Protein-Mediated Antagonism between HIV Reverse Transcriptase Ligands Nevirapine and MgATP

Abstract

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) play a central role in the treatment of AIDS, but their mechanisms of action are incompletely understood. The interaction of the NNRTI nevirapine (NVP) with HIV-1 reverse transcriptase (RT) is characterized by a preference for the open conformation of the fingers/thumb subdomains, and a reported variation of three orders of magnitude between the binding affinity of NVP for RT in the presence or absence of primer/template DNA. To investigate the relationship between conformation and ligand binding, we evaluated the use of methionine NMR probes positioned near the tip of the fingers or thumb subdomains. Such probes would be expected to be sensitive to changes in the local environment depending on the fractions of open and closed RT. Comparisons of the NMR spectra of three conservative mutations, I63M, L74M, and L289M, indicated that M63 showed the greatest shift sensitivity to the addition of NVP. The exchange kinetics of the M63 resonance are fast on the chemical shift timescale, but become slow in the presence of NVP due to the slow binding of RT with the inhibitor. The simplest model consistent with this behavior involves a rapid open/closed equilibrium coupled with a slow interaction of the inhibitor with the open conformation. Studies of RT in the presence of both NVP and MgATP indicate a strong negative cooperativity. Binding of MgATP reduces the fraction of RT bound to NVP, as indicated by the intensity of the NVP-perturbed M230 resonance, and enhances the dissociation rate constant of the NVP, resulting in an increase of the open/closed interconversion rate, so that the M63 resonance moves into the fast/intermediate-exchange regime. Protein-mediated interactions appear to explain most of the affinity variation of NVP for RT.

NMR characterization of HIV-1 reverse transcriptase binding to various non-nucleoside reverse transcriptase inhibitors with different activities

“…S1 ). To assign each methyl resonance, six different mutants of HIV-1 RT were prepared, substituting each methionine in the p66 subunit with leucine 16 17 . The 1 H- 13 C HSQC spectra of these mutants were compared with those of the wild type, thereby identifying peaks originating from M16, M184, and M357, because these peaks were missing in the spectra of the corresponding mutants ( supplementary Fig.…”

“…Although applying the NMR technique to analysis of large proteins remains challenging, this spectroscopic method provides valuable information regarding dynamic aspects of ligand binding. It has been reported that selective isotope labeling with 13 C at the methyl side chain of methionine offers useful spectroscopic probes for investigating the structures and dynamics of larger proteins 14 15 16 17 18 . Zheng et al previously reported heteronuclear single-quantum coherence (HSQC) spectra for observing signals from the methionine methyl groups of the HIV-1 RT p66 subunit in the absence and presence of nevirapine, with assignments based on the site-directed mutagenesis method 16 17 .…”

Mass Spectrometric Characterization of HIV-1 Reverse Transcriptase Interactions with Non-nucleoside Reverse Transcriptase Inhibitors

“…To characterize the interaction of HIV-1 RT with NNRTIs, various biophysical approaches have been applied, including X-ray crystallography, 8,[10][11][12][13] NMR spectroscopy, [14][15][16][17] isothermal titration calorimetry (ITC), [18][19][20][21] and surface plasmon resonance (SPR), 20,22) as well as computational methods. 13,23,24) However, these techniques have often been hampered by the extremely low water solubility of NNRTIs, especially the current most promising diarylpyrimidine-based inhibitors, 25) such as rilpivirine and etravirine.…”

Structural Maturation of HIV-1 Reverse Transcriptase—A Metamorphic Solution to Genomic Instability

“…These interactions will in general not be synergistic, since the ligands often select different active site conformations, resulting in negative binding cooperativity (e.g., [80,90]). In addition, double-stranded nucleotides can contact both subunits of the RT heterodimer, providing an additional basis for stabilization.…”

“…Agopian et al [140] have developed several thumb-derived peptides that inhibit RT maturation and block viral maturation at subnanomolar concentrations. However, this peptide apparently does not bind in such a way that it significantly interferes with the stability of the mature RT heterodimer or alters the open/closed ratio of the fingers:thumb [90]. Wendeler et al [141] and Chung et al [142] have described vinylogous ureas that interfere with RNase H activity, possibly by interacting with the p51 thumb' domain and altering the dimer interface [141,143], and Masaoka et al [144] have described thumb domain-targeted thienopyrimidinones that are predicted to bind near the RH:thumb’ interface and destabilize the heterodimer, decreasing the melting temperature by 5 °C.…”