Comportements conformationnels et antigénicités de l'Ag-O de Shigella flexneri et de ses mimes

Dans le contexte de la conception de nouvelles stratégies vaccinales contre les shigelloses, nous nous sommes appliqués à décrire les comportements conformationnels des polysaccharides antigéniques (Ag-O) de Shigella flexneri ainsi que de leurs mimes synthétiques. Nous avons étudié par des méthodes biophysiques complémentaires (cristallographie, RMN, modélisation moléculaire par informatique, ITC, SPR, etc..) les caractéristiques structurales des molécules mimes et de leur reconnaissance par des anticorps spécifiques de l'antigène naturel. Nous avons de terminé les comportements conformationnels adoptés par l'Ag-O de la plupart des sérotypes de S. flexneri. Pour l'un de ces sérotypes, nous avons identifié les interactions intramoléculaires qui influencent le comportement conformationnel de l'Ag-O et de ses oligosaccharides mimes, ainsi que les conformations reconnues par le système immunitaire. Ces travaux permettront de mieux appréhender la conception de vaccins saccharidiques.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

La RMN, un couteau suisse pour disséquer la chimie cellulaire à toutes les échelles.

International audienceCet article présente deux développements récents sur la physico-chimie du vivant utilisant la résonance magnétique nucléaire : la structure des protéines en cellule et la détection d’activité enzymatique avec l’IRM d’agents de contrastes dits « intelligents »

Paramagnetic relaxation enhancement to improve sensitivity of fast NMR methods: application to intrinsically disordered proteins

Abstract We report enhanced sensitivity NMR measurements of intrinsically disordered proteins in the presence of paramagnetic relaxation enhancement (PRE) agents such as Ni 2? -chelated DO2A. In proton-detected 1 H-15 N SOFAST-HMQC and carbon-detected (H-flip) 13 CO-15 N experiments, faster longitudinal relaxation enables the usage of even shorter interscan delays. This results in higher NMR signal intensities per units of experimental time, without adverse line broadening effects. At 40 mmolÁL -1 of the PRE agent, we obtain a 1.7-to 1.9-fold larger signal to noise (S/N) for the respective 2D NMR experiments. High solvent accessibility of intrinsically disordered protein (IDP) residues renders this class of proteins particularly amenable to the outlined approach

1H, 13C and 15N backbone resonance assignment of the intrinsically disordered region of the nuclear envelope protein emerin.

International audienceHuman emerin is an inner nuclear membrane protein involved in the response of the nucleus to mechanical stress. It contributes to the physical connection between the cytoskeleton and the nucleoskeleton. It is also involved in chromatin organization. Its N-terminal region is nucleoplasmic and comprises a globular LEM domain from residue 1 to residue 43. The three-dimensional structure of this LEM domain in complex with the chromatin BAF protein was solved from NMR data. Apart from the LEM domain, the nucleoplasmic region of emerin, from residue 44 to residue 221, is predicted to be intrinsically disordered. Mutations in this region impair binding to several emerin partners as lamin A, actin or HDAC3. However the molecular details of these recognition defects are unknown. Here we report (1)H, (15)N, (13)CO, (13)Cα and (13)Cβ NMR chemical shift assignments of the emerin fragment from residue 67 to residue 170, which is sufficient for nuclear localization and involved in lamin A binding. Chemical shift analysis confirms that this fragment is intrinsically disordered in 0 and 8 M urea

1H, 13C and 15N backbone resonance assignment of the human BRCA2 N-terminal region

International audienceThe Breast Cancer susceptibility protein 2 (BRCA2) is involved in mechanisms that maintain genome stability, including DNA repair, replication and cell division. These functions are ensured by the folded C-terminal DNA binding domain of BRCA2 but also by its large regions predicted to be disordered. Several studies have shown that disordered regions of BRCA2 are subjected to phosphorylation, thus regulating BRCA2 interactions through the cell cycle. The N-terminal region of BRCA2 contains two highly conserved clusters of phosphorylation sites between amino acids 75 and 210. Upon phosphorylation by CDK, the cluster 1 is known to become a docking site for the kinase PLK1. The cluster 2 is phosphorylated by PLK1 at least at two positions. Both of these phosphorylation clusters are important for mitosis progression, in particular for chromosome segregation and cytokinesis. In order to identify the phosphorylated residues and to characterize the phosphorylation sites preferences and their functional consequences within BRCA2 N-terminus, we have produced and analyzed the BRCA2 fragment from amino acid 48 to amino acid 284 (BRCA248-284). Here, we report the assignment of 1H, 15N, 13CO, 13Cα and 13Cβ NMR chemical shifts of this region. Analysis of these chemical shifts confirmed that BRCA248-284 shows no stable fold: it is intrinsically disordered, with only short, transient α-helices

H-1, C-13 and N-15 backbone resonance assignment of the lamin C-terminal region specific to prelamin A

International audienceLamins are the main components of the nucleoskeleton. They form a protein meshwork that underlies the inner nuclear membrane. Mutations in the LMNA gene coding for A-type lamins (lamins A and C) cause a large panel of human diseases, referred to as laminopathies. These diseases include muscular dystrophies, lipodystrophies and premature aging diseases. Lamin A exhibits a C-terminal region that is different from lamin C and is post-translationally modified. It is produced as prelamin A and it is then farnesylated, cleaved, carboxymethylated and cleaved again in order to become mature lamin A. In patients with the severe Hutchinson-Gilford progeria syndrome, a specific single point mutation in LMNA leads to an aberrant splicing of the LMNA gene preventing the post-translational processing of prelamin A. This leads to the accumulation of a permanently farnesylated lamin A mutant lacking 50 amino acids named progerin. We here report the NMR H-1, N-15, (CO)-C-13, C-13 and C-13 chemical shift assignment of the C-terminal region that is specific to prelamin A, from amino acid 567 to amino acid 664. We also report the NMR H-1, N-15, (CO)-C-13, C-13 and C-13 chemical shift assignment of the C-terminal region of the progerin variant, from amino acid 567 to amino acid 614. Analysis of these chemical shift data confirms that both prelamin A and progerin C-terminal domains are largely disordered and identifies a common partially populated -helix from amino acid 576 to amino acid 585. This helix is well conserved from fishes to mammals

Effects of backbone substitutions on the conformational behavior of Shigella flexneri O-antigens: implications for vaccine strategy.

International audienceThe O-antigen (O-Ag), the polysaccharide part of the lipopolysaccharide, is the major target of the serotype-specific protective humoral response elicited upon host infection by Shigella flexneri, the main causal agent of the endemic form of bacillary dysentery. The O-Ag repeat units (RUs) of 12 S. flexneri serotypes share the tetrasaccharide backbone →2)-α-l-Rhap-(1 → 2)-α-l-Rhap-(1 → 3)-α-l-Rhap-(1 → 3)-β-d-GlcpNAc-(1→, with site-selective glucosylation(s) and/or O-acetylation defining the serotypes. To investigate the conformational basis of serotype specificity, we sampled conformational behaviors during 60 ns of molecular dynamic simulations for oligosaccharides representing three RUs of each one of the O-Ags corresponding to serotypes 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, X and Y, respectively. The calculated trajectories were checked by nuclear magnetic resonance (NMR) for 1a, 2a, 3a and 5a O-Ags. The simulations predict that in all O-Ags, but 1a and 1b, serotype-specific substitutions of the backbone do not induce any new backbone conformations compared with the linear type O-Ag Y, although they restrain locally the accessible conformational space. Moreover, the influence of any given substituent on the backbone is independent of the eventual presence of other substituents. Finally, only slight differences in conformational behavior between terminal and inner RUs were observed. These results suggest that the reported serotype-specificity of the protective immune response is not due to recognition of distinct backbone conformations, but to binding of the serotype-defining substituents in the O-Ag context. The gained knowledge is discussed in terms of impact on the development of a broad-serotype coverage vaccine

Thermodynamics of protein destabilization in live cells

Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a ß-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37°C but with a conspicuous twist While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.Fil: Danielsson, Jens. Stockholms Universitet; SueciaFil: Mu, Xin. Stockholms Universitet; SueciaFil: Lang, Lisa. Stockholms Universitet; SueciaFil: Wang, Huabing. Stockholms Universitet; SueciaFil: Binolfi, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Leibniz Institute of Molecular Pharmacology; AlemaniaFil: Theillet, François Xavier. Leibniz Institute of Molecular Pharmacology; AlemaniaFil: Bekei, Beata. Leibniz Institute of Molecular Pharmacology; AlemaniaFil: Logan, Derek T.. Lund University; SueciaFil: Selenko, Philipp. Leibniz Institute of Molecular Pharmacology; AlemaniaFil: Wennerström, Håkan. Lund University; SueciaFil: Oliveberg, Mikael. Stockholms Universitet; Sueci

Structural mimicry of O-antigen by a peptide revealed in a complex with an antibody raised against Shigella flexneri serotype 2a.

International audienceThe use of carbohydrate-mimicking peptides to induce immune responses against surface polysaccharides of pathogenic bacteria offers a novel approach to vaccine development. Factors governing antigenic and immunogenic mimicry, however, are complex and poorly understood. We have addressed this question using the anti-lipopolysaccharide monoclonal antibody F22-4, which was raised against Shigella flexneri serotype 2a and shown to protect against homologous infection in a mouse model. In a previous crystallographic study, we described F22-4 in complex with two synthetic fragments of the O-antigen, the serotype-specific saccharide moiety of lipopolysaccharide. Here, we present a crystallographic and NMR study of the interaction of F22-4 with a dodecapeptide selected by phage display using the monoclonal antibody. Like the synthetic decasaccharide, the peptide binds to F22-4 with micromolar affinity. Although the peptide and decasaccharide use very similar regions of the antigen-binding site, indicating good antigenic mimicry, immunogenic mimicry by the peptide was not observed. The F22-4-antigen interaction is significantly more hydrophobic with the peptide than with oligosaccharides; nonetheless, all hydrogen bonds formed between the peptide and F22-4 have equivalents in the oligosaccharide complex. Two bridging water molecules are also in common, adding to partial structural mimicry. Whereas the bound peptide is entirely helical, its structure in solution, as shown by NMR, is helical in the central region only. Moreover, docking the NMR structure into the antigen-binding site shows that steric hindrance would occur, revealing poor complementarity between the major solution conformation and the antibody that could contribute to the absence of immunogenic mimicry

Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells.

Levels of residual structure in disordered interaction domains determine in vitro binding affinities, but whether they exert similar roles in cells is not known. Here, we show that increasing residual p53 helicity results in stronger Mdm2 binding, altered p53 dynamics, impaired target gene expression and failure to induce cell cycle arrest upon DNA damage. These results establish that residual structure is an important determinant of signaling fidelity in cells