IIBBA   05544
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE BUENOS AIRES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Insights into intrinsic disorder in a viral RNA polymerase phosphoprotein cofactor.
Autor/es:
MARIA GABRIELA NOVAL; ESPERANTE SEBASTIÁN A.; TAMARA A ALTIERI; MARTIN BANCHERO; DE PRAT-GAY GONZALO
Lugar:
EASTON, MA
Reunión:
Conferencia; Intrinsically Disordered Proteins Gordon Research Seminar and Conference; 2014
Institución organizadora:
GORDON RESEARCH CONFERENCES
Resumen:
Because of the multiple functions and promiscuous molecular partnering found in viral proteins mostly related to the evolutionary necessity of gene information economy, intrinsic disorder and structural plasticity are features frequently found in viral genomes. Paramyuxoviruses are a family of non-segmented negative RNA viruses which include several human pathogens such as measles, mumps, parainfluenza and respiratory syncytial virus (RSV), among others. The replicative complex of these viruses is composed mainly of three proteins: RNA dependent RNA polymerase (RdRp) L, the nucleocapsid protein N and the phosphoprotein P, an essential cofactor that helps the RdRp accessing to the nucleocapsid for initiation of RNA synthesis and transcription processivity. RSV P is the smallest of its kind within the family, keeping a minimum of sequence elements required for its function. Sequence analysis and experiments showed a coiled-coil domain conserved even among viruses from other families, flanked by two domains (P-Nterm and P-Cterm) classified as intrinsically disordered by prediction algorithms, where each interact with L and N. We tackled an experimental approach which showed that RSV P has a modular unfolding behavior, limited by the independent and stable tetramerization domain (P-TET), with at least one more structural elements with low stability, but with otherwise clear indication of secondary structure. P is reversibly denatured by temperature, as indicated by the loss in alpha helix content, but the P-TET remains intact up to 70 °C. A weak but otherwise cooperative secondary structure transition between 10 and 40 °C indicates that there is at least one structural element that is very sensitive to temperature, with an apparent Tm of 23 °C. A similar metastable transition is also found in Guanidine Chloride unfolding (up to 2.0 M). Through a series of constructs of different combinations of the P domains we dissect the intrinsic disorder or marginally stable structures of these domains and whether they are independent ?folding units?. The species containing P-TET plus P-Cterm shows a 10 °C shift in the Tm, which suggests that the weak but significative structure present in the full-length P, has components from both C- and N- terminal domains. Structural disorder is known to be at the center of P-N and P-L interactions that modulate RNA synthesis, and thus replication and transcription of Paramixoviridae. We discuss the general implications of temperature sensitive metastable structural elements among other members within this family of important pathogens.