nils-herrmann/clean_scirepeval huggingface dataset:
Identification and structural characterization of two 14-3-3 binding sites in the human peptidylarginine deiminase type VI.
Venue: Journal of structural biology

The regulation and function of peptidylarginine deiminase isoform VI (PAD6), which is a highly abundant protein associated with the cytoplasmic lattices in mammalian oocytes, is poorly understood so far. It has been shown previously, that 14-3-3 proteins, a class of regulatory adapter proteins ubiquitous in eukaryotes, bind to PAD6 in vivo in a phosphorylation dependent manner. Here we identify possible 14-3-3 binding sites in human PAD6 by in silico methods, looking for conserved, surface exposed serine residues. Two of these sites were confirmed as 14-3-3 binding sites by fluorescence polarization competition and X-ray crystallography. We furthermore suggest a role of RSK-type kinases in the phosphorylation of one of these two binding sites and provide evidence in the form of in vitro kinase assays with p70S6 kinase and RSK1.
How many packing contacts are observed in protein crystals?
Venue: Journal of structural biology

Most of the protein three-dimensional structures were determined with crystallographic methods in the solid state, where each molecule (therein after referred to as the "reference molecule") is surrounded by several other molecules that can influence its structure. The number of molecules that surround the reference molecule in protein crystal structures is examined on the basis of data extracted from the protein data bank. It is observed that there are as few as three and as many as 18 molecules in contact with the reference molecule, with an average number of about 10. The number of molecules around the reference molecule is smaller at higher symmetries than at lower symmetries and does not depend on the protein shape and dimension. The results reported here are a benchmark that may be useful the detection of potential anomalies and for the analyses of structural information both stored in databases or novel.
Structural characterization of the PliG lysozyme inhibitor family.
Venue: Journal of structural biology

Several Gram-negative bacteria protect themselves against the lytic action of host lysozymes by producing specific proteinaceous inhibitors. So far, four different families of lysozyme inhibitors have been identified including Ivy (Inhibitor of vertebrate lysozyme), MliC/PliC (Membrane associated/periplasmic inhibitor of C-type lysozyme), PliI and PliG (periplasmic inhibitors of I- and G-type lysozymes, respectively). Here we provide the first crystallographic description of the PliG family. Crystal structures were obtained for the PliG homologues from Escherichia coli, Salmonella enterica serotype Typhimurium and Aeromonas hydrophila. These structures show that the fold of the PliG family is very distinct from that of all other families of lysozyme inhibitors. Small-angle X-ray scattering studies reveal that PliG is monomeric in solution as opposed to the dimeric PliC and PliI. The PliG family shares a highly conserved SG(x)xY sequence motif with the MliC/PliC and PliI families where it was shown to reside on a loop that blocks the active site of lysozyme leading to inhibition. Surprisingly, we found that in PliG this motif is not well exposed and not involved in the inhibitory action. Instead, we could identify a distinct cluster of surface residues that are conserved across the PliG family and are essential for efficient G-type lysozyme inhibition, as evidenced by mutagenesis studies.
Optimal noise reduction in 3D reconstructions of single particles using a volume-normalized filter.
Venue: Journal of structural biology

The high noise level found in single-particle electron cryo-microscopy (cryo-EM) image data presents a special challenge for three-dimensional (3D) reconstruction of the imaged molecules. The spectral signal-to-noise ratio (SSNR) and related Fourier shell correlation (FSC) functions are commonly used to assess and mitigate the noise-generated error in the reconstruction. Calculation of the SSNR and FSC usually includes the noise in the solvent region surrounding the particle and therefore does not accurately reflect the signal in the particle density itself. Here we show that the SSNR in a reconstructed 3D particle map is linearly proportional to the fractional volume occupied by the particle. Using this relationship, we devise a novel filter (the "single-particle Wiener filter") to minimize the error in a reconstructed particle map, if the particle volume is known. Moreover, we show how to approximate this filter even when the volume of the particle is not known, by optimizing the signal within a representative interior region of the particle. We show that the new filter improves on previously proposed error-reduction schemes, including the conventional Wiener filter as well as figure-of-merit weighting, and quantify the relationship between all of these methods by theoretical analysis as well as numeric evaluation of both simulated and experimentally collected data. The single-particle Wiener filter is applicable across a broad range of existing 3D reconstruction techniques, but is particularly well suited to the Fourier inversion method, leading to an efficient and accurate implementation.
Hydration-layer models for cryo-EM image simulation.
Venue: Journal of structural biology

To compare cryo-EM images and 3D reconstructions with atomic structures in a quantitative way it is essential to model the electron scattering by solvent (water or ice) that surrounds protein assemblies. The most rigorous method for determining the density of solvating water atoms for this purpose has been to perform molecular-dynamics (MD) simulations of the protein-water system. In this paper we adapt the ideas of bulk-water modeling that are used in the refinement of X-ray crystal structures to the cryo-EM solvent-modeling problem. We present a continuum model for solvent density which matches MD-based results to within sampling errors. However, we also find that the simple binary-mask model of Jiang and Brünger (1994) performs nearly as well as the new model. We conclude that several methods are now available for rapid and accurate modeling of cryo-EM images and maps of solvated proteins.
Quantitative assessment of forward and backward second harmonic three dimensional images of collagen Type I matrix remodeling in a stimulated cellular environment.
Venue: Journal of structural biology

The structural remodeling of collagens is important in several biological processes including wound healing, tendon repair and adaptation, fibrosis and morphogenesis. Multiphoton microscopy is efficient in the induction of highly specific second harmonic generation (SHG) signal from non-centrosymmetric macromolecules such as fibrillar collagens. Although the detectors in the reflection geometry have been normally employed for capturing the backward scattered SHG considering the wide range of engineered thick tissue applications, there are still questions about the generated 3D collagen structures because of the directional pattern of SHG signals. The present study dealt with an in vitro collagen-fibroblast raft or bioartificial tendon model where the stimulation of fibroblast cells induced lateral orientation of collagen Type I fibers. The SHG signals originating from 3D collagen matrix were captured simultaneously in both forward and backward scattering directions. Our structural analysis indicates that collagen fibers formed in such in vitro model systems are predominantly of uniform sizes and are aligned preferentially in the lateral direction. The criss-cross arrangements of laterally oriented fibers are evident in the initial stages of contraction but eventually those laterally oriented collagen fibers are found to be aligned in parallel to each other as well as to the fibroblasts after an extended period of contraction. Our comprehensive quantitative assessment of simultaneously captured forward and backward 3D SHG image datasets, which includes the SHG signal decay, fiber diameter, cell dimensions, colocalization profiles, the 3D voxel volumes and Fourier analysis, indicates strong correlation of structural features identified in forward and backward directions.
Protein interactions in 3D: from interface evolution to drug discovery.
Venue: Journal of structural biology

Over the past 10years, much research has been dedicated to the understanding of protein interactions. Large-scale experiments to elucidate the global structure of protein interaction networks have been complemented by detailed studies of protein interaction interfaces. Understanding the evolution of interfaces allows one to identify convergently evolved interfaces which are evolutionary unrelated but share a few key residues and hence have common binding partners. Understanding interaction interfaces and their evolution is an important basis for pharmaceutical applications in drug discovery. Here, we review the algorithms and databases on 3D protein interactions and discuss in detail applications in interface evolution, drug discovery, and interface prediction.
ClpP: a structurally dynamic protease regulated by AAA+ proteins.
Venue: Journal of structural biology

Proteolysis is an important process for many aspects of bacterial physiology. Clp proteases carry out a large proportion of protein degradation in bacteria. These enzymes assemble in complexes that combine the protease ClpP and the unfoldase, ClpA or ClpX. ClpP oligomerizes as two stacked heptameric rings enclosing a central chamber containing the proteolytic sites. ClpX and ClpA assemble into hexameric rings that bind both axial surfaces of the ClpP tetradecamer forming a barrel-like complex. ClpP requires association with ClpA or ClpX to unfold and thread protein substrates through the axial pore into the inner chamber where degradation occurs. A gating mechanism regulated by the ATPase exists at the entry of the ClpP axial pore and involves the N-terminal regions of the ClpP protomers. These gating motifs are located at the axial regions of the tetradecamer but in most crystal structures they are not visible. We also lack structural information about the ClpAP or ClpXP complexes. Therefore, the structural details of how the axial gate in ClpP is regulated by the ATPases are unknown. Here, we review our current understanding of the conformational changes that ClpA or ClpX induce in ClpP to open the axial gate and increase substrate accessibility into the degradation chamber. Most of this knowledge comes from the recent crystal structures of ClpP in complex with acyldepsipeptides (ADEP) antibiotics. These small molecules are providing new insights into the gating mechanism of this protease because they imitate the interaction of ClpA/ClpX with ClpP and activate its protease activity.
Electron microscopy of biotinylated protein complexes bound to streptavidin monolayer crystals.
Venue: Journal of structural biology

Chemical biotinylation of protein complexes followed by binding to two-dimensional (monolayer) crystals of streptavidin is shown to be an effective way to prepare cryo-EM specimens from samples at low protein concentration. Three different multiprotein complexes are used to demonstrate the generality of this method. In addition, native thermosomes, purified from Sulfolobus solfataricus P2, are used to demonstrate that a uniform distribution of Euler angles is produced, even though this particle is known to adopt a preferred orientation when other methods of cryo-EM specimen preparation are used.
Chaperone networks in protein disaggregation and prion propagation.
Venue: Journal of structural biology

The oligomeric AAA+ chaperones Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 cooperate with cognate Hsp70/Hsp40 chaperone machineries in the reactivation of aggregated proteins in E. coli and S. cerevisiae. In addition, Hsp104 and Hsp70/Hsp40 are crucial for the maintenance of prion aggregates in yeast cells. While the bichaperone system efficiently solubilizes stress-generated amorphous aggregates, structurally highly ordered prion fibrils are only partially processed, resulting in the generation of fragmented prion seeds that can be transmitted to daughter cells for stable inheritance. Here, we describe and discuss the most recent mechanistic findings on yeast Hsp104 and Hsp70/Hsp40 cooperation in the remodeling of both types of aggregates, emphasizing similarities in the mechanism but also differences in the sensitivities towards chaperone activities.
Cdc48p/p97-mediated regulation of mitochondrial morphology is Vms1p-independent.
Venue: Journal of structural biology

Cdc48p/p97 is a cytosolic essential AAA chaperone, which regulates multiple cellular reactions in a ubiquitin-dependent manner. We have recently shown that Cdc48p exhibits positively cooperative ATPase activity and loss of the positive cooperativity results in yeast cell death. Here we show that loss of the positive cooperativity of the yeast Cdc48p ATPase activity led to severe mitochondrial aggregation. The actin cytoskeleton and distribution of the ER-mitochondria tethering complex (ERMES) were eliminated from the cause of the mitochondrial aggregation. Instead, a mitochondrial outer membrane protein Fzo1p, which is required for mitochondrial fusion, and components of ERMES, which is involved in mitochondrial morphology, were remarkably stabilized in the Cdc48p mutants. In the last couple of years, it was shown that Vms1p functions as a cofactor of Cdc48p for the function of protein degradation of mitochondrial outer membrane proteins. Nevertheless, we found that Vms1p was not involved in the Cdc48p-dependent mitochondrial aggregation and loss of Vms1p did not significantly affect degradation rates of proteins anchored to the mitochondrial outer membrane. These results suggest that Cdc48p controls mitochondrial morphology by regulating turnover of proteins involved in mitochondrial morphology in a Vms1p-independent manner.
The combination of hydrogen/deuterium exchange or chemical cross-linking techniques with mass spectrometry: mapping of human 14-3-3ζ homodimer interface.
Venue: Journal of structural biology

Hydrogen/deuterium (H/D) exchange or chemical cross-linking by soluble carbodiimide (EDC) was employed in combination with high-resolution mass spectrometry (MS) to extend our knowledge about contact surface regions involved in the well-characterized model of interaction between two molecules of human 14-3-3ζ regulatory protein. The H/D exchange experiment provided low resolution mapping of interaction in the homodimeric 14-3-3ζ complex. A lower level of deuteration, suggesting structural protection, of two sequential segments has been demonstrated for dimeric 14-3-3ζ wild type relative to the monomeric mutant 14-3-3ζ S58D. The N-terminal sequence (the first 27 residues) from one subunit interacts with region αC'and αD'-helices (residues 45-98) of the other molecule across the dimer interface. To identify interacting amino acid residues within the studied complex, a chemical cross-linking reaction was carried out to produce the covalent homodimer, which was detected by SDS-PAGE. The MS analysis (following tryptic in-gel digestion) employing both high resolution and tandem mass spectrometry revealed cross-linked amino acid residues. Two alternative salt bridges between Glu81 and either Lys9 or the N-terminal amino group have been found to participate in transient interactions of the 14-3-3ζ isotype homodimerization. The data obtained, which have never previously been reported, were used to modify the published 14-3-3 crystal structure using molecular modeling. Based on our findings, utilization of this combination of experimental approaches, which preserve protein native structures, is suitable for mapping the contact between two proteins and also allows for the description of transient interactions or of regions with flexible structure in the studied protein complexes.
Transient transfection coupled to baculovirus infection for rapid protein expression screening in insect cells.
Venue: Journal of structural biology

Baculovirus infected insect cells are widely used for heterologous protein expression. Despite the power of this system, the use of baculovirus techniques for protein expression screening is hampered by the time and resources needed to generate each recombinant baculovirus. Here, we show that a transfection/infection based expression system is suitable for screening of expression constructs in insect cells and represents a valid alternative to other traditional screening methodologies using recombinant baculovirus. The described method is based on gene delivery by transfection coupled to the induction of protein expression by non-recombinant baculovirus infection. Vectors that control expression by a combination of the baculovirus promoters ie1 and p10 and the enhancer element hr5 are among the ones suitable for this method. Infection with non-recombinant baculovirus drastically increases the basal activity of these elements, leading to protein over-expression. Multiple vectors can be simultaneously co-transfected/infected, making transfection/infection amenable for screening of multiple co-expressed proteins and protein complexes. Taken together, our results prove that the transfection/infection protocol is a valid and innovative approach for increasing speed and reducing costs of protein expression screening for structural and functional studies.
Modeling of the major gas vesicle protein, GvpA: from protein sequence to vesicle wall structure.
Venue: Journal of structural biology

The structure and assembly process of gas vesicles have received significant attention in recent decades, although relatively little is still known. This work combines state-of-the-art computational methods to develop a model for the major gas vesicle protein, GvpA, and explore its structure within the assembled vesicle. Elucidating this protein's structure has been challenging due to its adherent and aggregative nature, which has so far precluded in-depth biochemical analyses. Moreover, GvpA has extremely low similarity with any known protein structure, which renders homology modeling methods ineffective. Thus, alternate approaches were used to model its tertiary structure. Starting with the sequence from haloarchaeon Halobacterium sp. NRC-1, we performed ab initio modeling and threading to acquire a multitude of structure decoys, which were equilibrated and ranked using molecular dynamics and mechanics, respectively. The highest ranked predictions exhibited an α-β-β-α secondary structure in agreement with earlier experimental findings, as well as with our own secondary structure predictions. Afterwards, GvpA subunits were docked in a quasi-periodic arrangement to investigate the assembly of the vesicle wall and to conduct simulations of contact-mode atomic force microscopy imaging, which allowed us to reconcile the structure predictions with the available experimental data. Finally, the GvpA structure for two representative organisms, Anabaena flos-aquae and Calothrix sp. PCC 7601, was also predicted, which reproduced the major features of our GvpA model, supporting the expectation that homologous GvpA sequences synthesized by different organisms should exhibit similar structures.
Structural and functional deviations in disease-associated p97 mutants.
Venue: Journal of structural biology

Missense mutations that occur at the interface between two functional domains in the AAA protein p97 lead to suboptimal performance in its enzymatic activity and impaired intracellular functions, causing human disorders such as inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia (IBMPFD). Much progress has been made in characterizing these mutants at cellular, sub-cellular and molecular levels, gaining a substantial understanding of the involvement of p97 in various cellular pathways. At the tissue level, patient biopsies revealed co-localization of p97 with pathologic proteineous inclusions and rimmed vacuoles, which can be reproduced in various cellular and animal models of IBMPFD. At the subcellular level, alterations in p97's ability to bind various adaptor proteins have been demonstrated for some but not all binding partners. Biochemical and biophysical characterizations of pathogenic p97 revealed altered nucleotide binding properties in the D1-domains compared to the wild type. Structural studies showed that mutant p97 are capable of undergoing a uniform transition in the N-domain from a Down- to an Up-conformation in the presence of ATPγS, while in the wild-type p97, this conformational change can only be demonstrated in solutions but not in crystals. These structural and biochemical analyses of IBMPFD mutants shed new light into the mechanism of p97 function.
Stable nucleotide binding to DnaA requires a specific glutamic acid residue within the AAA+ box II motif.
Venue: Journal of structural biology

In complex with ATP, but not ADP, DnaA protein multimers unwind a specific region of duplex DNA within the chromosomal replication origin, oriC, triggering a series of reactions that result in initiation of DNA replication. Following replication initiation, ATP hydrolysis, which is coupled to DNA replication, results in the generation of initiation-incompetent ADP-DnaA. Suppression of overinitiation of replication requires that ADP-DnaA complexes be stably maintained until the next round of replication. Thus, the functional and structural requirements that ensure stable nucleotide binding to DnaA are crucial for proper regulation of replication. Here, we demonstrate that Glu143 of DnaA, located within the AAA+ box II N-linker motif, is a key residue involved in stable nucleotide binding. A Glu143 substitution variant of DnaA (DnaA E143A) bound to ADP on ice with an affinity similar to wild-type DnaA, but the resultant ADP-DnaA E143A complex was more labile at 37 °C than wild-type ADP-DnaA complexes. Consistent with this, conversion of ADP-DnaA E143A to ATP-DnaA E143A was stimulated at 37°C in the presence of ATP, which also stimulated replication of a minichromosome in an in vitro reconstitution reaction. Expression of DnaA E143A in vivo inhibited cell growth in an oriC-dependent manner, suggesting that DnaA E143A caused over-initiation of replication, consistent with the in vitro results. Glu is a highly conserved residue at the corresponding position of γ-proteobacterial DnaA orthologs. Our finding of the novel role for the DnaA N-linker region may represent a conserved function of this motif among those DnaA orthologs.