Arabinoxylan in Water through SANS: Single-Chain Conformation, Chain Overlap, and Clustering.

Using small-angle neutron scattering (SANS), we examine the structure and conformational behavior of wheat arabinoxylan (AX) prepared at various concentrations in a sodium phosphate aqueous buffer. As for another major hemicellulose, xyloglucan, we observe a small number of large clusters surrounded by AX chains that behave exactly as a polymer in good solvent with a Flory exponent ν = 0.588. The fit of the data at high q-values to a standard worm-like chain model gives the persistence length lp = 45 Å and cross section of the chains 2Rc = 11-12 Å. In addition, using a dedicated modeling approach, we extract from the SANS data at the intermediate q-range the correlation length ξ of the solutions in the semidilute regime. The decay of ξ with concentration follows a scaling law that further confirms the self-avoiding statistical behavior of the AX chains. This first comprehensive study about the properties of water-soluble AX at different length scales may help in the development of products and processes involving AX as a substitute for fossil carbon molecules.

Biochemical characterization of the respiratory syncytial virus N0-P complex in solution.

As all the viruses belonging to the Mononegavirales order, the nonsegmented negative-strand RNA genome of respiratory syncytial virus (RSV) is encapsidated by the viral nucleoprotein N. N protein polymerizes along the genomic and anti-genomic RNAs during replication. This requires the maintenance of the neosynthesized N protein in a monomeric and RNA-free form by the viral phosphoprotein P that plays the role of a chaperone protein, forming a soluble N0-P complex. We have previously demonstrated that residues 1-30 of P specifically bind to N0 Here, to isolate a stable N0-P complex suitable for structural studies, we used the N-terminal peptide of P (P40) to purify truncated forms of the N protein. We show that to purify a stable N0-P-like complex, a deletion of the first 30 N-terminal residues of N (NΔ30) is required to impair N oligomerization, whereas the presence of a full-length C-arm of N is required to inhibit RNA binding. We generated structural models of the RSV N0-P with biophysical approaches, including hydrodynamic measurements and small-angle X-ray scattering (SAXS), coupled with biochemical and functional analyses of human RSV (hRSV) NΔ30 mutants. These models suggest a strong structural homology between the hRSV and the human metapneumovirus (hMPV) N0-P complexes. In both complexes, the P40-binding sites on N0 appear to be similar, and the C-arm of N provides a high flexibility and a propensity to interact with the N RNA groove. These findings reveal two potential sites to target on N0-P for the development of RSV antivirals.

Pearl-necklace assembly of human serum albumin with the poly(acrylic acid) polyelectrolyte investigated using small angle X-ray scattering (SAXS).

In this comprehensive study, the interaction of human serum albumin (HSA) with poly(acrylic acid) (PAA) was explored using small angle X-ray scattering (SAXS) combined with chromatography. The results revealed the formation of a complex between HSA macromolecules and PAA chains but solely under some specific conditions of the ionic strength and pH of the medium. In fact, this binding was found to take place only at pH close to 5 and at low ionic strength (0.15 M). Otherwise, for a higher pH and a salt concentration of 0.75 M the HSA-PAA complex tends to dissociate completely showing the reversibility of the complexation. The assessment of the influence of the HSA/PAA molar ratio on the radius of gyration of the complex suggests that 4 HSA molecules could bind to each 100 kDa PAA chain. In addition, the Porod volume evaluation for the same range of the HSA/PAA ratio confirms this assumption. Finally, an all-atom SAXS modelling study using the BUNCH program was conducted to find a compatible model that fits the HSA-PAA complex scattering data. This model allows us to portray the HSA/PAA complex as a pearl-necklace assembly with 4 HSA molecules on the 100 kDa PAA chain.

Characterisation of the Effect of the Spatial Organisation of Hemicellulases on the Hydrolysis of Plant Biomass Polymer.

Synergism between enzymes is of crucial importance in cell metabolism. This synergism occurs often through a spatial organisation favouring proximity and substrate channelling. In this context, we developed a strategy for evaluating the impact of the geometry between two enzymes involved in nature in the recycling of the carbon derived from plant cell wall polymers. By using an innovative covalent association process using two protein fragments, Jo and In, we produced two bi-modular chimeric complexes connecting a xylanase and a xylosidase, involved in the deconstruction of xylose-based plant cell wall polymer. We first show that the intrinsic activity of the individual enzymes was preserved. Small Angle X-rays Scattering (SAXS) analysis of the complexes highlighted two different spatial organisations in solution, affecting both the distance between the enzymes (53 Å and 28 Å) and the distance between the catalytic pockets (94 Å and 75 Å). Reducing sugar and HPAEC-PAD analysis revealed different behaviour regarding the hydrolysis of Beechwood xylan. After 24 h of hydrolysis, one complex was able to release a higher amount of reducing sugar compare to the free enzymes (i.e., 15,640 and 14,549 µM of equivalent xylose, respectively). However, more interestingly, the two complexes were able to release variable percentages of xylooligosaccharides compared to the free enzymes. The structure of the complexes revealed some putative steric hindrance, which impacted both enzymatic efficiency and the product profile. This report shows that controlling the spatial geometry between two enzymes would help to better investigate synergism effect within complex multi-enzymatic machinery and control the final product.

Dystrophin's central domain forms a complex filament that becomes disorganized by in-frame deletions.

Dystrophin, encoded by the DMD gene, is critical for maintaining plasma membrane integrity during muscle contraction events. Mutations in the DMD gene disrupting the reading frame prevent dystrophin production and result in severe Duchenne muscular dystrophy (DMD); in-frame internal deletions allow production of partly functional internally deleted dystrophin and result in less severe Becker muscular dystrophy (BMD). Many known BMD deletions occur in dystrophin's central domain, generally considered to be a monotonous rod-shaped domain based on the knowledge of spectrin family proteins. However, the effects caused by these deletions, ranging from asymptomatic to severe BMD, argue against the central domain serving only as a featureless scaffold. We undertook structural studies combining small-angle X-ray scattering and molecular modeling in an effort to uncover the structure of the central domain, as dystrophin has been refractory to characterization. We show that this domain appears to be a tortuous and complex filament that is profoundly disorganized by the most severe BMD deletion (loss of exons 45-47). Despite the preservation of large parts of the binding site for neuronal nitric oxide synthase (nNOS) in this deletion, computational approaches failed to recreate the association of dystrophin with nNOS. This observation is in agreement with a strong decrease of nNOS immunolocalization in muscle biopsies, a parameter related to the severity of BMD phenotypes. The structural description of the whole dystrophin central domain we present here is a first necessary step to improve the design of microdystrophin constructs toward the goal of a successful gene therapy for DMD.

Biophysical analysis of Arabidopsis protein-only RNase P alone and in complex with tRNA provides a refined model of tRNA binding.

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.

The Crystal Structure of Gurmarin, a Sweet Taste-Suppressing Protein: Identification of the Amino Acid Residues Essential for Inhibition.

Gurmarin is a highly specific sweet taste-suppressing protein in rodents that is isolated from the Indian plant Gymnema sylvestre. Gurmarin consists of 35 amino acid residues containing 3 intramolecular disulfide bridges that form a cystine knot. Here, we report the crystal structure of gurmarin at a 1.45 Å resolution and compare it with previously reported nuclear magnetic resonance solution structures. The atomic structure at this resolution allowed us to identify a very flexible region consisting of hydrophobic residues. Some of these amino acid residues had been identified as a putative binding site for the rat sweet taste receptor in a previous study. By combining alanine-scanning mutagenesis of the gurmarin molecule and a functional cell-based receptor assay, we confirmed that some single point mutations in these positions drastically affect sweet taste receptor inhibition by gurmarin.

In-Situ Quantitative and Multiscale Structural Study of Starch-Based Biomaterials Immersed in Water.

The behavior upon immersion in water of two types of starchy materials of biomedical relevance, amorphous potato starch and glycerol-plasticized potato starch, is analyzed in depth. Synchrotron X-ray scattering, specifically wide-angle X-ray scattering (WAXS), and magnetic resonance microimaging (MRµI) are used as very precise and nondestructive quantitative methods to monitor water transfers and structure changes in the samples, with refined spatial and kinetics results. The ingress of water in the cylinder-shaped samples can be inferred from both techniques, and from this, a diffusion mechanism is deduced for each sample type. Qualitatively, scattering and imaging give comparable results: plasticized samples are shown to behave close to a Fickian diffusion case, amorphous samples close to a case II. WAXS results also provide an in-depth knowledge of the crystalline structures associated to each step of the water ingress, and these are in turn correlated to water diffusion. To refine these observations, a recrystallized starch sample is also analyzed via WAXS. This study gives better insight into the structure of a material with a huge biomedical potential (as implants, for example), and for such applications, the behavior upon immersion in water is particularly relevant.

C-terminal region of bacterial Ku controls DNA bridging, DNA threading and recruitment of DNA ligase D for double strand breaks repair.

Non-homologous end joining is a ligation process repairing DNA double strand breaks in eukaryotes and many prokaryotes. The ring structured eukaryotic Ku binds DNA ends and recruits other factors which can access DNA ends through the threading of Ku inward the DNA, making this protein a key ingredient for the scaffolding of the NHEJ machinery. However, this threading ability seems unevenly conserved among bacterial Ku. As bacterial Ku differ mainly by their C-terminus, we evaluate the role of this region in the loading and the threading abilities of Bacillus subtilis Ku and the stimulation of the DNA ligase LigD. We identify two distinct sub-regions: a ubiquitous minimal C-terminal region and a frequent basic C-terminal extension. We show that truncation of one or both of these sub-regions in Bacillus subtilis Ku impairs the stimulation of the LigD end joining activity in vitro. We further demonstrate that the minimal C-terminus is required for the Ku-LigD interaction, whereas the basic extension controls the threading and DNA bridging abilities of Ku. We propose that the Ku basic C-terminal extension increases the concentration of Ku near DNA ends, favoring the recruitment of LigD at the break, thanks to the minimal C-terminal sub-region.

Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners.

YabA negatively regulates initiation of DNA replication in low-GC Gram-positive bacteria. The protein exerts its control through interactions with the initiator protein DnaA and the sliding clamp DnaN. Here, we combined X-ray crystallography, X-ray scattering (SAXS), modeling and biophysical approaches, with in vivo experimental data to gain insight into YabA function. The crystal structure of the N-terminal domain (NTD) of YabA solved at 2.7 Å resolution reveals an extended α-helix that contributes to an intermolecular four-helix bundle. Homology modeling and biochemical analysis indicates that the C-terminal domain (CTD) of YabA is a small Zn-binding domain. Multi-angle light scattering and SAXS demonstrate that YabA is a tetramer in which the CTDs are independent and connected to the N-terminal four-helix bundle via flexible linkers. While YabA can simultaneously interact with both DnaA and DnaN, we found that an isolated CTD can bind to either DnaA or DnaN, individually. Site-directed mutagenesis and yeast-two hybrid assays identified DnaA and DnaN binding sites on the YabA CTD that partially overlap and point to a mutually exclusive mode of interaction. Our study defines YabA as a novel structural hub and explains how the protein tetramer uses independent CTDs to bind multiple partners to orchestrate replication initiation in the bacterial cell.

A higher-order entity formed by the flexible assembly of RAP1 with TRF2.

Telomere integrity is essential to maintain genome stability, and telomeric dysfunctions are associated with cancer and aging pathologies. In human, the shelterin complex binds TTAGGG DNA repeats and provides capping to chromosome ends. Within shelterin, RAP1 is recruited through its interaction with TRF2, and TRF2 is required for telomere protection through a network of nucleic acid and protein interactions. RAP1 is one of the most conserved shelterin proteins although one unresolved question is how its interaction may influence TRF2 properties and regulate its capacity to bind multiple proteins. Through a combination of biochemical, biophysical and structural approaches, we unveiled a unique mode of assembly between RAP1 and TRF2. The complete interaction scheme between the full-length proteins involves a complex biphasic interaction of RAP1 that directly affects the binding properties of the assembly. These results reveal how a non-DNA binding protein can influence the properties of a DNA-binding partner by mutual conformational adjustments.

Structural insight into the mechanism of stabilization of the 7SK small nuclear RNA by LARP7.

The non-coding RNA 7SK is the scaffold for a small nuclear ribonucleoprotein (7SKsnRNP) which regulates the function of the positive transcription elongation factor P-TEFb in the control of RNA polymerase II elongation in metazoans. The La-related protein LARP7 is a component of the 7SKsnRNP required for stability and function of the RNA. To address the function of LARP7 we determined the crystal structure of its La module, which binds a stretch of uridines at the 3'-end of 7SK. The structure shows that the penultimate uridine is tethered by the two domains, the La-motif and the RNA-recognition motif (RRM1), and reveals that the RRM1 is significantly smaller and more exposed than in the La protein. Sequence analysis suggests that this impacts interaction with 7SK. Binding assays, footprinting and small-angle scattering experiments show that a second RRM domain located at the C-terminus binds the apical loop of the 3' hairpin of 7SK, while the N-terminal domains bind at its foot. Our results suggest that LARP7 uses both its N- and C-terminal domains to stabilize 7SK in a closed structure, which forms by joining conserved sequences at the 5'-end with the foot of the 3' hairpin and has thus functional implications.

Speciation of a group I intron into a lariat capping ribozyme.

The lariat-capping (LC) ribozyme is a natural ribozyme isolated from eukaryotic microorganisms. Despite apparent structural similarity to group I introns, the LC ribozyme catalyzes cleavage by a 2',5' branching reaction, leaving the 3' product with a 3-nt lariat cap that functionally substitutes for a conventional mRNA cap in the downstream pre-mRNA encoding a homing endonuclease. We describe the crystal structures of the precleavage and postcleavage LC ribozymes, which suggest that structural features inherited from group I ribozymes have undergone speciation due to profound changes in molecular selection pressure, ultimately giving rise to an original branching ribozyme family. The structures elucidate the role of key elements that regulate the activity of the LC ribozyme by conformational switching and suggest a mechanism by which the signal for branching is transmitted to the catalytic core. The structures also show how conserved interactions twist residues, forming the lariat to join chemical groups involved in branching.

Characterization of Intersubunit Communication in the Virginiamycin trans-Acyl Transferase Polyketide Synthase.

Modular polyketide synthases (PKSs) direct the biosynthesis of clinically valuable secondary metabolites in bacteria. The fidelity of chain growth depends on specific recognition between successive subunits in each assembly line: interactions mediated by C- and N-terminal "docking domains" (DDs). We have identified a new family of DDs in trans-acyl transferase PKSs, exemplified by a matched pair from the virginiamycin (Vir) system. In the absence of C-terminal partner (VirA (C)DD) or a downstream catalytic domain, the N-terminal DD (VirFG (N)DD) exhibits multiple characteristics of an intrinsically disordered protein. Fusion of the two docking domains results in a stable fold for VirFG (N)DD and an overall protein-protein complex of unique topology whose structure we support by site-directed mutagenesis. Furthermore, using small-angle X-ray scattering (SAXS), the positions of the flanking acyl carrier protein and ketosynthase domains have been identified, allowing modeling of the complete intersubunit interface.

How a single residue in individual ß-thymosin/WH2 domains controls their functions in actin assembly.

ß-Thymosin (ßT) and WH2 domains are widespread, intrinsically disordered actin-binding peptides that display significant sequence variability and different regulations of actin self-assembly in motile and morphogenetic processes. Here, we reveal the structural mechanisms by which, in their 1:1 stoichiometric complexes with actin, they either inhibit assembly by sequestering actin monomers like Thymosin-ß4, or enhance motility by directing polarized filament assembly like Ciboulot ßT. We combined mutational, functional or structural analysis by X-ray crystallography, SAXS (small angle X-ray scattering) and NMR on Thymosin-ß4, Ciboulot, TetraThymosinß and the long WH2 domain of WASP-interacting protein. The latter sequesters G-actin with the same molecular mechanisms as Thymosin-ß4. Functionally different ßT/WH2 domains differ by distinct dynamics of their C-terminal half interactions with G-actin pointed face. These C-terminal interaction dynamics are controlled by the strength of electrostatic interactions with G-actin. At physiological ionic strength, a single salt bridge with actin located next to their central LKKT/V motif induces G-actin sequestration in both isolated long ßT and WH2 domains. The results open perspectives for elucidating the functions of ßT/WH2 domains in other modular proteins.

Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor.

Translation initiation factor 2 (IF2) promotes 30S initiation complex (IC) formation and 50S subunit joining, which produces the 70S IC. The architecture of full-length IF2, determined by small angle X-ray diffraction and cryo electron microscopy, reveals a more extended conformation of IF2 in solution and on the ribosome than in the crystal. The N-terminal domain is only partially visible in the 30S IC, but in the 70S IC, it stabilizes interactions between IF2 and the L7/L12 stalk of the 50S, and on its deletion, proper N-formyl-methionyl(fMet)-tRNA(fMet) positioning and efficient transpeptidation are affected. Accordingly, fast kinetics and single-molecule fluorescence data indicate that the N terminus promotes 70S IC formation by stabilizing the productive sampling of the 50S subunit during 30S IC joining. Together, our data highlight the dynamics of IF2-dependent ribosomal subunit joining and the role played by the N terminus of IF2 in this process.

A Salmonella type three secretion effector/chaperone complex adopts a hexameric ring-like structure.

Many bacterial pathogens use type three secretion systems (T3SS) to inject virulence factors, named effectors, directly into the cytoplasm of target eukaryotic cells. Most of the T3SS components are conserved among plant and animal pathogens, suggesting a common mechanism of recognition and secretion of effectors. However, no common motif has yet been identified for effectors allowing T3SS recognition. In this work, we performed a biochemical and structural characterization of the Salmonella SopB/SigE chaperone/effector complex by small-angle X-ray scattering (SAXS). Our results showed that the SopB/SigE complex is assembled in dynamic homohexameric-ring-shaped structures with an internal tunnel. In this ring, the chaperone maintains a disordered N-terminal end of SopB molecules, in a good position to be reached and processed by the T3SS. This ring dimensionally fits the ring-organized molecules of the injectisome, including ATPase hexameric rings; this organization suggests that this structural feature is important for ATPase recognition by T3SS. Our work constitutes the first evidence of the oligomerization of an effector, analogous to the organization of the secretion machinery, obtained in solution. As effectors share neither sequence nor structural identity, the quaternary oligomeric structure could constitute a strategy evolved to promote the specificity and efficiency of T3SS recognition.

Structural bases for N-glycan processing by mannoside phosphorylase.

The first crystal structure of Uhgb_MP, a ß-1,4-mannopyranosyl-chitobiose phosphorylase belonging to the GH130 family which is involved in N-glycan degradation by human gut bacteria, was solved at 1.85 Å resolution in the apo form and in complex with mannose and N-acetylglucosamine. SAXS and crystal structure analysis revealed a hexameric structure, a specific feature of GH130 enzymes among other glycoside phosphorylases. Mapping of the -1 and +1 subsites in the presence of phosphate confirmed the conserved Asp104 as the general acid/base catalytic residue, which is in agreement with a single-step reaction mechanism involving Man O3 assistance for proton transfer. Analysis of this structure, the first to be solved for a member of the GH130_2 subfamily, revealed Met67, Phe203 and the Gly121-Pro125 loop as the main determinants of the specificity of Uhgb_MP and its homologues towards the N-glycan core oligosaccharides and mannan, and the molecular bases of the key role played by GH130 enzymes in the catabolism of dietary fibre and host glycans.

In vitro digestion of emulsions: high spatiotemporal resolution using synchrotron SAXS.

Although the biochemical processes of lipid digestion are well-known, the biophysical ones, responsible for the assembly of molecules into functional structures, lack studies resolving both time and space scales. About 35 years ago, the seminal microscopy study of Patton and Carey constituted a major advance to reach this goal. Nowadays, new perspectives arise from the availability of large facilities scattering techniques, able to monitor the dynamics of multi-scale assemblies with unprecedented resolutions. The present small angle X-ray scattering (SAXS) study focused on the roles of the emulsifier and triglyceride in the formation of lipid assemblies during emulsion digestion in vitro. By developing several interpretations of the data in the whole space range (qualitative, shape-dependent and shape-independent models), the characteristic size of the assemblies and their transition times were obtained, which depended on the triglyceride, but not on the emulsifier. The major assembly formed was found to be a spherical mixed micelle, but vesicle was also found to coexist throughout the digestion, although in a lower proportion. The quantitative determination of the sizes and proportions of these assemblies, as well as the evolution of these characteristics during digestion are precious information for nutritional sciences, as these assemblies are the vehicles of lipophilic nutrients and micronutrients towards their absorption site.

The structural organization of the N-terminus domain of SopB, a virulence factor of Salmonella, depends on the nature of its protein partners.

The TTSS is used by Salmonella and many bacterial pathogens to inject virulence factors directly into the cytoplasm of target eukaryotic cells. Once translocated these so-called effector proteins hijack a vast array of crucial cellular functions to the benefit of the bacteria. In the bacterial cytoplasm, some effectors are stabilized and maintained in a secretion competent state by interaction with specific type III chaperones. In this work we studied the conformation of the Chaperone Binding Domain of the effector named Salmonella Outer protein B (SopB) alone and in complex with its cognate chaperone SigE by a combination of biochemical, biophysical and structural approaches. Our results show that the N-terminus part of SopB is mainly composed by α-helices and unfolded regions whose organization/stabilization depends on their interaction with the different partners. This suggests that the partially unfolded state of this N-terminal region, which confers the adaptability of the effector to bind very different partners during the infection cycle, allows the bacteria to modulate numerous host cells functions limiting the number of translocated effectors.