Methyl Relaxation Measurements Reveal Patterns of Fast Dynamics in a Viral RNA-Directed RNA Polymerase

摘要: Molecular dynamics (MD) simulations combined with biochemical studies have suggested the presence of long-range networks functionally relevant conformational flexibility on nanosecond time scale in single-subunit RNA polymerases many viruses. However, experimental verification these at a sufficient level detail has been lacking. Here we describe fast, picosecond to an archetypal viral RNA-directed polymerase (RdRp), 75 kDa P2 protein from cystovirus ϕ12, using analyses (1)H-(1)H dipole-dipole cross-correlated relaxation methyl positions Ile (δ1), Leu, Val, and Met residues. Our results, which represent most detailed characterization fast RdRp until date, reveal highly connected dynamic network as predicted by MD related systems. results suggest that entry portals for template substrate NTPs are relatively disordered, while conserved motifs involved metal binding, nucleotide selection, catalysis display greater rigidity. Perturbations active site through binding or functional mutation affect not only immediate vicinity but also remote regions. Comparison limited extensive silico available homologous systems suggests conservation overall pattern RdRps.