Conformational dynamics plays a significant role in HIV reverse transcriptase resistance and substrate selection

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2012-12

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Nguyen, Virginia Myanh

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Abstract

Human immunodeficiency virus reverse transcriptase (HIV RT) is a virally encoded polymerase responsible for replicating the HIV genome. Most HIV treatments include nucleotide RT inhibitors (NRTIs) which inhibit HIV RT replication by serving as a substrate for the polymerase reaction but then blocks subsequent polymerization after incorporation. However, resistance to these NRTIs may occur through specific mutations in HIV RT that increase the discrimination of HIV RT for natural nucleotides over NRTIs. The role of enzyme conformational dynamics in specificity and substrate selection was studied using transient kinetic methods on HIV RT enzymes that have been site-specifically labeled with a conformationally sensitive fluorophore, to measure the rates of binding and catalysis. First, HIV RT with the mutation of lysine to arginine at the residue position 65 (K65R) was examined for its resistance against the NRTI tenofovir diphosphate (TFV), an acyclic deoxyadenosine triphosphate (dATP) analog. It was found that HIV RT K65R resistance to TFV was achieved through decreased rates of catalysis and increased rates of dissociation for TFV over dATP when compared with the kinetics of wild-type HIV RT. Moreover, global fitting analysis confirmed a mechanism where a large conformational change, after initial ground state binding of the substrate, contributed significantly to enzyme specificity. This led to our investigation of the molecular basis for enzyme specificity using HIV RT as a model system. Again, transient kinetic methods were applied with the addition of molecular dynamics simulations. The simulated results were substantiated by the corroborating experimental results. It was found that a substrate-induced conformational change in the transition of HIV RT from an open nucleotide-bound state to a closed nucleotide-bound state was the major determinant in enzyme specificity. The molecular basis for substrate selection resulted from the molecular alignments of the substrate in the active-site, which induced the conformational change. When the correct nucleotide was bound, optimal molecular interactions in the active-site yielded a stably closed complex, which promoted nucleotide incorporation. In contrast, when an incorrect nucleotide was bound, the molecular interactions at the active-site were not ideal, which yielded an unstable closed complex, which promoted substrate dissociation rather than incorporation.

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