ClusPro FMFT-SAXS: Ultra-fast Filtering Using Small-Angle X-ray Scattering Data in Protein Docking

We have recently demonstrated that incorporation of small-angle X-ray scattering (SAXS)-based filtering in our heavily used docking server ClusPro improves docking results. However, the filtering step is time consuming, since ≈ 10⁵ conformations have to be sequentially processed. At the same time, we have demonstrated the possibility of ultra-fast systematic energy evaluation for all rigid body orientations of two proteins, by sampling using Fast Manifold Fourier Transform (FMFT), if energies are represented as a combination of convolution-like expressions. Here we present a novel FMFT-based algorithm FMFT-SAXS for massive SAXS computation on multiple conformations of a protein complex. This algorithm exploits the convolutional form of SAXS calculation function. FMFT-SAXS allows computation of SAXS profiles for millions of conformations in a matter of minutes, providing an opportunity to explore the whole conformational space of two interacting proteins. We demonstrate the application of the new FMFT-SAXS approach to significantly speed up SAXS filtering step in our current docking protocol (1 to 2 orders of magnitude faster, running in several minutes on a modern 16-core CPU) without loss of accuracy. This is demonstrated on the benchmark set as well as on the experimental data. The new approach is available as a part of ClusPro server (https://beta.cluspro.org) and as an open source C library (https://bitbucket.org/abc-group/libfmftsaxs).     

Improving small-angle X-ray scattering data for structural analyses of the RNA world

Defining the shape, conformation, or assembly state of an RNA in solution often requires multiple investigative tools ranging from nucleotide analog interference mapping to X-ray crystallography. A key addition to this toolbox is small-angle X-ray scattering (SAXS). SAXS provides direct structural information regarding the size, shape, and flexibility of the particle in solution and has proven powerful for analyses of RNA structures with minimal requirements for sample concentration and volumes. In principle, SAXS can provide reliable data on small and large RNA molecules. In practice, SAXS investigations of RNA samples can show inconsistencies that suggest limitations in the SAXS experimental analyses or problems with the samples. Here, we show through investigations on the SAM-I riboswitch, the Group I intron P4-P6 domain, 30S ribosomal subunit from Sulfolobus solfataricus (30S), brome mosaic virus tRNA-like structure (BMV TLS), Thermotoga maritima asd lysine riboswitch, the recombinant tRNA^val, and yeast tRNA^phe that many problems with SAXS experiments on RNA samples derive from heterogeneity of the folded RNA. Furthermore, we propose and test a general approach to reducing these sample limitations for accurate SAXS analyses of RNA. Together our method and results show that SAXS with synchrotron radiation has great potential to provide accurate RNA shapes, conformations, and assembly states in solution that inform RNA biological functions in fundamental ways.     

Structural transitions and thermodynamics of a glycine-dependent riboswitch from Vibrio cholerae

Riboswitches are complex folded RNA domains found in noncoding regions of mRNA that regulate gene expression upon small molecule binding. Recently, Breaker and coworkers reported a tandem aptamer riboswitch (VCI-II) that binds glycine cooperatively. Here, we use hydroxyl radical footprinting and small-angle X-ray scattering (SAXS) to study the conformations of this tandem aptamer as a function of Mg(2+) and glycine concentration. We fit a simple three-state thermodynamic model that describes the energetic coupling between magnesium-induced folding and glycine binding. Furthermore, we characterize the structural conformations of each of the three states: In low salt with no magnesium present, the VCI-II construct has an extended overall conformation, presumably representing unfolded structures. Addition of millimolar concentrations of Mg(2+) in the absence of glycine leads to a significant compaction and partial folding as judged by hydroxyl radical protections. In the presence of millimolar Mg(2+) concentrations, the tandem aptamer binds glycine cooperatively. The glycine binding transition involves a further compaction, additional tertiary packing interactions and further uptake of magnesium ions relative to the state in high Mg(2+) but no glycine. Employing density reconstruction algorithms, we obtain low resolution 3-D structures for all three states from the SAXS measurements. These data provide a first glimpse into the structural conformations of the VCI-II aptamer, establish rigorous constraints for further modeling, and provide a framework for future mechanistic studies.     

Domain Organization of the Monomeric Form of the Tom70 Mitochondrial Import Receptor

Tom70 is a mitochondrial protein import receptor composed of 11 tetratricopeptide repeats (TPRs). The first three TPRs form an N-terminal domain that recruits heat shock protein family chaperones, while the eight C-terminal TPRs form a domain that receives, from the bound chaperone, mitochondrial precursor proteins destined for import. Analytical ultracentrifugation and solution small-angle X-ray scattering (SAXS) analysis characterized Tom70 as an elongated monomer. A model for the Tom70 monomer was proposed based on the alternate interpretation of the domain pairings observed in the crystal structure of the Tom70 dimer and refined against the SAXS data. In this “open” model of the Tom70 monomer, the chaperone- and precursor-binding sites are exposed and lay side by side on one face of the molecule. Fluorescence anisotropy measurements indicated that monomeric Tom70 can bind both chaperone and precursor peptides and that chaperone peptide binding does not alter the affinity of Tom70 for the precursor peptide. SAXS was unable to detect any shape change in Tom70 upon chaperone binding. However, molecular modeling indicated that chaperone binding is incompatible with Tom70 dimer formation. It is proposed that the Tom70 monomer is the functional unit mediating initial chaperone docking and precursor recognition.     

On the Calculation of SAXS Profiles of Folded and Intrinsically Disordered Proteins from Computer Simulations

Solution techniques such as small-angle X-ray scattering (SAXS) play a central role in structural studies of intrinsically disordered proteins (IDPs); yet, due to low resolution, it is generally necessary to combine SAXS with additional experimental sources of data and to use molecular simulations. Computational methods for the calculation of theoretical SAXS intensity profiles can be separated into two groups, depending on whether the solvent is modeled implicitly as continuous electron density or considered explicitly. The former offers reduced computational cost but requires the definition of a number of free parameters to account for, for example, the excess density of the solvation layer. Overfitting can thus be an issue, particularly when the structural ensemble is unknown. Here, we investigate and show how small variations of the contrast of the hydration shell, δρ, severely affect the outcome, analysis and interpretation of computed SAXS profiles for folded and disordered proteins. For both the folded and disordered proteins studied here, using a default δρ may, in some cases, result in the calculation of non-representative SAXS profiles, leading to an overestimation of their size and a misinterpretation of their structural nature. The solvation layer of the different IDP simulations also impacts their size estimates differently, depending on the protein force field used. The same is not true for the folded protein simulations, suggesting differences in the solvation of the two classes of proteins, and indicating that different force fields optimized for IDPs may cause expansion of the polypeptide chain through different physical mechanisms.     

Macromolecular docking restrained by a small angle X-ray scattering profile

While many structures of single protein components are becoming available, structural characterization of their complexes remains challenging. Methods for modeling assembly structures from individual components frequently suffer from large errors, due to protein flexibility and inaccurate scoring functions. However, when additional information is available, it may be possible to reduce the errors and compute near-native complex structures. One such type of information is a small angle X-ray scattering (SAXS) profile that can be collected in a high-throughput fashion from a small amount of sample in solution. Here, we present an efficient method for protein–protein docking with a SAXS profile (FoXSDock): generation of complex models by rigid global docking with PatchDock, filtering of the models based on the SAXS profile, clustering of the models, and refining the interface by flexible docking with FireDock. FoXSDock is benchmarked on 124 protein complexes with simulated SAXS profiles, as well as on 6 complexes with experimentally determined SAXS profiles. When induced fit is less than 1.5 Å interface C_α RMSD and the fraction residues of missing from the component structures is less than 3%, FoXSDock can find a model close to the native structure within the top 10 predictions in 77% of the cases; in comparison, docking alone succeeds in only 34% of the cases. Thus, the integrative approach significantly improves on molecular docking alone. The improvement arises from an increased resolution of rigid docking sampling and more accurate scoring.     

SAXSDom: Modeling multidomain protein structures using small-angle X-ray scattering data

Many proteins are composed of several domains that pack together into a complex tertiary structure. Multidomain proteins can be challenging for protein structure modeling, particularly those for which templates can be found for individual domains but not for the entire sequence. In such cases, homology modeling can generate high quality models of the domains but not for the orientations between domains. Small-angle X-ray scattering (SAXS) reports the structural properties of entire proteins and has the potential for guiding homology modeling of multidomain proteins. In this article, we describe a novel multidomain protein assembly modeling method, SAXSDom that integrates experimental knowledge from SAXS with probabilistic Input-Output Hidden Markov model to assemble the structures of individual domains together. Four SAXS-based scoring functions were developed and tested, and the method was evaluated on multidomain proteins from two public datasets. Incorporation of SAXS information improved the accuracy of domain assembly for 40 out of 46 critical assessment of protein structure prediction multidomain protein targets and 45 out of 73 multidomain protein targets from the ab initio domain assembly dataset. The results demonstrate that SAXS data can provide useful information to improve the accuracy of domain-domain assembly. The source code and tool packages are available at https://github.com/jianlin-cheng/SAXSDom .     

The Ligand-Free State of the TPP Riboswitch: A Partially Folded RNA Structure

Riboswitches are elements of mRNA that regulate gene expression by undergoing structural changes upon binding of small ligands. Although the structures of several riboswitches have been solved with their ligands bound, the ligand-free states of only a few riboswitches have been characterized. The ligand-free state is as important for the functionality of the riboswitch as the ligand-bound form, but the ligand-free state is often a partially folded structure of the RNA, with conformational heterogeneity that makes it particularly challenging to study. Here, we present models of the ligand-free state of a thiamine pyrophosphate riboswitch that are derived from a combination of complementary experimental and computational modeling approaches. We obtain a global picture of the molecule using small-angle X-ray scattering data and use an RNA structure modeling software, MC-Sym, to fit local structural details to these data on an atomic scale. We have used two different approaches to obtaining these models. Our first approach develops a model of the RNA from the structures of its constituent junction fragments in isolation. The second approach treats the RNA as a single entity, without bias from the structure of its individual constituents. We find that both approaches give similar models for the ligand-free form, but the ligand-bound models differ for the two approaches, and only the models from the second approach agree with the ligand-bound structure known previously from X-ray crystallography. Our models provide a picture of the conformational changes that may occur in the riboswitch upon binding of its ligand. Our results also demonstrate the power of combining experimental small-angle X-ray scattering data with theoretical structure prediction tools in the determination of RNA structures beyond riboswitches.     

Integration of small-angle X-ray scattering data into structural modeling of proteins and their assemblies

A major challenge in structural biology is to determine the configuration of domains and proteins in multidomain proteins and assemblies, respectively. All available data should be considered to maximize the accuracy and precision of these models. Small-angle X-ray scattering (SAXS) efficiently provides low-resolution experimental data about the shapes of proteins and their assemblies. Thus, we integrated SAXS profiles into our software for modeling proteins and their assemblies by satisfaction of spatial restraints. Specifically, we modeled the quaternary structures of multidomain proteins with structurally defined rigid domains as well as quaternary structures of binary complexes of structurally defined rigid proteins. In addition to SAXS profiles and the component structures, we used stereochemical restraints and an atomic distance-dependent statistical potential. The scoring function is optimized by a biased Monte Carlo protocol, including quasi-Newton and simulated annealing schemes. The final prediction corresponds to the best scoring solution in the largest cluster of many independently calculated solutions. To quantify how well the quaternary structures are determined based on their SAXS profiles, we used a benchmark of 12 simulated examples as well as an experimental SAXS profile of the homotetramer d-xylose isomerase. Optimization of the SAXS-dependent scoring function generally results in accurate models if sufficiently precise approximations for the constituent rigid bodies are available; otherwise, the best scoring models can have significant errors. Thus, SAXS profiles can play a useful role in the structural characterization of proteins and assemblies if they are combined with additional data and used judiciously. Our integration of a SAXS profile into modeling by satisfaction of spatial restraints will facilitate further integration of different kinds of data for structure determination of proteins and their assemblies.     

Solution conformation and heparin-induced dimerization of the full-length extracellular domain of the human amyloid precursor protein

Proteolytic cleavage of the amyloid precursor protein (APP) by beta and gamma-secretases gives rise to the beta-amyloid peptide, considered to be a causal factor in Alzheimer's disease. Conversely, the soluble extracellular domain of APP (sAPPalpha), released upon its cleavage by alpha-secretase, plays a number of important physiological functions. Several APP fragments have been structurally characterized at atomic resolution, but the structures of intact APP and of full-length sAPPalpha have not been determined. Here, ab initio reconstruction of molecular models from high-resolution solution X-ray scattering (SAXS) data for the two main isoforms of sAPPalpha (sAPPalpha(695) and sAPPalpha(770)) provided models of sufficiently high resolution to identify distinct structural domains of APP. The fragments for which structures are known at atomic resolution were fitted within the solution models of full-length sAPPalpha, allowing localization of important functional sites (i.e. glycosylation, protease inhibitory and heparin-binding sites). Furthermore, combined results from SAXS, analytical ultracentrifugation (AUC) and size-exclusion chromatography (SEC) analysis indicate that both sAPPalpha isoforms are monomeric in solution. On the other hand, SEC, bis-ANS fluorescence, AUC and SAXS measurements showed that sAPPalpha forms a 2:1 complex with heparin. A conformational model for the sAPPalpha:heparin complex was also derived from the SAXS data. Possible implications of such complex formation for the physiological dimerization of APP and biological signaling are discussed in terms of the structural models proposed.     

Docking and small angle X-ray scattering studies of purine nucleoside phosphorylase

Docking simulations have been used to assess protein complexes with some success. Small angle X-ray scattering (SAXS) is a well-established technique to investigate protein spatial configuration. This work describes the integration of geometric docking with SAXS to investigate the quaternary structure of recombinant human purine nucleoside phosphorylase (PNP). This enzyme catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for PNP causes gradual decrease in T-cell immunity. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant rejection, rheumatoid arthritis, lupus, and T-cell lymphomas. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. The present analysis confirms the trimeric structure observed in the crystal. The potential application of the present procedure to other systems is discussed.     

The ATPase domain of SecA can form a tetramer in solution.

Preprotein translocase is a general and essential system for bacterial protein export, the minimal components of which are SecA and SecYEG. SecA is a peripheral ATPase that associates with nucleotide, preprotein, and the membrane integral SecYEG to form a translocation-competent complex. SecA can be separated into two domains: an N-terminal 68 kDa ATPase domain (N68) that binds preprotein and catalyzes ATP hydrolysis, and a 34 kDa C-terminal domain that regulates the ATPase activity of N68 and mediates dimerization. We have carried out gel filtration chromatography, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS) to demonstrate that isolated N68 self-associates to form a tetramer in solution, indicating that removal of the C-terminal domain facilitates the formation of a higher-order SecA structure. The associative process is best modelled as a monomer-tetramer equilibrium, with a K(D) value of 63 microM(3) (where K(D)=[monomer](4)/[tetramer]) so that at moderate concentrations (10 microM and above), the tetramer is the major species in solution. Hydrodynamic properties of the N68 monomer indicate that it is almost globular in shape, but the N68 tetramer has a more ellipsoidal structure. Analysis of SAXS data indicates that the N68 tetramer is a flattened, bi-lobed structure with dimensions of approximately 13.5 nm x 9.0 nm x 6.5 nm, that appears to contain a central pore.     

A novel procedure for preparation of submicron liposomes-lyophilization of oil-in-water emulsions

A novel liposome preparation method is described involving the freeze-drying of oil-in-water emulsions containing sucrose in the aqueous phase as a lyoprotectant and phospholipids in the oil as emulsifiers. The oil-in-water emulsions can be prepared by emulsification and then lyophilized to obtain dry products that will, upon rehydration, form unilamellar liposomes with a mean size of less than 200 nm, as proved by dynamic light scattering, atomic force micrographs, freeze-fracture electron micrographs, and small-angle X-ray scattering (SAXS). With or without combination with active loading, this method can be used successfully to encapsulate amphiphilic or lipophilic drugs into liposomes, although it cannot be applied to hydrophilic drugs. The lyophilized products are stable and can be rehydrated to form liposomes, even after a storage period of 10 months. Based on the encapsulation efficiency for hydrophilic drugs, as well as the scanning electron micrograph and SAXS of the freeze-dried products, a unique liposome formation mechanism is proposed.     

Combining NMR and small angle X-ray and neutron scattering in the structural analysis of a ternary protein-RNA complex

Many processes in the regulation of gene expression and signaling involve the formation of protein complexes involving multi-domain proteins. Individual domains that mediate protein-protein and protein-nucleic acid interactions are typically connected by flexible linkers, which contribute to conformational dynamics and enable the formation of complexes with distinct binding partners. Solution techniques are therefore required for structural analysis and to characterize potential conformational dynamics. Nuclear magnetic resonance spectroscopy (NMR) provides such information but often only sparse data are obtained with increasing molecular weight of the complexes. It is therefore beneficial to combine NMR data with additional structural restraints from complementary solution techniques. Small angle X-ray/neutron scattering (SAXS/SANS) data can be efficiently combined with NMR-derived information, either for validation or by providing additional restraints for structural analysis. Here, we show that the combination of SAXS and SANS data can help to refine structural models obtained from data-driven docking using HADDOCK based on sparse NMR data. The approach is demonstrated with the ternary protein-protein-RNA complex involving two RNA recognition motif (RRM) domains of Sex-lethal, the N-terminal cold shock domain of Upstream-to-N-Ras, and msl-2 mRNA. Based on chemical shift perturbations we have mapped protein-protein and protein-RNA interfaces and complemented this NMR-derived information with SAXS data, as well as SANS measurements on subunit-selectively deuterated samples of the ternary complex. Our results show that, while the use of SAXS data is beneficial, the additional combination with contrast variation in SANS data resolves remaining ambiguities and improves the docking based on chemical shift perturbations of the ternary protein-RNA complex.     

Tetrameric structure of thermostable direct hemolysin from vibrio parahaemolyticus revealed by ultracentrifugation, small-angle X-ray scattering and electron microscopy

The thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus. We have characterized the conformational properties of TDH by small-angle X-ray scattering (SAXS), ultracentrifugation and transmission electron microscopy. Sedimentation equilibrium and velocity studies revealed that the protein is tetrameric in aqueous solvents. The Guinier plot derived from SAXS data provided a radius of gyration of 29.0 Å. The elongated pattern with a shoulder of a pair distance distribution function derived from SAXS data suggested the presence of molecules with an anisotropic shape having a maximum diameter of 98 Å. Electron microscopic image analysis of the negatively stained TDH oligomer showed the presence of C₄ symmetric particles with edge and diagonal lengths of 65 Å and 80 Å, respectively. Shape reconstruction was carried out by ab initio calculations using the SAXS data with a C₄ symmetric approximation. These results suggested that the tetrameric TDH assumes an oblate structure. The hydrodynamic parameters predicted from the ab initio model differed slightly from the experimental values, suggesting the presence of flexible segments.     

Dynamic Motion and Communication in the Streptococcal C1 Phage Lysin,PlyC

The growing problem of antibiotic resistance underlies the critical need to develop new treatments to prevent and control resistant bacterial infection. Exogenous application of bacteriophage lysins results in rapid and specific destruction of Gram-positive bacteria and therefore lysins represent novel antibacterial agents. The PlyC phage lysin is the most potent lysin characterized to date and can rapidly lyse Group A, C and E streptococci. Previously, we have determined the X-ray crystal structure of PlyC, revealing a complicated and unique arrangement of nine proteins. The scaffold features a multimeric cell-wall docking assembly bound to two catalytic domains that communicate and work synergistically. However, the crystal structure appeared to be auto-inhibited and raised important questions as to the mechanism underlying its extreme potency. Here we use small angle X-ray scattering (SAXS) and reveal that the conformational ensemble of PlyC in solution is different to that in the crystal structure. We also investigated the flexibility of the enzyme using both normal mode (NM) analysis and molecular dynamics (MD) simulations. Consistent with our SAXS data, MD simulations show rotational dynamics of both catalytic domains, and implicate inter-domain communication in achieving a substrate-ready conformation required for enzyme function. Our studies therefore provide insights into how the domains in the PlyC holoenzyme may act together to achieve its extraordinary potency.     

Determination of protein oligomeric structure from small‐angle X‐ray scattering

Small‐angle X‐ray scattering (SAXS) is useful for determining the oligomeric states and quaternary structures of proteins in solution. The average molecular mass in solution can be calculated directly from a single SAXS curve collected on an arbitrary scale from a sample of unknown protein concentration without the need for beamline calibration or protein standards. The quaternary structure in solution can be deduced by comparing the experimental SAXS curve to theoretical curves calculated from proposed models of the oligomer. This approach is especially robust when the crystal structure of the target protein is known, and the candidate oligomer models are derived from the crystal lattice. When SAXS data are obtained at multiple protein concentrations, this analysis can provide insight into dynamic self‐association equilibria. Herein, we summarize the computational methods that are used to determine protein molecular mass and quaternary structure from SAXS data. These methods are organized into a workflow and demonstrated with four case studies using experimental SAXS data from the published literature.     

Molecular dynamics simulations of aqueous pullulan oligomers

Molecular dynamics (MD) simulations have been used to model small-angle X-ray scattering (SAXS) data on aqueous solutions of four oligomeric segments of the glucan pullulan: the trimer G(3) (comprising one polymer repeating unit), the hexamer (G(3))(2), the nonamer (G(3))(3), and the dodecamer (G(3))(4). The AMBER force field was used in conjunction with the GB/SA continuum solvation model to calculate both the mean global dimensions of the oligomers from the limiting small angle scattering behavior and the shorter range structural information implicit in the Debye scattering function at larger scattering angles. This same force field and solvation treatment were employed earlier by Liu et al. (Macromolecules 1999, 32, 8611-8620) with apparent success in a rotational isomeric state (RIS) treatment of the same experimental data. The present work discloses that, despite numerical success in modeling the SAXS data, the RIS treatment, which includes only the interactions within dimeric segments of the polymer chain, fails to account accurately for excluded volume effects at the range of 3-12 sugar residues in the polymer backbone. It is suggested that MD simulations using continuum solvation models can be used to circumvent errors inherent in the computationally efficient RIS treatments of polymer nano- and picosecond dynamics while at the same time avoiding the heavy computational requirements of all-atom methods.