Efavirenz binding site in HIV-1 reverse transcriptase monomers

Abstract

Efavirenz (EFV) is a potent nonnucleoside reverse transcriptase inhibitor (NNRTI) used in the treatment of AIDS. NNRTIs bind in a hydrophobic pocket located in the p66 subunit of reverse transcriptase (RT), which is not present in crystal structures of RT without an inhibitor. Recent studies showed that monomeric forms of the p66 and p51 subunits bind efavirenz with micromolar affinity. The effect of efavirenz on the solution conformations of p66 and p51 monomers was studied by hydrogen-deuterium exchange mass spectrometry (HXMS) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). HXMS data reveal that five peptides, four of which contain efavirenz contact residues seen in the crystal structure of the RT-EFV complex, exhibit a reduced level of exchange in monomer-EFV complexes. Moreover, peptide 232-246 undergoes slow cooperative unfolding-refolding in the bound monomers, but at a rate much slower than that observed in the p66 subunit of the RT heterodimer [Seckler, J. M., Howard, K. J., Barkley, M. D., and Wintrode, P. L. (2009) Biochemistry 48, 7646-7655]. These results suggest that the efavirenz binding site on p66 and p51 monomers is similar to the NNRTI binding pocket in the p66 subunit of RT. Nanoelectrospray ionization FT-ICR mass spectra indicate that the intact monomers each have (at least) two different conformations. In the presence of efavirenz, the mass spectra change significantly and suggest that p51 adopts a single, more compact conformation, whereas p66 undergoes facile, electrospray-induced cleavage. The population shift is consistent with a selected-fit binding mechanism.

FIGURE 1

Crystal structure of unliganded HIV-1 RT (1DLO). Four subdomains of the polymerase domain in p66 and p51 subunits: (blue) fingers, (pink) palm, (green) thumb, and (orange) connection; (grey) RNase H domain of p66 subunit; (black) efavirenz contact residues with side chains in p66 and p51 subunits.

FIGURE 2

Percent exchange of peptides in p66^W401A monomer in (upper) absence and (lower) presence of efavirenz. Color of amino acid sequence indicates subdomains: (blue) fingers, (red) palm, (green) thumb, and (orange) connection; (magenta) RNase H domain; (black) efavirenz contact residues. Colored bars below sequence from top to bottom give exchange at 10, 50, 100, 500, 1000, and 5000 s.

FIGURE 3

Percent exchange of peptides in p51^W401A monomer in (upper) absence and (lower) presence of efavirenz. Color of amino acid sequence indicates subdomains: (blue) fingers, (red) palm, (green) thumb, and (orange) connection; (black) efavirenz contact residues. Colored bars below sequence from top to bottom give exchange at 10, 50, 100, 500, 1000, and 5000 s.

FIGURE 4

Difference in number of deuteria exchanged in bound and unbound p66 and p51. Difference calculated by subtracting the exchange in unbound monomer from the exchange in the monomer–EFV complex. Differences are shown for (black) p66^W401A and (grey) p51^W401A after (left) 10 s, (middle) 100 s, and (right) 1000 s.

FIGURE 5

Mass spectra of peptide 232–246 in (left) p51^W401A and (right) p51^W401A–EFV complex after different incubations times in RT buffer D-D₂O. Low and high m/z peaks for p51^W401A–EFV complex fit to Gaussian distributions (dashed lines).

FIGURE 6

Nano-ESI mass spectra of (a) p51^W401A, (b) p51^W401A–EFV complex, (c) p66^W401A, (d) p66^W401A–EFV complex upon initiation of nano-ESI, and (e) p66^W401A–EFV complex after 3 min of nano-ESI. For p51^W401A in the absence and presence of EFV, the deconvoluted mass is 52,790 Da. Deconvoluted masses for p66^W401A are (▲) 66,210 Da, (●) 65,890 Da, and (▽) 65,550 Da. The peaks marked with (*) correspond to a 47 kDa truncated protein.

FIGURE 7

Five peptides stabilized in p66^W401A– and p51^W401A–EFV complexes shown in black on the subunits of p66/p51–EFV complex: (left) p66 subunit and (right) p51 subunit from crystal structure of HIV-1 RT–EFV complex (1FK9). Four subdomains of the polymerase domain: (blue) fingers, (pink) palm, (green) thumb, and (orange) connection; (grey) RNase H domain of p66.