Etude, par resonance magnetique nucleaire, de la structure d'une petite proteine riche en ponts disulfure: la charybdotoxine, une toxine extraite du venin du scorpion Leiurus Quinquestriatus hebraeus

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : TD 83677 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

New insights into structural disorder in human respiratory syncytial virus phosphoprotein and implications for binding of protein partners

Phosphoprotein is the main cofactor of the viral RNA polymerase of Mononegavirales. It is involved in multiple interactions that are essential for the polymerase function. Most prominently it positions the polymerase complex onto the nucleocapsid, but also acts as a chaperone for the nucleoprotein. Mononegavirales phosphoproteins lack sequence conservation, but contain all large disordered regions. We show here that Nand Cterminal intrinsically disordered regions account for 80% of the phosphoprotein of the respiratory syncytial virus. But these regions display marked dynamic heterogeneity. Whereas almost stable helices are formed C terminally to the oligomerization domain, extremely transient helices are present in the N terminal region. They all mediate internal long range contacts in this non globular protein. Transient secondary elements together with fully disordered regions also provide protein binding sites recognized by the respiratory syncytial virus nucleoprotein and compatible with weak interactions required for the processivity of the polymerase

Crystal Structure of the Pestivirus Envelope Glycoprotein E(rns) and Mechanistic Analysis of Its Ribonuclease Activity.

International audiencePestiviruses, which belong to the Flaviviridae family of RNA viruses, are important agents of veterinary diseases causing substantial economical losses in animal farming worldwide. Pestivirus particles display three envelope glycoproteins at their surface: E(rns), E1, and E2. We report here the crystal structure of the catalytic domain of E(rns), the ribonucleolytic activity of which is believed to counteract the innate immunity of the host. The structure reveals a three-dimensional fold corresponding to T2 ribonucleases from plants and fungi. Cocrystallization experiments with mono- and oligonucleotides revealed the structural basis for substrate recognition at two binding sites previously identified for T2 RNases. A detailed analysis of poly-U cleavage products using (31)P-NMR and size exclusion chromatography, together with molecular docking studies, provides a comprehensive mechanistic picture of E(rns) activity on its substrates and reveals the presence of at least one additional nucleotide binding site

A Druggable Pocket at the Nucleocapsid/Phosphoprotein Interaction Site of Human Respiratory Syncytial Virus

Presently, respiratory syncytial virus (RSV), the main cause of severe respiratory infections in infants, cannot be treated efficiently with antivirals. However, its RNA-dependent polymerase complex offers potential targets for RSV-specific drugs. This includes the recognition of its template, the ribonucleoprotein complex (RNP), consisting of genomic RNA encapsidated by the RSV nucleoprotein, N. This recognition proceeds via interaction between the phosphoprotein P, which is the main polymerase cofactor, and N. The determinant role of the C terminus of P, and more particularly of the last residue, F241, in RNP binding and viral RNA synthesis has been assessed previously. Here, we provide detailed structural insight into this crucial interaction for RSV polymerase activity. We solved the crystallographic structures of complexes between the N-terminal domain of N (N-NTD) and C-terminal peptides of P and characterized binding by biophysical approaches. Our results provide a rationale for the pivotal role of F241, which inserts into a well-defined N-NTD pocket. This primary binding site is completed by transient contacts with upstream P residues outside the pocket. Based on the structural information of the N-NTD:P complex, we identified inhibitors of this interaction, selected by in silico screening of small compounds, that efficiently bind to N and compete with P in vitro. One of the compounds displayed inhibitory activity on RSV replication, thereby strengthening the relevance of N-NTD for structure-based design of RSV-specific antivirals