Publications by authors named "Maxim V Petoukhov"

56 Publications

: expanded functionality and new tools for small-angle scattering data analysis.

J Appl Crystallogr 2021 Feb 1;54(Pt 1):343-355. Epub 2021 Feb 1.

European Molecular Biology Laboratory, Hamburg Site, Notkestrasse 85, Building 25 A, Hamburg, 22607, Germany.

The software suite encompasses a number of programs for the processing, visualization, analysis and modelling of small-angle scattering data, with a focus on the data measured from biological macromolecules. Here, new developments in the package are described. They include , for simulating isotropic 2D scattering patterns; , to perform operations on 2D images and masks; , a method for variance estimation of structural invariants through parametric resampling; , which computes the pair distance distribution function by a direct Fourier transform of the scattering data; , to compute the scattering data from a pair distance distribution function, allowing comparison with the experimental data; a new module in for Bayesian consensus-based concentration-independent molecular weight estimation; , an shape analysis method that optimizes the search model directly against the scattering data; , an application to set up the initial search volume for multiphase modelling of membrane proteins; , to perform quasi-atomistic modelling of liposomes with elliptical shapes; , which models conformational changes in nucleic acid structures through normal mode analysis in torsion angle space; , which reconstructs the shape of an unknown intermediate in an evolving system; and and , for modelling multilamellar and asymmetric lipid vesicles, respectively. In addition, technical updates were deployed to facilitate maintainability of the package, which include porting the graphical interface to Qt5, updating - a plugin to run a subset of tools - to be both Python 2 and 3 compatible, and adding utilities to facilitate mmCIF compatibility in future releases. All these features are implemented in , freely available for academic users at https://www.embl-hamburg.de/biosaxs/software.html.
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http://dx.doi.org/10.1107/S1600576720013412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941305PMC
February 2021

The Structure of the Potato Virus A Particles Elucidated by Small Angle X-Ray Scattering and Complementary Techniques.

Biochemistry (Mosc) 2021 Feb;86(2):230-240

Shubnikov Institute of Crystallography of Federal Scientific Research Centre "Crystallography and Photonics" of the Russian Academy of Sciences, Moscow, 119333, Russia.

Potato virus A (PVA) protein coat contains on its surface partially unstructured N-terminal domain of the viral coat protein (CP), whose structural and functional characteristics are important for understanding the mechanism of plant infection with this virus. In this work, we investigated the properties and the structure of intact PVA and partially trypsinized PVAΔ32 virions using small-angle X-ray scattering (SAXS) and complimentary methods. It was shown that after the removal of 32 N-terminal amino acids of the CP, the virion did not disintegrate and remained compact, but the helical pitch of the CP packing changed. To determine the nature of these changes, we performed ab initio modeling, including the multiphase procedure, with the geometric bodies (helices) and restoration of the PVA structure in solution using available high-resolution structures of the homologous CP from the PVY potyvirus, based on the SAXS data. As a result, for the first time, a low-resolution structure of the filamentous PVA virus, both intact and partially degraded, was elucidated under conditions close to natural. The far-UV circular dichroism spectra of the PVA and PVAΔ32 samples differed significantly in the amplitude and position of the main negative maximum. The extent of thermal denaturation of these samples in the temperature range of 20-55°C was also different. The data of transmission electron microscopy showed that the PVAΔ32 virions were mostly rod-shaped, in contrast to the flexible filamentous particles typical of the intact virus, which correlated well with the SAXS results. In general, structural analysis indicates an importance of the CP N-terminal domain for the vital functions of PVA, which can be used to develop a strategy for combating this plant pathogen.
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http://dx.doi.org/10.1134/S0006297921020115DOI Listing
February 2021

Comment on the Optimal Parameters to Derive Intrinsically Disordered Protein Conformational Ensembles from Small-Angle X-ray Scattering Data Using the Ensemble Optimization Method.

J Chem Theory Comput 2021 Apr 16;17(4):2014-2021. Epub 2021 Mar 16.

Centre de Biologie Structurale (CBS), INSERM, CNRS, Université de Montpellier, 29, rue de Navacelles, 34090 Montpellier, France.

The Ensemble Optimization Method (EOM) is a popular approach to describe small-angle X-ray scattering (SAXS) data from highly disordered proteins. The EOM algorithm selects subensembles of coexisting states from large pools of randomized conformations to fit the SAXS data. Based on the unphysical bimodal radius of gyration () distribution of conformations resulting from the EOM analysis, a recent article (Fagerberg et al. 2019, 15 (12), 6968-6983) concluded that this approach inadequately described the SAXS data measured for human Histatin 5 (Hst5), a peptide with antifungal properties. Using extensive experimental and synthetic data, we explored the origin of this observation. We found that the one-bead-per-residue coarse-grained representation with averaged scattering form factors (provided in the EOM as an add-on to represent disordered missing loops or domains) may not be appropriate for EOM analyses of scattering data from short (below 50 residues) proteins/peptides. The method of choice for these proteins is to employ atomistic models (e.g., from molecular dynamics simulations) to sample the protein conformational landscape. As a convenient alternative, we have also improved the coarse-grained approach by introducing amino acid specific form factors in the calculations. We also found that, for small proteins, the search for relatively large subensembles of 20-50 conformers (as implemented in the original EOM version) more adequately describes the conformational space sampled in solution than the procedures optimizing the ensemble size. Our observations have been added as recommendations into the information for EOM users to promote the proper utilization of the program for ensemble-based modeling of SAXS data for all types of disordered systems.
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http://dx.doi.org/10.1021/acs.jctc.1c00014DOI Listing
April 2021

Spatial organization of Dps and DNA-Dps complexes.

J Mol Biol 2021 05 11;433(10):166930. Epub 2021 Mar 11.

A.N. Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, 31/4 Leninskiy prospekt, Moscow 119071, Russia.

DNA co-crystallization with Dps family proteins is a fundamental mechanism, which preserves DNA in bacteria from harsh conditions. Though many aspects of this phenomenon are well characterized, the spatial organization of DNA in DNA-Dps co-crystals is not completely understood, and existing models need further clarification. To advance in this problem we have utilized atomic force microscopy (AFM) as the main structural tool, and small-angle X-scattering (SAXS) to characterize Dps as a key component of the DNA-protein complex. SAXS analysis in the presence of EDTA indicates a significantly larger radius of gyration for Dps than would be expected for the core of the dodecamer, consistent with the N-terminal regions extending out into solution and being accessible for interaction with DNA. In AFM experiments, both Dps protein molecules and DNA-Dps complexes adsorbed on mica or highly oriented pyrolytic graphite (HOPG) surfaces form densely packed hexagonal structures with a characteristic size of about 9 nm. To shed light on the peculiarities of DNA interaction with Dps molecules, we have characterized individual DNA-Dps complexes. Contour length evaluation has confirmed the non-specific character of Dps binding with DNA and revealed that DNA does not wrap Dps molecules in DNA-Dps complexes. Angle analysis has demonstrated that in DNA-Dps complexes a Dps molecule contacts with a DNA segment of ~6 nm in length. Consideration of DNA condensation upon complex formation with small Dps quasi-crystals indicates that DNA may be arranged along the rows of ordered protein molecules on a Dps sheet.
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http://dx.doi.org/10.1016/j.jmb.2021.166930DOI Listing
May 2021

Hybrid Polycarbosilane-Siloxane Dendrimers: Synthesis and Properties.

Polymers (Basel) 2021 Feb 17;13(4). Epub 2021 Feb 17.

Enikolopov Institute of Synthetic Polymeric Materials of Russian Academy of Sciences (ISPM RAS), 117393 Moscow, Russia.

A series of carbosilane dendrimers of the 4th, 6th, and 7th generations with a terminal trimethylsilylsiloxane layer was synthesized. Theoretical models of these dendrimers were developed, and equilibrium dendrimer conformations obtained via molecular dynamics simulations were in a good agreement with experimental small-angle X-ray scattering (SAXS) data demonstrating molecule monodispersity and an almost spherical shape. It was confirmed that the glass transition temperature is independent of the dendrimer generation, but is greatly affected by the chemical nature of the dendrimer terminal groups. A sharp increase in the zero-shear viscosity of dendrimer melts was found between the 5th and the 7th dendrimer generations, which was qualitatively identical to that previously reported for polycarbosilane dendrimers with butyl terminal groups. The viscoelastic properties of high-generation dendrimers seem to follow some general trends with an increase in the generation number, which are determined by the regular branching structure of dendrimers.
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http://dx.doi.org/10.3390/polym13040606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7922192PMC
February 2021

Structural peculiarities of lysozyme - PLURONIC complexes at the aqueous-air and liquid-liquid interfaces and in the bulk of aqueous solution.

Int J Biol Macromol 2020 May 5;158:721-731. Epub 2020 May 5.

Dpt. Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia.

Interaction between proteins and synthetic polymers that represent a perspective potential in drug delivery or/and already used in medicine plays a key role in biological functioning of both molecules along with a system as a whole. In present study association between hen egg white lysozyme and Pluronic triblock-copolymers (L121, P123 and F127) in the bulk of the solution as well as at the aqueous-air and liquid-liquid interfaces was analyzed by means of spectroscopic and radiochemical assay. In protein-Pluronic complexes lysozyme keeps the secondary structure (CD and SAXS data results), while fluorescence and UV-analysis indicates changes in the local surrounding of fluorophoric amino acid residues. Radiochemical assay in combination with molecular docking reveals the formation of the complexes, in which proline residues turned to the interface between water and hydrophobic medium.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.04.221DOI Listing
May 2020

The dimeric ectodomain of the alkali-sensing insulin receptor-related receptor (ectoIRR) has a droplike shape.

J Biol Chem 2019 11 15;294(47):17790-17798. Epub 2019 Oct 15.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia.

Insulin receptor-related receptor (IRR) is a receptor tyrosine kinase of the insulin receptor family and functions as an extracellular alkali sensor that controls metabolic alkalosis in the regulation of the acid-base balance. In the present work, we sought to analyze structural features of IRR by comparing them with those of the insulin receptor, which is its closest homolog but does not respond to pH changes. Using small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM), we investigated the overall conformation of the recombinant soluble IRR ectodomain (ectoIRR) at neutral and alkaline pH. In contrast to the well-known inverted U-shaped (or λ-shaped) conformation of the insulin receptor, the structural models reconstructed at different pH values revealed that the ectoIRR organization has a "droplike" shape with a shorter distance between the fibronectin domains of the disulfide-linked dimer subunits within ectoIRR. We detected no large-scale pH-dependent conformational changes of ectoIRR in both SAXS and AFM experiments, an observation that agreed well with previous biochemical and functional analyses of IRR. Our findings indicate that ectoIRR's sensing of alkaline conditions involves additional molecular mechanisms, for example engagement of receptor juxtamembrane regions or the surrounding lipid environment.
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http://dx.doi.org/10.1074/jbc.RA119.010390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879334PMC
November 2019

Protective Dps-DNA co-crystallization in stressed cells: an in vitro structural study by small-angle X-ray scattering and cryo-electron tomography.

FEBS Lett 2019 06 28;593(12):1360-1371. Epub 2019 May 28.

Shubnikov Institute of Crystallography of Federal Scientific Research Centre "Crystallography and Photonics", Russian Academy of Sciences, Moscow, Russia.

Under severe or prolonged stress, bacteria produce a nonspecific DNA-binding protein (Dps), which effectively protects DNA against damaging agents both in vitro and in vivo by forming intracellular biocrystals. The phenomenon of protective crystallization of DNA in living cells has been intensively investigated during the last two decades; however, the results of studies are somewhat contradictory, and up to now, there has been no direct determination of a Dps-DNA crystal structure. Here, we report the in vitro analysis of the vital process of Dps-DNA co-crystallization using two complementary structural methods: synchrotron small-angle X-ray scattering in solution and cryo-electron tomography. Importantly, for the first time, the DNA in the co-crystals was visualized, and the lattice parameters of the crystalline Dps-DNA complex were determined.
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http://dx.doi.org/10.1002/1873-3468.13439DOI Listing
June 2019

Human MICAL1: Activation by the small GTPase Rab8 and small-angle X-ray scattering studies on the oligomerization state of MICAL1 and its complex with Rab8.

Protein Sci 2019 01 31;28(1):150-166. Epub 2018 Oct 31.

Department of Biosciences, University of Milan, Via Celoria 26, 20133, Milan, Italy.

Human MICAL1 is a member of a recently discovered family of multidomain proteins that couple a FAD-containing monooxygenase-like domain to typical protein interaction domains. Growing evidence implicates the NADPH oxidase reaction catalyzed by the flavoprotein domain in generation of hydrogen peroxide as a second messenger in an increasing number of cell types and as a specific modulator of actin filaments stability. Several proteins of the Rab families of small GTPases are emerging as regulators of MICAL activity by binding to its C-terminal helical domain presumably shifting the equilibrium from the free - auto-inhibited - conformation to the active one. We here extend the characterization of the MICAL1-Rab8 interaction and show that indeed Rab8, in the active GTP-bound state, stabilizes the active MICAL1 conformation causing a specific four-fold increase of k of the NADPH oxidase reaction. Kinetic data and small-angle X-ray scattering (SAXS) measurements support the formation of a 1:1 complex between full-length MICAL1 and Rab8 with an apparent dissociation constant of approximately 8 μM. This finding supports the hypothesis that Rab8 is a physiological regulator of MICAL1 activity and shows how the protein region preceding the C-terminal Rab-binding domain may mask one of the Rab-binding sites detected with the isolated C-terminal fragment. SAXS-based modeling allowed us to propose the first model of the free full-length MICAL1, which is consistent with an auto-inhibited conformation in which the C-terminal region prevents catalysis by interfering with the conformational changes that are predicted to occur during the catalytic cycle.
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http://dx.doi.org/10.1002/pro.3512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6295892PMC
January 2019

Robo1 Forms a Compact Dimer-of-Dimers Assembly.

Structure 2018 02 4;26(2):320-328.e4. Epub 2018 Jan 4.

European Molecular Biology Laboratory, Grenoble Outstation, 71 avenue des Martyrs, 38042 Grenoble, France. Electronic address:

Roundabout (Robo) receptors provide an essential repulsive cue in neuronal development following Slit ligand binding. This important signaling pathway can also be hijacked in numerous cancers, making Slit-Robo an attractive therapeutic target. However, little is known about how Slit binding mediates Robo activation. Here we present the crystal structure of Robo1 Ig1-4 and Robo1 Ig5, together with a negative stain electron microscopy reconstruction of the Robo1 ectodomain. These results show how the Robo1 ectodomain is arranged as compact dimers, mainly mediated by the central Ig domains, which can further interact in a "back-to-back" fashion to generate a tetrameric assembly. We also observed no change in Robo1 oligomerization upon interaction with the dimeric Slit2-N ligand using fluorescent imaging. Taken together with previous studies we propose that Slit2-N binding results in a conformational change of Robo1 to trigger cell signaling.
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http://dx.doi.org/10.1016/j.str.2017.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807052PMC
February 2018

SAS-Based Structural Modelling and Model Validation.

Adv Exp Med Biol 2017 ;1009:87-105

Hamburg Unit, European Molecular Biology Laboratory, EMBL c/o DESY, Notkestrasse 85, 22607, Hamburg, Germany.

Small angle scattering of X-rays (SAXS) and neutrons (SANS) is a structural technique to study disordered systems with chaotic orientations of scattering inhomogeneities at low resolution. An important example of such systems are solutions of biological macromolecules. Rapid development in the methodology for solution scattering data interpretation and model building during the last two decades brought the analysis far beyond the determination of just few overall structural parameters (which was the only possibility in the past) and ensured SAS a firm position in the methods palette of the modern life sciences. The advances in the methodology include ab initio approaches for shape and domain structure restoration from scattering curves without a priori structural knowledge, classification and validation of the models, evaluation of potential ambiguity associated with the reconstruction. In rigid body and hybrid modelling approaches, solution scattering is synergistically used with other structural techniques utilizing the complementary information such as atomic models of the components, intramolecular contacts, subunits orientations etc. for the reconstruction of complex systems. The usual requirement of the sample monodispersity has been loosed recently and the technique can now address such systems as weakly bound oligomers and transient complexes. These state-of-the-art methods are described together with the examples of their applications and the possible ways of post-processing of the models.
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http://dx.doi.org/10.1007/978-981-10-6038-0_6DOI Listing
June 2018

Structural insights of RmXyn10A - A prebiotic-producing GH10 xylanase with a non-conserved aglycone binding region.

Biochim Biophys Acta Proteins Proteom 2018 Feb 14;1866(2):292-306. Epub 2017 Nov 14.

Biotechnology, Department of Chemistry, Lund University, PO Box 124, SE-221 00 Lund, Sweden.

Hydrolysis of arabinoxylan (AX) by glycoside hydrolase family 10 (GH10) xylanases produces xylo- and arabinoxylo-oligosaccharides ((A)XOS) which have shown prebiotic effects. The thermostable GH10 xylanase RmXyn10A has shown great potential to produce (A)XOS. In this study, the structure of RmXyn10A was investigated, the catalytic module by homology modelling and site-directed mutagenesis and the arrangement of its five domains by small-angle X-ray scattering (SAXS). Substrate specificity was explored in silico by manual docking and molecular dynamic simulations. It has been shown in the literature that the glycone subsites of GH10 xylanases are well conserved and our results suggest that RmXyn10A is no exception. The aglycone subsites are less investigated, and the modelled structure of RmXyn10A suggests that loop βα in the aglycone part of the active site contains a non-conserved α-helix, which blocks the otherwise conserved space of subsite +2. This structural feature has only been observed for one other GH10 xylanase. In RmXyn10A, docking revealed two alternative binding regions, one on either side of the α-helix. However, only one was able to accommodate arabinose-substitutions and the mutation study suggests that the same region is responsible for binding XOS. Several non-conserved structural features are most likely to be responsible for providing affinity for arabinose-substitutions in subsites +1 and +2. The SAXS rigid model of the modular arrangement of RmXyn10A displays the catalytic module close to the cell-anchoring domain while the carbohydrate binding modules are further away, likely explaining the observed lack of contribution of the CBMs to activity.
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http://dx.doi.org/10.1016/j.bbapap.2017.11.006DOI Listing
February 2018

A Spring-Loaded Mechanism Governs the Clamp-like Dynamics of the Skp Chaperone.

Structure 2017 07 22;25(7):1079-1088.e3. Epub 2017 Jun 22.

Bioinformatics Institute (A∗STAR), 30 Biopolis Street, #07-01 Matrix, 138671 Singapore, Singapore; Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, 117543 Singapore, Singapore. Electronic address:

The trimeric periplasmic holdase chaperone Skp binds and stabilizes unfolded outer membrane proteins (OMPs) as part of bacterial OMP biogenesis. Skp binds client proteins in its central cavity, thereby reducing its backbone dynamics, but the molecular mechanisms that govern Skp dynamics and adaptation to differently sized clients remains unknown. Here, we employ a combination of microsecond timescale molecular dynamics simulation, small-angle X-ray scattering, and nuclear magnetic resonance spectroscopy to reveal that Skp is remarkably flexible, and features a molecular spring-loaded mechanism in its "tentacle" arms that enables switching between two distinct conformations on sub-millisecond timescales. The conformational switch is executed around a conserved pivot element within the coiled-coil structures of the tentacles, allowing expansion of the cavity and thus accommodation of differently sized clients. The spring-loaded mechanism shows how a chaperone can efficiently modulate its structure and function in an ATP-independent manner.
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http://dx.doi.org/10.1016/j.str.2017.05.018DOI Listing
July 2017

Proteins involved in sleep homeostasis: Biophysical characterization of INC and its partners.

Biochimie 2016 Dec 24;131:106-114. Epub 2016 Sep 24.

Institute of Biostructures and Bioimaging, C.N.R., Via Mezzocannone 16, 80134 Napoli, Italy. Electronic address:

The insomniac protein of Drosophila melanogaster (INC) has a crucial role in sleep homeostasis as flies lacking the inc gene exhibit strikingly reduced and poorly consolidated sleep. Nevertheless, in vitro characterizations of INC biophysical properties and partnerships have not been yet reported. Here we report the heterologous expression of the protein and its characterization using a number of different techniques. Present data indicate that INC is endowed with a remarkable stability, which results from the cooperation of the two protein domains. Moreover, we also demonstrated and quantified the ability of INC to recognize its potential partners Cul3 and dGRASP. Taking into account the molecular organization of the protein, these two partners may be anchored simultaneously. Although there is no evident relationship between the reported INC functions and dGRASP binding, our data suggest that INC may cooperate as ligase adaptor to dGRASP ubiquitination. SAXS data collected on the complex between INC and Cul3, which represent the first structural characterization of this type of assemblies, clearly highlight the highly dynamic nature of these complexes. This strongly suggests that the functional behavior of these proteins cannot be understood if dynamic effects are not considered. Finally, the strict analogy of the biochemical/biophysical properties of INC and of its human homolog KCTD5 may reliably indicate that this latter protein and/or the closely related proteins KCTD2/KCTD17 may play important roles in human sleep regulation.
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http://dx.doi.org/10.1016/j.biochi.2016.09.013DOI Listing
December 2016

Rapid automated superposition of shapes and macromolecular models using spherical harmonics.

J Appl Crystallogr 2016 Jun 16;49(Pt 3):953-960. Epub 2016 May 16.

Hamburg Outstation, European Molecular Biology Laboratory , Notkestrasse 85, Hamburg, 22607, Germany.

A rapid algorithm to superimpose macromolecular models in Fourier space is proposed and implemented (). The method uses a normalized integrated cross-term of the scattering amplitudes as a proximity measure between two three-dimensional objects. The reciprocal-space algorithm allows for direct matching of heterogeneous objects including high- and low-resolution models represented by atomic coordinates, beads or dummy residue chains as well as electron microscopy density maps and inhomogeneous multi-phase models ( of protein-nucleic acid complexes). Using spherical harmonics for the computation of the amplitudes, the method is up to an order of magnitude faster than the real-space algorithm implemented in by Kozin & Svergun [ (2001 ▸), , 33-41]. The utility of the new method is demonstrated in a number of test cases and compared with the results of . The spherical harmonics algorithm is best suited for low-resolution shape models, . those provided by solution scattering experiments, but also facilitates a rapid cross-validation against structural models obtained by other methods.
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http://dx.doi.org/10.1107/S1600576716005793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4886985PMC
June 2016

Solution Behavior of the Intrinsically Disordered N-Terminal Domain of Retinoid X Receptor α in the Context of the Full-Length Protein.

Biochemistry 2016 Mar 15;55(12):1741-1748. Epub 2016 Mar 15.

Department of Integrative Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de la Santé et de la Recherche Médicale (INSERM) U964 / Centre National de la Recherche Scientifique (CNRS) UMR 7104 / Université de Strasbourg, 67404 Illkirch, France.

Retinoid X receptors (RXRs) are transcription factors with important functions in embryonic development, metabolic processes, differentiation, and apoptosis. A particular feature of RXRs is their ability to act as obligatory heterodimerization partners of class II nuclear receptors. At the same time, these receptors are also able to form homodimers that bind to direct repeat separated by one nucleotide hormone response elements. Since the discovery of RXRs, most of the studies focused on its ligand binding and DNA binding domains, while its N-terminal domain (NTD) harboring a ligand-independent activation function remained poorly characterized. Here, we investigated the solution properties of the NTD of RXRα alone and in the context of the full-length receptor using small-angle X-ray scattering and nuclear magnetic resonance spectroscopy. We report the solution structure of the full-length homodimeric RXRα on DNA and show that the NTD remains highly flexible within this complex.
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http://dx.doi.org/10.1021/acs.biochem.5b01122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968620PMC
March 2016

Conformational plasticity of RepB, the replication initiator protein of promiscuous streptococcal plasmid pMV158.

Sci Rep 2016 Feb 15;6:20915. Epub 2016 Feb 15.

Institute for Research in Biomedicine (IRB Barcelona), Barcelona, 08028 Spain.

DNA replication initiation is a vital and tightly regulated step in all replicons and requires an initiator factor that specifically recognizes the DNA replication origin and starts replication. RepB from the promiscuous streptococcal plasmid pMV158 is a hexameric ring protein evolutionary related to viral initiators. Here we explore the conformational plasticity of the RepB hexamer by i) SAXS, ii) sedimentation experiments, iii) molecular simulations and iv) X-ray crystallography. Combining these techniques, we derive an estimate of the conformational ensemble in solution showing that the C-terminal oligomerisation domains of the protein form a rigid cylindrical scaffold to which the N-terminal DNA-binding/catalytic domains are attached as highly flexible appendages, featuring multiple orientations. In addition, we show that the hinge region connecting both domains plays a pivotal role in the observed plasticity. Sequence comparisons and a literature survey show that this hinge region could exists in other initiators, suggesting that it is a common, crucial structural element for DNA binding and manipulation.
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http://dx.doi.org/10.1038/srep20915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753449PMC
February 2016

KSHV but not MHV-68 LANA induces a strong bend upon binding to terminal repeat viral DNA.

Nucleic Acids Res 2015 Nov 30;43(20):10039-54. Epub 2015 Sep 30.

Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras 2700-157, Portugal

Latency-associated nuclear antigen (LANA) is central to episomal tethering, replication and transcriptional regulation of γ2-herpesviruses. LANA binds cooperatively to the terminal repeat (TR) region of the viral episome via adjacent LANA binding sites (LBS), but the molecular mechanism by which LANA assembles on the TR remains elusive. We show that KSHV LANA and MHV-68 LANA proteins bind LBS DNA using strikingly different modes. Solution structure of LANA complexes revealed that while kLANA tetramer is intrinsically bent both in the free and bound state to LBS1-2 DNA, mLANA oligomers instead adopt a rigid linear conformation. In addition, we report a novel non-ring kLANA structure that displays more flexibility at its assembly interface than previously demonstrated. We identified a hydrophobic pivot point located at the dimer-dimer assembly interface, which gives rotational freedom for kLANA to adopt variable conformations to accommodate both LBS1-2 and LBS2-1-3 DNA. Alterations in the arrangement of LBS within TR or at the tetramer assembly interface have a drastic effect on the ability of kLANA binding. We also show kLANA and mLANA DNA binding functions can be reciprocated. Although KSHV and MHV-68 are closely related, the findings provide new insights into how the structure, oligomerization, and DNA binding of LANA have evolved differently to assemble on the TR DNA.
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http://dx.doi.org/10.1093/nar/gkv987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4787769PMC
November 2015

Ambiguity assessment of small-angle scattering curves from monodisperse systems.

Acta Crystallogr D Biol Crystallogr 2015 May 24;71(Pt 5):1051-8. Epub 2015 Apr 24.

European Molecular Biology Laboratory, Hamburg Unit, EMBL c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany.

A novel approach is presented for an a priori assessment of the ambiguity associated with spherically averaged single-particle scattering. The approach is of broad interest to the structural biology community, allowing the rapid and model-independent assessment of the inherent non-uniqueness of three-dimensional shape reconstruction from scattering experiments on solutions of biological macromolecules. One-dimensional scattering curves recorded from monodisperse systems are nowadays routinely utilized to generate low-resolution particle shapes, but the potential ambiguity of such reconstructions remains a major issue. At present, the (non)uniqueness can only be assessed by a posteriori comparison and averaging of repetitive Monte Carlo-based shape-determination runs. The new a priori ambiguity measure is based on the number of distinct shape categories compatible with a given data set. For this purpose, a comprehensive library of over 14,000 shape topologies has been generated containing up to seven beads closely packed on a hexagonal grid. The computed scattering curves rescaled to keep only the shape topology rather than the overall size information provide a `scattering map' of this set of shapes. For a given scattering data set, one rapidly obtains the number of neighbours in the map and the associated shape topologies such that in addition to providing a quantitative ambiguity measure the algorithm may also serve as an alternative shape-analysis tool. The approach has been validated in model calculations on geometrical bodies and its usefulness is further demonstrated on a number of experimental X-ray scattering data sets from proteins in solution. A quantitative ambiguity score (a-score) is introduced to provide immediate and convenient guidance to the user on the uniqueness of the ab initio shape reconstruction from the given data set.
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http://dx.doi.org/10.1107/S1399004715002576DOI Listing
May 2015

The C-terminal random coil region tunes the Ca²⁺-binding affinity of S100A4 through conformational activation.

PLoS One 2014 15;9(5):e97654. Epub 2014 May 15.

Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden.

S100A4 interacts with many binding partners upon Ca2+ activation and is strongly associated with increased metastasis formation. In order to understand the role of the C-terminal random coil for the protein function we examined how small angle X-ray scattering of the wild-type S100A4 and its C-terminal deletion mutant (residues 1-88, Δ13) changes upon Ca2+ binding. We found that the scattering intensity of wild-type S100A4 changes substantially in the 0.15-0.25 Å-1 q-range whereas a similar change is not visible in the C-terminus deleted mutant. Ensemble optimization SAXS modeling indicates that the entire C-terminus is extended when Ca2+ is bound. Pulsed field gradient NMR measurements provide further support as the hydrodynamic radius in the wild-type protein increases upon Ca2+ binding while the radius of Δ13 mutant does not change. Molecular dynamics simulations provide a rational explanation of the structural transition: the positively charged C-terminal residues associate with the negatively charged residues of the Ca2+-free EF-hands and these interactions loosen up considerably upon Ca2+-binding. As a consequence the Δ13 mutant has increased Ca2+ affinity and is constantly loaded at Ca2+ concentration ranges typically present in cells. The activation of the entire C-terminal random coil may play a role in mediating interaction with selected partner proteins of S100A4.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0097654PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4022583PMC
January 2015

Crystal structures of substrate-bound chitinase from the psychrophilic bacterium Moritella marina and its structure in solution.

Acta Crystallogr D Biol Crystallogr 2014 Mar 15;70(Pt 3):676-84. Epub 2014 Feb 15.

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.

The four-domain structure of chitinase 60 from Moritella marina (MmChi60) is outstanding in its complexity. Many glycoside hydrolases, such as chitinases and cellulases, have multi-domain structures, but only a few have been solved. The flexibility of the hinge regions between the domains apparently makes these proteins difficult to crystallize. The analysis of an active-site mutant of MmChi60 in an unliganded form and in complex with the substrates NAG4 and NAG5 revealed significant differences in the substrate-binding site compared with the previously determined complexes of most studied chitinases. A SAXS experiment demonstrated that in addition to the elongated state found in the crystal, the protein can adapt other conformations in solution ranging from fully extended to compact.
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http://dx.doi.org/10.1107/S1399004713032264DOI Listing
March 2014

Endophilin-A1 BAR domain interaction with arachidonyl CoA.

Front Mol Biosci 2014 28;1:20. Epub 2014 Oct 28.

Hamburg Unit, European Molecular Biology Laboratory c/o DESY Hamburg, Germany.

Endophilin-A1 belongs to the family of BAR domain containing proteins that catalyze membrane remodeling processes via sensing, inducing and stabilizing membrane curvature. We show that the BAR domain of endophilin-A1 binds arachidonic acid and molds its coenzyme A (CoA) activated form, arachidonyl-CoA into a defined structure. We studied low resolution structures of endophilin-A1-BAR and its complex with arachidonyl-CoA in solution using synchrotron small-angle X-ray scattering (SAXS). The free endophilin-A1-BAR domain is shown to be dimeric at lower concentrations but builds tetramers and higher order complexes with increasing concentrations. Extensive titration SAXS studies revealed that the BAR domain produces a homogenous complex with the lipid micelles. The structural model of the complexes revealed two arachidonyl-CoA micelles bound to the distal arms of an endophilin-A1-BAR dimer. Intriguingly, the radius of the bound micelles significantly decreases compared to that of the free micelles, and this structural result may provide hints on the potential biological relevance of the endophilin-A1-BAR interaction with arachidonyl CoA.
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http://dx.doi.org/10.3389/fmolb.2014.00020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4428356PMC
May 2015

Small angle X-ray scattering studies of mitochondrial glutaminase C reveal extended flexible regions, and link oligomeric state with enzyme activity.

PLoS One 2013 30;8(9):e74783. Epub 2013 Sep 30.

Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.

Glutaminase C is a key metabolic enzyme, which is unregulated in many cancer systems and believed to play a central role in the Warburg effect, whereby cancer cells undergo changes to an altered metabolic profile. A long-standing hypothesis links enzymatic activity to the protein oligomeric state, hence the study of the solution behavior in general and the oligomer state in particular of glutaminase C is important for the understanding of the mechanism of protein activation and inhibition. In this report, this is extensively investigated in correlation to enzyme concentration or phosphate level, using a high-throughput microfluidic-mixing chip for the SAXS data collection, and we confirm that the oligomeric state correlates with activity. The in-depth solution behavior analysis further reveals the structural behavior of flexible regions of the protein in the dimeric, tetrameric and octameric state and investigates the C-terminal influence on the enzyme solution behavior. Our data enable SAXS-based rigid body modeling of the full-length tetramer states, thereby presenting the first ever experimentally derived structural model of mitochondrial glutaminase C including the N- and C-termini of the enzyme.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074783PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3787022PMC
May 2014

Reconstruction of quaternary structure from X-ray scattering by equilibrium mixtures of biological macromolecules.

Biochemistry 2013 Oct 19;52(39):6844-55. Epub 2013 Sep 19.

European Molecular Biology Laboratory, Hamburg Outstation , Notkestrasse 85, Hamburg 22607, Germany.

A recent renaissance in small-angle X-ray scattering (SAXS) made this technique a major tool for the low-resolution structural characterization of biological macromolecules in solution. The major limitation of existing methods for reconstructing 3D models from SAXS is imposed by the requirement of solute monodispersity. We present a novel approach that couples low-resolution 3D SAXS reconstruction with composition analysis of mixtures. The approach is applicable to polydisperse and difficult to purify systems, including weakly associated oligomers and transient complexes. Ab initio shape analysis is possible for symmetric homo-oligomers, whereas rigid body modeling is applied also to dissociating complexes when atomic structures of the individual subunits are available. In both approaches, the sample is considered as an equilibrium mixture of intact complexes/oligomers with their dissociation products or free subunits. The algorithms provide the 3D low-resolution model (for ab initio modeling, also the shape of the monomer) and the volume fractions of the bound and free state(s). The simultaneous fitting of multiple scattering data sets collected under different conditions allows one to restrain the modeling further. The possibilities of the approach are illustrated in simulated and experimental SAXS data from protein oligomers and multisubunit complexes including nucleoproteins. Using this approach, new structural insights are provided in the association behavior and conformations of estrogen-related receptors ERRα and ERRγ. The possibility of 3D modeling from the scattering by mixtures significantly widens the range of applicability of SAXS and opens novel avenues in the analysis of oligomeric mixtures and assembly/dissociation processes.
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http://dx.doi.org/10.1021/bi400731uDOI Listing
October 2013

The asymmetric binding of PGC-1α to the ERRα and ERRγ nuclear receptor homodimers involves a similar recognition mechanism.

PLoS One 2013 9;8(7):e67810. Epub 2013 Jul 9.

Department of Integrative Structural Biology, Institute of Genetics and Molecular and Cellular Biology (IGBMC), Centre National de la Recherche Scientifique (CNRS), UMR 7104, Institut National de la Santé et de la Recherche Médicale (INSERM) U964, Université de Strasbourg (UdS), Illkirch, France.

Background: PGC-1α is a crucial regulator of cellular metabolism and energy homeostasis that functionally acts together with the estrogen-related receptors (ERRα and ERRγ) in the regulation of mitochondrial and metabolic gene networks. Dimerization of the ERRs is a pre-requisite for interactions with PGC-1α and other coactivators, eventually leading to transactivation. It was suggested recently (Devarakonda et al) that PGC-1α binds in a strikingly different manner to ERRγ ligand-binding domains (LBDs) compared to its mode of binding to ERRα and other nuclear receptors (NRs), where it interacts directly with the two ERRγ homodimer subunits.

Methods/principal Findings: Here, we show that PGC-1α receptor interacting domain (RID) binds in an almost identical manner to ERRα and ERRγ homodimers. Microscale thermophoresis demonstrated that the interactions between PGC-1α RID and ERR LBDs involve a single receptor subunit through high-affinity, ERR-specific L3 and low-affinity L2 interactions. NMR studies further defined the limits of PGC-1α RID that interacts with ERRs. Consistent with these findings, the solution structures of PGC-1α/ERRα LBDs and PGC-1α/ERRγ LBDs complexes share an identical architecture with an asymmetric binding of PGC-1α to homodimeric ERR.

Conclusions/significance: These studies provide the molecular determinants for the specificity of interactions between PGC-1α and the ERRs, whereby negative cooperativity prevails in the binding of the coactivators to these receptors. Our work indicates that allosteric regulation may be a general mechanism controlling the binding of the coactivators to homodimers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0067810PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706463PMC
February 2014

Ceruloplasmin: macromolecular assemblies with iron-containing acute phase proteins.

PLoS One 2013 3;8(7):e67145. Epub 2013 Jul 3.

Institute of Crystallography RAS, Moscow, Russia.

Copper-containing ferroxidase ceruloplasmin (Cp) forms binary and ternary complexes with cationic proteins lactoferrin (Lf) and myeloperoxidase (Mpo) during inflammation. We present an X-ray crystal structure of a 2Cp-Mpo complex at 4.7 Å resolution. This structure allows one to identify major protein-protein interaction areas and provides an explanation for a competitive inhibition of Mpo by Cp and for the activation of p-phenylenediamine oxidation by Mpo. Small angle X-ray scattering was employed to construct low-resolution models of the Cp-Lf complex and, for the first time, of the ternary 2Cp-2Lf-Mpo complex in solution. The SAXS-based model of Cp-Lf supports the predicted 1:1 stoichiometry of the complex and demonstrates that both lobes of Lf contact domains 1 and 6 of Cp. The 2Cp-2Lf-Mpo SAXS model reveals the absence of interaction between Mpo and Lf in the ternary complex, so Cp can serve as a mediator of protein interactions in complex architecture. Mpo protects antioxidant properties of Cp by isolating its sensitive loop from proteases. The latter is important for incorporation of Fe(3+) into Lf, which activates ferroxidase activity of Cp and precludes oxidation of Cp substrates. Our models provide the structural basis for possible regulatory role of these complexes in preventing iron-induced oxidative damage.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0067145PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3700992PMC
February 2014

Using stable MutS dimers and tetramers to quantitatively analyze DNA mismatch recognition and sliding clamp formation.

Nucleic Acids Res 2013 Sep 1;41(17):8166-81. Epub 2013 Jul 1.

Division of Biochemistry and CancerGenomiCs.nl, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands, European Molecular Biology Laboratory, Hamburg Outstation, EMBL c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany, Institute for Biochemistry, Justus Liebig University, Heinrich-Buff Ring 58, D-35392, Giessen, Germany, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA, Department of Cell Biology and Genetics, Cancer Genomics Center, Erasmus Medical Center, PO Box 2040, 3000 CA Rotterdam, the Netherlands and Department of Radiation Oncology, Erasmus Medical Center, PO Box 2040, 3000 CA Rotterdam, the Netherlands.

The process of DNA mismatch repair is initiated when MutS recognizes mismatched DNA bases and starts the repair cascade. The Escherichia coli MutS protein exists in an equilibrium between dimers and tetramers, which has compromised biophysical analysis. To uncouple these states, we have generated stable dimers and tetramers, respectively. These proteins allowed kinetic analysis of DNA recognition and structural analysis of the full-length protein by X-ray crystallography and small angle X-ray scattering. Our structural data reveal that the tetramerization domains are flexible with respect to the body of the protein, resulting in mostly extended structures. Tetrameric MutS has a slow dissociation from DNA, which can be due to occasional bending over and binding DNA in its two binding sites. In contrast, the dimer dissociation is faster, primarily dependent on a combination of the type of mismatch and the flanking sequence. In the presence of ATP, we could distinguish two kinetic groups: DNA sequences where MutS forms sliding clamps and those where sliding clamps are not formed efficiently. Interestingly, this inability to undergo a conformational change rather than mismatch affinity is correlated with mismatch repair.
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http://dx.doi.org/10.1093/nar/gkt582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3783165PMC
September 2013

Applications of small-angle X-ray scattering to biomacromolecular solutions.

Int J Biochem Cell Biol 2013 Feb 9;45(2):429-37. Epub 2012 Nov 9.

European Molecular Biology Laboratory, Hamburg Outstation, Notkestrasse 85, 22607 Hamburg, Germany.

Small-angle scattering of X-rays (SAXS) is an established method for low-resolution structural characterization of biological macromolecules in solution. Being complementary to the high resolution methods (X-ray crystallography and NMR), SAXS is often used in combination with them. The technique provides overall three-dimensional structures using ab initio reconstructions and hybrid modeling, and allows one to quantitatively characterize equilibrium mixtures as well as flexible systems. Recent progress in SAXS instrumentation, most notably, high brilliance synchrotron sources, has paved the way for high throughput automated SAXS studies allowing screening of external conditions (pH, temperature, ligand binding etc.). The modern approaches for SAXS data analysis are presented in this review including rapid characterization of macromolecular solutions in terms of low-resolution shapes, validation of high-resolution models in close-to-native conditions, quaternary structure analysis of complexes and quantitative description of the oligomeric composition in mixtures. Practical aspects of SAXS as a standalone tool and its combinations with other structural, biophysical or bioinformatics methods are reviewed. The capabilities of the technique are illustrated by a selection of recent applications for the studies of biological molecules. Future perspectives on SAXS and its potential impact to structural molecular biology are discussed.
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http://dx.doi.org/10.1016/j.biocel.2012.10.017DOI Listing
February 2013

MaxOcc: a web portal for maximum occurrence analysis.

J Biomol NMR 2012 Aug 26;53(4):271-80. Epub 2012 May 26.

Magnetic Resonance Center (CERM), University of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino, FI, Italy.

The MaxOcc web portal is presented for the characterization of the conformational heterogeneity of two-domain proteins, through the calculation of the Maximum Occurrence that each protein conformation can have in agreement with experimental data. Whatever the real ensemble of conformations sampled by a protein, the weight of any conformation cannot exceed the calculated corresponding Maximum Occurrence value. The present portal allows users to compute these values using any combination of restraints like pseudocontact shifts, paramagnetism-based residual dipolar couplings, paramagnetic relaxation enhancements and small angle X-ray scattering profiles, given the 3D structure of the two domains as input. MaxOcc is embedded within the NMR grid services of the WeNMR project and is available via the WeNMR gateway at http://py-enmr.cerm.unifi.it/access/index/maxocc . It can be used freely upon registration to the grid with a digital certificate.
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http://dx.doi.org/10.1007/s10858-012-9638-1DOI Listing
August 2012

New developments in the program package for small-angle scattering data analysis.

J Appl Crystallogr 2012 Apr 15;45(Pt 2):342-350. Epub 2012 Mar 15.

European Molecular Biology Laboratory, Hamburg Unit, EMBL c/o DESY, Notkestrasse 85, Hamburg 22607, Germany.

New developments in the program package (version 2.4) for the processing and analysis of isotropic small-angle X-ray and neutron scattering data are described. They include (i) multiplatform data manipulation and display tools, (ii) programs for automated data processing and calculation of overall parameters, (iii) improved usage of high- and low-resolution models from other structural methods, (iv) new algorithms to build three-dimensional models from weakly interacting oligomeric systems and complexes, and (v) enhanced tools to analyse data from mixtures and flexible systems. The new release includes installers for current major platforms (Windows, Linux and Mac OSX) and provides improved indexed user documentation. The web-related developments, including a user discussion forum and a widened online access to run programs, are also presented.
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http://dx.doi.org/10.1107/S0021889812007662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4233345PMC
April 2012