100, 106, "hybrid", "protein_state" ], [ 107, 111, "tRNA", "chemical" ] ] }, { "sid": 41, "sent": "In the rotated pre-translocation ribosome, the peptidyl-tRNA binds the A site of the small subunit with its ASL and the P site of the large subunit with the CCA 3\u2019 end (A/P hybrid state).", "section": "INTRO", "ner": [ [ 7, 14, "rotated", "protein_state" ], [ 15, 32, "pre-translocation", "protein_state" ], [ 33, 41, "ribosome", "complex_assembly" ], [ 47, 60, "peptidyl-tRNA", "chemical" ], [ 71, 77, "A site", "site" ], [ 85, 98, "small subunit", "structure_element" ], [ 108, 111, "ASL", "structure_element" ], [ 120, 126, "P site", "site" ], [ 134, 147, "large subunit", "structure_element" ], [ 169, 179, "A/P hybrid", "protein_state" ] ] }, { "sid": 42, "sent": "Concurrently, the deacyl-tRNA interacts with the P site of the small subunit and the E site of the large subunit (P/E hybrid state).", "section": "INTRO", "ner": [ [ 18, 29, "deacyl-tRNA", "chemical" ], [ 49, 55, "P site", "site" ], [ 63, 76, "small subunit", "structure_element" ], [ 85, 91, "E site", "site" ], [ 99, 112, "large subunit", "structure_element" ], [ 114, 124, "P/E hybrid", "protein_state" ] ] }, { "sid": 43, "sent": "The ribosome can undergo spontaneous, thermally-driven forward-reverse rotation that shifts the two tRNAs between the hybrid and 'classical' states while the anticodon stem loops remain non-translocated.", "section": "INTRO", "ner": [ [ 4, 12, "ribosome", "complex_assembly" ], [ 100, 105, "tRNAs", "chemical" ], [ 118, 124, "hybrid", "protein_state" ], [ 130, 139, "classical", "protein_state" ], [ 158, 178, "anticodon stem loops", "structure_element" ], [ 186, 202, "non-translocated", "protein_state" ] ] }, { "sid": 44, "sent": "Binding of EF-G next to the A site and reverse rotation of the small subunit results in translocation of both ASLs on the small subunit.", "section": "INTRO", "ner": [ [ 11, 15, "EF-G", "protein" ], [ 28, 34, "A site", "site" ], [ 63, 76, "small subunit", "structure_element" ], [ 110, 114, "ASLs", "structure_element" ], [ 122, 135, "small subunit", "structure_element" ] ] }, { "sid": 45, "sent": "EF-G is thought to 'unlock' the pre-translocation ribosome, allowing movement of the 2tRNA\u2022mRNA complex, however the structural details of this unlocking are not known.", "section": "INTRO", "ner": [ [ 0, 4, "EF-G", "protein" ], [ 32, 49, "pre-translocation", "protein_state" ], [ 50, 58, "ribosome", "complex_assembly" ], [ 85, 95, "2tRNA\u2022mRNA", "complex_assembly" ] ] }, { "sid": 46, "sent": "The second large-scale rearrangement involves rotation, or swiveling, of the head of the small subunit relative to the body.", "section": "INTRO", "ner": [ [ 77, 81, "head", "structure_element" ], [ 89, 102, "small subunit", "structure_element" ], [ 119, 123, "body", "structure_element" ] ] }, { "sid": 47, "sent": "The head can rotate by up to ~20\u00b0 around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. F\u00f6rster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA\u2022mRNA.", "section": "INTRO", "ner": [ [ 4, 8, "head", "structure_element" ], [ 109, 119, "absence of", "protein_state" ], [ 120, 124, "tRNA", "chemical" ], [ 135, 146, "presence of", "protein_state" ], [ 156, 157, "P", "site" ], [ 158, 159, "E", "site" ], [ 160, 164, "tRNA", "chemical" ], [ 169, 173, "eEF2", "protein" ], [ 177, 181, "EF-G", "protein" ], [ 183, 216, "F\u00f6rster resonance energy transfer", "experimental_method" ], [ 218, 222, "FRET", "experimental_method" ], [ 242, 246, "head", "structure_element" ], [ 261, 268, "rotated", "protein_state" ], [ 269, 282, "small subunit", "structure_element" ], [ 295, 299, "EF-G", "protein" ], [ 321, 331, "2tRNA\u2022mRNA", "complex_assembly" ] ] }, { "sid": 48, "sent": "Structures of the 70S\u2022EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18\u00b0 to 21\u00b0 head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S\u20222tRNA\u2022EF-G structures.", "section": "INTRO", "ner": [ [ 0, 10, "Structures", "evidence" ], [ 18, 26, "70S\u2022EF-G", "complex_assembly" ], [ 35, 45, "bound with", "protein_state" ], [ 50, 69, "nearly translocated", "protein_state" ], [ 70, 75, "tRNAs", "chemical" ], [ 104, 108, "head", "structure_element" ], [ 121, 132, "mid-rotated", "protein_state" ], [ 133, 140, "subunit", "structure_element" ], [ 153, 157, "head", "structure_element" ], [ 184, 197, "fully rotated", "protein_state" ], [ 198, 215, "pre-translocation", "protein_state" ], [ 226, 237, "non-rotated", "protein_state" ], [ 238, 256, "post-translocation", "protein_state" ], [ 257, 271, "70S\u20222tRNA\u2022EF-G", "complex_assembly" ], [ 272, 282, "structures", "evidence" ] ] }, { "sid": 49, "sent": "The structural role of head swivel is not fully understood.", "section": "INTRO", "ner": [ [ 23, 27, "head", "structure_element" ] ] }, { "sid": 50, "sent": "The head swivel was proposed to facilitate transition of the tRNA from the P to E site by widening a constriction between these sites on the 30S subunit.", "section": "INTRO", "ner": [ [ 4, 8, "head", "structure_element" ], [ 61, 65, "tRNA", "chemical" ], [ 75, 86, "P to E site", "site" ], [ 101, 113, "constriction", "site" ], [ 141, 144, "30S", "complex_assembly" ], [ 145, 152, "subunit", "structure_element" ], [ 145, 152, "subunit", "structure_element" ] ] }, { "sid": 51, "sent": "This widening\u00a0allows the ASL to sample positions between the P and E sites.", "section": "INTRO", "ner": [ [ 25, 28, "ASL", "structure_element" ], [ 61, 74, "P and E sites", "site" ] ] }, { "sid": 52, "sent": "Whether and how the head swivel mediates tRNA transition from the A to P site remains unknown.", "section": "INTRO", "ner": [ [ 20, 24, "head", "structure_element" ], [ 41, 45, "tRNA", "chemical" ], [ 66, 77, "A to P site", "site" ] ] }, { "sid": 53, "sent": "Comparison of 70S\u20222tRNA\u2022mRNA and 80S\u2022IRES translocation complexes.", "section": "FIG", "ner": [ [ 14, 28, "70S\u20222tRNA\u2022mRNA", "complex_assembly" ], [ 33, 41, "80S\u2022IRES", "complex_assembly" ] ] }, { "sid": 54, "sent": "(a) Structures of bacterial 70S\u20222tRNA\u2022mRNA translocation complexes, ordered according to the position of the translocating A->P tRNA (orange).", "section": "FIG", "ner": [ [ 4, 14, "Structures", "evidence" ], [ 18, 27, "bacterial", "taxonomy_domain" ], [ 28, 42, "70S\u20222tRNA\u2022mRNA", "complex_assembly" ], [ 123, 127, "A->P", "site" ], [ 128, 132, "tRNA", "chemical" ] ] }, { "sid": 55, "sent": "The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body), elongation factor G (EF-G) is shown in green.", "section": "FIG", "ner": [ [ 20, 27, "subunit", "structure_element" ], [ 50, 63, "small subunit", "structure_element" ], [ 81, 85, "head", "structure_element" ], [ 105, 109, "body", "structure_element" ], [ 112, 131, "elongation factor G", "protein" ], [ 133, 137, "EF-G", "protein" ] ] }, { "sid": 56, "sent": "Nucleotides C1054, G966 and G693 of 16S rRNA are shown in black to denote the A, P and E sites, respectively.", "section": "FIG", "ner": [ [ 12, 17, "C1054", "residue_name_number" ], [ 19, 23, "G966", "residue_name_number" ], [ 28, 32, "G693", "residue_name_number" ], [ 36, 44, "16S rRNA", "chemical" ], [ 78, 94, "A, P and E sites", "site" ] ] }, { "sid": 57, "sent": "The extents of the 30S subunit rotation and head swivel relative to their positions in the post-translocation structure are shown with arrows.", "section": "FIG", "ner": [ [ 19, 22, "30S", "complex_assembly" ], [ 23, 30, "subunit", "structure_element" ], [ 44, 48, "head", "structure_element" ], [ 91, 109, "post-translocation", "protein_state" ], [ 110, 119, "structure", "evidence" ] ] }, { "sid": 58, "sent": "References and PDB codes of the structures are shown.", "section": "FIG", "ner": [ [ 32, 42, "structures", "evidence" ] ] }, { "sid": 59, "sent": "(b) Structures of the 80S\u2022IRES complexes in the absence and presence of eEF2 (this work).", "section": "FIG", "ner": [ [ 4, 14, "Structures", "evidence" ], [ 22, 30, "80S\u2022IRES", "complex_assembly" ], [ 48, 55, "absence", "protein_state" ], [ 60, 71, "presence of", "protein_state" ], [ 72, 76, "eEF2", "protein" ] ] }, { "sid": 60, "sent": "The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green.", "section": "FIG", "ner": [ [ 20, 27, "subunit", "structure_element" ], [ 50, 63, "small subunit", "structure_element" ], [ 81, 85, "head", "structure_element" ], [ 105, 109, "body", "structure_element" ], [ 116, 119, "TSV", "species" ], [ 120, 124, "IRES", "site" ], [ 133, 137, "eEF2", "protein" ] ] }, { "sid": 61, "sent": "Nucleotides C1274, U1191 of the 40S head and G904 of the platform (corresponding to C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively.", "section": "FIG", "ner": [ [ 12, 17, "C1274", "residue_name_number" ], [ 19, 24, "U1191", "residue_name_number" ], [ 32, 35, "40S", "complex_assembly" ], [ 36, 40, "head", "structure_element" ], [ 45, 49, "G904", "residue_name_number" ], [ 57, 65, "platform", "structure_element" ], [ 84, 89, "C1054", "residue_name_number" ], [ 91, 95, "G966", "residue_name_number" ], [ 100, 104, "G693", "residue_name_number" ], [ 108, 115, "E. coli", "species" ], [ 116, 124, "16S rRNA", "chemical" ], [ 159, 175, "A, P and E sites", "site" ] ] }, { "sid": 62, "sent": "Unresolved regions of the IRES in densities for Structures III and V are shown in gray.", "section": "FIG", "ner": [ [ 26, 30, "IRES", "site" ], [ 34, 43, "densities", "evidence" ], [ 48, 68, "Structures III and V", "evidence" ], [ 26, 30, "IRES", "site" ], [ 34, 43, "densities", "evidence" ], [ 48, 68, "Structures III and V", "evidence" ] ] }, { "sid": 63, "sent": "The extents of the 40S subunit rotation and head swivel relative to their positions in the post-translocation structure are shown with arrows.", "section": "FIG", "ner": [ [ 19, 22, "40S", "complex_assembly" ], [ 23, 30, "subunit", "structure_element" ], [ 44, 48, "head", "structure_element" ], [ 91, 109, "post-translocation", "protein_state" ], [ 110, 119, "structure", "evidence" ] ] }, { "sid": 64, "sent": "Schematic of cryo-EM refinement and classification procedures.", "section": "FIG", "ner": [ [ 13, 20, "cryo-EM", "experimental_method" ] ] }, { "sid": 65, "sent": "All particles were initially aligned to a single model.", "section": "FIG", "ner": [ [ 4, 13, "particles", "experimental_method" ] ] }, { "sid": 66, "sent": "3D classification using a 3D mask around the 40S head, TSV IRES and eEF2, of the 4x binned stack was used to identify particles containing both the IRES and eEF2.", "section": "FIG", "ner": [ [ 0, 17, "3D classification", "experimental_method" ], [ 26, 33, "3D mask", "evidence" ], [ 45, 48, "40S", "complex_assembly" ], [ 49, 53, "head", "structure_element" ], [ 55, 58, "TSV", "species" ], [ 59, 63, "IRES", "site" ], [ 68, 72, "eEF2", "protein" ], [ 91, 96, "stack", "bond_interaction" ], [ 118, 127, "particles", "experimental_method" ], [ 148, 152, "IRES", "site" ], [ 157, 161, "eEF2", "protein" ] ] }, { "sid": 67, "sent": "Subsequent 3D classification using a 2D mask comprising PKI and domain IV of eEF2 yielded 5 'purified' classes representing Structures I through V. Sub-classification of each class did not yield additional classes, but helped improve density in the PKI region of class III (estimated resolution and percentage of particles in the sub-classified reconstruction are shown in parentheses).", "section": "FIG", "ner": [ [ 11, 28, "3D classification", "experimental_method" ], [ 37, 44, "2D mask", "evidence" ], [ 56, 59, "PKI", "structure_element" ], [ 71, 73, "IV", "structure_element" ], [ 77, 81, "eEF2", "protein" ], [ 124, 146, "Structures I through V", "evidence" ], [ 148, 166, "Sub-classification", "experimental_method" ], [ 234, 241, "density", "evidence" ], [ 249, 252, "PKI", "structure_element" ], [ 313, 322, "particles", "experimental_method" ], [ 330, 344, "sub-classified", "experimental_method" ], [ 345, 359, "reconstruction", "evidence" ] ] }, { "sid": 68, "sent": "Cryo-EM density of Structures I-V.", "section": "FIG", "ner": [ [ 0, 7, "Cryo-EM", "experimental_method" ], [ 8, 15, "density", "evidence" ], [ 19, 33, "Structures I-V", "evidence" ] ] }, { "sid": 69, "sent": "In panels (a-e), the maps are segmented and colored as in Figure 1.", "section": "FIG", "ner": [ [ 21, 25, "maps", "evidence" ] ] }, { "sid": 70, "sent": "The maps in all panels were B-softened by applying a B-factor of 30 \u00c52.", "section": "FIG", "ner": [ [ 4, 8, "maps", "evidence" ] ] }, { "sid": 71, "sent": "(a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency \u00c5-1 and with \u00c5. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC').", "section": "FIG", "ner": [ [ 6, 13, "Cryo-EM", "experimental_method" ], [ 14, 17, "map", "evidence" ], [ 21, 52, "Structures I, II, III, IV and V", "evidence" ], [ 104, 111, "cryo-EM", "experimental_method" ], [ 112, 127, "reconstructions", "evidence" ], [ 144, 151, "Blocres", "experimental_method" ], [ 180, 211, "Structures I, II, III, IV and V", "evidence" ], [ 219, 226, "Cryo-EM", "experimental_method" ], [ 227, 234, "density", "evidence" ], [ 243, 246, "TSV", "species" ], [ 247, 251, "IRES", "site" ], [ 268, 272, "eEF2", "protein" ], [ 290, 321, "Structures I, II, III, IV and V", "evidence" ], [ 327, 352, "Fourier shell correlation", "evidence" ], [ 354, 357, "FSC", "evidence" ], [ 359, 365, "curves", "evidence" ], [ 370, 384, "Structures I-V", "evidence" ], [ 508, 511, "FSC", "evidence" ], [ 570, 578, "FREALIGN", "experimental_method" ], [ 587, 590, "FSC", "evidence" ] ] }, { "sid": 72, "sent": "Cryo-EM structures of the 80S\u2022TSV IRES bound with eEF2\u2022GDP\u2022sordarin.", "section": "FIG", "ner": [ [ 0, 7, "Cryo-EM", "experimental_method" ], [ 8, 18, "structures", "evidence" ], [ 26, 38, "80S\u2022TSV IRES", "complex_assembly" ], [ 39, 49, "bound with", "protein_state" ], [ 50, 67, "eEF2\u2022GDP\u2022sordarin", "complex_assembly" ] ] }, { "sid": 73, "sent": "(a)\u00a0Structures I through V. In all panels, the large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green.", "section": "FIG", "ner": [ [ 4, 26, "Structures I through V", "evidence" ], [ 47, 70, "large ribosomal subunit", "structure_element" ], [ 93, 106, "small subunit", "structure_element" ], [ 124, 128, "head", "structure_element" ], [ 148, 152, "body", "structure_element" ], [ 159, 162, "TSV", "species" ], [ 163, 167, "IRES", "site" ], [ 176, 180, "eEF2", "protein" ] ] }, { "sid": 74, "sent": "Nucleotides C1274, U1191 of the 40S head and G904 of the platform (C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively.", "section": "FIG", "ner": [ [ 12, 17, "C1274", "residue_name_number" ], [ 19, 24, "U1191", "residue_name_number" ], [ 32, 35, "40S", "complex_assembly" ], [ 36, 40, "head", "structure_element" ], [ 45, 49, "G904", "residue_name_number" ], [ 57, 65, "platform", "site" ], [ 67, 72, "C1054", "residue_name_number" ], [ 74, 78, "G966", "residue_name_number" ], [ 83, 87, "G693", "residue_name_number" ], [ 91, 98, "E. coli", "species" ], [ 99, 107, "16S rRNA", "chemical" ], [ 142, 158, "A, P and E sites", "site" ] ] }, { "sid": 75, "sent": "(b) Schematic representation of the structures shown in panel a, denoting the conformations of the small subunit relative to the large subunit.", "section": "FIG", "ner": [ [ 36, 46, "structures", "evidence" ], [ 99, 112, "small subunit", "structure_element" ], [ 129, 142, "large subunit", "structure_element" ] ] }, { "sid": 76, "sent": "A, P and E sites are shown as rectangles.", "section": "FIG", "ner": [ [ 0, 16, "A, P and E sites", "site" ] ] }, { "sid": 77, "sent": "All measurements are relative to the non-rotated 80S\u20222tRNA\u2022mRNA structure.", "section": "FIG", "ner": [ [ 37, 48, "non-rotated", "protein_state" ], [ 49, 63, "80S\u20222tRNA\u2022mRNA", "complex_assembly" ], [ 64, 73, "structure", "evidence" ], [ 37, 48, "non-rotated", "protein_state" ], [ 49, 63, "80S\u20222tRNA\u2022mRNA", "complex_assembly" ], [ 64, 73, "structure", "evidence" ] ] }, { "sid": 78, "sent": "We sought to address the following questions by structural visualization of 80S\u2022IRES\u2022eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation?", "section": "INTRO", "ner": [ [ 48, 72, "structural visualization", "experimental_method" ], [ 76, 89, "80S\u2022IRES\u2022eEF2", "complex_assembly" ], [ 136, 140, "IRES", "site" ], [ 141, 144, "RNA", "chemical" ], [ 202, 205, "PKI", "structure_element" ], [ 215, 226, "A to P site", "site" ], [ 234, 247, "small subunit", "structure_element" ], [ 262, 266, "eEF2", "protein" ], [ 275, 279, "IRES", "site" ], [ 304, 308, "IRES", "site" ], [ 359, 367, "ribosome", "complex_assembly" ], [ 380, 384, "tRNA", "chemical" ], [ 445, 448, "40S", "complex_assembly" ], [ 449, 453, "head", "structure_element" ], [ 466, 470, "IRES", "site" ] ] }, { "sid": 79, "sent": "We used cryo-EM to visualize 80S\u2022TSV IRES complexes formed in the presence of eEF2\u2022GTP and the translation inhibitor sordarin, which stabilizes eEF2 on the ribosome.", "section": "INTRO", "ner": [ [ 8, 15, "cryo-EM", "experimental_method" ], [ 29, 41, "80S\u2022TSV IRES", "complex_assembly" ], [ 66, 77, "presence of", "protein_state" ], [ 78, 86, "eEF2\u2022GTP", "complex_assembly" ], [ 117, 125, "sordarin", "chemical" ], [ 144, 148, "eEF2", "protein" ], [ 156, 164, "ribosome", "complex_assembly" ] ] }, { "sid": 80, "sent": "Although the mechanism of sordarin action is not fully understood, the inhibitor does not affect the conformation of eEF2\u2022GDPNP on the ribosome, rendering it an excellent tool in translocation studies.", "section": "INTRO", "ner": [ [ 26, 34, "sordarin", "chemical" ], [ 117, 127, "eEF2\u2022GDPNP", "complex_assembly" ], [ 135, 143, "ribosome", "complex_assembly" ] ] }, { "sid": 81, "sent": "Maximum-likelihood classification using FREALIGN identified five IRES-eEF2-bound ribosome structures within a single sample (Figures 1 and 2).", "section": "INTRO", "ner": [ [ 0, 33, "Maximum-likelihood classification", "experimental_method" ], [ 40, 48, "FREALIGN", "experimental_method" ], [ 65, 80, "IRES-eEF2-bound", "protein_state" ], [ 81, 89, "ribosome", "complex_assembly" ], [ 90, 100, "structures", "evidence" ] ] }, { "sid": 82, "sent": "The structures differ in the positions and conformations of ribosomal subunits (Figures 1b and 2), IRES RNA (Figures 3 and 4) and eEF2 (Figures 5 and 6).", "section": "INTRO", "ner": [ [ 4, 14, "structures", "evidence" ], [ 99, 103, "IRES", "site" ], [ 104, 107, "RNA", "chemical" ], [ 130, 134, "eEF2", "protein" ] ] }, { "sid": 83, "sent": "This ensemble of structures allowed us to reconstruct a sequence of steps in IRES translocation induced by eEF2.", "section": "INTRO", "ner": [ [ 17, 27, "structures", "evidence" ], [ 77, 81, "IRES", "site" ], [ 107, 111, "eEF2", "protein" ] ] }, { "sid": 84, "sent": "We used single-particle cryo-EM and maximum-likelihood image classification in FREALIGN to obtain three-dimensional density maps from a single specimen.", "section": "RESULTS", "ner": [ [ 8, 31, "single-particle cryo-EM", "experimental_method" ], [ 36, 75, "maximum-likelihood image classification", "experimental_method" ], [ 79, 87, "FREALIGN", "experimental_method" ], [ 116, 128, "density maps", "evidence" ] ] }, { "sid": 85, "sent": "The translocation complex was formed using S. cerevisiae 80S ribosomes, Taura syndrome virus IRES, and S. cerevisiae eEF2 in the presence of GTP and the eEF2-binding translation inhibitor sordarin.", "section": "RESULTS", "ner": [ [ 43, 56, "S. cerevisiae", "species" ], [ 57, 70, "80S ribosomes", "complex_assembly" ], [ 72, 92, "Taura syndrome virus", "species" ], [ 93, 97, "IRES", "site" ], [ 103, 116, "S. cerevisiae", "species" ], [ 117, 121, "eEF2", "protein" ], [ 129, 140, "presence of", "protein_state" ], [ 141, 144, "GTP", "chemical" ], [ 153, 157, "eEF2", "protein" ], [ 188, 196, "sordarin", "chemical" ] ] }, { "sid": 86, "sent": "Unsupervised cryo-EM data classification was combined with the use of three-dimensional and two-dimensional masking around the ribosomal A site (Figure 1\u2014figure supplement 2).", "section": "RESULTS", "ner": [ [ 0, 40, "Unsupervised cryo-EM data classification", "experimental_method" ], [ 70, 115, "three-dimensional and two-dimensional masking", "experimental_method" ], [ 137, 143, "A site", "site" ] ] }, { "sid": 87, "sent": "This approach revealed five 80S\u2022IRES\u2022eEF2\u2022GDP structures at average resolutions of 3.5 to 4.2\u2009\u00c5, sufficient to locate IRES domains and to resolve individual residues in the core regions of the ribosome and eEF2 (Figures 3c,d, and\u00a05f,h; see also Figure 1\u2014figure supplement 2 and Figure 5\u2014figure supplement 2), including the post-translational modification diphthamide 699 (Figure 3c).", "section": "RESULTS", "ner": [ [ 28, 45, "80S\u2022IRES\u2022eEF2\u2022GDP", "complex_assembly" ], [ 46, 56, "structures", "evidence" ], [ 118, 122, "IRES", "site" ], [ 193, 201, "ribosome", "complex_assembly" ], [ 206, 210, "eEF2", "protein" ], [ 355, 370, "diphthamide 699", "ptm" ] ] }, { "sid": 88, "sent": "Large-scale rearrangements in Structures I through V, coupled with the movement of PKI from the A to P site and eEF2 entry into the A site.", "section": "FIG", "ner": [ [ 30, 52, "Structures I through V", "evidence" ], [ 83, 86, "PKI", "structure_element" ], [ 96, 107, "A to P site", "site" ], [ 112, 116, "eEF2", "protein" ], [ 132, 138, "A site", "site" ], [ 30, 52, "Structures I through V", "evidence" ], [ 83, 86, "PKI", "structure_element" ], [ 96, 107, "A to P site", "site" ], [ 112, 116, "eEF2", "protein" ], [ 132, 138, "A site", "site" ] ] }, { "sid": 89, "sent": "(a) Rotational states of the 40S subunit in the 80S\u2022IRES structure (INIT; PDB 3J6Y) and in 80S\u2022IRES\u2022eEF2 Structures I, II, III, IV and V (this work).", "section": "FIG", "ner": [ [ 29, 32, "40S", "complex_assembly" ], [ 33, 40, "subunit", "structure_element" ], [ 48, 56, "80S\u2022IRES", "complex_assembly" ], [ 57, 66, "structure", "evidence" ], [ 68, 72, "INIT", "complex_assembly" ], [ 91, 104, "80S\u2022IRES\u2022eEF2", "complex_assembly" ], [ 105, 136, "Structures I, II, III, IV and V", "evidence" ] ] }, { "sid": 90, "sent": "For each structure, the triangle outlines the contours of the 40S body; the lower angle illustrates the extent of intersubunit (body) rotation.", "section": "FIG", "ner": [ [ 9, 18, "structure", "evidence" ], [ 62, 65, "40S", "complex_assembly" ], [ 66, 70, "body", "structure_element" ], [ 128, 132, "body", "structure_element" ] ] }, { "sid": 91, "sent": "The sizes of the arrows correspond to the extent of the head swivel (yellow) and subunit rotation (black).", "section": "FIG", "ner": [ [ 56, 60, "head", "structure_element" ], [ 81, 88, "subunit", "structure_element" ] ] }, { "sid": 92, "sent": "The views were obtained by structural alignment of the 25S rRNAs; the sarcin-ricin loop (SRL) of 25S rRNA is shown in gray for reference.", "section": "FIG", "ner": [ [ 27, 47, "structural alignment", "experimental_method" ], [ 55, 64, "25S rRNAs", "chemical" ], [ 70, 87, "sarcin-ricin loop", "structure_element" ], [ 89, 92, "SRL", "structure_element" ], [ 97, 105, "25S rRNA", "chemical" ] ] }, { "sid": 93, "sent": "(b) Solvent view (opposite from that shown in (a)) of the 40S subunit in the 80S\u2022IRES structure (INIT; PDB 3J6Y) and in 80S\u2022IRES\u2022eEF2 Structures I, II, III, IV and V (this work).", "section": "FIG", "ner": [ [ 58, 61, "40S", "complex_assembly" ], [ 62, 69, "subunit", "structure_element" ], [ 77, 85, "80S\u2022IRES", "complex_assembly" ], [ 86, 95, "structure", "evidence" ], [ 97, 101, "INIT", "complex_assembly" ], [ 120, 133, "80S\u2022IRES\u2022eEF2", "complex_assembly" ], [ 134, 165, "Structures I, II, III, IV and V", "evidence" ] ] }, { "sid": 94, "sent": "The structures are colored as in Figure 1.", "section": "FIG", "ner": [ [ 4, 14, "structures", "evidence" ] ] }, { "sid": 95, "sent": "(a) Comparison of the 40S-subunit rotational states in Structures I through V, sampling a ~10\u00b0 range between Structure I (fully rotated) and Structure V (non-rotated).", "section": "FIG", "ner": [ [ 22, 25, "40S", "complex_assembly" ], [ 26, 33, "subunit", "structure_element" ], [ 55, 77, "Structures I through V", "evidence" ], [ 109, 120, "Structure I", "evidence" ], [ 122, 135, "fully rotated", "protein_state" ], [ 141, 152, "Structure V", "evidence" ], [ 154, 165, "non-rotated", "protein_state" ] ] }, { "sid": 96, "sent": "18S ribosomal RNA is shown and ribosomal proteins are omitted for clarity.", "section": "FIG", "ner": [ [ 0, 17, "18S ribosomal RNA", "chemical" ] ] }, { "sid": 97, "sent": "The superpositions of Structures I-V were performed by structural alignments of the 25S ribosomal RNAs.", "section": "FIG", "ner": [ [ 4, 18, "superpositions", "experimental_method" ], [ 22, 36, "Structures I-V", "evidence" ], [ 55, 76, "structural alignments", "experimental_method" ], [ 84, 102, "25S ribosomal RNAs", "chemical" ] ] }, { "sid": 98, "sent": "(b)\u00a0Bar graph of the angles characterizing the 40S rotational and 40S head swiveling states in Structures I through V. Measurements for the two 80S\u2022IRES (INIT) structures are included for comparison.", "section": "FIG", "ner": [ [ 47, 50, "40S", "complex_assembly" ], [ 66, 69, "40S", "complex_assembly" ], [ 70, 74, "head", "structure_element" ], [ 95, 117, "Structures I through V", "evidence" ], [ 144, 152, "80S\u2022IRES", "complex_assembly" ], [ 154, 158, "INIT", "complex_assembly" ], [ 160, 170, "structures", "evidence" ] ] }, { "sid": 99, "sent": "(c) Comparison of the 40S conformations in Structures I through V shows distinct positions of the head relative to the body of the 40S subunit (head swivel).", "section": "FIG", "ner": [ [ 22, 25, "40S", "complex_assembly" ], [ 43, 65, "Structures I through V", "evidence" ], [ 98, 102, "head", "structure_element" ], [ 119, 123, "body", "structure_element" ], [ 131, 134, "40S", "complex_assembly" ], [ 135, 142, "subunit", "structure_element" ], [ 144, 148, "head", "structure_element" ] ] }, { "sid": 100, "sent": "Conformation of the non-swiveled 40S subunit in the S. cerevisiae 80S ribosome bound with two tRNAs is shown for reference (blue).", "section": "FIG", "ner": [ [ 20, 32, "non-swiveled", "protein_state" ], [ 33, 36, "40S", "complex_assembly" ], [ 37, 44, "subunit", "structure_element" ], [ 52, 65, "S. cerevisiae", "species" ], [ 66, 78, "80S ribosome", "complex_assembly" ], [ 79, 89, "bound with", "protein_state" ], [ 94, 99, "tRNAs", "chemical" ] ] }, { "sid": 101, "sent": "(d) Comparison of conformations of the L1 and P stalks of the large subunit in Structures I through V with those in the 80S\u2022IRES and tRNA-bound 80S structures.", "section": "FIG", "ner": [ [ 39, 41, "L1", "structure_element" ], [ 46, 54, "P stalks", "structure_element" ], [ 62, 75, "large subunit", "structure_element" ], [ 79, 101, "Structures I through V", "evidence" ], [ 120, 128, "80S\u2022IRES", "complex_assembly" ], [ 133, 143, "tRNA-bound", "protein_state" ], [ 144, 147, "80S", "complex_assembly" ], [ 148, 158, "structures", "evidence" ] ] }, { "sid": 102, "sent": "Superpositions were performed by structural alignments of 25S ribosomal RNAs.", "section": "FIG", "ner": [ [ 0, 14, "Superpositions", "experimental_method" ], [ 33, 54, "structural alignments", "experimental_method" ], [ 58, 76, "25S ribosomal RNAs", "chemical" ] ] }, { "sid": 103, "sent": "The central protuberance (CP) is labeled.", "section": "FIG", "ner": [ [ 4, 24, "central protuberance", "structure_element" ], [ 26, 28, "CP", "structure_element" ] ] }, { "sid": 104, "sent": "\u00a0(e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g)\u00a0Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow.", "section": "FIG", "ner": [ [ 35, 38, "PKI", "structure_element" ], [ 50, 52, "IV", "structure_element" ], [ 56, 60, "eEF2", "protein" ], [ 77, 83, "P site", "site" ], [ 100, 104, "head", "structure_element" ], [ 106, 111, "U1191", "residue_name_number" ], [ 117, 121, "body", "structure_element" ], [ 123, 128, "C1637", "residue_name_number" ], [ 133, 155, "Structures I through V", "evidence" ], [ 206, 219, "A and P sites", "site" ], [ 229, 239, "initiation", "protein_state" ], [ 247, 251, "INIT", "complex_assembly" ], [ 273, 291, "post-translocation", "protein_state" ], [ 292, 303, "Structure V", "evidence" ], [ 398, 401, "40S", "complex_assembly" ], [ 402, 406, "body", "structure_element" ], [ 428, 431, "40S", "complex_assembly" ], [ 432, 436, "head", "structure_element" ] ] }, { "sid": 105, "sent": "The superpositions of structures were performed by structural alignments of the 18S ribosomal RNAs excluding the head region (nt 1150\u20131620).", "section": "FIG", "ner": [ [ 4, 18, "superpositions", "experimental_method" ], [ 22, 32, "structures", "evidence" ], [ 51, 72, "structural alignments", "experimental_method" ], [ 80, 98, "18S ribosomal RNAs", "chemical" ], [ 113, 117, "head", "structure_element" ], [ 129, 138, "1150\u20131620", "residue_range" ] ] }, { "sid": 106, "sent": "Our structures represent hitherto uncharacterized translocation complexes of the TSV IRES captured within globally distinct 80S conformations (Figures 1b and 2).", "section": "RESULTS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 81, 84, "TSV", "species" ], [ 85, 89, "IRES", "site" ], [ 124, 127, "80S", "complex_assembly" ] ] }, { "sid": 107, "sent": "We numbered the structures from I to V, according to the position of the tRNA-mRNA-like PKI on the 40S subunit (Figure 2\u2014source data 1).", "section": "RESULTS", "ner": [ [ 16, 38, "structures from I to V", "evidence" ], [ 73, 82, "tRNA-mRNA", "complex_assembly" ], [ 88, 91, "PKI", "structure_element" ], [ 99, 102, "40S", "complex_assembly" ], [ 103, 110, "subunit", "structure_element" ] ] }, { "sid": 108, "sent": "Specifically, PKI is partially withdrawn from the A site in Structure I, and fully translocated to the P site in Structure V (Figure 4; see also Figure 3\u2014figure supplement 1).", "section": "RESULTS", "ner": [ [ 14, 17, "PKI", "structure_element" ], [ 50, 56, "A site", "site" ], [ 60, 71, "Structure I", "evidence" ], [ 77, 95, "fully translocated", "protein_state" ], [ 103, 109, "P site", "site" ], [ 113, 124, "Structure V", "evidence" ] ] }, { "sid": 109, "sent": "Thus Structures I to IV represent different positions of PKI between the A and P sites (Figure 2\u2014source data 1), suggesting that these structures describe intermediate states of translocation.", "section": "RESULTS", "ner": [ [ 5, 23, "Structures I to IV", "evidence" ], [ 57, 60, "PKI", "structure_element" ], [ 73, 86, "A and P sites", "site" ], [ 135, 145, "structures", "evidence" ] ] }, { "sid": 110, "sent": "Structure V corresponds to the post-translocation state.", "section": "RESULTS", "ner": [ [ 0, 11, "Structure V", "evidence" ], [ 31, 49, "post-translocation", "protein_state" ] ] }, { "sid": 111, "sent": "Changes in ribosome conformation and eEF2 positions are coupled with IRES movement through the ribosome", "section": "RESULTS", "ner": [ [ 11, 19, "ribosome", "complex_assembly" ], [ 37, 41, "eEF2", "protein" ], [ 69, 73, "IRES", "site" ], [ 95, 103, "ribosome", "complex_assembly" ] ] }, { "sid": 112, "sent": "Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S\u20222tRNA\u2022mRNA), in which both the subunit rotation and the head-body swivel are 0\u00b0, we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1\u2014figure supplement 1 and Figure 2\u2014source data 1).", "section": "RESULTS", "ner": [ [ 10, 28, "post-translocation", "protein_state" ], [ 29, 42, "S. cerevisiae", "species" ], [ 43, 55, "80S ribosome", "complex_assembly" ], [ 56, 66, "bound with", "protein_state" ], [ 71, 83, "P and E site", "site" ], [ 84, 89, "tRNAs", "chemical" ], [ 106, 120, "80S\u20222tRNA\u2022mRNA", "complex_assembly" ], [ 141, 148, "subunit", "structure_element" ], [ 166, 170, "head", "structure_element" ], [ 171, 175, "body", "structure_element" ], [ 209, 217, "ribosome", "complex_assembly" ], [ 265, 287, "Structures I through V", "evidence" ] ] }, { "sid": 113, "sent": "Structure I comprises the most rotated ribosome conformation (~10\u00b0), characteristic of pre-translocation hybrid-tRNA states.", "section": "RESULTS", "ner": [ [ 0, 11, "Structure I", "evidence" ], [ 26, 38, "most rotated", "protein_state" ], [ 39, 47, "ribosome", "complex_assembly" ], [ 87, 104, "pre-translocation", "protein_state" ], [ 105, 116, "hybrid-tRNA", "protein_state" ] ] }, { "sid": 114, "sent": "From Structure I to V, the body of the small subunit undergoes backward (reverse) rotation (Figure 2b; see also Figure 1\u2014figure supplement 2 and Figure 2\u2014figure supplement 1).", "section": "RESULTS", "ner": [ [ 5, 21, "Structure I to V", "evidence" ], [ 27, 31, "body", "structure_element" ], [ 39, 52, "small subunit", "structure_element" ] ] }, { "sid": 115, "sent": "Structures II and III are in mid-rotation conformations (~5\u00b0).", "section": "RESULTS", "ner": [ [ 0, 21, "Structures II and III", "evidence" ], [ 29, 41, "mid-rotation", "protein_state" ] ] }, { "sid": 116, "sent": "Structure IV adopts a slightly rotated conformation (~1\u00b0).", "section": "RESULTS", "ner": [ [ 0, 12, "Structure IV", "evidence" ], [ 22, 38, "slightly rotated", "protein_state" ] ] }, { "sid": 117, "sent": "Structure V is in a nearly non-rotated conformation (0.5\u00b0), very similar to that of post-translocation ribosome-tRNA complexes.", "section": "RESULTS", "ner": [ [ 0, 11, "Structure V", "evidence" ], [ 27, 38, "non-rotated", "protein_state" ], [ 84, 102, "post-translocation", "protein_state" ], [ 103, 116, "ribosome-tRNA", "complex_assembly" ] ] }, { "sid": 118, "sent": "Thus, intersubunit rotation of ~9\u00b0 from Structure I to V covers a nearly complete range of relative subunit positions, similar to what was reported for tRNA-bound yeast, bacterial and mammalian ribosomes.", "section": "RESULTS", "ner": [ [ 40, 56, "Structure I to V", "evidence" ], [ 100, 107, "subunit", "structure_element" ], [ 152, 162, "tRNA-bound", "protein_state" ], [ 163, 168, "yeast", "taxonomy_domain" ], [ 170, 179, "bacterial", "taxonomy_domain" ], [ 184, 193, "mammalian", "taxonomy_domain" ], [ 194, 203, "ribosomes", "complex_assembly" ] ] }, { "sid": 119, "sent": "40S head swivel", "section": "RESULTS", "ner": [ [ 0, 3, "40S", "complex_assembly" ], [ 4, 8, "head", "structure_element" ] ] }, { "sid": 120, "sent": "The pattern of 40S head swivel between the structures is different from that of intersubunit rotation (Figures 2c and d; see also Figure 2\u2014source data 1).", "section": "RESULTS", "ner": [ [ 15, 18, "40S", "complex_assembly" ], [ 19, 23, "head", "structure_element" ], [ 43, 53, "structures", "evidence" ] ] }, { "sid": 121, "sent": "As with the intersubunit rotation, the small head swivel (~1\u00b0) in the non-rotated Structure V is closest to that in the 80S\u20222tRNA\u2022mRNA post-translocation ribosome.", "section": "RESULTS", "ner": [ [ 45, 49, "head", "structure_element" ], [ 70, 81, "non-rotated", "protein_state" ], [ 82, 93, "Structure V", "evidence" ], [ 120, 134, "80S\u20222tRNA\u2022mRNA", "complex_assembly" ], [ 135, 153, "post-translocation", "protein_state" ], [ 154, 162, "ribosome", "complex_assembly" ] ] }, { "sid": 122, "sent": "However in the pre-translocation intermediates (from Structure I to IV), the beak of the head domain first turns toward the large subunit and then backs off (Figure 2\u2014figure supplement 1).", "section": "RESULTS", "ner": [ [ 15, 32, "pre-translocation", "protein_state" ], [ 53, 70, "Structure I to IV", "evidence" ], [ 89, 93, "head", "structure_element" ], [ 124, 137, "large subunit", "structure_element" ] ] }, { "sid": 123, "sent": "The head samples a mid-swiveled position in Structure I (12\u00b0), then a highly-swiveled position in Structures II and III (17\u00b0) and a less swiveled position in Structure IV (14\u00b0).", "section": "RESULTS", "ner": [ [ 4, 8, "head", "structure_element" ], [ 19, 31, "mid-swiveled", "protein_state" ], [ 44, 55, "Structure I", "evidence" ], [ 70, 85, "highly-swiveled", "protein_state" ], [ 98, 119, "Structures II and III", "evidence" ], [ 132, 145, "less swiveled", "protein_state" ], [ 158, 170, "Structure IV", "evidence" ] ] }, { "sid": 124, "sent": "The maximum head swivel is observed in the mid-rotated complexes II and III, in which PKI transitions from the A to P site, while eEF2 occupies the A site partially.", "section": "RESULTS", "ner": [ [ 12, 16, "head", "structure_element" ], [ 43, 54, "mid-rotated", "protein_state" ], [ 65, 75, "II and III", "evidence" ], [ 86, 89, "PKI", "structure_element" ], [ 111, 122, "A to P site", "site" ], [ 130, 134, "eEF2", "protein" ], [ 148, 154, "A site", "site" ] ] }, { "sid": 125, "sent": "By comparison, the similarly mid-rotated (4\u00b0) 80S\u2022TSV IRES initiation complex, in the absence of eEF2, adopts a mid-swiveled position (~10\u00b0) (Figure 2c).", "section": "RESULTS", "ner": [ [ 29, 40, "mid-rotated", "protein_state" ], [ 46, 58, "80S\u2022TSV IRES", "complex_assembly" ], [ 59, 69, "initiation", "protein_state" ], [ 86, 96, "absence of", "protein_state" ], [ 97, 101, "eEF2", "protein" ], [ 112, 124, "mid-swiveled", "protein_state" ] ] }, { "sid": 126, "sent": "These observations suggest that eEF2 is necessary for inducing or stabilizing the large head swivel of the 40S subunit characteristic for IRES translocation intermediates.", "section": "RESULTS", "ner": [ [ 32, 36, "eEF2", "protein" ], [ 88, 92, "head", "structure_element" ], [ 107, 110, "40S", "complex_assembly" ], [ 111, 118, "subunit", "structure_element" ], [ 138, 142, "IRES", "site" ] ] }, { "sid": 127, "sent": "IRES rearrangements", "section": "RESULTS", "ner": [ [ 0, 4, "IRES", "site" ] ] }, { "sid": 128, "sent": "Comparison of the TSV IRES and eEF2 positions in Structures I through V.", "section": "FIG", "ner": [ [ 18, 21, "TSV", "species" ], [ 22, 26, "IRES", "site" ], [ 31, 35, "eEF2", "protein" ], [ 49, 71, "Structures I through V", "evidence" ] ] }, { "sid": 129, "sent": "(a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown).", "section": "FIG", "ner": [ [ 21, 25, "IRES", "site" ], [ 30, 34, "eEF2", "protein" ], [ 42, 52, "initiation", "protein_state" ], [ 54, 71, "pre-translocation", "protein_state" ], [ 73, 74, "I", "evidence" ], [ 80, 98, "post-translocation", "protein_state" ], [ 100, 101, "V", "evidence" ], [ 127, 131, "body", "structure_element" ], [ 139, 142, "40S", "complex_assembly" ], [ 143, 150, "subunit", "structure_element" ], [ 143, 150, "subunit", "structure_element" ], [ 184, 188, "IRES", "site" ], [ 193, 197, "eEF2", "protein" ], [ 205, 215, "initiation", "protein_state" ], [ 223, 227, "INIT", "complex_assembly" ], [ 270, 284, "II, III and IV", "evidence" ], [ 303, 307, "body", "structure_element" ], [ 315, 318, "40S", "complex_assembly" ], [ 319, 326, "subunit", "structure_element" ], [ 319, 326, "subunit", "structure_element" ] ] }, { "sid": 130, "sent": "\u00a0Superpositions were obtained by structural alignments of the 18S rRNAs excluding the head domains (nt 1150\u20131620).", "section": "FIG", "ner": [ [ 33, 54, "structural alignments", "experimental_method" ], [ 62, 71, "18S rRNAs", "chemical" ], [ 86, 90, "head", "structure_element" ], [ 103, 112, "1150\u20131620", "residue_range" ] ] }, { "sid": 131, "sent": "Positions of the IRES relative to proteins uS7, uS11 and eS25.", "section": "FIG", "ner": [ [ 17, 21, "IRES", "site" ], [ 43, 46, "uS7", "protein" ], [ 48, 52, "uS11", "protein" ], [ 57, 61, "eS25", "protein" ] ] }, { "sid": 132, "sent": "(a) Intra-IRES rearrangements from the 80S*IRES initiation structure (INIT; PDB 3J6Y,) to Structures I through V. For each structure (shown in red), the conformation from a preceding structure is shown in light red for comparison.", "section": "FIG", "ner": [ [ 10, 14, "IRES", "site" ], [ 39, 47, "80S*IRES", "complex_assembly" ], [ 48, 58, "initiation", "protein_state" ], [ 59, 68, "structure", "evidence" ], [ 70, 74, "INIT", "complex_assembly" ], [ 90, 112, "Structures I through V", "evidence" ], [ 123, 132, "structure", "evidence" ], [ 183, 192, "structure", "evidence" ] ] }, { "sid": 133, "sent": "Superpositions were obtained by structural alignments of 18S rRNA.", "section": "FIG", "ner": [ [ 0, 14, "Superpositions", "experimental_method" ], [ 32, 53, "structural alignments", "experimental_method" ], [ 57, 65, "18S rRNA", "chemical" ] ] }, { "sid": 134, "sent": "(b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S\u2022tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5\u2032 domain of the IRES in the initiation state (left panel).", "section": "FIG", "ner": [ [ 21, 25, "IRES", "site" ], [ 30, 34, "eEF2", "protein" ], [ 66, 79, "P- and E-site", "site" ], [ 80, 85, "tRNAs", "chemical" ], [ 93, 101, "80S\u2022tRNA", "complex_assembly" ], [ 132, 136, "IRES", "site" ], [ 158, 162, "uS11", "protein" ], [ 164, 176, "40S platform", "site" ], [ 182, 185, "uS7", "protein" ], [ 190, 194, "eS25", "protein" ], [ 196, 199, "40S", "complex_assembly" ], [ 200, 204, "head", "structure_element" ], [ 231, 240, "5\u2032 domain", "structure_element" ], [ 248, 252, "IRES", "site" ], [ 260, 270, "initiation", "protein_state" ] ] }, { "sid": 135, "sent": "In all panels, superpositions were obtained by structural alignments of the 18S rRNAs.", "section": "FIG", "ner": [ [ 15, 29, "superpositions", "experimental_method" ], [ 47, 68, "structural alignments", "experimental_method" ], [ 76, 85, "18S rRNAs", "chemical" ] ] }, { "sid": 136, "sent": "Ribosomal proteins of the initiation state are shown in gray for comparison.", "section": "FIG", "ner": [ [ 26, 36, "initiation", "protein_state" ] ] }, { "sid": 137, "sent": "Positions of the L1stalk, tRNA and TSV IRES relative to proteins uS7 and eS25, in 80S\u2022tRNA structures and 80S\u2022IRES structures I and V (this work).", "section": "FIG", "ner": [ [ 17, 24, "L1stalk", "structure_element" ], [ 26, 30, "tRNA", "chemical" ], [ 35, 38, "TSV", "species" ], [ 39, 43, "IRES", "site" ], [ 65, 68, "uS7", "protein" ], [ 73, 77, "eS25", "protein" ], [ 82, 90, "80S\u2022tRNA", "complex_assembly" ], [ 91, 101, "structures", "evidence" ], [ 106, 114, "80S\u2022IRES", "complex_assembly" ], [ 115, 133, "structures I and V", "evidence" ] ] }, { "sid": 138, "sent": "The view shows the vicinity of the ribosomal E site.", "section": "FIG", "ner": [ [ 45, 51, "E site", "site" ] ] }, { "sid": 139, "sent": "Loop 1.1 and stem loops 4 and 5 of the IRES are labeled.", "section": "FIG", "ner": [ [ 0, 8, "Loop 1.1", "structure_element" ], [ 13, 31, "stem loops 4 and 5", "structure_element" ], [ 39, 43, "IRES", "site" ] ] }, { "sid": 140, "sent": "Interactions of the stem loops 4 and 5 of the TSV with proteins uS7 and eS25.", "section": "FIG", "ner": [ [ 20, 38, "stem loops 4 and 5", "structure_element" ], [ 46, 49, "TSV", "species" ], [ 64, 67, "uS7", "protein" ], [ 72, 76, "eS25", "protein" ] ] }, { "sid": 141, "sent": "Position and interactions of loop 3 (variable loop region) of the PKI domain in Structure V (this work) resembles those of the anticodon stem loop of the E-site tRNA (blue) in the 80S\u20222tRNA\u2022mRNA complex.", "section": "FIG", "ner": [ [ 29, 35, "loop 3", "structure_element" ], [ 37, 57, "variable loop region", "structure_element" ], [ 66, 69, "PKI", "structure_element" ], [ 80, 91, "Structure V", "evidence" ], [ 127, 146, "anticodon stem loop", "structure_element" ], [ 154, 160, "E-site", "site" ], [ 161, 165, "tRNA", "chemical" ], [ 180, 194, "80S\u20222tRNA\u2022mRNA", "complex_assembly" ] ] }, { "sid": 142, "sent": "Positions of tRNAs and the TSV IRES relative to the A-site finger (blue, nt 1008\u20131043 of 25S rRNA) and the P site of the large subunit, comprising helix 84 of 25S rRNA (nt.", "section": "FIG", "ner": [ [ 13, 18, "tRNAs", "chemical" ], [ 27, 30, "TSV", "species" ], [ 31, 35, "IRES", "site" ], [ 52, 65, "A-site finger", "structure_element" ], [ 76, 85, "1008\u20131043", "residue_range" ], [ 89, 97, "25S rRNA", "chemical" ], [ 107, 113, "P site", "site" ], [ 121, 134, "large subunit", "structure_element" ], [ 147, 155, "helix 84", "structure_element" ], [ 159, 167, "25S rRNA", "chemical" ] ] }, { "sid": 143, "sent": "2668\u20132687) and protein uL5 (collectively labeled as central protuberance, CP, in the upper-row first figure, and individually labeled in the lower-row first figure).", "section": "FIG", "ner": [ [ 0, 9, "2668\u20132687", "residue_range" ], [ 23, 26, "uL5", "protein" ], [ 52, 72, "central protuberance", "structure_element" ], [ 74, 76, "CP", "structure_element" ] ] }, { "sid": 144, "sent": "Structures of translocation complexes of the bacterial 70S ribosome bound with two tRNAs and yeast 80S complexes with tRNAs are shown in the upper row and labeled.", "section": "FIG", "ner": [ [ 0, 10, "Structures", "evidence" ], [ 45, 54, "bacterial", "taxonomy_domain" ], [ 55, 67, "70S ribosome", "complex_assembly" ], [ 68, 78, "bound with", "protein_state" ], [ 83, 88, "tRNAs", "chemical" ], [ 93, 98, "yeast", "taxonomy_domain" ], [ 99, 102, "80S", "complex_assembly" ], [ 103, 117, "complexes with", "protein_state" ], [ 118, 123, "tRNAs", "chemical" ] ] }, { "sid": 145, "sent": "Structures of 80S\u2022IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the lower row and labeled.", "section": "FIG", "ner": [ [ 0, 10, "Structures", "evidence" ], [ 14, 22, "80S\u2022IRES", "complex_assembly" ], [ 40, 50, "absence of", "protein_state" ], [ 51, 55, "eEF2", "protein" ], [ 57, 61, "INIT", "complex_assembly" ], [ 85, 96, "presence of", "protein_state" ], [ 97, 101, "eEF2", "protein" ] ] }, { "sid": 146, "sent": "Interactions of the TSV IRES with uL5 and eL42.", "section": "FIG", "ner": [ [ 20, 23, "TSV", "species" ], [ 24, 28, "IRES", "site" ], [ 34, 37, "uL5", "protein" ], [ 42, 46, "eL42", "protein" ] ] }, { "sid": 147, "sent": "Structures of 80S\u2022IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the upper row and labeled.", "section": "FIG", "ner": [ [ 0, 10, "Structures", "evidence" ], [ 14, 22, "80S\u2022IRES", "complex_assembly" ], [ 40, 50, "absence of", "protein_state" ], [ 51, 55, "eEF2", "protein" ], [ 57, 61, "INIT", "complex_assembly" ], [ 85, 96, "presence of", "protein_state" ], [ 97, 101, "eEF2", "protein" ] ] }, { "sid": 148, "sent": "Structures of the 80S complexes with tRNAs are shown in the lower row in a view similar to that for the 80S\u2022IRES complex.", "section": "FIG", "ner": [ [ 0, 10, "Structures", "evidence" ], [ 18, 21, "80S", "complex_assembly" ], [ 22, 36, "complexes with", "protein_state" ], [ 37, 42, "tRNAs", "chemical" ], [ 104, 112, "80S\u2022IRES", "complex_assembly" ] ] }, { "sid": 149, "sent": "Positions of the IRES relative to eEF2 and elements of the ribosome in Structures I through V.", "section": "FIG", "ner": [ [ 17, 21, "IRES", "site" ], [ 34, 38, "eEF2", "protein" ], [ 59, 67, "ribosome", "complex_assembly" ], [ 71, 93, "Structures I through V", "evidence" ] ] }, { "sid": 150, "sent": "(a) Secondary structure of the TSV IRES.", "section": "FIG", "ner": [ [ 14, 23, "structure", "evidence" ], [ 31, 34, "TSV", "species" ], [ 35, 39, "IRES", "site" ] ] }, { "sid": 151, "sent": "\u00a0The TSV IRES comprises two domains: the 5' domain (blue) and the PKI domain (red).", "section": "FIG", "ner": [ [ 5, 8, "TSV", "species" ], [ 9, 13, "IRES", "site" ], [ 41, 50, "5' domain", "structure_element" ], [ 66, 69, "PKI", "structure_element" ] ] }, { "sid": 152, "sent": "The open reading frame (gray) is immediately following pseudoknot I (PKI).", "section": "FIG", "ner": [ [ 4, 22, "open reading frame", "structure_element" ], [ 55, 67, "pseudoknot I", "structure_element" ], [ 69, 72, "PKI", "structure_element" ] ] }, { "sid": 153, "sent": "(b) Three-dimensional structure of the TSV IRES (Structure II).", "section": "FIG", "ner": [ [ 22, 31, "structure", "evidence" ], [ 39, 42, "TSV", "species" ], [ 43, 47, "IRES", "site" ], [ 49, 61, "Structure II", "evidence" ] ] }, { "sid": 154, "sent": "Pseudoknots and stem loops are labeled and colored as in (a).", "section": "FIG", "ner": [ [ 16, 26, "stem loops", "structure_element" ] ] }, { "sid": 155, "sent": "(c) Positions of the IRES and eEF2 on the small subunit in Structures I to V. The initiation-state IRES is shown in gray.", "section": "FIG", "ner": [ [ 21, 25, "IRES", "site" ], [ 30, 34, "eEF2", "protein" ], [ 42, 55, "small subunit", "structure_element" ], [ 59, 76, "Structures I to V", "evidence" ], [ 82, 92, "initiation", "protein_state" ], [ 99, 103, "IRES", "site" ] ] }, { "sid": 156, "sent": "\u00a0The insert shows density for interaction of diphthamide 699 (eEF2; green) with the codon-anticodon-like helix (PKI; red) in Structure V. (d and e) Density of the P site in Structure V shows that interactions of PKI with the 18S rRNA nucleotides (c) are nearly identical to those in the P site of the 2tRNA\u2022mRNA-bound 70S ribosome (d).", "section": "FIG", "ner": [ [ 61, 65, "eEF2", "protein" ], [ 111, 114, "PKI", "structure_element" ] ] }, { "sid": 157, "sent": "In each structure, the TSV IRES adopts a distinct conformation in the intersubunit space of the ribosome (Figures 3 and 4).", "section": "RESULTS", "ner": [ [ 8, 17, "structure", "evidence" ], [ 23, 26, "TSV", "species" ], [ 27, 31, "IRES", "site" ], [ 96, 104, "ribosome", "complex_assembly" ] ] }, { "sid": 158, "sent": "The IRES (nt 6758\u20136952) consists of two globular parts (Figure 3a): the 5\u2019-region (domains I and II, nt 6758\u20136888) and the PKI domain (domain III, nt 6889\u20136952).", "section": "RESULTS", "ner": [ [ 4, 8, "IRES", "site" ], [ 13, 22, "6758\u20136952", "residue_range" ], [ 72, 81, "5\u2019-region", "structure_element" ], [ 91, 92, "I", "structure_element" ], [ 97, 99, "II", "structure_element" ], [ 104, 113, "6758\u20136888", "residue_range" ], [ 123, 126, "PKI", "structure_element" ], [ 142, 145, "III", "structure_element" ], [ 150, 159, "6889\u20136952", "residue_range" ] ] }, { "sid": 159, "sent": "We collectively term domains I and II the 5\u2019 domain.", "section": "RESULTS", "ner": [ [ 29, 30, "I", "structure_element" ], [ 35, 37, "II", "structure_element" ], [ 42, 51, "5\u2019 domain", "structure_element" ] ] }, { "sid": 160, "sent": "The PKI domain comprises PKI and stem loop 3 (SL3), which stacks on top of the stem of PKI.", "section": "RESULTS", "ner": [ [ 4, 7, "PKI", "structure_element" ], [ 25, 28, "PKI", "structure_element" ], [ 33, 44, "stem loop 3", "structure_element" ], [ 46, 49, "SL3", "structure_element" ], [ 87, 90, "PKI", "structure_element" ] ] }, { "sid": 161, "sent": "The 6953GCU triplet immediately following the PKI domain is the first codon of the open reading frame.", "section": "RESULTS", "ner": [ [ 46, 49, "PKI", "structure_element" ], [ 83, 101, "open reading frame", "structure_element" ] ] }, { "sid": 162, "sent": "In the eEF2-free 80S\u2022IRES initiation complex (INIT), the bulk of the 5\u2019-domain (nt.", "section": "RESULTS", "ner": [ [ 7, 16, "eEF2-free", "protein_state" ], [ 17, 25, "80S\u2022IRES", "complex_assembly" ], [ 26, 36, "initiation", "protein_state" ], [ 46, 50, "INIT", "complex_assembly" ], [ 69, 78, "5\u2019-domain", "structure_element" ] ] }, { "sid": 163, "sent": "6758\u20136888) binds near the E site, contacting the ribosome mostly by means of three protruding structural elements: the L1.1 region and stem loops 4 and 5 (SL4 and SL5).", "section": "RESULTS", "ner": [ [ 0, 9, "6758\u20136888", "residue_range" ], [ 26, 32, "E site", "site" ], [ 49, 57, "ribosome", "complex_assembly" ], [ 119, 130, "L1.1 region", "structure_element" ], [ 135, 153, "stem loops 4 and 5", "structure_element" ], [ 155, 158, "SL4", "structure_element" ], [ 163, 166, "SL5", "structure_element" ] ] }, { "sid": 164, "sent": "In Structures I to IV, these contacts remain as in the initiation complex (Figure 1a).", "section": "RESULTS", "ner": [ [ 3, 21, "Structures I to IV", "evidence" ], [ 55, 73, "initiation complex", "complex_assembly" ] ] }, { "sid": 165, "sent": "Specifically, the L1.1 region interacts with the L1 stalk of the large subunit, while SL4 and SL5 bind at the side of the 40S head and interact with proteins uS7, uS11 and eS25 (Figure 3\u2014figure supplement 2 and Figure 3\u2014figure supplement 3; ribosomal proteins are termed according to).", "section": "RESULTS", "ner": [ [ 18, 29, "L1.1 region", "structure_element" ], [ 49, 57, "L1 stalk", "structure_element" ], [ 65, 78, "large subunit", "structure_element" ], [ 86, 89, "SL4", "structure_element" ], [ 94, 97, "SL5", "structure_element" ], [ 122, 125, "40S", "complex_assembly" ], [ 126, 130, "head", "structure_element" ], [ 158, 161, "uS7", "protein" ], [ 163, 167, "uS11", "protein" ], [ 172, 176, "eS25", "protein" ] ] }, { "sid": 166, "sent": "In Structures I-IV, the minor groove of SL4 (at nt 6840\u20136846) binds next to an \u03b1-helix of uS7, which is rich in positively charged residues (K212, K213, R219 and K222).", "section": "RESULTS", "ner": [ [ 3, 18, "Structures I-IV", "evidence" ], [ 24, 36, "minor groove", "site" ], [ 40, 43, "SL4", "structure_element" ], [ 51, 60, "6840\u20136846", "residue_range" ], [ 79, 86, "\u03b1-helix", "structure_element" ], [ 90, 93, "uS7", "protein" ], [ 141, 145, "K212", "residue_name_number" ], [ 147, 151, "K213", "residue_name_number" ], [ 153, 157, "R219", "residue_name_number" ], [ 162, 166, "K222", "residue_name_number" ] ] }, { "sid": 167, "sent": "The tip of SL4 binds in the vicinity of R157 in the \u03b2-hairpin of uS7 and of Y58 in uS11.", "section": "RESULTS", "ner": [ [ 11, 14, "SL4", "structure_element" ], [ 40, 44, "R157", "residue_name_number" ], [ 52, 61, "\u03b2-hairpin", "structure_element" ], [ 65, 68, "uS7", "protein" ], [ 76, 79, "Y58", "residue_name_number" ], [ 83, 87, "uS11", "protein" ] ] }, { "sid": 168, "sent": "The minor groove of SL5 (at nt 6862\u20136868) contacts the positively charged region of eS25 (R49, R58 and R68) (Figure 3\u2014figure supplement 4).", "section": "RESULTS", "ner": [ [ 4, 16, "minor groove", "site" ], [ 20, 23, "SL5", "structure_element" ], [ 31, 40, "6862\u20136868", "residue_range" ], [ 84, 88, "eS25", "protein" ], [ 90, 93, "R49", "residue_name_number" ], [ 95, 98, "R58", "residue_name_number" ], [ 103, 106, "R68", "residue_name_number" ] ] }, { "sid": 169, "sent": "In Structure V, however, the density for SL5 is missing suggesting that SL5 is mobile, while weak SL4 density suggests that SL4 is shifted along the surface of uS7, ~20\u2009\u00c5 away from its initial position (Figure 3\u2014figure supplement 2c).", "section": "RESULTS", "ner": [ [ 3, 14, "Structure V", "evidence" ], [ 29, 36, "density", "evidence" ], [ 41, 44, "SL5", "structure_element" ], [ 72, 75, "SL5", "structure_element" ], [ 79, 85, "mobile", "protein_state" ], [ 98, 101, "SL4", "structure_element" ], [ 102, 109, "density", "evidence" ], [ 124, 127, "SL4", "structure_element" ], [ 160, 163, "uS7", "protein" ] ] }, { "sid": 170, "sent": "The L1.1 region remains in contact with the L1 stalk (Figure 3\u2014figure supplement 3).", "section": "RESULTS", "ner": [ [ 4, 15, "L1.1 region", "structure_element" ], [ 44, 52, "L1 stalk", "structure_element" ] ] }, { "sid": 171, "sent": "Inchworm-like translocation of the TSV IRES.", "section": "FIG", "ner": [ [ 0, 8, "Inchworm", "protein_state" ], [ 35, 38, "TSV", "species" ], [ 39, 43, "IRES", "site" ] ] }, { "sid": 172, "sent": "Conformations and positions of the IRES in the initiation state and in Structures I-V are shown relative to those of the A-, P- and E-site tRNAs.", "section": "FIG", "ner": [ [ 35, 39, "IRES", "site" ], [ 47, 57, "initiation", "protein_state" ], [ 71, 85, "Structures I-V", "evidence" ], [ 121, 138, "A-, P- and E-site", "site" ], [ 139, 144, "tRNAs", "chemical" ] ] }, { "sid": 173, "sent": "The view was obtained by structural alignment of the body domains of 18S rRNAs of the corresponding 80S structures.", "section": "FIG", "ner": [ [ 25, 45, "structural alignment", "experimental_method" ], [ 53, 57, "body", "structure_element" ], [ 69, 78, "18S rRNAs", "chemical" ], [ 100, 103, "80S", "complex_assembly" ], [ 104, 114, "structures", "evidence" ] ] }, { "sid": 174, "sent": "Distances between nucleotides 6848 and 6913 in SL4 and PKI, respectively, are shown (see also Figure 2\u2014source data 1).", "section": "FIG", "ner": [ [ 30, 34, "6848", "residue_number" ], [ 39, 43, "6913", "residue_number" ], [ 47, 50, "SL4", "structure_element" ], [ 55, 58, "PKI", "structure_element" ] ] }, { "sid": 175, "sent": "The shape of the IRES changes considerably from the initiation state to Structures I through V, from an extended to compact to extended conformation (Figure 4; see also Figure 3\u2014figure supplement 2a).", "section": "RESULTS", "ner": [ [ 17, 21, "IRES", "site" ], [ 52, 62, "initiation", "protein_state" ], [ 72, 94, "Structures I through V", "evidence" ], [ 104, 112, "extended", "protein_state" ], [ 116, 123, "compact", "protein_state" ], [ 127, 135, "extended", "protein_state" ] ] }, { "sid": 176, "sent": "Because in Structures I to IV the PKI domain shifts toward the P site, while the 5\u2019 remains unchanged near the E site, the distance between the domains shortens (Figure 4).", "section": "RESULTS", "ner": [ [ 11, 29, "Structures I to IV", "evidence" ], [ 34, 37, "PKI", "structure_element" ], [ 63, 69, "P site", "site" ], [ 111, 117, "E site", "site" ] ] }, { "sid": 177, "sent": "In the 80S\u2022IRES initiation state, the A-site-bound PKI is separated from SL4 by almost 50\u2009\u00c5 (Figure 4).", "section": "RESULTS", "ner": [ [ 7, 15, "80S\u2022IRES", "complex_assembly" ], [ 16, 26, "initiation", "protein_state" ], [ 38, 50, "A-site-bound", "protein_state" ], [ 51, 54, "PKI", "structure_element" ], [ 73, 76, "SL4", "structure_element" ] ] }, { "sid": 178, "sent": "In Structures I and II, the PKI is partially retracted from the A site and the distance from SL4 shortens to ~35\u2009\u00c5. As PKI moves toward the P site in Structures III and IV, the PKI domain approaches to within ~25\u2009\u00c5 of SL4.", "section": "RESULTS", "ner": [ [ 3, 22, "Structures I and II", "evidence" ], [ 28, 31, "PKI", "structure_element" ], [ 64, 70, "A site", "site" ], [ 93, 96, "SL4", "structure_element" ], [ 119, 122, "PKI", "structure_element" ], [ 140, 146, "P site", "site" ], [ 150, 171, "Structures III and IV", "evidence" ], [ 177, 180, "PKI", "structure_element" ], [ 218, 221, "SL4", "structure_element" ] ] }, { "sid": 179, "sent": "Because the 5\u2019-domain in the following structure (V) moves by ~20\u2009\u00c5 along the 40S head, the IRES returns to an extended conformation (~45\u2009\u00c5) that is similar to that in the 80S\u2022IRES initiation complex.", "section": "RESULTS", "ner": [ [ 12, 21, "5\u2019-domain", "structure_element" ], [ 39, 52, "structure (V)", "evidence" ], [ 78, 81, "40S", "complex_assembly" ], [ 82, 86, "head", "structure_element" ], [ 92, 96, "IRES", "site" ], [ 111, 119, "extended", "protein_state" ], [ 172, 180, "80S\u2022IRES", "complex_assembly" ], [ 181, 191, "initiation", "protein_state" ] ] }, { "sid": 180, "sent": "Rearrangements of the IRES involve restructuring of several interactions with the ribosome.", "section": "RESULTS", "ner": [ [ 22, 26, "IRES", "site" ], [ 82, 90, "ribosome", "complex_assembly" ] ] }, { "sid": 181, "sent": "In Structure I, SL3 of the PKI domain is positioned between the A-site finger (nt 1008\u20131043 of 25S rRNA) and the P site of the 60S subunit, comprising helix 84 of 25S rRNA (nt.", "section": "RESULTS", "ner": [ [ 3, 14, "Structure I", "evidence" ], [ 16, 19, "SL3", "structure_element" ], [ 27, 30, "PKI", "structure_element" ], [ 64, 77, "A-site finger", "structure_element" ], [ 82, 91, "1008\u20131043", "residue_range" ], [ 95, 103, "25S rRNA", "chemical" ], [ 113, 119, "P site", "site" ], [ 127, 130, "60S", "complex_assembly" ], [ 131, 138, "subunit", "structure_element" ], [ 151, 159, "helix 84", "structure_element" ], [ 163, 171, "25S rRNA", "chemical" ] ] }, { "sid": 182, "sent": "2668\u20132687) and protein uL5 (Figure 3\u2014figure supplement 6).", "section": "RESULTS", "ner": [ [ 0, 9, "2668\u20132687", "residue_range" ], [ 23, 26, "uL5", "protein" ] ] }, { "sid": 183, "sent": "This position of SL3 is ~25\u2009\u00c5 away from that in the 80S\u2022IRES initiation state, in which PKI and SL3 closely mimic the ASL and elbow of the A-site tRNA, respectively.", "section": "RESULTS", "ner": [ [ 17, 20, "SL3", "structure_element" ], [ 52, 60, "80S\u2022IRES", "complex_assembly" ], [ 61, 71, "initiation", "protein_state" ], [ 88, 91, "PKI", "structure_element" ], [ 96, 99, "SL3", "structure_element" ], [ 118, 121, "ASL", "structure_element" ], [ 126, 131, "elbow", "structure_element" ], [ 139, 145, "A-site", "site" ], [ 146, 150, "tRNA", "chemical" ] ] }, { "sid": 184, "sent": "As such, the transition from the initiation state to Structure I involves repositioning of SL3 around the A-site finger, resembling the transition between the pre-translocation A/P and A/P* tRNA.", "section": "RESULTS", "ner": [ [ 33, 43, "initiation", "protein_state" ], [ 53, 64, "Structure I", "evidence" ], [ 91, 94, "SL3", "structure_element" ], [ 106, 119, "A-site finger", "structure_element" ], [ 159, 176, "pre-translocation", "protein_state" ], [ 177, 180, "A/P", "site" ], [ 185, 189, "A/P*", "site" ], [ 190, 194, "tRNA", "chemical" ] ] }, { "sid": 185, "sent": "The second set of major structural changes involves interaction of the P site region of the large subunit with the hinge point of the IRES bending between the 5\u00b4 domain and the PKI domain (nt. 6886\u20136890).", "section": "RESULTS", "ner": [ [ 71, 84, "P site region", "site" ], [ 92, 105, "large subunit", "structure_element" ], [ 115, 126, "hinge point", "structure_element" ], [ 134, 138, "IRES", "site" ], [ 159, 168, "5\u00b4 domain", "structure_element" ], [ 177, 180, "PKI", "structure_element" ], [ 193, 202, "6886\u20136890", "residue_range" ] ] }, { "sid": 186, "sent": "In the highly bent Structures III and IV, the hinge region interacts with the universally conserved uL5 and the C-terminal tail of eL42 (Figure 3\u2014figure supplement 7).", "section": "RESULTS", "ner": [ [ 7, 18, "highly bent", "protein_state" ], [ 19, 40, "Structures III and IV", "evidence" ], [ 46, 58, "hinge region", "structure_element" ], [ 78, 99, "universally conserved", "protein_state" ], [ 100, 103, "uL5", "protein" ], [ 112, 127, "C-terminal tail", "structure_element" ], [ 131, 135, "eL42", "protein" ] ] }, { "sid": 187, "sent": "However, in the extended conformations, these parts of the IRES and the 60S subunit are separated by more than 10\u2009\u00c5, suggesting that an interaction between them stabilizes the bent conformations but not the extended ones.", "section": "RESULTS", "ner": [ [ 16, 24, "extended", "protein_state" ], [ 59, 63, "IRES", "site" ], [ 72, 75, "60S", "complex_assembly" ], [ 76, 83, "subunit", "structure_element" ], [ 176, 180, "bent", "protein_state" ], [ 207, 215, "extended", "protein_state" ] ] }, { "sid": 188, "sent": "Another local rearrangement concerns loop 3, also known as the variable loop region\u00a0, which connects the ASL- and mRNA-like parts of PKI.", "section": "RESULTS", "ner": [ [ 37, 43, "loop 3", "structure_element" ], [ 63, 83, "variable loop region", "structure_element" ], [ 105, 129, "ASL- and mRNA-like parts", "structure_element" ], [ 133, 136, "PKI", "structure_element" ] ] }, { "sid": 189, "sent": "This loop is poorly resolved in Structures I through IV, suggesting conformational flexibility in agreement with structural studies of the isolated PKI and biochemical studies of unbound IRESs.", "section": "RESULTS", "ner": [ [ 5, 9, "loop", "structure_element" ], [ 32, 55, "Structures I through IV", "evidence" ], [ 113, 131, "structural studies", "experimental_method" ], [ 139, 147, "isolated", "protein_state" ], [ 148, 151, "PKI", "structure_element" ], [ 156, 175, "biochemical studies", "experimental_method" ], [ 179, 186, "unbound", "protein_state" ], [ 187, 192, "IRESs", "site" ] ] }, { "sid": 190, "sent": "In Structure V, loop 3 is bound in the 40S E site and the backbone of loop 3 near the codon-like part of PKI (at nt.", "section": "RESULTS", "ner": [ [ 3, 14, "Structure V", "evidence" ], [ 16, 22, "loop 3", "structure_element" ], [ 26, 34, "bound in", "protein_state" ], [ 39, 42, "40S", "complex_assembly" ], [ 43, 49, "E site", "site" ], [ 70, 76, "loop 3", "structure_element" ], [ 86, 101, "codon-like part", "structure_element" ], [ 105, 108, "PKI", "structure_element" ] ] }, { "sid": 191, "sent": "6945\u20136946) interacts with R148 and R157 in \u03b2-hairpin of uS7.", "section": "RESULTS", "ner": [ [ 0, 9, "6945\u20136946", "residue_range" ], [ 26, 30, "R148", "residue_name_number" ], [ 35, 39, "R157", "residue_name_number" ], [ 43, 52, "\u03b2-hairpin", "structure_element" ], [ 56, 59, "uS7", "protein" ] ] }, { "sid": 192, "sent": "The interaction of loop 3 backbone with uS7 resembles that of the anticodon-stem loop of E-site tRNA in the post-translocation 80S\u20222tRNA\u2022mRNA structure (Figure 3\u2014figure supplement 5).", "section": "RESULTS", "ner": [ [ 19, 25, "loop 3", "structure_element" ], [ 40, 43, "uS7", "protein" ], [ 66, 85, "anticodon-stem loop", "structure_element" ], [ 89, 95, "E-site", "site" ], [ 96, 100, "tRNA", "chemical" ], [ 108, 126, "post-translocation", "protein_state" ], [ 127, 141, "80S\u20222tRNA\u2022mRNA", "complex_assembly" ], [ 142, 151, "structure", "evidence" ] ] }, { "sid": 193, "sent": "Ordering of loop 3 suggests that this flexible region contributes to the stabilization of the PKI domain in the post-translocation state.", "section": "RESULTS", "ner": [ [ 12, 18, "loop 3", "structure_element" ], [ 94, 97, "PKI", "structure_element" ], [ 112, 130, "post-translocation", "protein_state" ] ] }, { "sid": 194, "sent": "This interpretation is consistent with the recent observation that alterations in loop 3 of the CrPV IRES result in decreased efficiency of translocation.", "section": "RESULTS", "ner": [ [ 82, 88, "loop 3", "structure_element" ], [ 96, 100, "CrPV", "species" ], [ 101, 105, "IRES", "site" ] ] }, { "sid": 195, "sent": "eEF2 structures", "section": "RESULTS", "ner": [ [ 0, 4, "eEF2", "protein" ], [ 5, 15, "structures", "evidence" ] ] }, { "sid": 196, "sent": "Elements of the 80S ribosome that contact eEF2 in Structures I through V.", "section": "FIG", "ner": [ [ 16, 28, "80S ribosome", "complex_assembly" ], [ 42, 46, "eEF2", "protein" ], [ 50, 72, "Structures I through V", "evidence" ] ] }, { "sid": 197, "sent": "The view and colors are as in Figure 5b: eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal.", "section": "FIG", "ner": [ [ 41, 45, "eEF2", "protein" ], [ 65, 69, "IRES", "site" ], [ 70, 73, "RNA", "chemical" ], [ 82, 85, "40S", "complex_assembly" ], [ 86, 93, "subunit", "structure_element" ], [ 86, 93, "subunit", "structure_element" ], [ 114, 117, "60S", "complex_assembly" ] ] }, { "sid": 198, "sent": "Cryo-EM density of the GTPase region in Structures I and II.", "section": "FIG", "ner": [ [ 0, 7, "Cryo-EM", "experimental_method" ], [ 8, 15, "density", "evidence" ], [ 23, 36, "GTPase region", "structure_element" ], [ 40, 59, "Structures I and II", "evidence" ] ] }, { "sid": 199, "sent": "The switch loop I in Structure I is shown in blue.", "section": "FIG", "ner": [ [ 4, 17, "switch loop I", "structure_element" ], [ 21, 32, "Structure I", "evidence" ] ] }, { "sid": 200, "sent": "The putative position of the switch loop I, unresolved in the density of Structure II, is shown with a dashed line.", "section": "FIG", "ner": [ [ 29, 42, "switch loop I", "structure_element" ], [ 62, 69, "density", "evidence" ], [ 73, 85, "Structure II", "evidence" ] ] }, { "sid": 201, "sent": "Colors for the ribosome and eEF2 are as in Figure 1.", "section": "FIG", "ner": [ [ 15, 23, "ribosome", "complex_assembly" ], [ 28, 32, "eEF2", "protein" ] ] }, { "sid": 202, "sent": "Conformations and interactions of eEF2.", "section": "FIG", "ner": [ [ 34, 38, "eEF2", "protein" ] ] }, { "sid": 203, "sent": "(a) Conformations of eEF2 in Structures I-V and domain organization of eEF2 are shown.", "section": "FIG", "ner": [ [ 21, 25, "eEF2", "protein" ], [ 29, 43, "Structures I-V", "evidence" ], [ 71, 75, "eEF2", "protein" ] ] }, { "sid": 204, "sent": "Roman numerals denote eEF2 domains.", "section": "FIG", "ner": [ [ 22, 26, "eEF2", "protein" ] ] }, { "sid": 205, "sent": "Superposition was obtained by structural alignment of domains I and II.", "section": "FIG", "ner": [ [ 0, 13, "Superposition", "experimental_method" ], [ 30, 50, "structural alignment", "experimental_method" ], [ 62, 63, "I", "structure_element" ], [ 68, 70, "II", "structure_element" ] ] }, { "sid": 206, "sent": "(b) Elements of the 80S ribosome in Structures I and V that contact eEF2.", "section": "FIG", "ner": [ [ 20, 32, "80S ribosome", "complex_assembly" ], [ 36, 54, "Structures I and V", "evidence" ], [ 68, 72, "eEF2", "protein" ] ] }, { "sid": 207, "sent": "eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal.", "section": "FIG", "ner": [ [ 0, 4, "eEF2", "protein" ], [ 24, 28, "IRES", "site" ], [ 29, 32, "RNA", "chemical" ], [ 41, 44, "40S", "complex_assembly" ], [ 45, 52, "subunit", "structure_element" ], [ 73, 76, "60S", "complex_assembly" ] ] }, { "sid": 208, "sent": "(c) Comparison of conformations of eEF2\u2022sordarin in Structure I (light green) with those of free apo-eEF2 (magenta) and eEF2\u2022sordarin (teal).", "section": "FIG", "ner": [ [ 35, 48, "eEF2\u2022sordarin", "complex_assembly" ], [ 52, 63, "Structure I", "evidence" ], [ 92, 96, "free", "protein_state" ], [ 97, 100, "apo", "protein_state" ], [ 101, 105, "eEF2", "protein" ], [ 120, 133, "eEF2\u2022sordarin", "complex_assembly" ] ] }, { "sid": 209, "sent": "(d) Interactions of the GTPase domains with the 40S and 60S subunits in Structure I (colored in green/blue, eEF2; orange, 40S; cyan/teal, 60S) and in Structure II (gray).", "section": "FIG", "ner": [ [ 24, 38, "GTPase domains", "structure_element" ], [ 48, 51, "40S", "complex_assembly" ], [ 56, 59, "60S", "complex_assembly" ], [ 60, 68, "subunits", "structure_element" ], [ 72, 83, "Structure I", "evidence" ], [ 108, 112, "eEF2", "protein" ], [ 122, 125, "40S", "complex_assembly" ], [ 138, 141, "60S", "complex_assembly" ], [ 150, 162, "Structure II", "evidence" ] ] }, { "sid": 210, "sent": "Switch loop I (SWI) in Structure I is in blue; dashed line shows the putative location of unresolved switch loop I in Structure II.", "section": "FIG", "ner": [ [ 0, 13, "Switch loop I", "structure_element" ], [ 15, 18, "SWI", "structure_element" ], [ 23, 34, "Structure I", "evidence" ], [ 101, 114, "switch loop I", "structure_element" ], [ 118, 130, "Structure II", "evidence" ] ] }, { "sid": 211, "sent": "Superposition was obtained by structural alignment of the 25S rRNAs.", "section": "FIG", "ner": [ [ 0, 13, "Superposition", "experimental_method" ], [ 30, 50, "structural alignment", "experimental_method" ], [ 58, 67, "25S rRNAs", "chemical" ] ] }, { "sid": 212, "sent": "(e) Comparison of the GTP-like conformation of eEF2\u2022GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu\u2022GDPCP (teal) and EF-G\u2022GDP\u2022fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal).", "section": "FIG", "ner": [ [ 22, 30, "GTP-like", "protein_state" ], [ 47, 55, "eEF2\u2022GDP", "complex_assembly" ], [ 59, 70, "Structure I", "evidence" ], [ 99, 108, "70S-bound", "protein_state" ], [ 109, 127, "elongation factors", "protein_type" ], [ 128, 139, "EF-Tu\u2022GDPCP", "complex_assembly" ], [ 151, 172, "EF-G\u2022GDP\u2022fusidic acid", "complex_assembly" ], [ 212, 219, "Cryo-EM", "experimental_method" ], [ 220, 227, "density", "evidence" ], [ 236, 257, "guanosine diphosphate", "chemical" ], [ 258, 266, "bound in", "protein_state" ], [ 271, 284, "GTPase center", "site" ], [ 305, 322, "sarcin-ricin loop", "structure_element" ], [ 326, 334, "25S rRNA", "chemical" ], [ 345, 357, "Structure II", "evidence" ], [ 381, 403, "sordarin-binding sites", "site" ], [ 411, 425, "ribosome-bound", "protein_state" ], [ 440, 452, "Structure II", "evidence" ], [ 467, 471, "eEF2", "protein" ] ] }, { "sid": 213, "sent": "(h) Cryo-EM density showing the sordarin-binding pocket of eEF2 (Structure II).", "section": "FIG", "ner": [ [ 4, 11, "Cryo-EM", "experimental_method" ], [ 12, 19, "density", "evidence" ], [ 32, 55, "sordarin-binding pocket", "site" ], [ 59, 63, "eEF2", "protein" ], [ 65, 77, "Structure II", "evidence" ] ] }, { "sid": 214, "sent": "Sordarin is shown in pink with oxygen atoms in red.", "section": "FIG", "ner": [ [ 0, 8, "Sordarin", "chemical" ] ] }, { "sid": 215, "sent": "Elongation factor eEF2 in all five structures is bound with GDP and sordarin (Figure 5).", "section": "RESULTS", "ner": [ [ 0, 17, "Elongation factor", "protein_type" ], [ 18, 22, "eEF2", "protein" ], [ 35, 45, "structures", "evidence" ], [ 49, 59, "bound with", "protein_state" ], [ 60, 63, "GDP", "chemical" ], [ 68, 76, "sordarin", "chemical" ] ] }, { "sid": 216, "sent": "The elongation factor consists of three dynamic superdomains: an N-terminal globular superdomain formed by the G (GTPase) domain (domain I) and domain II; a linker domain III; and a C-terminal superdomain comprising domains IV and V (Figure 5a).", "section": "RESULTS", "ner": [ [ 4, 21, "elongation factor", "protein_type" ], [ 48, 60, "superdomains", "structure_element" ], [ 85, 96, "superdomain", "structure_element" ], [ 111, 128, "G (GTPase) domain", "structure_element" ], [ 137, 138, "I", "structure_element" ], [ 151, 153, "II", "structure_element" ], [ 157, 174, "linker domain III", "structure_element" ], [ 193, 204, "superdomain", "structure_element" ], [ 224, 226, "IV", "structure_element" ], [ 231, 232, "V", "structure_element" ] ] }, { "sid": 217, "sent": "Domain IV extends from the main body and is critical for translocation catalyzed by eEF2\u00a0or\u00a0EF-G. ADP-ribosylation of eEF2 at the tip of domain IV or deletion of domain IV from EF-G abrogate translocation.", "section": "RESULTS", "ner": [ [ 7, 9, "IV", "structure_element" ], [ 32, 36, "body", "structure_element" ], [ 84, 88, "eEF2", "protein" ], [ 92, 96, "EF-G", "protein" ], [ 98, 114, "ADP-ribosylation", "ptm" ], [ 118, 122, "eEF2", "protein" ], [ 144, 146, "IV", "structure_element" ], [ 150, 158, "deletion", "experimental_method" ], [ 169, 171, "IV", "structure_element" ], [ 177, 181, "EF-G", "protein" ] ] }, { "sid": 218, "sent": "In post-translocation-like 80S\u2022tRNA\u2022eEF2 complexes, domain IV binds in the 40S A site, suggesting direct involvement of domain IV in translocation of tRNA from the A to P site.", "section": "RESULTS", "ner": [ [ 3, 21, "post-translocation", "protein_state" ], [ 27, 40, "80S\u2022tRNA\u2022eEF2", "complex_assembly" ], [ 59, 61, "IV", "structure_element" ], [ 75, 78, "40S", "complex_assembly" ], [ 79, 85, "A site", "site" ], [ 127, 129, "IV", "structure_element" ], [ 150, 154, "tRNA", "chemical" ], [ 164, 175, "A to P site", "site" ] ] }, { "sid": 219, "sent": "GDP in our structures is bound in the GTPase center (Figures 5d, e and f) and sordarin is sandwiched between the \u03b2-platforms of domains III and V (Figures 5g and h), as in the structure of free eEF2\u2022sordarin complex.", "section": "RESULTS", "ner": [ [ 0, 3, "GDP", "chemical" ], [ 11, 21, "structures", "evidence" ], [ 25, 33, "bound in", "protein_state" ], [ 38, 51, "GTPase center", "site" ], [ 78, 86, "sordarin", "chemical" ], [ 113, 124, "\u03b2-platforms", "structure_element" ], [ 136, 139, "III", "structure_element" ], [ 144, 145, "V", "structure_element" ], [ 176, 185, "structure", "evidence" ], [ 189, 193, "free", "protein_state" ], [ 194, 207, "eEF2\u2022sordarin", "complex_assembly" ] ] }, { "sid": 220, "sent": "The global conformations of eEF2 (Figure 5a) are similar in these structures (all-atom RMSD \u2264\u00a02\u00a0\u00c5), but the positions of eEF2 relative to the 40S subunit differ substantially as a result of 40S subunit rotation (Figure 2\u2014source data 1).", "section": "RESULTS", "ner": [ [ 28, 32, "eEF2", "protein" ], [ 66, 76, "structures", "evidence" ], [ 87, 91, "RMSD", "evidence" ], [ 121, 125, "eEF2", "protein" ], [ 142, 145, "40S", "complex_assembly" ], [ 146, 153, "subunit", "structure_element" ], [ 190, 193, "40S", "complex_assembly" ], [ 194, 201, "subunit", "structure_element" ] ] }, { "sid": 221, "sent": "From Structure I to V, eEF2 is rigidly attached to the GTPase-associated center of the 60S subunit.", "section": "RESULTS", "ner": [ [ 5, 21, "Structure I to V", "evidence" ], [ 23, 27, "eEF2", "protein" ], [ 55, 79, "GTPase-associated center", "site" ], [ 87, 90, "60S", "complex_assembly" ], [ 91, 98, "subunit", "structure_element" ] ] }, { "sid": 222, "sent": "The GTPase-associated center comprises the P stalk (L11 and L7/L12 stalk in bacteria) and the sarcin-ricin loop (SRL, nt 3012\u20133042).", "section": "RESULTS", "ner": [ [ 4, 28, "GTPase-associated center", "site" ], [ 43, 50, "P stalk", "structure_element" ], [ 52, 55, "L11", "structure_element" ], [ 60, 62, "L7", "structure_element" ], [ 63, 66, "L12", "structure_element" ], [ 67, 72, "stalk", "structure_element" ], [ 76, 84, "bacteria", "taxonomy_domain" ], [ 94, 111, "sarcin-ricin loop", "structure_element" ], [ 113, 116, "SRL", "structure_element" ], [ 121, 130, "3012\u20133042", "residue_range" ] ] }, { "sid": 223, "sent": "The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An \u03b1\u03b2\u03b2 motif of the eukaryote-specific protein P0 (aa 126\u2013154) packs in the crevice between the long \u03b1-helix D (aa 172\u2013188) of the GTPase domain and the \u03b2-sheet region (aa 246\u2013263) of the GTPase domain insert (or G\u2019 insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same).", "section": "RESULTS", "ner": [ [ 12, 20, "25S rRNA", "chemical" ], [ 21, 38, "helices 43 and 44", "structure_element" ], [ 46, 53, "P stalk", "structure_element" ], [ 67, 72, "G1242", "residue_name_number" ], [ 77, 82, "A1270", "residue_name_number" ], [ 98, 103, "stack", "bond_interaction" ], [ 107, 111, "V754", "residue_name_number" ], [ 116, 120, "Y744", "residue_name_number" ], [ 131, 132, "V", "structure_element" ], [ 137, 146, "\u03b1\u03b2\u03b2 motif", "structure_element" ], [ 154, 163, "eukaryote", "taxonomy_domain" ], [ 181, 183, "P0", "protein" ], [ 188, 195, "126\u2013154", "residue_range" ], [ 230, 244, "long \u03b1-helix D", "structure_element" ], [ 249, 256, "172\u2013188", "residue_range" ], [ 265, 278, "GTPase domain", "structure_element" ], [ 287, 301, "\u03b2-sheet region", "structure_element" ], [ 306, 313, "246\u2013263", "residue_range" ], [ 322, 342, "GTPase domain insert", "structure_element" ], [ 347, 356, "G\u2019 insert", "structure_element" ], [ 361, 365, "eEF2", "protein" ], [ 405, 409, "eEF2", "protein" ], [ 414, 418, "EF-G", "protein" ] ] }, { "sid": 224, "sent": "Although the P/L11 stalk is known to be dynamic, its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223\u20131286) are within 2.5\u2009\u00c5. However, with respect to its position in the 80S\u2022IRES complex in the absence of eEF2 and in the 80S\u20222tRNA\u2022mRNA complex, the P stalk is shifted by ~13\u2009\u00c5 toward the A site (Figure 2d).", "section": "RESULTS", "ner": [ [ 13, 24, "P/L11 stalk", "structure_element" ], [ 85, 101, "Structure I to V", "evidence" ], [ 112, 140, "root-mean-square differences", "evidence" ], [ 149, 157, "25S rRNA", "chemical" ], [ 165, 172, "P stalk", "structure_element" ], [ 177, 186, "1223\u20131286", "residue_range" ], [ 251, 259, "80S\u2022IRES", "complex_assembly" ], [ 275, 285, "absence of", "protein_state" ], [ 286, 290, "eEF2", "protein" ], [ 302, 316, "80S\u20222tRNA\u2022mRNA", "complex_assembly" ], [ 330, 337, "P stalk", "structure_element" ], [ 369, 375, "A site", "site" ] ] }, { "sid": 225, "sent": "The sarcin-ricin loop interacts with the GTP-binding site of eEF2 (Figures 5d and f).", "section": "RESULTS", "ner": [ [ 4, 21, "sarcin-ricin loop", "structure_element" ], [ 41, 57, "GTP-binding site", "site" ], [ 61, 65, "eEF2", "protein" ] ] }, { "sid": 226, "sent": "While the overall mode of this interaction is similar to that seen in 70S\u2022EF-G crystal structures, there is an important local difference between Structure I and Structures II-V in switch loop I, as discussed below.", "section": "RESULTS", "ner": [ [ 70, 78, "70S\u2022EF-G", "complex_assembly" ], [ 79, 97, "crystal structures", "evidence" ], [ 146, 157, "Structure I", "evidence" ], [ 162, 177, "Structures II-V", "evidence" ], [ 181, 194, "switch loop I", "structure_element" ] ] }, { "sid": 227, "sent": "Repositioning (sliding) of the positively-charged cluster of domain IV of eEF2 over the phosphate backbone (red) of the 18S helices 33 and 34.", "section": "FIG", "ner": [ [ 31, 57, "positively-charged cluster", "site" ], [ 68, 70, "IV", "structure_element" ], [ 74, 78, "eEF2", "protein" ], [ 120, 141, "18S helices 33 and 34", "structure_element" ] ] }, { "sid": 228, "sent": "Structures I through V are shown.", "section": "FIG", "ner": [ [ 0, 22, "Structures I through V", "evidence" ] ] }, { "sid": 229, "sent": "Electrostatic surface of eEF2 is shown; negatively and positively charged regions are shown in red and blue, respectively.", "section": "FIG", "ner": [ [ 25, 29, "eEF2", "protein" ] ] }, { "sid": 230, "sent": "The view was obtained by structural alignment of the 18S rRNAs.", "section": "FIG", "ner": [ [ 25, 45, "structural alignment", "experimental_method" ], [ 53, 62, "18S rRNAs", "chemical" ] ] }, { "sid": 231, "sent": "Interactions of eEF2 with the 40S subunit.", "section": "FIG", "ner": [ [ 16, 20, "eEF2", "protein" ], [ 30, 33, "40S", "complex_assembly" ], [ 34, 41, "subunit", "structure_element" ] ] }, { "sid": 232, "sent": "(a)\u00a0eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b)\u00a0Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown.", "section": "FIG", "ner": [ [ 4, 8, "eEF2", "protein" ], [ 41, 45, "body", "structure_element" ], [ 49, 60, "Structure I", "evidence" ], [ 62, 66, "eEF2", "protein" ], [ 136, 140, "head", "structure_element" ], [ 145, 149, "body", "structure_element" ], [ 153, 176, "Structures II through V", "evidence" ], [ 220, 223, "40S", "complex_assembly" ], [ 257, 261, "eEF2", "protein" ], [ 315, 319, "eEF2", "protein" ], [ 329, 332, "40S", "complex_assembly" ], [ 333, 339, "A site", "site" ], [ 346, 367, "Structure I through V", "evidence" ], [ 386, 392, "A-site", "site" ], [ 406, 410, "eEF2", "protein" ], [ 412, 423, "Structure V", "evidence" ] ] }, { "sid": 233, "sent": "The view was obtained by superpositions of the body domains of 18S rRNAs.", "section": "FIG", "ner": [ [ 25, 39, "superpositions", "experimental_method" ], [ 47, 51, "body", "structure_element" ], [ 63, 72, "18S rRNAs", "chemical" ] ] }, { "sid": 234, "sent": "(c)\u00a0Rearrangements, from Structure I through V, of a positively charged cluster of eEF2 (K613, R617 and R631) positioned over the phosphate backbone of 18S helices 33 and 34, suggesting a role of electrostatic interactions in eEF2 diffusion over the 40S surface.", "section": "FIG", "ner": [ [ 25, 46, "Structure I through V", "evidence" ], [ 83, 87, "eEF2", "protein" ], [ 89, 93, "K613", "residue_name_number" ], [ 95, 99, "R617", "residue_name_number" ], [ 104, 108, "R631", "residue_name_number" ], [ 152, 173, "18S helices 33 and 34", "structure_element" ], [ 196, 222, "electrostatic interactions", "bond_interaction" ], [ 226, 230, "eEF2", "protein" ], [ 250, 253, "40S", "complex_assembly" ] ] }, { "sid": 235, "sent": "(d) Shift of the tip of domain III of eEF2, interacting with uS12 upon reverse subunit rotation from Structure I to Structure V. Structure I colored as in Figure 1, except uS12, which is in purple; Structure V is in gray.", "section": "FIG", "ner": [ [ 31, 34, "III", "structure_element" ], [ 38, 42, "eEF2", "protein" ], [ 61, 65, "uS12", "protein" ], [ 79, 86, "subunit", "structure_element" ], [ 101, 127, "Structure I to Structure V", "evidence" ], [ 129, 140, "Structure I", "evidence" ], [ 172, 176, "uS12", "protein" ], [ 198, 209, "Structure V", "evidence" ] ] }, { "sid": 236, "sent": "There are two modest but noticeable domain rearrangements between Structures I and V. Unlike in free eEF2, which can sample large movements of at least 50\u2009\u00c5 of the C-terminal superdomain relative to the N-terminal superdomain (Figure 5c), eEF2 undergoes moderate repositioning of domain IV (~3\u2009\u00c5; Figure 5a) and domain III (~5\u2009\u00c5; Figure 6d).", "section": "RESULTS", "ner": [ [ 66, 84, "Structures I and V", "evidence" ], [ 96, 100, "free", "protein_state" ], [ 101, 105, "eEF2", "protein" ], [ 175, 186, "superdomain", "structure_element" ], [ 214, 225, "superdomain", "structure_element" ], [ 239, 243, "eEF2", "protein" ], [ 287, 289, "IV", "structure_element" ], [ 319, 322, "III", "structure_element" ] ] }, { "sid": 237, "sent": "This limited flexibility of the ribosome-bound eEF2 is likely the result of simultaneous fixation of eEF2 superdomains, via domains I and V, by the GTPase-associated center of the large subunit.", "section": "RESULTS", "ner": [ [ 32, 46, "ribosome-bound", "protein_state" ], [ 47, 51, "eEF2", "protein" ], [ 101, 105, "eEF2", "protein" ], [ 106, 118, "superdomains", "structure_element" ], [ 132, 133, "I", "structure_element" ], [ 138, 139, "V", "structure_element" ], [ 148, 172, "GTPase-associated center", "site" ], [ 180, 193, "large subunit", "structure_element" ] ] }, { "sid": 238, "sent": "Domain IV of eEF2 binds at the 40S A site in Structures I to V but the mode of interaction differs in each complex (Figure 6).", "section": "RESULTS", "ner": [ [ 7, 9, "IV", "structure_element" ], [ 13, 17, "eEF2", "protein" ], [ 31, 34, "40S", "complex_assembly" ], [ 35, 41, "A site", "site" ], [ 45, 62, "Structures I to V", "evidence" ] ] }, { "sid": 239, "sent": "Because eEF2 is rigidly attached to the 60S subunit and does not undergo large inter-subunit rearrangements, gradual entry of domain IV into the A site between Structures I and V is due to 40S subunit rotation and head swivel.", "section": "RESULTS", "ner": [ [ 8, 12, "eEF2", "protein" ], [ 40, 43, "60S", "complex_assembly" ], [ 44, 51, "subunit", "structure_element" ], [ 85, 92, "subunit", "structure_element" ], [ 133, 135, "IV", "structure_element" ], [ 145, 151, "A site", "site" ], [ 160, 178, "Structures I and V", "evidence" ], [ 189, 192, "40S", "complex_assembly" ], [ 193, 200, "subunit", "structure_element" ], [ 214, 218, "head", "structure_element" ] ] }, { "sid": 240, "sent": "eEF2 settles into the A site from Structure I to V, as the tip of domain IV shifts by ~10\u2009\u00c5 relative to the body and by ~20\u2009\u00c5 relative to the swiveling head.", "section": "RESULTS", "ner": [ [ 0, 4, "eEF2", "protein" ], [ 22, 28, "A site", "site" ], [ 34, 50, "Structure I to V", "evidence" ], [ 73, 75, "IV", "structure_element" ], [ 108, 112, "body", "structure_element" ], [ 152, 156, "head", "structure_element" ] ] }, { "sid": 241, "sent": "Modest intra-eEF2 shifts of domain IV between Structures I to V outline a stochastic trajectory (Figure 5a), consistent with local adjustments of the domain in the A site.", "section": "RESULTS", "ner": [ [ 13, 17, "eEF2", "protein" ], [ 35, 37, "IV", "structure_element" ], [ 46, 63, "Structures I to V", "evidence" ], [ 164, 170, "A site", "site" ] ] }, { "sid": 242, "sent": "At the central region of eEF2, domains II and III contact the 40S body (mainly at nucleotides 48\u201352 and 429\u2013432 of 18S rRNA helix 5 and uS12).", "section": "RESULTS", "ner": [ [ 25, 29, "eEF2", "protein" ], [ 39, 41, "II", "structure_element" ], [ 46, 49, "III", "structure_element" ], [ 62, 65, "40S", "complex_assembly" ], [ 66, 70, "body", "structure_element" ], [ 94, 99, "48\u201352", "residue_range" ], [ 104, 111, "429\u2013432", "residue_range" ], [ 115, 123, "18S rRNA", "chemical" ], [ 124, 131, "helix 5", "structure_element" ], [ 136, 140, "uS12", "protein" ] ] }, { "sid": 243, "sent": "From Structure I to V, these central domains migrate by ~10\u2009\u00c5 along the 40S surface (Figure 6c).", "section": "RESULTS", "ner": [ [ 5, 21, "Structure I to V", "evidence" ], [ 72, 75, "40S", "complex_assembly" ] ] }, { "sid": 244, "sent": "Comparison of eEF2 conformations reveals that in Structure V, domain III is displaced as a result of interaction with uS12, as discussed below.", "section": "RESULTS", "ner": [ [ 14, 18, "eEF2", "protein" ], [ 49, 60, "Structure V", "evidence" ], [ 69, 72, "III", "structure_element" ], [ 118, 122, "uS12", "protein" ] ] }, { "sid": 245, "sent": "In summary, between Structures I and V, a step-wise translocation of PKI by ~15\u2009\u00c5 from the A to P site - within the 40S subunit \u2013 occurs simultaneously with the ~11\u2009\u00c5 side-way entry of domain IV into the A site coupled with ~3 to 5\u2009\u00c5 inter-domain rearrangements in eEF2.", "section": "RESULTS", "ner": [ [ 20, 38, "Structures I and V", "evidence" ], [ 69, 72, "PKI", "structure_element" ], [ 91, 102, "A to P site", "site" ], [ 116, 119, "40S", "complex_assembly" ], [ 120, 127, "subunit", "structure_element" ], [ 192, 194, "IV", "structure_element" ], [ 204, 210, "A site", "site" ], [ 265, 269, "eEF2", "protein" ] ] }, { "sid": 246, "sent": "These shifts occur during the reverse rotation of the 40S body coupled with the forward-then-reverse head swivel.", "section": "RESULTS", "ner": [ [ 54, 57, "40S", "complex_assembly" ], [ 58, 62, "body", "structure_element" ], [ 101, 105, "head", "structure_element" ] ] }, { "sid": 247, "sent": "To elucidate the detailed structural mechanism of IRES translocation and the roles of eEF2 and ribosome rearrangements, we describe in the following sections the interactions of PKI and eEF2 with the ribosomal A and P sites in Structures I through V (Figure 2g; see also Figure 1\u2014figure supplement 1).", "section": "RESULTS", "ner": [ [ 50, 54, "IRES", "site" ], [ 86, 90, "eEF2", "protein" ], [ 95, 103, "ribosome", "complex_assembly" ], [ 178, 181, "PKI", "structure_element" ], [ 186, 190, "eEF2", "protein" ], [ 210, 223, "A and P sites", "site" ], [ 227, 249, "Structures I through V", "evidence" ] ] }, { "sid": 248, "sent": "Structure I represents a pre-translocation IRES and initial entry of eEF2 in a GTP-like state", "section": "RESULTS", "ner": [ [ 0, 11, "Structure I", "evidence" ], [ 25, 42, "pre-translocation", "protein_state" ], [ 43, 47, "IRES", "site" ], [ 69, 73, "eEF2", "protein" ], [ 79, 82, "GTP", "chemical" ] ] }, { "sid": 249, "sent": "In the fully rotated Structure I, PKI is shifted toward the P site by ~3\u2009\u00c5 relative to its position in the initiation complex but maintains interactions with the partially swiveled head.", "section": "RESULTS", "ner": [ [ 7, 20, "fully rotated", "protein_state" ], [ 21, 32, "Structure I", "evidence" ], [ 34, 37, "PKI", "structure_element" ], [ 60, 66, "P site", "site" ], [ 107, 125, "initiation complex", "complex_assembly" ], [ 162, 180, "partially swiveled", "protein_state" ], [ 181, 185, "head", "structure_element" ] ] }, { "sid": 250, "sent": "At the head, C1274 of the 18S rRNA (C1054 in E. coli) base pairs with the first nucleotide of the ORF immediately downstream of PKI.", "section": "RESULTS", "ner": [ [ 7, 11, "head", "structure_element" ], [ 13, 18, "C1274", "residue_name_number" ], [ 26, 34, "18S rRNA", "chemical" ], [ 36, 41, "C1054", "residue_name_number" ], [ 45, 52, "E. coli", "species" ], [ 98, 101, "ORF", "structure_element" ], [ 128, 131, "PKI", "structure_element" ] ] }, { "sid": 251, "sent": "The C1274:G6953 base pair provides a stacking platform for the codon-anticodon\u2013like helix of PKI.", "section": "RESULTS", "ner": [ [ 4, 9, "C1274", "residue_name_number" ], [ 10, 15, "G6953", "residue_name_number" ], [ 37, 54, "stacking platform", "site" ], [ 63, 89, "codon-anticodon\u2013like helix", "structure_element" ], [ 93, 96, "PKI", "structure_element" ] ] }, { "sid": 252, "sent": "We therefore define C1274 as the foundation of the 'head A site'.", "section": "RESULTS", "ner": [ [ 20, 25, "C1274", "residue_name_number" ], [ 52, 56, "head", "structure_element" ], [ 57, 63, "A site", "site" ] ] }, { "sid": 253, "sent": "Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below.", "section": "RESULTS", "ner": [ [ 20, 25, "U1191", "residue_name_number" ], [ 27, 31, "G966", "residue_name_number" ], [ 35, 42, "E. coli", "species" ], [ 48, 53, "C1637", "residue_name_number" ], [ 55, 60, "C1400", "residue_name_number" ], [ 64, 71, "E. coli", "species" ], [ 105, 109, "head", "structure_element" ], [ 110, 116, "P site", "site" ], [ 123, 127, "body", "structure_element" ], [ 128, 134, "P site", "site" ], [ 224, 242, "fully translocated", "protein_state" ], [ 243, 258, "mRNA-tRNA helix", "structure_element" ], [ 262, 272, "tRNA-bound", "protein_state" ], [ 273, 283, "structures", "evidence" ], [ 295, 313, "post-translocation", "protein_state" ], [ 314, 325, "Structure V", "evidence" ] ] }, { "sid": 254, "sent": "Interactions of the residues at the eEF2 tip with the decoding center of the IRES-bound ribosome.", "section": "FIG", "ner": [ [ 36, 40, "eEF2", "protein" ], [ 54, 69, "decoding center", "site" ], [ 77, 87, "IRES-bound", "protein_state" ], [ 88, 96, "ribosome", "complex_assembly" ] ] }, { "sid": 255, "sent": "Key elements of the decoding center of the 'locked' initiation structure, 'unlocked' Structure I, and post-translocation Structure V (this work) are shown.", "section": "FIG", "ner": [ [ 20, 35, "decoding center", "site" ], [ 44, 50, "locked", "protein_state" ], [ 52, 62, "initiation", "protein_state" ], [ 63, 72, "structure", "evidence" ], [ 75, 83, "unlocked", "protein_state" ], [ 85, 96, "Structure I", "evidence" ], [ 102, 120, "post-translocation", "protein_state" ], [ 121, 132, "Structure V", "evidence" ] ] }, { "sid": 256, "sent": "The histidine-diphthamide tip of eEF2 is shown in green.", "section": "FIG", "ner": [ [ 4, 29, "histidine-diphthamide tip", "site" ], [ 33, 37, "eEF2", "protein" ] ] }, { "sid": 257, "sent": "The codon-anticodon-like helix of PKI is shown in red, the downstream first codon of the ORF in magenta.", "section": "FIG", "ner": [ [ 4, 30, "codon-anticodon-like helix", "structure_element" ], [ 34, 37, "PKI", "structure_element" ], [ 89, 92, "ORF", "structure_element" ] ] }, { "sid": 258, "sent": "Nucleotides of the 18S rRNA body are in orange and head in yellow; 25S rRNA nucleotide A2256 is blue.", "section": "FIG", "ner": [ [ 19, 27, "18S rRNA", "chemical" ], [ 28, 32, "body", "structure_element" ], [ 51, 55, "head", "structure_element" ], [ 67, 75, "25S rRNA", "chemical" ], [ 87, 92, "A2256", "residue_name_number" ] ] }, { "sid": 259, "sent": "A and P sites are schematically demarcated by dotted lines.", "section": "FIG", "ner": [ [ 0, 13, "A and P sites", "site" ] ] }, { "sid": 260, "sent": "The interaction of PKI with the 40S body is substantially rearranged relative to that in the initiation state.", "section": "RESULTS", "ner": [ [ 19, 22, "PKI", "structure_element" ], [ 32, 35, "40S", "complex_assembly" ], [ 36, 40, "body", "structure_element" ], [ 93, 103, "initiation", "protein_state" ] ] }, { "sid": 261, "sent": "In the latter, PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 ('body A site'), as in the A-site tRNA bound complexes.", "section": "RESULTS", "ner": [ [ 15, 18, "PKI", "structure_element" ], [ 58, 79, "universally conserved", "protein_state" ], [ 80, 95, "decoding-center", "site" ], [ 108, 112, "G577", "residue_name_number" ], [ 114, 119, "A1755", "residue_name_number" ], [ 124, 129, "A1756", "residue_name_number" ], [ 132, 136, "body", "structure_element" ], [ 137, 143, "A site", "site" ], [ 157, 163, "A-site", "site" ], [ 164, 174, "tRNA bound", "protein_state" ] ] }, { "sid": 262, "sent": "In Structure I, PKI does not contact these nucleotides (Figures 2g and 7).", "section": "RESULTS", "ner": [ [ 3, 14, "Structure I", "evidence" ], [ 16, 19, "PKI", "structure_element" ] ] }, { "sid": 263, "sent": "The position of eEF2 on the 40S subunit of Structure I is markedly distinct from those in Structures II to V. The translocase interacts with the 40S body but does not contact the head (Figures 5b and 6a; Figure 5\u2014figure supplement 1).", "section": "RESULTS", "ner": [ [ 16, 20, "eEF2", "protein" ], [ 28, 31, "40S", "complex_assembly" ], [ 32, 39, "subunit", "structure_element" ], [ 32, 39, "subunit", "structure_element" ], [ 43, 54, "Structure I", "evidence" ], [ 90, 108, "Structures II to V", "evidence" ], [ 114, 125, "translocase", "protein_type" ], [ 145, 148, "40S", "complex_assembly" ], [ 149, 153, "body", "structure_element" ], [ 179, 183, "head", "structure_element" ] ] }, { "sid": 264, "sent": "Domain IV is partially engaged with the body A site.", "section": "RESULTS", "ner": [ [ 7, 9, "IV", "structure_element" ], [ 40, 44, "body", "structure_element" ], [ 45, 51, "A site", "site" ] ] }, { "sid": 265, "sent": "The tip of domain IV is wedged between PKI and decoding-center nucleotides A1755 and A1756, which are bulged out of h44.", "section": "RESULTS", "ner": [ [ 18, 20, "IV", "structure_element" ], [ 39, 42, "PKI", "structure_element" ], [ 47, 62, "decoding-center", "site" ], [ 75, 80, "A1755", "residue_name_number" ], [ 85, 90, "A1756", "residue_name_number" ] ] }, { "sid": 266, "sent": "This tip contains the histidine-diphthamide triad (H583, H694 and Diph699), which interacts with the codon-anticodon-like helix of PKI and A1756 (Figure 7).", "section": "RESULTS", "ner": [ [ 22, 49, "histidine-diphthamide triad", "site" ], [ 51, 55, "H583", "residue_name_number" ], [ 57, 61, "H694", "residue_name_number" ], [ 66, 73, "Diph699", "ptm" ], [ 101, 127, "codon-anticodon-like helix", "structure_element" ], [ 131, 134, "PKI", "structure_element" ], [ 139, 144, "A1756", "residue_name_number" ] ] }, { "sid": 267, "sent": "Histidines 583 and 694 interact with the phosphate backbone of the anticodon-like strand (at G6907 and C6908).", "section": "RESULTS", "ner": [ [ 0, 22, "Histidines 583 and 694", "residue_name_number" ], [ 67, 88, "anticodon-like strand", "structure_element" ], [ 93, 98, "G6907", "residue_name_number" ], [ 103, 108, "C6908", "residue_name_number" ] ] }, { "sid": 268, "sent": "Diphthamide is a unique posttranslational modification conserved in archaeal and eukaryotic EF2 (at residue 699 in S. cerevisiae) and involves addition of a ~7-\u00c5 long 3-carboxyamido-3-(trimethylamino)-propyl moiety to the histidine imidazole ring at CE1.", "section": "RESULTS", "ner": [ [ 0, 11, "Diphthamide", "ptm" ], [ 55, 64, "conserved", "protein_state" ], [ 68, 76, "archaeal", "taxonomy_domain" ], [ 81, 91, "eukaryotic", "taxonomy_domain" ], [ 92, 95, "EF2", "protein" ], [ 108, 111, "699", "residue_number" ], [ 115, 128, "S. cerevisiae", "species" ], [ 222, 231, "histidine", "residue_name" ] ] }, { "sid": 269, "sent": "The trimethylamino end of Diph699 packs over A1756 (Figure 7).", "section": "RESULTS", "ner": [ [ 26, 33, "Diph699", "ptm" ], [ 45, 50, "A1756", "residue_name_number" ] ] }, { "sid": 270, "sent": "The opposite surface of the tail is oriented toward the minor-groove side of the second base pair of the codon-anticodon helix (G6906:C6951).", "section": "RESULTS", "ner": [ [ 56, 68, "minor-groove", "site" ], [ 105, 126, "codon-anticodon helix", "structure_element" ], [ 128, 133, "G6906", "residue_name_number" ], [ 134, 139, "C6951", "residue_name_number" ] ] }, { "sid": 271, "sent": "Thus, in comparison with the initiation state, the histidine-diphthamide tip of eEF2 replaces the codon-anticodon\u2013like helix of PKI.", "section": "RESULTS", "ner": [ [ 29, 39, "initiation", "protein_state" ], [ 51, 76, "histidine-diphthamide tip", "site" ], [ 80, 84, "eEF2", "protein" ], [ 98, 124, "codon-anticodon\u2013like helix", "structure_element" ], [ 128, 131, "PKI", "structure_element" ] ] }, { "sid": 272, "sent": "The splitting of the interaction of A1755-A1756 and PKI is achieved by providing the histidine-diphthamine tip as a binding partner for both A1756 and the minor groove of the codon-anticodon helix (Figure 7).", "section": "RESULTS", "ner": [ [ 36, 41, "A1755", "residue_name_number" ], [ 42, 47, "A1756", "residue_name_number" ], [ 52, 55, "PKI", "structure_element" ], [ 85, 110, "histidine-diphthamine tip", "site" ], [ 141, 146, "A1756", "residue_name_number" ], [ 155, 167, "minor groove", "site" ], [ 175, 196, "codon-anticodon helix", "structure_element" ] ] }, { "sid": 273, "sent": "Unlike in Structures II to V, the conformation of the eEF2 GTPase center in Structure I resembles that of a GTP-bound translocase (Figure 5e).", "section": "RESULTS", "ner": [ [ 10, 28, "Structures II to V", "evidence" ], [ 54, 58, "eEF2", "protein" ], [ 59, 72, "GTPase center", "site" ], [ 76, 87, "Structure I", "evidence" ], [ 108, 117, "GTP-bound", "protein_state" ], [ 118, 129, "translocase", "protein_type" ] ] }, { "sid": 274, "sent": "In translational GTPases, switch loops I and II are involved in the GTPase activity (reviewed in).", "section": "RESULTS", "ner": [ [ 3, 24, "translational GTPases", "protein_type" ], [ 26, 47, "switch loops I and II", "structure_element" ], [ 68, 74, "GTPase", "protein_type" ] ] }, { "sid": 275, "sent": "Switch loop II (aa 105\u2013110), which carries the catalytic H108 (H92 in E. coli EF-G; is well resolved in all five structures.", "section": "RESULTS", "ner": [ [ 0, 14, "Switch loop II", "structure_element" ], [ 19, 26, "105\u2013110", "residue_range" ], [ 47, 56, "catalytic", "protein_state" ], [ 57, 61, "H108", "residue_name_number" ], [ 63, 66, "H92", "residue_name_number" ], [ 70, 77, "E. coli", "species" ], [ 78, 82, "EF-G", "protein" ], [ 113, 123, "structures", "evidence" ] ] }, { "sid": 276, "sent": "The histidine resides next to the backbone of G3028 of the sarcin-ricin loop and near the diphosphate of GDP (Figure 5e).", "section": "RESULTS", "ner": [ [ 4, 13, "histidine", "residue_name" ], [ 46, 51, "G3028", "residue_name_number" ], [ 59, 76, "sarcin-ricin loop", "structure_element" ], [ 105, 108, "GDP", "chemical" ] ] }, { "sid": 277, "sent": "By contrast, switch loop I (aa 50\u201370 in S. cerevisiae eEF2) is resolved only in Structure I (Figure 5\u2014figure supplement 2).", "section": "RESULTS", "ner": [ [ 13, 26, "switch loop I", "structure_element" ], [ 31, 36, "50\u201370", "residue_range" ], [ 40, 53, "S. cerevisiae", "species" ], [ 54, 58, "eEF2", "protein" ], [ 80, 91, "Structure I", "evidence" ] ] }, { "sid": 278, "sent": "The N-terminal part of the loop (aa 50\u201360) is sandwiched between the tip of helix 14 (415CAAA418) of the 18S rRNA of the 40S subunit and helix A (aa 32\u201342) of eEF2 (Figure 5d).", "section": "RESULTS", "ner": [ [ 27, 31, "loop", "structure_element" ], [ 36, 41, "50\u201360", "residue_range" ], [ 76, 84, "helix 14", "structure_element" ], [ 86, 96, "415CAAA418", "structure_element" ], [ 105, 113, "18S rRNA", "chemical" ], [ 121, 124, "40S", "complex_assembly" ], [ 125, 132, "subunit", "structure_element" ], [ 125, 132, "subunit", "structure_element" ], [ 137, 144, "helix A", "structure_element" ], [ 149, 154, "32\u201342", "residue_range" ], [ 159, 163, "eEF2", "protein" ] ] }, { "sid": 279, "sent": "Bulged A416 interacts with the switch loop in the vicinity of D53.", "section": "RESULTS", "ner": [ [ 0, 6, "Bulged", "protein_state" ], [ 7, 11, "A416", "residue_name_number" ], [ 31, 42, "switch loop", "structure_element" ], [ 62, 65, "D53", "residue_name_number" ] ] }, { "sid": 280, "sent": "Next to GDP, the C-terminal part of the switch loop (aa 61\u201367) adopts a helical fold.", "section": "RESULTS", "ner": [ [ 8, 11, "GDP", "chemical" ], [ 40, 51, "switch loop", "structure_element" ], [ 56, 61, "61\u201367", "residue_range" ], [ 72, 84, "helical fold", "protein_state" ] ] }, { "sid": 281, "sent": "As such, the conformations of SWI and the GTPase center in general are similar to those observed in ribosome-bound EF-Tu and EF-G in the presence of GTP analogs.", "section": "RESULTS", "ner": [ [ 30, 33, "SWI", "structure_element" ], [ 42, 55, "GTPase center", "site" ], [ 100, 114, "ribosome-bound", "protein_state" ], [ 115, 120, "EF-Tu", "protein" ], [ 125, 129, "EF-G", "protein" ], [ 137, 148, "presence of", "protein_state" ], [ 149, 152, "GTP", "chemical" ] ] }, { "sid": 282, "sent": "Structure II reveals PKI between the body A and P sites and eEF2 partially advanced into the A site", "section": "RESULTS", "ner": [ [ 0, 12, "Structure II", "evidence" ], [ 21, 24, "PKI", "structure_element" ], [ 37, 41, "body", "structure_element" ], [ 42, 55, "A and P sites", "site" ], [ 60, 64, "eEF2", "protein" ], [ 93, 99, "A site", "site" ] ] }, { "sid": 283, "sent": "In Structure II, relative to Structure I, PKI is further shifted along the 40S body, traversing ~4\u2009\u00c5 toward the P site (Figures 2e, f, and g), while stacking on C1274 at the head A site.", "section": "RESULTS", "ner": [ [ 3, 15, "Structure II", "evidence" ], [ 29, 40, "Structure I", "evidence" ], [ 42, 45, "PKI", "structure_element" ], [ 75, 78, "40S", "complex_assembly" ], [ 79, 83, "body", "structure_element" ], [ 112, 118, "P site", "site" ], [ 149, 157, "stacking", "bond_interaction" ], [ 161, 166, "C1274", "residue_name_number" ], [ 174, 178, "head", "structure_element" ], [ 179, 185, "A site", "site" ] ] }, { "sid": 284, "sent": "Thus, the intermediate position of PKI is possible due to a large swivel of the head relative to the body, which brings the head A site close to the body P site.", "section": "RESULTS", "ner": [ [ 35, 38, "PKI", "structure_element" ], [ 80, 84, "head", "structure_element" ], [ 101, 105, "body", "structure_element" ], [ 124, 128, "head", "structure_element" ], [ 129, 135, "A site", "site" ], [ 149, 153, "body", "structure_element" ], [ 154, 160, "P site", "site" ] ] }, { "sid": 285, "sent": "Domain IV of eEF2 is further entrenched in the A site by ~3\u2009\u00c5 relative to the body and ~8\u2009\u00c5 relative to the head, preserving its interactions with PKI.", "section": "RESULTS", "ner": [ [ 7, 9, "IV", "structure_element" ], [ 13, 17, "eEF2", "protein" ], [ 47, 53, "A site", "site" ], [ 78, 82, "body", "structure_element" ], [ 108, 112, "head", "structure_element" ], [ 147, 150, "PKI", "structure_element" ] ] }, { "sid": 286, "sent": "The decoding center residues A1755 and A1756 are rearranged to pack inside helix 44, making room for eEF2.", "section": "RESULTS", "ner": [ [ 4, 19, "decoding center", "site" ], [ 29, 34, "A1755", "residue_name_number" ], [ 39, 44, "A1756", "residue_name_number" ], [ 75, 83, "helix 44", "structure_element" ], [ 101, 105, "eEF2", "protein" ] ] }, { "sid": 287, "sent": "This conformation of decoding center residues is also observed in the absence of A-site ligands.", "section": "RESULTS", "ner": [ [ 21, 36, "decoding center", "site" ], [ 70, 80, "absence of", "protein_state" ], [ 81, 87, "A-site", "site" ] ] }, { "sid": 288, "sent": "The head interface of domain IV interacts with the 40S head (Figure 6a).", "section": "RESULTS", "ner": [ [ 4, 18, "head interface", "site" ], [ 29, 31, "IV", "structure_element" ], [ 51, 54, "40S", "complex_assembly" ], [ 55, 59, "head", "structure_element" ] ] }, { "sid": 289, "sent": "Here, a positively charged surface of\u00a0eEF2, formed by K613, R617 and R631 contacts the phosphate backbone of helix 33 (Figures 6c; see also Figure 6\u2014figure supplement 1).", "section": "RESULTS", "ner": [ [ 8, 34, "positively charged surface", "site" ], [ 38, 42, "eEF2", "protein" ], [ 54, 58, "K613", "residue_name_number" ], [ 60, 64, "R617", "residue_name_number" ], [ 69, 73, "R631", "residue_name_number" ], [ 109, 117, "helix 33", "structure_element" ] ] }, { "sid": 290, "sent": "Structure III represents a highly bent IRES with PKI captured between the head A and P sites", "section": "RESULTS", "ner": [ [ 0, 13, "Structure III", "evidence" ], [ 27, 38, "highly bent", "protein_state" ], [ 39, 43, "IRES", "site" ], [ 49, 52, "PKI", "structure_element" ], [ 74, 78, "head", "structure_element" ], [ 79, 92, "A and P sites", "site" ] ] }, { "sid": 291, "sent": "Consistent with the similar head swivels in Structure III and Structure II, relative positions of the 40S\u00a0head A site and body P site remain as in Structure II.", "section": "RESULTS", "ner": [ [ 28, 32, "head", "structure_element" ], [ 44, 57, "Structure III", "evidence" ], [ 62, 74, "Structure II", "evidence" ], [ 102, 105, "40S", "complex_assembly" ], [ 106, 110, "head", "structure_element" ], [ 111, 117, "A site", "site" ], [ 122, 126, "body", "structure_element" ], [ 127, 133, "P site", "site" ], [ 147, 159, "Structure II", "evidence" ] ] }, { "sid": 292, "sent": "Among the five structures, the PKI domain is least ordered in Structure III and lacks density for SL3.", "section": "RESULTS", "ner": [ [ 15, 25, "structures", "evidence" ], [ 31, 34, "PKI", "structure_element" ], [ 62, 75, "Structure III", "evidence" ], [ 86, 93, "density", "evidence" ], [ 98, 101, "SL3", "structure_element" ] ] }, { "sid": 293, "sent": "The map allows placement of PKI at the body P site (Figure 1\u2014figure supplement 3).", "section": "RESULTS", "ner": [ [ 4, 7, "map", "evidence" ], [ 28, 31, "PKI", "structure_element" ], [ 39, 43, "body", "structure_element" ], [ 44, 50, "P site", "site" ] ] }, { "sid": 294, "sent": "Thus, in Structure III, PKI has translocated along the 40S body, but the head remains fully swiveled so that PKI is between the head A and P sites.", "section": "RESULTS", "ner": [ [ 9, 22, "Structure III", "evidence" ], [ 24, 27, "PKI", "structure_element" ], [ 55, 58, "40S", "complex_assembly" ], [ 59, 63, "body", "structure_element" ], [ 73, 77, "head", "structure_element" ], [ 86, 100, "fully swiveled", "protein_state" ], [ 109, 112, "PKI", "structure_element" ], [ 128, 132, "head", "structure_element" ], [ 133, 146, "A and P sites", "site" ] ] }, { "sid": 295, "sent": "Lower resolution of the map in this region suggests that PKI is somewhat destabilized in the vicinity of the body P site in the absence of stacking with the foundations of the head A site (C1274) or P site (U1191).", "section": "RESULTS", "ner": [ [ 24, 27, "map", "evidence" ], [ 57, 60, "PKI", "structure_element" ], [ 109, 113, "body", "structure_element" ], [ 114, 120, "P site", "site" ], [ 128, 138, "absence of", "protein_state" ], [ 139, 147, "stacking", "bond_interaction" ], [ 176, 180, "head", "structure_element" ], [ 181, 187, "A site", "site" ], [ 189, 194, "C1274", "residue_name_number" ], [ 199, 205, "P site", "site" ], [ 207, 212, "U1191", "residue_name_number" ] ] }, { "sid": 296, "sent": "The position of eEF2 is similar to that in Structure II.", "section": "RESULTS", "ner": [ [ 16, 20, "eEF2", "protein" ], [ 43, 55, "Structure II", "evidence" ] ] }, { "sid": 297, "sent": "Structure IV represents a highly bent IRES with PKI partially accommodated in the P site", "section": "RESULTS", "ner": [ [ 0, 12, "Structure IV", "evidence" ], [ 26, 37, "highly bent", "protein_state" ], [ 38, 42, "IRES", "site" ], [ 48, 51, "PKI", "structure_element" ], [ 82, 88, "P site", "site" ] ] }, { "sid": 298, "sent": "In Structure IV, the 40S subunit is almost non-rotated relative to the 60S subunit, and the 40S head is mid-swiveled.", "section": "RESULTS", "ner": [ [ 3, 15, "Structure IV", "evidence" ], [ 21, 24, "40S", "complex_assembly" ], [ 25, 32, "subunit", "structure_element" ], [ 25, 32, "subunit", "structure_element" ], [ 43, 54, "non-rotated", "protein_state" ], [ 71, 74, "60S", "complex_assembly" ], [ 75, 82, "subunit", "structure_element" ], [ 92, 95, "40S", "complex_assembly" ], [ 96, 100, "head", "structure_element" ], [ 104, 116, "mid-swiveled", "protein_state" ] ] }, { "sid": 299, "sent": "Unwinding of the head moves the head P-site residue U1191 and body P-site residue C1637 closer together, resulting in a partially restored 40S P site.", "section": "RESULTS", "ner": [ [ 17, 21, "head", "structure_element" ], [ 32, 36, "head", "structure_element" ], [ 37, 43, "P-site", "site" ], [ 52, 57, "U1191", "residue_name_number" ], [ 62, 66, "body", "structure_element" ], [ 67, 73, "P-site", "site" ], [ 82, 87, "C1637", "residue_name_number" ], [ 139, 142, "40S", "complex_assembly" ], [ 143, 149, "P site", "site" ] ] }, { "sid": 300, "sent": "Whereas C1637 forms a stacking platform for the last base pair of PKI, U1191 does not yet stack on PKI because the head remains partially swiveled.", "section": "RESULTS", "ner": [ [ 8, 13, "C1637", "residue_name_number" ], [ 22, 39, "stacking platform", "site" ], [ 66, 69, "PKI", "structure_element" ], [ 71, 76, "U1191", "residue_name_number" ], [ 90, 95, "stack", "bond_interaction" ], [ 99, 102, "PKI", "structure_element" ], [ 115, 119, "head", "structure_element" ] ] }, { "sid": 301, "sent": "This renders PKI partially accommodated in the P site (Figure 2g).", "section": "RESULTS", "ner": [ [ 13, 16, "PKI", "structure_element" ], [ 47, 53, "P site", "site" ] ] }, { "sid": 302, "sent": "Unwinding of the 40S head also positions the head A site closer to the body A site.", "section": "RESULTS", "ner": [ [ 17, 20, "40S", "complex_assembly" ], [ 21, 25, "head", "structure_element" ], [ 45, 49, "head", "structure_element" ], [ 50, 56, "A site", "site" ], [ 71, 75, "body", "structure_element" ], [ 76, 82, "A site", "site" ] ] }, { "sid": 303, "sent": "This results in rearrangements of eEF2 interactions with the head, allowing eEF2 to advance further into the A site.", "section": "RESULTS", "ner": [ [ 34, 38, "eEF2", "protein" ], [ 61, 65, "head", "structure_element" ], [ 76, 80, "eEF2", "protein" ], [ 109, 115, "A site", "site" ] ] }, { "sid": 304, "sent": "To this end, the head-interacting interface of domain IV slides along the surface of the head by 5\u2009\u00c5. Helix A of domain IV is positioned next to the backbone of h34, with positively charged residues K613, R617 and R631 rearranged from the backbone of h33 (Figure 6c; see also Figure 6\u2014figure supplement 1).", "section": "RESULTS", "ner": [ [ 17, 43, "head-interacting interface", "site" ], [ 54, 56, "IV", "structure_element" ], [ 89, 93, "head", "structure_element" ], [ 102, 109, "Helix A", "structure_element" ], [ 120, 122, "IV", "structure_element" ], [ 161, 164, "h34", "structure_element" ], [ 199, 203, "K613", "residue_name_number" ], [ 205, 209, "R617", "residue_name_number" ], [ 214, 218, "R631", "residue_name_number" ], [ 251, 254, "h33", "structure_element" ] ] }, { "sid": 305, "sent": "Structure V represents an extended IRES with PKI fully accommodated in the P site and domain IV of eEF2 in the A site", "section": "RESULTS", "ner": [ [ 0, 11, "Structure V", "evidence" ], [ 26, 34, "extended", "protein_state" ], [ 35, 39, "IRES", "site" ], [ 45, 48, "PKI", "structure_element" ], [ 75, 81, "P site", "site" ], [ 93, 95, "IV", "structure_element" ], [ 99, 103, "eEF2", "protein" ], [ 111, 117, "A site", "site" ] ] }, { "sid": 306, "sent": "In the nearly non-rotated and non-swiveled ribosome conformation in Structure V closely resembling that of the post-translocation 80S\u20222tRNA\u2022mRNA complex, PKI is fully accommodated in the P site.", "section": "RESULTS", "ner": [ [ 7, 25, "nearly non-rotated", "protein_state" ], [ 30, 42, "non-swiveled", "protein_state" ], [ 43, 51, "ribosome", "complex_assembly" ], [ 68, 79, "Structure V", "evidence" ], [ 111, 129, "post-translocation", "protein_state" ], [ 130, 144, "80S\u20222tRNA\u2022mRNA", "complex_assembly" ], [ 154, 157, "PKI", "structure_element" ], [ 187, 193, "P site", "site" ] ] }, { "sid": 307, "sent": "The codon-anticodon\u2013like helix is stacked on P-site residues U1191 and C1637 (Figure 3d), analogous to stacking of the tRNA-mRNA helix (Figure 3e).", "section": "RESULTS", "ner": [ [ 4, 30, "codon-anticodon\u2013like helix", "structure_element" ], [ 45, 51, "P-site", "site" ], [ 61, 66, "U1191", "residue_name_number" ], [ 71, 76, "C1637", "residue_name_number" ], [ 103, 111, "stacking", "bond_interaction" ], [ 119, 128, "tRNA-mRNA", "complex_assembly" ], [ 129, 134, "helix", "structure_element" ] ] }, { "sid": 308, "sent": "A notable conformational change in eEF2 from that in the preceding Structures is visible in the position of domain III, which contacts uS12 (Figure 6d).", "section": "RESULTS", "ner": [ [ 35, 39, "eEF2", "protein" ], [ 67, 77, "Structures", "evidence" ], [ 115, 118, "III", "structure_element" ], [ 135, 139, "uS12", "protein" ] ] }, { "sid": 309, "sent": "In Structure V, protein uS12 is shifted along with the 40S body as a result of intersubunit rotation.", "section": "RESULTS", "ner": [ [ 3, 14, "Structure V", "evidence" ], [ 24, 28, "uS12", "protein" ], [ 55, 58, "40S", "complex_assembly" ], [ 59, 63, "body", "structure_element" ] ] }, { "sid": 310, "sent": "In this position, uS12 forms extensive interactions with eEF2 domains II and III.", "section": "RESULTS", "ner": [ [ 18, 22, "uS12", "protein" ], [ 57, 61, "eEF2", "protein" ], [ 70, 72, "II", "structure_element" ], [ 77, 80, "III", "structure_element" ] ] }, { "sid": 311, "sent": "Specifically, the C-terminal tail of uS12 packs against the \u03b2-barrel of domain II, while the \u03b2-barrel of uS12 packs against helix A of domain III.", "section": "RESULTS", "ner": [ [ 18, 33, "C-terminal tail", "structure_element" ], [ 37, 41, "uS12", "protein" ], [ 60, 68, "\u03b2-barrel", "structure_element" ], [ 79, 81, "II", "structure_element" ], [ 93, 101, "\u03b2-barrel", "structure_element" ], [ 105, 109, "uS12", "protein" ], [ 124, 131, "helix A", "structure_element" ], [ 142, 145, "III", "structure_element" ] ] }, { "sid": 312, "sent": "This shifts the tip of helix A of domain III (at aa 500) by ~5\u2009\u00c5 (relative to that in Structure I) toward domain I. Although domain III remains in contact with domain V, the shift occurs in the direction that could eventually disconnect the \u03b2-platforms of these domains.", "section": "RESULTS", "ner": [ [ 23, 30, "helix A", "structure_element" ], [ 41, 44, "III", "structure_element" ], [ 52, 55, "500", "residue_number" ], [ 86, 97, "Structure I", "evidence" ], [ 113, 114, "I", "structure_element" ], [ 132, 135, "III", "structure_element" ], [ 167, 168, "V", "structure_element" ], [ 241, 252, "\u03b2-platforms", "structure_element" ] ] }, { "sid": 313, "sent": "Domain IV of eEF2 is fully accommodated in the A site.", "section": "RESULTS", "ner": [ [ 7, 9, "IV", "structure_element" ], [ 13, 17, "eEF2", "protein" ], [ 47, 53, "A site", "site" ] ] }, { "sid": 314, "sent": "The first codon of the open reading frame is also positioned in the A site, with bases exposed toward eEF2 (Figure 7), resembling the conformations of the A-site codons in EF-G-bound 70S complexes.", "section": "RESULTS", "ner": [ [ 23, 41, "open reading frame", "structure_element" ], [ 68, 74, "A site", "site" ], [ 102, 106, "eEF2", "protein" ], [ 155, 161, "A-site", "site" ], [ 172, 182, "EF-G-bound", "protein_state" ], [ 183, 186, "70S", "complex_assembly" ] ] }, { "sid": 315, "sent": "As in the preceding Structures, the histidine-diphthamide tip is bound in the minor groove of the P-site codon-anticodon helix.", "section": "RESULTS", "ner": [ [ 20, 30, "Structures", "evidence" ], [ 36, 61, "histidine-diphthamide tip", "site" ], [ 65, 73, "bound in", "protein_state" ], [ 78, 90, "minor groove", "site" ], [ 98, 104, "P-site", "site" ], [ 105, 126, "codon-anticodon helix", "structure_element" ] ] }, { "sid": 316, "sent": "Diph699 slightly rearranges, relative to that in Structure I (Figure 7), and interacts with four out of six codon-anticodon nucleotides.", "section": "RESULTS", "ner": [ [ 0, 7, "Diph699", "ptm" ], [ 49, 60, "Structure I", "evidence" ] ] }, { "sid": 317, "sent": "The imidazole moiety stacks on G6907 (corresponding to nt 36 in the tRNA anticodon) and hydrogen bonds with O2\u2019 of G6906 (nt 35 of tRNA).", "section": "RESULTS", "ner": [ [ 31, 36, "G6907", "residue_name_number" ], [ 68, 72, "tRNA", "chemical" ], [ 88, 102, "hydrogen bonds", "bond_interaction" ], [ 115, 120, "G6906", "residue_name_number" ], [ 131, 135, "tRNA", "chemical" ] ] }, { "sid": 318, "sent": "The amide at the diphthamide end interacts with N2 of G6906 and O2 and O2\u2019 of C6951 (corresponding to nt 2 of the codon).", "section": "RESULTS", "ner": [ [ 17, 28, "diphthamide", "ptm" ], [ 54, 59, "G6906", "residue_name_number" ], [ 78, 83, "C6951", "residue_name_number" ] ] }, { "sid": 319, "sent": "The trimethylamino-group is positioned over the ribose of C6952 (codon nt 3).", "section": "RESULTS", "ner": [ [ 58, 63, "C6952", "residue_name_number" ] ] }, { "sid": 320, "sent": "IRES translocation mechanism", "section": "DISCUSS", "ner": [ [ 0, 4, "IRES", "site" ] ] }, { "sid": 321, "sent": "Animation showing the transition from the initiation 80S\u2022TSV IRES structures (Koh et al., 2014) to eEF2-bound Structures I through V (this work).", "section": "FIG", "ner": [ [ 42, 52, "initiation", "protein_state" ], [ 53, 65, "80S\u2022TSV IRES", "complex_assembly" ], [ 66, 76, "structures", "evidence" ], [ 99, 109, "eEF2-bound", "protein_state" ], [ 110, 132, "Structures I through V", "evidence" ] ] }, { "sid": 322, "sent": "Four views (scenes) are shown: (1) A view down the intersubunit space, with the head of the 40S subunit oriented toward a viewer, as in Figure 1a; (2) A view at the solvent side of the 40S subunit, with the 40S head shown at the top, as in Figure 2\u2014figure supplement 1; (3) A view down at the subunit interface of the 40S subunit; (4) A close-up view of the decoding center (A site) and the P site, as in Figure 2g. Each scene is shown twice.", "section": "FIG", "ner": [ [ 80, 84, "head", "structure_element" ], [ 92, 95, "40S", "complex_assembly" ], [ 96, 103, "subunit", "structure_element" ], [ 185, 188, "40S", "complex_assembly" ], [ 189, 196, "subunit", "structure_element" ], [ 207, 210, "40S", "complex_assembly" ], [ 211, 215, "head", "structure_element" ], [ 318, 321, "40S", "complex_assembly" ], [ 322, 329, "subunit", "structure_element" ], [ 358, 373, "decoding center", "site" ], [ 375, 381, "A site", "site" ], [ 391, 397, "P site", "site" ] ] }, { "sid": 323, "sent": "In scenes 1, 2 and 3, nucleotides C1274, U1191 of the 40S head and G904 of the 40S platform are shown in black to denote the A, P and E sites, respectively.", "section": "FIG", "ner": [ [ 34, 39, "C1274", "residue_name_number" ], [ 41, 46, "U1191", "residue_name_number" ], [ 54, 57, "40S", "complex_assembly" ], [ 58, 62, "head", "structure_element" ], [ 67, 71, "G904", "residue_name_number" ], [ 79, 91, "40S platform", "site" ], [ 125, 141, "A, P and E sites", "site" ] ] }, { "sid": 324, "sent": "In scene 4, C1274 and U1191 are labeled and shown in yellow; G577, A1755 and A1756 of the 40S body A site and C1637 of the body P site are labeled and shown in orange.", "section": "FIG", "ner": [ [ 12, 17, "C1274", "residue_name_number" ], [ 22, 27, "U1191", "residue_name_number" ], [ 61, 65, "G577", "residue_name_number" ], [ 67, 72, "A1755", "residue_name_number" ], [ 77, 82, "A1756", "residue_name_number" ], [ 90, 93, "40S", "complex_assembly" ], [ 94, 98, "body", "structure_element" ], [ 99, 105, "A site", "site" ], [ 110, 115, "C1637", "residue_name_number" ], [ 123, 127, "body", "structure_element" ], [ 128, 134, "P site", "site" ] ] }, { "sid": 325, "sent": "In this work we have captured the structures of the TSV IRES, whose PKI samples positions between the A and P sites (Structures I\u2013IV), as well as in the P site (Structure V).", "section": "DISCUSS", "ner": [ [ 34, 44, "structures", "evidence" ], [ 52, 55, "TSV", "species" ], [ 56, 60, "IRES", "site" ], [ 68, 71, "PKI", "structure_element" ], [ 102, 115, "A and P sites", "site" ], [ 117, 132, "Structures I\u2013IV", "evidence" ], [ 153, 159, "P site", "site" ], [ 161, 172, "Structure V", "evidence" ] ] }, { "sid": 326, "sent": "We propose that together with the previously reported initiation state, these structures represent the trajectory of eEF2-induced IRES translocation (shown as an animation in http://labs.umassmed.edu/korostelevlab/msc/iresmovie.gif and Video 1).", "section": "DISCUSS", "ner": [ [ 54, 64, "initiation", "protein_state" ], [ 78, 88, "structures", "evidence" ], [ 117, 121, "eEF2", "protein" ], [ 130, 134, "IRES", "site" ] ] }, { "sid": 327, "sent": "Our structures reveal previously unseen intermediate states of eEF2 or EF-G engagement with the A site, providing the structural basis for the mechanism of translocase action.", "section": "DISCUSS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 63, 67, "eEF2", "protein" ], [ 71, 75, "EF-G", "protein" ], [ 96, 102, "A site", "site" ], [ 156, 167, "translocase", "protein_type" ] ] }, { "sid": 328, "sent": "Furthermore, they provide insight into the mechanism of eEF2\u2022GTP association with the pre-translocation ribosome and eEF2\u2022GDP dissociation from the post-translocation ribosome, also delineating the mechanism of translation inhibition by the antifungal drug sordarin.", "section": "DISCUSS", "ner": [ [ 56, 64, "eEF2\u2022GTP", "complex_assembly" ], [ 86, 103, "pre-translocation", "protein_state" ], [ 104, 112, "ribosome", "complex_assembly" ], [ 117, 125, "eEF2\u2022GDP", "complex_assembly" ], [ 148, 166, "post-translocation", "protein_state" ], [ 167, 175, "ribosome", "complex_assembly" ], [ 257, 265, "sordarin", "chemical" ] ] }, { "sid": 329, "sent": "In summary, the reported ensemble of structures substantially enhances our understanding of the translocation mechanism, including that of tRNAs as discussed below.", "section": "DISCUSS", "ner": [ [ 37, 47, "structures", "evidence" ], [ 139, 144, "tRNAs", "chemical" ] ] }, { "sid": 330, "sent": "Translocation of the TSV IRES on the 40S subunit globally resembles a step of an inchworm (Figure 4; see also Figure 3\u2014figure supplement 2).", "section": "DISCUSS", "ner": [ [ 21, 24, "TSV", "species" ], [ 25, 29, "IRES", "site" ], [ 37, 40, "40S", "complex_assembly" ], [ 41, 48, "subunit", "structure_element" ], [ 81, 89, "inchworm", "protein_state" ] ] }, { "sid": 331, "sent": "At the start (initiation state), the IRES adopts an extended conformation (extended inchworm).", "section": "DISCUSS", "ner": [ [ 14, 24, "initiation", "protein_state" ], [ 37, 41, "IRES", "site" ], [ 52, 60, "extended", "protein_state" ], [ 75, 92, "extended inchworm", "protein_state" ] ] }, { "sid": 332, "sent": "The front 'legs' (SL4 and SL5) of the 5\u2019-domain (front end) are attached to the 40S head proteins uS7, uS11 and eS25 (Figure 3\u2014figure supplement 2).", "section": "DISCUSS", "ner": [ [ 4, 15, "front 'legs", "structure_element" ], [ 18, 21, "SL4", "structure_element" ], [ 26, 29, "SL5", "structure_element" ], [ 38, 47, "5\u2019-domain", "structure_element" ], [ 49, 58, "front end", "structure_element" ], [ 80, 83, "40S", "complex_assembly" ], [ 84, 88, "head", "structure_element" ], [ 98, 101, "uS7", "protein" ], [ 103, 107, "uS11", "protein" ], [ 112, 116, "eS25", "protein" ] ] }, { "sid": 333, "sent": "PKI, representing the hind end, is bound in the A site.", "section": "DISCUSS", "ner": [ [ 0, 3, "PKI", "structure_element" ], [ 22, 30, "hind end", "structure_element" ], [ 35, 43, "bound in", "protein_state" ], [ 48, 54, "A site", "site" ] ] }, { "sid": 334, "sent": "In the first sub-step (Structures I to IV), the hind end advances from the A to the P site and approaches the front end, which remains attached to the 40S surface.", "section": "DISCUSS", "ner": [ [ 23, 41, "Structures I to IV", "evidence" ], [ 48, 56, "hind end", "structure_element" ], [ 75, 90, "A to the P site", "site" ], [ 110, 119, "front end", "structure_element" ], [ 151, 154, "40S", "complex_assembly" ] ] }, { "sid": 335, "sent": "This shortens the distance between PKI and SL4 by up to 20\u2009\u00c5 relative to the initiating IRES structure, resulting in a bent IRES conformation (bent inchworm).", "section": "DISCUSS", "ner": [ [ 35, 38, "PKI", "structure_element" ], [ 43, 46, "SL4", "structure_element" ], [ 88, 92, "IRES", "site" ], [ 93, 102, "structure", "evidence" ], [ 119, 123, "bent", "protein_state" ], [ 124, 128, "IRES", "site" ], [ 143, 156, "bent inchworm", "protein_state" ] ] }, { "sid": 336, "sent": "Finally (Structures IV to V), as the hind end is accommodated in the P site, the front 'legs' advance by departing from their initial binding sites.", "section": "DISCUSS", "ner": [ [ 9, 27, "Structures IV to V", "evidence" ], [ 37, 45, "hind end", "structure_element" ], [ 69, 75, "P site", "site" ], [ 81, 93, "front 'legs'", "structure_element" ], [ 126, 147, "initial binding sites", "site" ] ] }, { "sid": 337, "sent": "This converts the IRES into an extended conformation, rendering the inchworm prepared for the next translocation step.", "section": "DISCUSS", "ner": [ [ 18, 22, "IRES", "site" ], [ 31, 39, "extended", "protein_state" ], [ 68, 76, "inchworm", "protein_state" ] ] }, { "sid": 338, "sent": "Notably, at all steps, the head of the IRES inchworm (L1.1 region) is supported by the mobile L1 stalk.", "section": "DISCUSS", "ner": [ [ 27, 31, "head", "structure_element" ], [ 39, 43, "IRES", "site" ], [ 44, 52, "inchworm", "protein_state" ], [ 54, 65, "L1.1 region", "structure_element" ], [ 87, 93, "mobile", "protein_state" ], [ 94, 102, "L1 stalk", "structure_element" ] ] }, { "sid": 339, "sent": "In the post-translocation CrPV IRES structure, the 5\u2019-domain similarly protrudes between the subunits and interacts with the L1 stalk, as in the initiation state for this IRES.", "section": "DISCUSS", "ner": [ [ 7, 25, "post-translocation", "protein_state" ], [ 26, 30, "CrPV", "species" ], [ 31, 35, "IRES", "site" ], [ 36, 45, "structure", "evidence" ], [ 51, 60, "5\u2019-domain", "structure_element" ], [ 125, 133, "L1 stalk", "structure_element" ], [ 145, 155, "initiation", "protein_state" ], [ 171, 175, "IRES", "site" ] ] }, { "sid": 340, "sent": "This underlines structural similarity for the TSV and CrPV IRES translocation mechanisms.", "section": "DISCUSS", "ner": [ [ 46, 49, "TSV", "species" ], [ 54, 58, "CrPV", "species" ], [ 59, 63, "IRES", "site" ] ] }, { "sid": 341, "sent": "Upon translocation, the GCU start codon is positioned in the A site (Structure V), ready for interaction with Ala-tRNAAla upon eEF2 departure.", "section": "DISCUSS", "ner": [ [ 61, 67, "A site", "site" ], [ 69, 80, "Structure V", "evidence" ], [ 110, 121, "Ala-tRNAAla", "chemical" ], [ 127, 131, "eEF2", "protein" ] ] }, { "sid": 342, "sent": "Recent studies have shown that in some cases a fraction of IGR IRES-driven translation results from an alternative reading frame, which is shifted by one nucleotide relative to the normal ORF.", "section": "DISCUSS", "ner": [ [ 59, 62, "IGR", "structure_element" ], [ 63, 67, "IRES", "site" ], [ 188, 191, "ORF", "structure_element" ] ] }, { "sid": 343, "sent": "One of the mechanistic scenarios (discussed in) involves binding of the first aminoacyl-tRNA to the post-translocated IRES mRNA frame shifted by one nucleotide (predominantly a +1 frame shift).", "section": "DISCUSS", "ner": [ [ 78, 92, "aminoacyl-tRNA", "chemical" ], [ 100, 117, "post-translocated", "protein_state" ], [ 118, 122, "IRES", "site" ], [ 123, 127, "mRNA", "chemical" ] ] }, { "sid": 344, "sent": "In our structures, the IRES presents to the decoding center a pre-translocated or fully translocated ORF, rather than a +1 (more translocated) ORF, suggesting that eEF2 does not induce a highly populated fraction of +1 shifted IRES mRNAs.", "section": "DISCUSS", "ner": [ [ 7, 17, "structures", "evidence" ], [ 23, 27, "IRES", "site" ], [ 44, 59, "decoding center", "site" ], [ 62, 78, "pre-translocated", "protein_state" ], [ 82, 100, "fully translocated", "protein_state" ], [ 101, 104, "ORF", "structure_element" ], [ 143, 146, "ORF", "structure_element" ], [ 164, 168, "eEF2", "protein" ], [ 227, 231, "IRES", "site" ], [ 232, 237, "mRNAs", "chemical" ] ] }, { "sid": 345, "sent": "It is likely that alternative frame setting occurs following eEF2 release and that this depends on transient displacement of the start codon in the decoding center, allowing binding of the corresponding amino acyl-tRNA to an off-frame codon.", "section": "DISCUSS", "ner": [ [ 61, 65, "eEF2", "protein" ], [ 148, 163, "decoding center", "site" ], [ 203, 218, "amino acyl-tRNA", "chemical" ] ] }, { "sid": 346, "sent": "Further structural studies involving 80S\u2022IRES\u2022tRNA complexes are necessary to understand the mechanisms underlying alternative reading frame selection.", "section": "DISCUSS", "ner": [ [ 8, 26, "structural studies", "experimental_method" ], [ 37, 50, "80S\u2022IRES\u2022tRNA", "complex_assembly" ] ] }, { "sid": 347, "sent": "The presence of several translocation complexes in a single sample suggests that the structures represent equilibrium states of forward and reverse translocation of the IRES, which interconvert among each other.", "section": "DISCUSS", "ner": [ [ 4, 15, "presence of", "protein_state" ], [ 85, 95, "structures", "evidence" ], [ 169, 173, "IRES", "site" ] ] }, { "sid": 348, "sent": "This is consistent with the observations that the intergenic IRESs are prone to reverse translocation.", "section": "DISCUSS", "ner": [ [ 61, 66, "IRESs", "site" ] ] }, { "sid": 349, "sent": "Specifically, biochemical toe-printing studies in the presence of eEF2\u2022GTP identified IRES in a non-translocated position unless eEF1a\u2022aa-tRNA is also present.", "section": "DISCUSS", "ner": [ [ 14, 46, "biochemical toe-printing studies", "experimental_method" ], [ 54, 65, "presence of", "protein_state" ], [ 66, 74, "eEF2\u2022GTP", "complex_assembly" ], [ 86, 90, "IRES", "site" ], [ 96, 112, "non-translocated", "protein_state" ], [ 129, 142, "eEF1a\u2022aa-tRNA", "complex_assembly" ] ] }, { "sid": 350, "sent": "These findings indicate that IRES translocation by eEF2 is futile: the IRES returns to the A site upon releasing eEF2\u2022GDP unless an amino-acyl tRNA enters the A site and blocks IRES back-translocation.", "section": "DISCUSS", "ner": [ [ 29, 33, "IRES", "site" ], [ 51, 55, "eEF2", "protein" ], [ 71, 75, "IRES", "site" ], [ 91, 97, "A site", "site" ], [ 113, 121, "eEF2\u2022GDP", "complex_assembly" ], [ 132, 147, "amino-acyl tRNA", "chemical" ], [ 159, 165, "A site", "site" ], [ 177, 181, "IRES", "site" ] ] }, { "sid": 351, "sent": "This contrasts with the post-translocated 2tRNA\u2022mRNA complex, in which the classical P and E-site tRNAs are stabilized in the non-rotated ribosome after translocase release.", "section": "DISCUSS", "ner": [ [ 24, 41, "post-translocated", "protein_state" ], [ 42, 52, "2tRNA\u2022mRNA", "complex_assembly" ], [ 85, 97, "P and E-site", "site" ], [ 98, 103, "tRNAs", "chemical" ], [ 126, 137, "non-rotated", "protein_state" ], [ 138, 146, "ribosome", "complex_assembly" ], [ 153, 164, "translocase", "protein_type" ] ] }, { "sid": 352, "sent": "Thus, the meta-stability of the post-translocation IRES is likely due to the absence of stabilizing structural features present in the 2tRNA\u2022mRNA complex.", "section": "DISCUSS", "ner": [ [ 32, 50, "post-translocation", "protein_state" ], [ 51, 55, "IRES", "site" ], [ 77, 87, "absence of", "protein_state" ], [ 135, 145, "2tRNA\u2022mRNA", "complex_assembly" ] ] }, { "sid": 353, "sent": "In the initiation state, the IRES resembles a pre-translocation 2tRNA\u2022mRNA complex reduced to the A/P-tRNA anticodon-stem loop and elbow in the A site and the P/E-tRNA elbow contacting the L1 stalk.", "section": "DISCUSS", "ner": [ [ 7, 17, "initiation", "protein_state" ], [ 29, 33, "IRES", "site" ], [ 46, 63, "pre-translocation", "protein_state" ], [ 64, 74, "2tRNA\u2022mRNA", "complex_assembly" ], [ 98, 101, "A/P", "site" ], [ 102, 106, "tRNA", "chemical" ], [ 107, 126, "anticodon-stem loop", "structure_element" ], [ 131, 136, "elbow", "structure_element" ], [ 144, 150, "A site", "site" ], [ 159, 162, "P/E", "site" ], [ 163, 167, "tRNA", "chemical" ], [ 168, 173, "elbow", "structure_element" ], [ 189, 197, "L1 stalk", "structure_element" ] ] }, { "sid": 354, "sent": "Because the anticodon-stem loop of the A-tRNA is sufficient for translocation completion, we ascribe the meta-stability of the post-translocation IRES to the absence of the P/E-tRNA elements, either the ASL or the acceptor arm, or both.", "section": "DISCUSS", "ner": [ [ 12, 31, "anticodon-stem loop", "structure_element" ], [ 39, 40, "A", "site" ], [ 41, 45, "tRNA", "chemical" ], [ 127, 145, "post-translocation", "protein_state" ], [ 146, 150, "IRES", "site" ], [ 158, 168, "absence of", "protein_state" ], [ 173, 176, "P/E", "site" ], [ 177, 181, "tRNA", "chemical" ], [ 203, 206, "ASL", "structure_element" ] ] }, { "sid": 355, "sent": "Furthermore, interactions of SL4 and SL5 with the 40S subunit likely contribute to stabilization of pre-translocation structures.", "section": "DISCUSS", "ner": [ [ 29, 32, "SL4", "structure_element" ], [ 37, 40, "SL5", "structure_element" ], [ 50, 53, "40S", "complex_assembly" ], [ 54, 61, "subunit", "structure_element" ], [ 100, 117, "pre-translocation", "protein_state" ], [ 118, 128, "structures", "evidence" ] ] }, { "sid": 356, "sent": "Partitioned roles of 40S subunit rearrangements", "section": "DISCUSS", "ner": [ [ 21, 24, "40S", "complex_assembly" ], [ 25, 32, "subunit", "structure_element" ] ] }, { "sid": 357, "sent": "Our structures delineate the mechanistic functions for intersubunit rotation and head swivel in translocation.", "section": "DISCUSS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 81, 85, "head", "structure_element" ] ] }, { "sid": 358, "sent": "Specifically, intersubunit rotation allows eEF2 entry into the A site, while the head swivel mediates PKI translocation.", "section": "DISCUSS", "ner": [ [ 43, 47, "eEF2", "protein" ], [ 63, 69, "A site", "site" ], [ 81, 85, "head", "structure_element" ], [ 102, 105, "PKI", "structure_element" ] ] }, { "sid": 359, "sent": "Various degrees of intersubunit rotation have been observed in cryo-EM studies of the 80S\u2022IRES initiation complexes.", "section": "DISCUSS", "ner": [ [ 63, 78, "cryo-EM studies", "experimental_method" ], [ 86, 94, "80S\u2022IRES", "complex_assembly" ], [ 95, 105, "initiation", "protein_state" ] ] }, { "sid": 360, "sent": "This suggests that the subunits are capable of spontaneous rotation, as is the case for tRNA-bound pre-translocation complexes.", "section": "DISCUSS", "ner": [ [ 23, 31, "subunits", "structure_element" ], [ 88, 98, "tRNA-bound", "protein_state" ], [ 99, 116, "pre-translocation", "protein_state" ] ] }, { "sid": 361, "sent": "The pre-translocation Structure I with eEF2 least advanced into the A site adopts a fully rotated conformation.", "section": "DISCUSS", "ner": [ [ 4, 21, "pre-translocation", "protein_state" ], [ 22, 33, "Structure I", "evidence" ], [ 39, 43, "eEF2", "protein" ], [ 68, 74, "A site", "site" ], [ 84, 110, "fully rotated conformation", "protein_state" ] ] }, { "sid": 362, "sent": "Reverse intersubunit rotation from Structure I to V shifts the translocation tunnel (the tunnel between the A, P and E sites) toward eEF2, which is rigidly attached to the 60S subunit.", "section": "DISCUSS", "ner": [ [ 35, 51, "Structure I to V", "evidence" ], [ 63, 83, "translocation tunnel", "site" ], [ 89, 95, "tunnel", "site" ], [ 108, 124, "A, P and E sites", "site" ], [ 133, 137, "eEF2", "protein" ], [ 172, 175, "60S", "complex_assembly" ], [ 176, 183, "subunit", "structure_element" ] ] }, { "sid": 363, "sent": "This allows eEF2 to move into the A site.", "section": "DISCUSS", "ner": [ [ 12, 16, "eEF2", "protein" ], [ 34, 40, "A site", "site" ] ] }, { "sid": 364, "sent": "As such, reverse intersubunit rotation facilitates full docking of eEF2 in the A site.", "section": "DISCUSS", "ner": [ [ 67, 71, "eEF2", "protein" ], [ 79, 85, "A site", "site" ] ] }, { "sid": 365, "sent": "Because the histidine-diphthamide tip of eEF2 (H583, H694 and Diph699) attaches to the codon-anticodon-like helix of PKI, eEF2 appears to directly force PKI out of the A site.", "section": "DISCUSS", "ner": [ [ 12, 37, "histidine-diphthamide tip", "site" ], [ 41, 45, "eEF2", "protein" ], [ 47, 51, "H583", "residue_name_number" ], [ 53, 57, "H694", "residue_name_number" ], [ 62, 69, "Diph699", "ptm" ], [ 87, 113, "codon-anticodon-like helix", "structure_element" ], [ 117, 120, "PKI", "structure_element" ], [ 122, 126, "eEF2", "protein" ], [ 153, 156, "PKI", "structure_element" ], [ 168, 174, "A site", "site" ] ] }, { "sid": 366, "sent": "The head swivel allows gradual translocation of PKI to the P site, first with respect to the body and then to the head.", "section": "DISCUSS", "ner": [ [ 4, 8, "head", "structure_element" ], [ 48, 51, "PKI", "structure_element" ], [ 59, 65, "P site", "site" ], [ 93, 97, "body", "structure_element" ], [ 114, 118, "head", "structure_element" ] ] }, { "sid": 367, "sent": "The fully swiveled conformations of Structures II and III represent the mid-point of translocation, in which PKI relocates between the head A site and body P site.", "section": "DISCUSS", "ner": [ [ 4, 18, "fully swiveled", "protein_state" ], [ 36, 57, "Structures II and III", "evidence" ], [ 109, 112, "PKI", "structure_element" ], [ 135, 139, "head", "structure_element" ], [ 140, 146, "A site", "site" ], [ 151, 155, "body", "structure_element" ], [ 156, 162, "P site", "site" ] ] }, { "sid": 368, "sent": "We note that such mid-states have not been observed for 2tRNA\u2022mRNA, but their formation can explain the formation of subsequent pe/E hybrid and ap/P chimeric structures (Figure 1\u2014figure supplement 1).", "section": "DISCUSS", "ner": [ [ 56, 66, "2tRNA\u2022mRNA", "complex_assembly" ], [ 128, 139, "pe/E hybrid", "protein_state" ], [ 144, 157, "ap/P chimeric", "protein_state" ], [ 158, 168, "structures", "evidence" ] ] }, { "sid": 369, "sent": "Reverse swivel from Structure III to V brings the head to the non-swiveled position, restoring the A and P sites on the small subunit.", "section": "DISCUSS", "ner": [ [ 20, 38, "Structure III to V", "evidence" ], [ 50, 54, "head", "structure_element" ], [ 62, 74, "non-swiveled", "protein_state" ], [ 99, 112, "A and P sites", "site" ], [ 120, 133, "small subunit", "structure_element" ] ] }, { "sid": 370, "sent": "The functions of eEF2 in translocation", "section": "DISCUSS", "ner": [ [ 17, 21, "eEF2", "protein" ] ] }, { "sid": 371, "sent": "To our knowledge, our work provides the first high-resolution view of the dynamics of a ribosomal translocase that is inferred from an ensemble of structures sampled under uniform conditions.", "section": "DISCUSS", "ner": [ [ 88, 109, "ribosomal translocase", "protein_type" ], [ 147, 157, "structures", "evidence" ] ] }, { "sid": 372, "sent": "The structures, therefore, offer a unique opportunity to address the role of the elongation factors during translocation.", "section": "DISCUSS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 81, 99, "elongation factors", "protein_type" ] ] }, { "sid": 373, "sent": "Translocases are efficient enzymes.", "section": "DISCUSS", "ner": [ [ 0, 12, "Translocases", "protein_type" ] ] }, { "sid": 374, "sent": "While the ribosome itself has the capacity to translocate in the absence of the translocase, spontaneous translocation is slow.", "section": "DISCUSS", "ner": [ [ 10, 18, "ribosome", "complex_assembly" ], [ 65, 75, "absence of", "protein_state" ], [ 80, 91, "translocase", "protein_type" ] ] }, { "sid": 375, "sent": "EF-G enhances the translocation rate by several orders of magnitude, aided by an additional 2- to 50-fold boost from GTP hydrolysis.", "section": "DISCUSS", "ner": [ [ 0, 4, "EF-G", "protein" ], [ 117, 120, "GTP", "chemical" ] ] }, { "sid": 376, "sent": "Due to the lack of structures of translocation intermediates, the mechanistic role of eEF2/EF-G is not fully understood.", "section": "DISCUSS", "ner": [ [ 19, 29, "structures", "evidence" ], [ 86, 90, "eEF2", "protein" ], [ 91, 95, "EF-G", "protein" ] ] }, { "sid": 377, "sent": "The 80S\u2022IRES\u2022eEF2 structures reported here suggest two main roles for eEF2 in translocation.", "section": "DISCUSS", "ner": [ [ 4, 17, "80S\u2022IRES\u2022eEF2", "complex_assembly" ], [ 18, 28, "structures", "evidence" ], [ 70, 74, "eEF2", "protein" ] ] }, { "sid": 378, "sent": "As discussed above, the first role is to directly shift PKI out of the A site upon spontaneous reverse intersubunit rotation.", "section": "DISCUSS", "ner": [ [ 56, 59, "PKI", "structure_element" ], [ 71, 77, "A site", "site" ] ] }, { "sid": 379, "sent": "In our structures, the tip of domain IV docks next to PKI, with diphthamide 699 fit into the minor groove of the codon-anticodon-like helix of PKI (Figure 7).", "section": "DISCUSS", "ner": [ [ 7, 17, "structures", "evidence" ], [ 37, 39, "IV", "structure_element" ], [ 54, 57, "PKI", "structure_element" ], [ 64, 79, "diphthamide 699", "ptm" ], [ 93, 105, "minor groove", "site" ], [ 113, 139, "codon-anticodon-like helix", "structure_element" ], [ 143, 146, "PKI", "structure_element" ] ] }, { "sid": 380, "sent": "This arrangement rationalizes inactivation of eEF2 by diphtheria toxin, which catalyzes ADP-ribosylation of the diphthamide (reviewed in).", "section": "DISCUSS", "ner": [ [ 46, 50, "eEF2", "protein" ], [ 54, 70, "diphtheria toxin", "protein_type" ], [ 88, 104, "ADP-ribosylation", "ptm" ], [ 112, 123, "diphthamide", "ptm" ] ] }, { "sid": 381, "sent": "The enzyme ADP-ribosylates the NE2 atom of the imidazole ring, which in our structures interacts with the first two residues of the anticodon-like strand of PKI.", "section": "DISCUSS", "ner": [ [ 11, 26, "ADP-ribosylates", "ptm" ], [ 76, 86, "structures", "evidence" ], [ 132, 153, "anticodon-like strand", "structure_element" ], [ 157, 160, "PKI", "structure_element" ] ] }, { "sid": 382, "sent": "The bulky ADP-ribosyl moiety at this position would disrupt the interaction, rendering eEF2 unable to bind to the A site and/or stalled on ribosomes in a non-productive conformation.", "section": "DISCUSS", "ner": [ [ 10, 13, "ADP", "chemical" ], [ 87, 91, "eEF2", "protein" ], [ 114, 120, "A site", "site" ], [ 139, 148, "ribosomes", "complex_assembly" ] ] }, { "sid": 383, "sent": "As eEF2 shifts PKI toward the P site in the course of reverse intersubunit rotation, the 60S-attached translocase migrates along the surface of the 40S subunit, guided by electrostatic interactions.", "section": "DISCUSS", "ner": [ [ 3, 7, "eEF2", "protein" ], [ 15, 18, "PKI", "structure_element" ], [ 30, 36, "P site", "site" ], [ 89, 101, "60S-attached", "protein_state" ], [ 102, 113, "translocase", "protein_type" ], [ 148, 151, "40S", "complex_assembly" ], [ 152, 159, "subunit", "structure_element" ], [ 171, 197, "electrostatic interactions", "bond_interaction" ] ] }, { "sid": 384, "sent": "Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively.", "section": "DISCUSS", "ner": [ [ 0, 26, "Positively-charged patches", "site" ], [ 38, 40, "II", "structure_element" ], [ 45, 48, "III", "structure_element" ], [ 50, 54, "R391", "residue_name_number" ], [ 56, 60, "K394", "residue_name_number" ], [ 62, 66, "R433", "residue_name_number" ], [ 68, 72, "R510", "residue_name_number" ], [ 78, 80, "IV", "structure_element" ], [ 82, 86, "K613", "residue_name_number" ], [ 88, 92, "R617", "residue_name_number" ], [ 94, 98, "R609", "residue_name_number" ], [ 100, 104, "R631", "residue_name_number" ], [ 106, 110, "K651", "residue_name_number" ], [ 123, 127, "rRNA", "chemical" ], [ 135, 138, "40S", "complex_assembly" ], [ 139, 143, "body", "structure_element" ], [ 145, 147, "h5", "structure_element" ], [ 153, 157, "head", "structure_element" ], [ 159, 162, "h18", "structure_element" ], [ 167, 170, "h33", "structure_element" ], [ 171, 174, "h34", "structure_element" ] ] }, { "sid": 385, "sent": "The Structures reveal hopping of the positive clusters over rRNA helices.", "section": "DISCUSS", "ner": [ [ 4, 14, "Structures", "evidence" ], [ 60, 64, "rRNA", "chemical" ], [ 65, 72, "helices", "structure_element" ] ] }, { "sid": 386, "sent": "For example, between Structures II and V, the K613/R617/R631 cluster of domain IV hops by ~19\u2009\u00c5 (for C\u03b1 of R617) from the phosphate backbone of h33 (at nt 1261\u20131264) to that of the neighboring h34 (at nt 1442\u20131445).", "section": "DISCUSS", "ner": [ [ 21, 40, "Structures II and V", "evidence" ], [ 46, 50, "K613", "residue_name_number" ], [ 51, 55, "R617", "residue_name_number" ], [ 56, 60, "R631", "residue_name_number" ], [ 79, 81, "IV", "structure_element" ], [ 107, 111, "R617", "residue_name_number" ], [ 144, 147, "h33", "structure_element" ], [ 155, 164, "1261\u20131264", "residue_range" ], [ 193, 196, "h34", "structure_element" ], [ 204, 213, "1442\u20131445", "residue_range" ] ] }, { "sid": 387, "sent": "Thus, sliding of eEF2 involves reorganization of electrostatic, perhaps isoenergetic interactions, echoing those implied in extraordinarily fast ribosome inactivation rates by the small-protein ribotoxins and in fast protein association and diffusion along DNA.", "section": "DISCUSS", "ner": [ [ 17, 21, "eEF2", "protein" ], [ 49, 97, "electrostatic, perhaps isoenergetic interactions", "bond_interaction" ], [ 145, 153, "ribosome", "complex_assembly" ] ] }, { "sid": 388, "sent": "Comparison of our structures with the 80S\u2022IRES initiation structure reveals the structural basis for the second key function of the translocase: 'unlocking' of intrasubunit rearrangements that are required for step-wise translocation of PKI on the small subunit.", "section": "DISCUSS", "ner": [ [ 0, 10, "Comparison", "experimental_method" ], [ 18, 28, "structures", "evidence" ], [ 38, 46, "80S\u2022IRES", "complex_assembly" ], [ 47, 57, "initiation", "protein_state" ], [ 58, 67, "structure", "evidence" ], [ 132, 143, "translocase", "protein_type" ], [ 237, 240, "PKI", "structure_element" ], [ 248, 261, "small subunit", "structure_element" ] ] }, { "sid": 389, "sent": "The unlocking model of the ribosome\u20222tRNA\u2022mRNA pre-translocation complex has been proposed decades ago and functional requirement of the translocase in this process has been implicated.", "section": "DISCUSS", "ner": [ [ 27, 46, "ribosome\u20222tRNA\u2022mRNA", "complex_assembly" ], [ 47, 64, "pre-translocation", "protein_state" ], [ 137, 148, "translocase", "protein_type" ] ] }, { "sid": 390, "sent": "However, the structural and mechanistic definitions of the locked and unlocked states have remained unclear, ranging from the globally distinct ribosome conformations to unknown local rearrangements, e.g. those in the decoding center.", "section": "DISCUSS", "ner": [ [ 59, 65, "locked", "protein_state" ], [ 70, 78, "unlocked", "protein_state" ], [ 144, 152, "ribosome", "complex_assembly" ], [ 218, 233, "decoding center", "site" ] ] }, { "sid": 391, "sent": "FRET data indicate that translocation of 2tRNA\u2022mRNA on the 70S ribosome requires a forward-and-reverse head swivel, which may be related to the unlocking phenomenon.", "section": "DISCUSS", "ner": [ [ 0, 9, "FRET data", "evidence" ], [ 41, 51, "2tRNA\u2022mRNA", "complex_assembly" ], [ 59, 71, "70S ribosome", "complex_assembly" ], [ 103, 107, "head", "structure_element" ] ] }, { "sid": 392, "sent": "Whereas intersubunit rotation of the pre-translocation complex occurs spontaneously, the head swivel is induced by the eEF2/EF-G translocase, consistent with requirement of eEF2 for unlocking.", "section": "DISCUSS", "ner": [ [ 37, 54, "pre-translocation", "protein_state" ], [ 89, 93, "head", "structure_element" ], [ 119, 123, "eEF2", "protein" ], [ 124, 128, "EF-G", "protein" ], [ 129, 140, "translocase", "protein_type" ], [ 173, 177, "eEF2", "protein" ] ] }, { "sid": 393, "sent": "Structural studies revealed large head swivels in various 70S\u2022tRNA\u2022EF-G and 80S\u2022tRNA\u2022eEF2 complexes, but not in 'locked' complexes with the A site occupied by the tRNA in the absence of the translocase.", "section": "DISCUSS", "ner": [ [ 0, 18, "Structural studies", "experimental_method" ], [ 34, 38, "head", "structure_element" ], [ 58, 71, "70S\u2022tRNA\u2022EF-G", "complex_assembly" ], [ 76, 89, "80S\u2022tRNA\u2022eEF2", "complex_assembly" ], [ 113, 119, "locked", "protein_state" ], [ 121, 135, "complexes with", "protein_state" ], [ 140, 146, "A site", "site" ], [ 163, 167, "tRNA", "chemical" ], [ 175, 185, "absence of", "protein_state" ], [ 190, 201, "translocase", "protein_type" ] ] }, { "sid": 394, "sent": "Our structures suggest that eEF2 induces head swivel by 'unlocking' the head-body interactions (Figure 7).", "section": "DISCUSS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 28, 32, "eEF2", "protein" ], [ 41, 45, "head", "structure_element" ], [ 72, 76, "head", "structure_element" ], [ 77, 81, "body", "structure_element" ] ] }, { "sid": 395, "sent": "Binding of the ASL to the A site is known from structural studies of bacterial ribosomes to result in 'domain closure' of the small subunit, i.e. closer association of the head, shoulder and body domains.", "section": "DISCUSS", "ner": [ [ 15, 18, "ASL", "structure_element" ], [ 26, 32, "A site", "site" ], [ 47, 65, "structural studies", "experimental_method" ], [ 69, 78, "bacterial", "taxonomy_domain" ], [ 79, 88, "ribosomes", "complex_assembly" ], [ 103, 117, "domain closure", "protein_state" ], [ 126, 139, "small subunit", "structure_element" ], [ 172, 176, "head", "structure_element" ], [ 178, 186, "shoulder", "structure_element" ], [ 191, 195, "body", "structure_element" ] ] }, { "sid": 396, "sent": "The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli).", "section": "DISCUSS", "ner": [ [ 35, 39, "tRNA", "chemical" ], [ 47, 53, "A site", "site" ], [ 58, 66, "stacking", "bond_interaction" ], [ 74, 78, "head", "structure_element" ], [ 79, 85, "A site", "site" ], [ 87, 92, "C1274", "residue_name_number" ], [ 96, 109, "S. cerevisiae", "species" ], [ 113, 118, "C1054", "residue_name_number" ], [ 122, 129, "E. coli", "species" ], [ 157, 161, "body", "structure_element" ], [ 162, 168, "A-site", "site" ], [ 181, 186, "A1755", "residue_name_number" ], [ 191, 196, "A1756", "residue_name_number" ], [ 198, 203, "A1492", "residue_name_number" ], [ 208, 213, "A1493", "residue_name_number" ], [ 217, 224, "E. coli", "species" ] ] }, { "sid": 397, "sent": "This 'locked' state is identical to that observed for PKI in the 80S\u2022IRES initiation structures in the absence of eEF2.", "section": "DISCUSS", "ner": [ [ 6, 12, "locked", "protein_state" ], [ 54, 57, "PKI", "structure_element" ], [ 65, 73, "80S\u2022IRES", "complex_assembly" ], [ 74, 84, "initiation", "protein_state" ], [ 85, 95, "structures", "evidence" ], [ 103, 113, "absence of", "protein_state" ], [ 114, 118, "eEF2", "protein" ] ] }, { "sid": 398, "sent": "Structure I demonstrates that at an early pre-translocation step, the histidine-diphthamide tip of eEF2 is wedged between A1755 and A1756 and PKI.", "section": "DISCUSS", "ner": [ [ 0, 11, "Structure I", "evidence" ], [ 42, 59, "pre-translocation", "protein_state" ], [ 70, 95, "histidine-diphthamide tip", "site" ], [ 99, 103, "eEF2", "protein" ], [ 122, 127, "A1755", "residue_name_number" ], [ 132, 137, "A1756", "residue_name_number" ], [ 142, 145, "PKI", "structure_element" ] ] }, { "sid": 399, "sent": "This destabilization allows PKI to detach from the body A site upon spontaneous reverse 40S body rotation, while maintaining interactions with the head A site.", "section": "DISCUSS", "ner": [ [ 28, 31, "PKI", "structure_element" ], [ 51, 55, "body", "structure_element" ], [ 56, 62, "A site", "site" ], [ 88, 91, "40S", "complex_assembly" ], [ 92, 96, "body", "structure_element" ], [ 147, 151, "head", "structure_element" ], [ 152, 158, "A site", "site" ] ] }, { "sid": 400, "sent": "Destabilization of the head-bound PKI at the body A site thus allows mobility of the head relative to the body.", "section": "DISCUSS", "ner": [ [ 23, 33, "head-bound", "protein_state" ], [ 34, 37, "PKI", "structure_element" ], [ 45, 49, "body", "structure_element" ], [ 50, 56, "A site", "site" ], [ 85, 89, "head", "structure_element" ], [ 106, 110, "body", "structure_element" ] ] }, { "sid": 401, "sent": "The histidine-diphthamide-induced disengagement of PKI from A1755 and A1756 therefore provides the structural definition for the 'unlocking' mode of eEF2 action.", "section": "DISCUSS", "ner": [ [ 4, 25, "histidine-diphthamide", "ptm" ], [ 51, 54, "PKI", "structure_element" ], [ 60, 65, "A1755", "residue_name_number" ], [ 70, 75, "A1756", "residue_name_number" ], [ 149, 153, "eEF2", "protein" ] ] }, { "sid": 402, "sent": "In summary, our structures are consistent with a model of eEF2-induced translocation in which both PKI and eEF2 passively migrate into the P and A site, respectively, during spontaneous 40S body rotation and head swivel, the latter being allowed by 'unlocking' of the A site by eEF2.", "section": "DISCUSS", "ner": [ [ 16, 26, "structures", "evidence" ], [ 58, 62, "eEF2", "protein" ], [ 99, 102, "PKI", "structure_element" ], [ 107, 111, "eEF2", "protein" ], [ 139, 151, "P and A site", "site" ], [ 186, 189, "40S", "complex_assembly" ], [ 190, 194, "body", "structure_element" ], [ 208, 212, "head", "structure_element" ], [ 268, 274, "A site", "site" ], [ 278, 282, "eEF2", "protein" ] ] }, { "sid": 403, "sent": "Observation of different PKI conformations sampling a range of positions between the A and P sites in the presence of eEF2\u2022GDP implies that thermal fluctuations of the 40S head domain are sufficient for translocation along the energetically flat trajectory.", "section": "DISCUSS", "ner": [ [ 25, 28, "PKI", "structure_element" ], [ 85, 98, "A and P sites", "site" ], [ 106, 117, "presence of", "protein_state" ], [ 118, 126, "eEF2\u2022GDP", "complex_assembly" ], [ 168, 171, "40S", "complex_assembly" ], [ 172, 176, "head", "structure_element" ] ] }, { "sid": 404, "sent": "Insights into eEF2 association with and dissociation from the ribosome", "section": "DISCUSS", "ner": [ [ 14, 18, "eEF2", "protein" ], [ 62, 70, "ribosome", "complex_assembly" ] ] }, { "sid": 405, "sent": "The conformational rearrangements in eEF2 from Structure I through Structure V provide insights into the mechanisms of eEF2 association with the pre-translocation ribosome and dissociation from the post-translocation ribosome.", "section": "DISCUSS", "ner": [ [ 37, 41, "eEF2", "protein" ], [ 47, 58, "Structure I", "evidence" ], [ 67, 78, "Structure V", "evidence" ], [ 119, 123, "eEF2", "protein" ], [ 145, 162, "pre-translocation", "protein_state" ], [ 163, 171, "ribosome", "complex_assembly" ], [ 198, 216, "post-translocation", "protein_state" ], [ 217, 225, "ribosome", "complex_assembly" ] ] }, { "sid": 406, "sent": "In all five structures, the GTPase domain is attached to the P stalk and the sarcin-ricin loop.", "section": "DISCUSS", "ner": [ [ 12, 22, "structures", "evidence" ], [ 28, 41, "GTPase domain", "structure_element" ], [ 61, 68, "P stalk", "structure_element" ], [ 77, 94, "sarcin-ricin loop", "structure_element" ] ] }, { "sid": 407, "sent": "In the fully-rotated pre-translocation-like Structure I, an additional interaction exists.", "section": "DISCUSS", "ner": [ [ 7, 20, "fully-rotated", "protein_state" ], [ 21, 38, "pre-translocation", "protein_state" ], [ 44, 55, "Structure I", "evidence" ] ] }, { "sid": 408, "sent": "Here, switch loop I interacts with helix 14 (415CAAA418) of the 18S rRNA.", "section": "DISCUSS", "ner": [ [ 6, 19, "switch loop I", "structure_element" ], [ 35, 43, "helix 14", "structure_element" ], [ 45, 55, "415CAAA418", "structure_element" ], [ 64, 72, "18S rRNA", "chemical" ] ] }, { "sid": 409, "sent": "This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S\u2022eEF2 complex bound with a transition-state mimic GDP\u2022AlF4\u2013. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens).", "section": "DISCUSS", "ner": [ [ 31, 44, "GTPase center", "site" ], [ 56, 65, "GTP-bound", "protein_state" ], [ 115, 136, "translational GTPases", "protein_type" ], [ 144, 155, "presence of", "protein_state" ], [ 156, 159, "GTP", "chemical" ], [ 179, 187, "80S\u2022eEF2", "complex_assembly" ], [ 196, 206, "bound with", "protein_state" ], [ 232, 241, "GDP\u2022AlF4\u2013", "complex_assembly" ], [ 247, 258, "switch loop", "structure_element" ], [ 280, 284, "A416", "residue_name_number" ], [ 286, 296, "invariable", "protein_state" ], [ 297, 301, "A344", "residue_name_number" ], [ 305, 312, "E. coli", "species" ], [ 317, 321, "A463", "residue_name_number" ], [ 325, 335, "H. sapiens", "species" ] ] }, { "sid": 410, "sent": "Mutations of residues flanking A344 in E. coli 16S rRNA modestly inhibit translation but do not specifically affect EF-G-mediated translocation.", "section": "DISCUSS", "ner": [ [ 0, 9, "Mutations", "experimental_method" ], [ 31, 35, "A344", "residue_name_number" ], [ 39, 46, "E. coli", "species" ], [ 47, 55, "16S rRNA", "chemical" ], [ 116, 120, "EF-G", "protein" ] ] }, { "sid": 411, "sent": "However, the effect of A344 mutation on translation was not addressed in that study, leaving the question open whether this residue is critical for eEF2/EF-G function.", "section": "DISCUSS", "ner": [ [ 23, 27, "A344", "residue_name_number" ], [ 28, 36, "mutation", "experimental_method" ], [ 148, 152, "eEF2", "protein" ], [ 153, 157, "EF-G", "protein" ] ] }, { "sid": 412, "sent": "The interaction between h14 and switch loop I is not resolved in Structures II to V, in all of which the small subunit is partially rotated or non-rotated, so that helix 14 is placed at least 6\u2009\u00c5 farther from eEF2 (Figure 5d).", "section": "DISCUSS", "ner": [ [ 24, 27, "h14", "structure_element" ], [ 32, 45, "switch loop I", "structure_element" ], [ 65, 83, "Structures II to V", "evidence" ], [ 105, 118, "small subunit", "structure_element" ], [ 122, 139, "partially rotated", "protein_state" ], [ 143, 154, "non-rotated", "protein_state" ], [ 164, 172, "helix 14", "structure_element" ], [ 209, 213, "eEF2", "protein" ] ] }, { "sid": 413, "sent": "We conclude that unlike other conformations of the ribosome, the fully rotated 40S subunit of the pre-translocation ribosome provides an interaction surface, complementing the P stalk and SRL, for binding of the GTP-bound translocase.", "section": "DISCUSS", "ner": [ [ 51, 59, "ribosome", "complex_assembly" ], [ 65, 78, "fully rotated", "protein_state" ], [ 79, 82, "40S", "complex_assembly" ], [ 83, 90, "subunit", "structure_element" ], [ 98, 115, "pre-translocation", "protein_state" ], [ 116, 124, "ribosome", "complex_assembly" ], [ 137, 156, "interaction surface", "site" ], [ 176, 183, "P stalk", "structure_element" ], [ 188, 191, "SRL", "structure_element" ], [ 212, 221, "GTP-bound", "protein_state" ], [ 222, 233, "translocase", "protein_type" ] ] }, { "sid": 414, "sent": "This structural basis rationalizes the observation of transient stabilization of the rotated 70S ribosome upon EF-G\u2022GTP binding and prior to translocation.", "section": "DISCUSS", "ner": [ [ 85, 92, "rotated", "protein_state" ], [ 93, 105, "70S ribosome", "complex_assembly" ], [ 111, 119, "EF-G\u2022GTP", "complex_assembly" ] ] }, { "sid": 415, "sent": "The least rotated conformation of the post-translocation Structure V suggests conformational changes that may trigger eEF2 release from the ribosome at the end of translocation.", "section": "DISCUSS", "ner": [ [ 4, 17, "least rotated", "protein_state" ], [ 38, 56, "post-translocation", "protein_state" ], [ 57, 68, "Structure V", "evidence" ], [ 118, 122, "eEF2", "protein" ], [ 140, 148, "ribosome", "complex_assembly" ] ] }, { "sid": 416, "sent": "The most pronounced inter-domain rearrangement in eEF2 involves movement of domain III.", "section": "DISCUSS", "ner": [ [ 50, 54, "eEF2", "protein" ], [ 83, 86, "III", "structure_element" ] ] }, { "sid": 417, "sent": "In the rotated or mid-rotated Structures I through III, this domain remains rigidly associated with domain V and the N-terminal superdomain and does not undergo noticeable rearrangements.", "section": "DISCUSS", "ner": [ [ 7, 14, "rotated", "protein_state" ], [ 18, 29, "mid-rotated", "protein_state" ], [ 30, 54, "Structures I through III", "evidence" ], [ 107, 108, "V", "structure_element" ], [ 128, 139, "superdomain", "structure_element" ] ] }, { "sid": 418, "sent": "In Structure V, however, the tip of helix A of domain III is displaced toward domain I by ~5\u2009\u00c5 relative to that in mid-rotated or fully rotated structures.", "section": "DISCUSS", "ner": [ [ 3, 14, "Structure V", "evidence" ], [ 36, 43, "helix A", "structure_element" ], [ 54, 57, "III", "structure_element" ], [ 85, 86, "I", "structure_element" ], [ 115, 126, "mid-rotated", "protein_state" ], [ 130, 143, "fully rotated", "protein_state" ], [ 144, 154, "structures", "evidence" ] ] }, { "sid": 419, "sent": "This displacement is caused by the 8\u2009\u00c5 movement of the 40S body protein uS12 upon reverse intersubunit rotation from Structure I to V (Figure 6d).", "section": "DISCUSS", "ner": [ [ 55, 58, "40S", "complex_assembly" ], [ 59, 63, "body", "structure_element" ], [ 72, 76, "uS12", "protein" ], [ 117, 133, "Structure I to V", "evidence" ] ] }, { "sid": 420, "sent": "We propose that the shift of domain III by uS12 initiates intra-domain rearrangements in eEF2, which unstack the \u03b2-platform of domain III from that of domain V. This would result in a conformation characteristic of free eEF2 and EF-G in which the \u03b2-platforms are nearly perpendicular.", "section": "DISCUSS", "ner": [ [ 36, 39, "III", "structure_element" ], [ 43, 47, "uS12", "protein" ], [ 89, 93, "eEF2", "protein" ], [ 113, 123, "\u03b2-platform", "structure_element" ], [ 134, 137, "III", "structure_element" ], [ 158, 159, "V", "structure_element" ], [ 215, 219, "free", "protein_state" ], [ 220, 224, "eEF2", "protein" ], [ 229, 233, "EF-G", "protein" ], [ 247, 258, "\u03b2-platforms", "structure_element" ] ] }, { "sid": 421, "sent": "As we discuss below, Structure V captures a 'pre-unstacking' state due to stabilization of the interface between domains III and V by sordarin.", "section": "DISCUSS", "ner": [ [ 21, 32, "Structure V", "evidence" ], [ 45, 59, "pre-unstacking", "protein_state" ], [ 95, 104, "interface", "site" ], [ 121, 124, "III", "structure_element" ], [ 129, 130, "V", "structure_element" ], [ 134, 142, "sordarin", "chemical" ] ] }, { "sid": 422, "sent": "Sordarin stabilizes GDP-bound eEF2 on the ribosome", "section": "DISCUSS", "ner": [ [ 0, 8, "Sordarin", "chemical" ], [ 20, 29, "GDP-bound", "protein_state" ], [ 30, 34, "eEF2", "protein" ], [ 42, 50, "ribosome", "complex_assembly" ] ] }, { "sid": 423, "sent": "Sordarin is a potent antifungal antibiotic that inhibits translation.", "section": "DISCUSS", "ner": [ [ 0, 8, "Sordarin", "chemical" ] ] }, { "sid": 424, "sent": "Based on biochemical experiments, two alternative mechanisms of action were proposed: sordarin either prevents eEF2 departure by inhibiting GTP hydrolysis or acts after GTP hydrolysis.", "section": "DISCUSS", "ner": [ [ 9, 32, "biochemical experiments", "experimental_method" ], [ 86, 94, "sordarin", "chemical" ], [ 111, 115, "eEF2", "protein" ], [ 140, 143, "GTP", "chemical" ], [ 169, 172, "GTP", "chemical" ] ] }, { "sid": 425, "sent": "Although our complex was assembled using eEF2\u2022GTP, density maps clearly show GDP and Mg2+ in each structure (Figure 5g).", "section": "DISCUSS", "ner": [ [ 41, 49, "eEF2\u2022GTP", "complex_assembly" ], [ 51, 63, "density maps", "evidence" ], [ 77, 80, "GDP", "chemical" ], [ 85, 89, "Mg2+", "chemical" ], [ 98, 107, "structure", "evidence" ] ] }, { "sid": 426, "sent": "Our structures therefore indicate that sordarin stalls eEF2 on the ribosome in the GDP-bound form, i.e. following GTP hydrolysis and phosphate release.", "section": "DISCUSS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 39, 47, "sordarin", "chemical" ], [ 55, 59, "eEF2", "protein" ], [ 67, 75, "ribosome", "complex_assembly" ], [ 83, 92, "GDP-bound", "protein_state" ], [ 114, 117, "GTP", "chemical" ] ] }, { "sid": 427, "sent": "The mechanism of stalling is suggested by comparison of pre-translocation and post-translocation structures in our ensemble.", "section": "DISCUSS", "ner": [ [ 56, 73, "pre-translocation", "protein_state" ], [ 78, 96, "post-translocation", "protein_state" ], [ 97, 107, "structures", "evidence" ] ] }, { "sid": 428, "sent": "In all five structures, sordarin is bound between domains III and V of eEF2, stabilized by hydrophobic interactions identical to those in the isolated eEF2\u2022sordarin complex (Figures 5g and h).", "section": "DISCUSS", "ner": [ [ 12, 22, "structures", "evidence" ], [ 24, 32, "sordarin", "chemical" ], [ 36, 41, "bound", "protein_state" ], [ 58, 61, "III", "structure_element" ], [ 66, 67, "V", "structure_element" ], [ 71, 75, "eEF2", "protein" ], [ 91, 115, "hydrophobic interactions", "bond_interaction" ], [ 142, 150, "isolated", "protein_state" ], [ 151, 164, "eEF2\u2022sordarin", "complex_assembly" ] ] }, { "sid": 429, "sent": "In the nearly non-rotated post-translocation Structure V, the tip of domain III is shifted, however the interface between domains III and V remains unchanged, suggesting strong stabilization of this interface by sordarin.", "section": "DISCUSS", "ner": [ [ 7, 25, "nearly non-rotated", "protein_state" ], [ 26, 44, "post-translocation", "protein_state" ], [ 45, 56, "Structure V", "evidence" ], [ 76, 79, "III", "structure_element" ], [ 104, 113, "interface", "site" ], [ 130, 133, "III", "structure_element" ], [ 138, 139, "V", "structure_element" ], [ 199, 208, "interface", "site" ], [ 212, 220, "sordarin", "chemical" ] ] }, { "sid": 430, "sent": "We note that Structure V is slightly more rotated than the 80S\u20222tRNA\u2022mRNA complex in the absence of eEF2\u2022sordarin, implying that sordarin interferes with the final stages of reverse rotation of the post-translocation ribosome.", "section": "DISCUSS", "ner": [ [ 13, 24, "Structure V", "evidence" ], [ 59, 73, "80S\u20222tRNA\u2022mRNA", "complex_assembly" ], [ 89, 99, "absence of", "protein_state" ], [ 100, 113, "eEF2\u2022sordarin", "complex_assembly" ], [ 129, 137, "sordarin", "chemical" ], [ 198, 216, "post-translocation", "protein_state" ], [ 217, 225, "ribosome", "complex_assembly" ] ] }, { "sid": 431, "sent": "We propose that sordarin acts to prevent full reverse rotation and release of eEF2\u2022GDP by stabilizing the interdomain interface and thus blocking uS12-induced disengagement of domain III from domain V.", "section": "DISCUSS", "ner": [ [ 16, 24, "sordarin", "chemical" ], [ 78, 86, "eEF2\u2022GDP", "complex_assembly" ], [ 106, 127, "interdomain interface", "site" ], [ 146, 150, "uS12", "protein" ], [ 183, 186, "III", "structure_element" ], [ 199, 200, "V", "structure_element" ] ] }, { "sid": 432, "sent": "Implications for tRNA and mRNA translocation during translation", "section": "DISCUSS", "ner": [ [ 17, 21, "tRNA", "chemical" ], [ 26, 30, "mRNA", "chemical" ] ] }, { "sid": 433, "sent": "Because translocation of tRNA must involve large-scale dynamics, this step has long been regarded as the most puzzling step of translation.", "section": "DISCUSS", "ner": [ [ 25, 29, "tRNA", "chemical" ] ] }, { "sid": 434, "sent": "Intersubunit rearrangements and tRNA hybrid states have been proposed to play key roles half a century ago.", "section": "DISCUSS", "ner": [ [ 32, 36, "tRNA", "chemical" ], [ 37, 43, "hybrid", "protein_state" ] ] }, { "sid": 435, "sent": "Despite an impressive body of biochemical, fluorescence and structural data accumulated since then, translocation remains the least understood step of elongation.", "section": "DISCUSS", "ner": [ [ 22, 26, "body", "structure_element" ], [ 30, 75, "biochemical, fluorescence and structural data", "evidence" ] ] }, { "sid": 436, "sent": "The structural understanding of ribosome and tRNA dynamics has been greatly aided by a wealth of X-ray and cryo-EM structures (reviewed in).", "section": "DISCUSS", "ner": [ [ 32, 40, "ribosome", "complex_assembly" ], [ 45, 49, "tRNA", "chemical" ], [ 97, 102, "X-ray", "experimental_method" ], [ 107, 114, "cryo-EM", "experimental_method" ], [ 115, 125, "structures", "evidence" ] ] }, { "sid": 437, "sent": "However, visualization of the eEF2/EF-G-induced translocation is confined to very early pre-EF-G-entry states and late (almost translocated or fully translocated) states, leaving most of the path from the A to the P site uncharacterized (Figure 1\u2014figure supplement 1).", "section": "DISCUSS", "ner": [ [ 30, 34, "eEF2", "protein" ], [ 35, 39, "EF-G", "protein" ], [ 88, 102, "pre-EF-G-entry", "protein_state" ], [ 120, 139, "almost translocated", "protein_state" ], [ 143, 161, "fully translocated", "protein_state" ], [ 205, 220, "A to the P site", "site" ] ] }, { "sid": 438, "sent": "Our study provides new insights into the structural understanding of tRNA translocation.", "section": "DISCUSS", "ner": [ [ 69, 73, "tRNA", "chemical" ] ] }, { "sid": 439, "sent": "First, we propose that tRNA and IRES translocations occur via the same general trajectory.", "section": "DISCUSS", "ner": [ [ 23, 27, "tRNA", "chemical" ], [ 32, 36, "IRES", "site" ] ] }, { "sid": 440, "sent": "This is evident from the fact that ribosome rearrangements in translocation are inherent to the ribosome and likely occur in similar ways in both cases.", "section": "DISCUSS", "ner": [ [ 35, 43, "ribosome", "complex_assembly" ], [ 96, 104, "ribosome", "complex_assembly" ] ] }, { "sid": 441, "sent": "Furthermore, the step-wise coupling of ribosome dynamics with IRES translocation is overall consistent with that observed for 2tRNA\u2022mRNA translocation in solution.", "section": "DISCUSS", "ner": [ [ 39, 47, "ribosome", "complex_assembly" ], [ 62, 66, "IRES", "site" ], [ 126, 136, "2tRNA\u2022mRNA", "complex_assembly" ] ] }, { "sid": 442, "sent": "For example, fluorescence and biochemical studies revealed that the early pre-translocation EF-G-bound ribosomes are fully rotated and translocation of the tRNA-mRNA complex occurs during reverse rotation of the small subunit, coupled with head swivel.", "section": "DISCUSS", "ner": [ [ 13, 49, "fluorescence and biochemical studies", "experimental_method" ], [ 74, 91, "pre-translocation", "protein_state" ], [ 92, 102, "EF-G-bound", "protein_state" ], [ 103, 112, "ribosomes", "complex_assembly" ], [ 117, 130, "fully rotated", "protein_state" ], [ 156, 165, "tRNA-mRNA", "complex_assembly" ], [ 212, 225, "small subunit", "structure_element" ], [ 240, 244, "head", "structure_element" ] ] }, { "sid": 443, "sent": "The sequence of ribosome rearrangements during IRES translocation also agrees with that inferred from 70S\u2022EF-G structures, including those in which the A-to-P-site translocating tRNA was not present.", "section": "DISCUSS", "ner": [ [ 16, 24, "ribosome", "complex_assembly" ], [ 47, 51, "IRES", "site" ], [ 102, 110, "70S\u2022EF-G", "complex_assembly" ], [ 111, 121, "structures", "evidence" ], [ 152, 163, "A-to-P-site", "site" ], [ 178, 182, "tRNA", "chemical" ] ] }, { "sid": 444, "sent": "Specifically, an earlier translocation intermediate ribosome (TIpre) was proposed to adopt a rotated (7\u20139\u00b0) body and a partly rotated head (5\u20137.5\u00b0), in agreement with the conformation of our Structure I. The most swiveled head (18\u201321\u00b0) was observed in a mid-rotated ribosome (3\u20135\u00b0) of a later translocation intermediate TIpost, similar to the conformation of our Structure III.", "section": "DISCUSS", "ner": [ [ 52, 60, "ribosome", "complex_assembly" ], [ 93, 100, "rotated", "protein_state" ], [ 108, 112, "body", "structure_element" ], [ 119, 133, "partly rotated", "protein_state" ], [ 134, 138, "head", "structure_element" ], [ 191, 202, "Structure I", "evidence" ], [ 208, 221, "most swiveled", "protein_state" ], [ 222, 226, "head", "structure_element" ], [ 254, 265, "mid-rotated", "protein_state" ], [ 266, 274, "ribosome", "complex_assembly" ], [ 363, 376, "Structure III", "evidence" ] ] }, { "sid": 445, "sent": "Overall, these correlations suggest that the intermediate locations of the elusive A-to-P-site translocating tRNA are similar to those of PKI in our structures.", "section": "DISCUSS", "ner": [ [ 83, 94, "A-to-P-site", "site" ], [ 109, 113, "tRNA", "chemical" ], [ 138, 141, "PKI", "structure_element" ], [ 149, 159, "structures", "evidence" ] ] }, { "sid": 446, "sent": "Second, the structures clarify the structural basis of the often-used but structurally undefined terms 'locking' and 'unlocking' with respect to the pre-translocation complex (Figure 6f).", "section": "DISCUSS", "ner": [ [ 12, 22, "structures", "evidence" ], [ 149, 166, "pre-translocation", "protein_state" ] ] }, { "sid": 447, "sent": "We deem the pre-translocation complex locked, because the A-site bound ASL-mRNA is stabilized by interactions with the decoding center.", "section": "DISCUSS", "ner": [ [ 12, 29, "pre-translocation", "protein_state" ], [ 38, 44, "locked", "protein_state" ], [ 58, 70, "A-site bound", "protein_state" ], [ 75, 79, "mRNA", "chemical" ], [ 119, 134, "decoding center", "site" ] ] }, { "sid": 448, "sent": "These interactions are maintained for the classical- and hybrid-state tRNAs in the spontaneously sampled non-rotated and rotated ribosomes, respectively.", "section": "DISCUSS", "ner": [ [ 42, 51, "classical", "protein_state" ], [ 57, 63, "hybrid", "protein_state" ], [ 70, 75, "tRNAs", "chemical" ], [ 105, 116, "non-rotated", "protein_state" ], [ 121, 128, "rotated", "protein_state" ], [ 129, 138, "ribosomes", "complex_assembly" ] ] }, { "sid": 449, "sent": "Unlocking involves separation of the codon-anticodon helix from the decoding center residues by the protruding tip of eEF2/EF-G (Figure 7), occurring in the fully rotated ribosome at an early pre-translocation step.", "section": "DISCUSS", "ner": [ [ 37, 58, "codon-anticodon helix", "structure_element" ], [ 68, 83, "decoding center", "site" ], [ 118, 122, "eEF2", "protein" ], [ 123, 127, "EF-G", "protein" ], [ 157, 170, "fully rotated", "protein_state" ], [ 171, 179, "ribosome", "complex_assembly" ], [ 192, 209, "pre-translocation", "protein_state" ] ] }, { "sid": 450, "sent": "This unlatches the head, allowing creation of hitherto elusive intermediate tRNA positions during spontaneous reverse body rotation.", "section": "DISCUSS", "ner": [ [ 19, 23, "head", "structure_element" ], [ 76, 80, "tRNA", "chemical" ], [ 118, 122, "body", "structure_element" ] ] }, { "sid": 451, "sent": "Third, our findings uncover a new role of the head swivel.", "section": "DISCUSS", "ner": [ [ 46, 50, "head", "structure_element" ] ] }, { "sid": 452, "sent": "Previous studies showed that this movement widens the constriction ('gate') between the P and E sites, thus allowing the P-tRNA passage to the E site.", "section": "DISCUSS", "ner": [ [ 54, 66, "constriction", "site" ], [ 69, 73, "gate", "site" ], [ 88, 101, "P and E sites", "site" ], [ 121, 122, "P", "site" ], [ 123, 127, "tRNA", "chemical" ], [ 143, 149, "E site", "site" ] ] }, { "sid": 453, "sent": "In addition to the 'gate-opening' role, we now show that the head swivel brings the head A site to the body P site, allowing a step-wise conveying of the codon-anticodon helix between the A and P sites.", "section": "DISCUSS", "ner": [ [ 20, 24, "gate", "site" ], [ 61, 65, "head", "structure_element" ], [ 84, 88, "head", "structure_element" ], [ 89, 95, "A site", "site" ], [ 103, 107, "body", "structure_element" ], [ 108, 114, "P site", "site" ], [ 154, 175, "codon-anticodon helix", "structure_element" ], [ 188, 201, "A and P sites", "site" ] ] }, { "sid": 454, "sent": "Finally, the similar populations of particles (within a 2X range) in our 80S\u2022IRES\u2022eEF2 reconstructions (Figure 1\u2014figure supplement 2) suggest that the intermediate translocation states sample several energetically similar and interconverting conformations.", "section": "DISCUSS", "ner": [ [ 36, 45, "particles", "experimental_method" ], [ 73, 86, "80S\u2022IRES\u2022eEF2", "complex_assembly" ], [ 87, 102, "reconstructions", "evidence" ] ] }, { "sid": 455, "sent": "This is consistent with the idea of a rather flat energy landscape of translocation, suggested by recent work that measured mechanical work produced by the ribosome during translocation.", "section": "DISCUSS", "ner": [ [ 156, 164, "ribosome", "complex_assembly" ] ] }, { "sid": 456, "sent": "Our findings implicate, however, that the energy landscape is not completely flat and contains local minima for transient positions of the codon-anticodon helix between the A and P sites.", "section": "DISCUSS", "ner": [ [ 139, 160, "codon-anticodon helix", "structure_element" ], [ 173, 186, "A and P sites", "site" ] ] }, { "sid": 457, "sent": "The shift of the PKI with respect to the body occurs during forward head swivel in two major sub-steps of ~4\u2009\u00c5 each (initiation complex to I, and I to II), after which PKI undergoes small shifts to settle in the body P site in Structures III, IV and V (Figure 2\u2014source data 1).", "section": "DISCUSS", "ner": [ [ 17, 20, "PKI", "structure_element" ], [ 41, 45, "body", "structure_element" ], [ 68, 72, "head", "structure_element" ], [ 117, 135, "initiation complex", "complex_assembly" ], [ 139, 140, "I", "evidence" ], [ 146, 147, "I", "evidence" ], [ 151, 153, "II", "evidence" ], [ 168, 171, "PKI", "structure_element" ], [ 212, 216, "body", "structure_element" ], [ 217, 223, "P site", "site" ], [ 227, 251, "Structures III, IV and V", "evidence" ] ] }, { "sid": 458, "sent": "Movement of PKI relative to the head occurs during the subsequent reverse swivel in three 3\u20137\u2009\u00c5 sub-steps (II to III to IV to V).", "section": "DISCUSS", "ner": [ [ 12, 15, "PKI", "structure_element" ], [ 32, 36, "head", "structure_element" ], [ 107, 127, "II to III to IV to V", "evidence" ] ] }, { "sid": 459, "sent": "We note that four of our near-atomic resolution maps comprised ~30,000 particles each, the minimum number required for a near-atomic-resolution reconstruction of the ribosome.", "section": "DISCUSS", "ner": [ [ 48, 52, "maps", "evidence" ], [ 71, 80, "particles", "experimental_method" ], [ 121, 158, "near-atomic-resolution reconstruction", "evidence" ], [ 166, 174, "ribosome", "complex_assembly" ] ] }, { "sid": 460, "sent": "Translation of viral mRNA", "section": "DISCUSS", "ner": [ [ 15, 20, "viral", "taxonomy_domain" ], [ 21, 25, "mRNA", "chemical" ] ] }, { "sid": 461, "sent": "Our work sheds light on the dynamic mechanism of cap-independent translation by IGR IRESs, tightly coupled with the universally conserved dynamic properties of the ribosome.", "section": "DISCUSS", "ner": [ [ 80, 83, "IGR", "structure_element" ], [ 84, 89, "IRESs", "site" ], [ 116, 137, "universally conserved", "protein_state" ], [ 164, 172, "ribosome", "complex_assembly" ] ] }, { "sid": 462, "sent": "The cryo-EM structures demonstrate that the TSV IRES structurally and dynamically represents a chimera of the 2tRNA\u2022mRNA translocating complex (A/P-tRNA \u2022 P/E-tRNA \u2022 mRNA).", "section": "DISCUSS", "ner": [ [ 4, 11, "cryo-EM", "experimental_method" ], [ 12, 22, "structures", "evidence" ], [ 44, 47, "TSV", "species" ], [ 48, 52, "IRES", "site" ], [ 110, 120, "2tRNA\u2022mRNA", "complex_assembly" ], [ 144, 170, "A/P-tRNA \u2022 P/E-tRNA \u2022 mRNA", "complex_assembly" ] ] }, { "sid": 463, "sent": "Like in the 2tRNA\u2022mRNA translocating complex in which the two tRNAs move independently of each other, the PKI domain moves relative to the 5\u00b4-domain, causing the IRES to undergo an inchworm-walk translocation.", "section": "DISCUSS", "ner": [ [ 12, 22, "2tRNA\u2022mRNA", "complex_assembly" ], [ 62, 67, "tRNAs", "chemical" ], [ 106, 109, "PKI", "structure_element" ], [ 139, 148, "5\u00b4-domain", "structure_element" ], [ 162, 166, "IRES", "site" ], [ 181, 189, "inchworm", "protein_state" ] ] }, { "sid": 464, "sent": "A large structural difference between the IRES and the 2tRNA\u2022mRNA complex exists, however, in that the IRES lacks three out of six tRNA-like domains involved in tRNA translocation.", "section": "DISCUSS", "ner": [ [ 42, 46, "IRES", "site" ], [ 55, 65, "2tRNA\u2022mRNA", "complex_assembly" ], [ 103, 107, "IRES", "site" ], [ 108, 113, "lacks", "protein_state" ], [ 131, 148, "tRNA-like domains", "structure_element" ], [ 161, 165, "tRNA", "chemical" ] ] }, { "sid": 465, "sent": "This difference likely accounts for the inefficient translocation of the IRES, which is difficult to stabilize in the post-translocation state and therefore is prone to reverse translocation.", "section": "DISCUSS", "ner": [ [ 73, 77, "IRES", "site" ], [ 118, 136, "post-translocation", "protein_state" ] ] }, { "sid": 466, "sent": "Although structurally handicapped, the TSV IRES manages to translocate by employing ribosome dynamics that are remarkably similar to that in 2tRNA\u2022mRNA translocation.", "section": "DISCUSS", "ner": [ [ 39, 42, "TSV", "species" ], [ 43, 47, "IRES", "site" ], [ 84, 92, "ribosome", "complex_assembly" ], [ 141, 151, "2tRNA\u2022mRNA", "complex_assembly" ] ] }, { "sid": 467, "sent": "The uniformity of ribosome dynamics underscores the idea that translocation is an inherent and structurally-optimized property of the ribosome, supported also by translocation activity in the absence of the elongation factor.", "section": "DISCUSS", "ner": [ [ 18, 26, "ribosome", "complex_assembly" ], [ 134, 142, "ribosome", "complex_assembly" ], [ 192, 202, "absence of", "protein_state" ], [ 207, 224, "elongation factor", "protein_type" ] ] }, { "sid": 468, "sent": "This property is rendered by the relative mobility of the three major building blocks, the 60S subunit and the 40S head and body, assisted by ligand-interacting extensions including the L1 stalk and the P stalk.", "section": "DISCUSS", "ner": [ [ 91, 94, "60S", "complex_assembly" ], [ 95, 102, "subunit", "structure_element" ], [ 111, 114, "40S", "complex_assembly" ], [ 115, 119, "head", "structure_element" ], [ 124, 128, "body", "structure_element" ], [ 142, 171, "ligand-interacting extensions", "structure_element" ], [ 186, 194, "L1 stalk", "structure_element" ], [ 203, 210, "P stalk", "structure_element" ] ] }, { "sid": 469, "sent": "Intergenic IRESs, in turn, represent a striking example of convergent molecular evolution.", "section": "DISCUSS", "ner": [ [ 11, 16, "IRESs", "site" ] ] }, { "sid": 470, "sent": "Viral mRNAs have evolved to adopt an atypical structure to employ the inherent ribosome dynamics, to be able to hijack the host translational machinery in a simple fashion.", "section": "DISCUSS", "ner": [ [ 0, 5, "Viral", "taxonomy_domain" ], [ 6, 11, "mRNAs", "chemical" ], [ 46, 55, "structure", "evidence" ], [ 79, 87, "ribosome", "complex_assembly" ] ] }, { "sid": 471, "sent": "Ensemble cryo-EM", "section": "DISCUSS", "ner": [ [ 9, 16, "cryo-EM", "experimental_method" ] ] }, { "sid": 472, "sent": "Our current understanding of macromolecular machines, such as the ribosome, is often limited by a gap between biophysical/biochemical studies and structural studies.", "section": "DISCUSS", "ner": [ [ 66, 74, "ribosome", "complex_assembly" ], [ 110, 141, "biophysical/biochemical studies", "experimental_method" ], [ 146, 164, "structural studies", "experimental_method" ] ] }, { "sid": 473, "sent": "For example, F\u00f6rster resonance energy transfer can provide insight into the macromolecular dynamics of an assembly at the single-molecule level but is limited to specifically labeled locations within the assembly.", "section": "DISCUSS", "ner": [ [ 13, 46, "F\u00f6rster resonance energy transfer", "experimental_method" ] ] }, { "sid": 474, "sent": "High-resolution crystal structures, on the other hand, can provide static images of an assembly, and the structural dynamics can only be inferred by comparing structures that are usually obtained in different experiments and under different, often non-native, conditions.", "section": "DISCUSS", "ner": [ [ 16, 34, "crystal structures", "evidence" ], [ 159, 169, "structures", "evidence" ] ] }, { "sid": 475, "sent": "Cryo-EM offers the possibility of obtaining integrated information of both structure and dynamics as demonstrated in lower-resolution studies of bacterial ribosome complexes.", "section": "DISCUSS", "ner": [ [ 0, 7, "Cryo-EM", "experimental_method" ], [ 75, 84, "structure", "evidence" ], [ 145, 154, "bacterial", "taxonomy_domain" ], [ 155, 163, "ribosome", "complex_assembly" ] ] }, { "sid": 476, "sent": "This is presumably one of the reasons why most recent studies of ribosome complexes have focused on a single high-resolution structure despite the non-uniform local resolution of the maps that likely reflects structural heterogeneity.", "section": "DISCUSS", "ner": [ [ 65, 73, "ribosome", "complex_assembly" ], [ 125, 134, "structure", "evidence" ], [ 183, 187, "maps", "evidence" ] ] }, { "sid": 477, "sent": "The computational efficiency of FREALIGN has allowed us to classify a relatively large dataset (1.1 million particles) into 15 classes (Figure 1\u2014figure supplement 2) and obtain eight near-atomic-resolution structures from it.", "section": "DISCUSS", "ner": [ [ 32, 40, "FREALIGN", "experimental_method" ], [ 108, 117, "particles", "experimental_method" ], [ 206, 216, "structures", "evidence" ] ] }, { "sid": 478, "sent": "The classification, which followed an initial alignment of all particles to a single reference, required about 130,000 CPU hours or about five to six full days on a 1000-CPU cluster.", "section": "DISCUSS", "ner": [ [ 63, 72, "particles", "experimental_method" ] ] }, { "sid": 479, "sent": "Therefore, cryo-EM has the potential to become a standard tool for uncovering detailed dynamic pathways of complex macromolecular machines.", "section": "DISCUSS", "ner": [ [ 11, 18, "cryo-EM", "experimental_method" ] ] } ] }, "PMC4792962": { "annotations": [ { "sid": 0, "sent": "A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome", "section": "TITLE", "ner": [ [ 40, 64, "autocatalytic activation", "ptm" ], [ 72, 86, "20S proteasome", "complex_assembly" ] ] }, { "sid": 1, "sent": "Biogenesis of the 20S proteasome is tightly regulated.", "section": "ABSTRACT", "ner": [ [ 18, 32, "20S proteasome", "complex_assembly" ] ] }, { "sid": 2, "sent": "The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly.", "section": "ABSTRACT", "ner": [ [ 15, 26, "propeptides", "structure_element" ], [ 42, 53, "active-site", "site" ], [ 54, 64, "threonines", "residue_name" ], [ 69, 86, "autocatalytically", "ptm" ] ] }, { "sid": 3, "sent": "However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic.", "section": "ABSTRACT", "ner": [ [ 68, 87, "strict conservation", "protein_state" ], [ 91, 100, "threonine", "residue_name" ] ] }, { "sid": 4, "sent": "Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad.", "section": "ABSTRACT", "ner": [ [ 12, 23, "mutagenesis", "experimental_method" ], [ 25, 46, "X-ray crystallography", "experimental_method" ], [ 51, 69, "biochemical assays", "experimental_method" ], [ 86, 91, "Lys33", "residue_name_number" ], [ 129, 139, "propeptide", "structure_element" ], [ 161, 165, "Thr1", "residue_name_number" ], [ 218, 223, "Asp17", "residue_name_number" ], [ 238, 253, "catalytic triad", "site" ] ] }, { "sid": 5, "sent": "Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome.", "section": "ABSTRACT", "ner": [ [ 0, 12, "Substitution", "experimental_method" ], [ 16, 20, "Thr1", "residue_name_number" ], [ 24, 27, "Cys", "residue_name" ], [ 58, 63, "Lys33", "residue_name_number" ], [ 68, 79, "inactivates", "protein_state" ], [ 84, 94, "proteasome", "complex_assembly" ] ] }, { "sid": 6, "sent": "Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates.", "section": "ABSTRACT", "ner": [ [ 11, 18, "Thr1Ser", "mutant" ], [ 19, 25, "mutant", "protein_state" ], [ 29, 35, "active", "protein_state" ], [ 72, 81, "wild type", "protein_state" ], [ 125, 129, "Ser1", "residue_name_number" ] ] }, { "sid": 7, "sent": "This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease.", "section": "ABSTRACT", "ner": [ [ 72, 92, "propeptide autolysis", "ptm" ], [ 147, 157, "proteasome", "complex_assembly" ], [ 170, 188, "threonine protease", "protein_type" ] ] }, { "sid": 8, "sent": " The proteasome, an essential molecular machine, is a threonine protease, but the evolution and the components of its proteolytic centre are unclear.", "section": "ABSTRACT", "ner": [ [ 5, 15, "proteasome", "complex_assembly" ], [ 54, 72, "threonine protease", "protein_type" ] ] }, { "sid": 9, "sent": "Here, the authors use structural biology and biochemistry to investigate the role of proteasome active site residues on maturation and activity.", "section": "ABSTRACT", "ner": [ [ 85, 95, "proteasome", "complex_assembly" ], [ 96, 107, "active site", "site" ] ] }, { "sid": 10, "sent": "The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells.", "section": "INTRO", "ner": [ [ 4, 32, "20S proteasome core particle", "complex_assembly" ], [ 34, 36, "CP", "complex_assembly" ], [ 49, 71, "non-lysosomal protease", "protein_type" ], [ 75, 85, "eukaryotic", "taxonomy_domain" ] ] }, { "sid": 11, "sent": "Its seven different \u03b1 and seven different \u03b2 subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder.", "section": "INTRO", "ner": [ [ 20, 21, "\u03b1", "protein" ], [ 42, 52, "\u03b2 subunits", "protein" ], [ 72, 82, "heptameric", "oligomeric_state" ], [ 83, 88, "rings", "structure_element" ], [ 130, 145, "hollow cylinder", "structure_element" ] ] }, { "sid": 12, "sent": "While the inactive \u03b1 subunits build the two outer rings, the \u03b2 subunits form the inner rings.", "section": "INTRO", "ner": [ [ 10, 18, "inactive", "protein_state" ], [ 19, 29, "\u03b1 subunits", "protein" ], [ 50, 55, "rings", "structure_element" ], [ 61, 71, "\u03b2 subunits", "protein" ], [ 87, 92, "rings", "structure_element" ] ] }, { "sid": 13, "sent": "Only three out of the seven different \u03b2 subunits, namely \u03b21, \u03b22 and \u03b25, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides.", "section": "INTRO", "ner": [ [ 38, 48, "\u03b2 subunits", "protein" ], [ 57, 59, "\u03b21", "protein" ], [ 61, 63, "\u03b22", "protein" ], [ 68, 70, "\u03b25", "protein" ], [ 88, 114, "proteolytic active centres", "site" ], [ 127, 129, "CP", "complex_assembly" ], [ 164, 175, "propeptides", "structure_element" ] ] }, { "sid": 14, "sent": "In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1).", "section": "INTRO", "ner": [ [ 21, 23, "CP", "complex_assembly" ], [ 40, 51, "prosegments", "structure_element" ], [ 56, 81, "autocatalytically removed", "ptm" ], [ 117, 136, "active site residue", "site" ], [ 137, 141, "Thr1", "residue_name_number" ], [ 182, 189, "Gly(-1)", "residue_name_number" ] ] }, { "sid": 15, "sent": "Release of the propeptides creates a functionally active CP that cleaves proteins into short peptides.", "section": "INTRO", "ner": [ [ 15, 26, "propeptides", "structure_element" ], [ 50, 56, "active", "protein_state" ], [ 57, 59, "CP", "complex_assembly" ] ] }, { "sid": 16, "sent": "Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active \u03b2 subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds.", "section": "INTRO", "ner": [ [ 36, 61, "substrate-binding channel", "site" ], [ 129, 135, "active", "protein_state" ], [ 136, 146, "\u03b2 subunits", "protein" ], [ 152, 164, "active sites", "site" ] ] }, { "sid": 17, "sent": "Nucleophilic attack of Thr1O\u03b3 on the carbonyl carbon atom of the scissile peptide bond creates a first cleavage product and a covalent acyl-enzyme intermediate.", "section": "INTRO", "ner": [ [ 23, 27, "Thr1", "residue_name_number" ] ] }, { "sid": 18, "sent": "Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment.", "section": "INTRO", "ner": [ [ 19, 26, "complex", "complex_assembly" ], [ 61, 66, "water", "chemical" ], [ 92, 98, "enzyme", "complex_assembly" ], [ 123, 130, "peptide", "chemical" ] ] }, { "sid": 19, "sent": "The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1O\u03b3 for peptide-bond cleavage.", "section": "INTRO", "ner": [ [ 4, 14, "proteasome", "complex_assembly" ], [ 40, 80, "N-terminal nucleophilic (Ntn) hydrolases", "protein_type" ], [ 90, 94, "free", "protein_state" ], [ 121, 125, "Thr1", "residue_name_number" ], [ 158, 162, "Thr1", "residue_name_number" ], [ 205, 209, "Thr1", "residue_name_number" ] ] }, { "sid": 20, "sent": "This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed.", "section": "INTRO", "ner": [ [ 40, 74, "autocatalytic precursor processing", "ptm" ], [ 90, 98, "immature", "protein_state" ], [ 99, 111, "active sites", "site" ], [ 113, 117, "Thr1", "residue_name_number" ], [ 152, 159, "Gly(-1)", "residue_name_number" ] ] }, { "sid": 21, "sent": "An alternative candidate for deprotonating the Thr1 hydroxyl group is the side chain of Lys33 as it is within hydrogen-bonding distance to Thr1OH (2.7\u2009\u00c5).", "section": "INTRO", "ner": [ [ 47, 51, "Thr1", "residue_name_number" ], [ 88, 93, "Lys33", "residue_name_number" ], [ 110, 126, "hydrogen-bonding", "bond_interaction" ], [ 139, 143, "Thr1", "residue_name_number" ] ] }, { "sid": 22, "sent": "In principle it could function as the general base during both autocatalytic removal of the propeptide and protein substrate cleavage.", "section": "INTRO", "ner": [ [ 63, 84, "autocatalytic removal", "ptm" ], [ 92, 102, "propeptide", "structure_element" ] ] }, { "sid": 23, "sent": "Here we provide experimental evidences for this distinct view of the proteasome active-site mechanism.", "section": "INTRO", "ner": [ [ 69, 79, "proteasome", "complex_assembly" ], [ 80, 91, "active-site", "site" ] ] }, { "sid": 24, "sent": "Data from biochemical and structural analyses of proteasome variants with mutations in the \u03b25 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome.", "section": "INTRO", "ner": [ [ 10, 45, "biochemical and structural analyses", "experimental_method" ], [ 91, 93, "\u03b25", "protein" ], [ 94, 104, "propeptide", "structure_element" ], [ 113, 124, "active site", "site" ], [ 228, 252, "autocatalytic activation", "ptm" ], [ 260, 270, "proteasome", "complex_assembly" ] ] }, { "sid": 25, "sent": "Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays.", "section": "INTRO", "ner": [ [ 44, 47, "Thr", "residue_name" ], [ 53, 56, "Cys", "residue_name" ], [ 60, 63, "Ser", "residue_name" ], [ 101, 122, "X-ray crystallography", "experimental_method" ], [ 137, 167, "activity and inhibition assays", "experimental_method" ] ] }, { "sid": 26, "sent": "Inactivation of proteasome subunits by T1A mutations", "section": "RESULTS", "ner": [ [ 16, 26, "proteasome", "complex_assembly" ], [ 27, 35, "subunits", "protein" ], [ 39, 42, "T1A", "mutant" ], [ 43, 52, "mutations", "experimental_method" ] ] }, { "sid": 27, "sent": "Proteasome-mediated degradation of cell-cycle regulators and potentially toxic misfolded proteins is required for the viability of eukaryotic cells.", "section": "RESULTS", "ner": [ [ 0, 10, "Proteasome", "complex_assembly" ], [ 131, 141, "eukaryotic", "taxonomy_domain" ] ] }, { "sid": 28, "sent": "Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites.", "section": "RESULTS", "ner": [ [ 20, 31, "active site", "site" ], [ 32, 36, "Thr1", "residue_name_number" ], [ 40, 51, "mutation to", "experimental_method" ], [ 52, 55, "Ala", "residue_name" ], [ 126, 136, "proteasome", "complex_assembly" ], [ 137, 149, "active sites", "site" ] ] }, { "sid": 29, "sent": "Yeast strains carrying the single mutations \u03b21-T1A or \u03b22-T1A, or both, are viable, even though one or two of the three distinct catalytic \u03b2 subunits are disabled and carry remnants of their N-terminal propeptides (Table 1).", "section": "RESULTS", "ner": [ [ 0, 5, "Yeast", "taxonomy_domain" ], [ 44, 50, "\u03b21-T1A", "mutant" ], [ 54, 60, "\u03b22-T1A", "mutant" ], [ 128, 137, "catalytic", "protein_state" ], [ 138, 148, "\u03b2 subunits", "protein" ], [ 153, 161, "disabled", "protein_state" ], [ 166, 183, "carry remnants of", "protein_state" ], [ 201, 212, "propeptides", "structure_element" ] ] }, { "sid": 30, "sent": "These results indicate that the \u03b21 and \u03b22 proteolytic activities are not essential for cell survival.", "section": "RESULTS", "ner": [ [ 32, 34, "\u03b21", "protein" ], [ 39, 41, "\u03b22", "protein" ] ] }, { "sid": 31, "sent": "By contrast, the T1A mutation in subunit \u03b25 has been reported to be lethal or nearly so.", "section": "RESULTS", "ner": [ [ 17, 20, "T1A", "mutant" ], [ 41, 43, "\u03b25", "protein" ] ] }, { "sid": 32, "sent": "Viability is restored if the \u03b25-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (\u03b25-T1A pp trans), although substantial phenotypic impairment remains (Table 1).", "section": "RESULTS", "ner": [ [ 29, 35, "\u03b25-T1A", "mutant" ], [ 52, 62, "propeptide", "structure_element" ], [ 64, 66, "pp", "chemical" ], [ 68, 100, "deleted but expressed separately", "experimental_method" ], [ 104, 109, "trans", "protein_state" ], [ 111, 117, "\u03b25-T1A", "mutant" ], [ 118, 120, "pp", "chemical" ], [ 121, 126, "trans", "protein_state" ] ] }, { "sid": 33, "sent": "Our present crystallographic analysis of the \u03b25-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed \u03b25 propeptide is not bound in the \u03b25 substrate-binding channel (Supplementary Fig. 1a).", "section": "RESULTS", "ner": [ [ 12, 37, "crystallographic analysis", "experimental_method" ], [ 45, 51, "\u03b25-T1A", "mutant" ], [ 52, 54, "pp", "chemical" ], [ 55, 60, "trans", "protein_state" ], [ 61, 67, "mutant", "protein_state" ], [ 90, 98, "mutation", "experimental_method" ], [ 138, 159, "catalytic active site", "site" ], [ 173, 188, "trans-expressed", "experimental_method" ], [ 189, 191, "\u03b25", "protein" ], [ 192, 202, "propeptide", "structure_element" ], [ 206, 215, "not bound", "protein_state" ], [ 223, 225, "\u03b25", "protein" ], [ 226, 251, "substrate-binding channel", "site" ] ] }, { "sid": 34, "sent": "The extremely weak growth of the \u03b25-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy.", "section": "RESULTS", "ner": [ [ 33, 39, "\u03b25-T1A", "mutant" ], [ 40, 46, "mutant", "protein_state" ], [ 47, 49, "pp", "chemical" ], [ 50, 53, "cis", "protein_state" ] ] }, { "sid": 35, "sent": "Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the \u03b25-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4\u20135 days for initial colony formation (Table 1 and Supplementary Methods).", "section": "RESULTS", "ner": [ [ 0, 26, "Sequencing of the plasmids", "experimental_method" ], [ 59, 64, "yeast", "taxonomy_domain" ], [ 88, 113, "site-directed mutagenesis", "experimental_method" ], [ 132, 138, "\u03b25-T1A", "mutant" ], [ 139, 145, "mutant", "protein_state" ], [ 146, 148, "pp", "chemical" ], [ 149, 152, "cis", "protein_state" ] ] }, { "sid": 36, "sent": "We also identified an additional point mutation K81R in subunit \u03b25 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits \u03b14, \u03b24 and \u03b25 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts.", "section": "RESULTS", "ner": [ [ 48, 52, "K81R", "mutant" ], [ 64, 66, "\u03b25", "protein" ], [ 112, 143, "This single amino-acid exchange", "experimental_method" ], [ 162, 171, "interface", "site" ], [ 188, 190, "\u03b14", "protein" ], [ 192, 194, "\u03b24", "protein" ], [ 199, 201, "\u03b25", "protein" ], [ 251, 253, "CP", "complex_assembly" ] ] }, { "sid": 37, "sent": "The slightly better growth of the \u03b25-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the \u03b25-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1).", "section": "RESULTS", "ner": [ [ 34, 45, "\u03b25-T1A-K81R", "mutant" ], [ 46, 52, "mutant", "protein_state" ], [ 77, 94, "crystal structure", "evidence" ], [ 100, 105, "yeast", "taxonomy_domain" ], [ 106, 116, "proteasome", "complex_assembly" ], [ 118, 121, "yCP", "complex_assembly" ], [ 132, 138, "\u03b25-T1A", "mutant" ] ] }, { "sid": 38, "sent": "Propeptide conformation and triggering of autolysis", "section": "RESULTS", "ner": [ [ 0, 10, "Propeptide", "structure_element" ], [ 42, 51, "autolysis", "ptm" ] ] }, { "sid": 39, "sent": "In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature \u03b2-subunit domains.", "section": "RESULTS", "ner": [ [ 22, 32, "proteasome", "complex_assembly" ], [ 49, 60, "propeptides", "structure_element" ], [ 65, 90, "autocatalytically cleaved", "ptm" ], [ 100, 106, "mature", "protein_state" ], [ 107, 124, "\u03b2-subunit domains", "protein" ] ] }, { "sid": 40, "sent": "For subunit \u03b21, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2).", "section": "RESULTS", "ner": [ [ 12, 14, "\u03b21", "protein" ], [ 73, 83, "propeptide", "structure_element" ], [ 104, 108, "(-2)", "residue_number" ], [ 147, 168, "S1 specificity pocket", "site" ], [ 176, 201, "substrate-binding channel", "site" ], [ 223, 225, "45", "residue_number" ] ] }, { "sid": 41, "sent": "Furthermore, it was observed that the prosegment forms an antiparallel \u03b2-sheet in the active site, and that Gly(-1) adopts a \u03b3-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.).", "section": "RESULTS", "ner": [ [ 38, 48, "prosegment", "structure_element" ], [ 58, 78, "antiparallel \u03b2-sheet", "structure_element" ], [ 86, 97, "active site", "site" ], [ 108, 115, "Gly(-1)", "residue_name_number" ], [ 125, 144, "\u03b3-turn conformation", "structure_element" ], [ 188, 201, "hydrogen bond", "bond_interaction" ], [ 210, 217, "Leu(-2)", "residue_name_number" ], [ 223, 227, "Thr1", "residue_name_number" ] ] }, { "sid": 42, "sent": "Here we again analysed the \u03b21-T1A mutant crystallographically but in addition determined the structures of the \u03b22-T1A single and \u03b21-T1A-\u03b22-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1).", "section": "RESULTS", "ner": [ [ 27, 33, "\u03b21-T1A", "mutant" ], [ 34, 40, "mutant", "protein_state" ], [ 41, 61, "crystallographically", "experimental_method" ], [ 93, 103, "structures", "evidence" ], [ 111, 117, "\u03b22-T1A", "mutant" ], [ 129, 142, "\u03b21-T1A-\u03b22-T1A", "mutant" ] ] }, { "sid": 43, "sent": "In subunit \u03b21, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a).", "section": "RESULTS", "ner": [ [ 11, 13, "\u03b21", "protein" ], [ 29, 36, "Gly(-1)", "residue_name_number" ], [ 52, 62, "sharp turn", "structure_element" ], [ 81, 100, "prosegment cleavage", "ptm" ] ] }, { "sid": 44, "sent": "However, the \u03b3-turn conformation and the associated hydrogen bond initially proposed is for geometric and chemical reasons inappropriate and would not perfectly position the carbonyl carbon atom of Gly(-1) for nucleophilic attack by Thr1.", "section": "RESULTS", "ner": [ [ 13, 32, "\u03b3-turn conformation", "structure_element" ], [ 52, 65, "hydrogen bond", "bond_interaction" ], [ 198, 205, "Gly(-1)", "residue_name_number" ], [ 233, 237, "Thr1", "residue_name_number" ] ] }, { "sid": 45, "sent": "Regarding the \u03b22 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in \u03b21, which might be due to the rather large \u03b22-S1 pocket created by Gly45.", "section": "RESULTS", "ner": [ [ 14, 16, "\u03b22", "protein" ], [ 17, 27, "propeptide", "structure_element" ], [ 29, 36, "Thr(-2)", "residue_name_number" ], [ 50, 59, "S1 pocket", "site" ], [ 102, 109, "Leu(-2)", "residue_name_number" ], [ 113, 115, "\u03b21", "protein" ], [ 156, 158, "\u03b22", "protein" ], [ 159, 168, "S1 pocket", "site" ], [ 180, 185, "Gly45", "residue_name_number" ] ] }, { "sid": 46, "sent": "Thr(-2) positions Gly(-1)O via hydrogen bonding (\u223c2.8\u2009\u00c5) in a perfect trajectory for the nucleophilic attack by Thr1O\u03b3 (Fig. 1b and Supplementary Fig. 2b).", "section": "RESULTS", "ner": [ [ 0, 7, "Thr(-2)", "residue_name_number" ], [ 18, 25, "Gly(-1)", "residue_name_number" ], [ 31, 47, "hydrogen bonding", "bond_interaction" ], [ 112, 116, "Thr1", "residue_name_number" ] ] }, { "sid": 47, "sent": "Next, we examined the position of the \u03b25 propeptide in the \u03b25-T1A-K81R mutant.", "section": "RESULTS", "ner": [ [ 38, 40, "\u03b25", "protein" ], [ 41, 51, "propeptide", "structure_element" ], [ 59, 70, "\u03b25-T1A-K81R", "mutant" ], [ 71, 77, "mutant", "protein_state" ] ] }, { "sid": 48, "sent": "Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel \u03b2-sheet.", "section": "RESULTS", "ner": [ [ 14, 21, "Gly(-1)", "residue_name_number" ], [ 60, 69, "histidine", "residue_name" ], [ 93, 97, "(-2)", "residue_number" ], [ 112, 114, "S2", "site" ], [ 130, 139, "S1 pocket", "site" ], [ 164, 184, "antiparallel \u03b2-sheet", "structure_element" ] ] }, { "sid": 49, "sent": "Nonetheless, the carbonyl carbon of Gly(-1) would be ideally placed for nucleophilic attack by Thr1O\u03b3 (Fig. 1c and Supplementary Fig. 2c,d).", "section": "RESULTS", "ner": [ [ 36, 43, "Gly(-1)", "residue_name_number" ], [ 95, 99, "Thr1", "residue_name_number" ] ] }, { "sid": 50, "sent": "As the K81R mutation is located far from the active site (Thr1C\u03b1\u2013Arg81C\u03b1: 24\u2009\u00c5), any influence on propeptide conformation can be excluded.", "section": "RESULTS", "ner": [ [ 7, 11, "K81R", "mutant" ], [ 45, 56, "active site", "site" ], [ 58, 62, "Thr1", "residue_name_number" ], [ 65, 70, "Arg81", "residue_name_number" ], [ 98, 108, "propeptide", "structure_element" ] ] }, { "sid": 51, "sent": "Instead, the plasticity of the \u03b25 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis.", "section": "RESULTS", "ner": [ [ 31, 33, "\u03b25", "protein" ], [ 34, 43, "S1 pocket", "site" ], [ 84, 89, "Met45", "residue_name_number" ], [ 128, 135, "His(-2)", "residue_name_number" ], [ 143, 150, "S1 site", "site" ], [ 201, 210, "autolysis", "ptm" ] ] }, { "sid": 52, "sent": "Processing of \u03b2-subunit precursors requires deprotonation of Thr1OH; however, the general base initiating autolysis is unknown.", "section": "RESULTS", "ner": [ [ 61, 65, "Thr1", "residue_name_number" ], [ 106, 115, "autolysis", "ptm" ] ] }, { "sid": 53, "sent": "Remarkably, eukaryotic proteasomal \u03b25 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a).", "section": "RESULTS", "ner": [ [ 12, 22, "eukaryotic", "taxonomy_domain" ], [ 35, 37, "\u03b25", "protein" ], [ 54, 57, "His", "residue_name" ], [ 78, 82, "(-2)", "residue_number" ], [ 90, 100, "propeptide", "structure_element" ] ] }, { "sid": 54, "sent": "As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the \u03b25 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30\u2009\u00b0C, but suffered from growth defects at 37\u2009\u00b0C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1).", "section": "RESULTS", "ner": [ [ 3, 12, "histidine", "residue_name" ], [ 59, 75, "catalytic triads", "site" ], [ 79, 95, "serine proteases", "protein_type" ], [ 125, 132, "His(-2)", "residue_name_number" ], [ 154, 156, "\u03b25", "protein" ], [ 157, 167, "propeptide", "structure_element" ], [ 171, 188, "exchanging it for", "experimental_method" ], [ 189, 192, "Asn", "residue_name" ], [ 194, 197, "Lys", "residue_name" ], [ 199, 202, "Phe", "residue_name" ], [ 207, 210, "Ala", "residue_name" ], [ 216, 221, "yeast", "taxonomy_domain" ], [ 303, 309, "H(-2)N", "mutant" ], [ 314, 320, "H(-2)F", "mutant" ] ] }, { "sid": 55, "sent": "In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c).", "section": "RESULTS", "ner": [ [ 56, 62, "H(-2)N", "mutant" ], [ 67, 73, "H(-2)F", "mutant" ], [ 74, 80, "mutant", "protein_state" ], [ 81, 85, "yCPs", "complex_assembly" ] ] }, { "sid": 56, "sent": "Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO\u2013FC electron densities.", "section": "RESULTS", "ner": [ [ 0, 19, "Structural analyses", "experimental_method" ], [ 38, 49, "propeptides", "structure_element" ], [ 57, 63, "mutant", "protein_state" ], [ 64, 68, "yCPs", "complex_assembly" ], [ 85, 110, "2FO\u2013FC electron densities", "evidence" ] ] }, { "sid": 57, "sent": "Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned.", "section": "RESULTS", "ner": [ [ 0, 7, "Gly(-1)", "residue_name_number" ], [ 12, 15, "Phe", "residue_name" ], [ 16, 23, "Lys(-2)", "residue_name_number" ], [ 64, 67, "Ala", "residue_name" ], [ 68, 75, "Asn(-2)", "residue_name_number" ] ] }, { "sid": 58, "sent": "This observation indicates a mixture of processed and unprocessed \u03b25 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base.", "section": "RESULTS", "ner": [ [ 40, 49, "processed", "protein_state" ], [ 54, 65, "unprocessed", "protein_state" ], [ 66, 68, "\u03b25", "protein" ], [ 101, 110, "autolysis", "ptm" ], [ 160, 164, "(-2)", "residue_number" ] ] }, { "sid": 59, "sent": "Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions.", "section": "RESULTS", "ner": [ [ 40, 44, "(-2)", "residue_number" ], [ 71, 81, "propeptide", "structure_element" ], [ 85, 114, "creating mutants that combine", "experimental_method" ], [ 119, 122, "T1A", "mutant" ], [ 124, 128, "K81R", "mutant" ], [ 130, 141, "mutation(s)", "experimental_method" ], [ 147, 153, "H(-2)L", "mutant" ], [ 155, 161, "H(-2)T", "mutant" ], [ 165, 171, "H(-2)A", "mutant" ], [ 172, 185, "substitutions", "experimental_method" ] ] }, { "sid": 60, "sent": "Leu(-2) is encoded in the yeast \u03b21 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of \u03b22-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the \u03b25-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate \u2018\u03b21-like' propeptide positioning.", "section": "RESULTS", "ner": [ [ 0, 7, "Leu(-2)", "residue_name_number" ], [ 26, 31, "yeast", "taxonomy_domain" ], [ 32, 34, "\u03b21", "protein" ], [ 78, 85, "Thr(-2)", "residue_name_number" ], [ 107, 109, "\u03b22", "protein" ], [ 110, 121, "propeptides", "structure_element" ], [ 151, 158, "Ala(-2)", "residue_name_number" ], [ 183, 185, "\u03b25", "protein" ], [ 186, 195, "S1 pocket", "site" ], [ 239, 244, "Met45", "residue_name_number" ] ] }, { "sid": 61, "sent": "As expected from \u03b25-T1A mutants, the yeasts show severe growth phenotypes, with minor variations (Supplementary Fig. 4a and Table 1).", "section": "RESULTS", "ner": [ [ 17, 23, "\u03b25-T1A", "mutant" ], [ 37, 43, "yeasts", "taxonomy_domain" ] ] }, { "sid": 62, "sent": "We determined crystal structures of the \u03b25-H(-2)L-T1A, \u03b25-H(-2)T-T1A and the \u03b25-H(-2)A-T1A-K81R mutants (Supplementary Table 1).", "section": "RESULTS", "ner": [ [ 14, 32, "crystal structures", "evidence" ], [ 40, 53, "\u03b25-H(-2)L-T1A", "mutant" ], [ 55, 68, "\u03b25-H(-2)T-T1A", "mutant" ], [ 77, 95, "\u03b25-H(-2)A-T1A-K81R", "mutant" ] ] }, { "sid": 63, "sent": "For the \u03b25-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d).", "section": "RESULTS", "ner": [ [ 8, 26, "\u03b25-H(-2)A-T1A-K81R", "mutant" ], [ 54, 61, "Gly(-1)", "residue_name_number" ], [ 66, 73, "Ala(-2)", "residue_name_number" ], [ 111, 118, "Ala(-2)", "residue_name_number" ], [ 162, 172, "propeptide", "structure_element" ], [ 180, 205, "substrate-binding channel", "site" ] ] }, { "sid": 64, "sent": "By contrast, the prosegments of the \u03b25-H(-2)L-T1A and the \u03b25-H(-2)T-T1A mutants were significantly better resolved in the 2FO\u2013FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable.", "section": "RESULTS", "ner": [ [ 17, 28, "prosegments", "structure_element" ], [ 36, 49, "\u03b25-H(-2)L-T1A", "mutant" ], [ 58, 71, "\u03b25-H(-2)T-T1A", "mutant" ], [ 122, 150, "2FO\u2013FC electron-density maps", "evidence" ], [ 258, 268, "propeptide", "structure_element" ], [ 277, 284, "His(-2)", "residue_name_number" ] ] }, { "sid": 65, "sent": "Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f\u2013h).", "section": "RESULTS", "ner": [ [ 19, 26, "Leu(-2)", "residue_name_number" ], [ 31, 38, "Thr(-2)", "residue_name_number" ], [ 64, 85, "S1 specificity pocket", "site" ], [ 96, 101, "Met45", "residue_name_number" ] ] }, { "sid": 66, "sent": "This result proves that the naturally occurring His(-2) of the \u03b25 propeptide does not stably fit into the S1 site.", "section": "RESULTS", "ner": [ [ 48, 55, "His(-2)", "residue_name_number" ], [ 63, 65, "\u03b25", "protein" ], [ 66, 76, "propeptide", "structure_element" ], [ 106, 113, "S1 site", "site" ] ] }, { "sid": 67, "sent": "Since Gly(-1) adopts the same position in both wild-type (WT) and mutant \u03b25 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1O\u03b3 (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel \u03b2-sheet is essential for autolysis of the propeptide.", "section": "RESULTS", "ner": [ [ 6, 13, "Gly(-1)", "residue_name_number" ], [ 47, 56, "wild-type", "protein_state" ], [ 58, 60, "WT", "protein_state" ], [ 66, 72, "mutant", "protein_state" ], [ 73, 75, "\u03b25", "protein" ], [ 76, 87, "propeptides", "structure_element" ], [ 179, 183, "Thr1", "residue_name_number" ], [ 240, 244, "(-2)", "residue_number" ], [ 252, 261, "S1 pocket", "site" ], [ 283, 303, "antiparallel \u03b2-sheet", "structure_element" ], [ 321, 330, "autolysis", "ptm" ], [ 338, 348, "propeptide", "structure_element" ] ] }, { "sid": 68, "sent": "Next, we determined the crystal structure of a chimeric yCP having the yeast \u03b21-propeptide replaced by its \u03b25 counterpart.", "section": "RESULTS", "ner": [ [ 24, 41, "crystal structure", "evidence" ], [ 47, 55, "chimeric", "protein_state" ], [ 56, 59, "yCP", "complex_assembly" ], [ 71, 76, "yeast", "taxonomy_domain" ], [ 77, 79, "\u03b21", "protein" ], [ 80, 90, "propeptide", "structure_element" ], [ 91, 102, "replaced by", "experimental_method" ], [ 107, 109, "\u03b25", "protein" ], [ 110, 121, "counterpart", "structure_element" ] ] }, { "sid": 69, "sent": "Although we observed fragments of 2FO\u2013FC electron density in the \u03b21 active site, the data were not interpretable.", "section": "RESULTS", "ner": [ [ 34, 57, "2FO\u2013FC electron density", "evidence" ], [ 65, 67, "\u03b21", "protein" ], [ 68, 79, "active site", "site" ] ] }, { "sid": 70, "sent": "Bearing in mind that in contrast to Thr(-2) in \u03b22, Leu(-2) in subunit \u03b21 is not conserved among species (Supplementary Fig. 3a), we created a \u03b22-T(-2)V proteasome mutant.", "section": "RESULTS", "ner": [ [ 36, 43, "Thr(-2)", "residue_name_number" ], [ 47, 49, "\u03b22", "protein" ], [ 51, 58, "Leu(-2)", "residue_name_number" ], [ 70, 72, "\u03b21", "protein" ], [ 76, 89, "not conserved", "protein_state" ], [ 132, 139, "created", "experimental_method" ], [ 142, 151, "\u03b22-T(-2)V", "mutant" ], [ 152, 162, "proteasome", "complex_assembly" ], [ 163, 169, "mutant", "protein_state" ] ] }, { "sid": 71, "sent": "As proven by the \u03b22-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the \u03b25-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in \u03b22, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j).", "section": "RESULTS", "ner": [ [ 17, 23, "\u03b22-T1A", "mutant" ], [ 24, 42, "crystal structures", "evidence" ], [ 44, 51, "Thr(-2)", "residue_name_number" ], [ 52, 66, "hydrogen bonds", "bond_interaction" ], [ 70, 77, "Gly(-1)", "residue_name_number" ], [ 131, 144, "\u03b25-H(-2)T-T1A", "mutant" ], [ 145, 151, "mutant", "protein_state" ], [ 191, 199, "exchange", "experimental_method" ], [ 203, 210, "Thr(-2)", "residue_name_number" ], [ 214, 217, "Val", "residue_name" ], [ 221, 223, "\u03b22", "protein" ] ] }, { "sid": 72, "sent": "Notably, the 2FO\u2013FC electron-density map displays a different orientation for the \u03b22 propeptide than has been observed for the \u03b22-T1A proteasome.", "section": "RESULTS", "ner": [ [ 13, 40, "2FO\u2013FC electron-density map", "evidence" ], [ 82, 84, "\u03b22", "protein" ], [ 85, 95, "propeptide", "structure_element" ], [ 127, 133, "\u03b22-T1A", "mutant" ], [ 134, 144, "proteasome", "complex_assembly" ] ] }, { "sid": 73, "sent": "In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8\u2009\u00c5), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k).", "section": "RESULTS", "ner": [ [ 15, 22, "Val(-2)", "residue_name_number" ], [ 45, 52, "S1 site", "site" ], [ 57, 64, "Gly(-1)", "residue_name_number" ], [ 176, 180, "Thr1", "residue_name_number" ] ] }, { "sid": 74, "sent": "These results further confirm that correct positioning of the active-site residues and Gly(-1) is decisive for the maturation of the proteasome.", "section": "RESULTS", "ner": [ [ 62, 82, "active-site residues", "site" ], [ 87, 94, "Gly(-1)", "residue_name_number" ], [ 133, 143, "proteasome", "complex_assembly" ] ] }, { "sid": 75, "sent": "The active site of the proteasome", "section": "RESULTS", "ner": [ [ 4, 15, "active site", "site" ], [ 23, 33, "proteasome", "complex_assembly" ] ] }, { "sid": 76, "sent": "Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis.", "section": "RESULTS", "ner": [ [ 38, 49, "active site", "site" ], [ 50, 54, "Thr1", "residue_name_number" ], [ 60, 64, "Thr1", "residue_name_number" ], [ 87, 92, "water", "chemical" ] ] }, { "sid": 77, "sent": "However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1O\u03b3.", "section": "RESULTS", "ner": [ [ 16, 24, "immature", "protein_state" ], [ 25, 33, "particle", "complex_assembly" ], [ 34, 38, "Thr1", "residue_name_number" ], [ 60, 70, "propeptide", "structure_element" ], [ 91, 95, "Thr1", "residue_name_number" ] ] }, { "sid": 78, "sent": "Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1O\u03b3 (2.7\u2009\u00c5) in all catalytically active \u03b2 subunits (Fig. 3a,b), was proposed to serve as the proton acceptor.", "section": "RESULTS", "ner": [ [ 9, 14, "Lys33", "residue_name_number" ], [ 31, 47, "hydrogen-bonding", "bond_interaction" ], [ 60, 64, "Thr1", "residue_name_number" ], [ 82, 102, "catalytically active", "protein_state" ], [ 103, 113, "\u03b2 subunits", "protein" ] ] }, { "sid": 79, "sent": "A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17O\u03b4, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site.", "section": "RESULTS", "ner": [ [ 11, 27, "catalytic tetrad", "site" ], [ 44, 48, "Thr1", "residue_name_number" ], [ 52, 56, "Thr1", "residue_name_number" ], [ 61, 66, "Lys33", "residue_name_number" ], [ 74, 79, "Asp17", "residue_name_number" ], [ 109, 114, "water", "chemical" ], [ 208, 219, "active site", "site" ] ] }, { "sid": 80, "sent": "Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base.", "section": "RESULTS", "ner": [ [ 39, 42, "yCP", "complex_assembly" ], [ 43, 59, "X-ray structures", "evidence" ], [ 98, 109, "active site", "site" ], [ 117, 127, "proteasome", "complex_assembly" ] ] }, { "sid": 81, "sent": "Mutation of \u03b25-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1).", "section": "RESULTS", "ner": [ [ 0, 8, "Mutation", "experimental_method" ], [ 12, 14, "\u03b25", "protein" ], [ 15, 20, "Lys33", "residue_name_number" ], [ 24, 27, "Ala", "residue_name" ], [ 82, 117, "structural and biochemical analyses", "experimental_method" ], [ 162, 181, "propeptide cleavage", "ptm" ] ] }, { "sid": 82, "sent": "The phenotype of the \u03b25-K33A mutant was however less pronounced than for the \u03b25-T1A-K81R yeast (Fig. 4a).", "section": "RESULTS", "ner": [ [ 21, 28, "\u03b25-K33A", "mutant" ], [ 29, 35, "mutant", "protein_state" ], [ 77, 88, "\u03b25-T1A-K81R", "mutant" ], [ 89, 94, "yeast", "taxonomy_domain" ] ] }, { "sid": 83, "sent": "This discrepancy in growth was traced to an additional point mutation L(-49)S in the \u03b25-propeptide of the \u03b25-K33A mutant (see also Supplementary Note 1).", "section": "RESULTS", "ner": [ [ 70, 77, "L(-49)S", "mutant" ], [ 85, 87, "\u03b25", "protein" ], [ 88, 98, "propeptide", "structure_element" ], [ 106, 113, "\u03b25-K33A", "mutant" ], [ 114, 120, "mutant", "protein_state" ] ] }, { "sid": 84, "sent": "Structural comparison of the \u03b25-L(-49)S-K33A and \u03b25-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide.", "section": "RESULTS", "ner": [ [ 0, 21, "Structural comparison", "experimental_method" ], [ 29, 44, "\u03b25-L(-49)S-K33A", "mutant" ], [ 49, 60, "\u03b25-T1A-K81R", "mutant" ], [ 61, 73, "active sites", "site" ], [ 88, 96, "mutation", "experimental_method" ], [ 100, 105, "Lys33", "residue_name_number" ], [ 109, 112, "Ala", "residue_name" ], [ 150, 154, "Thr1", "residue_name_number" ], [ 171, 181, "propeptide", "structure_element" ] ] }, { "sid": 85, "sent": "This structural alteration destroys active-site integrity and abolishes catalytic activity of the \u03b25 active site (Supplementary Fig. 5a).", "section": "RESULTS", "ner": [ [ 36, 47, "active-site", "site" ], [ 98, 100, "\u03b25", "protein" ], [ 101, 112, "active site", "site" ] ] }, { "sid": 86, "sent": "Additional proof for the key function of Lys33 was obtained from the \u03b25-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans).", "section": "RESULTS", "ner": [ [ 41, 46, "Lys33", "residue_name_number" ], [ 69, 76, "\u03b25-K33A", "mutant" ], [ 77, 83, "mutant", "protein_state" ], [ 94, 104, "propeptide", "structure_element" ], [ 105, 125, "expressed separately", "experimental_method" ], [ 149, 151, "pp", "chemical" ], [ 152, 157, "trans", "protein_state" ] ] }, { "sid": 87, "sent": "The Thr1 N terminus of this mutant is not blocked by the propeptide, yet its catalytic activity is reduced by \u223c83% (Supplementary Fig. 6b).", "section": "RESULTS", "ner": [ [ 4, 8, "Thr1", "residue_name_number" ], [ 28, 34, "mutant", "protein_state" ], [ 57, 67, "propeptide", "structure_element" ] ] }, { "sid": 88, "sent": "Consistent with this, the crystal structure of the \u03b25-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the \u03b25 active sites (Supplementary Fig. 5b and Supplementary Table 1).", "section": "RESULTS", "ner": [ [ 26, 43, "crystal structure", "evidence" ], [ 51, 58, "\u03b25-K33A", "mutant" ], [ 59, 61, "pp", "chemical" ], [ 62, 67, "trans", "protein_state" ], [ 68, 74, "mutant", "protein_state" ], [ 75, 90, "in complex with", "protein_state" ], [ 91, 102, "carfilzomib", "chemical" ], [ 154, 156, "\u03b25", "protein" ], [ 157, 169, "active sites", "site" ] ] }, { "sid": 89, "sent": "Since no acetylation of the Thr1 N terminus was observed for the \u03b25-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH.", "section": "RESULTS", "ner": [ [ 9, 20, "acetylation", "ptm" ], [ 28, 32, "Thr1", "residue_name_number" ], [ 65, 72, "\u03b25-K33A", "mutant" ], [ 73, 75, "pp", "chemical" ], [ 76, 81, "trans", "protein_state" ], [ 82, 85, "apo", "protein_state" ], [ 86, 103, "crystal structure", "evidence" ], [ 177, 182, "Lys33", "residue_name_number" ], [ 199, 203, "Thr1", "residue_name_number" ], [ 235, 239, "Thr1", "residue_name_number" ] ] }, { "sid": 90, "sent": "Furthermore, the crystal structure of the \u03b25-K33A pp trans mutant without inhibitor revealed that Thr1O\u03b3 strongly coordinates a well-defined water molecule (\u223c2\u2009\u00c5; Fig. 3c and Supplementary Fig. 5c,d).", "section": "RESULTS", "ner": [ [ 17, 34, "crystal structure", "evidence" ], [ 42, 49, "\u03b25-K33A", "mutant" ], [ 50, 52, "pp", "chemical" ], [ 53, 58, "trans", "protein_state" ], [ 59, 65, "mutant", "protein_state" ], [ 66, 83, "without inhibitor", "protein_state" ], [ 98, 102, "Thr1", "residue_name_number" ], [ 114, 125, "coordinates", "bond_interaction" ], [ 141, 146, "water", "chemical" ] ] }, { "sid": 91, "sent": "This water hydrogen bonds also to Arg19O (\u223c3.0\u2009\u00c5) and Asp17O\u03b4 (\u223c3.0\u2009\u00c5), and thereby presumably enables residual activity of the mutant.", "section": "RESULTS", "ner": [ [ 5, 10, "water", "chemical" ], [ 11, 25, "hydrogen bonds", "bond_interaction" ], [ 34, 39, "Arg19", "residue_name_number" ], [ 54, 59, "Asp17", "residue_name_number" ], [ 128, 134, "mutant", "protein_state" ] ] }, { "sid": 92, "sent": "Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis.", "section": "RESULTS", "ner": [ [ 73, 78, "Lys33", "residue_name_number" ], [ 89, 91, "WT", "protein_state" ], [ 92, 102, "proteasome", "complex_assembly" ], [ 103, 112, "structure", "evidence" ], [ 168, 173, "Lys33", "residue_name_number" ], [ 198, 207, "autolysis", "ptm" ] ] }, { "sid": 93, "sent": "Conservative substitution of Lys33 by Arg delays autolysis of the \u03b25 precursor and impairs yeast growth (for details see Supplementary Note 1).", "section": "RESULTS", "ner": [ [ 0, 25, "Conservative substitution", "experimental_method" ], [ 29, 34, "Lys33", "residue_name_number" ], [ 38, 41, "Arg", "residue_name" ], [ 49, 58, "autolysis", "ptm" ], [ 66, 68, "\u03b25", "protein" ], [ 91, 96, "yeast", "taxonomy_domain" ] ] }, { "sid": 94, "sent": "While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the \u03b25 active site (Supplementary Fig. 5e).", "section": "RESULTS", "ner": [ [ 6, 10, "Thr1", "residue_name_number" ], [ 44, 46, "WT", "protein_state" ], [ 47, 51, "yCPs", "complex_assembly" ], [ 53, 58, "Arg33", "residue_name_number" ], [ 72, 85, "hydrogen bond", "bond_interaction" ], [ 89, 94, "Asp17", "residue_name_number" ], [ 121, 123, "\u03b25", "protein" ], [ 124, 135, "active site", "site" ] ] }, { "sid": 95, "sent": "The conservative mutation of Asp17 to Asn in subunit \u03b25 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1).", "section": "RESULTS", "ner": [ [ 4, 25, "conservative mutation", "experimental_method" ], [ 29, 34, "Asp17", "residue_name_number" ], [ 38, 41, "Asn", "residue_name" ], [ 53, 55, "\u03b25", "protein" ], [ 63, 66, "yCP", "complex_assembly" ] ] }, { "sid": 96, "sent": "Notably, only with the additional point mutation L(-49)S present in the \u03b25 propeptide could we purify a small amount of the \u03b25-D17N mutant yCP.", "section": "RESULTS", "ner": [ [ 49, 56, "L(-49)S", "mutant" ], [ 72, 74, "\u03b25", "protein" ], [ 75, 85, "propeptide", "structure_element" ], [ 124, 131, "\u03b25-D17N", "mutant" ], [ 132, 138, "mutant", "protein_state" ], [ 139, 142, "yCP", "complex_assembly" ] ] }, { "sid": 97, "sent": "As determined by crystallographic analysis, this mutant \u03b25 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits \u03b21 and \u03b22, and with WT \u03b25 (Supplementary Fig. 7a).", "section": "RESULTS", "ner": [ [ 17, 42, "crystallographic analysis", "experimental_method" ], [ 49, 55, "mutant", "protein_state" ], [ 56, 58, "\u03b25", "protein" ], [ 71, 90, "partially processed", "protein_state" ], [ 147, 157, "proteasome", "complex_assembly" ], [ 168, 179, "carfilzomib", "chemical" ], [ 207, 209, "\u03b21", "protein" ], [ 214, 216, "\u03b22", "protein" ], [ 227, 229, "WT", "protein_state" ], [ 230, 232, "\u03b25", "protein" ] ] }, { "sid": 98, "sent": "In contrast to the cis-construct, expression of the \u03b25 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome.", "section": "RESULTS", "ner": [ [ 19, 22, "cis", "protein_state" ], [ 34, 44, "expression", "experimental_method" ], [ 52, 54, "\u03b25", "protein" ], [ 55, 65, "propeptide", "structure_element" ], [ 69, 74, "trans", "protein_state" ], [ 99, 108, "isolation", "experimental_method" ], [ 113, 128, "crystallization", "experimental_method" ], [ 136, 140, "D17N", "mutant" ], [ 141, 147, "mutant", "protein_state" ], [ 148, 158, "proteasome", "complex_assembly" ] ] }, { "sid": 99, "sent": "The ChT-L activity of the \u03b25-D17N pp in trans CP towards the canonical \u03b25 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome.", "section": "RESULTS", "ner": [ [ 26, 33, "\u03b25-D17N", "mutant" ], [ 34, 36, "pp", "chemical" ], [ 40, 45, "trans", "protein_state" ], [ 46, 48, "CP", "complex_assembly" ], [ 71, 73, "\u03b25", "protein" ], [ 91, 142, "N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin", "chemical" ], [ 144, 156, "Suc-LLVY-AMC", "chemical" ], [ 162, 205, "carboxybenzyl-Gly-Gly-Leu-para-nitroanilide", "chemical" ], [ 207, 216, "Z-GGL-pNA", "chemical" ], [ 280, 285, "Asp17", "residue_name_number" ], [ 349, 355, "mature", "protein_state" ], [ 356, 366, "proteasome", "complex_assembly" ] ] }, { "sid": 100, "sent": "Even though the \u03b25-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the \u03b1\u2032, \u03b2\u2032 epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the \u03b25 active site (Supplementary Fig. 7a).", "section": "RESULTS", "ner": [ [ 16, 23, "\u03b25-D17N", "mutant" ], [ 24, 26, "pp", "chemical" ], [ 27, 32, "trans", "protein_state" ], [ 33, 36, "yCP", "complex_assembly" ], [ 37, 54, "crystal structure", "evidence" ], [ 81, 83, "WT", "protein_state" ], [ 84, 87, "yCP", "complex_assembly" ], [ 117, 137, "co-crystal structure", "evidence" ], [ 147, 165, "\u03b1\u2032, \u03b2\u2032 epoxyketone", "chemical" ], [ 176, 187, "carfilzomib", "chemical" ], [ 243, 245, "\u03b25", "protein" ], [ 246, 257, "active site", "site" ] ] }, { "sid": 101, "sent": "This observation is consistent with a strongly reduced reactivity of \u03b25-Thr1 and the crystal structure of the \u03b25-D17N pp cis mutant in complex with carfilzomib.", "section": "RESULTS", "ner": [ [ 69, 71, "\u03b25", "protein" ], [ 72, 76, "Thr1", "residue_name_number" ], [ 85, 102, "crystal structure", "evidence" ], [ 110, 117, "\u03b25-D17N", "mutant" ], [ 118, 120, "pp", "chemical" ], [ 121, 124, "cis", "protein_state" ], [ 125, 131, "mutant", "protein_state" ], [ 132, 147, "in complex with", "protein_state" ], [ 148, 159, "carfilzomib", "chemical" ] ] }, { "sid": 102, "sent": "Autolysis and residual catalytic activity of the \u03b25-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1.", "section": "RESULTS", "ner": [ [ 0, 9, "Autolysis", "ptm" ], [ 49, 56, "\u03b25-D17N", "mutant" ], [ 106, 111, "Asn17", "residue_name_number" ], [ 163, 168, "Lys33", "residue_name_number" ], [ 191, 195, "Thr1", "residue_name_number" ] ] }, { "sid": 103, "sent": "In agreement, an E17A mutant in the proteasomal \u03b2-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis.", "section": "RESULTS", "ner": [ [ 17, 21, "E17A", "mutant" ], [ 22, 28, "mutant", "protein_state" ], [ 48, 57, "\u03b2-subunit", "protein" ], [ 65, 73, "archaeon", "taxonomy_domain" ], [ 74, 98, "Thermoplasma acidophilum", "species" ], [ 108, 117, "autolysis", "ptm" ] ] }, { "sid": 104, "sent": "Strikingly, although the X-ray data on the \u03b25-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b).", "section": "RESULTS", "ner": [ [ 25, 35, "X-ray data", "evidence" ], [ 43, 50, "\u03b25-D17N", "mutant" ], [ 51, 57, "mutant", "protein_state" ], [ 67, 77, "propeptide", "structure_element" ], [ 78, 87, "expressed", "experimental_method" ], [ 91, 94, "cis", "protein_state" ], [ 102, 107, "trans", "protein_state" ] ] }, { "sid": 105, "sent": "On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d).", "section": "RESULTS", "ner": [ [ 47, 50, "CPs", "complex_assembly" ], [ 82, 97, "catalytic triad", "site" ], [ 112, 116, "Thr1", "residue_name_number" ], [ 118, 123, "Lys33", "residue_name_number" ], [ 128, 131, "Asp", "residue_name" ], [ 132, 137, "Glu17", "residue_name_number" ], [ 147, 181, "autocatalytic precursor processing", "ptm" ] ] }, { "sid": 106, "sent": "This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1O\u03b3 to Thr1NH2.", "section": "RESULTS", "ner": [ [ 60, 65, "water", "chemical" ], [ 94, 100, "mature", "protein_state" ], [ 101, 103, "WT", "protein_state" ], [ 116, 127, "active site", "site" ], [ 163, 167, "Thr1", "residue_name_number" ], [ 173, 177, "Thr1", "residue_name_number" ] ] }, { "sid": 107, "sent": "To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast \u03b25 subunit.", "section": "RESULTS", "ner": [ [ 16, 27, "active-site", "site" ], [ 46, 69, "exchanged the conserved", "experimental_method" ], [ 70, 76, "Asp166", "residue_name_number" ], [ 89, 92, "Asn", "residue_name" ], [ 100, 105, "yeast", "taxonomy_domain" ], [ 106, 108, "\u03b25", "protein" ] ] }, { "sid": 108, "sent": "Asp166O\u03b4 is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis.", "section": "RESULTS", "ner": [ [ 0, 6, "Asp166", "residue_name_number" ], [ 12, 27, "hydrogen-bonded", "bond_interaction" ], [ 31, 35, "Thr1", "residue_name_number" ], [ 43, 49, "Ser129", "residue_name_number" ], [ 56, 62, "Ser169", "residue_name_number" ] ] }, { "sid": 109, "sent": "The \u03b25-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1).", "section": "RESULTS", "ner": [ [ 4, 12, "\u03b25-D166N", "mutant" ], [ 13, 15, "pp", "chemical" ], [ 16, 19, "cis", "protein_state" ], [ 20, 25, "yeast", "taxonomy_domain" ], [ 26, 32, "mutant", "protein_state" ] ] }, { "sid": 110, "sent": "X-ray data on the \u03b25-D166N mutant indicate that the \u03b25 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166O\u03b4 is disrupted (Supplementary Fig. 8a).", "section": "RESULTS", "ner": [ [ 0, 10, "X-ray data", "evidence" ], [ 18, 26, "\u03b25-D166N", "mutant" ], [ 27, 33, "mutant", "protein_state" ], [ 52, 54, "\u03b25", "protein" ], [ 55, 65, "propeptide", "structure_element" ], [ 109, 115, "Ser129", "residue_name_number" ], [ 140, 146, "Asn166", "residue_name_number" ] ] }, { "sid": 111, "sent": "Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing.", "section": "RESULTS", "ner": [ [ 11, 16, "water", "chemical" ], [ 29, 37, "bound to", "protein_state" ], [ 38, 44, "Ser129", "residue_name_number" ], [ 51, 55, "Thr1", "residue_name_number" ], [ 101, 121, "precursor processing", "ptm" ] ] }, { "sid": 112, "sent": "The hydrogen bonds involving Ser169OH are intact and may account for residual substrate turnover.", "section": "RESULTS", "ner": [ [ 4, 18, "hydrogen bonds", "bond_interaction" ], [ 29, 35, "Ser169", "residue_name_number" ] ] }, { "sid": 113, "sent": "Soaking the \u03b25-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c).", "section": "RESULTS", "ner": [ [ 0, 7, "Soaking", "experimental_method" ], [ 12, 20, "\u03b25-D166N", "mutant" ], [ 21, 29, "crystals", "experimental_method" ], [ 35, 46, "carfilzomib", "chemical" ], [ 51, 56, "MG132", "chemical" ], [ 94, 98, "Thr1", "residue_name_number" ] ] }, { "sid": 114, "sent": "In the carfilzomib complex structure, Thr1O\u03b3 and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation.", "section": "RESULTS", "ner": [ [ 7, 26, "carfilzomib complex", "complex_assembly" ], [ 27, 36, "structure", "evidence" ], [ 38, 42, "Thr1", "residue_name_number" ], [ 49, 53, "Thr1", "residue_name_number" ], [ 104, 110, "Ser129", "residue_name_number" ], [ 122, 124, "WT", "protein_state" ] ] }, { "sid": 115, "sent": "In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166.", "section": "RESULTS", "ner": [ [ 7, 24, "MG132-bound state", "protein_state" ], [ 26, 30, "Thr1", "residue_name_number" ], [ 35, 45, "unmodified", "protein_state" ], [ 73, 79, "Ser129", "residue_name_number" ], [ 83, 98, "hydrogen-bonded", "bond_interaction" ], [ 104, 109, "water", "chemical" ], [ 130, 136, "Asn166", "residue_name_number" ] ] }, { "sid": 116, "sent": "Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis.", "section": "RESULTS", "ner": [ [ 8, 11, "Asn", "residue_name" ], [ 39, 45, "Asp166", "residue_name_number" ], [ 91, 94, "Ala", "residue_name" ], [ 138, 147, "autolysis", "ptm" ] ] }, { "sid": 117, "sent": "These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis.", "section": "RESULTS", "ner": [ [ 27, 33, "Asp166", "residue_name_number" ], [ 38, 44, "Ser129", "residue_name_number" ], [ 110, 114, "Thr1", "residue_name_number" ], [ 123, 132, "autolysis", "ptm" ] ] }, { "sid": 118, "sent": "Substitution of the active-site Thr1 by Cys", "section": "RESULTS", "ner": [ [ 0, 12, "Substitution", "experimental_method" ], [ 20, 31, "active-site", "site" ], [ 32, 36, "Thr1", "residue_name_number" ], [ 40, 43, "Cys", "residue_name" ] ] }, { "sid": 119, "sent": "Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum.", "section": "RESULTS", "ner": [ [ 0, 8, "Mutation", "experimental_method" ], [ 12, 16, "Thr1", "residue_name_number" ], [ 20, 23, "Cys", "residue_name" ], [ 40, 54, "20S proteasome", "complex_assembly" ], [ 64, 72, "archaeon", "taxonomy_domain" ], [ 73, 87, "T. acidophilum", "species" ] ] }, { "sid": 120, "sent": "In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure.", "section": "RESULTS", "ner": [ [ 3, 8, "yeast", "taxonomy_domain" ], [ 15, 23, "mutation", "experimental_method" ], [ 90, 100, "propeptide", "structure_element" ], [ 137, 152, "X-ray structure", "evidence" ] ] }, { "sid": 121, "sent": "In one of the two \u03b25 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c).", "section": "RESULTS", "ner": [ [ 18, 20, "\u03b25", "protein" ], [ 53, 60, "cleaved", "protein_state" ], [ 61, 71, "propeptide", "structure_element" ], [ 72, 83, "still bound", "protein_state" ], [ 91, 116, "substrate-binding channel", "site" ] ] }, { "sid": 122, "sent": "His(-2) occupies the S2 pocket like observed for the \u03b25-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel \u03b2-sheet conformation as known from inhibitors like MG132 (Fig. 4c\u2013e and Supplementary Fig. 9b).", "section": "RESULTS", "ner": [ [ 0, 7, "His(-2)", "residue_name_number" ], [ 21, 30, "S2 pocket", "site" ], [ 53, 64, "\u03b25-T1A-K81R", "mutant" ], [ 65, 71, "mutant", "protein_state" ], [ 108, 118, "propeptide", "structure_element" ], [ 126, 129, "T1C", "mutant" ], [ 130, 136, "mutant", "protein_state" ], [ 147, 167, "antiparallel \u03b2-sheet", "structure_element" ], [ 211, 216, "MG132", "chemical" ] ] }, { "sid": 123, "sent": "On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization.", "section": "RESULTS", "ner": [ [ 37, 40, "T1C", "mutant" ], [ 41, 47, "mutant", "protein_state" ], [ 56, 66, "propeptide", "structure_element" ], [ 93, 104, "active site", "site" ], [ 122, 131, "autolysis", "ptm" ], [ 183, 198, "crystallization", "experimental_method" ] ] }, { "sid": 124, "sent": "Owing to the unequal positions of the two \u03b25 subunits within the CP in the crystal lattice, maturation and propeptide displacement may occur at different timescales in the two subunits.", "section": "RESULTS", "ner": [ [ 42, 44, "\u03b25", "protein" ], [ 65, 67, "CP", "complex_assembly" ], [ 107, 117, "propeptide", "structure_element" ] ] }, { "sid": 125, "sent": "Despite propeptide hydrolysis, the \u03b25-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a).", "section": "RESULTS", "ner": [ [ 8, 29, "propeptide hydrolysis", "ptm" ], [ 35, 41, "\u03b25-T1C", "mutant" ], [ 42, 53, "active site", "site" ], [ 57, 79, "catalytically inactive", "protein_state" ] ] }, { "sid": 126, "sent": "In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the \u03b21 and \u03b22 active sites, while leaving the \u03b25-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant.", "section": "RESULTS", "ner": [ [ 14, 30, "soaking crystals", "experimental_method" ], [ 40, 42, "CP", "complex_assembly" ], [ 54, 64, "bortezomib", "chemical" ], [ 68, 79, "carfilzomib", "chemical" ], [ 98, 100, "\u03b21", "protein" ], [ 105, 107, "\u03b22", "protein" ], [ 108, 120, "active sites", "site" ], [ 140, 146, "\u03b25-T1C", "mutant" ], [ 147, 166, "proteolytic centres", "site" ], [ 167, 177, "unmodified", "protein_state" ], [ 230, 237, "cleaved", "protein_state" ], [ 238, 248, "propeptide", "structure_element" ] ] }, { "sid": 127, "sent": "Moreover, the structural data reveal that the thiol group of Cys1 is rotated by 74\u00b0 with respect to the hydroxyl side chain of Thr1 (Fig. 4f and Supplementary Fig. 9b).", "section": "RESULTS", "ner": [ [ 14, 29, "structural data", "evidence" ], [ 61, 65, "Cys1", "residue_name_number" ], [ 127, 131, "Thr1", "residue_name_number" ] ] }, { "sid": 128, "sent": "Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1.", "section": "RESULTS", "ner": [ [ 18, 31, "hydrogen bond", "bond_interaction" ], [ 73, 78, "Lys33", "residue_name_number" ], [ 82, 84, "WT", "protein_state" ], [ 85, 88, "CPs", "complex_assembly" ], [ 104, 108, "Cys1", "residue_name_number" ] ] }, { "sid": 129, "sent": "Notably, the 2FO\u2013FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered.", "section": "RESULTS", "ner": [ [ 13, 40, "2FO\u2013FC electron-density map", "evidence" ], [ 48, 51, "T1C", "mutant" ], [ 52, 58, "mutant", "protein_state" ], [ 79, 84, "Lys33", "residue_name_number" ], [ 91, 101, "disordered", "protein_state" ] ] }, { "sid": 130, "sent": "Together, these observations suggest that efficient peptide-bond hydrolysis requires that Lys33NH2 hydrogen bonds to the active site nucleophile.", "section": "RESULTS", "ner": [ [ 90, 95, "Lys33", "residue_name_number" ], [ 99, 113, "hydrogen bonds", "bond_interaction" ] ] }, { "sid": 131, "sent": "The benefit of Thr over Ser as the active-site nucleophile", "section": "RESULTS", "ner": [ [ 15, 18, "Thr", "residue_name" ], [ 24, 27, "Ser", "residue_name" ] ] }, { "sid": 132, "sent": "All proteasomes strictly employ threonine as the active-site residue instead of serine.", "section": "RESULTS", "ner": [ [ 4, 15, "proteasomes", "complex_assembly" ], [ 16, 31, "strictly employ", "protein_state" ], [ 32, 41, "threonine", "residue_name" ], [ 49, 68, "active-site residue", "site" ], [ 80, 86, "serine", "residue_name" ] ] }, { "sid": 133, "sent": "To investigate the reason for this singularity, we analysed a \u03b25-T1S mutant, which is viable but suffers from growth defects (Fig. 4a and Table 1).", "section": "RESULTS", "ner": [ [ 62, 68, "\u03b25-T1S", "mutant" ], [ 69, 75, "mutant", "protein_state" ] ] }, { "sid": 134, "sent": "Activity assays with the \u03b25-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40\u201345% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c).", "section": "RESULTS", "ner": [ [ 0, 15, "Activity assays", "experimental_method" ], [ 25, 27, "\u03b25", "protein" ], [ 47, 59, "Suc-LLVY-AMC", "chemical" ], [ 104, 107, "T1S", "mutant" ], [ 108, 114, "mutant", "protein_state" ], [ 150, 152, "WT", "protein_state" ], [ 153, 164, "proteasomes", "complex_assembly" ] ] }, { "sid": 135, "sent": "By contrast, turnover of the substrate Z-GGL-pNA, used to monitor ChT-L activity in situ but in a less quantitative fashion, is not detectably impaired (Supplementary Fig. 9a).", "section": "RESULTS", "ner": [ [ 39, 48, "Z-GGL-pNA", "chemical" ] ] }, { "sid": 136, "sent": "Crystal structure analysis of the \u03b25-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5).", "section": "RESULTS", "ner": [ [ 0, 17, "Crystal structure", "evidence" ], [ 34, 40, "\u03b25-T1S", "mutant" ], [ 41, 47, "mutant", "protein_state" ], [ 58, 78, "precursor processing", "ptm" ], [ 94, 108, "ligand-complex", "complex_assembly" ], [ 109, 119, "structures", "evidence" ], [ 125, 135, "bortezomib", "chemical" ], [ 140, 151, "carfilzomib", "chemical" ], [ 197, 201, "Ser1", "residue_name_number" ] ] }, { "sid": 137, "sent": "However, the apo crystal structure revealed that Ser1O\u03b3 is turned away from the substrate-binding channel (Fig. 4g).", "section": "RESULTS", "ner": [ [ 13, 16, "apo", "protein_state" ], [ 17, 34, "crystal structure", "evidence" ], [ 49, 53, "Ser1", "residue_name_number" ], [ 80, 105, "substrate-binding channel", "site" ] ] }, { "sid": 138, "sent": "Compared with Thr1O\u03b3 in WT CP structures, Ser1O\u03b3 is rotated by 60\u00b0.", "section": "RESULTS", "ner": [ [ 14, 18, "Thr1", "residue_name_number" ], [ 24, 26, "WT", "protein_state" ], [ 27, 29, "CP", "complex_assembly" ], [ 30, 40, "structures", "evidence" ], [ 42, 46, "Ser1", "residue_name_number" ] ] }, { "sid": 139, "sent": "Because both conformations of Ser1O\u03b3 are hydrogen-bonded to Lys33NH2 (Fig. 4h), the relay system is capable of hydrolysing peptide substrates, albeit at lower rates compared with Thr1.", "section": "RESULTS", "ner": [ [ 30, 34, "Ser1", "residue_name_number" ], [ 41, 56, "hydrogen-bonded", "bond_interaction" ], [ 60, 65, "Lys33", "residue_name_number" ], [ 179, 183, "Thr1", "residue_name_number" ] ] }, { "sid": 140, "sent": "The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h).", "section": "RESULTS", "ner": [ [ 4, 23, "active-site residue", "site" ], [ 24, 28, "Thr1", "residue_name_number" ], [ 89, 113, "hydrophobic interactions", "bond_interaction" ], [ 119, 123, "Thr3", "residue_name_number" ], [ 128, 133, "Ala46", "residue_name_number" ] ] }, { "sid": 141, "sent": "Consequently, the hydroxyl group of Thr1 requires no reorientation before substrate cleavage and is thus more catalytically efficient than Ser1.", "section": "RESULTS", "ner": [ [ 36, 40, "Thr1", "residue_name_number" ], [ 139, 143, "Ser1", "residue_name_number" ] ] }, { "sid": 142, "sent": "In agreement, at an elevated growing temperature of 37\u2009\u00b0C the T1S mutant is unable to grow (Fig. 4a).", "section": "RESULTS", "ner": [ [ 62, 65, "T1S", "mutant" ], [ 66, 72, "mutant", "protein_state" ] ] }, { "sid": 143, "sent": "In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5).", "section": "RESULTS", "ner": [ [ 14, 20, "mutant", "protein_state" ], [ 21, 31, "proteasome", "complex_assembly" ], [ 55, 65, "proteasome", "complex_assembly" ], [ 80, 90, "bortezomib", "chemical" ], [ 106, 117, "carfilzomib", "chemical" ] ] }, { "sid": 144, "sent": "Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors.", "section": "RESULTS", "ner": [ [ 14, 31, "inhibitor complex", "complex_assembly" ], [ 32, 42, "structures", "evidence" ], [ 94, 96, "WT", "protein_state" ], [ 97, 100, "yCP", "complex_assembly" ], [ 101, 111, "structures", "evidence" ], [ 113, 137, "with the same inhibitors", "protein_state" ] ] }, { "sid": 145, "sent": "Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein.", "section": "RESULTS", "ner": [ [ 13, 21, "affinity", "evidence" ], [ 42, 53, "carfilzomib", "chemical" ], [ 106, 131, "substrate-binding channel", "site" ], [ 151, 161, "bortezomib", "chemical" ] ] }, { "sid": 146, "sent": "Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased.", "section": "RESULTS", "ner": [ [ 11, 30, "mean residence time", "evidence" ], [ 34, 45, "carfilzomib", "chemical" ], [ 53, 64, "active site", "site" ], [ 123, 127, "Ser1", "residue_name_number" ] ] }, { "sid": 147, "sent": "Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.).", "section": "RESULTS", "ner": [ [ 89, 98, "threonine", "residue_name" ], [ 133, 144, "proteasomes", "complex_assembly" ], [ 189, 214, "proteasome-like proteases", "protein_type" ], [ 223, 227, "HslV", "protein" ] ] }, { "sid": 148, "sent": "The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized.", "section": "DISCUSS", "ner": [ [ 4, 18, "20S proteasome", "complex_assembly" ], [ 19, 21, "CP", "complex_assembly" ], [ 35, 57, "non-lysosomal protease", "protein_type" ], [ 61, 71, "eukaryotic", "taxonomy_domain" ] ] }, { "sid": 149, "sent": "The \u03b2-subunit propeptides, particularly that of \u03b25, are key factors that help drive proper assembly of the CP complex.", "section": "DISCUSS", "ner": [ [ 4, 13, "\u03b2-subunit", "protein" ], [ 14, 25, "propeptides", "structure_element" ], [ 48, 50, "\u03b25", "protein" ], [ 107, 109, "CP", "complex_assembly" ] ] }, { "sid": 150, "sent": "In addition, they prevent irreversible inactivation of the Thr1 N terminus by N-acetylation.", "section": "DISCUSS", "ner": [ [ 59, 63, "Thr1", "residue_name_number" ], [ 78, 91, "N-acetylation", "ptm" ] ] }, { "sid": 151, "sent": "By contrast, the prosegments of \u03b2 subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli.", "section": "DISCUSS", "ner": [ [ 17, 28, "prosegments", "structure_element" ], [ 32, 42, "\u03b2 subunits", "protein" ], [ 63, 71, "archaeal", "taxonomy_domain" ], [ 72, 82, "proteasome", "complex_assembly" ], [ 107, 131, "heterologously expressed", "experimental_method" ], [ 135, 151, "Escherichia coli", "species" ] ] }, { "sid": 152, "sent": "In eukaryotes, deletion of or failure to cleave the \u03b21 and \u03b22 propeptides is well tolerated.", "section": "DISCUSS", "ner": [ [ 3, 13, "eukaryotes", "taxonomy_domain" ], [ 52, 54, "\u03b21", "protein" ], [ 59, 61, "\u03b22", "protein" ], [ 62, 73, "propeptides", "structure_element" ] ] }, { "sid": 153, "sent": "However, removal of the \u03b25 prosegment or any interference with its cleavage causes severe phenotypic defects.", "section": "DISCUSS", "ner": [ [ 9, 19, "removal of", "experimental_method" ], [ 24, 26, "\u03b25", "protein" ], [ 27, 37, "prosegment", "structure_element" ] ] }, { "sid": 154, "sent": "These observations highlight the unique function and importance of the \u03b25 propeptide as well as the \u03b25 active site for maturation and function of the eukaryotic CP.", "section": "DISCUSS", "ner": [ [ 71, 73, "\u03b25", "protein" ], [ 74, 84, "propeptide", "structure_element" ], [ 100, 102, "\u03b25", "protein" ], [ 103, 114, "active site", "site" ], [ 150, 160, "eukaryotic", "taxonomy_domain" ], [ 161, 163, "CP", "complex_assembly" ] ] }, { "sid": 155, "sent": "Here we have described the atomic structures of various \u03b25-T1A mutants, which allowed for the first time visualization of the residual \u03b25 propeptide.", "section": "DISCUSS", "ner": [ [ 27, 44, "atomic structures", "evidence" ], [ 56, 62, "\u03b25-T1A", "mutant" ], [ 135, 137, "\u03b25", "protein" ], [ 138, 148, "propeptide", "structure_element" ] ] }, { "sid": 156, "sent": "Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1O\u03b3, although it does not adopt the tight turn observed for the prosegment of subunit \u03b21.", "section": "DISCUSS", "ner": [ [ 17, 21, "(-2)", "residue_number" ], [ 50, 60, "propeptide", "structure_element" ], [ 80, 87, "Gly(-1)", "residue_name_number" ], [ 98, 108, "structures", "evidence" ], [ 158, 162, "Thr1", "residue_name_number" ], [ 197, 207, "tight turn", "structure_element" ], [ 225, 235, "prosegment", "structure_element" ], [ 247, 249, "\u03b21", "protein" ] ] }, { "sid": 157, "sent": "From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis.", "section": "DISCUSS", "ner": [ [ 57, 64, "Gly(-1)", "residue_name_number" ], [ 69, 73, "Thr1", "residue_name_number" ], [ 118, 129, "active site", "site" ], [ 147, 156, "autolysis", "ptm" ] ] }, { "sid": 158, "sent": "In this regard, inappropriate N-acetylation of the Thr1 N terminus cannot be removed by Thr1O\u03b3 due to the rotational freedom and flexibility of the acetyl group.", "section": "DISCUSS", "ner": [ [ 30, 43, "N-acetylation", "ptm" ], [ 51, 55, "Thr1", "residue_name_number" ], [ 88, 92, "Thr1", "residue_name_number" ] ] }, { "sid": 159, "sent": "The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2).", "section": "DISCUSS", "ner": [ [ 4, 14, "propeptide", "structure_element" ], [ 43, 68, "substrate-binding channel", "site" ], [ 90, 97, "Gly(-1)", "residue_name_number" ], [ 162, 166, "(-2)", "residue_number" ] ] }, { "sid": 160, "sent": "Autolytic activation of the CP constitutes one of the final steps of proteasome biogenesis, but the trigger for propeptide cleavage had remained enigmatic.", "section": "DISCUSS", "ner": [ [ 28, 30, "CP", "complex_assembly" ], [ 112, 131, "propeptide cleavage", "ptm" ] ] }, { "sid": 161, "sent": "On the basis of the numerous CP:ligand complexes solved during the past 18 years and in the current study, we provide a revised interpretation of proteasome active-site architecture.", "section": "DISCUSS", "ner": [ [ 29, 38, "CP:ligand", "complex_assembly" ], [ 146, 156, "proteasome", "complex_assembly" ], [ 157, 181, "active-site architecture", "site" ] ] }, { "sid": 162, "sent": "We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits.", "section": "DISCUSS", "ner": [ [ 13, 28, "catalytic triad", "site" ], [ 37, 48, "active site", "site" ], [ 56, 58, "CP", "complex_assembly" ], [ 82, 86, "Thr1", "residue_name_number" ], [ 88, 93, "Lys33", "residue_name_number" ], [ 98, 101, "Asp", "residue_name" ], [ 102, 107, "Glu17", "residue_name_number" ], [ 162, 172, "eukaryotic", "taxonomy_domain" ], [ 174, 183, "bacterial", "taxonomy_domain" ], [ 188, 196, "archaeal", "taxonomy_domain" ], [ 197, 207, "proteasome", "complex_assembly" ] ] }, { "sid": 163, "sent": "Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17O\u03b4 orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+\u2013Asp17O\u03b4: 2.9\u2009\u00c5).", "section": "DISCUSS", "ner": [ [ 0, 5, "Lys33", "residue_name_number" ], [ 58, 79, "autocatalytic removal", "ptm" ], [ 87, 98, "propeptides", "structure_element" ], [ 147, 152, "Asp17", "residue_name_number" ], [ 163, 168, "Lys33", "residue_name_number" ], [ 231, 244, "hydrogen bond", "bond_interaction" ], [ 255, 260, "Lys33", "residue_name_number" ], [ 265, 270, "Asp17", "residue_name_number" ] ] }, { "sid": 164, "sent": "Analogously to the proteasome, a Thr\u2013Lys\u2013Asp triad is also found in L-asparaginase.", "section": "DISCUSS", "ner": [ [ 19, 29, "proteasome", "complex_assembly" ], [ 33, 50, "Thr\u2013Lys\u2013Asp triad", "site" ], [ 68, 82, "L-asparaginase", "protein_type" ] ] }, { "sid": 165, "sent": "Thus, specific protein surroundings can significantly alter the chemical properties of amino acids such as Lys to function as an acid\u2013base catalyst.", "section": "DISCUSS", "ner": [ [ 107, 110, "Lys", "residue_name" ] ] }, { "sid": 166, "sent": "In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1O\u03b3 (Fig. 3d).", "section": "DISCUSS", "ner": [ [ 36, 47, "active site", "site" ], [ 72, 76, "Thr1", "residue_name_number" ], [ 90, 104, "hydrogen bonds", "bond_interaction" ], [ 207, 231, "endoproteolytic cleavage", "ptm" ], [ 235, 239, "Thr1", "residue_name_number" ] ] }, { "sid": 167, "sent": "Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), \u03b1-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.).", "section": "DISCUSS", "ner": [ [ 51, 55, "Thr1", "residue_name_number" ], [ 81, 95, "hydrogen bonds", "bond_interaction" ], [ 127, 140, "fellutamide B", "chemical" ], [ 149, 161, "\u03b1-ketoamides", "chemical" ], [ 163, 179, "homobelactosin C", "chemical" ], [ 191, 208, "salinosporamide A", "chemical" ] ] }, { "sid": 168, "sent": "Furthermore, opening of the \u03b2-lactone compound omuralide by Thr1 creates a C3-hydroxyl group, whose proton originates from Thr1NH3+.", "section": "DISCUSS", "ner": [ [ 47, 56, "omuralide", "chemical" ], [ 60, 64, "Thr1", "residue_name_number" ], [ 123, 127, "Thr1", "residue_name_number" ] ] }, { "sid": 169, "sent": "The resulting uncharged Thr1NH2 is hydrogen-bridged to the C3-OH group.", "section": "DISCUSS", "ner": [ [ 24, 28, "Thr1", "residue_name_number" ], [ 35, 51, "hydrogen-bridged", "bond_interaction" ] ] }, { "sid": 170, "sent": "In agreement, acetylation of the Thr1 N terminus irreversibly blocks hydrolytic activity, and binding of substrates is prevented for steric reasons.", "section": "DISCUSS", "ner": [ [ 14, 25, "acetylation", "ptm" ], [ 33, 37, "Thr1", "residue_name_number" ] ] }, { "sid": 171, "sent": "By acting as a proton donor during catalysis, the Thr1 N terminus may also favour cleavage of substrate peptide bonds (Fig. 3d).", "section": "DISCUSS", "ner": [ [ 50, 54, "Thr1", "residue_name_number" ] ] }, { "sid": 172, "sent": "Cleavage of the scissile peptide bond requires protonation of the emerging free amine, and in the proteasome, the Thr1 amine group is likely to assume this function.", "section": "DISCUSS", "ner": [ [ 98, 108, "proteasome", "complex_assembly" ], [ 114, 118, "Thr1", "residue_name_number" ] ] }, { "sid": 173, "sent": "Analogously, Thr1NH3+ might promote the bivalent reaction mode of epoxyketone inhibitors by protonating the epoxide moiety to create a positively charged trivalent oxygen atom that is subsequently nucleophilically attacked by Thr1NH2.", "section": "DISCUSS", "ner": [ [ 13, 17, "Thr1", "residue_name_number" ], [ 226, 230, "Thr1", "residue_name_number" ] ] }, { "sid": 174, "sent": "During autolysis the Thr1 N terminus is engaged in a hydroxyoxazolidine ring intermediate (Fig. 3d), which is unstable and short-lived.", "section": "DISCUSS", "ner": [ [ 7, 16, "autolysis", "ptm" ], [ 21, 25, "Thr1", "residue_name_number" ] ] }, { "sid": 175, "sent": "Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+.", "section": "DISCUSS", "ner": [ [ 60, 64, "Thr1", "residue_name_number" ], [ 98, 115, "aspartic acid 166", "residue_name_number" ], [ 120, 126, "Ser129", "residue_name_number" ], [ 138, 142, "Thr1", "residue_name_number" ] ] }, { "sid": 176, "sent": "The residues Ser129 and Asp166 are expected to increase the pKa value of Thr1N, thereby favouring its charged state.", "section": "DISCUSS", "ner": [ [ 13, 19, "Ser129", "residue_name_number" ], [ 24, 30, "Asp166", "residue_name_number" ], [ 73, 77, "Thr1", "residue_name_number" ] ] }, { "sid": 177, "sent": "Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%.", "section": "DISCUSS", "ner": [ [ 67, 75, "mutation", "experimental_method" ], [ 76, 81, "D166A", "mutant" ], [ 91, 100, "autolysis", "ptm" ], [ 108, 116, "archaeal", "taxonomy_domain" ], [ 117, 119, "CP", "complex_assembly" ], [ 128, 136, "exchange", "experimental_method" ], [ 137, 142, "D166N", "mutant" ], [ 177, 182, "yeast", "taxonomy_domain" ], [ 183, 185, "CP", "complex_assembly" ] ] }, { "sid": 178, "sent": "The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2).", "section": "DISCUSS", "ner": [ [ 4, 12, "mutation", "experimental_method" ], [ 13, 18, "D166N", "mutant" ], [ 37, 41, "Thr1", "residue_name_number" ], [ 116, 120, "Thr1", "residue_name_number" ] ] }, { "sid": 179, "sent": "This interpretation agrees with the strongly reduced catalytic activity of the \u03b25-D166N mutant on the one hand, and the ability to react readily with carfilzomib on the other.", "section": "DISCUSS", "ner": [ [ 79, 87, "\u03b25-D166N", "mutant" ], [ 88, 94, "mutant", "protein_state" ], [ 150, 161, "carfilzomib", "chemical" ] ] }, { "sid": 180, "sent": "Hence, the proteasome can be viewed as having a second triad that is essential for efficient proteolysis.", "section": "DISCUSS", "ner": [ [ 11, 21, "proteasome", "complex_assembly" ], [ 48, 60, "second triad", "site" ] ] }, { "sid": 181, "sent": "While Lys33NH2 and Asp17O\u03b4 are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1.", "section": "DISCUSS", "ner": [ [ 6, 11, "Lys33", "residue_name_number" ], [ 19, 24, "Asp17", "residue_name_number" ], [ 59, 63, "Thr1", "residue_name_number" ], [ 85, 91, "Ser129", "residue_name_number" ], [ 98, 104, "Asp166", "residue_name_number" ], [ 156, 160, "Thr1", "residue_name_number" ] ] }, { "sid": 182, "sent": "In accord with the proposed Thr1\u2013Lys33\u2013Asp17 catalytic triad, crystallographic data on the proteolytically inactive \u03b25-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken.", "section": "DISCUSS", "ner": [ [ 28, 32, "Thr1", "residue_name_number" ], [ 33, 38, "Lys33", "residue_name_number" ], [ 39, 44, "Asp17", "residue_name_number" ], [ 45, 60, "catalytic triad", "site" ], [ 62, 83, "crystallographic data", "evidence" ], [ 91, 115, "proteolytically inactive", "protein_state" ], [ 116, 122, "\u03b25-T1C", "mutant" ], [ 123, 129, "mutant", "protein_state" ], [ 166, 171, "Lys33", "residue_name_number" ], [ 179, 183, "Cys1", "residue_name_number" ] ] }, { "sid": 183, "sent": "However, owing to Cys being a strong nucleophile, the propeptide can still be cleaved off over time.", "section": "DISCUSS", "ner": [ [ 18, 21, "Cys", "residue_name" ], [ 54, 64, "propeptide", "structure_element" ], [ 78, 85, "cleaved", "protein_state" ] ] }, { "sid": 184, "sent": "While only one single turnover is necessary for autolysis, continuous enzymatic activity is required for significant and detectable substrate hydrolysis.", "section": "DISCUSS", "ner": [ [ 48, 57, "autolysis", "ptm" ] ] }, { "sid": 185, "sent": "Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing.", "section": "DISCUSS", "ner": [ [ 16, 29, "Ntn hydrolase", "protein_type" ], [ 30, 48, "penicillin acylase", "protein_type" ], [ 50, 62, "substitution", "experimental_method" ], [ 70, 79, "catalytic", "protein_state" ], [ 91, 94, "Ser", "residue_name" ], [ 106, 109, "Cys", "residue_name" ], [ 115, 126, "inactivates", "protein_state" ], [ 131, 137, "enzyme", "protein_type" ], [ 156, 176, "precursor processing", "ptm" ] ] }, { "sid": 186, "sent": "To investigate why the CP specifically employs threonine as its active-site residue, we used a \u03b25-T1S mutant of the yCP and characterized it biochemically and structurally.", "section": "DISCUSS", "ner": [ [ 23, 25, "CP", "complex_assembly" ], [ 47, 56, "threonine", "residue_name" ], [ 64, 83, "active-site residue", "site" ], [ 95, 101, "\u03b25-T1S", "mutant" ], [ 102, 108, "mutant", "protein_state" ], [ 116, 119, "yCP", "complex_assembly" ], [ 141, 171, "biochemically and structurally", "experimental_method" ] ] }, { "sid": 187, "sent": "Activity assays with the \u03b25-T1S mutant revealed reduced turnover of Suc-LLVY-AMC.", "section": "DISCUSS", "ner": [ [ 0, 15, "Activity assays", "experimental_method" ], [ 25, 31, "\u03b25-T1S", "mutant" ], [ 32, 38, "mutant", "protein_state" ], [ 68, 80, "Suc-LLVY-AMC", "chemical" ] ] }, { "sid": 188, "sent": "We also observed slightly lower affinity of the \u03b25-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib.", "section": "DISCUSS", "ner": [ [ 48, 54, "\u03b25-T1S", "mutant" ], [ 55, 61, "mutant", "protein_state" ], [ 62, 65, "yCP", "complex_assembly" ], [ 112, 122, "proteasome", "complex_assembly" ], [ 134, 144, "bortezomib", "chemical" ], [ 149, 160, "carfilzomib", "chemical" ] ] }, { "sid": 189, "sent": "Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes.", "section": "DISCUSS", "ner": [ [ 0, 19, "Structural analyses", "evidence" ], [ 52, 55, "T1S", "mutant" ], [ 56, 62, "mutant", "protein_state" ], [ 98, 111, "strict use of", "protein_state" ], [ 112, 115, "Thr", "residue_name" ], [ 128, 139, "proteasomes", "complex_assembly" ] ] }, { "sid": 190, "sent": "Thr1 is well anchored in the active site by hydrophobic interactions of its C\u03b3 methyl group with Ala46 (C\u03b2), Lys33 (carbon side chain) and Thr3 (C\u03b3).", "section": "DISCUSS", "ner": [ [ 0, 4, "Thr1", "residue_name_number" ], [ 29, 40, "active site", "site" ], [ 44, 68, "hydrophobic interactions", "bond_interaction" ], [ 97, 102, "Ala46", "residue_name_number" ], [ 109, 114, "Lys33", "residue_name_number" ], [ 139, 143, "Thr3", "residue_name_number" ] ] }, { "sid": 191, "sent": "Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the C\u03b3 for fixing the position of the nucleophilic Thr1.", "section": "DISCUSS", "ner": [ [ 9, 31, "proteolytically active", "protein_state" ], [ 32, 42, "proteasome", "complex_assembly" ], [ 57, 64, "archaea", "taxonomy_domain" ], [ 66, 71, "yeast", "taxonomy_domain" ], [ 76, 83, "mammals", "taxonomy_domain" ], [ 161, 164, "Thr", "residue_name" ], [ 168, 171, "Ile", "residue_name" ], [ 184, 185, "3", "residue_number" ], [ 267, 271, "Thr1", "residue_name_number" ] ] }, { "sid": 192, "sent": "In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h).", "section": "DISCUSS", "ner": [ [ 15, 19, "Thr1", "residue_name_number" ], [ 43, 47, "Ser1", "residue_name_number" ], [ 77, 81, "Thr1", "residue_name_number" ], [ 107, 109, "WT", "protein_state" ], [ 110, 116, "enzyme", "complex_assembly" ] ] }, { "sid": 193, "sent": "Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity.", "section": "DISCUSS", "ner": [ [ 16, 35, "threonine aspartase", "protein_type" ], [ 36, 44, "Taspase1", "protein" ], [ 46, 54, "mutation", "experimental_method" ], [ 62, 73, "active-site", "site" ], [ 74, 80, "Thr234", "residue_name_number" ], [ 84, 87, "Ser", "residue_name" ], [ 154, 160, "Thr234", "residue_name_number" ], [ 168, 170, "WT", "protein_state" ] ] }, { "sid": 194, "sent": "Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site.", "section": "DISCUSS", "ner": [ [ 24, 30, "serine", "residue_name" ], [ 31, 37, "mutant", "protein_state" ], [ 41, 47, "active", "protein_state" ], [ 49, 58, "threonine", "residue_name" ], [ 99, 109, "proteasome", "complex_assembly" ], [ 110, 121, "active site", "site" ] ] }, { "sid": 195, "sent": "The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes.", "section": "DISCUSS", "ner": [ [ 27, 36, "threonine", "residue_name" ], [ 45, 55, "proteasome", "complex_assembly" ], [ 56, 67, "active site", "site" ], [ 172, 184, "conservation", "protein_state" ], [ 192, 196, "Thr1", "residue_name_number" ], [ 212, 223, "proteasomes", "complex_assembly" ], [ 229, 237, "bacteria", "taxonomy_domain" ], [ 241, 251, "eukaryotes", "taxonomy_domain" ] ] }, { "sid": 196, "sent": "Conformation of proteasomal propeptides.", "section": "FIG", "ner": [ [ 28, 39, "propeptides", "structure_element" ] ] }, { "sid": 197, "sent": "(a) Structural superposition of the \u03b21-T1A propeptide and the matured WT \u03b21 active-site Thr1.", "section": "FIG", "ner": [ [ 4, 28, "Structural superposition", "experimental_method" ], [ 36, 42, "\u03b21-T1A", "mutant" ], [ 43, 53, "propeptide", "structure_element" ], [ 62, 69, "matured", "protein_state" ], [ 70, 72, "WT", "protein_state" ], [ 73, 75, "\u03b21", "protein" ], [ 76, 87, "active-site", "site" ], [ 88, 92, "Thr1", "residue_name_number" ] ] }, { "sid": 198, "sent": "Only the residues (-5) to (-1) of the \u03b21-T1A propeptide are displayed.", "section": "FIG", "ner": [ [ 18, 30, "(-5) to (-1)", "residue_range" ], [ 38, 44, "\u03b21-T1A", "mutant" ], [ 45, 55, "propeptide", "structure_element" ] ] }, { "sid": 199, "sent": "The major determinant of the S1 specificity pocket, residue 45, is depicted.", "section": "FIG", "ner": [ [ 29, 50, "S1 specificity pocket", "site" ], [ 60, 62, "45", "residue_number" ], [ 29, 50, "S1 specificity pocket", "site" ], [ 60, 62, "45", "residue_number" ] ] }, { "sid": 200, "sent": "Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow).", "section": "FIG", "ner": [ [ 31, 38, "Gly(-1)", "residue_name_number" ], [ 43, 47, "Ala1", "residue_name_number" ], [ 55, 65, "propeptide", "structure_element" ], [ 75, 80, "G(-1)", "residue_name_number" ], [ 133, 142, "processed", "protein_state" ], [ 143, 145, "WT", "protein_state" ], [ 146, 157, "active-site", "site" ], [ 158, 162, "Thr1", "residue_name_number" ] ] }, { "sid": 201, "sent": "The black arrow indicates the attack of Thr1O\u03b3 onto the carbonyl carbon atom of Gly(-1).", "section": "FIG", "ner": [ [ 40, 44, "Thr1", "residue_name_number" ], [ 80, 87, "Gly(-1)", "residue_name_number" ] ] }, { "sid": 202, "sent": "(b) Structural superposition of the \u03b21-T1A propeptide and the \u03b22-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well.", "section": "FIG", "ner": [ [ 4, 28, "Structural superposition", "experimental_method" ], [ 36, 42, "\u03b21-T1A", "mutant" ], [ 43, 53, "propeptide", "structure_element" ], [ 62, 68, "\u03b22-T1A", "mutant" ], [ 69, 79, "propeptide", "structure_element" ], [ 155, 159, "Ala1", "residue_name_number" ], [ 164, 171, "Gly(-1)", "residue_name_number" ] ] }, { "sid": 203, "sent": "Thr(-2)OH is hydrogen-bonded to Gly(-1)O (\u223c2.8\u2009\u00c5; black dashed line).", "section": "FIG", "ner": [ [ 0, 7, "Thr(-2)", "residue_name_number" ], [ 13, 28, "hydrogen-bonded", "bond_interaction" ], [ 32, 39, "Gly(-1)", "residue_name_number" ] ] }, { "sid": 204, "sent": "(c) Structural superposition of the \u03b21-T1A, the \u03b22-T1A and the \u03b25-T1A-K81R propeptide remnants depict their differences in conformation.", "section": "FIG", "ner": [ [ 4, 28, "Structural superposition", "experimental_method" ], [ 36, 42, "\u03b21-T1A", "mutant" ], [ 48, 54, "\u03b22-T1A", "mutant" ], [ 63, 74, "\u03b25-T1A-K81R", "mutant" ], [ 75, 85, "propeptide", "structure_element" ] ] }, { "sid": 205, "sent": "While residue (-2) of the \u03b21 and \u03b22 prosegments fit the S1 pocket, His(-2) of the \u03b25 propeptide occupies the S2 pocket.", "section": "FIG", "ner": [ [ 14, 18, "(-2)", "residue_number" ], [ 26, 28, "\u03b21", "protein" ], [ 33, 35, "\u03b22", "protein" ], [ 36, 47, "prosegments", "structure_element" ], [ 56, 65, "S1 pocket", "site" ], [ 67, 74, "His(-2)", "residue_name_number" ], [ 82, 84, "\u03b25", "protein" ], [ 85, 95, "propeptide", "structure_element" ], [ 109, 118, "S2 pocket", "site" ] ] }, { "sid": 206, "sent": "Nonetheless, in all mutants the carbonyl carbon atom of Gly(-1) is ideally placed for the nucleophilic attack by Thr1O\u03b3.", "section": "FIG", "ner": [ [ 56, 63, "Gly(-1)", "residue_name_number" ], [ 113, 117, "Thr1", "residue_name_number" ] ] }, { "sid": 207, "sent": "The hydrogen bond between Thr(-2)OH and Gly(-1)O (\u223c2.8\u2009\u00c5) is indicated by a black dashed line.", "section": "FIG", "ner": [ [ 4, 17, "hydrogen bond", "bond_interaction" ], [ 26, 33, "Thr(-2)", "residue_name_number" ], [ 40, 47, "Gly(-1)", "residue_name_number" ] ] }, { "sid": 208, "sent": "Mutations of residue (-2) and their influence on propeptide conformation and autolysis.", "section": "FIG", "ner": [ [ 0, 9, "Mutations", "experimental_method" ], [ 21, 25, "(-2)", "residue_number" ], [ 49, 59, "propeptide", "structure_element" ], [ 77, 86, "autolysis", "ptm" ] ] }, { "sid": 209, "sent": "(a) Structural superposition of the \u03b21-T1A propeptide and the \u03b25-H(-2)L-T1A mutant propeptide.", "section": "FIG", "ner": [ [ 4, 28, "Structural superposition", "experimental_method" ], [ 36, 42, "\u03b21-T1A", "mutant" ], [ 43, 53, "propeptide", "structure_element" ], [ 62, 75, "\u03b25-H(-2)L-T1A", "mutant" ], [ 76, 82, "mutant", "protein_state" ], [ 83, 93, "propeptide", "structure_element" ] ] }, { "sid": 210, "sent": "The (-2) residues of both prosegments point into the S1 pocket.", "section": "FIG", "ner": [ [ 4, 8, "(-2)", "residue_number" ], [ 26, 37, "prosegments", "structure_element" ], [ 53, 62, "S1 pocket", "site" ] ] }, { "sid": 211, "sent": "(b) Structural superposition of the \u03b25 propeptides in the \u03b25-H(-2)L-T1A, \u03b25-H(-2)T-T1A, \u03b25-(H-2)A-T1A-K81R and \u03b25-T1A-K81R mutant proteasomes.", "section": "FIG", "ner": [ [ 4, 28, "Structural superposition", "experimental_method" ], [ 36, 38, "\u03b25", "protein" ], [ 39, 50, "propeptides", "structure_element" ], [ 58, 71, "\u03b25-H(-2)L-T1A", "mutant" ], [ 73, 86, "\u03b25-H(-2)T-T1A", "mutant" ], [ 88, 106, "\u03b25-(H-2)A-T1A-K81R", "mutant" ], [ 111, 122, "\u03b25-T1A-K81R", "mutant" ], [ 123, 129, "mutant", "protein_state" ], [ 130, 141, "proteasomes", "complex_assembly" ] ] }, { "sid": 212, "sent": "While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions.", "section": "FIG", "ner": [ [ 19, 31, "(-2) to (-4)", "residue_range" ], [ 60, 67, "Gly(-1)", "residue_name_number" ], [ 72, 76, "Ala1", "residue_name_number" ], [ 96, 106, "structures", "evidence" ] ] }, { "sid": 213, "sent": "(c) Structural superposition of the \u03b22-T1A propeptide and the \u03b25-H(-2)T-T1A mutant propeptide.", "section": "FIG", "ner": [ [ 4, 28, "Structural superposition", "experimental_method" ], [ 36, 42, "\u03b22-T1A", "mutant" ], [ 43, 53, "propeptide", "structure_element" ], [ 62, 75, "\u03b25-H(-2)T-T1A", "mutant" ], [ 76, 82, "mutant", "protein_state" ], [ 83, 93, "propeptide", "structure_element" ] ] }, { "sid": 214, "sent": "The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of \u03b22 forms a hydrogen bridge to Gly(-1)O (black dashed line).", "section": "FIG", "ner": [ [ 4, 8, "(-2)", "residue_number" ], [ 26, 37, "prosegments", "structure_element" ], [ 53, 62, "S1 pocket", "site" ], [ 73, 80, "Thr(-2)", "residue_name_number" ], [ 86, 88, "\u03b22", "protein" ], [ 97, 112, "hydrogen bridge", "bond_interaction" ], [ 116, 123, "Gly(-1)", "residue_name_number" ] ] }, { "sid": 215, "sent": "(d) Structural superposition of the matured \u03b22 active site, the WT \u03b22-T1A propeptide and the \u03b22-T(-2)V mutant propeptide.", "section": "FIG", "ner": [ [ 4, 28, "Structural superposition", "experimental_method" ], [ 36, 43, "matured", "protein_state" ], [ 44, 46, "\u03b22", "protein" ], [ 47, 58, "active site", "site" ], [ 64, 66, "WT", "protein_state" ], [ 67, 73, "\u03b22-T1A", "mutant" ], [ 74, 84, "propeptide", "structure_element" ], [ 93, 102, "\u03b22-T(-2)V", "mutant" ], [ 103, 109, "mutant", "protein_state" ], [ 110, 120, "propeptide", "structure_element" ] ] }, { "sid": 216, "sent": "Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1O\u03b3 on the carbonyl carbon atom of Gly(-1).", "section": "FIG", "ner": [ [ 9, 16, "Val(-2)", "residue_name_number" ], [ 51, 60, "S1 pocket", "site" ], [ 98, 105, "Gly(-1)", "residue_name_number" ], [ 144, 148, "Thr1", "residue_name_number" ], [ 182, 189, "Gly(-1)", "residue_name_number" ] ] }, { "sid": 217, "sent": "Architecture and proposed reaction mechanism of the proteasomal active site.", "section": "FIG", "ner": [ [ 64, 75, "active site", "site" ] ] }, { "sid": 218, "sent": "(a) Hydrogen-bonding network at the mature WT \u03b25 proteasomal active site (dotted lines).", "section": "FIG", "ner": [ [ 4, 28, "Hydrogen-bonding network", "site" ], [ 36, 42, "mature", "protein_state" ], [ 43, 45, "WT", "protein_state" ], [ 46, 48, "\u03b25", "protein" ], [ 61, 72, "active site", "site" ] ] }, { "sid": 219, "sent": "Thr1OH is hydrogen-bonded to Lys33NH2 (2.7\u2009\u00c5), which in turn interacts with Asp17O\u03b4.", "section": "FIG", "ner": [ [ 0, 4, "Thr1", "residue_name_number" ], [ 10, 25, "hydrogen-bonded", "bond_interaction" ], [ 29, 34, "Lys33", "residue_name_number" ], [ 76, 81, "Asp17", "residue_name_number" ] ] }, { "sid": 220, "sent": "The Thr1 N terminus is engaged in hydrogen bonds with Ser129O\u03b3, the carbonyl oxygen of residue 168, Ser169O\u03b3 and Asp166O\u03b4. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the \u03b2 subunits.", "section": "FIG", "ner": [ [ 4, 8, "Thr1", "residue_name_number" ], [ 34, 48, "hydrogen bonds", "bond_interaction" ], [ 54, 60, "Ser129", "residue_name_number" ], [ 95, 98, "168", "residue_number" ], [ 100, 106, "Ser169", "residue_name_number" ], [ 113, 119, "Asp166", "residue_name_number" ], [ 151, 171, "active-site residues", "site" ], [ 184, 200, "hydrogen bonding", "bond_interaction" ], [ 205, 223, "strictly conserved", "protein_state" ], [ 232, 250, "proteolytic centre", "site" ], [ 264, 277, "superposition", "experimental_method" ], [ 285, 295, "\u03b2 subunits", "protein" ] ] }, { "sid": 221, "sent": "(c) Structural superposition of the WT \u03b25 and the \u03b25-K33A pp trans mutant active site.", "section": "FIG", "ner": [ [ 4, 28, "Structural superposition", "experimental_method" ], [ 36, 38, "WT", "protein_state" ], [ 39, 41, "\u03b25", "protein" ], [ 50, 57, "\u03b25-K33A", "mutant" ], [ 58, 60, "pp", "chemical" ], [ 61, 66, "trans", "protein_state" ], [ 67, 73, "mutant", "protein_state" ], [ 74, 85, "active site", "site" ] ] }, { "sid": 222, "sent": "In the latter, a water molecule (red sphere) is found at the position where in the WT structure the side chain amine group of Lys33 is located.", "section": "FIG", "ner": [ [ 17, 22, "water", "chemical" ], [ 83, 85, "WT", "protein_state" ], [ 126, 131, "Lys33", "residue_name_number" ] ] }, { "sid": 223, "sent": "Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17O\u03b4 and Thr1OH.", "section": "FIG", "ner": [ [ 13, 18, "Lys33", "residue_name_number" ], [ 24, 29, "water", "chemical" ], [ 39, 53, "hydrogen bonds", "bond_interaction" ], [ 57, 62, "Arg19", "residue_name_number" ], [ 65, 70, "Asp17", "residue_name_number" ], [ 77, 81, "Thr1", "residue_name_number" ] ] }, { "sid": 224, "sent": "Note, the strong interaction with the water molecule causes a minor shift of Thr1, while all other active-site residues remain in place.", "section": "FIG", "ner": [ [ 38, 43, "water", "chemical" ], [ 77, 81, "Thr1", "residue_name_number" ], [ 99, 119, "active-site residues", "site" ] ] }, { "sid": 225, "sent": "(d) Proposed chemical reaction mechanism for autocatalytic precursor processing and proteolysis in the proteasome.", "section": "FIG", "ner": [ [ 45, 79, "autocatalytic precursor processing", "ptm" ], [ 103, 113, "proteasome", "complex_assembly" ] ] }, { "sid": 226, "sent": "The active-site Thr1 is depicted in blue, the propeptide segment and the peptide substrate are coloured in green, whereas the scissile peptide bond is highlighted in red.", "section": "FIG", "ner": [ [ 4, 15, "active-site", "site" ], [ 16, 20, "Thr1", "residue_name_number" ], [ 46, 56, "propeptide", "structure_element" ] ] }, { "sid": 227, "sent": "Autolysis (left set of structures) is initiated by deprotonation of Thr1OH via Lys33NH2 and the formation of a tetrahedral transition state.", "section": "FIG", "ner": [ [ 0, 9, "Autolysis", "ptm" ], [ 68, 72, "Thr1", "residue_name_number" ], [ 79, 84, "Lys33", "residue_name_number" ] ] }, { "sid": 228, "sent": "The strictly conserved oxyanion hole Gly47NH stabilizing the negatively charged intermediate is illustrated as a semicircle.", "section": "FIG", "ner": [ [ 4, 22, "strictly conserved", "protein_state" ], [ 37, 42, "Gly47", "residue_name_number" ] ] }, { "sid": 229, "sent": "Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH.", "section": "FIG", "ner": [ [ 43, 47, "Thr1", "residue_name_number" ], [ 102, 112, "propeptide", "structure_element" ], [ 151, 157, "Asp166", "residue_name_number" ], [ 164, 170, "Ser129", "residue_name_number" ] ] }, { "sid": 230, "sent": "Next, Thr1NH2 polarizes a water molecule for the nucleophilic attack of the acyl-enzyme intermediate.", "section": "FIG", "ner": [ [ 6, 10, "Thr1", "residue_name_number" ], [ 26, 31, "water", "chemical" ] ] }, { "sid": 231, "sent": "On hydrolysis of the latter, the active-site Thr1 is ready for catalysis (right set of structures).", "section": "FIG", "ner": [ [ 33, 44, "active-site", "site" ], [ 45, 49, "Thr1", "residue_name_number" ] ] }, { "sid": 232, "sent": "The charged Thr1 N terminus may engage in the orientation of the amide moiety and donate a proton to the emerging N terminus of the C-terminal cleavage product.", "section": "FIG", "ner": [ [ 12, 16, "Thr1", "residue_name_number" ] ] }, { "sid": 233, "sent": "The resulting deprotonated Thr1NH2 finally activates a water molecule for hydrolysis of the acyl-enzyme.", "section": "FIG", "ner": [ [ 27, 31, "Thr1", "residue_name_number" ], [ 55, 60, "water", "chemical" ] ] }, { "sid": 234, "sent": "The proteasome favours threonine as the active-site nucleophile.", "section": "FIG", "ner": [ [ 4, 14, "proteasome", "complex_assembly" ], [ 23, 32, "threonine", "residue_name" ] ] }, { "sid": 235, "sent": "(a) Growth tests by serial dilution of WT and pre2 (\u03b25) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation.", "section": "FIG", "ner": [ [ 4, 35, "Growth tests by serial dilution", "experimental_method" ], [ 39, 41, "WT", "protein_state" ], [ 52, 54, "\u03b25", "protein" ], [ 56, 62, "mutant", "protein_state" ], [ 63, 68, "yeast", "taxonomy_domain" ], [ 107, 118, "active-site", "site" ], [ 119, 126, "mutants", "experimental_method" ] ] }, { "sid": 236, "sent": "(b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (\u03b25) using the substrate Suc-LLVY-AMC.", "section": "FIG", "ner": [ [ 13, 15, "WT", "protein_state" ], [ 20, 26, "mutant", "protein_state" ], [ 27, 38, "proteasomes", "complex_assembly" ], [ 89, 91, "\u03b25", "protein" ], [ 113, 125, "Suc-LLVY-AMC", "chemical" ] ] }, { "sid": 237, "sent": "(c) Illustration of the 2FO\u2013FC electron-density map (blue mesh contoured at 1\u03c3) for the \u03b25-T1C propeptide fragment.", "section": "FIG", "ner": [ [ 24, 51, "2FO\u2013FC electron-density map", "evidence" ], [ 88, 94, "\u03b25-T1C", "mutant" ], [ 95, 105, "propeptide", "structure_element" ] ] }, { "sid": 238, "sent": "The prosegment is cleaved but still bound in the substrate-binding channel.", "section": "FIG", "ner": [ [ 4, 14, "prosegment", "structure_element" ], [ 18, 25, "cleaved", "protein_state" ], [ 30, 41, "still bound", "protein_state" ], [ 49, 74, "substrate-binding channel", "site" ] ] }, { "sid": 239, "sent": "Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the \u03b25-T1A-K81R mutant.", "section": "FIG", "ner": [ [ 9, 16, "His(-2)", "residue_name_number" ], [ 37, 46, "S1 pocket", "site" ], [ 57, 62, "Met45", "residue_name_number" ], [ 101, 112, "\u03b25-T1A-K81R", "mutant" ], [ 113, 119, "mutant", "protein_state" ] ] }, { "sid": 240, "sent": "(d) Structural superposition of the \u03b25-T1A-K81R and the \u03b25-T1C mutant subunits onto the WT \u03b25 subunit. (e) Structural superposition of the \u03b25-T1C propeptide onto the \u03b21-T1A active site (blue) and the WT \u03b25 active site in complex with the proteasome inhibitor MG132 (ref.).", "section": "FIG", "ner": [ [ 4, 28, "Structural superposition", "experimental_method" ], [ 36, 47, "\u03b25-T1A-K81R", "mutant" ], [ 56, 62, "\u03b25-T1C", "mutant" ], [ 63, 69, "mutant", "protein_state" ], [ 88, 90, "WT", "protein_state" ], [ 91, 93, "\u03b25", "protein" ], [ 107, 131, "Structural superposition", "experimental_method" ], [ 139, 145, "\u03b25-T1C", "mutant" ], [ 146, 156, "propeptide", "structure_element" ], [ 166, 172, "\u03b21-T1A", "mutant" ], [ 173, 184, "active site", "site" ], [ 200, 202, "WT", "protein_state" ], [ 203, 205, "\u03b25", "protein" ], [ 206, 217, "active site", "site" ], [ 218, 233, "in complex with", "protein_state" ], [ 238, 248, "proteasome", "complex_assembly" ], [ 259, 264, "MG132", "chemical" ] ] }, { "sid": 241, "sent": "The inhibitor as well as the propeptides adopt similar conformations in the substrate-binding channel.", "section": "FIG", "ner": [ [ 4, 13, "inhibitor", "chemical" ], [ 29, 40, "propeptides", "structure_element" ], [ 76, 101, "substrate-binding channel", "site" ] ] }, { "sid": 242, "sent": "(f) Structural superposition of the WT \u03b25 and \u03b25-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1.", "section": "FIG", "ner": [ [ 4, 28, "Structural superposition", "experimental_method" ], [ 36, 38, "WT", "protein_state" ], [ 39, 41, "\u03b25", "protein" ], [ 46, 52, "\u03b25-T1C", "mutant" ], [ 53, 59, "mutant", "protein_state" ], [ 60, 72, "active sites", "site" ], [ 137, 141, "Thr1", "residue_name_number" ], [ 170, 174, "Cys1", "residue_name_number" ] ] }, { "sid": 243, "sent": "(g) Structural superposition of the WT \u03b25 and \u03b25-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively.", "section": "FIG", "ner": [ [ 4, 28, "Structural superposition", "experimental_method" ], [ 36, 38, "WT", "protein_state" ], [ 39, 41, "\u03b25", "protein" ], [ 46, 52, "\u03b25-T1S", "mutant" ], [ 53, 59, "mutant", "protein_state" ], [ 60, 72, "active sites", "site" ], [ 130, 134, "Thr1", "residue_name_number" ], [ 139, 143, "Ser1", "residue_name_number" ] ] }, { "sid": 244, "sent": "The 2FO\u2013FC electron-density map for Ser1 (blue mesh contoured at 1\u03c3) is illustrated.", "section": "FIG", "ner": [ [ 4, 31, "2FO\u2013FC electron-density map", "evidence" ], [ 36, 40, "Ser1", "residue_name_number" ] ] }, { "sid": 245, "sent": "(h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46C\u03b2 and Thr3C\u03b3.", "section": "FIG", "ner": [ [ 24, 28, "Thr1", "residue_name_number" ], [ 44, 68, "hydrophobic interactions", "bond_interaction" ], [ 74, 79, "Ala46", "residue_name_number" ], [ 86, 90, "Thr3", "residue_name_number" ] ] }, { "sid": 246, "sent": "Ser1 lacks this stabilization and is therefore rotated by 60\u00b0.", "section": "FIG", "ner": [ [ 0, 4, "Ser1", "residue_name_number" ], [ 5, 10, "lacks", "protein_state" ] ] }, { "sid": 247, "sent": "Inhibition of WT and mutant \u03b25-T1S proteasomes by bortezomib and carfilzomib.", "section": "FIG", "ner": [ [ 14, 16, "WT", "protein_state" ], [ 21, 27, "mutant", "protein_state" ], [ 28, 34, "\u03b25-T1S", "mutant" ], [ 35, 46, "proteasomes", "complex_assembly" ], [ 50, 60, "bortezomib", "chemical" ], [ 65, 76, "carfilzomib", "chemical" ] ] }, { "sid": 248, "sent": "Inhibition assays (left panel).", "section": "FIG", "ner": [ [ 0, 17, "Inhibition assays", "experimental_method" ] ] }, { "sid": 249, "sent": "Purified yeast proteasomes were tested for the susceptibility of their ChT-L (\u03b25) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC.", "section": "FIG", "ner": [ [ 9, 14, "yeast", "taxonomy_domain" ], [ 15, 26, "proteasomes", "complex_assembly" ], [ 78, 80, "\u03b25", "protein" ], [ 108, 118, "bortezomib", "chemical" ], [ 123, 134, "carfilzomib", "chemical" ], [ 155, 167, "Suc-LLVY-AMC", "chemical" ] ] }, { "sid": 250, "sent": "IC50 values were determined in triplicate; s.d.'s are indicated by error bars.", "section": "FIG", "ner": [ [ 0, 11, "IC50 values", "evidence" ] ] }, { "sid": 251, "sent": "Note that IC50 values depend on time and enzyme concentration.", "section": "FIG", "ner": [ [ 10, 21, "IC50 values", "evidence" ] ] }, { "sid": 252, "sent": "Proteasomes (final concentration: 66\u2009nM) were incubated with inhibitor for 45\u2009min before substrate addition (final concentration: 200\u2009\u03bcM).", "section": "FIG", "ner": [ [ 0, 11, "Proteasomes", "complex_assembly" ] ] }, { "sid": 253, "sent": "Structures of the \u03b25-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel).", "section": "FIG", "ner": [ [ 0, 10, "Structures", "evidence" ], [ 18, 24, "\u03b25-T1S", "mutant" ], [ 25, 31, "mutant", "protein_state" ], [ 35, 60, "complex with both ligands", "complex_assembly" ], [ 93, 97, "Ser1", "residue_name_number" ] ] }, { "sid": 254, "sent": "The 2FO\u2013FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1\u03c3.", "section": "FIG", "ner": [ [ 4, 32, "2FO\u2013FC electron-density maps", "evidence" ], [ 49, 53, "Ser1", "residue_name_number" ], [ 112, 119, "P1 site", "site" ], [ 121, 125, "Leu1", "residue_name_number" ] ] }, { "sid": 255, "sent": "The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib.", "section": "FIG", "ner": [ [ 4, 6, "WT", "protein_state" ], [ 7, 35, "proteasome:inhibitor complex", "complex_assembly" ], [ 36, 46, "structures", "evidence" ], [ 67, 71, "Thr1", "residue_name_number" ], [ 86, 98, "superimposed", "experimental_method" ], [ 120, 128, "mutation", "experimental_method" ], [ 132, 136, "Thr1", "residue_name_number" ], [ 140, 143, "Ser", "residue_name" ], [ 180, 190, "bortezomib", "chemical" ], [ 194, 205, "carfilzomib", "chemical" ] ] } ] }, "PMC4871749": { "annotations": [ { "sid": 0, "sent": "The Taf14 YEATS domain is a reader of histone crotonylation", "section": "TITLE", "ner": [ [ 4, 9, "Taf14", "protein" ], [ 10, 22, "YEATS domain", "structure_element" ], [ 38, 45, "histone", "protein_type" ], [ 46, 59, "crotonylation", "ptm" ] ] }, { "sid": 1, "sent": "The discovery of new histone modifications is unfolding at startling rates, however, the identification of effectors capable of interpreting these modifications has lagged behind.", "section": "ABSTRACT", "ner": [ [ 21, 28, "histone", "protein_type" ] ] }, { "sid": 2, "sent": "Here we report the YEATS domain as an effective reader of histone lysine crotonylation \u2013 an epigenetic signature associated with active transcription.", "section": "ABSTRACT", "ner": [ [ 19, 31, "YEATS domain", "structure_element" ], [ 58, 65, "histone", "protein_type" ], [ 66, 72, "lysine", "residue_name" ], [ 73, 86, "crotonylation", "ptm" ] ] }, { "sid": 3, "sent": "We show that the Taf14 YEATS domain engages crotonyllysine via a unique \u03c0-\u03c0-\u03c0-stacking mechanism and that other YEATS domains have crotonyllysine binding activity.", "section": "ABSTRACT", "ner": [ [ 17, 22, "Taf14", "protein" ], [ 23, 35, "YEATS domain", "structure_element" ], [ 44, 58, "crotonyllysine", "residue_name" ], [ 72, 86, "\u03c0-\u03c0-\u03c0-stacking", "bond_interaction" ], [ 112, 125, "YEATS domains", "structure_element" ], [ 131, 145, "crotonyllysine", "residue_name" ] ] }, { "sid": 4, "sent": "Crotonylation of lysine residues (crotonyllysine, Kcr) has emerged as one of the fundamental histone post-translational modifications (PTMs) found in mammalian chromatin.", "section": "INTRO", "ner": [ [ 0, 13, "Crotonylation", "ptm" ], [ 17, 23, "lysine", "residue_name" ], [ 34, 48, "crotonyllysine", "residue_name" ], [ 50, 53, "Kcr", "residue_name" ], [ 93, 100, "histone", "protein_type" ], [ 150, 159, "mammalian", "taxonomy_domain" ] ] }, { "sid": 5, "sent": "The crotonyllysine mark on histone H3K18 is produced by p300, a histone acetyltransferase also responsible for acetylation of histones.", "section": "INTRO", "ner": [ [ 4, 18, "crotonyllysine", "residue_name" ], [ 27, 34, "histone", "protein_type" ], [ 35, 37, "H3", "protein_type" ], [ 37, 40, "K18", "residue_name_number" ], [ 56, 60, "p300", "protein" ], [ 64, 89, "histone acetyltransferase", "protein_type" ], [ 111, 122, "acetylation", "ptm" ] ] }, { "sid": 6, "sent": "Owing to some differences in their genomic distribution, the crotonyllysine and acetyllysine (Kac) modifications have been linked to distinct functional outcomes.", "section": "INTRO", "ner": [ [ 61, 75, "crotonyllysine", "residue_name" ], [ 80, 92, "acetyllysine", "residue_name" ], [ 94, 97, "Kac", "residue_name" ] ] }, { "sid": 7, "sent": "p300-catalyzed histone crotonylation, which is likely metabolically regulated, stimulates transcription to a greater degree than p300-catalyzed acetylation.", "section": "INTRO", "ner": [ [ 0, 4, "p300", "protein" ], [ 15, 22, "histone", "protein_type" ], [ 23, 36, "crotonylation", "ptm" ], [ 129, 133, "p300", "protein" ], [ 144, 155, "acetylation", "ptm" ] ] }, { "sid": 8, "sent": "The discovery of individual biological roles for the crotonyllysine and acetyllysine marks suggests that these PTMs can be read by distinct readers.", "section": "INTRO", "ner": [ [ 53, 67, "crotonyllysine", "residue_name" ], [ 72, 84, "acetyllysine", "residue_name" ] ] }, { "sid": 9, "sent": "While a number of acetyllysine readers have been identified and characterized, a specific reader of the crotonyllysine mark remains unknown (reviewed in).", "section": "INTRO", "ner": [ [ 18, 30, "acetyllysine", "residue_name" ], [ 104, 118, "crotonyllysine", "residue_name" ] ] }, { "sid": 10, "sent": "A recent survey of bromodomains (BDs) demonstrates that only one BD associates very weakly with a crotonylated peptide, however it binds more tightly to acetylated peptides, inferring that bromodomains do not possess physiologically relevant crotonyllysine binding activity.", "section": "INTRO", "ner": [ [ 19, 31, "bromodomains", "structure_element" ], [ 33, 36, "BDs", "structure_element" ], [ 65, 67, "BD", "structure_element" ], [ 98, 110, "crotonylated", "protein_state" ], [ 153, 163, "acetylated", "protein_state" ], [ 189, 201, "bromodomains", "structure_element" ], [ 242, 256, "crotonyllysine", "residue_name" ] ] }, { "sid": 11, "sent": "The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac.", "section": "INTRO", "ner": [ [ 14, 26, "acetyllysine", "residue_name" ], [ 81, 86, "YEATS", "structure_element" ], [ 88, 92, "Yaf9", "protein" ], [ 94, 97, "ENL", "protein" ], [ 99, 102, "AF9", "protein" ], [ 104, 109, "Taf14", "protein" ], [ 111, 115, "Sas5", "protein" ], [ 128, 133, "human", "species" ], [ 134, 137, "AF9", "protein" ], [ 142, 147, "yeast", "taxonomy_domain" ], [ 148, 153, "Taf14", "protein" ], [ 185, 192, "histone", "protein_type" ], [ 198, 200, "H3", "protein_type" ], [ 200, 204, "K9ac", "ptm" ] ] }, { "sid": 12, "sent": "The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription.", "section": "INTRO", "ner": [ [ 4, 16, "acetyllysine", "residue_name" ], [ 41, 44, "AF9", "protein" ], [ 45, 57, "YEATS domain", "structure_element" ], [ 98, 123, "histone methyltransferase", "protein_type" ], [ 124, 129, "DOT1L", "protein" ], [ 133, 135, "H3", "protein_type" ], [ 135, 139, "K9ac", "ptm" ], [ 169, 174, "DOT1L", "protein" ], [ 184, 186, "H3", "protein_type" ], [ 186, 189, "K79", "residue_name_number" ], [ 190, 201, "methylation", "ptm" ] ] }, { "sid": 13, "sent": "Similarly, activation of a subset of genes and DNA damage repair in yeast require the acetyllysine binding activity of the Taf14 YEATS domain.", "section": "INTRO", "ner": [ [ 68, 73, "yeast", "taxonomy_domain" ], [ 86, 98, "acetyllysine", "residue_name" ], [ 123, 128, "Taf14", "protein" ], [ 129, 141, "YEATS domain", "structure_element" ] ] }, { "sid": 14, "sent": "Consistent with its role in gene regulation, Taf14 was identified as a core component of the transcription factor complexes TFIID and TFIIF.", "section": "INTRO", "ner": [ [ 45, 50, "Taf14", "protein" ], [ 124, 129, "TFIID", "complex_assembly" ], [ 134, 139, "TFIIF", "complex_assembly" ] ] }, { "sid": 15, "sent": "However, Taf14 is also found in a number of chromatin-remodeling complexes (i.e., INO80, SWI/SNF and RSC) and the histone acetyltransferase complex NuA3, indicating a multifaceted role of Taf14 in transcriptional regulation and chromatin biology.", "section": "INTRO", "ner": [ [ 9, 14, "Taf14", "protein" ], [ 82, 87, "INO80", "complex_assembly" ], [ 89, 96, "SWI/SNF", "complex_assembly" ], [ 101, 104, "RSC", "complex_assembly" ], [ 114, 139, "histone acetyltransferase", "protein_type" ], [ 148, 152, "NuA3", "complex_assembly" ], [ 188, 193, "Taf14", "protein" ] ] }, { "sid": 16, "sent": "In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism.", "section": "INTRO", "ner": [ [ 33, 38, "Taf14", "protein" ], [ 39, 51, "YEATS domain", "structure_element" ], [ 67, 81, "crotonyllysine", "residue_name" ], [ 96, 103, "histone", "protein_type" ], [ 104, 106, "H3", "protein_type" ], [ 107, 119, "crotonylated", "protein_state" ], [ 123, 131, "lysine 9", "residue_name_number" ], [ 133, 135, "H3", "protein_type" ], [ 135, 139, "K9cr", "ptm" ] ] }, { "sid": 17, "sent": "We found that H3K9cr is present in yeast and is dynamically regulated.", "section": "INTRO", "ner": [ [ 14, 16, "H3", "protein_type" ], [ 16, 20, "K9cr", "ptm" ], [ 35, 40, "yeast", "taxonomy_domain" ] ] }, { "sid": 18, "sent": "To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5\u201313 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1).", "section": "INTRO", "ner": [ [ 56, 58, "H3", "protein_type" ], [ 58, 62, "K9cr", "ptm" ], [ 83, 100, "crystal structure", "evidence" ], [ 108, 113, "Taf14", "protein" ], [ 114, 126, "YEATS domain", "structure_element" ], [ 127, 142, "in complex with", "protein_state" ], [ 143, 153, "H3K9cr5-13", "chemical" ], [ 164, 168, "5\u201313", "residue_range" ], [ 172, 174, "H3", "protein_type" ] ] }, { "sid": 19, "sent": "The Taf14 YEATS domain adopts an immunoglobin-like \u03b2 sandwich fold containing eight anti-parallel \u03b2 strands linked by short loops that form a binding site for H3K9cr (Fig. 1b).", "section": "INTRO", "ner": [ [ 4, 9, "Taf14", "protein" ], [ 10, 22, "YEATS domain", "structure_element" ], [ 33, 66, "immunoglobin-like \u03b2 sandwich fold", "structure_element" ], [ 84, 107, "anti-parallel \u03b2 strands", "structure_element" ], [ 124, 129, "loops", "structure_element" ], [ 142, 154, "binding site", "site" ], [ 159, 161, "H3", "protein_type" ], [ 161, 165, "K9cr", "ptm" ] ] }, { "sid": 20, "sent": "The H3K9cr peptide lays in an extended conformation in an orientation orthogonal to the \u03b2 strands and is stabilized through an extensive network of direct and water-mediated hydrogen bonds and a salt bridge (Fig. 1c).", "section": "INTRO", "ner": [ [ 4, 6, "H3", "protein_type" ], [ 6, 10, "K9cr", "ptm" ], [ 30, 51, "extended conformation", "protein_state" ], [ 88, 97, "\u03b2 strands", "structure_element" ], [ 159, 164, "water", "chemical" ], [ 174, 188, "hydrogen bonds", "bond_interaction" ], [ 195, 206, "salt bridge", "bond_interaction" ] ] }, { "sid": 21, "sent": "The most striking feature of the crotonyllysine recognition mechanism is the unique coordination of crotonylated lysine residue.", "section": "INTRO", "ner": [ [ 33, 47, "crotonyllysine", "residue_name" ], [ 100, 112, "crotonylated", "protein_state" ], [ 113, 119, "lysine", "residue_name" ] ] }, { "sid": 22, "sent": "The fully extended side chain of K9cr transverses the narrow tunnel, crossing the \u03b2 sandwich at right angle in a corkscrew-like manner (Fig. 1b and Supplementary Figure 1b).", "section": "INTRO", "ner": [ [ 33, 37, "K9cr", "ptm" ], [ 82, 92, "\u03b2 sandwich", "structure_element" ] ] }, { "sid": 23, "sent": "The planar crotonyl group is inserted between Trp81 and Phe62 of the protein, the aromatic rings of which are positioned strictly parallel to each other and at equal distance from the crotonyl group, yielding a novel aromatic-amide/aliphatic-aromatic \u03c0-\u03c0-\u03c0-stacking system that, to our knowledge, has not been reported previously for any protein-protein interaction (Fig. 1d and Supplementary Fig. 1c).", "section": "INTRO", "ner": [ [ 11, 19, "crotonyl", "chemical" ], [ 46, 51, "Trp81", "residue_name_number" ], [ 56, 61, "Phe62", "residue_name_number" ], [ 184, 192, "crotonyl", "chemical" ], [ 251, 265, "\u03c0-\u03c0-\u03c0-stacking", "bond_interaction" ] ] }, { "sid": 24, "sent": "The side chain of Trp81 appears to adopt two conformations, one of which provides maximum \u03c0-stacking with the alkene functional group while the other rotamer affords maximum \u03c0-stacking with the amide \u03c0 electrons (Supplementary Fig. 1c).", "section": "INTRO", "ner": [ [ 18, 23, "Trp81", "residue_name_number" ], [ 90, 100, "\u03c0-stacking", "bond_interaction" ], [ 174, 184, "\u03c0-stacking", "bond_interaction" ] ] }, { "sid": 25, "sent": "The dual conformation of Trp81 is likely due to the conjugated nature of the C=C and C=O \u03c0-orbitals within the crotonyl functional group.", "section": "INTRO", "ner": [ [ 25, 30, "Trp81", "residue_name_number" ], [ 111, 119, "crotonyl", "chemical" ] ] }, { "sid": 26, "sent": "In addition to \u03c0-\u03c0-\u03c0 stacking, the crotonyl group is stabilized by a set of hydrogen bonds and electrostatic interactions.", "section": "INTRO", "ner": [ [ 15, 29, "\u03c0-\u03c0-\u03c0 stacking", "bond_interaction" ], [ 35, 43, "crotonyl", "chemical" ], [ 76, 90, "hydrogen bonds", "bond_interaction" ], [ 95, 121, "electrostatic interactions", "bond_interaction" ] ] }, { "sid": 27, "sent": "The \u03c0 bond conjugation of the crotonyl group gives rise to a dipole moment of the alkene moiety, resulting in a partial positive charge on the \u03b2-carbon (C\u03b2) and a partial negative charge on the \u03b1-carbon (C\u03b1).", "section": "INTRO", "ner": [ [ 4, 10, "\u03c0 bond", "bond_interaction" ], [ 30, 38, "crotonyl", "chemical" ] ] }, { "sid": 28, "sent": "This provides the capability for the alkene moiety to form electrostatic contacts, as C\u03b1 and C\u03b2 lay within electrostatic interaction distances of the carbonyl oxygen of Gln79 and of the hydroxyl group of Thr61, respectively.", "section": "INTRO", "ner": [ [ 59, 81, "electrostatic contacts", "bond_interaction" ], [ 107, 132, "electrostatic interaction", "bond_interaction" ], [ 169, 174, "Gln79", "residue_name_number" ], [ 204, 209, "Thr61", "residue_name_number" ] ] }, { "sid": 29, "sent": "The hydroxyl group of Thr61 also participates in a hydrogen bond with the amide nitrogen of the K9cr side chain (Fig. 1d).", "section": "INTRO", "ner": [ [ 22, 27, "Thr61", "residue_name_number" ], [ 51, 64, "hydrogen bond", "bond_interaction" ], [ 96, 100, "K9cr", "ptm" ] ] }, { "sid": 30, "sent": "The fixed position of the Thr61 hydroxyl group, which facilitates interactions with both the amide and C\u03b1 of K9cr, is achieved through a hydrogen bond with imidazole ring of His59.", "section": "INTRO", "ner": [ [ 26, 31, "Thr61", "residue_name_number" ], [ 109, 113, "K9cr", "ptm" ], [ 137, 150, "hydrogen bond", "bond_interaction" ], [ 174, 179, "His59", "residue_name_number" ] ] }, { "sid": 31, "sent": "Extra stabilization of K9cr is attained by a hydrogen bond formed between its carbonyl oxygen and the backbone nitrogen of Trp81, as well as a water-mediated hydrogen bond with the backbone carbonyl group of Gly82 (Fig 1d).", "section": "INTRO", "ner": [ [ 23, 27, "K9cr", "ptm" ], [ 45, 58, "hydrogen bond", "bond_interaction" ], [ 123, 128, "Trp81", "residue_name_number" ], [ 143, 148, "water", "chemical" ], [ 158, 171, "hydrogen bond", "bond_interaction" ], [ 208, 213, "Gly82", "residue_name_number" ] ] }, { "sid": 32, "sent": "This distinctive mechanism was corroborated through mapping the Taf14 YEATS-H3K9cr binding interface in solution using NMR chemical shift perturbation analysis (Supplementary Fig. 2a, b).", "section": "INTRO", "ner": [ [ 64, 69, "Taf14", "protein" ], [ 70, 100, "YEATS-H3K9cr binding interface", "site" ], [ 119, 159, "NMR chemical shift perturbation analysis", "experimental_method" ] ] }, { "sid": 33, "sent": "Binding of the Taf14 YEATS domain to H3K9cr is robust.", "section": "INTRO", "ner": [ [ 15, 20, "Taf14", "protein" ], [ 21, 33, "YEATS domain", "structure_element" ], [ 37, 39, "H3", "protein_type" ], [ 39, 43, "K9cr", "ptm" ] ] }, { "sid": 34, "sent": "The dissociation constant (Kd) for the Taf14 YEATS-H3K9cr5-13 complex was found to be 9.5 \u03bcM, as measured by fluorescence spectroscopy (Supplementary Fig. 2c).", "section": "INTRO", "ner": [ [ 4, 25, "dissociation constant", "evidence" ], [ 27, 29, "Kd", "evidence" ], [ 39, 61, "Taf14 YEATS-H3K9cr5-13", "complex_assembly" ], [ 109, 134, "fluorescence spectroscopy", "experimental_method" ] ] }, { "sid": 35, "sent": "This value is in the range of binding affinities exhibited by the majority of histone readers, thus attesting to the physiological relevance of the H3K9cr recognition by Taf14.", "section": "INTRO", "ner": [ [ 30, 48, "binding affinities", "evidence" ], [ 148, 150, "H3", "protein_type" ], [ 150, 154, "K9cr", "ptm" ], [ 170, 175, "Taf14", "protein" ] ] }, { "sid": 36, "sent": "To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2).", "section": "INTRO", "ner": [ [ 21, 23, "H3", "protein_type" ], [ 23, 27, "K9cr", "ptm" ], [ 42, 47, "yeast", "taxonomy_domain" ], [ 62, 81, "whole cell extracts", "experimental_method" ], [ 111, 116, "yeast", "taxonomy_domain" ], [ 145, 166, "Western blot analysis", "experimental_method" ], [ 201, 203, "H3", "protein_type" ], [ 203, 207, "K9cr", "ptm" ], [ 209, 211, "H3", "protein_type" ], [ 211, 215, "K9ac", "ptm" ], [ 220, 222, "H3", "protein_type" ] ] }, { "sid": 37, "sent": "Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast.", "section": "INTRO", "ner": [ [ 5, 7, "H3", "protein_type" ], [ 7, 11, "K9cr", "ptm" ], [ 16, 18, "H3", "protein_type" ], [ 18, 22, "K9ac", "ptm" ], [ 40, 45, "yeast", "taxonomy_domain" ], [ 46, 54, "histones", "protein_type" ], [ 102, 104, "H3", "protein_type" ], [ 104, 108, "K9cr", "ptm" ], [ 122, 127, "yeast", "taxonomy_domain" ] ] }, { "sid": 38, "sent": "We next asked if H3K9cr is regulated by the actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs).", "section": "INTRO", "ner": [ [ 17, 19, "H3", "protein_type" ], [ 19, 23, "K9cr", "ptm" ], [ 55, 81, "histone acetyltransferases", "protein_type" ], [ 83, 87, "HATs", "protein_type" ], [ 93, 113, "histone deacetylases", "protein_type" ], [ 115, 120, "HDACs", "protein_type" ] ] }, { "sid": 39, "sent": "Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted.", "section": "INTRO", "ner": [ [ 50, 55, "yeast", "taxonomy_domain" ], [ 77, 82, "yeast", "taxonomy_domain" ], [ 83, 87, "HATs", "protein_type" ], [ 89, 93, "HAT1", "protein" ], [ 95, 99, "Gcn5", "protein" ], [ 105, 111, "Rtt109", "protein" ], [ 116, 121, "HDACs", "protein_type" ], [ 123, 127, "Rpd3", "protein" ], [ 129, 133, "Hos1", "protein" ], [ 139, 143, "Hos2", "protein" ], [ 150, 157, "deleted", "experimental_method" ] ] }, { "sid": 40, "sent": "As shown in Figure 2a, b and Supplementary Fig. 3e, H3K9cr levels were abolished or reduced considerably in the HAT deletion strains, whereas they were dramatically increased in the HDAC deletion strains.", "section": "INTRO", "ner": [ [ 52, 54, "H3", "protein_type" ], [ 54, 58, "K9cr", "ptm" ], [ 112, 115, "HAT", "protein_type" ], [ 116, 124, "deletion", "experimental_method" ], [ 182, 186, "HDAC", "protein_type" ], [ 187, 195, "deletion", "experimental_method" ] ] }, { "sid": 41, "sent": "Furthermore, fluctuations in the H3K9cr levels were more substantial than fluctuations in the corresponding H3K9ac levels.", "section": "INTRO", "ner": [ [ 33, 35, "H3", "protein_type" ], [ 35, 39, "K9cr", "ptm" ], [ 108, 110, "H3", "protein_type" ], [ 110, 114, "K9ac", "ptm" ] ] }, { "sid": 42, "sent": "Together, these results reveal that H3K9cr is a dynamic mark of chromatin in yeast and suggest an important role for this modification in transcription as it is regulated by HATs and HDACs.", "section": "INTRO", "ner": [ [ 36, 38, "H3", "protein_type" ], [ 38, 42, "K9cr", "ptm" ], [ 77, 82, "yeast", "taxonomy_domain" ], [ 174, 178, "HATs", "protein_type" ], [ 183, 188, "HDACs", "protein_type" ] ] }, { "sid": 43, "sent": "We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter.", "section": "INTRO", "ner": [ [ 36, 46, "acetylated", "protein_state" ], [ 47, 54, "histone", "protein_type" ], [ 66, 71, "Taf14", "protein" ], [ 72, 84, "YEATS domain", "structure_element" ], [ 93, 103, "acetylated", "protein_state" ], [ 104, 106, "H3", "protein_type" ], [ 106, 108, "K9", "residue_name_number" ], [ 163, 165, "H3", "protein_type" ], [ 165, 169, "K9cr", "ptm" ] ] }, { "sid": 44, "sent": "The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b).", "section": "INTRO", "ner": [ [ 19, 24, "Taf14", "protein" ], [ 33, 47, "crotonyllysine", "residue_name" ], [ 69, 80, "1H,15N HSQC", "experimental_method" ], [ 110, 120, "H3K9cr5-13", "chemical" ], [ 124, 134, "H3K9ac5-13", "chemical" ], [ 147, 155, "titrated", "experimental_method" ], [ 165, 176, "15N-labeled", "protein_state" ], [ 177, 182, "Taf14", "protein" ], [ 183, 195, "YEATS domain", "structure_element" ] ] }, { "sid": 45, "sent": "Binding of H3K9cr induced resonance changes in slow exchange regime on the NMR time scale, indicative of strong interaction.", "section": "INTRO", "ner": [ [ 11, 13, "H3", "protein_type" ], [ 13, 17, "K9cr", "ptm" ], [ 26, 43, "resonance changes", "evidence" ], [ 75, 78, "NMR", "experimental_method" ] ] }, { "sid": 46, "sent": "In contrast, binding of H3K9ac resulted in an intermediate exchange, which is characteristic of a weaker association.", "section": "INTRO", "ner": [ [ 24, 26, "H3", "protein_type" ], [ 26, 30, "K9ac", "ptm" ] ] }, { "sid": 47, "sent": "Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a).", "section": "INTRO", "ner": [ [ 13, 23, "crosspeaks", "evidence" ], [ 27, 32, "Gly80", "residue_name_number" ], [ 37, 42, "Trp81", "residue_name_number" ], [ 50, 62, "YEATS domain", "structure_element" ], [ 90, 92, "H3", "protein_type" ], [ 92, 96, "K9cr", "ptm" ], [ 101, 103, "H3", "protein_type" ], [ 103, 107, "K9ac", "ptm" ], [ 171, 218, "crotonyllysine and acetyllysine binding pockets", "site" ] ] }, { "sid": 48, "sent": "These differences support our model that Trp81 adopts two conformations upon complex formation with the H3K9cr mark as compared to H3K9ac (Supplementary Figs. 1c, d and 4c).", "section": "INTRO", "ner": [ [ 41, 46, "Trp81", "residue_name_number" ], [ 104, 106, "H3", "protein_type" ], [ 106, 110, "K9cr", "ptm" ], [ 131, 133, "H3", "protein_type" ], [ 133, 137, "K9ac", "ptm" ] ] }, { "sid": 49, "sent": "One of the conformations, characterized by the \u03c0 stacking involving two aromatic residues and the alkene group, is observed only in the YEATS-H3K9cr complex.", "section": "INTRO", "ner": [ [ 136, 148, "YEATS-H3K9cr", "complex_assembly" ] ] }, { "sid": 50, "sent": "To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1\u201320 of H3).", "section": "INTRO", "ner": [ [ 25, 30, "Taf14", "protein" ], [ 31, 43, "YEATS domain", "structure_element" ], [ 91, 101, "acyllysine", "residue_name" ], [ 122, 147, "solution pull-down assays", "experimental_method" ], [ 154, 156, "H3", "protein_type" ], [ 166, 176, "acetylated", "protein_state" ], [ 178, 191, "propionylated", "protein_state" ], [ 193, 204, "butyrylated", "protein_state" ], [ 210, 222, "crotonylated", "protein_state" ], [ 226, 234, "lysine 9", "residue_name_number" ], [ 245, 249, "1\u201320", "residue_range" ], [ 253, 255, "H3", "protein_type" ] ] }, { "sid": 51, "sent": "As shown in Figure 2d and Supplementary Fig. 5a, the Taf14 YEATS domain binds more strongly to H3K9cr1-20, as compared to other acylated histone peptides.", "section": "INTRO", "ner": [ [ 53, 58, "Taf14", "protein" ], [ 59, 71, "YEATS domain", "structure_element" ], [ 95, 105, "H3K9cr1-20", "chemical" ], [ 128, 136, "acylated", "protein_state" ] ] }, { "sid": 52, "sent": "The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments.", "section": "INTRO", "ner": [ [ 19, 21, "H3", "protein_type" ], [ 21, 25, "K9cr", "ptm" ], [ 31, 33, "H3", "protein_type" ], [ 33, 37, "K9ac", "ptm" ], [ 39, 41, "H3", "protein_type" ], [ 41, 45, "K9pr", "ptm" ], [ 50, 52, "H3", "protein_type" ], [ 52, 56, "K9bu", "ptm" ], [ 74, 107, "1H,15N HSQC titration experiments", "experimental_method" ] ] }, { "sid": 53, "sent": "Addition of H3K9ac1-20, H3K9pr1-20, and H3K9bu1-20 peptides caused chemical shift perturbations in the Taf14 YEATS domain in intermediate exchange regime, implying that these interactions are weaker compared to the interaction with the H3K9cr1-20 peptide (Supplementary Fig. 5b).", "section": "INTRO", "ner": [ [ 12, 22, "H3K9ac1-20", "chemical" ], [ 24, 34, "H3K9pr1-20", "chemical" ], [ 40, 50, "H3K9bu1-20", "chemical" ], [ 67, 95, "chemical shift perturbations", "evidence" ], [ 103, 108, "Taf14", "protein" ], [ 109, 121, "YEATS domain", "structure_element" ], [ 236, 246, "H3K9cr1-20", "chemical" ] ] }, { "sid": 54, "sent": "We concluded that H3K9cr is the preferred target of this domain.", "section": "INTRO", "ner": [ [ 18, 20, "H3", "protein_type" ], [ 20, 24, "K9cr", "ptm" ] ] }, { "sid": 55, "sent": "From comparative structural analysis of the YEATS complexes, Gly80 emerged as candidate residue potentially responsible for the preference for crotonyllysine.", "section": "INTRO", "ner": [ [ 5, 36, "comparative structural analysis", "experimental_method" ], [ 61, 66, "Gly80", "residue_name_number" ], [ 143, 157, "crotonyllysine", "residue_name" ] ] }, { "sid": 56, "sent": "In attempt to generate a mutant capable of accommodating a short acetyl moiety but discriminating against a longer, planar crotonyl moiety, we mutated Gly80 to more bulky residues, however all mutants of Gly80 lost their binding activities towards either acylated peptide, suggesting that Gly80 is absolutely required for the interaction.", "section": "INTRO", "ner": [ [ 123, 131, "crotonyl", "chemical" ], [ 143, 150, "mutated", "protein_state" ], [ 151, 156, "Gly80", "residue_name_number" ], [ 193, 203, "mutants of", "protein_state" ], [ 204, 209, "Gly80", "residue_name_number" ], [ 255, 263, "acylated", "protein_state" ], [ 289, 294, "Gly80", "residue_name_number" ] ] }, { "sid": 57, "sent": "In contrast, mutation of Val24, a residue located on another side of Trp81, had no effect on binding (Fig. 2d and Supplementary Fig. 5a, c).", "section": "INTRO", "ner": [ [ 13, 21, "mutation", "experimental_method" ], [ 25, 30, "Val24", "residue_name_number" ], [ 69, 74, "Trp81", "residue_name_number" ] ] }, { "sid": 58, "sent": "To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6).", "section": "INTRO", "ner": [ [ 31, 45, "crotonyllysine", "residue_name" ], [ 49, 58, "conserved", "protein_state" ], [ 70, 75, "human", "species" ], [ 76, 89, "YEATS domains", "structure_element" ], [ 93, 114, "pull-down experiments", "experimental_method" ], [ 141, 151, "acetylated", "protein_state" ], [ 153, 166, "propionylated", "protein_state" ], [ 168, 179, "butyrylated", "protein_state" ], [ 185, 197, "crotonylated", "protein_state" ], [ 198, 205, "histone", "protein_type" ] ] }, { "sid": 59, "sent": "We found that all YEATS domains tested are capable of binding to crotonyllysine peptides, though they display variable preferences for the acyl moieties.", "section": "INTRO", "ner": [ [ 18, 31, "YEATS domains", "structure_element" ], [ 65, 79, "crotonyllysine", "residue_name" ] ] }, { "sid": 60, "sent": "While YEATS2 and ENL showed selectivity for the crotonylated peptides, GAS41 and AF9 bound acylated peptides almost equally well.", "section": "INTRO", "ner": [ [ 6, 12, "YEATS2", "protein" ], [ 17, 20, "ENL", "protein" ], [ 48, 60, "crotonylated", "protein_state" ], [ 71, 76, "GAS41", "protein" ], [ 81, 84, "AF9", "protein" ], [ 91, 99, "acylated", "protein_state" ] ] }, { "sid": 61, "sent": "Unlike the YEATS domain, a known acetyllysine reader, bromodomain, does not recognize crotonyllysine.", "section": "INTRO", "ner": [ [ 11, 23, "YEATS domain", "structure_element" ], [ 33, 52, "acetyllysine reader", "protein_type" ], [ 54, 65, "bromodomain", "structure_element" ], [ 86, 100, "crotonyllysine", "residue_name" ] ] }, { "sid": 62, "sent": "We assayed a large set of BDs in pull-down experiments and found that this module is highly specific for acetyllysine and propionyllysine containing peptides (Supplementary Fig. 7).", "section": "INTRO", "ner": [ [ 26, 29, "BDs", "structure_element" ], [ 33, 54, "pull-down experiments", "experimental_method" ], [ 105, 117, "acetyllysine", "residue_name" ], [ 122, 137, "propionyllysine", "residue_name" ] ] }, { "sid": 63, "sent": "However, bromodomains did not interact (or associated very weakly) with longer acyl modifications, including crotonyllysine, as in the case of BDs of TAF1 and BRD2, supporting recent reports.", "section": "INTRO", "ner": [ [ 9, 21, "bromodomains", "structure_element" ], [ 109, 123, "crotonyllysine", "residue_name" ], [ 143, 146, "BDs", "structure_element" ], [ 150, 154, "TAF1", "protein" ], [ 159, 163, "BRD2", "protein" ] ] }, { "sid": 64, "sent": "These results demonstrate that the YEATS domain is currently the sole reader of crotonyllysine.", "section": "INTRO", "ner": [ [ 35, 47, "YEATS domain", "structure_element" ], [ 80, 94, "crotonyllysine", "residue_name" ] ] }, { "sid": 65, "sent": "In conclusion, we have identified the YEATS domain of Taf14 as the first reader of histone crotonylation.", "section": "INTRO", "ner": [ [ 38, 50, "YEATS domain", "structure_element" ], [ 54, 59, "Taf14", "protein" ], [ 83, 90, "histone", "protein_type" ], [ 91, 104, "crotonylation", "ptm" ] ] }, { "sid": 66, "sent": "The unique and previously unobserved aromatic-amide/aliphatic-aromatic \u03c0-\u03c0-\u03c0-stacking mechanism facilitates the specific recognition of the crotonyl moiety.", "section": "INTRO", "ner": [ [ 71, 85, "\u03c0-\u03c0-\u03c0-stacking", "bond_interaction" ], [ 140, 148, "crotonyl", "chemical" ] ] }, { "sid": 67, "sent": "We further demonstrate that H3K9cr exists in yeast and is dynamically regulated by HATs and HDACs.", "section": "INTRO", "ner": [ [ 28, 30, "H3", "protein_type" ], [ 30, 34, "K9cr", "ptm" ], [ 45, 50, "yeast", "taxonomy_domain" ], [ 83, 87, "HATs", "protein_type" ], [ 92, 97, "HDACs", "protein_type" ] ] }, { "sid": 68, "sent": "As we previously showed the importance of acyllysine binding by the Taf14 YEATS domain for the DNA damage response and gene transcription, it will be essential in the future to define the physiological role of crotonyllysine recognition and to differentiate the activities of Taf14 that are due to binding to crotonyllysine and acetyllysine modifications.", "section": "INTRO", "ner": [ [ 42, 52, "acyllysine", "residue_name" ], [ 68, 73, "Taf14", "protein" ], [ 74, 86, "YEATS domain", "structure_element" ], [ 210, 224, "crotonyllysine", "residue_name" ], [ 276, 281, "Taf14", "protein" ], [ 309, 323, "crotonyllysine", "residue_name" ], [ 328, 340, "acetyllysine", "residue_name" ] ] }, { "sid": 69, "sent": "Furthermore, the functional significance of crotonyllysine recognition by other YEATS proteins will be of great importance to elucidate and compare.", "section": "INTRO", "ner": [ [ 44, 58, "crotonyllysine", "residue_name" ], [ 80, 85, "YEATS", "protein_type" ] ] }, { "sid": 70, "sent": "The structural mechanism for the recognition of H3K9cr", "section": "FIG", "ner": [ [ 48, 50, "H3", "protein_type" ], [ 50, 54, "K9cr", "ptm" ] ] }, { "sid": 71, "sent": "(a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain.", "section": "FIG", "ner": [ [ 26, 40, "crotonyllysine", "residue_name" ], [ 50, 67, "crystal structure", "evidence" ], [ 75, 80, "Taf14", "protein" ], [ 81, 93, "YEATS domain", "structure_element" ], [ 102, 117, "in complex with", "protein_state" ], [ 122, 132, "H3K9cr5-13", "chemical" ], [ 154, 156, "H3", "protein_type" ], [ 156, 160, "K9cr", "ptm" ], [ 218, 254, "electrostatic and polar interactions", "bond_interaction" ], [ 264, 269, "Taf14", "protein" ], [ 270, 282, "YEATS domain", "structure_element" ] ] }, { "sid": 72, "sent": "(d) The \u03c0-\u03c0-\u03c0 stacking mechanism involving the alkene moiety of crotonyllysine.", "section": "FIG", "ner": [ [ 8, 22, "\u03c0-\u03c0-\u03c0 stacking", "bond_interaction" ], [ 64, 78, "crotonyllysine", "residue_name" ] ] }, { "sid": 73, "sent": "H3K9cr is a selective target of the Taf14 YEATS domain", "section": "FIG", "ner": [ [ 0, 2, "H3", "protein_type" ], [ 2, 6, "K9cr", "ptm" ], [ 36, 41, "Taf14", "protein" ], [ 42, 54, "YEATS domain", "structure_element" ] ] }, { "sid": 74, "sent": "(a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains.", "section": "FIG", "ner": [ [ 7, 19, "Western blot", "experimental_method" ], [ 53, 55, "H3", "protein_type" ], [ 55, 59, "K9cr", "ptm" ], [ 64, 66, "H3", "protein_type" ], [ 66, 70, "K9ac", "ptm" ], [ 74, 83, "wild type", "protein_state" ], [ 85, 87, "WT", "protein_state" ], [ 90, 93, "HAT", "protein_type" ], [ 107, 111, "HDAC", "protein_type" ], [ 112, 120, "deletion", "experimental_method" ], [ 121, 126, "yeast", "taxonomy_domain" ] ] }, { "sid": 75, "sent": "Total H3 was used as a loading control.", "section": "FIG", "ner": [ [ 6, 8, "H3", "protein_type" ] ] }, { "sid": 76, "sent": "(c) Superimposed 1H,15N HSQC spectra of Taf14 YEATS recorded as H3K9cr5-13 and H3K9ac5-13 peptides were titrated in.", "section": "FIG", "ner": [ [ 17, 28, "1H,15N HSQC", "experimental_method" ], [ 29, 36, "spectra", "evidence" ], [ 40, 45, "Taf14", "protein" ], [ 46, 51, "YEATS", "structure_element" ], [ 64, 74, "H3K9cr5-13", "chemical" ], [ 79, 89, "H3K9ac5-13", "chemical" ], [ 104, 112, "titrated", "experimental_method" ] ] }, { "sid": 77, "sent": "Spectra are color coded according to the protein:peptide molar ratio.", "section": "FIG", "ner": [ [ 0, 7, "Spectra", "evidence" ] ] }, { "sid": 78, "sent": "(d) Western blot analyses of peptide pull-down assays using wild-type and mutated Taf14 YEATS domains and indicated peptides.", "section": "FIG", "ner": [ [ 4, 16, "Western blot", "experimental_method" ], [ 29, 53, "peptide pull-down assays", "experimental_method" ], [ 60, 69, "wild-type", "protein_state" ], [ 74, 81, "mutated", "protein_state" ], [ 82, 87, "Taf14", "protein" ], [ 88, 101, "YEATS domains", "structure_element" ] ] } ] }, "PMC4781976": { "annotations": [ { "sid": 0, "sent": "Structure of the GAT domain of the endosomal adapter protein Tom1", "section": "TITLE", "ner": [ [ 0, 9, "Structure", "evidence" ], [ 17, 20, "GAT", "structure_element" ], [ 45, 60, "adapter protein", "protein_type" ], [ 61, 65, "Tom1", "protein" ] ] }, { "sid": 1, "sent": "Cellular homeostasis requires correct delivery of cell-surface receptor proteins (cargo) to their target subcellular compartments.", "section": "ABSTRACT", "ner": [ [ 50, 71, "cell-surface receptor", "protein_type" ] ] }, { "sid": 2, "sent": "The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments.", "section": "ABSTRACT", "ner": [ [ 4, 20, "adapter proteins", "protein_type" ], [ 21, 25, "Tom1", "protein" ], [ 30, 36, "Tollip", "protein" ], [ 64, 77, "ubiquitinated", "ptm" ] ] }, { "sid": 3, "sent": "Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain\u2019s association to Tollip\u2019s Tom1-binding domain (TBD).", "section": "ABSTRACT", "ner": [ [ 15, 19, "Tom1", "protein" ], [ 69, 72, "GAT", "structure_element" ], [ 97, 103, "Tollip", "protein" ], [ 106, 125, "Tom1-binding domain", "structure_element" ], [ 127, 130, "TBD", "structure_element" ] ] }, { "sid": 4, "sent": "In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain.", "section": "ABSTRACT", "ner": [ [ 36, 48, "solution NMR", "experimental_method" ], [ 57, 66, "structure", "evidence" ], [ 74, 78, "Tom1", "protein" ], [ 79, 82, "GAT", "structure_element" ] ] }, { "sid": 5, "sent": "The estimated protein structure exhibits a bundle of three helical elements.", "section": "ABSTRACT", "ner": [ [ 22, 31, "structure", "evidence" ] ] }, { "sid": 6, "sent": "We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states.", "section": "ABSTRACT", "ner": [ [ 3, 10, "compare", "experimental_method" ], [ 15, 19, "Tom1", "protein" ], [ 20, 23, "GAT", "structure_element" ], [ 24, 33, "structure", "evidence" ], [ 45, 55, "structures", "evidence" ], [ 77, 83, "Tollip", "protein" ], [ 84, 88, "TBD-", "protein_state" ], [ 93, 108, "ubiquitin-bound", "protein_state" ] ] }, { "sid": 7, "sent": "Subject area\tBiology\t \tMore specific subject area\tStructural biology\t \tType of data\tTable, text file, graph, figures\t \tHow data was acquired\tCircular dichroism and NMR.", "section": "TABLE", "ner": [ [ 141, 159, "Circular dichroism", "experimental_method" ], [ 164, 167, "NMR", "experimental_method" ] ] }, { "sid": 8, "sent": "NMR data was recorded using a Bruker 800\u00a0MHz\t \tData format\tPDB format text file.", "section": "TABLE", "ner": [ [ 0, 3, "NMR", "experimental_method" ] ] }, { "sid": 9, "sent": "Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol\t \tExperimental factors\tRecombinant human Tom1 GAT domain was purified to homogeneity before use\t \tExperimental features\tSolution structure of Tom1 GAT was determined from NMR chemical shift data\t \tData source location\tVirginia and Colorado, United States.", "section": "TABLE", "ner": [ [ 12, 22, "CS-Rosetta", "experimental_method" ], [ 24, 59, "Protein Structure Validation Server", "experimental_method" ], [ 61, 65, "PSVS", "experimental_method" ], [ 68, 75, "NMRPipe", "experimental_method" ], [ 77, 84, "NMRDraw", "experimental_method" ], [ 131, 136, "human", "species" ], [ 137, 141, "Tom1", "protein" ], [ 142, 145, "GAT", "structure_element" ], [ 216, 234, "Solution structure", "evidence" ], [ 238, 242, "Tom1", "protein" ], [ 243, 246, "GAT", "structure_element" ], [ 267, 270, "NMR", "experimental_method" ], [ 271, 285, "chemical shift", "evidence" ] ] }, { "sid": 10, "sent": "Tom1 GAT structural data is publicly available in the RCSB Protein Data Bank (http://www.rscb.org/) under the accession number PDB: 2n9d\t \t", "section": "TABLE", "ner": [ [ 5, 8, "GAT", "structure_element" ] ] }, { "sid": 11, "sent": "The Tom1 GAT domain solution structure will provide additional tools for modulating its biological function.", "section": "TABLE", "ner": [ [ 4, 8, "Tom1", "protein" ], [ 9, 12, "GAT", "structure_element" ], [ 20, 38, "solution structure", "evidence" ] ] }, { "sid": 12, "sent": "Tom1 GAT can adopt distinct conformations upon ligand binding.", "section": "TABLE", "ner": [ [ 0, 4, "Tom1", "protein" ], [ 5, 8, "GAT", "structure_element" ] ] }, { "sid": 13, "sent": "A conformational response of the Tom1 GAT domain upon Tollip TBD binding can serve as an example to explain mutually exclusive ligand binding events.", "section": "TABLE", "ner": [ [ 33, 37, "Tom1", "protein" ], [ 38, 41, "GAT", "structure_element" ], [ 54, 60, "Tollip", "protein" ], [ 61, 64, "TBD", "structure_element" ] ] }, { "sid": 14, "sent": "Analysis of the far-UV circular dichroism (CD) spectrum of the Tom 1 GAT domain (Fig. 1) predicts 58.7% \u03b1-helix, 3% \u03b2-strand, 15.5% turn, and 22.8% disordered regions.", "section": "TABLE", "ner": [ [ 16, 41, "far-UV circular dichroism", "experimental_method" ], [ 43, 45, "CD", "experimental_method" ], [ 47, 55, "spectrum", "evidence" ], [ 63, 68, "Tom 1", "protein" ], [ 69, 72, "GAT", "structure_element" ], [ 104, 111, "\u03b1-helix", "structure_element" ], [ 116, 124, "\u03b2-strand", "structure_element" ] ] }, { "sid": 15, "sent": "The Tom1 GAT structural restraints yielded ten helical structures (Fig. 2A,B) with a root mean square deviation (RMSD) of 0.9\u00a0\u00c5 for backbone and 1.3\u00a0\u00c5 for all heavy atoms (Table 1) and estimated the presence of three helices spanning residues Q216-E240 (\u03b1-helix 1), P248-Q274 (\u03b1-helix 2), and E278-T306 (\u03b1-helix 3).", "section": "TABLE", "ner": [ [ 4, 8, "Tom1", "protein" ], [ 9, 12, "GAT", "structure_element" ], [ 13, 34, "structural restraints", "evidence" ], [ 55, 65, "structures", "evidence" ], [ 85, 111, "root mean square deviation", "evidence" ], [ 113, 117, "RMSD", "evidence" ], [ 243, 252, "Q216-E240", "residue_range" ], [ 254, 263, "\u03b1-helix 1", "structure_element" ], [ 266, 275, "P248-Q274", "residue_range" ], [ 277, 286, "\u03b1-helix 2", "structure_element" ], [ 293, 302, "E278-T306", "residue_range" ], [ 304, 313, "\u03b1-helix 3", "structure_element" ] ] }, { "sid": 16, "sent": "Unlike ubiquitin binding, data suggest that conformational changes of the Tom1 GAT \u03b1-helices 1 and 2 occur upon Tollip TBD binding (Fig. 3A,B).", "section": "TABLE", "ner": [ [ 7, 16, "ubiquitin", "chemical" ], [ 74, 78, "Tom1", "protein" ], [ 79, 82, "GAT", "structure_element" ], [ 83, 100, "\u03b1-helices 1 and 2", "structure_element" ], [ 112, 118, "Tollip", "protein" ], [ 119, 122, "TBD", "structure_element" ] ] }, { "sid": 17, "sent": "Representative far-UV CD spectrum of the His-Tom1 GAT domain.", "section": "FIG", "ner": [ [ 15, 24, "far-UV CD", "experimental_method" ], [ 25, 33, "spectrum", "evidence" ], [ 41, 45, "His-", "experimental_method" ], [ 45, 49, "Tom1", "protein" ], [ 50, 53, "GAT", "structure_element" ] ] }, { "sid": 18, "sent": "(A) Stereo view displaying the best-fit backbone superposition of the refined structures for the Tom1 GAT domain.", "section": "FIG", "ner": [ [ 40, 62, "backbone superposition", "experimental_method" ], [ 78, 88, "structures", "evidence" ], [ 97, 101, "Tom1", "protein" ], [ 102, 105, "GAT", "structure_element" ] ] }, { "sid": 19, "sent": "Helices are shown in orange, whereas loops are colored in green. (B) Ribbon illustration of the Tom1 GAT domain.", "section": "FIG", "ner": [ [ 96, 100, "Tom1", "protein" ], [ 101, 104, "GAT", "structure_element" ] ] }, { "sid": 20, "sent": "(A) Two views of the superimposed structures of the Tom1 GAT domain in the free state (gray) with that in the Tollip TBD-bound state (red). (B) Two views of the superimposed structures of the Tom1 GAT domain (gray) with that in the Ub-bound state (green).", "section": "FIG", "ner": [ [ 21, 44, "superimposed structures", "experimental_method" ], [ 52, 56, "Tom1", "protein" ], [ 57, 60, "GAT", "structure_element" ], [ 75, 79, "free", "protein_state" ], [ 110, 116, "Tollip", "protein" ], [ 117, 126, "TBD-bound", "protein_state" ], [ 161, 184, "superimposed structures", "experimental_method" ], [ 192, 196, "Tom1", "protein" ], [ 197, 200, "GAT", "structure_element" ], [ 232, 240, "Ub-bound", "protein_state" ] ] }, { "sid": 21, "sent": "NMR and refinement statistics for the Tom1 GAT domain.", "section": "TABLE", "ner": [ [ 0, 3, "NMR", "experimental_method" ], [ 8, 29, "refinement statistics", "evidence" ], [ 38, 42, "Tom1", "protein" ], [ 43, 46, "GAT", "structure_element" ] ] }, { "sid": 22, "sent": "NMR structural statistics for lowest energy conformers of Tom1 GAT using PSVS.", "section": "TABLE", "ner": [ [ 0, 3, "NMR", "experimental_method" ], [ 4, 25, "structural statistics", "evidence" ], [ 58, 62, "Tom1", "protein" ], [ 63, 66, "GAT", "structure_element" ], [ 73, 77, "PSVS", "experimental_method" ] ] }, { "sid": 23, "sent": "deviations were obtained by superimposing residues 215\u2013309 of Tom1 GAT among 10 lowest energy refined structures.", "section": "TABLE", "ner": [ [ 28, 41, "superimposing", "experimental_method" ], [ 51, 58, "215\u2013309", "residue_range" ], [ 62, 66, "Tom1", "protein" ], [ 67, 70, "GAT", "structure_element" ], [ 102, 112, "structures", "evidence" ] ] } ] }, "PMC4802085": { "annotations": [ { "sid": 0, "sent": "Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance", "section": "TITLE", "ner": [ [ 0, 4, "Haem", "chemical" ], [ 15, 27, "dimerization", "oligomeric_state" ], [ 31, 37, "PGRMC1", "protein" ], [ 38, 45, "Sigma-2", "protein" ] ] }, { "sid": 1, "sent": "Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown.", "section": "ABSTRACT", "ner": [ [ 0, 42, "Progesterone-receptor membrane component 1", "protein" ], [ 44, 50, "PGRMC1", "protein" ], [ 51, 67, "Sigma-2 receptor", "protein" ], [ 74, 97, "haem-containing protein", "protein_type" ], [ 118, 150, "epidermal growth factor receptor", "protein_type" ], [ 152, 156, "EGFR", "protein_type" ], [ 162, 178, "cytochromes P450", "protein_type" ] ] }, { "sid": 2, "sent": "Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95\u2009\u00c5 resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules.", "section": "ABSTRACT", "ner": [ [ 5, 30, "crystallographic analyses", "experimental_method" ], [ 38, 44, "PGRMC1", "protein" ], [ 45, 61, "cytosolic domain", "structure_element" ], [ 106, 112, "stable", "protein_state" ], [ 113, 118, "dimer", "oligomeric_state" ], [ 127, 148, "stacking interactions", "bond_interaction" ], [ 167, 171, "haem", "chemical" ] ] }, { "sid": 3, "sent": "The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization.", "section": "ABSTRACT", "ner": [ [ 4, 8, "haem", "chemical" ], [ 9, 13, "iron", "chemical" ], [ 17, 36, "five-coordinated by", "bond_interaction" ], [ 37, 43, "Tyr113", "residue_name_number" ], [ 58, 65, "surface", "site" ], [ 73, 77, "haem", "chemical" ], [ 87, 99, "dimerization", "oligomeric_state" ] ] }, { "sid": 4, "sent": "Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem.", "section": "ABSTRACT", "ner": [ [ 0, 15, "Carbon monoxide", "chemical" ], [ 17, 19, "CO", "chemical" ], [ 37, 43, "PGRMC1", "protein" ], [ 44, 56, "dimerization", "oligomeric_state" ], [ 75, 98, "sixth coordination site", "site" ], [ 106, 110, "haem", "chemical" ] ] }, { "sid": 5, "sent": "Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells.", "section": "ABSTRACT", "ner": [ [ 0, 4, "Haem", "chemical" ], [ 14, 20, "PGRMC1", "protein" ], [ 21, 33, "dimerization", "oligomeric_state" ], [ 68, 72, "EGFR", "protein_type" ], [ 77, 93, "cytochromes P450", "protein_type" ], [ 201, 203, "CO", "chemical" ], [ 207, 211, "haem", "chemical" ] ] }, { "sid": 6, "sent": "This study demonstrates protein dimerization via haem\u2013haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer.", "section": "ABSTRACT", "ner": [ [ 32, 44, "dimerization", "oligomeric_state" ], [ 49, 67, "haem\u2013haem stacking", "bond_interaction" ], [ 96, 106, "eukaryotes", "taxonomy_domain" ] ] }, { "sid": 7, "sent": " PGRMC1 binds to EGFR and cytochromes P450, and is known to be involved in cancer proliferation and in drug resistance.", "section": "ABSTRACT", "ner": [ [ 1, 7, "PGRMC1", "protein" ], [ 17, 21, "EGFR", "protein_type" ], [ 26, 42, "cytochromes P450", "protein_type" ] ] }, { "sid": 8, "sent": "Here, the authors determine the structure of the cytosolic domain of PGRMC1, which forms a dimer via haem\u2013haem stacking, and propose how this interaction could be involved in its function.", "section": "ABSTRACT", "ner": [ [ 32, 41, "structure", "evidence" ], [ 49, 65, "cytosolic domain", "structure_element" ], [ 69, 75, "PGRMC1", "protein" ], [ 91, 96, "dimer", "oligomeric_state" ], [ 101, 119, "haem\u2013haem stacking", "bond_interaction" ] ] }, { "sid": 9, "sent": "Much attention has been paid to the roles of haem-iron in cancer development.", "section": "INTRO", "ner": [ [ 45, 49, "haem", "chemical" ], [ 50, 54, "iron", "chemical" ] ] }, { "sid": 10, "sent": "Increased dietary intake of haem is a risk factor for several types of cancer.", "section": "INTRO", "ner": [ [ 28, 32, "haem", "chemical" ] ] }, { "sid": 11, "sent": "Previous studies showed that deprivation of iron or haem suppresses tumourigenesis.", "section": "INTRO", "ner": [ [ 29, 43, "deprivation of", "protein_state" ], [ 44, 48, "iron", "chemical" ], [ 52, 56, "haem", "chemical" ] ] }, { "sid": 12, "sent": "On the other hand, carbon monoxide (CO), the gaseous mediator generated by oxidative degradation of haem via haem oxygenase (HO), inhibits tumour growth.", "section": "INTRO", "ner": [ [ 19, 34, "carbon monoxide", "chemical" ], [ 36, 38, "CO", "chemical" ], [ 100, 104, "haem", "chemical" ], [ 109, 123, "haem oxygenase", "protein_type" ], [ 125, 127, "HO", "protein_type" ] ] }, { "sid": 13, "sent": "Thus, a tenuous balance between free haem and CO plays key roles in cancer development and chemoresistance, although the underlying mechanisms are not fully understood.", "section": "INTRO", "ner": [ [ 37, 41, "haem", "chemical" ], [ 46, 48, "CO", "chemical" ] ] }, { "sid": 14, "sent": "To gain insight into the underlying mechanisms, we took chemical biological approaches using affinity nanobeads carrying haem and identified progesterone-receptor membrane component 1 (PGRMC1) as a haem-binding protein from mouse liver extracts (Supplementary Fig. 1).", "section": "INTRO", "ner": [ [ 93, 111, "affinity nanobeads", "experimental_method" ], [ 121, 125, "haem", "chemical" ], [ 141, 183, "progesterone-receptor membrane component 1", "protein" ], [ 185, 191, "PGRMC1", "protein" ], [ 198, 202, "haem", "chemical" ], [ 224, 229, "mouse", "taxonomy_domain" ] ] }, { "sid": 15, "sent": "PGRMC1 is a member of the membrane-associated progesterone receptor (MAPR) family with a cytochrome b5-like haem-binding region, and is known to be highly expressed in various types of cancers.", "section": "INTRO", "ner": [ [ 0, 6, "PGRMC1", "protein" ], [ 26, 67, "membrane-associated progesterone receptor", "protein_type" ], [ 69, 73, "MAPR", "protein_type" ], [ 89, 107, "cytochrome b5-like", "structure_element" ], [ 108, 127, "haem-binding region", "site" ], [ 148, 164, "highly expressed", "protein_state" ] ] }, { "sid": 16, "sent": "PGRMC1 is anchored to the cell membrane through the N-terminal transmembrane helix and interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 (ref).", "section": "INTRO", "ner": [ [ 0, 6, "PGRMC1", "protein" ], [ 63, 82, "transmembrane helix", "structure_element" ], [ 102, 134, "epidermal growth factor receptor", "protein_type" ], [ 136, 140, "EGFR", "protein_type" ], [ 146, 162, "cytochromes P450", "protein_type" ] ] }, { "sid": 17, "sent": "While PGRMC1 is implicated in cell proliferation and cholesterol biosynthesis, the structural basis on which PGRMC1 exerts its function remains largely unknown.", "section": "INTRO", "ner": [ [ 6, 12, "PGRMC1", "protein" ], [ 109, 115, "PGRMC1", "protein" ] ] }, { "sid": 18, "sent": "Here we show that PGRMC1 exhibits a unique haem-dependent dimerization.", "section": "INTRO", "ner": [ [ 18, 24, "PGRMC1", "protein" ], [ 43, 47, "haem", "chemical" ], [ 58, 70, "dimerization", "oligomeric_state" ] ] }, { "sid": 19, "sent": "The dimer binds to EGFR and cytochromes P450 to enhance tumour cell proliferation and chemoresistance.", "section": "INTRO", "ner": [ [ 4, 9, "dimer", "oligomeric_state" ], [ 19, 23, "EGFR", "protein_type" ], [ 28, 44, "cytochromes P450", "protein_type" ] ] }, { "sid": 20, "sent": "The dimer is dissociated to monomers by physiological levels of CO, suggesting that PGRMC1 serves as a CO-sensitive molecular switch regulating cancer cell proliferation.", "section": "INTRO", "ner": [ [ 4, 9, "dimer", "oligomeric_state" ], [ 28, 36, "monomers", "oligomeric_state" ], [ 64, 66, "CO", "chemical" ], [ 84, 90, "PGRMC1", "protein" ], [ 103, 105, "CO", "chemical" ] ] }, { "sid": 21, "sent": "X-ray crystal structure of PGRMC1", "section": "RESULTS", "ner": [ [ 0, 23, "X-ray crystal structure", "evidence" ], [ 27, 33, "PGRMC1", "protein" ] ] }, { "sid": 22, "sent": "We solved the crystal structure of the haem-bound PGRMC1 cytosolic domain (a.a.72\u2013195) at 1.95\u2009\u00c5 resolution (Supplementary Fig. 2).", "section": "RESULTS", "ner": [ [ 3, 9, "solved", "experimental_method" ], [ 14, 31, "crystal structure", "evidence" ], [ 39, 49, "haem-bound", "protein_state" ], [ 50, 56, "PGRMC1", "protein" ], [ 57, 73, "cytosolic domain", "structure_element" ], [ 79, 85, "72\u2013195", "residue_range" ] ] }, { "sid": 23, "sent": "In the presence of haem, PGRMC1 forms a dimeric structure largely through hydrophobic interactions between the haem moieties of two monomers (Fig. 1a, Table 1 and Supplementary Fig. 3; a stereo-structural image is shown in Supplementary Fig 4).", "section": "RESULTS", "ner": [ [ 7, 18, "presence of", "protein_state" ], [ 19, 23, "haem", "chemical" ], [ 25, 31, "PGRMC1", "protein" ], [ 40, 47, "dimeric", "oligomeric_state" ], [ 74, 98, "hydrophobic interactions", "bond_interaction" ], [ 111, 115, "haem", "chemical" ], [ 132, 140, "monomers", "oligomeric_state" ] ] }, { "sid": 24, "sent": "While the overall fold of PGRMC1 is similar to that of canonical cytochrome b5, their haem irons are coordinated differently.", "section": "RESULTS", "ner": [ [ 26, 32, "PGRMC1", "protein" ], [ 65, 78, "cytochrome b5", "protein_type" ], [ 86, 90, "haem", "chemical" ] ] }, { "sid": 25, "sent": "In cytochrome b5, the haem iron is six-coordinated by two axial histidine residues.", "section": "RESULTS", "ner": [ [ 3, 16, "cytochrome b5", "protein_type" ], [ 22, 26, "haem", "chemical" ], [ 27, 31, "iron", "chemical" ], [ 35, 53, "six-coordinated by", "bond_interaction" ], [ 64, 73, "histidine", "residue_name" ] ] }, { "sid": 26, "sent": "These histidines are missing in PGRMC1, and the haem iron is five-coordinated by Tyr113 (Y113) alone (Fig. 1b and Supplementary Fig. 3).", "section": "RESULTS", "ner": [ [ 6, 16, "histidines", "residue_name" ], [ 21, 28, "missing", "protein_state" ], [ 32, 38, "PGRMC1", "protein" ], [ 48, 52, "haem", "chemical" ], [ 53, 57, "iron", "chemical" ], [ 61, 80, "five-coordinated by", "bond_interaction" ], [ 81, 87, "Tyr113", "residue_name_number" ], [ 89, 93, "Y113", "residue_name_number" ], [ 95, 100, "alone", "protein_state" ] ] }, { "sid": 27, "sent": "A homologous helix that holds haem in cytochrome b5 is longer, shifts away from haem, and does not form a coordinate bond in PGRMC1 (Fig. 1c).", "section": "RESULTS", "ner": [ [ 2, 18, "homologous helix", "structure_element" ], [ 30, 34, "haem", "chemical" ], [ 38, 51, "cytochrome b5", "protein_type" ], [ 80, 84, "haem", "chemical" ], [ 125, 131, "PGRMC1", "protein" ] ] }, { "sid": 28, "sent": "Consequently, the five-coordinated haem of PGRMC1 has an open surface that allows its dimerization through hydrophobic haem\u2013haem stacking.", "section": "RESULTS", "ner": [ [ 35, 39, "haem", "chemical" ], [ 43, 49, "PGRMC1", "protein" ], [ 62, 69, "surface", "site" ], [ 86, 98, "dimerization", "oligomeric_state" ], [ 107, 137, "hydrophobic haem\u2013haem stacking", "bond_interaction" ] ] }, { "sid": 29, "sent": "Contrary to our finding, Kaluka et al. recently reported that Tyr164 of PGRMC1 is the axial ligand of haem because mutation of this residue impairs haem binding.", "section": "RESULTS", "ner": [ [ 62, 68, "Tyr164", "residue_name_number" ], [ 72, 78, "PGRMC1", "protein" ], [ 102, 106, "haem", "chemical" ], [ 115, 123, "mutation", "experimental_method" ], [ 148, 152, "haem", "chemical" ] ] }, { "sid": 30, "sent": "Our structural data revealed that Tyr164 and a few other residues such as Tyr107 and Lys163 are in fact hydrogen-bonded to haem propionates.", "section": "RESULTS", "ner": [ [ 4, 19, "structural data", "evidence" ], [ 34, 40, "Tyr164", "residue_name_number" ], [ 74, 80, "Tyr107", "residue_name_number" ], [ 85, 91, "Lys163", "residue_name_number" ], [ 104, 119, "hydrogen-bonded", "bond_interaction" ], [ 123, 127, "haem", "chemical" ] ] }, { "sid": 31, "sent": "This is consistent with observations by Min et al. that Tyr 107 and Tyr113 of PGRMC1 are involved in binding with haem.", "section": "RESULTS", "ner": [ [ 56, 63, "Tyr 107", "residue_name_number" ], [ 68, 74, "Tyr113", "residue_name_number" ], [ 78, 84, "PGRMC1", "protein" ], [ 114, 118, "haem", "chemical" ] ] }, { "sid": 32, "sent": "These amino acid residues are conserved among MAPR family members (Supplementary Fig. 5a), suggesting that these proteins share the ability to exhibit haem-dependent dimerization.", "section": "RESULTS", "ner": [ [ 30, 39, "conserved", "protein_state" ], [ 46, 50, "MAPR", "protein_type" ], [ 151, 155, "haem", "chemical" ], [ 166, 178, "dimerization", "oligomeric_state" ] ] }, { "sid": 33, "sent": "PGRMC1 exhibits haem-dependent dimerization in solution", "section": "RESULTS", "ner": [ [ 0, 6, "PGRMC1", "protein" ], [ 16, 20, "haem", "chemical" ], [ 31, 43, "dimerization", "oligomeric_state" ] ] }, { "sid": 34, "sent": "In the PGRMC1 crystal, two different types of crystal contacts (chain A\u2013A\u2033 and A\u2013B) were observed in addition to the haem-mediated dimer (chain A\u2013A\u2032) (Supplementary Figs 3 and 6a).", "section": "RESULTS", "ner": [ [ 7, 13, "PGRMC1", "protein" ], [ 14, 21, "crystal", "evidence" ], [ 117, 121, "haem", "chemical" ], [ 131, 136, "dimer", "oligomeric_state" ] ] }, { "sid": 35, "sent": "To confirm that haem-assisted dimerization of PGRMC1 occurs in solution, we analysed the structure of apo- and haem-bound PGMRC1 by two-dimensional nuclear magnetic resonance (NMR) using heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy (Supplementary Figs 6b and 7).", "section": "RESULTS", "ner": [ [ 16, 20, "haem", "chemical" ], [ 30, 42, "dimerization", "oligomeric_state" ], [ 46, 52, "PGRMC1", "protein" ], [ 89, 98, "structure", "evidence" ], [ 102, 105, "apo", "protein_state" ], [ 111, 121, "haem-bound", "protein_state" ], [ 122, 128, "PGMRC1", "protein" ], [ 132, 174, "two-dimensional nuclear magnetic resonance", "experimental_method" ], [ 176, 179, "NMR", "experimental_method" ], [ 187, 274, "heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy", "experimental_method" ] ] }, { "sid": 36, "sent": "NMR signals from some amino acid residues of PGRMC1 disappeared due to the paramagnetic relaxation effect of haem (Supplementary Figs 6b); these residues were located in the haem-binding region.", "section": "RESULTS", "ner": [ [ 0, 3, "NMR", "experimental_method" ], [ 45, 51, "PGRMC1", "protein" ], [ 109, 113, "haem", "chemical" ], [ 174, 193, "haem-binding region", "site" ] ] }, { "sid": 37, "sent": "When chemical shifts of apo- and haem-bound forms of PGMRC1 were compared, some amino acid residues close to those which disappeared because of the paramagnetic relaxation effect of haem exhibit notable chemical shifts (Supplementary Fig. 6a,b; dark yellow).", "section": "RESULTS", "ner": [ [ 5, 20, "chemical shifts", "evidence" ], [ 24, 27, "apo", "protein_state" ], [ 33, 43, "haem-bound", "protein_state" ], [ 53, 59, "PGMRC1", "protein" ], [ 182, 186, "haem", "chemical" ] ] }, { "sid": 38, "sent": "However, at the interfaces of the other possible dimeric structures (Supplementary Fig. 6a, chain A\u2013A\u2033; cyan and chain A\u2013B; violet), no significant difference was observed.", "section": "RESULTS", "ner": [ [ 16, 26, "interfaces", "site" ], [ 49, 56, "dimeric", "oligomeric_state" ], [ 57, 67, "structures", "evidence" ] ] }, { "sid": 39, "sent": "Furthermore, free energy of dissociation predicted by PISA suggested that the haem-mediated dimer is stable in solution while the other potential interactions are not.", "section": "RESULTS", "ner": [ [ 13, 40, "free energy of dissociation", "evidence" ], [ 54, 58, "PISA", "experimental_method" ], [ 78, 82, "haem", "chemical" ], [ 92, 97, "dimer", "oligomeric_state" ], [ 101, 107, "stable", "protein_state" ] ] }, { "sid": 40, "sent": "We also attempted to predict the secondary structure of PGRMC1 through NMR data by calculating with TALOS+ program (Supplementary Fig. 8); the prediction suggested that the overall secondary structure is comparable between apo- and haem-bound forms of PGRMC1 in solution.", "section": "RESULTS", "ner": [ [ 56, 62, "PGRMC1", "protein" ], [ 71, 74, "NMR", "experimental_method" ], [ 100, 114, "TALOS+ program", "experimental_method" ], [ 223, 226, "apo", "protein_state" ], [ 232, 242, "haem-bound", "protein_state" ], [ 252, 258, "PGRMC1", "protein" ] ] }, { "sid": 41, "sent": "We analysed the haem-dependent dimerization of the PGRMC1 cytosolic domain (a.a.44\u2013195) in solution (Fig. 2 and Table 2).", "section": "RESULTS", "ner": [ [ 16, 20, "haem", "chemical" ], [ 31, 43, "dimerization", "oligomeric_state" ], [ 51, 57, "PGRMC1", "protein" ], [ 58, 74, "cytosolic domain", "structure_element" ], [ 80, 86, "44\u2013195", "residue_range" ] ] }, { "sid": 42, "sent": "Mass spectrometry (MS) analyses under non-denaturing condition demonstrated that the apo-monomer PGRMC1 resulted in dimerization by binding with haem (Fig. 2a).", "section": "RESULTS", "ner": [ [ 0, 17, "Mass spectrometry", "experimental_method" ], [ 19, 21, "MS", "experimental_method" ], [ 38, 62, "non-denaturing condition", "experimental_method" ], [ 85, 88, "apo", "protein_state" ], [ 89, 96, "monomer", "oligomeric_state" ], [ 97, 103, "PGRMC1", "protein" ], [ 116, 128, "dimerization", "oligomeric_state" ], [ 145, 149, "haem", "chemical" ] ] }, { "sid": 43, "sent": "It should be noted that a disulfide bond between two Cys129 residues is observed in the crystal of PGRMC1 (Fig. 1a), while Cys129 is not conserved among the MAPR family proteins (Supplementary Fig. 5a).", "section": "RESULTS", "ner": [ [ 26, 40, "disulfide bond", "ptm" ], [ 53, 59, "Cys129", "residue_name_number" ], [ 88, 95, "crystal", "evidence" ], [ 99, 105, "PGRMC1", "protein" ], [ 123, 129, "Cys129", "residue_name_number" ], [ 133, 146, "not conserved", "protein_state" ], [ 157, 161, "MAPR", "protein_type" ] ] }, { "sid": 44, "sent": "This observation led us to examine whether or not the disulfide bond contributes to PGRMC1 dimerization.", "section": "RESULTS", "ner": [ [ 54, 68, "disulfide bond", "ptm" ], [ 84, 90, "PGRMC1", "protein" ], [ 91, 103, "dimerization", "oligomeric_state" ] ] }, { "sid": 45, "sent": "MS analyses under non-denaturing conditions clearly showed that the Cys129Ser (C129S) mutant is dimerized in the presence of haem, indicating that the haem-mediated dimerization of PGRMC1 occurs independently of the disulfide bond formation via Cys129 (Fig. 2a).", "section": "RESULTS", "ner": [ [ 0, 2, "MS", "experimental_method" ], [ 18, 43, "non-denaturing conditions", "experimental_method" ], [ 68, 77, "Cys129Ser", "mutant" ], [ 79, 84, "C129S", "mutant" ], [ 86, 92, "mutant", "protein_state" ], [ 96, 105, "dimerized", "protein_state" ], [ 113, 124, "presence of", "protein_state" ], [ 125, 129, "haem", "chemical" ], [ 151, 155, "haem", "chemical" ], [ 165, 177, "dimerization", "oligomeric_state" ], [ 181, 187, "PGRMC1", "protein" ], [ 216, 230, "disulfide bond", "ptm" ], [ 245, 251, "Cys129", "residue_name_number" ] ] }, { "sid": 46, "sent": "Supporting this, MS analyses under denaturing conditions showed that haem-mediated PGRMC1 dimer is completely dissociated into monomer, indicating that dimerization of this kind is not mediated by any covalent bond such as disulfide bond (Supplementary Fig. 9).", "section": "RESULTS", "ner": [ [ 17, 19, "MS", "experimental_method" ], [ 35, 56, "denaturing conditions", "experimental_method" ], [ 69, 73, "haem", "chemical" ], [ 83, 89, "PGRMC1", "protein" ], [ 90, 95, "dimer", "oligomeric_state" ], [ 127, 134, "monomer", "oligomeric_state" ], [ 152, 164, "dimerization", "oligomeric_state" ], [ 223, 237, "disulfide bond", "ptm" ] ] }, { "sid": 47, "sent": "We also analysed the haem-dependent dimerization of PGRMC1 by diffusion-ordered NMR spectroscopy (DOSY) analyses (Table 2, Supplementary Fig. 10).", "section": "RESULTS", "ner": [ [ 21, 25, "haem", "chemical" ], [ 36, 48, "dimerization", "oligomeric_state" ], [ 52, 58, "PGRMC1", "protein" ], [ 62, 96, "diffusion-ordered NMR spectroscopy", "experimental_method" ], [ 98, 102, "DOSY", "experimental_method" ] ] }, { "sid": 48, "sent": "The results suggested that the hydrodynamic radius of haem-bound PGRMC1 is larger than that of apo-PGRMC1.", "section": "RESULTS", "ner": [ [ 31, 50, "hydrodynamic radius", "evidence" ], [ 54, 64, "haem-bound", "protein_state" ], [ 65, 71, "PGRMC1", "protein" ], [ 95, 98, "apo", "protein_state" ], [ 99, 105, "PGRMC1", "protein" ] ] }, { "sid": 49, "sent": "To further evaluate changes in molecular weights in dimerization of PGRMC1, sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis was carried out.", "section": "RESULTS", "ner": [ [ 52, 64, "dimerization", "oligomeric_state" ], [ 68, 74, "PGRMC1", "protein" ], [ 76, 129, "sedimentation velocity analytical ultracentrifugation", "experimental_method" ], [ 131, 137, "SV-AUC", "experimental_method" ] ] }, { "sid": 50, "sent": "Whereas the wild-type (wt) apo-PGRMC1 appeared at a 1.9 S peak as monomer, the haem-binding PGRMC1 was converted into dimer at a 3.1 S peak (Fig. 2b).", "section": "RESULTS", "ner": [ [ 12, 21, "wild-type", "protein_state" ], [ 23, 25, "wt", "protein_state" ], [ 27, 30, "apo", "protein_state" ], [ 31, 37, "PGRMC1", "protein" ], [ 66, 73, "monomer", "oligomeric_state" ], [ 79, 83, "haem", "chemical" ], [ 92, 98, "PGRMC1", "protein" ], [ 118, 123, "dimer", "oligomeric_state" ] ] }, { "sid": 51, "sent": "Similarly, the C129S mutant of PGRMC1 converted from monomer to dimer by binding to haem (Fig. 2b).", "section": "RESULTS", "ner": [ [ 15, 20, "C129S", "mutant" ], [ 21, 27, "mutant", "protein_state" ], [ 31, 37, "PGRMC1", "protein" ], [ 53, 60, "monomer", "oligomeric_state" ], [ 64, 69, "dimer", "oligomeric_state" ], [ 84, 88, "haem", "chemical" ] ] }, { "sid": 52, "sent": "SV-AUC analyses also allowed us to examine the stability of haem/PGRMC1 dimer.", "section": "RESULTS", "ner": [ [ 0, 6, "SV-AUC", "experimental_method" ], [ 60, 64, "haem", "chemical" ], [ 65, 71, "PGRMC1", "protein" ], [ 72, 77, "dimer", "oligomeric_state" ] ] }, { "sid": 53, "sent": "To this end, we used different concentrations (3.5\u2013147\u2009\u03bcmol\u2009l\u22121) of haem-bound PGRMC1 protein (a.a. 72\u2013195), which were identical to that used in the crystallographic analysis.", "section": "RESULTS", "ner": [ [ 68, 78, "haem-bound", "protein_state" ], [ 79, 85, "PGRMC1", "protein" ], [ 100, 106, "72\u2013195", "residue_range" ], [ 150, 175, "crystallographic analysis", "experimental_method" ] ] }, { "sid": 54, "sent": "The sedimentation coefficients calculated on the basis of the crystal structure were 1.71 S for monomer and 2.56 S for dimer (Supplementary Fig. 11, upper panel).", "section": "RESULTS", "ner": [ [ 4, 30, "sedimentation coefficients", "evidence" ], [ 62, 79, "crystal structure", "evidence" ], [ 96, 103, "monomer", "oligomeric_state" ], [ 119, 124, "dimer", "oligomeric_state" ] ] }, { "sid": 55, "sent": "The results showed that the PGRMC1 dimer is not dissociated into monomer at all concentrations examined (Supplementary Fig. 11, lower panel), suggesting that the Kd value of haem-mediated dimer of PGRMC1 is under 3.5\u2009\u03bcmol\u2009l\u22121.", "section": "RESULTS", "ner": [ [ 28, 34, "PGRMC1", "protein" ], [ 35, 40, "dimer", "oligomeric_state" ], [ 65, 72, "monomer", "oligomeric_state" ], [ 162, 164, "Kd", "evidence" ], [ 174, 178, "haem", "chemical" ], [ 188, 193, "dimer", "oligomeric_state" ], [ 197, 203, "PGRMC1", "protein" ] ] }, { "sid": 56, "sent": "A value of this kind implies that the PGRMC1 dimer is more stable than other dimers of extracellular domain of membrane proteins such as Toll like receptor 9 (dimerization Kd of 20\u2009\u03bcmol\u2009l\u22121) (ref.) and plexin A2 receptor (dimerization Kd higher than 300\u2009\u03bcmol\u2009l\u22121) (ref.).", "section": "RESULTS", "ner": [ [ 38, 44, "PGRMC1", "protein" ], [ 45, 50, "dimer", "oligomeric_state" ], [ 77, 83, "dimers", "oligomeric_state" ], [ 87, 107, "extracellular domain", "structure_element" ], [ 111, 128, "membrane proteins", "protein_type" ], [ 137, 157, "Toll like receptor 9", "protein" ], [ 159, 171, "dimerization", "oligomeric_state" ], [ 172, 174, "Kd", "evidence" ], [ 202, 220, "plexin A2 receptor", "protein" ], [ 222, 234, "dimerization", "oligomeric_state" ], [ 235, 237, "Kd", "evidence" ] ] }, { "sid": 57, "sent": "The current analytical data confirmed that apo-PGRMC1 monomer converts into dimer by binding to haem in solution (Table 2).", "section": "RESULTS", "ner": [ [ 43, 46, "apo", "protein_state" ], [ 47, 53, "PGRMC1", "protein" ], [ 54, 61, "monomer", "oligomeric_state" ], [ 76, 81, "dimer", "oligomeric_state" ], [ 96, 100, "haem", "chemical" ] ] }, { "sid": 58, "sent": "We also showed by haem titration experiments that haem binding to PGRMC1 was of low affinity with a Kd value of 50\u2009nmol\u2009l\u22121; this is comparable with that of iron regulatory protein 2, which is known to be regulated by intracellular levels of haem (Fig. 2c and Supplementary Table 1).", "section": "RESULTS", "ner": [ [ 18, 44, "haem titration experiments", "experimental_method" ], [ 50, 54, "haem", "chemical" ], [ 66, 72, "PGRMC1", "protein" ], [ 100, 102, "Kd", "evidence" ], [ 157, 182, "iron regulatory protein 2", "protein" ], [ 242, 246, "haem", "chemical" ] ] }, { "sid": 59, "sent": "These results raised the possibility that the function of PGRMC1 is regulated by intracellular haem concentrations.", "section": "RESULTS", "ner": [ [ 58, 64, "PGRMC1", "protein" ], [ 95, 99, "haem", "chemical" ] ] }, { "sid": 60, "sent": "CO inhibits haem-dependent dimerization of PGRMC1", "section": "RESULTS", "ner": [ [ 0, 2, "CO", "chemical" ], [ 12, 16, "haem", "chemical" ], [ 27, 39, "dimerization", "oligomeric_state" ], [ 43, 49, "PGRMC1", "protein" ] ] }, { "sid": 61, "sent": "Crystallographic analyses revealed that Tyr113 of PGRMC1 is an axial ligand for haem and contributes to haem-dependent dimerization (Fig. 1a).", "section": "RESULTS", "ner": [ [ 0, 25, "Crystallographic analyses", "experimental_method" ], [ 40, 46, "Tyr113", "residue_name_number" ], [ 50, 56, "PGRMC1", "protein" ], [ 80, 84, "haem", "chemical" ], [ 104, 108, "haem", "chemical" ], [ 119, 131, "dimerization", "oligomeric_state" ] ] }, { "sid": 62, "sent": "Analysis of UV-visible spectra revealed that the heme of PGRMC1 is reducible from ferric to ferrous state, thus allowing CO binding (Fig. 3a).", "section": "RESULTS", "ner": [ [ 12, 30, "UV-visible spectra", "evidence" ], [ 49, 53, "heme", "chemical" ], [ 57, 63, "PGRMC1", "protein" ], [ 82, 88, "ferric", "protein_state" ], [ 92, 99, "ferrous", "protein_state" ], [ 121, 123, "CO", "chemical" ] ] }, { "sid": 63, "sent": "Furthermore, the UV-visible spectrum of the wild type PGRMC1 was the same as that of the C129S mutant of PGRMC1, and the R/Z ratio determined by the intensities between the Soret band (394\u2009nm) peak and the 274-nm peak showed that these proteins were fully loaded with haem (Supplementary Fig. 12).", "section": "RESULTS", "ner": [ [ 17, 36, "UV-visible spectrum", "evidence" ], [ 44, 53, "wild type", "protein_state" ], [ 54, 60, "PGRMC1", "protein" ], [ 89, 94, "C129S", "mutant" ], [ 95, 101, "mutant", "protein_state" ], [ 105, 111, "PGRMC1", "protein" ], [ 121, 130, "R/Z ratio", "evidence" ], [ 250, 267, "fully loaded with", "protein_state" ], [ 268, 272, "haem", "chemical" ] ] }, { "sid": 64, "sent": "Analysis of the ferric form of PGRMC1 using resonance Raman spectroscopy (Supplementary Fig. 13) showed that the relative intensity of oxidation and spin state marker bands (\u03bd4 and \u03bd3) is close to 1.0, which is consistent with it being a haem protein with a proximal Tyr coordination.", "section": "RESULTS", "ner": [ [ 16, 22, "ferric", "protein_state" ], [ 31, 37, "PGRMC1", "protein" ], [ 44, 72, "resonance Raman spectroscopy", "experimental_method" ], [ 238, 242, "haem", "chemical" ], [ 267, 270, "Tyr", "residue_name" ] ] }, { "sid": 65, "sent": "A specific Raman shift peaking at vFe\u2013CO=500\u2009cm\u22121 demonstrated that the CO-bound haem of PGRMC1 is six-coordinated (Supplementary Fig. 13).", "section": "RESULTS", "ner": [ [ 11, 22, "Raman shift", "evidence" ], [ 72, 80, "CO-bound", "protein_state" ], [ 81, 85, "haem", "chemical" ], [ 89, 95, "PGRMC1", "protein" ] ] }, { "sid": 66, "sent": "Since PGRMC1 dimerization involves the open surface of haem on the opposite side of the axial Tyr113, no space for CO binding is available in the dimeric structure (Fig. 3b).", "section": "RESULTS", "ner": [ [ 6, 12, "PGRMC1", "protein" ], [ 13, 25, "dimerization", "oligomeric_state" ], [ 44, 51, "surface", "site" ], [ 55, 59, "haem", "chemical" ], [ 94, 100, "Tyr113", "residue_name_number" ], [ 115, 117, "CO", "chemical" ], [ 146, 153, "dimeric", "oligomeric_state" ], [ 154, 163, "structure", "evidence" ] ] }, { "sid": 67, "sent": "This prompted us to ask if CO binding to haem causes dissociation of the PGRMC1 dimer.", "section": "RESULTS", "ner": [ [ 27, 29, "CO", "chemical" ], [ 41, 45, "haem", "chemical" ], [ 73, 79, "PGRMC1", "protein" ], [ 80, 85, "dimer", "oligomeric_state" ] ] }, { "sid": 68, "sent": "Analysis by gel filtration chromatography revealed that the relative molecular sizes of the wild-type and the C129S mutant of PGRMC1 are increased by adding haem to apo-PGRMC1 regardless of the oxidation state of the iron (Fig. 3c), which is in agreement with the results in Table 1.", "section": "RESULTS", "ner": [ [ 12, 41, "gel filtration chromatography", "experimental_method" ], [ 92, 101, "wild-type", "protein_state" ], [ 110, 115, "C129S", "mutant" ], [ 116, 122, "mutant", "protein_state" ], [ 126, 132, "PGRMC1", "protein" ], [ 157, 161, "haem", "chemical" ], [ 165, 168, "apo", "protein_state" ], [ 169, 175, "PGRMC1", "protein" ], [ 217, 221, "iron", "chemical" ] ] }, { "sid": 69, "sent": "CO application to ferrous PGRMC1 abolished the haem-dependent increase in its molecular size.", "section": "RESULTS", "ner": [ [ 0, 2, "CO", "chemical" ], [ 18, 25, "ferrous", "protein_state" ], [ 26, 32, "PGRMC1", "protein" ], [ 47, 51, "haem", "chemical" ] ] }, { "sid": 70, "sent": "Under this reducing condition in the presence of dithionite, analyses of UV-visible spectra indicated that CO-binding with haem-PGRMC1 is stable, showing only 20% reduction of the absorbance at 412\u2009nm within 2\u2009h (Supplementary Fig. 14).", "section": "RESULTS", "ner": [ [ 37, 48, "presence of", "protein_state" ], [ 49, 59, "dithionite", "chemical" ], [ 73, 91, "UV-visible spectra", "evidence" ], [ 107, 109, "CO", "chemical" ], [ 123, 134, "haem-PGRMC1", "complex_assembly" ], [ 138, 144, "stable", "protein_state" ] ] }, { "sid": 71, "sent": "Furthermore, the Tyr113Phe (Y113F) mutant of PGRMC1 was not responsive to haem.", "section": "RESULTS", "ner": [ [ 17, 26, "Tyr113Phe", "mutant" ], [ 28, 33, "Y113F", "mutant" ], [ 35, 41, "mutant", "protein_state" ], [ 45, 51, "PGRMC1", "protein" ], [ 74, 78, "haem", "chemical" ] ] }, { "sid": 72, "sent": "These results suggest that CO favours the six-coordinate form of haem and interferes with the haem-mediated dimerization of PGRMC1.", "section": "RESULTS", "ner": [ [ 27, 29, "CO", "chemical" ], [ 65, 69, "haem", "chemical" ], [ 94, 98, "haem", "chemical" ], [ 108, 120, "dimerization", "oligomeric_state" ], [ 124, 130, "PGRMC1", "protein" ] ] }, { "sid": 73, "sent": "To examine the inhibitory effects of CO on haem-mediated PGRMC1 dimerization, SV-AUC analysis was carried out.", "section": "RESULTS", "ner": [ [ 37, 39, "CO", "chemical" ], [ 43, 47, "haem", "chemical" ], [ 57, 63, "PGRMC1", "protein" ], [ 64, 76, "dimerization", "oligomeric_state" ], [ 78, 84, "SV-AUC", "experimental_method" ] ] }, { "sid": 74, "sent": "The peak corresponding to the haem/PGRMC1 dimer was detected under reducing conditions in the presence of dithionite (Supplementary Fig. 15, middle panel).", "section": "RESULTS", "ner": [ [ 30, 34, "haem", "chemical" ], [ 35, 41, "PGRMC1", "protein" ], [ 42, 47, "dimer", "oligomeric_state" ], [ 94, 105, "presence of", "protein_state" ], [ 106, 116, "dithionite", "chemical" ] ] }, { "sid": 75, "sent": "Under these circumstances, CO application induced dissociation of the haem-mediated dimers of PGRMC1 to generate a peak of monomers (Supplementary Fig. 15, lower panel).", "section": "RESULTS", "ner": [ [ 27, 29, "CO", "chemical" ], [ 70, 74, "haem", "chemical" ], [ 84, 90, "dimers", "oligomeric_state" ], [ 94, 100, "PGRMC1", "protein" ], [ 123, 131, "monomers", "oligomeric_state" ] ] }, { "sid": 76, "sent": "These observations raised the transition model for structural regulation of PGRMC1 in response to haem (Fig. 3d).", "section": "RESULTS", "ner": [ [ 76, 82, "PGRMC1", "protein" ], [ 98, 102, "haem", "chemical" ] ] }, { "sid": 77, "sent": "As mentioned above, apo-PGRMC1 exists as monomer.", "section": "RESULTS", "ner": [ [ 20, 23, "apo", "protein_state" ], [ 24, 30, "PGRMC1", "protein" ], [ 41, 48, "monomer", "oligomeric_state" ] ] }, { "sid": 78, "sent": "By binding with haem (binding Kd=50\u2009nmol\u2009l\u22121), PGRMC1 forms a stable dimer (dimerization Kd<<3.5\u2009\u03bcmol\u2009l\u22121) through stacking of the two open surfaces of the five-coordinated haem molecules in each monomer.", "section": "RESULTS", "ner": [ [ 16, 20, "haem", "chemical" ], [ 30, 32, "Kd", "evidence" ], [ 47, 53, "PGRMC1", "protein" ], [ 62, 68, "stable", "protein_state" ], [ 69, 74, "dimer", "oligomeric_state" ], [ 76, 88, "dimerization", "oligomeric_state" ], [ 89, 91, "Kd", "evidence" ], [ 115, 123, "stacking", "bond_interaction" ], [ 140, 148, "surfaces", "site" ], [ 173, 177, "haem", "chemical" ], [ 196, 203, "monomer", "oligomeric_state" ] ] }, { "sid": 79, "sent": "CO induces the dissociation of the haem-mediated dimer of PGRMC1 by interfering with the haem-stacking interface via formation of the six-coordinated CO-haem-PGRMC1 complex.", "section": "RESULTS", "ner": [ [ 0, 2, "CO", "chemical" ], [ 35, 39, "haem", "chemical" ], [ 49, 54, "dimer", "oligomeric_state" ], [ 58, 64, "PGRMC1", "protein" ], [ 89, 112, "haem-stacking interface", "site" ], [ 150, 164, "CO-haem-PGRMC1", "complex_assembly" ] ] }, { "sid": 80, "sent": "Such a dynamic structural regulation led us to further examine the regulation of PGRMC1 functions in cancer cells.", "section": "RESULTS", "ner": [ [ 81, 87, "PGRMC1", "protein" ] ] }, { "sid": 81, "sent": "PGRMC1 dimerization is required for binding to EGFR", "section": "RESULTS", "ner": [ [ 0, 6, "PGRMC1", "protein" ], [ 7, 19, "dimerization", "oligomeric_state" ], [ 47, 51, "EGFR", "protein_type" ] ] }, { "sid": 82, "sent": "Because PGRMC1 is known to interact with EGFR and to accelerate tumour progression, we examined the effect of haem-dependent dimerization of PGRMC1 on its interaction with EGFR by using purified proteins.", "section": "RESULTS", "ner": [ [ 8, 14, "PGRMC1", "protein" ], [ 41, 45, "EGFR", "protein_type" ], [ 110, 114, "haem", "chemical" ], [ 125, 137, "dimerization", "oligomeric_state" ], [ 141, 147, "PGRMC1", "protein" ], [ 172, 176, "EGFR", "protein_type" ] ] }, { "sid": 83, "sent": "As shown in Fig. 4a, the cytosolic domain of wild-type PGRMC1, but not the Y113F mutant, interacted with purified EGFR in a haem-dependent manner.", "section": "RESULTS", "ner": [ [ 25, 41, "cytosolic domain", "structure_element" ], [ 45, 54, "wild-type", "protein_state" ], [ 55, 61, "PGRMC1", "protein" ], [ 75, 80, "Y113F", "mutant" ], [ 81, 87, "mutant", "protein_state" ], [ 114, 118, "EGFR", "protein_type" ], [ 124, 128, "haem", "chemical" ] ] }, { "sid": 84, "sent": "This interaction was disrupted by the ruthenium-based CO-releasing molecule, CORM3, but not by RuCl3 as a control reagent (Fig. 4b).", "section": "RESULTS", "ner": [ [ 38, 47, "ruthenium", "chemical" ], [ 54, 56, "CO", "chemical" ], [ 77, 82, "CORM3", "chemical" ], [ 95, 100, "RuCl3", "chemical" ] ] }, { "sid": 85, "sent": "We further analysed the intracellular interaction between PGRMC1 and EGFR.", "section": "RESULTS", "ner": [ [ 58, 64, "PGRMC1", "protein" ], [ 69, 73, "EGFR", "protein_type" ] ] }, { "sid": 86, "sent": "FLAG-tagged PGRMC1 ectopically expressed in human colon cancer HCT116 cells was immunoprecipitated with anti-FLAG antibody, and co-immunoprecipitated EGFR and endogenous PGRMC1 binding to FLAG-PGRMC1 were detected by Western blotting (Fig. 4c).", "section": "RESULTS", "ner": [ [ 0, 11, "FLAG-tagged", "protein_state" ], [ 12, 18, "PGRMC1", "protein" ], [ 19, 40, "ectopically expressed", "experimental_method" ], [ 44, 49, "human", "species" ], [ 80, 98, "immunoprecipitated", "experimental_method" ], [ 128, 149, "co-immunoprecipitated", "experimental_method" ], [ 150, 154, "EGFR", "protein_type" ], [ 159, 169, "endogenous", "protein_state" ], [ 170, 176, "PGRMC1", "protein" ], [ 193, 199, "PGRMC1", "protein" ], [ 217, 233, "Western blotting", "experimental_method" ] ] }, { "sid": 87, "sent": "The C129S mutant of PGRMC1 also interacted with endogenous PGRMC1 and EGFR (Supplementary Fig. 16).", "section": "RESULTS", "ner": [ [ 4, 9, "C129S", "mutant" ], [ 10, 16, "mutant", "protein_state" ], [ 20, 26, "PGRMC1", "protein" ], [ 48, 58, "endogenous", "protein_state" ], [ 59, 65, "PGRMC1", "protein" ], [ 70, 74, "EGFR", "protein_type" ] ] }, { "sid": 88, "sent": "Whereas FLAG-tagged wild-type PGRMC1 interacted with endogenous PGRMC1 and EGFR, the Y113F mutant did not.", "section": "RESULTS", "ner": [ [ 8, 19, "FLAG-tagged", "protein_state" ], [ 20, 29, "wild-type", "protein_state" ], [ 30, 36, "PGRMC1", "protein" ], [ 53, 63, "endogenous", "protein_state" ], [ 64, 70, "PGRMC1", "protein" ], [ 75, 79, "EGFR", "protein_type" ], [ 85, 90, "Y113F", "mutant" ], [ 91, 97, "mutant", "protein_state" ] ] }, { "sid": 89, "sent": "We also examined the effect of succinylacetone (SA), an inhibitor of haem biosynthesis (Fig. 4d).", "section": "RESULTS", "ner": [ [ 31, 46, "succinylacetone", "chemical" ], [ 48, 50, "SA", "chemical" ], [ 69, 73, "haem", "chemical" ] ] }, { "sid": 90, "sent": "As expected, SA significantly reduced PGRMC1 dimerization and its interaction with EGFR (Fig. 4e), indicating that haem-mediated dimerization of PGMRC1 is critical for its binding to EGFR.", "section": "RESULTS", "ner": [ [ 13, 15, "SA", "chemical" ], [ 30, 37, "reduced", "protein_state" ], [ 38, 44, "PGRMC1", "protein" ], [ 45, 57, "dimerization", "oligomeric_state" ], [ 83, 87, "EGFR", "protein_type" ], [ 115, 119, "haem", "chemical" ], [ 129, 141, "dimerization", "oligomeric_state" ], [ 145, 151, "PGMRC1", "protein" ], [ 183, 187, "EGFR", "protein_type" ] ] }, { "sid": 91, "sent": "PGRMC1 dimer facilitates EGFR-mediated cancer growth", "section": "RESULTS", "ner": [ [ 0, 6, "PGRMC1", "protein" ], [ 7, 12, "dimer", "oligomeric_state" ], [ 25, 29, "EGFR", "protein_type" ] ] }, { "sid": 92, "sent": "Next, we investigated the functional significance of PGRMC1 dimerization in EGFR signaling.", "section": "RESULTS", "ner": [ [ 53, 59, "PGRMC1", "protein" ], [ 60, 72, "dimerization", "oligomeric_state" ], [ 76, 80, "EGFR", "protein_type" ] ] }, { "sid": 93, "sent": "EGF-induced phosphorylations of EGFR and its downstream targets AKT and ERK were decreased by PGRMC1 knockdown (PGRMC1-KD) (Fig. 4f).", "section": "RESULTS", "ner": [ [ 0, 3, "EGF", "protein_type" ], [ 12, 28, "phosphorylations", "ptm" ], [ 32, 36, "EGFR", "protein_type" ], [ 64, 67, "AKT", "protein_type" ], [ 72, 75, "ERK", "protein_type" ], [ 94, 100, "PGRMC1", "protein" ], [ 101, 110, "knockdown", "protein_state" ], [ 112, 121, "PGRMC1-KD", "mutant" ] ] }, { "sid": 94, "sent": "Similarly, EGFR signaling was suppressed by treatment of HCT116 cells with SA (Fig. 4g) or CORM3 (Fig. 4h).", "section": "RESULTS", "ner": [ [ 11, 15, "EGFR", "protein_type" ], [ 75, 77, "SA", "chemical" ], [ 91, 96, "CORM3", "chemical" ] ] }, { "sid": 95, "sent": "These results suggested that haem-mediated dimerization of PGRMC1 is critical for EGFR signaling.", "section": "RESULTS", "ner": [ [ 29, 33, "haem", "chemical" ], [ 43, 55, "dimerization", "oligomeric_state" ], [ 59, 65, "PGRMC1", "protein" ], [ 82, 86, "EGFR", "protein_type" ] ] }, { "sid": 96, "sent": "To further investigate the role of the dimerized form of PGRMC1 in cancer proliferation, we performed PGRMC1 knockdown-rescue experiments using FLAG-tagged wild-type and Y113F PGRMC1 expression vectors, in which silent mutations were introduced into the nucleotide sequence targeted by shRNA (Fig. 5a).", "section": "RESULTS", "ner": [ [ 39, 48, "dimerized", "protein_state" ], [ 57, 63, "PGRMC1", "protein" ], [ 102, 108, "PGRMC1", "protein" ], [ 109, 137, "knockdown-rescue experiments", "experimental_method" ], [ 144, 155, "FLAG-tagged", "protein_state" ], [ 156, 165, "wild-type", "protein_state" ], [ 170, 175, "Y113F", "mutant" ], [ 176, 182, "PGRMC1", "protein" ], [ 183, 201, "expression vectors", "experimental_method" ], [ 212, 228, "silent mutations", "experimental_method" ], [ 234, 244, "introduced", "experimental_method" ], [ 286, 291, "shRNA", "chemical" ] ] }, { "sid": 97, "sent": "While proliferation of HCT116 cells was not affected by knocking down PGRMC1, PGRMC1-KD cells were more sensitive to the EGFR inhibitor erlotinib than control HCT116 cells, and the knockdown effect was reversed by co-expression of shRNA-resistant wild-type PGRMC1 but not of the Y113F mutant (Fig. 5b).", "section": "RESULTS", "ner": [ [ 56, 69, "knocking down", "experimental_method" ], [ 70, 76, "PGRMC1", "protein" ], [ 78, 87, "PGRMC1-KD", "mutant" ], [ 121, 125, "EGFR", "protein_type" ], [ 136, 145, "erlotinib", "chemical" ], [ 214, 227, "co-expression", "experimental_method" ], [ 231, 246, "shRNA-resistant", "protein_state" ], [ 247, 256, "wild-type", "protein_state" ], [ 257, 263, "PGRMC1", "protein" ], [ 279, 284, "Y113F", "mutant" ], [ 285, 291, "mutant", "protein_state" ] ] }, { "sid": 98, "sent": "Chemosensitivity enhancement by two different shRNAs to PGRMC1 was seen also in HCT116 cells and human hepatoma HuH7 cells (Supplementary Fig. 17).", "section": "RESULTS", "ner": [ [ 46, 52, "shRNAs", "chemical" ], [ 56, 62, "PGRMC1", "protein" ], [ 97, 102, "human", "species" ] ] }, { "sid": 99, "sent": "Furthermore, PGRMC1-KD inhibited spheroid formation of HCT116 cells in culture, and this inhibition was reversed by co-expression of wild-type PGRMC1 but not of the Y113F mutant (Fig. 5c and Supplementary Fig. 18).", "section": "RESULTS", "ner": [ [ 13, 22, "PGRMC1-KD", "mutant" ], [ 116, 129, "co-expression", "experimental_method" ], [ 133, 142, "wild-type", "protein_state" ], [ 143, 149, "PGRMC1", "protein" ], [ 165, 170, "Y113F", "mutant" ], [ 171, 177, "mutant", "protein_state" ] ] }, { "sid": 100, "sent": "Thus, PGRMC1 dimerization is important for cancer cell proliferation and chemoresistance.", "section": "RESULTS", "ner": [ [ 6, 12, "PGRMC1", "protein" ], [ 13, 25, "dimerization", "oligomeric_state" ] ] }, { "sid": 101, "sent": "We examined the role of PGRMC1 in metastatic progression by xenograft transplantation assays using super-immunodeficient NOD/scid/\u03b3null (NOG) mice.", "section": "RESULTS", "ner": [ [ 24, 30, "PGRMC1", "protein" ], [ 60, 92, "xenograft transplantation assays", "experimental_method" ] ] }, { "sid": 102, "sent": "Ten days after intra-splenic implantation of HCT116 cells that were genetically tagged with a fluorescent protein Venus, the group implanted with PGRMC1-KD cells showed a significant decrease of liver metastasis in comparison with the control group (Fig. 5d).", "section": "RESULTS", "ner": [ [ 15, 41, "intra-splenic implantation", "experimental_method" ], [ 146, 155, "PGRMC1-KD", "mutant" ] ] }, { "sid": 103, "sent": "Interaction of PGRMC1 dimer with cytochromes P450", "section": "RESULTS", "ner": [ [ 15, 21, "PGRMC1", "protein" ], [ 22, 27, "dimer", "oligomeric_state" ], [ 33, 49, "cytochromes P450", "protein_type" ] ] }, { "sid": 104, "sent": "Since PGRMC1 has been shown to interact with cytochromes P450 (ref), we investigated whether the haem-mediated dimerization of PGRMC1 is necessary for their interactions.", "section": "RESULTS", "ner": [ [ 6, 12, "PGRMC1", "protein" ], [ 45, 61, "cytochromes P450", "protein_type" ], [ 97, 101, "haem", "chemical" ], [ 111, 123, "dimerization", "oligomeric_state" ], [ 127, 133, "PGRMC1", "protein" ] ] }, { "sid": 105, "sent": "Recombinant CYP1A2 and CYP3A4 including a microsomal formulation containing cytochrome b5 and cytochrome P450 reductase, drug-metabolizing cytochromes P450, interacted with wild-type PGRMC1, but not with the Y113F mutant, in a haem-dependent manner (Fig. 6a,b).", "section": "RESULTS", "ner": [ [ 12, 18, "CYP1A2", "protein" ], [ 23, 29, "CYP3A4", "protein" ], [ 76, 89, "cytochrome b5", "protein_type" ], [ 94, 119, "cytochrome P450 reductase", "protein" ], [ 139, 155, "cytochromes P450", "protein_type" ], [ 173, 182, "wild-type", "protein_state" ], [ 183, 189, "PGRMC1", "protein" ], [ 208, 213, "Y113F", "mutant" ], [ 214, 220, "mutant", "protein_state" ], [ 227, 231, "haem", "chemical" ] ] }, { "sid": 106, "sent": "Moreover, the interaction of PGRMC1 with CYP1A2 was blocked by CORM3 under reducing conditions (Fig. 6c), indicating that PGRMC1 dimerization is necessary for its interaction with cytochromes P450.", "section": "RESULTS", "ner": [ [ 29, 35, "PGRMC1", "protein" ], [ 41, 47, "CYP1A2", "protein" ], [ 63, 68, "CORM3", "chemical" ], [ 122, 128, "PGRMC1", "protein" ], [ 129, 141, "dimerization", "oligomeric_state" ], [ 180, 196, "cytochromes P450", "protein_type" ] ] }, { "sid": 107, "sent": "Doxorubicin is an anti-cancer reagent that is metabolized into inactive doxorubicinol by CYP2D6 and CYP3A4 (Fig. 6d).", "section": "RESULTS", "ner": [ [ 0, 11, "Doxorubicin", "chemical" ], [ 72, 85, "doxorubicinol", "chemical" ], [ 89, 95, "CYP2D6", "protein" ], [ 100, 106, "CYP3A4", "protein" ] ] }, { "sid": 108, "sent": "PGRMC1-KD significantly suppressed the conversion of doxorubicin to doxorubicinol (Fig. 6d) and increased sensitivity to doxorubicin (Fig. 6e).", "section": "RESULTS", "ner": [ [ 0, 9, "PGRMC1-KD", "mutant" ], [ 53, 64, "doxorubicin", "chemical" ], [ 68, 81, "doxorubicinol", "chemical" ], [ 121, 132, "doxorubicin", "chemical" ] ] }, { "sid": 109, "sent": "Enhanced doxorubicin sensitivity was modestly but significantly induced by PGRMC1-KD.", "section": "RESULTS", "ner": [ [ 9, 20, "doxorubicin", "chemical" ], [ 75, 84, "PGRMC1-KD", "mutant" ] ] }, { "sid": 110, "sent": "This effect was reversed by co-expression of the wild-type PGRMC1 but not of the Y113F mutant, suggesting that PGRMC1 enhances doxorubicin resistance of cancer cells by facilitating its degradation via cytochromes P450.", "section": "RESULTS", "ner": [ [ 28, 41, "co-expression", "experimental_method" ], [ 49, 58, "wild-type", "protein_state" ], [ 59, 65, "PGRMC1", "protein" ], [ 81, 86, "Y113F", "mutant" ], [ 87, 93, "mutant", "protein_state" ], [ 111, 117, "PGRMC1", "protein" ], [ 127, 138, "doxorubicin", "chemical" ], [ 202, 218, "cytochromes P450", "protein_type" ] ] }, { "sid": 111, "sent": "To gain further insight into the interaction between PGRMC1 and cytochromes P450, surface plasmon resonance analyses were conducted using recombinant CYP51 and PGRMC1.", "section": "RESULTS", "ner": [ [ 53, 59, "PGRMC1", "protein" ], [ 64, 80, "cytochromes P450", "protein_type" ], [ 82, 116, "surface plasmon resonance analyses", "experimental_method" ], [ 150, 155, "CYP51", "protein" ], [ 160, 166, "PGRMC1", "protein" ] ] }, { "sid": 112, "sent": "This was based on a previous study showing that PGRMC1 binds to CYP51 and enhances cholesterol biosynthesis by CYP51 (refs).", "section": "RESULTS", "ner": [ [ 48, 54, "PGRMC1", "protein" ], [ 64, 69, "CYP51", "protein" ], [ 111, 116, "CYP51", "protein" ] ] }, { "sid": 113, "sent": "CYP51 interacted with PGRMC1 in a concentration-dependent manner in the presence of haem, but not in its absence (Supplementary Fig. 19), suggesting the requirement for the haem-dependent dimerization of PGRMC1.", "section": "RESULTS", "ner": [ [ 0, 5, "CYP51", "protein" ], [ 22, 28, "PGRMC1", "protein" ], [ 72, 83, "presence of", "protein_state" ], [ 84, 88, "haem", "chemical" ], [ 105, 112, "absence", "protein_state" ], [ 173, 177, "haem", "chemical" ], [ 188, 200, "dimerization", "oligomeric_state" ], [ 204, 210, "PGRMC1", "protein" ] ] }, { "sid": 114, "sent": "The Kd value of PGRMC1 binding to CYP51 was in a micromolar range and comparable with those of other haem proteins, such as cytochrome P450 reductase and neuroglobin/G\u03b1i1 (ref.), suggesting that haem-dependent PGRMC1 interaction with CYP51 is biologically relevant.", "section": "RESULTS", "ner": [ [ 4, 6, "Kd", "evidence" ], [ 16, 22, "PGRMC1", "protein" ], [ 34, 39, "CYP51", "protein" ], [ 101, 105, "haem", "chemical" ], [ 124, 149, "cytochrome P450 reductase", "protein" ], [ 154, 165, "neuroglobin", "protein" ], [ 166, 170, "G\u03b1i1", "protein" ], [ 195, 199, "haem", "chemical" ], [ 210, 216, "PGRMC1", "protein" ], [ 234, 239, "CYP51", "protein" ] ] }, { "sid": 115, "sent": "In this study, we showed that PGRMC1 dimerizes by stacking interactions of haem molecules from each monomer.", "section": "DISCUSS", "ner": [ [ 30, 36, "PGRMC1", "protein" ], [ 37, 46, "dimerizes", "oligomeric_state" ], [ 50, 71, "stacking interactions", "bond_interaction" ], [ 75, 79, "haem", "chemical" ], [ 100, 107, "monomer", "oligomeric_state" ] ] }, { "sid": 116, "sent": "Recently, Lucas et al. reported that translationally-controlled tumour protein was dimerized by binding with haem, but its structural basis remains unclear.", "section": "DISCUSS", "ner": [ [ 37, 78, "translationally-controlled tumour protein", "protein_type" ], [ 83, 92, "dimerized", "protein_state" ], [ 109, 113, "haem", "chemical" ] ] }, { "sid": 117, "sent": "This is the report showing crystallographic evidence that indicates roles of the direct haem\u2013haem stacking in haem-mediated dimerization in eukaryotes, although a few examples are known in bacteria.", "section": "DISCUSS", "ner": [ [ 88, 106, "haem\u2013haem stacking", "bond_interaction" ], [ 110, 114, "haem", "chemical" ], [ 124, 136, "dimerization", "oligomeric_state" ], [ 140, 150, "eukaryotes", "taxonomy_domain" ], [ 189, 197, "bacteria", "taxonomy_domain" ] ] }, { "sid": 118, "sent": "Sequence alignments show that haem-binding residues (Tyr113, Tyr107, Lys163 and Tyr164) in PGRMC1 are conserved among MAPR proteins (Supplementary Fig. 5).", "section": "DISCUSS", "ner": [ [ 0, 19, "Sequence alignments", "experimental_method" ], [ 30, 51, "haem-binding residues", "site" ], [ 53, 59, "Tyr113", "residue_name_number" ], [ 61, 67, "Tyr107", "residue_name_number" ], [ 69, 75, "Lys163", "residue_name_number" ], [ 80, 86, "Tyr164", "residue_name_number" ], [ 91, 97, "PGRMC1", "protein" ], [ 102, 111, "conserved", "protein_state" ], [ 118, 122, "MAPR", "protein_type" ] ] }, { "sid": 119, "sent": "In the current study, the Y113 residue plays a crucial role for the haem-dependent dimerization of PGRMC1 and resultant regulation of cancer proliferation and chemoresistance (Figs 5c and 6e).", "section": "DISCUSS", "ner": [ [ 26, 30, "Y113", "residue_name_number" ], [ 68, 72, "haem", "chemical" ], [ 83, 95, "dimerization", "oligomeric_state" ], [ 99, 105, "PGRMC1", "protein" ] ] }, { "sid": 120, "sent": "Since the Y113 residue is involved in the putative consensus motif of phosphorylation by tyrosine kinases such as Abl and Lck, we investigated whether phosphorylated Y113 is present in HCT116 cells by ESI-MS analysis.", "section": "DISCUSS", "ner": [ [ 10, 14, "Y113", "residue_name_number" ], [ 51, 66, "consensus motif", "structure_element" ], [ 70, 85, "phosphorylation", "ptm" ], [ 89, 105, "tyrosine kinases", "protein_type" ], [ 114, 117, "Abl", "protein_type" ], [ 122, 125, "Lck", "protein_type" ], [ 151, 165, "phosphorylated", "protein_state" ], [ 166, 170, "Y113", "residue_name_number" ], [ 201, 207, "ESI-MS", "experimental_method" ] ] }, { "sid": 121, "sent": "Recently, Peluso et al. reported that PGRMC1 binds to PGRMC2, suggesting that MAPR family members may also undergo haem-mediated heterodimerization.", "section": "DISCUSS", "ner": [ [ 38, 44, "PGRMC1", "protein" ], [ 54, 60, "PGRMC2", "protein" ], [ 78, 82, "MAPR", "protein_type" ], [ 115, 119, "haem", "chemical" ] ] }, { "sid": 122, "sent": "We showed that the haem-mediated dimer of PGRMC1 enables interaction with different subclasses of cytochromes P450 (CYP) (Fig. 6).", "section": "DISCUSS", "ner": [ [ 19, 23, "haem", "chemical" ], [ 33, 38, "dimer", "oligomeric_state" ], [ 42, 48, "PGRMC1", "protein" ], [ 98, 114, "cytochromes P450", "protein_type" ], [ 116, 119, "CYP", "protein_type" ] ] }, { "sid": 123, "sent": "While the effects of PGRMC1 on cholesterol synthesis mediated by CYP51 have been well documented in yeast and human cells, it has not been clear whether drug-metabolizing CYP activities are regulated by PGRMC1.", "section": "DISCUSS", "ner": [ [ 21, 27, "PGRMC1", "protein" ], [ 65, 70, "CYP51", "protein" ], [ 100, 105, "yeast", "taxonomy_domain" ], [ 110, 115, "human", "species" ], [ 171, 174, "CYP", "protein_type" ], [ 203, 209, "PGRMC1", "protein" ] ] }, { "sid": 124, "sent": "Szczesna-Skorupa and Kemper reported that PGRMC1 exhibited an inhibitory effect on CYP3A4 drug metabolizing activity by competitively binding with cytochrome P450 reductase (CPR) in HEK293 or HepG2 cells.", "section": "DISCUSS", "ner": [ [ 42, 48, "PGRMC1", "protein" ], [ 83, 89, "CYP3A4", "protein" ], [ 147, 172, "cytochrome P450 reductase", "protein" ], [ 174, 177, "CPR", "protein" ] ] }, { "sid": 125, "sent": "On the other hand, Oda et al. reported that PGRMC1 had no effect to CYP2E1 and CYP3A4 activities in HepG2 cell.", "section": "DISCUSS", "ner": [ [ 44, 50, "PGRMC1", "protein" ], [ 68, 74, "CYP2E1", "protein" ], [ 79, 85, "CYP3A4", "protein" ] ] }, { "sid": 126, "sent": "Several other groups showed that PGRMC1 enhanced chemoresistance in several cancer cells such as uterine sarcoma, breast cancer, endometrial tumour and ovarian cancer; however, no evidence of PGRMC1-dependent regulation of CYP activity was provided.", "section": "DISCUSS", "ner": [ [ 33, 39, "PGRMC1", "protein" ], [ 192, 198, "PGRMC1", "protein" ], [ 223, 226, "CYP", "protein_type" ] ] }, { "sid": 127, "sent": "Our results showed that PGRMC1 contributes to enhancement of the doxorubicin metabolism, which is mediated by CYP2D6 or CYP3A4 in human colon cancer HCT116 cells (Fig. 6d).", "section": "DISCUSS", "ner": [ [ 24, 30, "PGRMC1", "protein" ], [ 65, 76, "doxorubicin", "chemical" ], [ 110, 116, "CYP2D6", "protein" ], [ 120, 126, "CYP3A4", "protein" ], [ 130, 135, "human", "species" ] ] }, { "sid": 128, "sent": "While the effects of structural diversity of CYP family proteins and interactions with different xenobiotic substrates should further be examined, the current results suggest that the interaction of drug-metabolizing CYPs with the haem-mediated dimer of PGRMC1 plays a crucial role in regulating their activities.", "section": "DISCUSS", "ner": [ [ 45, 48, "CYP", "protein_type" ], [ 217, 221, "CYPs", "protein_type" ], [ 231, 235, "haem", "chemical" ], [ 245, 250, "dimer", "oligomeric_state" ], [ 254, 260, "PGRMC1", "protein" ] ] }, { "sid": 129, "sent": "We showed that haem-mediated dimerization of PGRMC1 enhances proliferation and chemoresistance of cancer cells through binding to and regulating EGFR and cytochromes P450 (illustrated in Fig. 7).", "section": "DISCUSS", "ner": [ [ 15, 19, "haem", "chemical" ], [ 29, 41, "dimerization", "oligomeric_state" ], [ 45, 51, "PGRMC1", "protein" ], [ 145, 149, "EGFR", "protein_type" ], [ 154, 170, "cytochromes P450", "protein_type" ] ] }, { "sid": 130, "sent": "Since the haem-binding affinity of PGRMC1 is lower than those of constitutive haem-binding proteins such as myoglobin, PGMRC1 is probably interconverted between apo-monomer and haem-bound dimer forms in response to changes in the intracellular haem concentration.", "section": "DISCUSS", "ner": [ [ 10, 31, "haem-binding affinity", "evidence" ], [ 35, 41, "PGRMC1", "protein" ], [ 65, 77, "constitutive", "protein_state" ], [ 78, 99, "haem-binding proteins", "protein_type" ], [ 108, 117, "myoglobin", "protein" ], [ 119, 125, "PGMRC1", "protein" ], [ 161, 164, "apo", "protein_state" ], [ 165, 172, "monomer", "oligomeric_state" ], [ 177, 187, "haem-bound", "protein_state" ], [ 188, 193, "dimer", "oligomeric_state" ], [ 244, 248, "haem", "chemical" ] ] }, { "sid": 131, "sent": "Considering microenvironments in and around malignant tumours, the haem concentration in cancer cells is likely to be elevated through multiple mechanisms, such as (i) an increased intake of haem, (ii) mutation of enzymes in TCA cycle (for example, fumarate hydratase) that increases the level of succinyl CoA, a substrate for haem biosynthesis and (iii) metastasis to haem-rich organs such as liver, brain and bone marrow.", "section": "DISCUSS", "ner": [ [ 67, 71, "haem", "chemical" ], [ 191, 195, "haem", "chemical" ], [ 249, 267, "fumarate hydratase", "protein_type" ], [ 297, 309, "succinyl CoA", "chemical" ], [ 327, 331, "haem", "chemical" ], [ 369, 373, "haem", "chemical" ] ] }, { "sid": 132, "sent": "Moreover, exposure of cancer cells to stimuli such as hypoxia, radiation and chemotherapy causes cell damages and leads to protein degradation, resulting in increased levels of TCA cycle intermediates and in an enhanced haem biosynthesis.", "section": "DISCUSS", "ner": [ [ 220, 224, "haem", "chemical" ] ] }, { "sid": 133, "sent": "On the other hand, excessive haem induces HO-1, the enzyme that oxidatively degrades haem and generates CO.", "section": "DISCUSS", "ner": [ [ 29, 33, "haem", "chemical" ], [ 42, 46, "HO-1", "protein" ], [ 85, 89, "haem", "chemical" ], [ 104, 106, "CO", "chemical" ] ] }, { "sid": 134, "sent": "Thus, HO-1 induction in cancer cells may inhibit the haem-mediated dimerization of PGRMC1 through the production of CO and thereby suppress tumour progression.", "section": "DISCUSS", "ner": [ [ 6, 10, "HO-1", "protein" ], [ 53, 57, "haem", "chemical" ], [ 67, 79, "dimerization", "oligomeric_state" ], [ 83, 89, "PGRMC1", "protein" ], [ 116, 118, "CO", "chemical" ] ] }, { "sid": 135, "sent": "This idea is consistent with the observation that HO-1 induction or CO inhibits tumour growth.", "section": "DISCUSS", "ner": [ [ 50, 54, "HO-1", "protein" ], [ 68, 70, "CO", "chemical" ] ] }, { "sid": 136, "sent": "Besides the regulatory roles of PGRMC1/Sigma-2 receptor in proliferation and chemoresistance in cancer cells (ref.), recent reports show that PGRMC1 is able to bind to amyloid beta oligomer to enhance its neurotoxicity.", "section": "DISCUSS", "ner": [ [ 32, 38, "PGRMC1", "protein" ], [ 39, 46, "Sigma-2", "protein" ], [ 142, 148, "PGRMC1", "protein" ], [ 168, 180, "amyloid beta", "protein" ], [ 181, 189, "oligomer", "oligomeric_state" ] ] }, { "sid": 137, "sent": "Furthermore, Sigma-2 ligand-binding is decreased in transgenic amyloid beta deposition model APP/PS1 female mice.", "section": "DISCUSS", "ner": [ [ 13, 20, "Sigma-2", "protein" ] ] }, { "sid": 138, "sent": "These results suggest a possible involvement of PGRMC1 in Alzheimer's disease.", "section": "DISCUSS", "ner": [ [ 48, 54, "PGRMC1", "protein" ] ] }, { "sid": 139, "sent": "The roles of haem-dependent dimerization of PGRMC1 in the functional regulation of its target proteins deserve further studies to find evidence that therapeutic interventions to interfere with the function of the dimer may control varied disease conditions.", "section": "DISCUSS", "ner": [ [ 13, 17, "haem", "chemical" ], [ 28, 40, "dimerization", "oligomeric_state" ], [ 44, 50, "PGRMC1", "protein" ], [ 213, 218, "dimer", "oligomeric_state" ] ] }, { "sid": 140, "sent": "Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers II: Sigma-2/PGRMC1 receptors mediate Abeta 42 oligomer binding and synaptotoxicity", "section": "REF", "ner": [ [ 109, 117, "oligomer", "oligomeric_state" ] ] }, { "sid": 141, "sent": "X-ray crystal structure of PGRMC1.", "section": "FIG", "ner": [ [ 0, 23, "X-ray crystal structure", "evidence" ], [ 27, 33, "PGRMC1", "protein" ] ] }, { "sid": 142, "sent": "(a) Structure of the PGRMC1 dimer formed through stacked haems.", "section": "FIG", "ner": [ [ 21, 27, "PGRMC1", "protein" ], [ 28, 33, "dimer", "oligomeric_state" ], [ 57, 62, "haems", "chemical" ] ] }, { "sid": 143, "sent": "Two PGRMC1 subunits (blue and green ribbons) dimerize via stacking of the haem molecules.", "section": "FIG", "ner": [ [ 4, 10, "PGRMC1", "protein" ], [ 11, 19, "subunits", "structure_element" ], [ 45, 53, "dimerize", "oligomeric_state" ], [ 58, 66, "stacking", "bond_interaction" ], [ 74, 78, "haem", "chemical" ] ] }, { "sid": 144, "sent": "(b) Haem coordination of PGRMC1 with Tyr113.", "section": "FIG", "ner": [ [ 4, 8, "Haem", "chemical" ], [ 9, 21, "coordination", "bond_interaction" ], [ 25, 31, "PGRMC1", "protein" ], [ 37, 43, "Tyr113", "residue_name_number" ] ] }, { "sid": 145, "sent": "Comparison of PGRMC1 (blue) and cytochrome b5 (yellow, ID: 3NER). (c) PGRMC1 has a longer helix (a.a.147\u2013163), which is shifted away from the haem (arrow).", "section": "FIG", "ner": [ [ 14, 20, "PGRMC1", "protein" ], [ 32, 45, "cytochrome b5", "protein_type" ], [ 70, 76, "PGRMC1", "protein" ], [ 90, 95, "helix", "structure_element" ], [ 101, 108, "147\u2013163", "residue_range" ], [ 142, 146, "haem", "chemical" ] ] }, { "sid": 146, "sent": "PGRCM1 is dimerized by binding with haem.", "section": "FIG", "ner": [ [ 0, 6, "PGRCM1", "protein" ], [ 10, 19, "dimerized", "protein_state" ], [ 36, 40, "haem", "chemical" ] ] }, { "sid": 147, "sent": "(a) Mass spectrometric analyses of the wild-type (wt) PGRMC1 or the C129S mutant in the presence or absence of haem under non-denaturing condition.", "section": "FIG", "ner": [ [ 4, 22, "Mass spectrometric", "experimental_method" ], [ 39, 48, "wild-type", "protein_state" ], [ 50, 52, "wt", "protein_state" ], [ 54, 60, "PGRMC1", "protein" ], [ 68, 73, "C129S", "mutant" ], [ 74, 80, "mutant", "protein_state" ], [ 88, 96, "presence", "protein_state" ], [ 100, 110, "absence of", "protein_state" ], [ 111, 115, "haem", "chemical" ] ] }, { "sid": 148, "sent": "Both proteins had identical lengths (a.a.44\u2013195).", "section": "FIG", "ner": [ [ 41, 47, "44\u2013195", "residue_range" ] ] }, { "sid": 149, "sent": "(b) SV-AUC analyses of the wt-PGRMC1 and the C129S mutant (a.a.44\u2013195) in the presence or absence of haem.", "section": "FIG", "ner": [ [ 4, 10, "SV-AUC", "experimental_method" ], [ 27, 29, "wt", "protein_state" ], [ 30, 36, "PGRMC1", "protein" ], [ 45, 50, "C129S", "mutant" ], [ 51, 57, "mutant", "protein_state" ], [ 63, 69, "44\u2013195", "residue_range" ], [ 78, 86, "presence", "protein_state" ], [ 90, 100, "absence of", "protein_state" ], [ 101, 105, "haem", "chemical" ] ] }, { "sid": 150, "sent": "SV-AUC experiments were performed with 1.5\u2009mg\u2009ml\u22121 of PGRMC1 proteins.", "section": "FIG", "ner": [ [ 0, 6, "SV-AUC", "experimental_method" ], [ 54, 60, "PGRMC1", "protein" ] ] }, { "sid": 151, "sent": "The major peak with sedimentation coefficient S20,w of 1.9\u223c2.0 S (monomer) or 3.1 S (dimer) was detected.", "section": "FIG", "ner": [ [ 20, 45, "sedimentation coefficient", "evidence" ], [ 46, 51, "S20,w", "evidence" ], [ 66, 73, "monomer", "oligomeric_state" ], [ 85, 90, "dimer", "oligomeric_state" ] ] }, { "sid": 152, "sent": "(c) Difference absorption spectra of PGRMC1 (a.a.44\u2013195) titrated with haem (left panel).", "section": "FIG", "ner": [ [ 4, 33, "Difference absorption spectra", "evidence" ], [ 37, 43, "PGRMC1", "protein" ], [ 49, 55, "44\u2013195", "residue_range" ], [ 57, 70, "titrated with", "experimental_method" ], [ 71, 75, "haem", "chemical" ] ] }, { "sid": 153, "sent": "The titration curve of haem to PGRMC1 (right panel).", "section": "FIG", "ner": [ [ 4, 19, "titration curve", "evidence" ], [ 23, 27, "haem", "chemical" ], [ 31, 37, "PGRMC1", "protein" ] ] }, { "sid": 154, "sent": "The absorbance difference at 400\u2009nm is plotted against the haem concentration.", "section": "FIG", "ner": [ [ 4, 25, "absorbance difference", "evidence" ], [ 59, 63, "haem", "chemical" ] ] }, { "sid": 155, "sent": "Carbon monoxide inhibits haem-dependent PGRMC1 dimerization.", "section": "FIG", "ner": [ [ 0, 15, "Carbon monoxide", "chemical" ], [ 25, 29, "haem", "chemical" ], [ 40, 46, "PGRMC1", "protein" ], [ 47, 59, "dimerization", "oligomeric_state" ] ] }, { "sid": 156, "sent": "(a) UV-visible absorption spectra of PGRMC1 (a.a.44\u2013195).", "section": "FIG", "ner": [ [ 4, 33, "UV-visible absorption spectra", "evidence" ], [ 37, 43, "PGRMC1", "protein" ], [ 49, 55, "44\u2013195", "residue_range" ] ] }, { "sid": 157, "sent": "Measurements were performed in the presence of the oxidized form of haem (ferric), the reduced form of haem (ferrous) and the reduced form of haem plus CO gas (ferrous+CO).", "section": "FIG", "ner": [ [ 35, 46, "presence of", "protein_state" ], [ 51, 59, "oxidized", "protein_state" ], [ 68, 72, "haem", "chemical" ], [ 74, 80, "ferric", "protein_state" ], [ 87, 94, "reduced", "protein_state" ], [ 103, 107, "haem", "chemical" ], [ 109, 116, "ferrous", "protein_state" ], [ 126, 133, "reduced", "protein_state" ], [ 142, 146, "haem", "chemical" ], [ 152, 154, "CO", "chemical" ], [ 160, 167, "ferrous", "protein_state" ], [ 168, 170, "CO", "chemical" ] ] }, { "sid": 158, "sent": "(b) Close-up view of haem stacking.", "section": "FIG", "ner": [ [ 21, 34, "haem stacking", "bond_interaction" ] ] }, { "sid": 159, "sent": "(c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44\u2013195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO.", "section": "FIG", "ner": [ [ 4, 33, "Gel-filtration chromatography", "experimental_method" ], [ 46, 52, "PGRMC1", "protein" ], [ 58, 64, "44\u2013195", "residue_range" ], [ 66, 75, "wild-type", "protein_state" ], [ 77, 79, "wt", "protein_state" ], [ 89, 94, "Y113F", "mutant" ], [ 98, 103, "C129S", "mutant" ], [ 104, 110, "mutant", "protein_state" ], [ 118, 126, "presence", "protein_state" ], [ 130, 140, "absence of", "protein_state" ], [ 141, 145, "haem", "chemical" ], [ 147, 157, "dithionite", "chemical" ], [ 165, 167, "CO", "chemical" ], [ 219, 225, "PGRMC1", "protein" ], [ 241, 245, "haem", "chemical" ], [ 250, 252, "CO", "chemical" ] ] }, { "sid": 160, "sent": "Haem-dependent dimerization of PGRMC1 is necessary for tumour proliferation mediated by EGFR signalling.", "section": "FIG", "ner": [ [ 0, 4, "Haem", "chemical" ], [ 15, 27, "dimerization", "oligomeric_state" ], [ 31, 37, "PGRMC1", "protein" ], [ 88, 92, "EGFR", "protein_type" ] ] }, { "sid": 161, "sent": "(a) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44\u2013195), in either apo- or haem-bound form, were incubated with purified EGFR and co-immunoprecipitated with anti-FLAG antibody-conjugated beads.", "section": "FIG", "ner": [ [ 9, 15, "PGRMC1", "protein" ], [ 16, 25, "wild-type", "protein_state" ], [ 27, 29, "wt", "protein_state" ], [ 35, 40, "Y113F", "mutant" ], [ 41, 47, "mutant", "protein_state" ], [ 62, 68, "44\u2013195", "residue_range" ], [ 81, 84, "apo", "protein_state" ], [ 89, 99, "haem-bound", "protein_state" ], [ 111, 120, "incubated", "experimental_method" ], [ 135, 139, "EGFR", "protein_type" ], [ 144, 165, "co-immunoprecipitated", "experimental_method" ] ] }, { "sid": 162, "sent": "Input and bound proteins were detected by Western blotting.", "section": "FIG", "ner": [ [ 42, 58, "Western blotting", "experimental_method" ], [ 42, 58, "Western blotting", "experimental_method" ] ] }, { "sid": 163, "sent": "(b) In vitro binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt (a.a.44\u2013195) and purified EGFR with or without treatment of RuCl3 and CORM3.", "section": "FIG", "ner": [ [ 4, 26, "In vitro binding assay", "experimental_method" ], [ 57, 67, "haem-bound", "protein_state" ], [ 73, 79, "PGRMC1", "protein" ], [ 80, 82, "wt", "protein_state" ], [ 88, 94, "44\u2013195", "residue_range" ], [ 109, 113, "EGFR", "protein_type" ], [ 143, 148, "RuCl3", "chemical" ], [ 153, 158, "CORM3", "chemical" ] ] }, { "sid": 164, "sent": "(c) FLAG-PGRMC1 wt or Y113F (full length) was over-expressed in HCT116 cells and immunoprecipitated with anti-FLAG antibody-conjugated beads.", "section": "FIG", "ner": [ [ 9, 15, "PGRMC1", "protein" ], [ 16, 18, "wt", "protein_state" ], [ 22, 27, "Y113F", "mutant" ], [ 29, 40, "full length", "protein_state" ], [ 46, 60, "over-expressed", "experimental_method" ], [ 81, 99, "immunoprecipitated", "experimental_method" ] ] }, { "sid": 165, "sent": "Co-immunoprecipitated proteins (FLAG-PGRMC1, endogenous PGRMC1 and EGFR) were detected with Western blotting by using anti-PGRMC1 or anti-EGFR antibody.", "section": "FIG", "ner": [ [ 0, 21, "Co-immunoprecipitated", "experimental_method" ], [ 37, 43, "PGRMC1", "protein" ], [ 45, 55, "endogenous", "protein_state" ], [ 56, 62, "PGRMC1", "protein" ], [ 67, 71, "EGFR", "protein_type" ], [ 92, 108, "Western blotting", "experimental_method" ], [ 123, 129, "PGRMC1", "protein" ], [ 138, 142, "EGFR", "protein_type" ] ] }, { "sid": 166, "sent": "(d) HCT116 cells were treated with or without 250\u2009\u03bcmol\u2009l\u22121 of succinylacetone (SA) for 48\u2009h. The intracellular haem was extracted and quantified by reverse-phase HPLC.", "section": "FIG", "ner": [ [ 62, 77, "succinylacetone", "chemical" ], [ 79, 81, "SA", "chemical" ], [ 111, 115, "haem", "chemical" ], [ 148, 166, "reverse-phase HPLC", "experimental_method" ] ] }, { "sid": 167, "sent": "of four separate experiments. **P<0.01 using unpaired Student's t-test. (e) Co-immunoprecipitation assay was performed as in (c) with or without SA treatment in HCT116 cells.", "section": "FIG", "ner": [ [ 54, 70, "Student's t-test", "experimental_method" ], [ 76, 104, "Co-immunoprecipitation assay", "experimental_method" ], [ 145, 147, "SA", "chemical" ] ] }, { "sid": 168, "sent": "(f) HCT116 cells expressing control shRNA or those knocking down PGRMC1 (PGRMC1-KD) were treated with EGF or left untreated, and components of the EGFR signaling pathway were detected by Western blotting.", "section": "FIG", "ner": [ [ 36, 41, "shRNA", "chemical" ], [ 51, 64, "knocking down", "experimental_method" ], [ 65, 71, "PGRMC1", "protein" ], [ 73, 82, "PGRMC1-KD", "mutant" ], [ 102, 105, "EGF", "protein_type" ], [ 147, 151, "EGFR", "protein_type" ], [ 187, 203, "Western blotting", "experimental_method" ] ] }, { "sid": 169, "sent": "(g,h) HCT116 cells were treated with or without EGF, SA, RuCl3 and CORM3 as indicated, and components of the EGFR signaling pathway were detected by Western blotting.", "section": "FIG", "ner": [ [ 48, 51, "EGF", "protein_type" ], [ 53, 55, "SA", "chemical" ], [ 57, 62, "RuCl3", "chemical" ], [ 67, 72, "CORM3", "chemical" ], [ 109, 113, "EGFR", "protein_type" ], [ 149, 165, "Western blotting", "experimental_method" ] ] }, { "sid": 170, "sent": "Haem-dependent dimerization of PGRMC1 accelerates tumour growth through the EGFR signaling pathway.", "section": "FIG", "ner": [ [ 0, 4, "Haem", "chemical" ], [ 15, 27, "dimerization", "oligomeric_state" ], [ 31, 37, "PGRMC1", "protein" ], [ 76, 80, "EGFR", "protein_type" ] ] }, { "sid": 171, "sent": "(a) Nucleotide sequences of PGRMC1 targeted by shRNA and of the shRNA-resistant full length PGRMC1 expression vector.", "section": "FIG", "ner": [ [ 28, 34, "PGRMC1", "protein" ], [ 47, 52, "shRNA", "chemical" ], [ 64, 79, "shRNA-resistant", "protein_state" ], [ 80, 91, "full length", "protein_state" ], [ 92, 98, "PGRMC1", "protein" ] ] }, { "sid": 172, "sent": "Stable PGRMC1-knockdown (PGRMC1-KD) HCT116 cells were transiently transfected with the shRNA-resistant expression vector of wild-type PGRMC1 (wt) or the Y113F mutant (Y113F).", "section": "FIG", "ner": [ [ 7, 23, "PGRMC1-knockdown", "mutant" ], [ 25, 34, "PGRMC1-KD", "mutant" ], [ 54, 77, "transiently transfected", "experimental_method" ], [ 87, 102, "shRNA-resistant", "protein_state" ], [ 103, 120, "expression vector", "experimental_method" ], [ 124, 133, "wild-type", "protein_state" ], [ 134, 140, "PGRMC1", "protein" ], [ 142, 144, "wt", "protein_state" ], [ 153, 158, "Y113F", "mutant" ], [ 159, 165, "mutant", "protein_state" ], [ 167, 172, "Y113F", "mutant" ] ] }, { "sid": 173, "sent": "(b) Erlotinib was added to HCT116 (control) cells, PGRMC1-KD cells or PGRMC1-KD cells expressing shRNA-resistant PGRMC1 wt or Y113F, and cell viability was examined by MTT assay.", "section": "FIG", "ner": [ [ 4, 13, "Erlotinib", "chemical" ], [ 51, 60, "PGRMC1-KD", "mutant" ], [ 70, 79, "PGRMC1-KD", "mutant" ], [ 97, 112, "shRNA-resistant", "protein_state" ], [ 113, 119, "PGRMC1", "protein" ], [ 120, 122, "wt", "protein_state" ], [ 126, 131, "Y113F", "mutant" ], [ 168, 177, "MTT assay", "experimental_method" ] ] }, { "sid": 174, "sent": "of four separate experiments. *P<0.01 using ANOVA with Fischer's LSD test.", "section": "FIG", "ner": [ [ 30, 32, "*P", "evidence" ], [ 44, 49, "ANOVA", "experimental_method" ], [ 55, 73, "Fischer's LSD test", "experimental_method" ], [ 30, 32, "*P", "evidence" ], [ 44, 49, "ANOVA", "experimental_method" ], [ 55, 73, "Fischer's LSD test", "experimental_method" ] ] }, { "sid": 175, "sent": "(c) Spheroid formation in control and PGRMC1-KD HCT116 cells.", "section": "FIG", "ner": [ [ 38, 47, "PGRMC1-KD", "mutant" ] ] }, { "sid": 176, "sent": "The graph represents mean\u00b1s.e. of each spheroid size. *P<0.01 using ANOVA with Fischer's LSD test.", "section": "FIG", "ner": [ [ 54, 56, "*P", "evidence" ], [ 68, 73, "ANOVA", "experimental_method" ], [ 79, 97, "Fischer's LSD test", "experimental_method" ] ] }, { "sid": 177, "sent": "Scale bar: 0.1\u2009mm. (d) Tumour-bearing livers of NOG mice at 10 days after intrasplenic injection of HCT116 (control) or PGRMC1-KD cells.", "section": "FIG", "ner": [ [ 74, 96, "intrasplenic injection", "experimental_method" ], [ 120, 129, "PGRMC1-KD", "mutant" ] ] }, { "sid": 178, "sent": "of 10 separate experiments. *P<0.05 using unpaired Student's t-test.", "section": "FIG", "ner": [ [ 28, 30, "*P", "evidence" ], [ 51, 67, "Student's t-test", "experimental_method" ] ] }, { "sid": 179, "sent": "Haem-dependent PGRMC1 dimerization enhances tumour chemoresistance through interaction with cytochromes P450.", "section": "FIG", "ner": [ [ 0, 4, "Haem", "chemical" ], [ 15, 21, "PGRMC1", "protein" ], [ 22, 34, "dimerization", "oligomeric_state" ], [ 92, 108, "cytochromes P450", "protein_type" ] ] }, { "sid": 180, "sent": "(a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44\u2013195), in either apo or haem-bound form, were incubated with CYP1A2 (a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads.", "section": "FIG", "ner": [ [ 11, 17, "PGRMC1", "protein" ], [ 18, 27, "wild-type", "protein_state" ], [ 29, 31, "wt", "protein_state" ], [ 37, 42, "Y113F", "mutant" ], [ 43, 49, "mutant", "protein_state" ], [ 64, 70, "44\u2013195", "residue_range" ], [ 83, 86, "apo", "protein_state" ], [ 90, 100, "haem-bound", "protein_state" ], [ 112, 121, "incubated", "experimental_method" ], [ 127, 133, "CYP1A2", "protein" ], [ 141, 147, "CYP3A4", "protein" ], [ 156, 174, "immunoprecipitated", "experimental_method" ] ] }, { "sid": 181, "sent": "(c) Binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt and CYP1A2 with or without RuCl3 and CORM3.", "section": "FIG", "ner": [ [ 4, 17, "Binding assay", "experimental_method" ], [ 48, 58, "haem-bound", "protein_state" ], [ 64, 70, "PGRMC1", "protein" ], [ 71, 73, "wt", "protein_state" ], [ 78, 84, "CYP1A2", "protein" ], [ 101, 106, "RuCl3", "chemical" ], [ 111, 116, "CORM3", "chemical" ] ] }, { "sid": 182, "sent": "(d) Schematic illustration of doxorubicin metabolism is shown on the left.", "section": "FIG", "ner": [ [ 30, 41, "doxorubicin", "chemical" ] ] }, { "sid": 183, "sent": "Doxorubicin was incubated with HCT116 cells expressing control shRNA or shPGRMC1 (PGRMC1-KD), and the doxorubicinol/doxorubicin ratios in cell pellets were determined using LC-MS.", "section": "FIG", "ner": [ [ 0, 11, "Doxorubicin", "chemical" ], [ 16, 25, "incubated", "experimental_method" ], [ 63, 68, "shRNA", "chemical" ], [ 72, 80, "shPGRMC1", "chemical" ], [ 82, 91, "PGRMC1-KD", "mutant" ], [ 102, 115, "doxorubicinol", "chemical" ], [ 116, 127, "doxorubicin", "chemical" ], [ 173, 178, "LC-MS", "experimental_method" ] ] }, { "sid": 184, "sent": "of four separate experiments. **P<0.01 versus control using unpaired Student's t-test. (e) Indicated amounts of doxorubicin were added to HCT116 (control) cells, PGRMC1-KD cells, or PGRMC1-KD cells expressing shRNA-resistant full-length PGRMC1 wt or Y113F, and cell viability was examined by MTT assay.", "section": "FIG", "ner": [ [ 31, 33, "*P", "evidence" ], [ 69, 85, "Student's t-test", "experimental_method" ], [ 112, 123, "doxorubicin", "chemical" ], [ 162, 171, "PGRMC1-KD", "mutant" ], [ 182, 191, "PGRMC1-KD", "mutant" ], [ 209, 224, "shRNA-resistant", "protein_state" ], [ 225, 236, "full-length", "protein_state" ], [ 237, 243, "PGRMC1", "protein" ], [ 244, 246, "wt", "protein_state" ], [ 250, 255, "Y113F", "mutant" ], [ 292, 301, "MTT assay", "experimental_method" ] ] }, { "sid": 185, "sent": "Schematic diagram for the regulation of PGRMC1 functions.", "section": "FIG", "ner": [ [ 40, 46, "PGRMC1", "protein" ] ] }, { "sid": 186, "sent": "Apo-PGRMC1 exists as an inactive monomer.", "section": "FIG", "ner": [ [ 0, 3, "Apo", "protein_state" ], [ 4, 10, "PGRMC1", "protein" ], [ 24, 32, "inactive", "protein_state" ], [ 33, 40, "monomer", "oligomeric_state" ] ] }, { "sid": 187, "sent": "On binding to haem, PGRMC1 forms a dimer through stacking interactions between the haem moieties, which enables PGRMC1 to interact with EGFR and cytochromes P450, leading to an enhanced proliferation and chemoresistance of cancer cells.", "section": "FIG", "ner": [ [ 3, 13, "binding to", "protein_state" ], [ 14, 18, "haem", "chemical" ], [ 20, 26, "PGRMC1", "protein" ], [ 35, 40, "dimer", "oligomeric_state" ], [ 49, 70, "stacking interactions", "bond_interaction" ], [ 83, 87, "haem", "chemical" ], [ 112, 118, "PGRMC1", "protein" ], [ 136, 140, "EGFR", "protein_type" ], [ 145, 161, "cytochromes P450", "protein_type" ] ] }, { "sid": 188, "sent": "CO interferes with the stacking interactions of the haems and thereby inhibits PGRMC1 functions.", "section": "FIG", "ner": [ [ 0, 2, "CO", "chemical" ], [ 23, 44, "stacking interactions", "bond_interaction" ], [ 52, 57, "haems", "chemical" ], [ 79, 85, "PGRMC1", "protein" ] ] }, { "sid": 189, "sent": "PGRMC1 proteins exhibit haem-dependent dimerization in solution.", "section": "TABLE", "ner": [ [ 39, 51, "dimerization", "oligomeric_state" ] ] }, { "sid": 190, "sent": "\u00a0\tApo form\tHaem-bound form\t \t\u00a0\t\u00a0\tMass (Da)\t\u00a0\tMass (Da)\t \taPGRMC1 wt (a.a.44\u2013195)\t \t\u2003ESI-MS\t\u2014\t17,844.14\t\u2014\t36,920.19\t \t\u2003Theoretical\t\u00a0\t17,843.65\t\u00a0\t36,918.06\t \t\u00a0\tHydrodynamic radius 10\u22129 (m)\tMW (kDa)\tHydrodynamic radius 10\u22129 (m)\tMW (kDa)\t \t\u2003DOSY\t2.04\u20132.15\t20\t2.94\u20133.02\t42\t \t\u00a0\tS20,w (S)\tMW (kDa)\tS20,w (S)\tMW (kDa)\t \t\u2003SV-AUC\t1.9\t17.6\t3.1\t35.5\t \t\u00a0\t\u00a0\t\u00a0\t\u00a0\t\u00a0\t \tbPGRMC1 C129S (a.a.44\u2013195)\t \t\u2003ESI-MS\t\u2014\t17,827.91\t\u2014\t36,887.07\t \t\u2003Theoretical\t\u00a0\t17,827.59\t\u00a0\t36,885.6\t \t\u00a0\tS20,w (S)\tMW (kDa)\tS20,w (S)\tMW (kDa)\t \t\u2003SV-AUC\t2.0\t18.1\t3.1\t35.8\t \t", "section": "TABLE", "ner": [ [ 360, 365, "C129S", "mutant" ] ] }, { "sid": 191, "sent": "Differences in molecular weights of the wild-type (wt; a) and the C129S mutant (b) PGRMC1 proteins in the absence (apo form) or the presence of haem (haem-bound form).", "section": "TABLE", "ner": [ [ 66, 71, "C129S", "mutant" ] ] }, { "sid": 192, "sent": "The protein sizes of the wt and C129S PGRMC1 cytosolic domains (a.a.44\u2013195) in the presence or absence of haem were estimated by ESI-MS, DOSY and SV-AUC.", "section": "TABLE", "ner": [ [ 32, 37, "C129S", "mutant" ] ] } ] }, "PMC4887163": { "annotations": [ { "sid": 0, "sent": "Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity", "section": "TITLE", "ner": [ [ 19, 34, "T cell receptor", "protein_type" ], [ 66, 73, "insulin", "chemical" ] ] }, { "sid": 1, "sent": "However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide\u2013major histocompatibility complexes (pMHC) are unclear.", "section": "ABSTRACT", "ner": [ [ 46, 69, "T cell antigen receptor", "complex_assembly" ], [ 71, 74, "TCR", "complex_assembly" ], [ 108, 150, "peptide\u2013major histocompatibility complexes", "complex_assembly" ], [ 152, 156, "pMHC", "complex_assembly" ] ] }, { "sid": 2, "sent": "Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I\u2013restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides.", "section": "ABSTRACT", "ner": [ [ 49, 54, "human", "species" ], [ 56, 69, "preproinsulin", "protein" ], [ 80, 83, "MHC", "complex_assembly" ] ] }, { "sid": 3, "sent": "We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide.", "section": "ABSTRACT", "ner": [ [ 29, 39, "structures", "evidence" ], [ 47, 54, "1E6 TCR", "complex_assembly" ], [ 55, 63, "bound to", "protein_state" ], [ 66, 89, "altered peptide ligands", "chemical" ] ] }, { "sid": 4, "sent": "Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key\u2013like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot.", "section": "ABSTRACT", "ner": [ [ 20, 30, "structures", "evidence" ], [ 149, 156, "1E6 TCR", "complex_assembly" ] ] }, { "sid": 5, "sent": "Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand.", "section": "ABSTRACT", "ner": [ [ 30, 37, "1E6 TCR", "complex_assembly" ], [ 60, 94, "antipathogen-like binding affinity", "evidence" ] ] }, { "sid": 6, "sent": "T cells perform an essential role in adaptive immunity by interrogating the host proteome for anomalies, classically by recognizing peptides bound in major histocompatibility (MHC) molecules at the cell surface.", "section": "INTRO", "ner": [ [ 150, 174, "major histocompatibility", "complex_assembly" ], [ 176, 179, "MHC", "complex_assembly" ] ] }, { "sid": 7, "sent": "Recent data supports the notion that, to perform this role, the highly variable \u03b1\u03b2 T cell antigen receptor (TCR) must be able to recognize thousands, if not millions, of different peptide ligands.", "section": "INTRO", "ner": [ [ 64, 79, "highly variable", "protein_state" ], [ 80, 106, "\u03b1\u03b2 T cell antigen receptor", "complex_assembly" ], [ 108, 111, "TCR", "complex_assembly" ] ] }, { "sid": 8, "sent": "This ability is required to enable the estimated 25 million distinct TCRs expressed in humans to provide effective immune coverage against all possible foreign peptide antigens.", "section": "INTRO", "ner": [ [ 69, 73, "TCRs", "complex_assembly" ], [ 87, 93, "humans", "species" ] ] }, { "sid": 9, "sent": "Several mechanisms, by which TCRs could bind to a large number of different peptide-MHC (pMHC), have been proposed.", "section": "INTRO", "ner": [ [ 29, 33, "TCRs", "complex_assembly" ], [ 76, 87, "peptide-MHC", "complex_assembly" ], [ 89, 93, "pMHC", "complex_assembly" ] ] }, { "sid": 10, "sent": "Structures of unligated and ligated TCRs have shown that the TCR complementarity determining region (CDR) loops can be flexible, perhaps enabling peptide binding using different loop conformations.", "section": "INTRO", "ner": [ [ 0, 10, "Structures", "evidence" ], [ 14, 23, "unligated", "protein_state" ], [ 28, 35, "ligated", "protein_state" ], [ 36, 40, "TCRs", "complex_assembly" ], [ 61, 64, "TCR", "complex_assembly" ], [ 65, 99, "complementarity determining region", "structure_element" ], [ 101, 104, "CDR", "structure_element" ], [ 106, 111, "loops", "structure_element" ], [ 178, 182, "loop", "structure_element" ] ] }, { "sid": 11, "sent": "Both MHC and peptide have also been shown to undergo structural changes upon TCR binding, mediating an induced fit between the TCR and pMHC.", "section": "INTRO", "ner": [ [ 5, 8, "MHC", "complex_assembly" ], [ 13, 20, "peptide", "chemical" ], [ 77, 80, "TCR", "complex_assembly" ], [ 127, 130, "TCR", "complex_assembly" ], [ 135, 139, "pMHC", "complex_assembly" ] ] }, { "sid": 12, "sent": "Other studies, mainly in the murine system, have demonstrated that the same TCR can interact with different pMHCs using a common or divergent modality.", "section": "INTRO", "ner": [ [ 29, 35, "murine", "taxonomy_domain" ], [ 76, 79, "TCR", "complex_assembly" ], [ 108, 113, "pMHCs", "complex_assembly" ] ] }, { "sid": 13, "sent": "Recent studies in model murine systems demonstrate that TCR cross-reactivity can be governed by recognition of a conserved region in the peptide that allows tolerance of peptide sequence variation outside of this hotspot.", "section": "INTRO", "ner": [ [ 24, 30, "murine", "taxonomy_domain" ], [ 56, 59, "TCR", "complex_assembly" ] ] }, { "sid": 14, "sent": "We recently reported that the 1E6 human CD8+ T cell clone \u2014 which mediates the destruction of \u03b2 cells through the recognition of a major, HLA-A*0201\u2013restricted, preproinsulin signal peptide (ALWGPDPAAA15\u201324) \u2014 can recognize upwards of 1 million different peptides.", "section": "INTRO", "ner": [ [ 34, 39, "human", "species" ], [ 138, 148, "HLA-A*0201", "protein" ], [ 161, 174, "preproinsulin", "protein" ], [ 175, 189, "signal peptide", "structure_element" ], [ 191, 206, "ALWGPDPAAA15\u201324", "chemical" ] ] }, { "sid": 15, "sent": "CD8+ T cells that recognize HLA-A*0201\u2013ALWGPDPAAA have been shown to populate insulitic lesions in patients with type 1 diabetes (T1D).", "section": "INTRO", "ner": [ [ 28, 49, "HLA-A*0201\u2013ALWGPDPAAA", "complex_assembly" ] ] }, { "sid": 16, "sent": "We demonstrated that the TCR from the 1E6 T cell clone bound to HLA-A*0201\u2013ALWGPDPAAA using a limited footprint and very weak binding affinity.", "section": "INTRO", "ner": [ [ 25, 28, "TCR", "complex_assembly" ], [ 55, 63, "bound to", "protein_state" ], [ 64, 85, "HLA-A*0201\u2013ALWGPDPAAA", "complex_assembly" ], [ 126, 142, "binding affinity", "evidence" ] ] }, { "sid": 17, "sent": "This first experimental evidence of a high level of CD8+ T cell cross-reactivity in a human autoimmune disease system hinted toward molecular mimicry by a more potent pathogenic peptide as a potential mechanism leading to \u03b2 cell destruction.", "section": "INTRO", "ner": [ [ 86, 91, "human", "species" ] ] }, { "sid": 18, "sent": "Here, we solved the structure of the 1E6 TCR with 7 altered peptide ligands (APLs) determined by our previously published combinatorial peptide library (CPL) screening, 2 of which mapped within human pathogens.", "section": "INTRO", "ner": [ [ 9, 15, "solved", "experimental_method" ], [ 20, 29, "structure", "evidence" ], [ 37, 44, "1E6 TCR", "complex_assembly" ], [ 52, 75, "altered peptide ligands", "chemical" ], [ 77, 81, "APLs", "chemical" ], [ 122, 167, "combinatorial peptide library (CPL) screening", "experimental_method" ], [ 194, 199, "human", "species" ] ] }, { "sid": 19, "sent": "These APLs differed from the natural preproinsulin peptide by up to 7 of 10 residues.", "section": "INTRO", "ner": [ [ 6, 10, "APLs", "chemical" ], [ 37, 50, "preproinsulin", "protein" ] ] }, { "sid": 20, "sent": "We also solved the structure of each unligated APL to investigate whether structural changes occurred before or after binding \u2014 which, combined with an in-depth cellular and biophysical analysis of the 1E6 interaction with each APL, demonstrated the molecular mechanism mediating the high level of cross-reactivity exhibited by this preproinsulin-reactive human CD8+ T cell clone.", "section": "INTRO", "ner": [ [ 8, 14, "solved", "experimental_method" ], [ 19, 28, "structure", "evidence" ], [ 37, 46, "unligated", "protein_state" ], [ 47, 50, "APL", "chemical" ], [ 161, 194, "cellular and biophysical analysis", "experimental_method" ], [ 228, 231, "APL", "chemical" ], [ 333, 346, "preproinsulin", "protein" ], [ 356, 361, "human", "species" ] ] }, { "sid": 21, "sent": "The 1E6 T cell clone recognizes APLs across a large dynamic range.", "section": "RESULTS", "ner": [ [ 32, 36, "APLs", "chemical" ] ] }, { "sid": 22, "sent": "We have previously demonstrated that the 1E6 T cell clone can recognize over 1 million different peptides with a potency comparable with, or better than, the cognate preproinsulin peptide ALWGPDPAAA.", "section": "RESULTS", "ner": [ [ 166, 179, "preproinsulin", "protein" ], [ 188, 198, "ALWGPDPAAA", "chemical" ] ] }, { "sid": 23, "sent": "From this large functional scan, we selected 7 different APLs that activated the 1E6 T cell clone across a wide (4-log) functional range (Table 1).", "section": "RESULTS", "ner": [ [ 57, 61, "APLs", "chemical" ] ] }, { "sid": 24, "sent": "Two of these peptides, MVWGPDPLYV and RQFGPDWIVA (bold text signifies amino acids that are different from the index preproinsulin\u2013derived sequence), are contained within the proteomes of the human pathogens Bacteroides fragilis/thetaiotaomicron and Clostridium asparagiforme, respectively.", "section": "RESULTS", "ner": [ [ 23, 33, "MVWGPDPLYV", "chemical" ], [ 38, 48, "RQFGPDWIVA", "chemical" ], [ 116, 129, "preproinsulin", "protein" ], [ 191, 196, "human", "species" ], [ 207, 244, "Bacteroides fragilis/thetaiotaomicron", "species" ], [ 249, 274, "Clostridium asparagiforme", "species" ] ] }, { "sid": 25, "sent": "Competitive functional testing revealed that the preproinsulin-derived sequence ALWGPDPAAA was one of the least potent targets for 1E6, with only the MVWGPDPLYV and YLGGPDFPTI demonstrating a similar low-activity profile in MIP-1\u03b2 secretion and target killing assays (Figure 1, A and B).", "section": "RESULTS", "ner": [ [ 0, 30, "Competitive functional testing", "experimental_method" ], [ 49, 62, "preproinsulin", "protein" ], [ 80, 90, "ALWGPDPAAA", "chemical" ], [ 150, 160, "MVWGPDPLYV", "chemical" ], [ 165, 175, "YLGGPDFPTI", "chemical" ], [ 224, 230, "MIP-1\u03b2", "protein" ] ] }, { "sid": 26, "sent": "The RQFGPDWIVA sequence (present in C. asparagiforme) activated the 1E6 T cell with around 1 log\u2013greater potency compared with ALWGPDPAAA.", "section": "RESULTS", "ner": [ [ 4, 14, "RQFGPDWIVA", "chemical" ], [ 36, 52, "C. asparagiforme", "species" ], [ 127, 137, "ALWGPDPAAA", "chemical" ] ] }, { "sid": 27, "sent": "At the other end of the spectrum, the RQFGPDFPTI peptide stimulated MIP-1\u03b2 release and killing by 1E6 at an exogenous peptide concentration 2\u20133 logs lower compared with ALWGPDPAAA.", "section": "RESULTS", "ner": [ [ 38, 48, "RQFGPDFPTI", "chemical" ], [ 68, 74, "MIP-1\u03b2", "protein" ], [ 169, 179, "ALWGPDPAAA", "chemical" ] ] }, { "sid": 28, "sent": "The pattern of peptide potency was closely mirrored by pMHC tetramer staining experiments (Figure 1C and plots shown in Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI85679DS1).", "section": "RESULTS", "ner": [ [ 55, 59, "pMHC", "complex_assembly" ], [ 60, 77, "tetramer staining", "experimental_method" ] ] }, { "sid": 29, "sent": "Here, the A2-RQFGPDFPTI tetramer stained 1E6 with the greatest MFI, gradually decreasing to the weakest tetramers: A2-MVWGPDPLYV and -YLGGPDFPTI.", "section": "RESULTS", "ner": [ [ 10, 23, "A2-RQFGPDFPTI", "chemical" ], [ 24, 32, "tetramer", "oligomeric_state" ], [ 104, 113, "tetramers", "oligomeric_state" ], [ 115, 128, "A2-MVWGPDPLYV", "chemical" ], [ 134, 144, "YLGGPDFPTI", "chemical" ] ] }, { "sid": 30, "sent": "To parallel the functional analysis, we also performed thermal melt (Tm) experiments using synchrotron radiation circular dichroism (SRCD) to investigate the stability of each APL (Figure 1D).", "section": "RESULTS", "ner": [ [ 55, 67, "thermal melt", "experimental_method" ], [ 69, 71, "Tm", "evidence" ], [ 91, 131, "synchrotron radiation circular dichroism", "experimental_method" ], [ 133, 137, "SRCD", "experimental_method" ], [ 176, 179, "APL", "chemical" ] ] }, { "sid": 31, "sent": "The range of Tm was between 49.4\u00b0C (RQFGPDWIVA) and 60.3\u00b0C (YQFGPDFPIA), with an average approximately 55\u00b0C, similar to our previous findings.", "section": "RESULTS", "ner": [ [ 13, 15, "Tm", "evidence" ], [ 36, 46, "RQFGPDWIVA", "chemical" ], [ 60, 70, "YQFGPDFPIA", "chemical" ] ] }, { "sid": 32, "sent": "This pattern of stability did not correlate with the T cell activation or tetramer staining experiments, indicating that peptide binding to the MHC do not explain ligand potency.", "section": "RESULTS", "ner": [ [ 74, 91, "tetramer staining", "experimental_method" ], [ 144, 147, "MHC", "complex_assembly" ] ] }, { "sid": 33, "sent": "The 1E6 TCR can bind peptides with strong antipathogen-like affinities.", "section": "RESULTS", "ner": [ [ 4, 11, "1E6 TCR", "complex_assembly" ] ] }, { "sid": 34, "sent": "We, and others, have previously demonstrated that antipathogenic TCRs tend to bind with stronger affinity compared with self-reactive TCRs, likely a consequence of the deletion of T cells with high-affinity self-reactive TCR during thymic selection.", "section": "RESULTS", "ner": [ [ 65, 69, "TCRs", "complex_assembly" ], [ 97, 105, "affinity", "evidence" ], [ 134, 138, "TCRs", "complex_assembly" ], [ 198, 206, "affinity", "evidence" ], [ 221, 224, "TCR", "complex_assembly" ] ] }, { "sid": 35, "sent": "In accordance with this trend, the 1E6 TCR bound the natural preproinsulin peptide, ALWGPDPAAA, with the weakest affinity currently published for a human CD8+ T cell\u2013derived TCR with a biologically relevant ligand (KD > 200 \u03bcM; KD, equilibrium binding constant).", "section": "RESULTS", "ner": [ [ 35, 42, "1E6 TCR", "complex_assembly" ], [ 43, 48, "bound", "protein_state" ], [ 61, 74, "preproinsulin", "protein" ], [ 84, 94, "ALWGPDPAAA", "chemical" ], [ 113, 121, "affinity", "evidence" ], [ 148, 153, "human", "species" ], [ 174, 177, "TCR", "complex_assembly" ], [ 215, 217, "KD", "evidence" ], [ 228, 230, "KD", "evidence" ], [ 232, 260, "equilibrium binding constant", "evidence" ] ] }, { "sid": 36, "sent": "Surface plasmon resonance (SPR) analysis of the 1E6 TCR\u2013pMHC interaction for all 7 APLs (Figure 2, A\u2013H) demonstrated that stronger binding affinity (represented as \u0394G\u00b0, kcal/mol) correlated well with the EC50 values (peptide concentration required to reach half-maximal 1E6 T cell killing) for each ligand, demonstrated by a Pearson\u2019s correlation analysis value of 0.8 (P = 0.01) (Figure 2I).", "section": "RESULTS", "ner": [ [ 0, 25, "Surface plasmon resonance", "experimental_method" ], [ 27, 30, "SPR", "experimental_method" ], [ 48, 55, "1E6 TCR", "complex_assembly" ], [ 56, 60, "pMHC", "complex_assembly" ], [ 83, 87, "APLs", "chemical" ], [ 131, 147, "binding affinity", "evidence" ], [ 164, 167, "\u0394G\u00b0", "evidence" ], [ 204, 208, "EC50", "evidence" ], [ 325, 355, "Pearson\u2019s correlation analysis", "experimental_method" ] ] }, { "sid": 37, "sent": "It should be noted that this correlation, although consistent with the T cell killing experiments, uses only approximate affinities calculated for the 2 weakest ligands.", "section": "RESULTS", "ner": [ [ 121, 131, "affinities", "evidence" ] ] }, { "sid": 38, "sent": "First, the 1E6 T cell could still functionally respond to peptide when the TCR binding affinity was extremely weak, e.g., the 1E6 TCR binding affinity for the A2-MVWGPDPLYV peptide was KD = ~600 \u03bcM. Second, the 1E6 TCR bound to A2-RQFGPDFPTI with KD = 0.5 \u03bcM, equivalent to the binding affinity of the very strongest antipathogen TCRs.", "section": "RESULTS", "ner": [ [ 75, 95, "TCR binding affinity", "evidence" ], [ 126, 150, "1E6 TCR binding affinity", "evidence" ], [ 159, 172, "A2-MVWGPDPLYV", "chemical" ], [ 185, 187, "KD", "evidence" ], [ 211, 218, "1E6 TCR", "complex_assembly" ], [ 219, 227, "bound to", "protein_state" ], [ 228, 241, "A2-RQFGPDFPTI", "chemical" ], [ 247, 249, "KD", "evidence" ], [ 278, 294, "binding affinity", "evidence" ], [ 330, 334, "TCRs", "complex_assembly" ] ] }, { "sid": 39, "sent": "Third, the 1E6 TCR bound to A2-RQFGPDWIVA peptide, within the C. asparagiforme proteome, with approximately 4-fold stronger affinity than A2-ALWGPDPAAA, demonstrating the potential for a pathogen-derived antigen to initiate a response to the self-derived sequence.", "section": "RESULTS", "ner": [ [ 11, 18, "1E6 TCR", "complex_assembly" ], [ 19, 27, "bound to", "protein_state" ], [ 28, 41, "A2-RQFGPDWIVA", "chemical" ], [ 62, 78, "C. asparagiforme", "species" ], [ 124, 132, "affinity", "evidence" ], [ 138, 151, "A2-ALWGPDPAAA", "chemical" ] ] }, { "sid": 40, "sent": "Finally, these data demonstrate the largest range of binding affinities reported for a natural, endogenous human TCR of more than 3 logs of magnitude (A2-MVWGPDPLYV vs. A2-RQFGPDFPTI).", "section": "RESULTS", "ner": [ [ 53, 71, "binding affinities", "evidence" ], [ 96, 106, "endogenous", "protein_state" ], [ 107, 112, "human", "species" ], [ 113, 116, "TCR", "complex_assembly" ], [ 151, 164, "A2-MVWGPDPLYV", "chemical" ], [ 169, 182, "A2-RQFGPDFPTI", "chemical" ] ] }, { "sid": 41, "sent": "To confirm the affinity spread detected by SPR, and to evaluate whether experiments performed using soluble molecules were biologically relevant to events at the T cell surface, we determined the effective 2D affinity of each APL using an adhesion frequency assay in which a human rbc coated in pMHC acted as an adhesion sensor.", "section": "RESULTS", "ner": [ [ 15, 23, "affinity", "evidence" ], [ 43, 46, "SPR", "experimental_method" ], [ 206, 217, "2D affinity", "evidence" ], [ 226, 229, "APL", "chemical" ], [ 239, 263, "adhesion frequency assay", "experimental_method" ], [ 275, 280, "human", "species" ], [ 295, 299, "pMHC", "complex_assembly" ] ] }, { "sid": 42, "sent": "In agreement with SPR experiments, the range of 2D affinities we detected differed by around 3 logs, with the A2-MVWGPDPLYV generating the weakest 2D affinity (2.6 \u00d7 10\u20135 AcKa \u03bcm4) and A2-RQFGPDFPTI the strongest (4.5 \u00d7 10\u20132 AcKa \u03bcm4) (Figure 2J).", "section": "RESULTS", "ner": [ [ 18, 21, "SPR", "experimental_method" ], [ 48, 61, "2D affinities", "evidence" ], [ 110, 123, "A2-MVWGPDPLYV", "chemical" ], [ 147, 158, "2D affinity", "evidence" ], [ 171, 175, "AcKa", "evidence" ], [ 185, 198, "A2-RQFGPDFPTI", "chemical" ], [ 225, 229, "AcKa", "evidence" ] ] }, { "sid": 43, "sent": "As with the 3D affinity measurements, the 2D affinity measurements correlated well with the EC50 values for each ligand (Figure 2K) demonstrating a strong correlation (Pearson\u2019s correlation = 0.8, P = 0.01) between T cell antigen sensitivity and TCR binding affinity.", "section": "RESULTS", "ner": [ [ 12, 23, "3D affinity", "evidence" ], [ 42, 53, "2D affinity", "evidence" ], [ 92, 96, "EC50", "evidence" ], [ 168, 189, "Pearson\u2019s correlation", "evidence" ], [ 197, 198, "P", "evidence" ], [ 246, 266, "TCR binding affinity", "evidence" ] ] }, { "sid": 44, "sent": "Of note, these data demonstrate a close agreement between the 3D affinity values generated using SPR and 2D affinity values generated using adhesion frequency assays.", "section": "RESULTS", "ner": [ [ 62, 73, "3D affinity", "evidence" ], [ 97, 100, "SPR", "experimental_method" ], [ 105, 116, "2D affinity", "evidence" ] ] }, { "sid": 45, "sent": "The 1E6 TCR uses a consensus binding mode to engage multiple APLs.", "section": "RESULTS", "ner": [ [ 4, 11, "1E6 TCR", "complex_assembly" ], [ 61, 65, "APLs", "chemical" ] ] }, { "sid": 46, "sent": "Our previous structure of the 1E6-A2-ALWGPDPAAA complex demonstrated a limited binding footprint between the TCR and pMHC.", "section": "RESULTS", "ner": [ [ 13, 22, "structure", "evidence" ], [ 30, 47, "1E6-A2-ALWGPDPAAA", "complex_assembly" ], [ 79, 96, "binding footprint", "site" ], [ 109, 112, "TCR", "complex_assembly" ], [ 117, 121, "pMHC", "complex_assembly" ] ] }, { "sid": 47, "sent": "The low number of contacts between the 2 molecules most likely contributed to the weak binding affinity of the interaction.", "section": "RESULTS", "ner": [ [ 87, 103, "binding affinity", "evidence" ] ] }, { "sid": 48, "sent": "In order to examine the mechanism by which the 1E6 TCR engaged a wide range of peptides with divergent binding affinities, we solved the structure of the 1E6 TCR in complex with all 7 APLs used in Figure 2.", "section": "RESULTS", "ner": [ [ 47, 54, "1E6 TCR", "complex_assembly" ], [ 103, 121, "binding affinities", "evidence" ], [ 126, 132, "solved", "experimental_method" ], [ 137, 146, "structure", "evidence" ], [ 154, 161, "1E6 TCR", "complex_assembly" ], [ 162, 177, "in complex with", "protein_state" ], [ 184, 188, "APLs", "chemical" ] ] }, { "sid": 49, "sent": "All structures were solved in space group P1 to 2\u20133 \u00c5 resolution with crystallographic Rwork/Rfree ratios within accepted limits as shown in the theoretically expected distribution (ref. and Supplemental Table 1).", "section": "RESULTS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 20, 26, "solved", "experimental_method" ], [ 87, 105, "Rwork/Rfree ratios", "evidence" ] ] }, { "sid": 50, "sent": "The 1E6 TCR used a very similar overall binding modality to engage all of the APLs, with root mean square deviation ranging between 0.81 and 1.12 \u00c52 (compared with 1E6-A2-ALWGPDPAAA).", "section": "RESULTS", "ner": [ [ 4, 11, "1E6 TCR", "complex_assembly" ], [ 78, 82, "APLs", "chemical" ], [ 89, 115, "root mean square deviation", "evidence" ], [ 164, 181, "1E6-A2-ALWGPDPAAA", "complex_assembly" ] ] }, { "sid": 51, "sent": "The relatively broad range of buried surface areas (1,670\u20131,920 \u00c52) did not correlate well with TCR binding affinity (Pearson\u2019s correlation = 0.45, P = 0.2).", "section": "RESULTS", "ner": [ [ 96, 116, "TCR binding affinity", "evidence" ], [ 118, 139, "Pearson\u2019s correlation", "evidence" ], [ 148, 149, "P", "evidence" ] ] }, { "sid": 52, "sent": "The surface complementarity values (0.52\u20130.7) correlated slightly with affinity (Pearson\u2019s correlation = 0.7, P = 0.05) but could not explain all differences in binding (Figure 3A and Table 2).", "section": "RESULTS", "ner": [ [ 4, 34, "surface complementarity values", "evidence" ], [ 71, 79, "affinity", "evidence" ], [ 81, 102, "Pearson\u2019s correlation", "evidence" ], [ 110, 111, "P", "evidence" ] ] }, { "sid": 53, "sent": "The TCR CDR loops were in a very similar position in all complexes, apart from some slight deviations in the TCR \u03b2-chain (Figure 3B); the peptides were all presented in a similar conformation (Figure 3C); and there was minimal variation in crossing angles of the TCR (42.3\u00b0\u201345.6\u00b0) (Figure 3D).", "section": "RESULTS", "ner": [ [ 4, 7, "TCR", "complex_assembly" ], [ 8, 17, "CDR loops", "structure_element" ], [ 109, 112, "TCR", "complex_assembly" ], [ 113, 120, "\u03b2-chain", "structure_element" ], [ 263, 266, "TCR", "complex_assembly" ] ] }, { "sid": 54, "sent": "Overall, the 1E6 TCR used a canonical binding mode to engage each APL with the TCR \u03b1-chain positioned over the MHC class I (MHCI) \u03b12-helix and the TCR \u03b2-chain over the MHCI \u03b1-1 helix, straddling the peptide cargo.", "section": "RESULTS", "ner": [ [ 13, 20, "1E6 TCR", "complex_assembly" ], [ 66, 69, "APL", "chemical" ], [ 79, 82, "TCR", "complex_assembly" ], [ 83, 90, "\u03b1-chain", "structure_element" ], [ 111, 122, "MHC class I", "complex_assembly" ], [ 124, 128, "MHCI", "complex_assembly" ], [ 130, 138, "\u03b12-helix", "structure_element" ], [ 147, 150, "TCR", "complex_assembly" ], [ 151, 158, "\u03b2-chain", "structure_element" ], [ 168, 172, "MHCI", "complex_assembly" ], [ 173, 182, "\u03b1-1 helix", "structure_element" ] ] }, { "sid": 55, "sent": "However, subtle differences in the respective interfaces were apparent (discussed below) and resulted in altered binding affinities of the respective complexes.", "section": "RESULTS", "ner": [ [ 46, 56, "interfaces", "site" ], [ 113, 131, "binding affinities", "evidence" ] ] }, { "sid": 56, "sent": "Interactions between the 1E6 TCR and different APLs are focused around a conserved GPD peptide motif.", "section": "RESULTS", "ner": [ [ 25, 32, "1E6 TCR", "complex_assembly" ], [ 47, 51, "APLs", "chemical" ], [ 73, 82, "conserved", "protein_state" ], [ 83, 100, "GPD peptide motif", "structure_element" ] ] }, { "sid": 57, "sent": "We next performed an in-depth atomic analysis of the contacts between the 1E6 TCR and each APL to determine the structural basis for the altered T cell peptide sensitivities and TCR binding affinities (Table 2).", "section": "RESULTS", "ner": [ [ 30, 45, "atomic analysis", "experimental_method" ], [ 74, 81, "1E6 TCR", "complex_assembly" ], [ 91, 94, "APL", "chemical" ], [ 178, 200, "TCR binding affinities", "evidence" ] ] }, { "sid": 58, "sent": "Concomitant with our global analysis of 1E6 TCR binding to the APLs, we observed a common interaction element, consistent with our previous findings, that utilized TCR residues Tyr97\u03b1 and Trp97\u03b2, forming an aromatic cap over a central GPD motif that was present in all of the APLs (Figure 4).", "section": "RESULTS", "ner": [ [ 40, 47, "1E6 TCR", "complex_assembly" ], [ 63, 67, "APLs", "chemical" ], [ 164, 167, "TCR", "complex_assembly" ], [ 177, 183, "Tyr97\u03b1", "residue_name_number" ], [ 188, 194, "Trp97\u03b2", "residue_name_number" ], [ 207, 219, "aromatic cap", "structure_element" ], [ 235, 244, "GPD motif", "structure_element" ], [ 276, 280, "APLs", "chemical" ] ] }, { "sid": 59, "sent": "Interactions between these 2 TCR and 3 peptide residues accounted for 41%\u201350% of the total contacts across all complexes (Table 2), demonstrating the conserved peptide centric binding mode utilized by the 1E6 TCR.", "section": "RESULTS", "ner": [ [ 29, 32, "TCR", "complex_assembly" ], [ 150, 159, "conserved", "protein_state" ], [ 205, 212, "1E6 TCR", "complex_assembly" ] ] }, { "sid": 60, "sent": "This fixed anchoring between the 2 molecules was important for stabilization of the TCR-pMHC complex, as \u2014 although other peptides without the \u2018GDP\u2019 motif were tested and shown to activate the 1E6 T cell clone \u2014 we were unable to measure robust affinities using SPR (data not shown).", "section": "RESULTS", "ner": [ [ 84, 92, "TCR-pMHC", "complex_assembly" ], [ 143, 154, "\u2018GDP\u2019 motif", "structure_element" ], [ 245, 255, "affinities", "evidence" ], [ 262, 265, "SPR", "experimental_method" ] ] }, { "sid": 61, "sent": "These data support the requirement for a conserved interaction between the 1E6 TCR and the GPD motif, as we observed in our previously published 1E6-A2-ALWGPDPAAA structure.", "section": "RESULTS", "ner": [ [ 41, 50, "conserved", "protein_state" ], [ 75, 82, "1E6 TCR", "complex_assembly" ], [ 91, 100, "GPD motif", "structure_element" ], [ 145, 162, "1E6-A2-ALWGPDPAAA", "complex_assembly" ], [ 163, 172, "structure", "evidence" ] ] }, { "sid": 62, "sent": "Focused hotspot binding around a conserved GPD motif enables the 1E6 TCR to tolerate peptide degeneracy.", "section": "RESULTS", "ner": [ [ 33, 42, "conserved", "protein_state" ], [ 43, 52, "GPD motif", "structure_element" ], [ 65, 72, "1E6 TCR", "complex_assembly" ] ] }, { "sid": 63, "sent": "Although the 1E6 TCR formed a similar overall interaction with each APL, the stabilization between the TCR and the GPD motif enabled fine differences in the contact network with both the peptide and MHC surface that allowed discrimination between each ligand (Figure 5).", "section": "RESULTS", "ner": [ [ 13, 20, "1E6 TCR", "complex_assembly" ], [ 68, 71, "APL", "chemical" ], [ 103, 106, "TCR", "complex_assembly" ], [ 115, 124, "GPD motif", "structure_element" ], [ 157, 172, "contact network", "site" ], [ 187, 194, "peptide", "chemical" ], [ 199, 210, "MHC surface", "site" ] ] }, { "sid": 64, "sent": "For example, the 1E6 TCR made only 47 peptide contacts with A2-MVWGPDPLYV (KD = ~600 \u03bcM) compared with 63 and 57 contacts with A2-YQFGPDFPIA (KD = 7.4 \u03bcM) and A2-RQFGPDFPTI (KD = 0.5 \u03bcM), respectively.", "section": "RESULTS", "ner": [ [ 17, 24, "1E6 TCR", "complex_assembly" ], [ 60, 73, "A2-MVWGPDPLYV", "chemical" ], [ 75, 77, "KD", "evidence" ], [ 127, 140, "A2-YQFGPDFPIA", "chemical" ], [ 142, 144, "KD", "evidence" ], [ 159, 172, "A2-RQFGPDFPTI", "chemical" ], [ 174, 176, "KD", "evidence" ] ] }, { "sid": 65, "sent": "Although the number of peptide contacts was a good predictor of TCR binding affinity for some of the APLs, for others, the correlation was poor (Pearson\u2019s correlation = 0.045, P = 0.92), possibly because of different resolutions for each complex structure.", "section": "RESULTS", "ner": [ [ 64, 84, "TCR binding affinity", "evidence" ], [ 101, 105, "APLs", "chemical" ], [ 145, 166, "Pearson\u2019s correlation", "evidence" ], [ 246, 255, "structure", "evidence" ] ] }, { "sid": 66, "sent": "For example, the 1E6 TCR made 64 peptide contacts with A2-YLGGPDFPTI (KD = ~400 \u03bcM) compared with 43 contacts with A2-RQWGPDPAAV (KD = 7.8 \u03bcM).", "section": "RESULTS", "ner": [ [ 17, 24, "1E6 TCR", "complex_assembly" ], [ 55, 68, "A2-YLGGPDFPTI", "chemical" ], [ 70, 72, "KD", "evidence" ], [ 115, 128, "A2-RQWGPDPAAV", "chemical" ], [ 130, 132, "KD", "evidence" ] ] }, { "sid": 67, "sent": "The most important peptide modification in terms of generating new contacts was peptide position 1.", "section": "RESULTS", "ner": [ [ 97, 98, "1", "residue_number" ] ] }, { "sid": 68, "sent": "The stronger ligands all encoded larger side chains (Arg or Tyr) at peptide position 1 (Figure 5, E\u2013H), enabling interactions with 1E6 that were not present in the weaker APLs that lacked large side chains in this position (Figure 5, A, C, and D).", "section": "RESULTS", "ner": [ [ 53, 56, "Arg", "residue_name" ], [ 60, 63, "Tyr", "residue_name" ], [ 85, 86, "1", "residue_number" ], [ 171, 175, "APLs", "chemical" ] ] }, { "sid": 69, "sent": "We have previously shown that the 1E6 TCR uses a rigid lock-and-key mechanism during binding to A2-ALWGPDPAAA.", "section": "RESULTS", "ner": [ [ 34, 41, "1E6 TCR", "complex_assembly" ], [ 96, 109, "A2-ALWGPDPAAA", "chemical" ] ] }, { "sid": 70, "sent": "These data demonstrated that the unligated structure of the 1E6 TCR was virtually identical to its ligated counterparts.", "section": "RESULTS", "ner": [ [ 33, 42, "unligated", "protein_state" ], [ 43, 52, "structure", "evidence" ], [ 60, 67, "1E6 TCR", "complex_assembly" ], [ 99, 106, "ligated", "protein_state" ] ] }, { "sid": 71, "sent": "In order to determine whether any of the APLs required an induced fit mechanism during binding that could explain the difference in free binding energy (\u0394G) between each complex (Table 2), we solved the unligated structures of all 7 APLs (the A2-ALWGPDPAAA structure has been previously published and was used in this comparison, ref.) (Figure 6 and Supplemental Table 2).", "section": "RESULTS", "ner": [ [ 41, 45, "APLs", "chemical" ], [ 132, 151, "free binding energy", "evidence" ], [ 153, 155, "\u0394G", "evidence" ], [ 192, 198, "solved", "experimental_method" ], [ 203, 212, "unligated", "protein_state" ], [ 213, 223, "structures", "evidence" ], [ 233, 237, "APLs", "chemical" ], [ 243, 256, "A2-ALWGPDPAAA", "chemical" ], [ 257, 266, "structure", "evidence" ] ] }, { "sid": 72, "sent": "The unligated A2-MVWGPDPLYV (KD = ~600 \u03bcM) structure revealed that the side chain Tyr9 swung around 8 \u00c5 in the complex structure, subsequently making contacts with TCR residues Asp30\u03b2 and Asn51\u03b2 (Figure 6A and Figure 5A, respectively).", "section": "RESULTS", "ner": [ [ 4, 13, "unligated", "protein_state" ], [ 14, 27, "A2-MVWGPDPLYV", "chemical" ], [ 29, 31, "KD", "evidence" ], [ 43, 52, "structure", "evidence" ], [ 82, 86, "Tyr9", "residue_name_number" ], [ 119, 128, "structure", "evidence" ], [ 164, 167, "TCR", "complex_assembly" ], [ 177, 183, "Asp30\u03b2", "residue_name_number" ], [ 188, 194, "Asn51\u03b2", "residue_name_number" ] ] }, { "sid": 73, "sent": "This movement could result in an entropic penalty contributing to the weak TCR binding affinity we observed for this ligand.", "section": "RESULTS", "ner": [ [ 75, 95, "TCR binding affinity", "evidence" ] ] }, { "sid": 74, "sent": "Additional small movements in the C\u03b1 backbone of the peptide around peptide residue Asp6 were apparent in the A2-YLGGPDFPTI (KD = ~400 \u03bcM), A2-ALWGPDPAAA (KD = ~208 \u03bcM), and A2-RQFGPDWIVA (KD = 44.4 \u03bcM) structures (Figure 6, B, C, and E).", "section": "RESULTS", "ner": [ [ 84, 88, "Asp6", "residue_name_number" ], [ 110, 123, "A2-YLGGPDFPTI", "chemical" ], [ 125, 127, "KD", "evidence" ], [ 140, 153, "A2-ALWGPDPAAA", "chemical" ], [ 155, 157, "KD", "evidence" ], [ 174, 187, "A2-RQFGPDWIVA", "chemical" ], [ 189, 191, "KD", "evidence" ], [ 203, 213, "structures", "evidence" ] ] }, { "sid": 75, "sent": "The unligated structures of A2-AQWGPDAAA, A2-RQWGPDPAAV, A2-YQFGPDFPIA, and A2-RQFGPDFPTI were virtually identical when in complex with 1E6 (Figure 6, D and F\u2013H).", "section": "RESULTS", "ner": [ [ 4, 13, "unligated", "protein_state" ], [ 14, 24, "structures", "evidence" ], [ 28, 40, "A2-AQWGPDAAA", "chemical" ], [ 42, 55, "A2-RQWGPDPAAV", "chemical" ], [ 57, 70, "A2-YQFGPDFPIA", "chemical" ], [ 76, 89, "A2-RQFGPDFPTI", "chemical" ], [ 120, 135, "in complex with", "protein_state" ] ] }, { "sid": 76, "sent": "Apart from the case of A2-AQWGPDAAA (KD = 61.9 \u03bcM), these observations support the conclusion that the higher-affinity ligands required less conformational melding during binding, which could be energetically beneficial (lower entopic cost) during ligation with the 1E6 TCR.", "section": "RESULTS", "ner": [ [ 23, 35, "A2-AQWGPDAAA", "chemical" ], [ 37, 39, "KD", "evidence" ], [ 110, 118, "affinity", "evidence" ], [ 266, 273, "1E6 TCR", "complex_assembly" ] ] }, { "sid": 77, "sent": "Peptide modifications alter the interaction between the 1E6 TCR and the MHC surface.", "section": "RESULTS", "ner": [ [ 56, 63, "1E6 TCR", "complex_assembly" ], [ 72, 83, "MHC surface", "site" ] ] }, { "sid": 78, "sent": "In addition to changes between the TCR and peptide component, we also observed that different APLs had different knock-on effects between the TCR and MHC.", "section": "RESULTS", "ner": [ [ 35, 38, "TCR", "complex_assembly" ], [ 94, 98, "APLs", "chemical" ], [ 142, 145, "TCR", "complex_assembly" ], [ 150, 153, "MHC", "complex_assembly" ] ] }, { "sid": 79, "sent": "MHC residue Arg65 that forms part of the MHC restriction triad (Arg65, Ala69, and Gln155) played a central role in TCR-MHC contacts, with Gln155 playing a less important role and Ala69 playing no role in binding at the interface (Figure 7).", "section": "RESULTS", "ner": [ [ 0, 3, "MHC", "complex_assembly" ], [ 12, 17, "Arg65", "residue_name_number" ], [ 41, 62, "MHC restriction triad", "site" ], [ 64, 69, "Arg65", "residue_name_number" ], [ 71, 76, "Ala69", "residue_name_number" ], [ 82, 88, "Gln155", "residue_name_number" ], [ 115, 118, "TCR", "complex_assembly" ], [ 119, 122, "MHC", "complex_assembly" ], [ 138, 144, "Gln155", "residue_name_number" ], [ 179, 184, "Ala69", "residue_name_number" ], [ 219, 228, "interface", "site" ] ] }, { "sid": 80, "sent": "Generally, the weaker-affinity APLs made fewer contacts with the MHC surface (27\u201329 interactions) compared with the stronger-affinity APLs (29\u201335 contacts), consistent with a better Pearson\u2019s correlation value (0.55) compared with TCR-peptide interactions versus affinity (0.045).", "section": "RESULTS", "ner": [ [ 22, 30, "affinity", "evidence" ], [ 31, 35, "APLs", "chemical" ], [ 65, 68, "MHC", "complex_assembly" ], [ 125, 133, "affinity", "evidence" ], [ 134, 138, "APLs", "chemical" ], [ 182, 209, "Pearson\u2019s correlation value", "evidence" ], [ 231, 234, "TCR", "complex_assembly" ], [ 263, 271, "affinity", "evidence" ] ] }, { "sid": 81, "sent": "For instance, contacts were made between TCR residue Val53\u03b2 and MHC residue Gln72 in all APLs except for in the weakest affinity ligand pair, 1E6-A2-MVWGPDPLYV, in which a subtle change in TCR conformation \u2014 probably mediated by different peptide contacts \u2014 abrogated this interaction (Figure 7A).", "section": "RESULTS", "ner": [ [ 41, 44, "TCR", "complex_assembly" ], [ 53, 59, "Val53\u03b2", "residue_name_number" ], [ 64, 67, "MHC", "complex_assembly" ], [ 76, 81, "Gln72", "residue_name_number" ], [ 89, 93, "APLs", "chemical" ], [ 120, 128, "affinity", "evidence" ], [ 142, 159, "1E6-A2-MVWGPDPLYV", "complex_assembly" ], [ 189, 192, "TCR", "complex_assembly" ] ] }, { "sid": 82, "sent": "An energetic switch from unfavorable to favorable entropy (order-to-disorder) correlates with antigen potency.", "section": "RESULTS", "ner": [ [ 50, 57, "entropy", "evidence" ] ] }, { "sid": 83, "sent": "Our analysis of the contact network provided some clues that could explain the different antigen potencies and binding affinities between the 1E6 TCR and the different APLs.", "section": "RESULTS", "ner": [ [ 20, 35, "contact network", "site" ], [ 111, 129, "binding affinities", "evidence" ], [ 142, 149, "1E6 TCR", "complex_assembly" ], [ 168, 172, "APLs", "chemical" ] ] }, { "sid": 84, "sent": "For example, the 1E6 TCR bound to A2-RQWGPDPAAV with the third strongest affinity (KD = 7.8 \u03bcM) but made fewer contacts than with A2-ALWGPDPAAA (KD = ~208 \u03bcM) (Table 2).", "section": "RESULTS", "ner": [ [ 17, 24, "1E6 TCR", "complex_assembly" ], [ 25, 33, "bound to", "protein_state" ], [ 34, 47, "A2-RQWGPDPAAV", "chemical" ], [ 73, 81, "affinity", "evidence" ], [ 83, 85, "KD", "evidence" ], [ 130, 143, "A2-ALWGPDPAAA", "chemical" ], [ 145, 147, "KD", "evidence" ] ] }, { "sid": 85, "sent": "Thus, we performed an in-depth thermodynamic analysis of 6 of the ligands under investigation (Figure 8 and Supplemental Table 3).", "section": "RESULTS", "ner": [ [ 31, 53, "thermodynamic analysis", "experimental_method" ] ] }, { "sid": 86, "sent": "The weak binding affinity between 1E6 and A2-MVWGPDPLYV and A2-YLGGPDFPTI generated thermodynamic data that were not robust enough to gain insight into the enthalpic (\u0394H\u00b0) and entropic (T\u0394S\u00b0) changes that contributed to the different binding affinities/potencies for each APL.", "section": "RESULTS", "ner": [ [ 9, 25, "binding affinity", "evidence" ], [ 42, 55, "A2-MVWGPDPLYV", "chemical" ], [ 60, 73, "A2-YLGGPDFPTI", "chemical" ], [ 156, 165, "enthalpic", "evidence" ], [ 167, 170, "\u0394H\u00b0", "evidence" ], [ 176, 184, "entropic", "evidence" ], [ 186, 190, "T\u0394S\u00b0", "evidence" ], [ 234, 252, "binding affinities", "evidence" ], [ 272, 275, "APL", "chemical" ] ] }, { "sid": 87, "sent": "The overall free binding energies (\u0394G\u00b0) were between \u20134.4 and \u20138.6 kcal/mol, reflecting the wide range of TCR binding affinities we observed for the different APLs.", "section": "RESULTS", "ner": [ [ 12, 33, "free binding energies", "evidence" ], [ 35, 38, "\u0394G\u00b0", "evidence" ], [ 106, 128, "TCR binding affinities", "evidence" ], [ 159, 163, "APLs", "chemical" ] ] }, { "sid": 88, "sent": "The enthalpic contribution in each complex did not follow a clear trend with affinity, with all but the 1E6-A2-RQFGPDFPTI interaction (\u0394H\u00b0 = 6.3 kcal/mol) generating an energetically favorable enthalpy value (\u0394H\u00b0 = \u20133.7 to \u201311.4 kcal/mol); this indicated a net gain in electrostatic interactions during complex formation.", "section": "RESULTS", "ner": [ [ 77, 85, "affinity", "evidence" ], [ 104, 121, "1E6-A2-RQFGPDFPTI", "complex_assembly" ], [ 135, 138, "\u0394H\u00b0", "evidence" ], [ 193, 201, "enthalpy", "evidence" ], [ 209, 212, "\u0394H\u00b0", "evidence" ] ] }, { "sid": 89, "sent": "However, there was a clear switch in entropy between the weaker-affinity and stronger-affinity ligands, indicated by a strong Pearson\u2019s correlation value between entropy and affinity (Pearson\u2019s correlation value 0.93, P =0.007).", "section": "RESULTS", "ner": [ [ 37, 44, "entropy", "evidence" ], [ 64, 72, "affinity", "evidence" ], [ 86, 94, "affinity", "evidence" ], [ 126, 153, "Pearson\u2019s correlation value", "evidence" ], [ 162, 169, "entropy", "evidence" ], [ 174, 182, "affinity", "evidence" ], [ 184, 211, "Pearson\u2019s correlation value", "evidence" ], [ 218, 219, "P", "evidence" ] ] }, { "sid": 90, "sent": "For instance, the A2-ALWGPDPAAA, A2-AQWGPDAAA, and A2-RQFGPDWIVA (KD = ~208 \u03bcM, KD = 61.9 \u03bcM, and KD = 44.4 \u03bcM, respectively) were all entropically unfavorable (T\u0394S\u00b0 = \u20132.9 to \u20135.6 kcal/mol), indicating a net change from disorder to order.", "section": "RESULTS", "ner": [ [ 18, 31, "A2-ALWGPDPAAA", "chemical" ], [ 33, 45, "A2-AQWGPDAAA", "chemical" ], [ 51, 64, "A2-RQFGPDWIVA", "chemical" ], [ 66, 68, "KD", "evidence" ], [ 80, 82, "KD", "evidence" ], [ 98, 100, "KD", "evidence" ], [ 161, 165, "T\u0394S\u00b0", "evidence" ] ] }, { "sid": 91, "sent": "Conversely, the stronger-affinity ligands A2-RQWGPDPAAV (KD = 7.8 \u03bcM), A2-YQFGPDFPIA (KD = 7.4 \u03bcM), and A2-RQFGPDFPTI (KD = 0.5 \u03bcM) exhibited favorable entropy (T\u0394S\u00b0 = 2.2 to 14.9 kcal/mol), indicating an order-to-disorder change during binding, possibly through the expulsion of ordered water molecules.", "section": "RESULTS", "ner": [ [ 25, 33, "affinity", "evidence" ], [ 42, 55, "A2-RQWGPDPAAV", "chemical" ], [ 57, 59, "KD", "evidence" ], [ 71, 84, "A2-YQFGPDFPIA", "chemical" ], [ 86, 88, "KD", "evidence" ], [ 104, 117, "A2-RQFGPDFPTI", "chemical" ], [ 119, 121, "KD", "evidence" ], [ 152, 159, "entropy", "evidence" ], [ 161, 165, "T\u0394S\u00b0", "evidence" ] ] }, { "sid": 92, "sent": "Furthermore, the structures of the unligated pMHCs demonstrated that, for these stronger-affinity ligands, there was less conformational difference between the TCR ligated pMHCs compared with the weaker-affinity ligands (Figure 6).", "section": "RESULTS", "ner": [ [ 35, 44, "unligated", "protein_state" ], [ 45, 50, "pMHCs", "complex_assembly" ], [ 89, 97, "affinity", "evidence" ], [ 160, 163, "TCR", "complex_assembly" ], [ 164, 171, "ligated", "protein_state" ], [ 172, 177, "pMHCs", "complex_assembly" ], [ 203, 211, "affinity", "evidence" ] ] }, { "sid": 93, "sent": "The potential requirement for a larger degree of induced fit during binding to these weaker-affinity ligands is consistent with the larger entropic penalties observed for these interactions.", "section": "RESULTS", "ner": [ [ 92, 100, "affinity", "evidence" ] ] }, { "sid": 94, "sent": "Potential epitopes for 1E6 TCR occur commonly in the viral proteome.", "section": "RESULTS", "ner": [ [ 23, 30, "1E6 TCR", "complex_assembly" ], [ 53, 58, "viral", "taxonomy_domain" ] ] }, { "sid": 95, "sent": "We searched a database of over 1,924,572 unique decamer peptides from the proteome of viral pathogens that are known, or strongly suspected, to infect humans.", "section": "RESULTS", "ner": [ [ 56, 64, "peptides", "chemical" ], [ 86, 91, "viral", "taxonomy_domain" ], [ 151, 157, "humans", "species" ] ] }, { "sid": 96, "sent": "Three hundred forty-two of these decamers conformed to the motif xxxGPDxxxx.", "section": "RESULTS", "ner": [ [ 65, 75, "xxxGPDxxxx", "structure_element" ] ] }, { "sid": 97, "sent": "Of these, 53 peptides contained the motif xOxGPDxxxO, where O is one of the hydrophobic amino acid residues A,V, I, L, M, Y, F, and W that might allow binding to HLA-A*0201 (Supplemental Table 4).", "section": "RESULTS", "ner": [ [ 42, 52, "xOxGPDxxxO", "structure_element" ], [ 108, 109, "A", "residue_name" ], [ 110, 111, "V", "residue_name" ], [ 113, 114, "I", "residue_name" ], [ 116, 117, "L", "residue_name" ], [ 119, 120, "M", "residue_name" ], [ 122, 123, "Y", "residue_name" ], [ 125, 126, "F", "residue_name" ], [ 132, 133, "W", "residue_name" ], [ 162, 172, "HLA-A*0201", "protein" ] ] }, { "sid": 98, "sent": "Thus, there are many pathogen-encoded peptides that could act as agonists for the 1E6 T cell beyond the MVWGPDPLYV and RQFGPDWIVA sequences studied here.", "section": "RESULTS", "ner": [ [ 104, 114, "MVWGPDPLYV", "chemical" ], [ 119, 129, "RQFGPDWIVA", "chemical" ] ] }, { "sid": 99, "sent": "Extension of these analyses to include the larger genomes of bacterial pathogens would be expected to considerably increase these numbers.", "section": "RESULTS", "ner": [ [ 61, 70, "bacterial", "taxonomy_domain" ] ] }, { "sid": 100, "sent": "The binding affinity of the 1E6 TCR interaction with A2-RQFGPDWIVA is considerably higher than with the disease-implicated A2-ALWGPDPAAA sequence (KD = 44.4 \u03bcM and KD > 200 \u03bcM, respectively), highlighting how a pathogen-derived sequence might be capable of priming a 1E6-like T cell.", "section": "RESULTS", "ner": [ [ 4, 20, "binding affinity", "evidence" ], [ 28, 35, "1E6 TCR", "complex_assembly" ], [ 53, 66, "A2-RQFGPDWIVA", "chemical" ], [ 123, 136, "A2-ALWGPDPAAA", "chemical" ], [ 147, 149, "KD", "evidence" ], [ 164, 166, "KD", "evidence" ] ] }, { "sid": 101, "sent": "T cell antigen discrimination is governed by an interaction between the clonally expressed TCR and pMHC, mediated by the chemical characteristics of the interacting molecules.", "section": "DISCUSS", "ner": [ [ 91, 94, "TCR", "complex_assembly" ], [ 99, 103, "pMHC", "complex_assembly" ] ] }, { "sid": 102, "sent": "It has recently become clear that TCR cross-reactivity with large numbers of different pMHC ligands is essential to plug holes in T cell immune coverage that pathogens could exploit.", "section": "DISCUSS", "ner": [ [ 34, 37, "TCR", "complex_assembly" ], [ 87, 91, "pMHC", "complex_assembly" ] ] }, { "sid": 103, "sent": "Flexibility at the interface between the TCR and pMHC, demonstrated in various studies, has been suggested as a mechanism mediating T cell cross-reactivity with multiple distinct epitopes.", "section": "DISCUSS", "ner": [ [ 19, 28, "interface", "site" ], [ 41, 44, "TCR", "complex_assembly" ], [ 49, 53, "pMHC", "complex_assembly" ] ] }, { "sid": 104, "sent": "This notion is attractive because the CDR loops, which form the TCR antigen-binding site, are usually the most flexible part of the TCR and have the ability to mold around differently shaped ligands.", "section": "DISCUSS", "ner": [ [ 38, 47, "CDR loops", "structure_element" ], [ 64, 88, "TCR antigen-binding site", "site" ], [ 132, 135, "TCR", "complex_assembly" ] ] }, { "sid": 105, "sent": "Focused binding around a minimal peptide motif has also been implicated as an alternative mechanism enabling TCR cross-reactivity.", "section": "DISCUSS", "ner": [ [ 109, 112, "TCR", "complex_assembly" ] ] }, { "sid": 106, "sent": "Notably among these studies, Garcia and colleagues recently used the alloreactive murine TCR-MHC pair of the 42F3 TCR and H2-Ld to demonstrate recognition of a large number of different peptides via conserved hotspot contacts with prominent up-facing peptide residues.", "section": "DISCUSS", "ner": [ [ 69, 81, "alloreactive", "protein_state" ], [ 82, 88, "murine", "taxonomy_domain" ], [ 89, 92, "TCR", "complex_assembly" ], [ 93, 96, "MHC", "complex_assembly" ], [ 109, 113, "42F3", "protein" ], [ 114, 117, "TCR", "complex_assembly" ], [ 122, 127, "H2-Ld", "protein" ], [ 199, 208, "conserved", "protein_state" ], [ 209, 216, "hotspot", "site" ] ] }, { "sid": 107, "sent": "Sethi and colleagues recently demonstrated that the MHCII-restricted Hy.1B11 TCR, which was isolated from a patient with multiple sclerosis, could anchor into a deep pocket formed from peptide residues 2, 3, and 5 (from MBP85\u201399 bound to HLA-DQ1).", "section": "DISCUSS", "ner": [ [ 52, 57, "MHCII", "protein_type" ], [ 69, 76, "Hy.1B11", "protein" ], [ 77, 80, "TCR", "complex_assembly" ], [ 161, 172, "deep pocket", "site" ], [ 202, 203, "2", "residue_number" ], [ 205, 206, "3", "residue_number" ], [ 212, 213, "5", "residue_number" ], [ 220, 228, "MBP85\u201399", "protein" ], [ 229, 237, "bound to", "protein_state" ], [ 238, 245, "HLA-DQ1", "protein" ] ] }, { "sid": 108, "sent": "This motif was conserved in at least 2 potential foreign peptides, originating from Herpes simplex virus and Pseudomonas aeruginosa, enabling TCR recognition of foreign epitopes.", "section": "DISCUSS", "ner": [ [ 15, 24, "conserved", "protein_state" ], [ 84, 104, "Herpes simplex virus", "species" ], [ 109, 131, "Pseudomonas aeruginosa", "species" ], [ 142, 145, "TCR", "complex_assembly" ] ] }, { "sid": 109, "sent": "First, we currently know nothing about how human MHCI\u2013restricted TCRs mediate cross-reactivity in the context of a clinically relevant model of autoimmunity, thought to be a major pathway of disease initiation in several autoimmune diseases.", "section": "DISCUSS", "ner": [ [ 43, 48, "human", "species" ], [ 49, 53, "MHCI", "complex_assembly" ], [ 65, 69, "TCRs", "complex_assembly" ] ] }, { "sid": 110, "sent": "Second, molecular studies have not yet revealed a broad set of rules that determine TCR cross-reactivity because, with the exception of the allo\u2013TCR-MHC pair of the 42F3 TCR and H2-Ld that did not encounter each other during T cell development, studies have been limited to structures of a TCR with only 2 or 3 different ligands.", "section": "DISCUSS", "ner": [ [ 84, 87, "TCR", "complex_assembly" ], [ 140, 144, "allo", "protein_state" ], [ 145, 152, "TCR-MHC", "complex_assembly" ], [ 165, 169, "42F3", "protein" ], [ 170, 173, "TCR", "complex_assembly" ], [ 178, 183, "H2-Ld", "protein" ], [ 274, 284, "structures", "evidence" ], [ 290, 293, "TCR", "complex_assembly" ] ] }, { "sid": 111, "sent": "Here, we investigated a highly cross-reactive MHCI-restricted TCR isolated from a patient with T1D that recognizes an HLA-A*0201\u2013restricted preproinsulin signal peptide (ALWGPDPAAA15\u201324).", "section": "DISCUSS", "ner": [ [ 46, 50, "MHCI", "complex_assembly" ], [ 62, 65, "TCR", "complex_assembly" ], [ 118, 128, "HLA-A*0201", "protein" ], [ 140, 153, "preproinsulin", "protein" ], [ 154, 168, "signal peptide", "structure_element" ], [ 170, 185, "ALWGPDPAAA15\u201324", "chemical" ] ] }, { "sid": 112, "sent": "Human CD8+ T cell clones expressing TCRs with this specificity mediate the destruction of \u03b2 cells, have been found in islets early in infection, and are proposed to be a major driver of disease.", "section": "DISCUSS", "ner": [ [ 0, 5, "Human", "species" ], [ 36, 40, "TCRs", "complex_assembly" ] ] }, { "sid": 113, "sent": "We solved the structure of the 1E6 TCR with 7 APLs to enable a comprehensive analysis of the molecular basis of TCR degeneracy.", "section": "DISCUSS", "ner": [ [ 3, 9, "solved", "experimental_method" ], [ 14, 23, "structure", "evidence" ], [ 31, 38, "1E6 TCR", "complex_assembly" ], [ 46, 50, "APLs", "chemical" ], [ 112, 115, "TCR", "complex_assembly" ] ] }, { "sid": 114, "sent": "Overall, the difference in antigen potency correlated well with the binding energy (\u0394G\u00b0 kcal/mol) of the 1E6 TCR for the different epitopes, which ranged from values of \u0394G\u00b0 = ~\u20134.4 to \u20138.6 kcal/mol (calculated from 3D affinity data) or 2D affinity values of AcKa = 2.5 \u00d7 10\u20135 to 4.4 \u00d7 10\u20132 \u03bcm4.", "section": "DISCUSS", "ner": [ [ 68, 82, "binding energy", "evidence" ], [ 84, 87, "\u0394G\u00b0", "evidence" ], [ 105, 112, "1E6 TCR", "complex_assembly" ], [ 169, 172, "\u0394G\u00b0", "evidence" ], [ 215, 226, "3D affinity", "evidence" ], [ 236, 247, "2D affinity", "evidence" ], [ 258, 262, "AcKa", "evidence" ] ] }, { "sid": 115, "sent": "The weaker end of this spectrum extends our understanding of the limits in which T cells can functionally operate in terms of TCR 3D binding affinity and is in line with the types of very low affinity, yet fully functional self-reactive CD8+ T cells we have observed in tumor-infiltrating lymphocytes.", "section": "DISCUSS", "ner": [ [ 126, 149, "TCR 3D binding affinity", "evidence" ], [ 192, 200, "affinity", "evidence" ] ] }, { "sid": 116, "sent": "Previous studies of autoreactive TCRs have shown that their binding mode is generally atypical, either due to an unusual binding manner, weak TCR binding affinity, an unstable pMHC, or a combination of these factors.", "section": "DISCUSS", "ner": [ [ 33, 37, "TCRs", "complex_assembly" ], [ 142, 162, "TCR binding affinity", "evidence" ], [ 167, 175, "unstable", "protein_state" ], [ 176, 180, "pMHC", "complex_assembly" ] ] }, { "sid": 117, "sent": "Our data demonstrate the potential for an autoreactive TCR to bind with a conventional binding mode to a stable pMHC with antipathogen-like affinity (KD = 0.5 \u03bcM) depending on the peptide sequence.", "section": "DISCUSS", "ner": [ [ 55, 58, "TCR", "complex_assembly" ], [ 105, 111, "stable", "protein_state" ], [ 112, 116, "pMHC", "complex_assembly" ], [ 140, 148, "affinity", "evidence" ], [ 150, 152, "KD", "evidence" ] ] }, { "sid": 118, "sent": "Our structural analysis revealed that the 1E6 TCR bound with a conserved conformation across all APLs investigated.", "section": "DISCUSS", "ner": [ [ 4, 23, "structural analysis", "experimental_method" ], [ 42, 49, "1E6 TCR", "complex_assembly" ], [ 50, 55, "bound", "protein_state" ], [ 63, 85, "conserved conformation", "protein_state" ], [ 97, 101, "APLs", "chemical" ] ] }, { "sid": 119, "sent": "This binding orientation was mediated through a focused interaction with TCR residues Tyr97\u03b1 and Trp97\u03b2 that formed an aromatic cap over a central \u2018GDP\u2019 motif that was common to all APLs.", "section": "DISCUSS", "ner": [ [ 73, 76, "TCR", "complex_assembly" ], [ 86, 92, "Tyr97\u03b1", "residue_name_number" ], [ 97, 103, "Trp97\u03b2", "residue_name_number" ], [ 119, 131, "aromatic cap", "structure_element" ], [ 147, 158, "\u2018GDP\u2019 motif", "structure_element" ], [ 182, 186, "APLs", "chemical" ] ] }, { "sid": 120, "sent": "We have previously demonstrated the importance of the GPD motif using a peptide library scan, as well as a CPL scan approach.", "section": "DISCUSS", "ner": [ [ 54, 63, "GPD motif", "structure_element" ], [ 72, 92, "peptide library scan", "experimental_method" ], [ 107, 115, "CPL scan", "experimental_method" ] ] }, { "sid": 121, "sent": "Although the 1E6 T cell was able to activate weakly with peptides that lacked this motif, we were unable to robustly measure binding affinities or generate complex structures with these ligands, highlighting the central role of this interaction during 1E6 T cell antigen recognition.", "section": "DISCUSS", "ner": [ [ 71, 77, "lacked", "protein_state" ], [ 125, 143, "binding affinities", "evidence" ], [ 164, 174, "structures", "evidence" ] ] }, { "sid": 122, "sent": "This hotspot binding, defined as a localized cluster of interactions that dominate binding energy during protein-protein interactions, has been previously shown to contribute to TCR recognition of MHC as a mechanism that tunes T cell cross-reactivity by providing fixed anchor points that enable TCRs to tolerate a variable peptide cargo.", "section": "DISCUSS", "ner": [ [ 178, 181, "TCR", "complex_assembly" ], [ 197, 200, "MHC", "complex_assembly" ], [ 296, 300, "TCRs", "complex_assembly" ] ] }, { "sid": 123, "sent": "Alternatively, interactions between the TCR and peptide have been shown to dominate the energetic landscape during ligand engagement, ensuring that T cells retain peptide specificity.", "section": "DISCUSS", "ner": [ [ 40, 43, "TCR", "complex_assembly" ] ] }, { "sid": 124, "sent": "The binding mechanism utilized by the 1E6 TCR during pMHC recognition is consistent with both of these models.", "section": "DISCUSS", "ner": [ [ 38, 45, "1E6 TCR", "complex_assembly" ], [ 53, 57, "pMHC", "complex_assembly" ] ] }, { "sid": 125, "sent": "Ligand engagement is dominated by peptide interactions, but hotspot-like interactions with the central GPD motif enable the 1E6 TCR to tolerate peptide residues that vary outside of this region, explaining how T cells expressing this TCR may cross-react with a large number of different peptides.", "section": "DISCUSS", "ner": [ [ 103, 112, "GPD motif", "structure_element" ], [ 124, 131, "1E6 TCR", "complex_assembly" ], [ 234, 237, "TCR", "complex_assembly" ] ] }, { "sid": 126, "sent": "These findings are also analogous to the observed binding mode of the Hy.1B11 TCR, in which one aromatic residue of the TCR CDR3\u03b1 loop anchored into a pocket created by a conserved peptide motif.", "section": "DISCUSS", "ner": [ [ 70, 77, "Hy.1B11", "protein" ], [ 78, 81, "TCR", "complex_assembly" ], [ 120, 123, "TCR", "complex_assembly" ], [ 124, 134, "CDR3\u03b1 loop", "structure_element" ] ] }, { "sid": 127, "sent": "In both of these examples, self-recognition is mediated by TCR residues with aromatic side chains.", "section": "DISCUSS", "ner": [ [ 59, 62, "TCR", "complex_assembly" ] ] }, { "sid": 128, "sent": "Combined with evidence demonstrating that aromatic side chains are conserved in the CDR2 loops of TCRs from many species, we speculate that these aromatic residues could impart a level of \u201cstickiness\u201d to TCRs, which might be enriched in an autoimmune setting when the TCR often binds in a nonoptimal fashion.", "section": "DISCUSS", "ner": [ [ 84, 94, "CDR2 loops", "structure_element" ], [ 98, 102, "TCRs", "complex_assembly" ], [ 204, 208, "TCRs", "complex_assembly" ], [ 268, 271, "TCR", "complex_assembly" ] ] }, { "sid": 129, "sent": "Despite some weak statistical correlation between the surface complementarity (SC) and affinity, closer inspection of the interface revealed no obvious structural signature that could definitively explain the differences in antigen potency and TCR binding strength between the different ligands.", "section": "DISCUSS", "ner": [ [ 54, 77, "surface complementarity", "evidence" ], [ 79, 81, "SC", "evidence" ], [ 87, 95, "affinity", "evidence" ], [ 122, 131, "interface", "site" ], [ 224, 239, "antigen potency", "evidence" ], [ 244, 264, "TCR binding strength", "evidence" ] ] }, { "sid": 130, "sent": "However, similar to our findings in other systems, modifications to residues outside of the canonical central peptide bulge were important for generating new interactions.", "section": "DISCUSS", "ner": [ [ 102, 123, "central peptide bulge", "structure_element" ] ] }, { "sid": 131, "sent": "For example, all of the stronger ligands encoded larger side chains (Arg or Tyr) at peptide position 1 that enabled new interactions with 1E6 not present with the Ala at this position in the natural preproinsulin peptide.", "section": "DISCUSS", "ner": [ [ 69, 72, "Arg", "residue_name" ], [ 76, 79, "Tyr", "residue_name" ], [ 101, 102, "1", "residue_number" ], [ 163, 166, "Ala", "residue_name" ], [ 199, 212, "preproinsulin", "protein" ], [ 213, 220, "peptide", "chemical" ] ] }, { "sid": 132, "sent": "These data also explain our previous findings that alteration of the anchor residue at peptide position 2 (Leu-Gln) has a direct effect on 1E6 TCR binding affinity because our structural analysis demonstrated that 1E6 made 3 additional bonds with A2-AQWGPDPAAA compared with A2-ALWGPDPAAA, consistent with the >3-fold stronger binding affinity.", "section": "DISCUSS", "ner": [ [ 69, 83, "anchor residue", "structure_element" ], [ 104, 105, "2", "residue_number" ], [ 107, 114, "Leu-Gln", "mutant" ], [ 139, 163, "1E6 TCR binding affinity", "evidence" ], [ 176, 195, "structural analysis", "experimental_method" ], [ 247, 260, "A2-AQWGPDPAAA", "chemical" ], [ 275, 288, "A2-ALWGPDPAAA", "chemical" ], [ 327, 343, "binding affinity", "evidence" ] ] }, { "sid": 133, "sent": "We have recently demonstrated how a suboptimal position 2 anchor in a melanoma-derived antigen can improve TCR binding through a similar mechanism.", "section": "DISCUSS", "ner": [ [ 56, 57, "2", "residue_number" ], [ 58, 64, "anchor", "structure_element" ], [ 107, 110, "TCR", "complex_assembly" ] ] }, { "sid": 134, "sent": "These results challenge the notion that the most potent peptide antigens exhibit the greatest pMHC stability and have implications for the design of anchor residue\u2013modified heteroclitic peptides for vaccination.", "section": "DISCUSS", "ner": [ [ 94, 98, "pMHC", "complex_assembly" ] ] }, { "sid": 135, "sent": "Early thermodynamic analysis of TCR-pMHC interactions suggested a common energetic signature, driven by favorable enthalpy (generally mediated through an increase in electrostatic interactions) and unfavorable entropy (changes from disorder to order).", "section": "DISCUSS", "ner": [ [ 6, 28, "thermodynamic analysis", "experimental_method" ], [ 32, 40, "TCR-pMHC", "complex_assembly" ], [ 114, 122, "enthalpy", "evidence" ], [ 210, 217, "entropy", "evidence" ] ] }, { "sid": 136, "sent": "These parameters aligned well with structural data, demonstrating that TCRs engaged pMHC using an induced fit binding mode.", "section": "DISCUSS", "ner": [ [ 35, 50, "structural data", "evidence" ], [ 71, 75, "TCRs", "complex_assembly" ], [ 84, 88, "pMHC", "complex_assembly" ] ] }, { "sid": 137, "sent": "However, more recent data have shown that TCRs can utilize a range of energetic strategies during pMHC binding, currently with no obvious pattern in terms of TCR affinity, binding mechanism, or specificity (pathogen, cancer, or self-ligands).", "section": "DISCUSS", "ner": [ [ 42, 46, "TCRs", "complex_assembly" ], [ 98, 102, "pMHC", "complex_assembly" ], [ 158, 170, "TCR affinity", "evidence" ] ] }, { "sid": 138, "sent": "Although no energetic signature appears to exist for different TCRs, we used thermodynamic analysis here to explore whether changes in energetics could help explain ligand discrimination by a single TCR.", "section": "DISCUSS", "ner": [ [ 63, 67, "TCRs", "complex_assembly" ], [ 77, 99, "thermodynamic analysis", "experimental_method" ], [ 199, 202, "TCR", "complex_assembly" ] ] }, { "sid": 139, "sent": "This analysis demonstrated a strong relationship (according to the Pearson\u2019s correlation analysis) between the energetic signature used by the 1E6 TCR and the sensitivity of the 1E6 T cell clone to different APLs.", "section": "DISCUSS", "ner": [ [ 67, 97, "Pearson\u2019s correlation analysis", "experimental_method" ], [ 143, 150, "1E6 TCR", "complex_assembly" ], [ 208, 212, "APLs", "chemical" ] ] }, { "sid": 140, "sent": "The weaker APL ligands were characterized by favorable enthalpy and unfavorable entropy, whereas the stronger ligands progressively shifted to favorable entropy.", "section": "DISCUSS", "ner": [ [ 11, 14, "APL", "chemical" ], [ 55, 63, "enthalpy", "evidence" ], [ 80, 87, "entropy", "evidence" ], [ 153, 160, "entropy", "evidence" ] ] }, { "sid": 141, "sent": "These differences were consistent with a greater degree of movement between the unligated and ligated pMHCs for the weaker ligands, suggesting a greater requirement for disorder-to-order changes during TCR binding.", "section": "DISCUSS", "ner": [ [ 80, 89, "unligated", "protein_state" ], [ 94, 101, "ligated", "protein_state" ], [ 102, 107, "pMHCs", "complex_assembly" ], [ 202, 205, "TCR", "complex_assembly" ] ] }, { "sid": 142, "sent": "Thus, the enhanced antigen potency was probably mediated through a shift from an induced fit to a lock-and-key interaction between the stronger ligands (less requirement for energetically unfavorable disorder-to-order changes), resulting in a more energetically favorable \u0394G value.", "section": "DISCUSS", "ner": [ [ 272, 280, "\u0394G value", "evidence" ] ] }, { "sid": 143, "sent": "Importantly, the preproinsulin-derived epitope was one of the least potent peptides, demonstrating that the 1E6 T cell clone had the ability to respond to different peptide sequences with far greater potency.", "section": "DISCUSS", "ner": [ [ 17, 30, "preproinsulin", "protein" ] ] }, { "sid": 144, "sent": "The RQFGPDWIVA peptide, which was substantially more potent than the preproinsulin peptide, is within the proteome of a common human pathogen (C. asparagiforme), demonstrating the potential for an encounter between a naive 1E6-like T cell and a foreign peptide with a more potent ligand that might then break self-tolerance.", "section": "DISCUSS", "ner": [ [ 4, 14, "RQFGPDWIVA", "chemical" ], [ 69, 82, "preproinsulin", "protein" ], [ 127, 132, "human", "species" ], [ 143, 159, "C. asparagiforme", "species" ] ] }, { "sid": 145, "sent": "Indeed, we found over 50 decamer peptides from the proteome of likely, or known, human viral pathogens alone that contained both the conserved central GPD motif and anchor residues at positions 2 and 10 that would enable binding to HLA-A*02:01.", "section": "DISCUSS", "ner": [ [ 81, 86, "human", "species" ], [ 87, 92, "viral", "taxonomy_domain" ], [ 133, 142, "conserved", "protein_state" ], [ 151, 160, "GPD motif", "structure_element" ], [ 165, 180, "anchor residues", "structure_element" ], [ 194, 195, "2", "residue_number" ], [ 200, 202, "10", "residue_number" ], [ 232, 243, "HLA-A*02:01", "protein" ] ] }, { "sid": 146, "sent": "Further experiments will be required to determine whether any naturally presented, human pathogen\u2013derived peptides act as active ligands for 1E6, but our work presented here demonstrates that it is at least feasible for an autoimmune TCR to bind to a different peptide sequence that could be present in a pathogen proteome with substantially higher affinity and potency than the interaction it might use to attack self-tissue.", "section": "DISCUSS", "ner": [ [ 83, 88, "human", "species" ], [ 234, 237, "TCR", "complex_assembly" ], [ 349, 357, "affinity", "evidence" ] ] }, { "sid": 147, "sent": "In summary, this investigation into the molecular basis of T cell cross-reactivity using a clinically relevant cytotoxic CD8+ T cell clone that kills human pancreatic \u03b2 cells provides answers to a number of previously outstanding questions.", "section": "DISCUSS", "ner": [ [ 150, 155, "human", "species" ] ] }, { "sid": 148, "sent": "First, our data shows that a single TCR has the potential to functionally (assessed through T cell activation) bind to different ligands with affinities ranging across 3 orders of magnitude.", "section": "DISCUSS", "ner": [ [ 36, 39, "TCR", "complex_assembly" ], [ 142, 152, "affinities", "evidence" ] ] }, { "sid": 149, "sent": "Second, this is the first example in which ligands have been identified and characterized for a human autoreactive TCR that are substantially more potent than the natural self-ligand, demonstrating the potential for a pathogenic ligand to break self-tolerance and prime self-reactive T cells.", "section": "DISCUSS", "ner": [ [ 96, 101, "human", "species" ], [ 115, 118, "TCR", "complex_assembly" ] ] }, { "sid": 150, "sent": "Third, this first structural analysis of a cross-reactive human MHCI\u2013restricted autoimmune TCR showed that degeneracy was mediated through TCR-pMHC anchoring by a conserved minimal binding peptide motif.", "section": "DISCUSS", "ner": [ [ 18, 37, "structural analysis", "experimental_method" ], [ 58, 63, "human", "species" ], [ 64, 68, "MHCI", "complex_assembly" ], [ 91, 94, "TCR", "complex_assembly" ], [ 139, 147, "TCR-pMHC", "complex_assembly" ], [ 163, 172, "conserved", "protein_state" ], [ 173, 202, "minimal binding peptide motif", "structure_element" ] ] }, { "sid": 151, "sent": "Finally, TCR ligand discrimination was characterized by an energetic shift from an enthalpically to entropically driven interaction.", "section": "DISCUSS", "ner": [ [ 9, 12, "TCR", "complex_assembly" ] ] }, { "sid": 152, "sent": "Our demonstration of the molecular mechanism governing cross-reactivity by this preproinsulin reactive human CD8+ T cell clone supports the notion first put forward by Wucherpfennig and Strominger that molecular mimicry could mediate autoimmunity and has far-reaching implications for the complex nature of T cell antigen discrimination.", "section": "DISCUSS", "ner": [ [ 80, 93, "preproinsulin", "protein" ], [ 103, 108, "human", "species" ] ] }, { "sid": 153, "sent": "The 1E6 T cell clone reacts with a broad sensitivity range to APLs.", "section": "FIG", "ner": [ [ 62, 66, "APLs", "chemical" ] ] }, { "sid": 154, "sent": "(A and B) The 1E6 T cell clone was tested in a peptide dilution assay, in triplicate, with MVWGPDPLYV (gray), YLGGPDFPTI (red), ALWGPDPAAA (blue), AQWGPDPAAA (green), RQFGPDWIVA (dark blue), RQWGPDPAAV (purple), YQFGPDFPTA (yellow), and RQFGPDFPTI (cyan) peptides presented by HLA-A*0201\u2013expressing C1R cells for release of MIP-1\u03b2 (A) and killing (B).", "section": "FIG", "ner": [ [ 47, 69, "peptide dilution assay", "experimental_method" ], [ 91, 101, "MVWGPDPLYV", "chemical" ], [ 110, 120, "YLGGPDFPTI", "chemical" ], [ 128, 138, "ALWGPDPAAA", "chemical" ], [ 147, 157, "AQWGPDPAAA", "chemical" ], [ 167, 177, "RQFGPDWIVA", "chemical" ], [ 191, 201, "RQWGPDPAAV", "chemical" ], [ 212, 222, "YQFGPDFPTA", "chemical" ], [ 237, 247, "RQFGPDFPTI", "chemical" ], [ 277, 287, "HLA-A*0201", "protein" ], [ 324, 330, "MIP-1\u03b2", "protein" ] ] }, { "sid": 155, "sent": "(C) The 1E6 T cell clone was stained, in duplicate, with tetramers composed of each APL (colored as above) presented by HLA-A*0201. (D) The stability of each APL (colored as above) was tested, in duplicate, using CD by recording the peak at 218 nm absorbance from 5\u00b0C\u201390\u00b0C.", "section": "FIG", "ner": [ [ 57, 66, "tetramers", "oligomeric_state" ], [ 84, 87, "APL", "chemical" ], [ 120, 130, "HLA-A*0201", "protein" ], [ 158, 161, "APL", "chemical" ], [ 213, 215, "CD", "experimental_method" ] ] }, { "sid": 156, "sent": "Tm values were calculated using a Boltzmann fit to each set of data.", "section": "FIG", "ner": [ [ 0, 2, "Tm", "evidence" ], [ 34, 67, "Boltzmann fit to each set of data", "experimental_method" ] ] }, { "sid": 157, "sent": "3D and 2D binding analysis of the 1E6 TCR with A2-ALW and the APLs.", "section": "FIG", "ner": [ [ 0, 26, "3D and 2D binding analysis", "experimental_method" ], [ 34, 41, "1E6 TCR", "complex_assembly" ], [ 47, 53, "A2-ALW", "chemical" ], [ 62, 66, "APLs", "chemical" ] ] }, { "sid": 158, "sent": "(A\u2013H) Binding affinity of the 1E6 TCR interaction at 25\u00b0C using SPR.", "section": "FIG", "ner": [ [ 6, 22, "Binding affinity", "evidence" ], [ 30, 37, "1E6 TCR", "complex_assembly" ], [ 64, 67, "SPR", "experimental_method" ] ] }, { "sid": 159, "sent": "Eight serial dilutions of the 1E6 TCR were measured (shown in the inset); representative data from 3 independent experiments are plotted.", "section": "FIG", "ner": [ [ 30, 37, "1E6 TCR", "complex_assembly" ] ] }, { "sid": 160, "sent": "The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y= [P1x]/[P2 + X]).", "section": "FIG", "ner": [ [ 4, 32, "equilibrium binding constant", "evidence" ], [ 34, 36, "KD", "evidence" ], [ 69, 88, "nonlinear curve fit", "experimental_method" ] ] }, { "sid": 161, "sent": "In order to calculate each response, the 1E6 TCR was also injected over a control sample (HLA-A*0201\u2013ILAKFLHWL) that was deducted from the experimental data.", "section": "FIG", "ner": [ [ 41, 48, "1E6 TCR", "complex_assembly" ], [ 90, 110, "HLA-A*0201\u2013ILAKFLHWL", "complex_assembly" ] ] }, { "sid": 162, "sent": "(A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) \u0394G values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson\u2019s coefficient analysis (r) and P value (including approximate values from A and B).", "section": "FIG", "ner": [ [ 4, 21, "1E6-A2-MVWGPDPLYV", "complex_assembly" ], [ 47, 64, "1E6-A2-YLGGPDFPTI", "complex_assembly" ], [ 90, 107, "1E6-A2-ALWGPDPAAA", "complex_assembly" ], [ 113, 130, "1E6-A2-AQWGPDPAAA", "complex_assembly" ], [ 136, 153, "1E6-A2-RQFGPDWIVA", "complex_assembly" ], [ 159, 176, "1E6-A2-RQWGPDPAAV", "complex_assembly" ], [ 182, 199, "1E6-A2-YQFGPDFPTA", "complex_assembly" ], [ 209, 226, "1E6-A2-RQFGPDFPTI", "complex_assembly" ], [ 232, 241, "\u0394G values", "evidence" ], [ 259, 262, "SPR", "experimental_method" ], [ 294, 298, "EC50", "evidence" ], [ 396, 426, "Pearson\u2019s coefficient analysis", "experimental_method" ], [ 428, 429, "r", "evidence" ], [ 435, 436, "P", "evidence" ] ] }, { "sid": 163, "sent": "(J) Effective 2D affinity (AcKa) calculated using adhesion frequency assays, using at least 5 cell pairs, and calculated as an average of 100 cell cell contacts.", "section": "FIG", "ner": [ [ 4, 25, "Effective 2D affinity", "evidence" ], [ 27, 31, "AcKa", "evidence" ], [ 50, 75, "adhesion frequency assays", "experimental_method" ] ] }, { "sid": 164, "sent": "(K) Effective 2D affinity plotted against 1/EC50 showing Pearson\u2019s coefficient analysis (r) and P value.", "section": "FIG", "ner": [ [ 4, 25, "Effective 2D affinity", "evidence" ], [ 44, 48, "EC50", "evidence" ], [ 57, 87, "Pearson\u2019s coefficient analysis", "experimental_method" ], [ 89, 90, "r", "evidence" ], [ 96, 103, "P value", "evidence" ] ] }, { "sid": 165, "sent": "The 1E6 TCR uses a conserved binding mode to engage A2-ALWGPDPAAA and the APLs.", "section": "FIG", "ner": [ [ 4, 11, "1E6 TCR", "complex_assembly" ], [ 52, 65, "A2-ALWGPDPAAA", "chemical" ], [ 74, 78, "APLs", "chemical" ] ] }, { "sid": 166, "sent": "(A) Superposition of the 1E6 TCR (multicolored illustration) in complex with all 7 APLs (multicolored sticks) and the A2-ALWGPDPAAA ligand using the HLA-A*0201 (gray illustration) molecule to align all of the structures.", "section": "FIG", "ner": [ [ 4, 17, "Superposition", "experimental_method" ], [ 25, 32, "1E6 TCR", "complex_assembly" ], [ 61, 76, "in complex with", "protein_state" ], [ 83, 87, "APLs", "chemical" ], [ 118, 131, "A2-ALWGPDPAAA", "chemical" ], [ 149, 159, "HLA-A*0201", "protein" ], [ 192, 197, "align", "experimental_method" ], [ 209, 219, "structures", "evidence" ] ] }, { "sid": 167, "sent": "The 1E6 TCR and each peptide are colored according to the APL used in the complex as in Figure 1. (B) Position of the 1E6 TCR CDR loops (multicolored lines) in each complex.", "section": "FIG", "ner": [ [ 4, 11, "1E6 TCR", "complex_assembly" ], [ 58, 61, "APL", "chemical" ], [ 118, 125, "1E6 TCR", "complex_assembly" ], [ 126, 135, "CDR loops", "structure_element" ] ] }, { "sid": 168, "sent": "The ALWGPDPAAA peptide (green sticks) is shown in the HLA-A*0201 binding groove (gray surface). (C) The C\u03b1 backbone conformation of each APL (multicolored illustration) in the context of the HLA-A*0201 \u03b11 helices (gray illustration). (D) Crossing angle of the 1E6 TCR (multicolored lines) calculated using previously published parameters in the context of the ALWGPDPAAA peptide (green sticks) bound in the HLA-A*0201 binding groove (gray surface).", "section": "FIG", "ner": [ [ 4, 14, "ALWGPDPAAA", "chemical" ], [ 54, 79, "HLA-A*0201 binding groove", "site" ], [ 137, 140, "APL", "chemical" ], [ 191, 201, "HLA-A*0201", "protein" ], [ 202, 212, "\u03b11 helices", "structure_element" ], [ 238, 252, "Crossing angle", "evidence" ], [ 260, 267, "1E6 TCR", "complex_assembly" ], [ 360, 370, "ALWGPDPAAA", "chemical" ], [ 394, 402, "bound in", "protein_state" ], [ 407, 432, "HLA-A*0201 binding groove", "site" ] ] }, { "sid": 169, "sent": "A conserved interaction with a GPD motif underpins the 1E6 TCR interaction with the APLs.", "section": "FIG", "ner": [ [ 31, 40, "GPD motif", "structure_element" ], [ 55, 62, "1E6 TCR", "complex_assembly" ], [ 84, 88, "APLs", "chemical" ] ] }, { "sid": 170, "sent": "Interaction between 1E6 TCR (gray illustration) residues Tyr97\u03b1 and Tyr97\u03b2 (the position of these side chains in the TCR in complex with all 7 APLs, and the previously reported A2-ALWGPDPAAA epitope, is shown in multicolored sticks; ref.) and the GPD peptide motif (the position of these side chains in all 7 APLs and A2-ALWGPDPAAA in complex with the 1E6 TCR is shown in multicolored sticks).", "section": "FIG", "ner": [ [ 20, 27, "1E6 TCR", "complex_assembly" ], [ 57, 63, "Tyr97\u03b1", "residue_name_number" ], [ 68, 74, "Tyr97\u03b2", "residue_name_number" ], [ 117, 120, "TCR", "complex_assembly" ], [ 121, 136, "in complex with", "protein_state" ], [ 143, 147, "APLs", "chemical" ], [ 177, 190, "A2-ALWGPDPAAA", "chemical" ], [ 247, 264, "GPD peptide motif", "structure_element" ], [ 309, 313, "APLs", "chemical" ], [ 318, 331, "A2-ALWGPDPAAA", "chemical" ], [ 332, 347, "in complex with", "protein_state" ], [ 352, 359, "1E6 TCR", "complex_assembly" ] ] }, { "sid": 171, "sent": "The rest of the peptide, and the MHC\u03b11 helix, are shown as a gray illustration.", "section": "FIG", "ner": [ [ 33, 36, "MHC", "complex_assembly" ], [ 36, 44, "\u03b11 helix", "structure_element" ] ] }, { "sid": 172, "sent": "The 1E6 TCR makes distinct peptide contacts with peripheral APL residues.", "section": "FIG", "ner": [ [ 4, 11, "1E6 TCR", "complex_assembly" ], [ 60, 63, "APL", "chemical" ] ] }, { "sid": 173, "sent": "Interactions between the 1E6 TCR and peptide residues outside of the conserved GPD motif.", "section": "FIG", "ner": [ [ 25, 32, "1E6 TCR", "complex_assembly" ], [ 69, 78, "conserved", "protein_state" ], [ 79, 88, "GPD motif", "structure_element" ] ] }, { "sid": 174, "sent": "The MHC\u03b11 helix is shown in gray illustrations.", "section": "FIG", "ner": [ [ 4, 7, "MHC", "complex_assembly" ], [ 7, 15, "\u03b11 helix", "structure_element" ] ] }, { "sid": 175, "sent": "Hydrogen bonds are shown as red dotted lines; van der Waals (vdW) contacts are shown as black dotted lines.", "section": "FIG", "ner": [ [ 0, 14, "Hydrogen bonds", "bond_interaction" ], [ 46, 74, "van der Waals (vdW) contacts", "bond_interaction" ] ] }, { "sid": 176, "sent": "Boxes show total contacts between the 1E6 TCR and each peptide ligand.", "section": "FIG", "ner": [ [ 38, 45, "1E6 TCR", "complex_assembly" ] ] }, { "sid": 177, "sent": "(A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data.", "section": "FIG", "ner": [ [ 28, 35, "1E6 TCR", "complex_assembly" ], [ 72, 85, "A2-MVWGPDPLYV", "chemical" ], [ 147, 154, "1E6 TCR", "complex_assembly" ], [ 189, 202, "A2-YLGGPDFPTI", "chemical" ], [ 262, 269, "1E6 TCR", "complex_assembly" ], [ 305, 318, "A2-ALWGPDPAAA", "chemical" ], [ 28, 35, "1E6 TCR", "complex_assembly" ], [ 72, 85, "A2-MVWGPDPLYV", "chemical" ], [ 147, 154, "1E6 TCR", "complex_assembly" ], [ 189, 202, "A2-YLGGPDFPTI", "chemical" ], [ 262, 269, "1E6 TCR", "complex_assembly" ], [ 305, 318, "A2-ALWGPDPAAA", "chemical" ] ] }, { "sid": 178, "sent": "(D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks).", "section": "FIG", "ner": [ [ 28, 35, "1E6 TCR", "complex_assembly" ], [ 72, 85, "A2-AQWGPDPAAA", "chemical" ], [ 147, 154, "1E6 TCR", "complex_assembly" ], [ 195, 208, "A2-RQFGPDWIVA", "chemical" ], [ 274, 281, "1E6 TCR", "complex_assembly" ], [ 319, 332, "A2-RQWGPDPAAV", "chemical" ], [ 395, 402, "1E6 TCR", "complex_assembly" ], [ 440, 453, "A2-YQFGPDFPTA", "chemical" ], [ 516, 523, "1E6 TCR", "complex_assembly" ], [ 559, 572, "A2-RQFGPDFPTI", "chemical" ], [ 28, 35, "1E6 TCR", "complex_assembly" ], [ 72, 85, "A2-AQWGPDPAAA", "chemical" ], [ 147, 154, "1E6 TCR", "complex_assembly" ], [ 195, 208, "A2-RQFGPDWIVA", "chemical" ], [ 274, 281, "1E6 TCR", "complex_assembly" ], [ 319, 332, "A2-RQWGPDPAAV", "chemical" ], [ 395, 402, "1E6 TCR", "complex_assembly" ], [ 440, 453, "A2-YQFGPDFPTA", "chemical" ], [ 516, 523, "1E6 TCR", "complex_assembly" ], [ 559, 572, "A2-RQFGPDFPTI", "chemical" ] ] }, { "sid": 179, "sent": "Comparison of ligated and unligated APLs.", "section": "FIG", "ner": [ [ 14, 21, "ligated", "protein_state" ], [ 26, 35, "unligated", "protein_state" ], [ 36, 40, "APLs", "chemical" ] ] }, { "sid": 180, "sent": "Superposition of each APL in unligated form and ligated to the 1E6 TCR.", "section": "FIG", "ner": [ [ 0, 13, "Superposition", "experimental_method" ], [ 22, 25, "APL", "chemical" ], [ 29, 38, "unligated", "protein_state" ], [ 48, 55, "ligated", "protein_state" ], [ 63, 70, "1E6 TCR", "complex_assembly" ] ] }, { "sid": 181, "sent": "All unligated pMHCs are shown as light green illustrations.", "section": "FIG", "ner": [ [ 4, 13, "unligated", "protein_state" ], [ 14, 19, "pMHCs", "complex_assembly" ] ] }, { "sid": 182, "sent": "Peptide sequences are shown underneath each structure aligned with the peptide structure.", "section": "FIG", "ner": [ [ 44, 53, "structure", "evidence" ], [ 79, 88, "structure", "evidence" ] ] }, { "sid": 183, "sent": "(A) A2-MVWGPDPLYV (black sticks).", "section": "FIG", "ner": [ [ 4, 17, "A2-MVWGPDPLYV", "chemical" ] ] }, { "sid": 184, "sent": "A large conformational shift was observed for Tyr8 in the ligated versus unligated states (black circle). (B) A2-YLGGPDFPTI (red sticks). (C) A2-ALWGPDPAAA (blue sticks) reproduced from previous published data. (D) A2-AQWGPDPAAA (green sticks). (E) A2-RQFGPDWIVA (dark blue sticks). (F) A2-RQWGPDPAAV (purple sticks). (G) A2-YQFGPDFPTA (yellow sticks). (H) A2-RQFGPDFPTI (cyan sticks).", "section": "FIG", "ner": [ [ 46, 50, "Tyr8", "residue_name_number" ], [ 58, 65, "ligated", "protein_state" ], [ 73, 82, "unligated", "protein_state" ], [ 110, 123, "A2-YLGGPDFPTI", "chemical" ], [ 142, 155, "A2-ALWGPDPAAA", "chemical" ], [ 215, 228, "A2-AQWGPDPAAA", "chemical" ], [ 249, 262, "A2-RQFGPDWIVA", "chemical" ], [ 287, 300, "A2-RQWGPDPAAV", "chemical" ], [ 322, 335, "A2-YQFGPDFPTA", "chemical" ], [ 357, 370, "A2-RQFGPDFPTI", "chemical" ] ] }, { "sid": 185, "sent": "The 1E6 TCR makes distinct peptide contacts with the MHC surface depending on the peptide cargo.", "section": "FIG", "ner": [ [ 4, 11, "1E6 TCR", "complex_assembly" ], [ 53, 64, "MHC surface", "site" ] ] }, { "sid": 186, "sent": "Interactions between the 1E6 TCR and the MHC \u03b11 helix residues Arg65, Lys66, and Gln72.", "section": "FIG", "ner": [ [ 25, 32, "1E6 TCR", "complex_assembly" ], [ 41, 44, "MHC", "complex_assembly" ], [ 45, 53, "\u03b11 helix", "structure_element" ], [ 63, 68, "Arg65", "residue_name_number" ], [ 70, 75, "Lys66", "residue_name_number" ], [ 81, 86, "Gln72", "residue_name_number" ] ] }, { "sid": 187, "sent": "Hydrogen bonds are shown as red dotted lines; vdW contacts are shown as black dotted lines.", "section": "FIG", "ner": [ [ 0, 14, "Hydrogen bonds", "bond_interaction" ], [ 46, 49, "vdW", "bond_interaction" ] ] }, { "sid": 188, "sent": "MHC\u03b11 helix are shown in gray illustrations.", "section": "FIG", "ner": [ [ 0, 3, "MHC", "complex_assembly" ], [ 3, 11, "\u03b11 helix", "structure_element" ] ] }, { "sid": 189, "sent": "Boxes show total contacts between the 1E6 TCR and these key residues (green boxes are MHC residues; white boxes are TCR residues).", "section": "FIG", "ner": [ [ 38, 45, "1E6 TCR", "complex_assembly" ], [ 86, 89, "MHC", "complex_assembly" ], [ 116, 119, "TCR", "complex_assembly" ] ] }, { "sid": 190, "sent": "Thermodynamic analysis of the 1E6 TCR with A2-ALWGPDPAAA and the APLs.", "section": "FIG", "ner": [ [ 0, 22, "Thermodynamic analysis", "experimental_method" ], [ 30, 37, "1E6 TCR", "complex_assembly" ], [ 43, 56, "A2-ALWGPDPAAA", "chemical" ], [ 65, 69, "APLs", "chemical" ] ] }, { "sid": 191, "sent": "Eight serial dilutions of the 1E6 TCR were injected, in duplicate, over each immobilized APL and A2-ALW at 5\u00b0C, 13\u00b0C, 18\u00b0C, 25\u00b0C, 30\u00b0C, and 37\u00b0C.", "section": "FIG", "ner": [ [ 30, 37, "1E6 TCR", "complex_assembly" ], [ 89, 92, "APL", "chemical" ], [ 97, 103, "A2-ALW", "chemical" ] ] }, { "sid": 192, "sent": "The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y = [P1x]/[P2 + X]), and thermodynamic parameters were calculated according to the Gibbs-Helmholtz equation (\u0394G\u00b0 = \u0394H \u2212 T\u0394S\u00b0).", "section": "FIG", "ner": [ [ 4, 32, "equilibrium binding constant", "evidence" ], [ 34, 36, "KD", "evidence" ], [ 69, 88, "nonlinear curve fit", "experimental_method" ], [ 173, 197, "Gibbs-Helmholtz equation", "experimental_method" ], [ 199, 202, "\u0394G\u00b0", "evidence" ], [ 205, 207, "\u0394H", "evidence" ], [ 210, 214, "T\u0394S\u00b0", "evidence" ] ] }, { "sid": 193, "sent": "The binding free energies, \u0394G\u00b0 (\u0394G\u00b0 = RTlnKD), were plotted against temperature (K) using nonlinear regression to fit the 3-parameters van\u2019t Hoff equation (RT ln KD = \u0394H\u00b0 \u2013 T\u0394S\u00b0 + \u0394Cp\u00b0[T-T0] \u2013 T\u0394Cp\u00b0 ln [T/T0] with T0 = 298 K).", "section": "FIG", "ner": [ [ 4, 25, "binding free energies", "evidence" ], [ 27, 30, "\u0394G\u00b0", "evidence" ], [ 32, 35, "\u0394G\u00b0", "evidence" ], [ 90, 110, "nonlinear regression", "experimental_method" ], [ 135, 154, "van\u2019t Hoff equation", "experimental_method" ], [ 156, 164, "RT ln KD", "evidence" ], [ 167, 170, "\u0394H\u00b0", "evidence" ], [ 173, 177, "T\u0394S\u00b0", "evidence" ], [ 180, 184, "\u0394Cp\u00b0", "evidence" ], [ 193, 198, "T\u0394Cp\u00b0", "evidence" ] ] }, { "sid": 194, "sent": "(A) 1E6-A2-ALWGPDPAAA; (B) 1E6-A2-AQWGPDPAAA; (C) 1E6-A2-RQFGPDWIVA; (D) 1E6-A2-RQWGPDPAAV, (E) 1E6-A2-YQFGPDFPTA; and (F) 1E6-A2-RQFGPDFPTI.", "section": "FIG", "ner": [ [ 4, 21, "1E6-A2-ALWGPDPAAA", "complex_assembly" ], [ 27, 44, "1E6-A2-AQWGPDPAAA", "complex_assembly" ], [ 50, 67, "1E6-A2-RQFGPDWIVA", "complex_assembly" ], [ 73, 90, "1E6-A2-RQWGPDPAAV", "complex_assembly" ], [ 96, 113, "1E6-A2-YQFGPDFPTA", "complex_assembly" ], [ 123, 140, "1E6-A2-RQFGPDFPTI", "complex_assembly" ] ] }, { "sid": 195, "sent": "1E6 TCR-pMHC contacts, affinity measurements and thermodynamics", "section": "TABLE", "ner": [ [ 0, 12, "1E6 TCR-pMHC", "complex_assembly" ], [ 23, 44, "affinity measurements", "experimental_method" ], [ 49, 63, "thermodynamics", "experimental_method" ] ] } ] } }