Symmetry Restraints Research Articles

Ab initio structure solution of proteins at atomic resolution using charge-flipping techniques and cloud computing.

Large protein structures at atomic resolution can be solved in minutes using charge-flipping techniques operating on hundreds of virtual machines (computers) on the Amazon Web Services cloud-computing platform driven by the computer programs TOPAS or TOPAS-Academic at a small financial cost. The speed of operation has allowed charge-flipping techniques to be investigated and modified, leading to two strategies that can solve a large range of difficult protein structures at atomic resolution. Techniques include the use of space-group symmetry restraints on the electron density as well as increasing the intensity of a randomly chosen high-intensity electron-density peak. It is also shown that the use of symmetry restraints increases the chance of finding a solution for low-resolution data. Finally, a flipping strategy that negates `uranium atom solutions' has been developed for structures that exhibit such solutions during charge flipping.

Enhanced Nonlinear Light Generation in Oligomers of Silicon Nanoparticles under Vector Beam Illumination.

All-dielectric nanoparticle oligomers have recently emerged as promising candidates for nonlinear optical applications. Their highly resonant collective modes, however, are difficult to access by linearly polarized beams due to symmetry restraints. In this paper, we propose a new way to increase the efficiency of nonlinear processes in all-dielectric oligomers by tightly focused azimuthally polarized cylindrical vector beam illumination. We demonstrate two orders enhancement of the third-harmonic generation signal, governed by a collective optical mode represented by out-of-plane magnetic dipoles. Crucially, the collective mode is characterized by strong electromagnetic field localization in the bulk of the nonlinear material. For comparison, we measure third-harmonic generation in the same oligomer pumped with linearly and radially polarized fundamental beams, which both show significantly lower harmonic output. We also provide numerical analysis to describe and characterize the observed effect. Our findings open a new route to enhance and modulate the third-harmonic generation efficiency of Mie-resonant isolated nanostructures by tailoring the polarization of the pump beam.

An overview of data-driven HADDOCK strategies in CAPRI rounds 38-45.

Our information-driven docking approach HADDOCK has demonstrated a sustained performance since the start of its participation to CAPRI. This is due, in part, to its ability to integrate data into the modeling process, and to the robustness of its scoring function. We participated in CAPRI both as server and manual predictors. In CAPRI rounds 38-45, we have used various strategies depending on the available information. These ranged from imposing restraints to a few residues identified from literature as being important for the interaction, to binding pockets identified from homologous complexes or template-based refinement/CA-CA restraint-guided docking from identified templates. When relevant, symmetry restraints were used to limit the conformational sampling. We also tested for a large decamer target a new implementation of the MARTINI coarse-grained force field in HADDOCK. Overall, we obtained acceptable or better predictions for 13 and 11 server and manual submissions, respectively, out of the 22 interfaces. Our server performance (acceptable or higher-quality models when considering the top 10) was better (59%) than the manual (50%) one, in which we typically experiment with various combinations of protocols and data sources. Again, our simple scoring function based on a linear combination of intermolecular van der Waals and electrostatic energies and an empirical desolvation term demonstrated a good performance in the scoring experiment with a 63% success rate across all 22 interfaces. An analysis of model quality indicates that, while we are consistently performing well in generating acceptable models, there is room for improvement for generating/identifying higher quality models.

Less Is More: Coarse-Grained Integrative Modeling of Large Biomolecular Assemblies with HADDOCK.

Predicting the 3D structure of protein interactions remains a challenge in the field of computational structural biology. This is in part due to difficulties in sampling the complex energy landscape of multiple interacting flexible polypeptide chains. Coarse-graining approaches, which reduce the number of degrees of freedom of the system, help address this limitation by smoothing the energy landscape, allowing an easier identification of the global energy minimum. They also accelerate the calculations, allowing for modeling larger assemblies. Here, we present the implementation of the MARTINI coarse-grained force field for proteins into HADDOCK, our integrative modeling platform. Docking and refinement are performed at the coarse-grained level, and the resulting models are then converted back to atomistic resolution through a distance restraints-guided morphing procedure. Our protocol, tested on the largest complexes of the protein docking benchmark 5, shows an overall ∼7-fold speed increase compared to standard all-atom calculations, while maintaining a similar accuracy and yielding substantially more near-native solutions. To showcase the potential of our method, we performed simultaneous 7 body docking to model the 1:6 KaiC-KaiB complex, integrating mutagenesis and hydrogen/deuterium exchange data from mass spectrometry with symmetry restraints, and validated the resulting models against a recently published cryo-EM structure.

Structural insights into stressosome assembly.

The stressosome transduces environmental stress signals to SigB to upregulate SigB-dependent transcription, which is required for bacterial viability. The stressosome core is composed of RsbS and at least one of the RsbR paralogs. A previous cryo-electron microscopy (cryo-EM) structure of the RsbRA-RsbS complex determined under a D2 symmetry restraint showed that the stressosome core forms a pseudo-icosahedron consisting of 60 STAS domains of RsbRA and RsbS. However, it is still unclear how RsbS and one of the RsbR paralogs assemble into the stressosome. Here, an assembly model of the stressosome is presented based on the crystal structure of the RsbS icosahedron and cryo-EM structures of the RsbRA-RsbS complex determined under diverse symmetry restraints (nonsymmetric C1, dihedral D2 and icosahedral I envelopes). 60 monomers of the crystal structure of RsbS fitted well into the I-restrained cryo-EM structure determined at 4.1 Å resolution, even though the STAS domains in the I envelope were averaged. This indicates that RsbS and RsbRA share a highly conserved STAS fold. 22 protrusions observed in the C1 envelope, corresponding to dimers of the RsbRA N-domain, allowed the STAS domains of RsbRA and RsbS to be distinguished in the stressosome core. Based on these, the model of the stressosome core was reconstructed. The mutation of RsbRA residues at the binding interface in the model (R189A/Q191A) significantly reduced the interaction between RsbRA and RsbS. These results suggest that nonconserved residues in the conserved STAS folds between RsbS and RsbR paralogs determine stressosome assembly.

Determination of contrast factors for cubic slip-systems and their application in the microstructural characterization of binary Fm[formula omitted]m materials

Theoretical attempts to rationalize the strain anisotropy of crystalline systems in terms of dislocations often include the calculation of contrast factors. However, the evaluation of such parameters can be cumbersome because elastic properties and symmetry restraints must be simultaneously taken into account, especially when calculating the distortion tensor and the elastic contributions in slip coordinate systems. In this study, a dislocation-dependent coordinate system is introduced to obtain straightforward expressions for the evaluation of individual contrast factors by a first principles approach. Therein, we report the contrast factors for KCl and NaCl regarding edge and screw dislocations; a further analysis of their microstructure was carried out through the modified Williamson-Hall method.

PyDock scoring for the new modeling challenges in docking: Protein-peptide, homo-multimers, and domain-domain interactions.

The sixth CAPRI edition included new modeling challenges, such as the prediction of protein-peptide complexes, and the modeling of homo-oligomers and domain-domain interactions as part of the first joint CASP-CAPRI experiment. Other non-standard targets included the prediction of interfacial water positions and the modeling of the interactions between proteins and nucleic acids. We have participated in all proposed targets of this CAPRI edition both as predictors and as scorers, with new protocols to efficiently use our docking and scoring scheme pyDock in a large variety of scenarios. In addition, we have participated for the first time in the servers section, with our recently developed webserver, pyDockWeb. Excluding the CASP-CAPRI cases, we submitted acceptable models (or better) for 7 out of the 18 evaluated targets as predictors, 4 out of the 11 targets as scorers, and 6 out of the 18 targets as servers. The overall success rates were below those in past CAPRI editions. This shows the challenging nature of this last edition, with many difficult targets for which no participant submitted a single acceptable model. Interestingly, we submitted acceptable models for 83% of the evaluated protein-peptide targets. As for the 25 cases of the CASP-CAPRI experiment, in which we used a larger variety of modeling techniques (template-based, symmetry restraints, literature information, etc.), we submitted acceptable models for 56% of the targets. In summary, this CAPRI edition showed that pyDock scheme can be efficiently adapted to the increasing variety of problems that the protein interactions field is currently facing. Proteins 2017; 85:487-496. © 2016 Wiley Periodicals, Inc.

PyDockSAXS: protein-protein complex structure by SAXS and computational docking.

Structural characterization of protein–protein interactions at molecular level is essential to understand biological processes and identify new therapeutic opportunities. However, atomic resolution structural techniques cannot keep pace with current advances in interactomics. Low-resolution structural techniques, such as small-angle X-ray scattering (SAXS), can be applied at larger scale, but they miss atomic details. For efficient application to protein–protein complexes, low-resolution information can be combined with theoretical methods that provide energetic description and atomic details of the interactions. Here we present the pyDockSAXS web server (http://life.bsc.es/pid/pydocksaxs) that provides an automatic pipeline for modeling the structure of a protein–protein complex from SAXS data. The method uses FTDOCK to generate rigid-body docking models that are subsequently evaluated by a combination of pyDock energy-based scoring function and their capacity to describe SAXS data. The only required input files are structural models for the interacting partners and a SAXS curve. The server automatically provides a series of structural models for the complex, sorted by the pyDockSAXS scoring function. The user can also upload a previously computed set of docking poses, which opens the possibility to filter the docking solutions by potential interface residues or symmetry restraints. The server is freely available to all users without restriction.

A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase

The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production.

Three-dimensional single-particle imaging using angular correlations from X-ray laser data

Femtosecond X-ray pulses from X-ray free-electron laser sources make it feasible to conduct room-temperature solution scattering experiments far below molecular rotational diffusion timescales. Owing to the ultra-short duration of each snapshot in these fluctuation scattering experiments, the particles are effectively frozen in space during the X-ray exposure. In contrast to standard small-angle scattering experiments, the resulting scattering patterns are anisotropic. The intensity fluctuations observed in the diffraction images can be used to obtain structural information embedded in the average angular correlation of the Fourier transform of the scattering species, of which standard small-angle scattering data are a subset. The additional information contained in the data of these fluctuation scattering experiments can be used to determine the structure of macromolecules in solution without imposing symmetry or spatial restraints during model reconstruction, reducing ambiguities normally observed in solution scattering studies. In this communication, a method that utilizes fluctuation X-ray scattering data to determine low-resolution solution structures is presented. The method is validated with theoretical data calculated from several representative molecules and applied to the reconstruction of nanoparticles from experimental data collected at the Linac Coherent Light Source.

Structure solution of network materials by solid-state NMR without knowledge of the crystallographic space group

An algorithm is presented for solving the structures of silicate network materials such as zeolites or layered silicates from solid-state 29Si double-quantum NMR data for situations in which the crystallographic space group is not known. The algorithm is explained and illustrated in detail using a hypothetical two-dimensional network structure as a working example. The algorithm involves an atom-by-atom structure building process in which candidate partial structures are evaluated according to their agreement with SiOSi connectivity information, symmetry restraints, and fits to 29Si double quantum NMR curves followed by minimization of a cost function that incorporates connectivity, symmetry, and quality of fit to the double quantum curves. The two-dimensional network material is successfully reconstructed from hypothetical NMR data that can be reasonably expected to be obtained for real samples. This advance in “NMR crystallography” is expected to be important for structure determination of partially ordered silicate materials for which diffraction provides very limited structural information.

Asymmetric Mode of Ca2+-S100A4 Interaction with Nonmuscle Myosin IIA Generates Nanomolar Affinity Required for Filament Remodeling

SummaryFilament assembly of nonmuscle myosin IIA (NMIIA) is selectively regulated by the small Ca2+-binding protein, S100A4, which causes enhanced cell migration and metastasis in certain cancers. Our NMR structure shows that an S100A4 dimer binds to a single myosin heavy chain in an asymmetrical configuration. NMIIA in the complex forms a continuous helix that stretches across the surface of S100A4 and engages the Ca2+-dependent binding sites of each subunit in the dimer. Synergy between these sites leads to a very tight association (KD ∼1 nM) that is unique in the S100 family. Single-residue mutations that remove this synergy weaken binding and ameliorate the effects of S100A4 on NMIIA filament assembly and cell spreading in A431 human epithelial carcinoma cells. We propose a model for NMIIA filament disassembly by S100A4 in which initial binding to the unstructured NMIIA tail initiates unzipping of the coiled coil and disruption of filament packing.

Molecular Dynamics Flexible Fitting of Poliovirus Structural Transitions During Initiation of Infection at Atomic Resolution

Poliovirus attaches to the surface of a host cell by binding to the poliovirus receptor (PVR). Interaction of poliovirus with PVR triggers a conformational change that converts it from the 160S to the 135S state. The 135S poliovirus particle unbinds from PVR and directly associates with the membrane to initiate cell entry. Structures of the poliovirus-receptor complex and the 135S particle are only available as cryo-electron microscopy density maps that do not resolve atomic details. However, such maps permit atomic level structural assignment when x-ray structures are flexibly matched to them, for example through molecular dynamics simulation. The respective method is called Molecular Dynamics Flexible Fitting (MDFF). A complete atomic model of the poliovirus 160S particle bound to PVR has been determined by MDFF with explicit solvent and symmetry restraints based on a 2.2 A x-ray structure of poliovirus, 3.5 A x-ray structure of the D1-D2 domains of PVR, and an 8.0 A cryoEM structure of the 160S-PVR complex. PVR is found to form extensive interactions with the core beta strands, GH loop, and C-terminus of the VP1 capsid protein. A complete atomic structure of the poliovirus 135S cell entry intermediate was also determined by MDFF based on a 9.6 A cryo-EM density map. Flexible rearrangements of the VP1 GH and VP2 EF loops are observed. Molecular dynamics simulations based on the 160S-PVR and 135S atomic structures determined by MDFF provide an opportunity to identify the pathway of the 160S-to-135S structural transition and visualize the infection process of a non-enveloped virus in full atomic detail.

The Holo-Apoptosome: Activation of Procaspase-9 and Interactions with Caspase-3

Activation of procaspase-9 on the apoptosome is a pivotal step in the intrinsic cell death pathway. We now provide further evidence that caspase recruitment domains of pc-9 and Apaf-1 form a CARD-CARD disk that is flexibly tethered to the apoptosome. In addition, a 3D reconstruction of the pc-9 apoptosome was calculated without symmetry restraints. In this structure, p20 and p10 catalytic domains of a single pc-9 interact with nucleotide binding domains of adjacent Apaf-1 subunits. Together, disk assembly and pc-9 binding create an asymmetric proteolysis machine. We also show that CARD-p20 and p20-p10 linkers play important roles in pc-9 activation. Based on the data, we propose a proximity-induced association model for pc-9 activation on the apoptosome. We also show that pc-9 and caspase-3 have overlapping binding sites on the central hub. These binding sites may play a role in pc-3 activation and could allow the formation of hybrid apoptosomes with pc-9 and caspase-3 proteolytic activities.

Der p 5 Crystal Structure Provides Insight into the Group 5 Dust Mite Allergens

Group 5 allergens from house dust mites elicit strong IgE antibody binding in mite-allergic patients. The structure of Der p 5 was determined by x-ray crystallography to better understand the IgE epitopes, to investigate the biologic function in mites, and to compare with the conflicting published Blo t 5 structures, designated 2JMH and 2JRK in the Protein Data Bank. Der p 5 is a three-helical bundle similar to Blo t 5, but the interactions of the helices are more similar to 2JMH than 2JRK. The crystallographic asymmetric unit contains three dimers of Der p 5 that are not exactly alike. Solution scattering techniques were used to assess the multimeric state of Der p 5 in vitro and showed that the predominant state was monomeric, similar to Blo t 5, but larger multimeric species are also present. In the crystal, the formation of the Der p 5 dimer creates a large hydrophobic cavity of approximately 3000 A(3) that could be a ligand-binding site. Many allergens are known to bind hydrophobic ligands, which are thought to stimulate the innate immune system and have adjuvant-like effects on IgE-mediated inflammatory responses.

The Crystal Structure of Dynein Intermediate Chain-Light Chain Roadblock Complex Gives New Insights into Dynein Assembly

The roadblock/LC7 dynein light chain is a ubiquitous component of all dyneins and is essential for many diverse processes including proper axonal transport and dendrite growth. In addition, LC7 functions in non-dynein transcriptional activation of the transforming growth factor-beta complex. Crystal structures of Drosophila melanogaster LC7 in the apo form and in complex with a segment of the disordered N-terminal domain of dynein intermediate chain (IC) provide the first definitive identification of the IC sequence recognized by LC7. The site, confirmed by isothermal titration calorimetry studies, overlaps the IC sequence considered in the literature to be an IC self-association domain. The IC peptide binds as two amphipathic helices that lie along an extensive hydrophobic cleft on LC7 and ends with a polar side-chain interaction network that includes conserved residues from both proteins. The LC7 recognition sequence on IC and its interface with LC7 are well conserved and are, thus, likely representative of all IC x LC7 structures. Interestingly, the position of bound IC in the IC x LC7 complex mimics a helix that is integrated into the primary structure in distantly related LC7 homologs. The IC x LC7 structure further shows that the naturally occurring robl(Z) deletion mutation contains the majority of the IC binding site and suggests that promotion of IC binding by phosphorylation of LC7 is an indirect effect.

Mechanism of Aldolase Control of Sorting Nexin 9 Function in Endocytosis

Sorting nexin 9 (SNX9) functions in a complex with the GTPase dynamin-2 at clathrin-coated pits, where it provokes fission of vesicles to complete endocytosis. Here the SNX9.dynamin-2 complex binds to clathrin and adapter protein complex 2 (AP-2) that line these pits, and this occurs through interactions of the low complexity domain (LC4) of SNX9 with AP-2. Intriguingly, localization of the SNX9.dynamin-2 complex to clathrin-coated pits is blocked by interactions with the abundant glycolytic enzyme aldolase, which also binds to the LC4 domain of SNX9. The crystal structure of the LC4 motif of human SNX9 in complex with aldolase explains the biochemistry and biology of this interaction, where SNX9 binds near the active site of aldolase via residues 165-171 that are also required for the interactions of SNX9 with AP-2. Accordingly, SNX9 binding to aldolase is structurally precluded by the binding of substrate to the active site. Interactions of SNX9 with aldolase are far more extensive and differ from those of the actin-nucleating factor WASP with aldolase, indicating considerable plasticity in mechanisms that direct the functions of the aldolase as a scaffold protein.

Gentisic Acid, a Compound Associated with Plant Defense and a Metabolite of Aspirin, Heads a New Class of in Vivo Fibroblast Growth Factor Inhibitors

Fibroblast growth factors are key proteins in many intercellular signaling networks. They normally remain attached to the extracellular matrix, which confers on them a considerable stability. The unrestrained accumulation of fibroblast growth factors in the extracellular milieu, either due to uncontrolled synthesis or enzymatic release, contributes to the pathology of many diseases. Consequently, the neutralization of improperly mobilized fibroblast growth factors is of clear therapeutic interest. In pursuing described rules to identify potential inhibitors of these proteins, gentisic acid, a plant pest-controlling compound, an aspirin and vegetarian diet common catabolite, and a component of many traditional liquors and herbal remedies, was singled out as a powerful inhibitor of fibroblast growth factors. Gentisic acid was used as a lead to identify additional compounds with better inhibitory characteristics generating a new chemical class of fibroblast growth factor inhibitors that includes the agent responsible for alkaptonuria. Through low and high resolution approaches, using representative members of the fibroblast growth factor family and their cell receptors, it was shown that this class of inhibitors may employ two different mechanisms to interfere with the assembly of the signaling complexes that trigger fibroblast growth factor-driven mitogenesis. In addition, we obtained evidence from in vivo disease models that this group of inhibitors may be of interest to treat cancer and angiogenesis-dependent diseases.

Recent Developments of the Molecular Dynamics Flexible Fitting Method

Molecular Dynamics Flexible Fitting (MDFF) is a computational method that combines structural information from X-ray crystallography and cryo-electron microscopy (cryo-EM). Cryo-EM provides structural data on biomolecules in their functional states but not at atomic resolution, while X-ray crystallography yields atomic-resolution data but for molecules in crystalline form that renders them often non-functional. MDFF employs molecular dynamics (MD) simulations to bridge the two sets of data, by adding an attractive potential derived from cryo-EM maps to the MD force field, driving atoms toward high-density regions while maintaining a stereochemically correct conformation of the molecules. MDFF has already been successfully applied to study several macromolecular complexes such as the ribosome.To further advance MDFF, we have created a test set consisting of different types of biomolecules in different conformational states. Using this test set, MDFF has been optimized for different types of molecules and conformational changes, providing a useful guideline for users applying MDFF to different biological systems. Moreover, helical symmetry restraints have been incorporated into MDFF as many biological systems studied by MDFF have helical symmetry, such as the family of microbial nitrilases. Information about helical symmetry can now be included in MDFF, improving the quality of the fit on these kinds of systems.

Structure of a Eukaryotic Nonribosomal Peptide Synthetase Adenylation Domain That Activates a Large Hydroxamate Amino Acid in Siderophore Biosynthesis

Nonribosomal peptide synthetases (NRPSs) are large, multidomain proteins that are involved in the biosynthesis of an array of secondary metabolites. We report the structure of the third adenylation domain from the siderophore-synthesizing NRPS, SidN, from the endophytic fungus Neotyphodium lolii. This is the first structure of a eukaryotic NRPS domain, and it reveals a large binding pocket required to accommodate the unusual amino acid substrate, N(delta)-cis-anhydromevalonyl-N(delta)-hydroxy-L-ornithine (cis-AMHO). The specific activation of cis-AMHO was confirmed biochemically, and an AMHO moiety was unambiguously identified as a component of the fungal siderophore using mass spectroscopy. The protein structure shows that the substrate binding pocket is defined by 17 amino acid residues, in contrast to both prokaryotic adenylation domains and to previous predictions based on modeling. Existing substrate prediction methods for NRPS adenylation domains fail for domains from eukaryotes due to the divergence of their signature sequences from those of prokaryotes. Thus, this new structure will provide a basis for improving prediction methods for eukaryotic NRPS enzymes that play important and diverse roles in the biology of fungi.