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Biochemistry. 2015 Sep 29;54(38):5828-38. doi: 10.1021/acs.biochem.5b00828. Epub 2015 Sep 17.

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

Author information

1
Department of Chemistry, The City College of New York , 160 Convent Avenue, New York, New York 10031, United States.
2
New York Structural Biology Center , 89 Convent Avenue, New York, New York 10027, United States.
3
Graduate Center of the City University of New York , 365 Fifth Avenue, New York, New York 10016, United States.

Abstract

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

PMID:
26333183
PMCID:
PMC5259806
DOI:
10.1021/acs.biochem.5b00828
[Indexed for MEDLINE]
Free PMC Article

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