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. 2014 May;82(5):815-29.
doi: 10.1002/prot.24460. Epub 2013 Nov 22.

Molecular Dynamics Study of HIV-1 RT-DNA-nevirapine Complexes Explains NNRTI Inhibition and Resistance by Connection Mutations

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Free PMC article

Molecular Dynamics Study of HIV-1 RT-DNA-nevirapine Complexes Explains NNRTI Inhibition and Resistance by Connection Mutations

R S K Vijayan et al. Proteins. .
Free PMC article

Abstract

HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that is targeted by nucleoside analogs (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). NNRTIs are allosteric inhibitors of RT, and constitute an integral part of several highly active antiretroviral therapy regimens. Under selective pressure, HIV-1 acquires resistance against NNRTIs primarily by selecting mutations around the NNRTI pocket. Complete RT sequencing of clinical isolates revealed that spatially distal mutations arising in connection and the RNase H domain also confer NNRTI resistance and contribute to NRTI resistance. However, the precise structural mechanism by which the connection domain mutations confer NNRTI resistance is poorly understood. We performed 50-ns molecular dynamics (MD) simulations, followed by essential dynamics, free-energy landscape analyses, and network analyses of RT-DNA, RT-DNA-nevirapine (NVP), and N348I/T369I mutant RT-DNA-NVP complexes. MD simulation studies revealed altered global motions and restricted conformational landscape of RT upon NVP binding. Analysis of protein structure network parameters demonstrated a dissortative hub pattern in the RT-DNA complex and an assortative hub pattern in the RT-DNA-NVP complex suggesting enhanced rigidity of RT upon NVP binding. The connection subdomain mutations N348I/T369I did not induce any significant structural change; rather, these mutations modulate the conformational dynamics and alter the long-range allosteric communication network between the connection subdomain and NNRTI pocket. Insights from the present study provide a structural basis for the biochemical and clinical findings on drug resistance caused by the connection and RNase H mutations.

Keywords: N348I; RNase H; T369I; allosteric communication; connection mutations; drug resistance; protein structural network.

Figures

Figure 1
Figure 1
Opening and closing of the cleft between the fingers and thumb subdomains during the course of MD simulations. (A) The distance between the centers of mass (COM) of fingers and COM of thumb subdomains in the course of 50-ns MD simulations of RT-DNA (black), RT-DNA-NVP (red), and N348I/T369I RT-DNA-NVP (green) structures. (B) Distribution profile based on fingers-thumb separation distance. (C) Ensembles of fingers and thumb positions obtained from the MD simulation of the RT-DNA-NVP complex show the flexibility of the thumb and finger subdomains.
Figure 2
Figure 2
Probing the catalytically-competent states of RT as a function of its distance between the polymerase active site and the primer 3’-terminal nucleotide during the course of MD simulations. (A) The distance between the COM of the primer 3’-end nucleotide and the COM of the polymerase active site. (B) Polymerase active site of the RT-DNA complex is in a catalytically competent mode. In the RT-DNA-NVP complex (C) or N348I/T369I mutant RT-DNA-NVP complex (D) the distance between the polymerase active site and the primer terminal nucleotide is increased. The distances listed in figure panels B, C, and D are the average distance calculated from panel A. At any instance of the simulation, the nevirapine-bound complexes the distance did not attain a value comparable to that observed in a catalytically-competent structure.
Figure 3
Figure 3
Free energy landscape obtained by projecting the MD trajectories on the first two principal components (PCs). (A) Free energy landscape of RT-DNA (A), RT-DNA-NVP (B), and N348I/T369I mutant RT-DNA-NVP (C) complexes. Representative conformations of RT from each major low energy basin are plotted below.
Figure 4
Figure 4
Hubs, communities, and communication paths identified from protein network analysis. (A) Hub residues identified for all the three complexes. The hub residues are depicted as van der Waals spheres and color-coded by location (fingers blue; palm red; connection yellow; RNase H orange; p51 gray). (B) Calculated communities and the shortest communication paths between the polymerase and RNase H active sites in the three complexes; the communication pathway is represented by red lines. Community-forming residues are shown as van der Waals spheres and color-coded based on the subdomains.
Figure 5
Figure 5
A close view of the distributions of hub residues (red) in the palm subdomain that emerged around the polymerase active site region, ATP-binding site (as an excision agent), and NNRTI pocket in the N348I/T369I mutant RT-DNA-NVP structure. Increases in the number of hub-forming residues imply enhanced structural rigidity around these regions. The common NNRTI-resistance mutation sites are shown as cyan spheres surrounding nevirapine (green).

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