{ "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": "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": 67, "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": 68, "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": 69, "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": 70, "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": 71, "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": 72, "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": 73, "sent": "The apo structure is color ramped from blue to red.", "section": "FIG", "ner": [ [ 4, 7, "apo", "protein_state" ], [ 8, 17, "structure", "evidence" ] ] }, { "sid": 74, "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": 75, "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": 76, "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": 77, "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": 78, "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": 79, "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": 80, "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": 81, "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": 82, "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" ], [ 107, 115, "\u03b2-glucan", "chemical" ] ] }, { "sid": 83, "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": 84, "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": 85, "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": [ [ 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": 86, "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": 87, "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": 88, "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" ], [ 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": 89, "sent": "Binding thermodynamics are based on the concentration of the binding unit, XyGO2.", "section": "TABLE", "ner": [ [ 75, 80, "XyGO2", "chemical" ] ] }, { "sid": 90, "sent": "Weak binding represents a Ka of <500 M\u22121.", "section": "TABLE", "ner": [ [ 26, 28, "Ka", "evidence" ] ] }, { "sid": 91, "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": 92, "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": 93, "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": 94, "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": 95, "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": 96, "sent": "Such domains are not typically involved in carbohydrate binding.", "section": "RESULTS", "ner": [ [ 0, 12, "Such domains", "structure_element" ], [ 43, 55, "carbohydrate", "chemical" ] ] }, { "sid": 97, "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": 98, "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": 99, "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": 100, "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": 101, "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": 102, "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": 103, "sent": "Prolines between domains are indicated as spheres.", "section": "FIG", "ner": [ [ 0, 8, "Prolines", "residue_name" ] ] }, { "sid": 104, "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": 105, "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": 106, "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": 107, "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" ] ] }, { "sid": 108, "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": 109, "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": 110, "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": 111, "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": 112, "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": 113, "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": 114, "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": 115, "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": 116, "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": 117, "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": 118, "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": 119, "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": 120, "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" ], [ 21, 28, "xylosyl", "chemical" ], [ 40, 44, "Glc3", "residue_name_number" ], [ 52, 56, "Q407", "residue_name_number" ], [ 107, 114, "xylosyl", "chemical" ], [ 123, 127, "Xyl1", "residue_name_number" ] ] }, { "sid": 121, "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": 122, "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": 123, "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": 124, "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": 125, "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": 126, "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": 127, "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": 128, "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": 129, "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": 130, "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": 131, "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": 132, "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": 133, "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": 134, "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": 135, "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": 136, "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": 137, "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": 138, "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": 139, "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": 140, "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": 141, "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": 142, "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": 143, "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": 144, "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": 145, "sent": "The \u0394SGBP-A (\u0394Bacova_02651) strain (cf.", "section": "RESULTS", "ner": [ [ 4, 11, "\u0394SGBP-A", "mutant" ], [ 13, 26, "\u0394Bacova_02651", "mutant" ] ] }, { "sid": 146, "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": 147, "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": 148, "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": 149, "sent": "Similarly, the function of SGBP-A extends beyond glycan binding.", "section": "RESULTS", "ner": [ [ 27, 33, "SGBP-A", "protein" ], [ 49, 55, "glycan", "chemical" ] ] }, { "sid": 150, "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": 151, "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": 152, "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": 153, "sent": "The specific glycan signal that upregulates BoXyGUL is currently unknown.", "section": "RESULTS", "ner": [ [ 13, 19, "glycan", "chemical" ], [ 44, 51, "BoXyGUL", "gene" ] ] }, { "sid": 154, "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": 155, "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": 156, "sent": "Intriguingly, the \u0394SGBP-B strain (\u0394Bacova_02650) (cf.", "section": "RESULTS", "ner": [ [ 18, 25, "\u0394SGBP-B", "mutant" ], [ 34, 47, "\u0394Bacova_02650", "mutant" ] ] }, { "sid": 157, "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": 158, "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": 159, "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": 160, "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": 161, "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": 162, "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": 163, "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": 164, "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": 165, "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": 166, "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": 167, "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": 168, "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": 169, "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": 170, "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": 171, "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": 172, "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": 173, "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": 174, "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": 175, "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": 176, "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": 177, "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": 178, "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": 179, "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": 180, "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": 181, "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": 182, "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": 183, "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": 184, "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": 185, "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": 186, "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": 187, "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": 188, "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": 189, "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": 190, "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": 191, "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": 192, "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": 193, "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": 194, "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": 195, "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": 196, "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": 197, "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": 198, "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": 199, "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": 200, "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": 201, "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": 202, "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" ] ] }, { "sid": 203, "sent": "Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont", "section": "REF", "ner": [ [ 8, 14, "glycan", "chemical" ] ] } ] }, "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": [ [ 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" ] ] }, { "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" ], [ 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" ] ] } ] }, "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" ], [ 58, 61, "CTD", "structure_element" ], [ 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" ] ] } ] }, "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": [ [ 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": [ [ 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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ] ] }, { "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" ], [ 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" ] ] } ] }, "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", "mutant" ] ] }, { "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", "mutant" ], [ 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", "mutant" ], [ 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", "mutant" ], [ 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", "mutant" ], [ 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", "mutant" ], [ 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", "mutant" ], [ 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" ] ] } ] }, "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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ], [ 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": [ [ 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" ], [ 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" ], [ 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" ] ] } ] }, "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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ], [ 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": [ [ 33, 37, "K340", "residue_name_number" ], [ 53, 63, "nucleotide", "chemical" ], [ 86, 90, "G338", "residue_name_number" ], [ 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" ], [ 85, 89, "K329", "residue_name_number" ], [ 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" ], [ 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" ], [ 78, 84, "ribose", "chemical" ], [ 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": [ [ 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" ], [ 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" ], [ 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" ], [ 133, 148, "\u03b2-sheet surface", "structure_element" ], [ 152, 156, "RRM2", "structure_element" ], [ 180, 184, "RNP1", "structure_element" ], [ 185, 189, "F304", "residue_name_number" ], [ 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" ] ] }, { "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" ], [ 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" ] ] }, { "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" ], [ 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" ] ] }, { "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" ] ] } ] }, "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": [ [ 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" ], [ 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": [ [ 53, 64, "IDA peptide", "chemical" ] ] }, { "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, 127, "Hyp64\u2192Pro IDA", "mutant" ], [ 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, 104, "IDA Hyp64\u2192Pro", "mutant" ], [ 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", "residue_name_number" ], [ 17, 20, "IDA", "protein" ], [ 45, 51, "pocket", "site" ], [ 62, 67, "HAESA", "protein" ], [ 68, 77, "LRRs 8\u201310", "structure_element" ], [ 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", "residue_name_number" ], [ 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": [ [ 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" ], [ 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", "residue_name_number" ], [ 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": [ [ 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": "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": 128, "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": 129, "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": 130, "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" ], [ 75, 80, "Lys66", "residue_name_number" ], [ 80, 83, "IDA", "protein" ], [ 103, 120, "Arg-His-Asn motif", "structure_element" ], [ 124, 127, "IDA", "protein" ] ] }, { "sid": 131, "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": 132, "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": 133, "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": 134, "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": 135, "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": 136, "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": 137, "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": 138, "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": 139, "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": 140, "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": 141, "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": 142, "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": 143, "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": 144, "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": 145, "sent": "We thus assessed their contribution to HAESA \u2013 SERK1 complex formation.", "section": "RESULTS", "ner": [ [ 39, 52, "HAESA \u2013 SERK1", "complex_assembly" ] ] }, { "sid": 146, "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": 147, "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": 148, "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": 149, "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": 150, "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": 151, "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": 152, "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": 153, "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": 154, "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": 155, "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": 156, "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": 157, "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": 158, "sent": "It has been previously suggested that SERK1 can inhibit cell separation.", "section": "DISCUSS", "ner": [ [ 38, 43, "SERK1", "protein" ] ] }, { "sid": 159, "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": 160, "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": 161, "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": 162, "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": 163, "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": 164, "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": 165, "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": 166, "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": 167, "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": 168, "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": 169, "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": 170, "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": 171, "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": 172, "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": 173, "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": 174, "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": 175, "sent": "A ribbon diagram of SERK1 in the same orientation is shown alongside.", "section": "FIG", "ner": [ [ 20, 25, "SERK1", "protein" ] ] }, { "sid": 176, "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": 177, "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": 178, "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": 179, "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": 180, "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": 181, "sent": "In both cases however, the co-receptor completes the hormone binding pocket.", "section": "DISCUSS", "ner": [ [ 53, 75, "hormone binding pocket", "site" ] ] }, { "sid": 182, "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": 183, "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": 184, "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": 185, "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": 186, "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": 187, "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": 188, "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": 189, "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": 190, "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" ] ] } ] }, "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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ], [ 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": [ [ 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" ], [ 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": [ [ 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" ] ] }, { "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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ] ] }, { "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" ], [ 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" ], [ 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": [ [ 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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ], [ 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" ] ] } ] } }