HIV reverse transcriptase (RT) is a primary target for
drug intervention in the treatment of
AIDS. We report the first
solution NMR studies of [methyl-(13)C]
methionine HIV-1 RT, aimed at better understanding the conformational and dynamic characteristics of RT, both in the presence and absence of the non-
nucleoside RT inhibitor (NNRTI)
nevirapine. The selection of
methionine as a structural probe was based both on its favorable NMR characteristics, and on the presence of two important active site
methionine residues, M184(66) and M230(66). Observation of the M184 resonance is subunit dependent; in the p66 subunit the
solvent-exposed residue produces a readily observed signal with a characteristic resonance shift, while in the globular p51 subunit, the M184(51) resonance is shifted and broadened as M184 becomes buried in the
protein interior. In contrast, although structural data indicates that the environment of M230 is also strongly subunit dependent, the M230 resonances from both subunits have very similar shift and relaxation characteristics. A comparison of chemical shift and intensity data with model-based predictions gives reasonable agreement for M184(66), while M230(66), located on the beta-hairpin "primer grip", is more mobile and
solvent-exposed than suggested by crystal structures of the apo
enzyme which have a "closed" fingers-thumb conformation. This mobility of the primer grip is presumably important for binding of non-
nucleoside RT inhibitors (NNRTIs), since the NNRTI binding pocket is not observed in the absence of the inhibitors, requiring instead that the binding pocket be dynamically accessible. In the presence of the
nevirapine, both the M184(66) and M230(66) resonances are significantly perturbed, while none of the
methionine resonances in the p51 subunit is sensitive to this inhibitor. Site-directed mutagenesis indicates that both M16 and M357 produce two resonances in each subunit, and for both residues, the intensity ratio of the component peaks is strongly subunit dependent. Conformational features that might explain the multiple peaks are discussed.