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Huntington's disease is a neurodegenerative disorder caused by a CAG expansion in the first exon of the HTT gene, resulting in an extended polyglutamine (poly-Q) tract in huntingtin (httex1). The structural changes occurring to the poly-Q when increasing its length remain poorly understood due to its intrinsic flexibility and the strong compositional bias. The systematic application of site-specific isotopic labeling has enabled residue-specific NMR investigations of the poly-Q tract of pathogenic httex1 variants with 46 and 66 consecutive glutamines. Integrative data analysis reveals that the poly-Q tract adopts long α-helical conformations propagated and stabilized by glutamine side chain to backbone hydrogen bonds. We show that α-helical stability is a stronger signature in defining aggregation kinetics and the structure of the resulting fibrils than the number of glutamines. Our observations provide a structural perspective of the pathogenicity of expanded httex1 and pave the way to a deeper understanding of poly-Q-related diseases., We thank G. Otting (Australian National University, Canberra, Australia) for providing the BL21 (DE3) Star::RF1-CBD3 strain. This work was supported by the European Research Council under the European Union’s H2020 Framework Programme (2014–2020)/ERC grant agreement no. 648030 and Labex EpiGenMed, an Investissements d’avenir program (grant no. ANR-10-LABX-12-01) awarded to P.B., grant no. ANR-17-CE11-0022-01 awarded to N.S. and grant no. ANR-19-PI3A-0004 awarded to J.C. The Centre for Structural Biology (CBS) is a member of France-BioImaging (FBI) and the French Infrastructure for Integrated Structural Biology, two national infrastructures supported by the French National Research Agency (grant nos. ANR-10-INBS-04-01 and ANR-10-INBS-05, respectively). A.U. is supported by a grant from the Fondation pour la Recherche Médicale (grant no. SPF20150934061). D.S. acknowledges a grant from the Métropole Européenne de Lille (PUSHUP). G. Levy (Université de Lille) is thanked for help with sample preparation and the 19F-NMR experiments. This work benefited from the high-performance computing resources of CSUC and the CALMIP supercomputing center under the allocations 2016-P16032 and 2021-P21043. The 600 MHz spectrometer for 19F-NMR measurements is funded by the Nord Region Council, CNRS, Institut Pasteur de Lille, the European Community (European Regional Development Fund, ERDF), the French Ministry of Research and the Université de Lille and by the CTRL CPER cofunded by the European Union with the ERDF, by the Hauts-de-France Regional Council (contract no. 17003781), Métropole Européenne de Lille (contract no. 2016_ESR_05) and the French State (contract no .2017-R3-CTRL-Phase1). We thank the SWING beamline at the SOLEIL synchrotron, Saint-Aubin, France (proposal 20181386), and P12 beamline at PETRAIII, Hamburg, Germany, for beamtime allocation to the project and assistance during data collection.

Small-angle X-ray scattering (SAXS) of proteins in solution has become a key tool for biochemists and structural biologists, thanks especially to the availability of beamlines with high-throughput capabilities at synchrotron sources. Despite the large spectrum of scientific disciplines tackled on the SWING beamline since its opening in 2008, there has always been a strong commitment to offering state-of-the-art biological SAXS (BioSAXS) instrumentation and data reduction methods to the scientific community. The extremely reliable in-vacuum EigerX-4M detector allows collection of an unlimited number of frames without noise. A small beamstop including a diamond diode-based monitor enables measurements of the transmitted intensity with 0.1% precision as well as a q max/q min ratio as large as 140 at a single distance. The parasitic scattering has been strongly reduced by the installation of new hybrid blades. A new thermally controlled in-vacuum capillary holder including fibre-optics-based spectroscopic functionalities allows the simultaneous use of three spectroscopic techniques in addition to SAXS measurements. The addition of a second high-performance liquid chromatography (HPLC) circuit has virtually eliminated the waiting time associated with column equilibration. The easy in-line connection of a multi-angle light scattering spectrometer and a refractometer allows for an independent determination of the molecular mass and of the concentration of low-UV-absorption samples such as detergents and sugars, respectively. These instrumental improvements are combined with important software developments. The HPLC injection Agilent software is controlled by the SAXS beamline acquisition software, allowing a virtually unlimited series of automated SAXS measurements to be synchronized with the sample injections. All data-containing files and reports are automatically stored in the same folders, with names related to both the user and sample. In addition, all raw SAXS images are processed automatically on the fly, and the analysed data are stored in the ISPyB database and made accessible via a web page.

A monodispersed and ideal solution is a key requirement of BioSAXS to allow one to extract structural information from the recorded pattern. On-line size exclusion chromatography (SEC) marked a major breakthrough, separating particles present in solution according to their size. Scattering curves with identical shape under an elution peak can be averaged and further processed free from contamination. However, this is not always straightforward, separation is often incomplete. Software have been developed to deconvolve the contributions from the different species (molecules or oligomeric forms) within the sample. In this chapter, we present the general workflow of a SEC-SAXS experiment. We present recent instrumental and data analysis developments that have improved the quality of recorded data, extended the potential of SEC-SAXS and turned it into a mainstream approach. We report a comparative analysis of two macromolecular systems using various deconvolution approaches that have been developed over the last years. Parallel analysis appears to be the best cross-validation method to assess the reliability of the reconstructed isolated species patterns that can safely be used as a support for meaningful molecular modeling.

SummaryTranslationally Controlled Tumour Protein (TCTP) is a pro-survival factor in tumor cells. TCTP inhibits the mitochondrial apoptosis pathway by potentiating the anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xL. Specifically, TCTP binds Bcl-xL and inhibits the Bax-dependent Bcl-xL-induced cytochrome c release and TCTP reduces Mcl-1 turnover by inhibiting its ubiquitinylation, thus resulting in decreased Mcl-1 mediated apoptosis. TCTP owns a BH3-like motif forming a β-strand buried in the globular domain of the protein. The crystal structure of TCTP BH3-like peptide in complex with Bcl-xL highlighted the α-helical conformation of TCTP BH3-like motif, suggesting major changes in TCTP structure upon complex formation. However, the structural impact of the interaction on the full-length TCTP and the structural description of TCTP/Mcl-1 interaction are still lacking. Here using biophysical/biochemical methods (NMR, SAXS, circular dichroism, limited proteolysis), we provide an in-depth description of the TCTP/Mcl-1 complex. We demonstrate that full length TCTP binds to the BH3 binding groove of Mcl-1 via its BH3-like motif which interconverts between different binding modes at the micro- to milli-second timescale. As a consequence of the engagement of the BH3-like motif in the interface, the TCTP globular domain is destabilized into a molten-globule state. We also establish that the residue D16 in TCTP BH3-like motif is crucial for the stability and dynamics of the intermolecular interface. As a conclusion, we reveal here in details the structural plasticity of TCTP and discuss its implications for TCTP biology and for future anticancer drug design strategies aiming at targeting TCTP complexes.ContactEwen Lescop,ewen.lescop@cnrs.fr.Supplementary InformationSupplementary figures, tables and files.

Huntington’s Disease is a neurodegenerative disorder caused by a CAG expansion of the first exon of theHTTgene, resulting in an extended poly-glutamine (poly-Q) tract in the N-terminus of the protein huntingtin (httex1). The structural changes occurring to the poly-Q when increasing its length remain poorly understood mainly due to its intrinsic flexibility and the strong compositional bias of the protein. The systematic application of site-specific isotopic labeling has enabled residue-specific NMR investigations of the poly-Q tract of pathogenic httex1 variants with 46 and 66 consecutive glutamines. The integrative analysis of the data reveals that the poly-Q tract adopts long α-helical conformations stabilized by glutamine side-chain to backbone hydrogen bonds.19F-NMR of site-specifically incorporated fluoro-glutamines and molecular dynamics simulations demonstrate that the mechanism propagating α-helical conformations towards the poly-Q from the upstream N17 domain is independent of the poly-Q track length. Aggregation and atomic force microscopy experiments show that the presence of long and persistent α-helices in the poly-Q tract is a stronger signature in defining the aggregation kinetics and the structure of the resulting fibrils than the number of glutamines. The ensemble of our observations provides a structural perspective of the pathogenicity of expanded httex1 and paves the way to a deeper understanding of poly-Q related diseases.

Small-angle X-ray scattering (SAXS) has become an indispensable tool in structural biology, complementing atomic-resolution techniques. It is sensitive to the electron-density difference between solubilized biomacromolecules and the buffer, and provides information on molecular masses, particle dimensions and interactions, low-resolution conformations and pair distance-distribution functions. When SAXS data are recorded at multiple contrasts, i.e. at different solvent electron densities, it is possible to probe, in addition to their overall shape, the internal electron-density profile of biomacromolecular assemblies. Unfortunately, contrast-variation SAXS has been limited by the range of solvent electron densities attainable using conventional co-solutes (for example sugars, glycerol and salt) and by the fact that some biological systems are destabilized in their presence. Here, SAXS contrast data from an oligomeric protein and a protein–RNA complex are presented in the presence of iohexol and Gd-HPDO3A, two electron-rich molecules that are used in biomedical imaging and that belong to the families of iodinated and lanthanide-based complexes, respectively. Moderate concentrations of both molecules allowed solvent electron densities matching those of proteins to be attained. While iohexol yielded higher solvent electron densities (per mole), it interacted specifically with the oligomeric protein and precipitated the protein–RNA complex. Gd-HPDO3A, while less efficient (per mole), did not disrupt the structural integrity of either system, and atomic models could be compared with the SAXS data. Due to their elevated solubility and electron density, their chemical inertness, as well as the possibility of altering their physico-chemical properties, lanthanide-based complexes represent a class of molecules with promising potential for contrast-variation SAXS experiments on diverse biomacromolecular systems.

Stringent factors orchestrate bacterial cell reprogramming through increasing the level of the alarmones (p)ppGpp. In Beta- and Gammaproteobacteria, SpoT hydrolyses (p)ppGpp to counteract the synthetase activity of RelA. However, structural information about how SpoT controls the levels of (p)ppGpp is missing. Here we present the crystal structure of the hydrolase-only SpoT from Acinetobacter baumannii and uncover the mechanism of intra-molecular regulation of “long”-RSH factors. In contrast to ribosome-associated Rel/RelA that adopt an elongated structure, SpoT assumes a compact τ-shaped structure in which the regulatory domains wrap around a Core subdomain that controls the conformational state of the enzyme. The Core is key to the specialisation of long-RSHs towards either synthesis or hydrolysis: while the short and structured Core of SpoT stabilises the τ-state priming the HD domain for (p)ppGpp hydrolysis, the longer, more dynamic Core of RelA destabilises the τ-state precluding (p)ppGpp hydrolysis and priming RelA for synthesis

The Orange Carotenoid Protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection, by quenching of the excess of light harvested energy. The photoactivation mechanism remains elusive, in part due to absence of data pertaining to the timescales over which protein structural changes take place. It also remains unclear whether or not oligomerization of the dark-adapted and light-adapted OCP could play a role in the regulation of its energy quenching activity. Here, we probed photo-induced structural changes in OCP by a combination of static and time-resolved X-ray scattering and steady-state and transient optical spectroscopy in the visible range. Our results suggest that oligomerization partakes in regulation of the OCP photocycle, with different oligomers slowing down the overall thermal recovery of the dark-adapted state of OCP. They furthermore reveal that upon non-photoproductive excitation, a numbed-state forms, which remains in a non-photoexcitable structural state for at least ∼0.5 µs after absorption of a first photon.Significance StatementThe orange carotenoid protein (OCP) is a photoactivatable protein involved in cyanobacterial photoprotection. Upon photoactivation, OCP becomes able to quench the excess of energy uptaken by the light-harvesting antennae, thereby evading damage to the cells. It remains unclear, however, what is the exact OCP photoactivation mechanism, and whether or not oligomerization partakes in the regulation of the OCP function. Here, we investigated these issues by combining static and time-resolved (TR) X-ray scattering and optical spectroscopy. Our results show that OCP oligomerizes in both the dark-adapted inactive and light-adapted active states, suggesting a functional role for oligomerization. TR scattering data furthermore reveal that the first large-scale conformational changes associated with OCP photoactivation take place on the µs time scale.

A compact active x-ray beamstop has been developed for the SWING beamline at Synchrotron SOLEIL with two main functions, blocking the x-ray beam directly transmitted by the sample to protect the Dectris EigerX4M 2D detector and monitoring its intensity. The beamstop is composed of a sensor inserted in a well of tungsten carbide. The sensor is based on a piece of free-standing single crystal chemical vapor deposited diamond used in the ionization chamber mode. The beamstop has been installed on the beamline detector stage within the detection vacuum chamber, just upstream of the large 2D detector. The intensity monitoring performance (rms noise over signal) is shown to be better than 0.06% and the linearity is shown to be better than 2% for over more than five decades. The beamstop has been calibrated between 5 and 16 keV to provide the photon flux measurements in absolute units (ph/s). The specific design of the beamstop increases the small-angle x-ray scattering q-range by a factor of 1.5 in the low angle side, as compared to the previous active beamstop, based on a more standard commercial Si diode. The beamstop has been available for three years for SWING user operation (5–17 keV). It is fully compatible with the different beamline operating modes and fluxes, except in the microbeam mode, where the very divergent beam becomes too large at the beamstop position, and the previous, larger, beamstop is then more appropriate.

The polysaccharide lyase family 6 (PL6) represents one of the 41 polysaccharide lyase families classified in the CAZy database with the vast majority of its members being alginate lyases grouped into three subfamilies, PL6_1–3. To decipher the mode of recognition and action of the enzymes belonging to subfamily PL6_1, we solved the crystal structures of Pedsa0632, Patl3640, Pedsa3628 and Pedsa3807, which all show different substrate specificities and mode of action (endo-/exolyase). Thorough exploration of the structures of Pedsa0632 and Patl3640 in complex with their substrates as well as docking experiments confirms that the conserved residues in subsites −1 to +3 of the catalytic site form a common platform that can accommodate various types of alginate in a very similar manner but with a series of original adaptations bringing them their specificities of action. From comparative studies with existing structures of PL6_1 alginate lyases, we observe that in the right-handed parallel β-helix fold shared by all these enzymes, the substrate-binding site harbors the same overall conserved structures and organization. Despite this apparent similarity, it appears that members of the PL6_1 subfamily specifically accommodate and catalyze the degradation of different alginates suggesting that this common platform is actually a highly adaptable and specific tool.

International audience; In budding yeast, the MutL homolog heterodimer Mlh1-Mlh3 (MutLγ) plays a central role in the formation of meiotic crossovers. It is also involved in the repair of a subset of mismatches besides the main mismatch repair (MMR) endonuclease Mlh1-Pms1 (MutLα). The heterodimer interface and endonuclease sites of MutLγ and MutLα are located in their C-terminal domain (CTD). The molecular basis of MutLγ’s dual roles in MMR and meiosis is not known. To better understand the specificity of MutLγ, we characterized the crystal structure of Saccharomyces cerevisiae MutLγ(CTD). Although MutLγ(CTD) presents overall similarities with MutLα(CTD), it harbors some rearrangement of the surface surrounding the active site, which indicates altered substrate preference. The last amino acids of Mlh1 participate in the Mlh3 endonuclease site as previously reported for Pms1. We characterized mlh1 alleles and showed a critical role of this Mlh1 extreme C terminus both in MMR and in meiotic recombination. We showed that the MutLγ(CTD) preferentially binds Holliday junctions, contrary to MutLα(CTD). We characterized Mlh3 positions on the N-terminal domain (NTD) and CTD that could contribute to the positioning of the NTD close to the CTD in the context of the full-length MutLγ. Finally, crystal packing revealed an assembly of MutLγ(CTD) molecules in filament structures. Mutation at the corresponding interfaces reduced crossover formation, suggesting that these superstructures may contribute to the oligomer formation proposed for MutLγ. This study defines clear divergent features between the MutL homologs and identifies, at the molecular level, their specialization toward MMR or meiotic recombination functions.

International audience; TCTP protein is a pharmacological target in cancer and TCTP inhibitors such as sertraline have been evaluated in clinical trials. The direct interaction of TCTP with the drugs sertraline and thioridazine has been reported in vitro by SPR experiments to be in the ~30-50 µM Kd range (Amson et al. Nature Med 2012), supporting a TCTP-dependent mode of action of the drugs on tumor cells. However, the molecular details of the interaction remain elusive although they are crucial to improve the efforts of on-going medicinal chemistry. In addition, TCTP can be phosphorylated by the Plk-1 kinase, which is indicative of poor prognosis in several cancers. The impact of phosphorylation on TCTP structure/dynamics and binding with therapeutical ligands remains unexplored. Here, we combined NMR, TSA, SPR, BLI and ITC techniques to probe the molecular interactions between TCTP with the drugs sertraline and thioridazine. We reveal that drug binding is much weaker than reported with an apparent ~mM Kd and leads to protein destabilization that obscured the analysis of the published SPR data. We further demonstrate by NMR and SAXS that TCTP S46 phosphorylation does not promote tighter interaction between TCTP and sertraline. Accordingly, we question the supported model in which sertraline and thioridazine directly interact with isolated TCTP in tumor cells and discuss alternative modes of action for the drugs in light of current literature.

Over the past two decades small-angle X-ray scattering (SAXS) has become a popular method to characterize solutions of biomolecules including ribonucleic acid (RNA). In an integrative structural approach, SAXS is complementary to crystallography, NMR, and electron microscopy and provides information about RNA architecture and dynamics. This chapter highlights the practical advantages of combining size-exclusion chromatography and SAXS at synchrotron facilities. It is illustrated by practical case studies of samples ranging from single hairpins and tRNA to a large IRES. The emphasis is also put on sample preparation which is a critical step of SAXS analysis and on optimized protocols for in vitro RNA synthesis ensuring the production of mg amount of pure and homogeneous molecules.

Over the past two decades small-angle X-ray scattering (SAXS) has become a popular method to characterize solutions of biomolecules including ribonucleic acid (RNA). In an integrative structural approach, SAXS is complementary to crystallography, NMR, and electron microscopy and provides information about RNA architecture and dynamics. This chapter highlights the practical advantages of combining size-exclusion chromatography and SAXS at synchrotron facilities. It is illustrated by practical case studies of samples ranging from single hairpins and tRNA to a large IRES. The emphasis is also put on sample preparation which is a critical step of SAXS analysis and on optimized protocols for in vitro RNA synthesis ensuring the production of mg amount of pure and homogeneous molecules.

JIP3 and JIP4 (JNK-interacting proteins 3 and 4) are adaptors for cargo recruitment by dynein/dynactin and kinesin1 motors. Both are dimers that are stabilised by two sections of leucine zipper coiled coils. The N-terminal Leucine Zipper I (LZI) belongs to a section that binds dynein-DLIC and kinesin1-KHC, whilst the medial Leucine Zipper II (LZII) binds dynactin-p150glued and kinesin1-KLC. Structural data is available for the LZII, but the LZI section is still uncharacterized. Here we characterize the N-terminal part of JIP3/4 which consists of an RH1 (RILP homology 1) domain followed by the LZI coiled coil using bioinformatical, biophysical and structural approaches. The RH1-LZI tandem of JIP3 associates as a high affinity homodimer exhibiting elongated alpha-helical fold. 3D homology modelling of the RH1-LZI tandem reveals that the kinesin1-KHC binding site mainly overlaps with the RH1 domain. A sequence comparison search indicates that only one other protein family has RH1 domains similar to those of JIP3/4, the RILP (Rab-interacting lysosomal protein) family which consists of adaptor proteins linking Rab GTPases to cytoskeletal motors. RILPL2 is recruited through its RH1 domain by the myosin 5a motor. Here, we showed that the RH1 domain of JIP3 also interacts with myosin 5 A in vitro, highlighting JIP3/4 as possible myosin 5a adaptors. Finally, we propose that JIP3/4 and RILP family members define a unique RH1/RH2-architecture adaptor superfamily linking cytoskeletal motors and Rab GTPases.

RNase P is a universal enzyme that removes 5′ leader sequences from tRNA precursors. The enzyme is therefore essential for maturation of functional tRNAs and mRNA translation. RNase P represents a unique example of an enzyme that can occur either as ribonucleoprotein or as protein alone. The latter form of the enzyme, called protein-only RNase P (PRORP), is widespread in eukaryotes in which it can provide organellar or nuclear RNase P activities. Here, we have focused on Arabidopsis nuclear PRORP2 and its interaction with tRNA substrates. Affinity measurements helped assess the respective importance of individual pentatricopeptide repeat motifs in PRORP2 for RNA binding. We characterized the PRORP2 structure by X-ray crystallography and by small-angle X-ray scattering in solution as well as that of its complex with a tRNA precursor by small-angle X-ray scattering. Of note, our study reports the first structural data of a PRORP–tRNA complex. Combined with complementary biochemical and biophysical analyses, our structural data suggest that PRORP2 undergoes conformational changes to accommodate its substrate. In particular, the catalytic domain and the RNA-binding domain can move around a central hinge. Altogether, this work provides a refined model of the PRORP–tRNA complex that illustrates how protein-only RNase P enzymes specifically bind tRNA and highlights the contribution of protein dynamics to achieve this specific interaction.

International audience; Despite sharing common features, previous studies have shown that gyrases from different species have been modified throughout evolution to modulate their properties. Here, we report two crystal structures of Mycobacterium tuberculosis DNA gyrase, an apo and AMPPNP-bound form at 2.6-Å and 3.3-Å resolution, respectively. These structures provide high-resolution structural data on the quaternary organization and interdomain connections of a gyrase (full-length GyrB-GyrA57)2 thus providing crucial inputs on this essential drug target. Together with small-angle X-ray scattering studies, they revealed an "extremely open" N-gate state, which persists even in the DNA-free gyrase-AMPPNP complex and an unexpected connection between the ATPase and cleavage core domains mediated by two Corynebacteriales-specific motifs, respectively the C-loop and DEEE-loop. We show that the C-loop participates in the stabilization of this open conformation, explaining why this gyrase has a lower ATPase activity. Our results image a conformational state which might be targeted for drug discovery.

RNase P is a universal enzyme that removes 5′ leader sequences from tRNA precursors. The enzyme is therefore essential for maturation of functional tRNAs and mRNA translation. RNase P represents a unique example of an enzyme that can occur either as ribonucleoprotein or as protein alone. The latter form of the enzyme, called protein-only RNase P (PRORP), is widespread in eukaryotes in which it can provide organellar or nuclear RNase P activities. Here, we have focused on Arabidopsis nuclear PRORP2 and its interaction with tRNA substrates. Affinity measurements helped assess the respective importance of individual pentatricopeptide repeat motifs in PRORP2 for RNA binding. We characterized the PRORP2 structure by X-ray crystallography and by small-angle X-ray scattering in solution as well as that of its complex with a tRNA precursor by small-angle X-ray scattering. Of note, our study reports the first structural data of a PRORP–tRNA complex. Combined with complementary biochemical and biophysical analyses, our structural data suggest that PRORP2 undergoes conformational changes to accommodate its substrate. In particular, the catalytic domain and the RNA-binding domain can move around a central hinge. Altogether, this work provides a refined model of the PRORP–tRNA complex that illustrates how protein-only RNase P enzymes specifically bind tRNA and highlights the contribution of protein dynamics to achieve this specific interaction.

International audience; In the field of structural biology, Small Angle X-ray Scattering (SAXS) has undergone a tremendous evolution in the last two decades. From a craft reserved to a few experts in the late 80's, it has now turned into a high-throughput technique, following the same trend as macromolecular crystallography. Synchrotron radiation has played a key role in this evolution, by providing intense X-ray beams of high optical quality that made possible the recording of statistically meaningful data from weakly scattering biological solutions in a reasonable time. This, in turn, prompted the development of powerful and specific software for data analysis and modeling. In this mini-review, mainly addressed towards a broad readership, representing as many potential users, we try to summarize the latest aspects of evolution of BioSAXS, both conceptually and from the point of view of instrumentation. We emphasize the need for complementary experimental or computational techniques used in combination with SAXS. The great potential of these multi-pronged approaches is illustrated by a series of very recent studies covering the various ways and means of using BioSAXS.

SummaryArpin is a newly discovered regulator of actin polymerization at the cell leading edge, which steers cell migration by exerting a negative control on the Arp2/3 complex. Arpin proteins have an acidic tail homologous to the acidic motif of the VCA domain of nucleation-promoting factors (NPFs). This tail is predicted to compete with the VCA of NPFs for binding to the Arp2/3 complex, thereby mitigating activation and/or tethering of the complex to sites of actin branching. Here, we investigated the structure of full-length Arpin using synchrotron small-angle X-ray scattering, and of its acidic tail in complex with an ankyrin repeats domain using X-ray crystallography. The data were combined in a hybrid model in which the acidic tail extends from the globular core as a linear peptide and forms a primary epitope that is readily accessible in unbound Arpin and suffices to tether Arpin to interacting proteins with high affinity.

We address a major obstacle to macromolecular docking algorithms by presenting a new method that takes into account the induced conformational adjustment of flexible loops situated at a protein/macromolecule interface. The method, MC2, is based on a multiple copy representation of the loops, coupled with a Monte Carlo conformational search of the relative position of the macromolecules and their side chain conformations. The selection of optimal loop conformations takes place during Monte Carlo cycling by the iterative adjustment of the weight of each copy. We describe here the parameterization of the method and trials on a protein-DNA complex of known 3-D structure, involving the Drosophila prd paired domain protein and its target oligonucleotide Wenqing, X. et al., Cell1995, 80, 639. We demonstrate that our algorithm can correctly configure and position this protein, despite its relatively complex interactions with both grooves of DNA. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 1910–1920, 2003

International audience; The small GTPases Arf1 and Arf6 have nonoverlapping functions in cellular traffic despite their very high sequence and structural resemblance. Notably, the exquisite isoform specificity of their guanine nucleotide exchange factors and their distinctive sensitivity to the drug brefeldin A cannot be explained by any straightforward structural model. Here we integrated structural and spectroscopic methods to address this issue using Δ13Arf6-GDP, a truncated mutant that mimics membrane-bound Arf6-GDP. The crystal structure of Δ13Arf6-GDP reveals an unprecedented unfolding of the GTPase core β-strands, which is fully accounted for by small-angle X-ray scattering data in solution and by ab initio three-dimensional envelope calculation. NMR chemical shifts identify this structural disorder in Δ13Arf6-GDP, but not in the closely related Δ17Arf1-GDP, which is consistent with their comparative thermodynamic and hydrodynamic analyses. Taken together, these experiments suggest an unfolding model for the nucleotide switch of Arf6 and shed new light on its biochemical differences with Arf1.

International audience; Assignment of heteronuclear and homonuclear multidimensional NMR spectra permits determination of the first three-dimensional solution structure of a higher-plant thioredoxin h. The collection of 1906 distance restraints, 137 TALOS-derived dihedral restraints, and 66 hydrogen bonds was used in the restrained molecular dynamics protocol to calculate the structure of the reduced form of thioredoxin h1 from poplar with an atomic rmsd of 0.60 ± 0.12 Å. This enzyme exhibits an unusual active site with the sequence WCPPC and original properties in terms of stability and specificity. Compared to other known thioredoxin structures, thioredoxin h1 from poplar adopts the classical "Trx fold". Its atypical active site possesses a conformation similar to that of other common thioredoxins but appears to be more rigid. Moreover, the hydrogen bond network, stabilizing the in-core -sheet, is tighter than in Chlamydomonas reinhardtii, explaining the difference in thermostability.

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Phosphorylation of the acetylcholine receptor (AChR) seems to be responsible for triggering several effects including its desensitization and aggregation at the postsynaptic membrane and probably initiates a signal transduction pathway at the postsynaptic membrane. To study the structural and functional role of the tyrosine phosphorylation site of the AChR beta-subunit and contribute to the in-depth understanding of the structural basis of the ion channel function, we synthesized four peptides containing the phosphorylated and nonphosphorylated sequences (380-391) of the human and Torpedo AChR beta-subunits and studied their interaction with a monoclonal antibody (mAb 148) that is known to bind to this region and that is capable of blocking ion channel function. All four peptides were efficient inhibitors of mAb 148 binding to AChR, although the nonphosphorylated human peptide was considerably less effective than the three others. We then investigated the conformation acquired by all four peptides in their antibody-bound state, which possibly illustrates the local conformation of the corresponding sites on the intact AChR molecule. The phosphorylated human and Torpedo peptides adopted a distorted 3(10) helix conformation. The nonphosphorylated Torpedo peptide, which is also an efficient inhibitor, was also folded. In contrast, the nonphosphorylated human peptide (a less efficient inhibitor) presented an extended structure. It is concluded that the phosphorylation of the AChR at its beta-subunit Tyr site leads to a significant change in its conformation, which may affect several functions of the AChR.

International audience