nils-herrmann/clean_scirepeval huggingface dataset:
Coupling between inter-helical hydrogen bonding and water dynamics in a proton transporter.
Venue: Journal of structural biology

Long-distance proton transfers by proton pumps occurs in discrete steps that may involve the direct participation of protein sidechains and water molecules, and coupling of protonation changes to structural rearrangements of the protein matrix. Here we explore the role of inter-helical hydrogen bonding in long-distance protein conformational coupling and dynamics of internal water molecules. From molecular dynamics simulations of wild type and nine different bacteriorhodopsin mutants we find that both intra- and inter-helical hydrogen bonds are important determinants of the local protein structure, dynamics, and water interactions. Based on molecular dynamics and bioinformatics analyses, we identify an aspartate/threonine inter-helical hydrogen-bonding motif involved in controlling the local conformational dynamics. Perturbation of inter-helical hydrogen bonds can couple to rapid changes in water dynamics.
A soluble mutant of the transmembrane receptor Af1503 features strong changes in coiled-coil periodicity.
Venue: Journal of structural biology

Structures of full-length, membrane-bound proteins are essential for understanding transmembrane signaling mechanisms. However, in prokaryotic receptors no such structure has been reported, despite active research for many years. Here we present results of an alternative strategy, whereby a transmembrane receptor is made soluble by selective mutations to the membrane-spanning region, chosen by analysis of helix geometry in the transmembrane regions of chemotaxis receptors. We thus converted the receptor Af1503 from Archaeoglobus fulgidus to a soluble form by deleting transmembrane helix 1 and mutating the surface residues of transmembrane helix 2 to hydrophilic amino acids. Crystallization of this protein resulted in the structure of a tetrameric proteolytic fragment representing the modified transmembrane helices plus the cytoplasmic HAMP domain, a ubiquitous domain of prokaryotic signal transducers. The protein forms a tetramer via native parallel dimerization of the HAMP domain and non-native antiparallel dimerization of the modified transmembrane helices. The latter results in a four-helical coiled coil, characterized by unusually large changes in helix periodicity. The structure offers the first view of the junction between the transmembrane region and HAMP and explains the conservation of a key sequence motif in HAMP domains.
A simple Fourier filter for suppression of the missing wedge ray artefacts in single-axis electron tomographic reconstructions
Venue: Journal of structural biology

The limited specimen tilting range that is typically available in electron tomography gives rise to a region in the Fourier space of the reconstructed object where experimental data are unavailable – the missing wedge. Since this region is sharply delimited from the area of available data, the reconstructed signal is typically hampered by convolution with its impulse response, which gives rise to the well-known missing wedge artefacts in 3D reconstructions. Despite the recent progress in the field of reconstruction and regularization techniques, the missing wedge artefacts remain untreated in most current reconstruction workflows in structural biology. Therefore we have designed a simple Fourier angular filter that effectively suppresses the ray artefacts in the single-axis tilting projection acquisition scheme, making single-axis tomographic reconstructions easier to interpret in particular at low signal-to-noise ratio in acquired projections. The proposed filter can be easily incorporated into current electron tomographic reconstruction schemes.
The structure of the N-terminal domain of the Legionella protein SidC.
Venue: Journal of structural biology

The Gram-negative bacterium Legionella pneumophila is the causative agent of Legionnaires' disease. During infection of eukaryotic cells, the bacterium releases about 300 different bacterial effector molecules that aid in the establishment of the Legionella-containing vacuole (LCV) among which SidC is one of these secreted proteins. However, apart from membrane lipid binding the function of SidC remains elusive. In order to characterize SidC further, we have determined the crystal structure of the N-terminal domain of SidC (amino acids 1-609, referred to as SidC-N) at 2.4Å resolution. SidC-N reveals a novel fold in which 4 potential subdomains (A-D) are arranged in a crescent-like structure. None of these subdomains currently has any known structural homologues, raising the question of how this fold has evolved. These domains are highly interconnected, with a low degree of flexibility towards each other. Due to the extended arrangement of the subdomains, SidC-N may contain multiple binding sites for potential interaction partners.
Structural and functional characterization of enamel pigmentation in shrews.
Venue: Journal of structural biology

Pigmented tooth enamel occurs in several vertebrate clades, ranging from mammals to fish. Although an iron compound is associated with this orange to red colored pigmentation, its chemical and structural organization within the enamel is unknown. To determine the nature of the iron compound, we investigated heavily pigmented teeth of the northern short-tailed shrew Blarina brevicauda using combined characterization techniques such as scanning and transmission electron microscopy and synchrotron X-ray diffraction. We found that the pigmentation of the enamel with an iron content of around 8wt% results from a close to amorphous magnetite phase deposited around the nm-sized enamel crystals. Furthermore, the influence of the pigmentation on the enamel hardness was determined by nanoindentation measurements. Finally, the biomechanical function and biological context are discussed in light of the obtained results.
Structure of the pseudokinase domain of BIR2, a regulator of BAK1-mediated immune signaling in Arabidopsis.
Venue: Journal of structural biology

The BAK1-interacting receptor-like kinase 2 (BIR2) belongs to the large family of leucine-rich repeat receptor-like kinases (LRR-RLKs) that mediate development and innate immunity in plants and form a monophyletic gene family with the Drosophila Pelle and human interleukin-1 receptor-associated kinases (IRAK). BIR2 is a negative regulator of BAK1-mediated defense mechanisms and cell death responses, yet key residues that are typically required for kinase activity are not present in the BIR2 kinase domain. We have determined the crystal structure of the BIR2 cytosolic domain and show that its nucleotide binding site is occluded. NMR spectroscopy confirmed that neither wild type nor phosphorylation-mimicking mutants of BIR2 bind ATP-analogues in solution, suggesting that BIR2 is a genuine enzymatically inactive pseudokinase. BIR2 is, however, phosphorylated by its target of regulation, BAK1. Using nano LC-MS/MS analysis for site-specific analysis of phosphorylation, we found a high density of BAK1-transphosphorylation sites in the BIR2 juxta membrane domain, a region previously implicated in regulation of RLKs. Our findings provide a structural basis to better understand signaling through kinase-dead domains that are predicted to account for 20% of all Arabidopsis RLKs and 10% of all human kinases.
3D domain swapping in a chimeric c-Src SH3 domain takes place through two hinge loops.
Venue: Journal of structural biology

In the Src Homology 3 domain (SH3) the RT and n-Src loops form a pocket that accounts for the specificity and affinity in binding of proline rich motifs (PRMs), while the distal and diverging turns play a key role in the folding of the protein. We have solved the structure of a chimeric mutant c-Src-SH3 domain where specific residues at the RT- and n-Src-loops have been replaced by those present in the corresponding Abl-SH3 domain. Crystals of the chimeric protein show a single molecule in the asymmetric unit, which appears in an unfolded-like structure that upon generation of the symmetry related molecules reveals the presence of a domain swapped dimer where both, RT- and n-Src loops, act as hinge loops. In contrast, the fold of the diverging type II β-turn and the distal loop are well conserved. Our results are the first evidence for the presence of a structured diverging type II β-turn in an unfolded-like intermediate of the c-Src-SH3 domain, which can be stabilized by interactions from the β-strands of the same polypeptide chain or from a neighboring one. Futhermore, this crystallographic structure opens a unique opportunity to study the effect of the amino acid sequence of the hinge loops on the 3D domain swapping process of c-Src-SH3.
The macular degeneration-linked C1QTNF5 (S163) mutation causes higher-order structural rearrangements.
Venue: Journal of structural biology

The C1q-tumor necrosis factor 5 (C1QTNF5) protein plays a significant role in retinal pigmented epithelium (RPE) cellular adhesion. The C1QTNF5 gene is co-transcribed with the frizzled-related protein (MFRP) gene. A Ser-to-Arg mutation at site 163 (S163R) in C1QTNF5 is known to cause late-onset retinal macular degeneration (L-ORMD). Here we also found that C1QTNF5 monomers can multimerize into a bouquet-like octadecamer. We found that a novel intermolecular hydrogen-bond network of S163 that glues adjacent globular heads of C1QTNF5 together was weakened or abolished by the R163 pathogenic mutation. These findings could underlie the structural basis of this protein's adhesive function and relate to the pathogenesis of its S163R mutation. Additionally, the fact that C1QTNF5 immobilized to a resin selectively enriched detergent extracted membrane-bound MFRP, further confirmed their interaction, implying functions other than cellular adhesion for C1QTNF5.
Key structural arrangements at the C-terminus domain of CETP suggest a potential mechanism for lipid-transfer activity.
Venue: Journal of structural biology

The cholesteryl-ester transfer protein (CETP) promotes cholesteryl-ester and triglyceride transfer between lipoproteins. We evaluated the secondary structure stability of a series of small peptides derived from the C-terminus of CETP in a wide range of pH's and lipid mixtures, and studied their capability to carry out disorder-to-order secondary structure transitions dependent of lipids. We report that while a mixture of phosphatidylcholine/cholesteryl-esters forms large aggregated particles, the inclusion of a series of CETP carboxy-terminal peptides in a stable α-helix conformation, allows the formation of small homogeneous micelle-like structures. This phenomenon of lipid ordering was directly connected to secondary structural transitions at the C-terminus domain when lysophosphatidic acid and lysophosphatidylcholine lipids were employed. Circular dichroism, cosedimentation experiments, electron microscopy, as well as molecular dynamics simulations confirm this phenomenon. When purified CETP is studied, the same type of phenomenon occurs by promoting the reorganization of lipid from large to smaller particles. Our findings extend the emerging view for a novel mechanism of lipid transfer carried out by CETP, assigning its C-terminus domain the property to accomplish lipid ordering through secondary structure disorder-to-order transitions.
Three-dimensional architecture of macrofibrils in the human scalp hair cortex.
Venue: Journal of structural biology

Human scalp hairs are comprised of a central cortex enveloped by plate-like cuticle cells. The elongate cortex cells of mature fibres are composed primarily of macrofibrils-bundles of hard-keratin intermediate filaments (IFs) chemically cross-linked within a globular protein matrix. In wool, three cell types (ortho-, meso- and paracortex) contain macrofibrils with distinctly different filament arrangements and matrix fractions, but in human hair macrofibril-cell type relationships are less clear. Here we show that hair macrofibrils all have a similar matrix fraction (∼0.4) and are typically composed of a double-twist architecture in which a central IF is surrounded by concentric rings of tangentially-angled IFs. The defining parameter is the incremental angle increase (IF-increment) between IFs of successive rings. Unlike the wool orthocortex, hair double-twist macrofibrils have considerable inter-macrofibril variation in IF increment (0.05-0.35°/nm), and macrofibril size and IF increment are negatively correlated. Correspondingly, angular difference between central and outer-most IFs is up to 40° in small macrofibrils, but only 5-10° in large macrofibrils. Single cells were observed containing mixtures of macrofibrils with different diameters. These new observations advance our understanding of the nano-level and cell-level organisation of human hair, with implications for interpretation of structure with respect the potential roles of cortex cell types in defining the mechanical properties of hair.
Your personalized protein structure: Andrei N. Lupas fused to GCN4 adaptors.
Venue: Journal of structural biology

This work presents a protein structure that has been designed purely for aesthetic reasons, symbolizing decades of coiled-coil research and praising its most fundamental model system, the GCN4 leucine zipper. The GCN4 leucine zipper is a highly stable coiled coil which can be tuned to adopt different oligomeric states via mutation of its core residues. For these reasons it is used in structural studies as a stabilizing fusion adaptor. On the occasion of the 50th birthday of Andrei N. Lupas, we used it to create the first personalized protein structure: we fused the sequence ANDREI-N-LVPAS in heptad register to trimeric GCN4 adaptors and determined its structure by X-ray crystallography. The structure demonstrates the robustness and versatility of GCN4 as a fusion adaptor. We learn how proline can be accommodated in trimeric coiled coils, and put the structure into the context of the other GCN4-fusion structures known to date.
Computational study of the elastic properties of Rheum rhabarbarum tissues via surrogate models of tissue geometry.
Venue: Journal of structural biology

Plant petioles and stems are hierarchical cellular structures, displaying geometrical features defined at multiple length scales. One or more of the intermediate hierarchical levels consists of tissues in which the cellular distribution is quasi-random, a factor that affects the elastic properties of the tissues. The current work focuses on the finite element analysis (FEA) of the constituent tissues of the plant Rheum rhabarbarum (rhubarb). The geometric model is generated via a recently introduced method: the finite edge centroidal Voronoi tessellation (FECVT), which is capable to capture the gradients of cellularity and diversified pattern of cellular materials, as opposed to current approaches in literature. The effective stiffness of the tissues is obtained by using an accurate numerical homogenization technique via detailed finite element analysis of the models of sub-regions of the tissues. As opposed to a large-scale representative volume element (RVE), statistical volume elements (SVE) are considered in this work to model tissue microstructures that are highly random. 2D finite element analyses demonstrate that the distribution of cells in collenchyma and parenchyma tissue make them stiffer in two different directions, while the overall effect of the combined tissues results in approximately equal stiffness in both directions. The rhubarb tissues, on the other hand, are more compliant than periodic and quasi-uniform random cellular materials by a factor of up to 47% and 44%, respectively. The variations of the stiffness shows the stiffening role that cell shape, size, and graded cellular distribution play in the mechanics of the rhubarb tissue.
Super-resolution imaging of Escherichia coli nucleoids reveals highly structured and asymmetric segregation during fast growth.
Venue: Journal of structural biology

Bacterial replication and chromosome segregation are highly organized both in space and in time. However, spatial analysis is hampered by the resolution limit of conventional fluorescence microscopy. In this study, we incubate rapidly-growing Escherichia coli with 5-ethynyl-2'-deoxyuridine (EdU), label the resulting EdU-DNA with photoswitchable fluorophores, and image incorporated molecules with an average experimental precision of 13 nm. During the segregation process, nucleoids develop highly-defined and cell-cycle dependent hetero-structures, which contain discrete DNA fibers with diameters far below the diffraction limit. Strikingly, these structures appear temporally shifted between sister chromosomes, an asymmetry which accumulates for ongoing replication rounds. Moreover, nucleoid positioning and expansion along the bacterial length axis fit into an elongation-mediated segregation model in fast growing E. coli cultures. This is supported by close proximity of the nucleoids to the bacterial plasma membrane, the nature of the observed hetero-structures and recently found interactions of membrane-associated proteins with DNA.
Structural and mechanistic insights into the kynurenine aminotransferase-mediated excretion of kynurenic acid.
Venue: Journal of structural biology

Kynurenine aminotransferase (KAT) is a homodimeric pyridoxal protein that mediates the catalytic conversion of kynurenine (KYN) to kynurenic acid (KYA), an endogenous N-methyl-d-aspartate (NMDA) receptor antagonist. KAT is involved in the biosynthesis of glutamic and aspartic acid, functions as a neurotransmitter for the NMDA receptor in mammals, and is regulated by allosteric mechanisms. Its importance in various diseases such as schizophrenia makes KAT a highly attractive drug target. Here, we present the crystal structure of the Pyrococcus horikoshii KAT (PhKAT) in complex with pyridoxamine phosphates (PMP), KYN, and KYA. Surprisingly, the PMP was bound to the LYS-269 of phKAT by forming a covalent hydrazine bond. This crystal structure clearly shows that an amino group of KYN was transaminated to PLP, which forms a Schiff's base with the LYS-269 of the KYN. Thus, our structure confirms that the PMPs represent an intermediate state during the KAT reaction. Thus, PhKAT catalyzes the sequential conversion of KYN to KYA via the formation of an intermediate 4-(2-aminophenyl)-2,4-dioxobutanoate (4AD), which is spontaneously converted to KYA in the absence of an amino group acceptor. Furthermore, we identified the two entry and exit sites of the PhKAT homodimer for KYN and KYA, respectively. The structural data on PhKAT presented in this manuscript contributes to further the understanding of transaminase enzyme reaction mechanisms.
Automatic cryo-EM particle selection for membrane proteins in spherical liposomes.
Venue: Journal of structural biology

Random spherically constrained (RSC) single particle reconstruction is a method to obtain structures of membrane proteins embedded in lipid vesicles (liposomes). As in all single-particle cryo-EM methods, structure determination is greatly aided by reliable detection of protein "particles" in micrographs. After fitting and subtraction of the membrane density from a micrograph, normalized cross-correlation (NCC) and estimates of the particle signal amplitude are used to detect particles, using as references the projections of a 3D model. At each pixel position, the NCC is computed with only those references that are allowed by the geometric constraint of the particle's embedding in the spherical vesicle membrane. We describe an efficient algorithm for computing this position-dependent correlation, and demonstrate its application to selection of membrane-protein particles, GluA2 glutamate receptors, which present very different views from different projection directions.
Structure of MST2 SARAH domain provides insights into its interaction with RAPL.
Venue: Journal of structural biology

The STE20 kinases MST1 and MST2 are key players in mammalian Hippo pathway. The SARAH domains of MST1/2 act as a platform to mediate homodimerization and hetero-interaction with a range of adaptors including RASSFs and Salvador, which also possess SARAH domains. Here, we determined the crystal structure of human MST2 SARAH domain, which forms an antiparallel homodimeric coiled coil. Structural comparison indicates that SARAH domains of different proteins may utilize a shared dimerization module to form homodimer or heterodimer. Structure-guided mutational study identified specific interface residues critical for MST2 homodimerization. MST2 mutations disrupting its homodimerization also impaired its hetero-interaction with RAPL (also named RASSF5 and NORE1), which is mediated by their SARAH domains. Further biochemical and cellular assays indicated that SARAH domain-mediated homodimerization and hetero-interaction with RAPL are required for full activation of MST2 and therefore apoptotic functions in T cells.
Crystal structure of NTPDase2 in complex with the sulfoanthraquinone inhibitor PSB-071.
Venue: Journal of structural biology

In many vertebrate tissues CD39-like ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) act in concert with ecto-5'-nucleotidase (e5NT, CD73) to convert extracellular ATP to adenosine. Extracellular ATP is a cytotoxic, pro-inflammatory signalling molecule whereas its product adenosine constitutes a universal and potent immune suppressor. Interference with these ectonucleotidases by use of small molecule inhibitors or inhibitory antibodies appears to be an effective strategy to enhance anti-tumour immunity and suppress neoangiogenesis. Here we present the first crystal structures of an NTPDase catalytic ectodomain in complex with the Reactive Blue 2 (RB2)-derived inhibitor PSB-071. In both of the two crystal forms presented the inhibitor binds as a sandwich of two molecules at the nucleoside binding site. One of the molecules is well defined in its orientation. Specific hydrogen bonds are formed between the sulfonyl group and the nucleoside binding loop. The methylphenyl side chain functionality that improved NTPDase2-specificity is sandwiched between R245 and R394, the latter of which is exclusively found in NTPDase2. The second molecule exhibits great in-plane rotational freedom and could not be modelled in a specific orientation. In addition to this structural insight into NTPDase inhibition, the observation of the putative membrane interaction loop (MIL) in two different conformations related by a 10° rotation identifies the MIL as a dynamic section of NTPDases that is potentially involved in regulation of catalysis.