D, after targeted molecular dynamics (see text for details), base-pairing with the template base (labeled “+1”) is readily established. The representative snapshot from the major cluster 6 used for A is indicated by the cyan arrow. C, clustering of the Arg 158 conformation along the trajectory with a timeline ( top) and a distribution of clusters ( bottom). 5 C show a reversal of Arg 158 interaction with base P-1 as the salt bridge with Glu 143 is re-established. B, evolution of the same Arg 158 distances as in Fig. The P-1 primer base on which the incoming nucleotide is to stack and the +1 adenine with which it is to bp are labeled. A, initial UTP and Mg(A) positions ( yellow) superimposed on the snapshot ( cyan) defined by cyan arrows in B and C. A–C, unbiased simulation after Mg(A) addition. I(1GX6) is the triphosphate for the UTP at the “I” site reported previously (27).Īddition of Mg(A) at the noncatalytic site triggers a displacement of motif F3 unmasking the +1 base. E, close up of the density at the NTP tunnel entry also showing the locations of Lys 51, Lys 151, and Asp 387. D, the four stable positions reached by UTP from initial position 3, colored according to the energy constant applied beyond 30 Å. C, UTP density when considering the 45 4-ns simulations (nine initial positions times five energy constants) simultaneously, contoured at two levels to highlight regions of nucleotide binding ( gray mesh) and more localized binding sites ( red volumes). B, UTP distances from the active site during five 4-ns simulations starting from position 3 and with five energy constants of 0 (no bias, red curve), 0.02 ( blue), 0.05 ( green), 0.1 ( magenta), and 0.5 ( yellow) Kcal/mol/Å 2. The semitransparent gray sphere on the right panel signals the 30 Å distance from the active site under which no extra energy term is added. A, nine initial positions for UTP (together with Mg(B)) 40 Å from NS5B's active site on the side of the NTP tunnel (in pale yellow). NS5B is in the same orientation in panels A, left, and C–E. Molecular dynamics simulations with distance restraints on UTP. RNA virus RNA-dependent RNA polymerase (RdRp) Single-stranded, positive-sense RNA virus molecular dynamics nucleoside/nucleotide transport protein motif structural biology viral polymerase. The findings of our work suggest that at least some of these features are general to viral RdRps and provide further details on the original nucleotide selection mechanism operating in RdRps of RNA viruses. These dynamics are finely regulated by a second magnesium dication, thus coordinating the entry of a magnesium-bound nucleotide with shuttling of the second magnesium necessary for the two-metal ion catalysis. Dynamics of RdRp motifs F1 + F3 then allow the nucleotide to interrogate the RNA template base prior to nucleotide insertion into the active site. We observed that a magnesium-bound nucleotide first binds next to the tunnel entry, and interactions with the triphosphate moiety orient it such that its base moiety enters first. Tracing the possible passage of incoming UTP or GTP through the RdRp-specific entry tunnel, we found two successive checkpoints that regulate nucleotide traffic to the active site. Here, we used molecular modeling and molecular dynamics simulations, starting from the available crystal structures of HCV NS5B in ternary complex with template-primer duplexes and nucleotides, to address the question of ribonucleotide entry into the active site of viral RdRp. However, peculiarities in the architecture and dynamics of RdRps raise fundamental questions about access to their active site during RNA polymerization. As such, these enzymes are prime targets for antiviral therapy, as has recently been demonstrated for hepatitis C virus (HCV). RNA viruses synthesize new genomes in the infected host thanks to dedicated, virally-encoded RNA-dependent RNA polymerases (RdRps).
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |