The L76V Drug Resistance Mutation Decreases the Dimer Stability and Rate of Autoprocessing of HIV-1 Protease by Reducing Internal Hydrophobic Contacts.

The mature HIV-1 protease (PR) bearing the L76V drug resistance mutation (PRL76V) is significantly less stable, with a >7-fold higher dimer dissociation constant (Kd) of 71 ± 24 nM and twice the sensitivity to urea denaturation (UC50 = 0.85 M) relative to those of PR. Differential scanning calorimetry showed decreases in Tm of 12 °C for PRL76V in the absence of inhibitors and 5−7 °C in the presence of inhibitors darunavir (DRV), saquinavir (SQV), and lopinavir (LPV), relative to that of PR. Isothermal titration calorimetry gave a ligand dissociation constant of 0.8 nM for DRV, 160-fold higher than that of PR, consistent with DRV resistance. Crystal structures of PRL76V in complexes with DRV and SQV were determined at resolutions of 1.45−1.46 Å. Compared to the corresponding PR complexes, the mutated Val76 lacks hydrophobic interactions with Asp30, Lys45, Ile47, and Thr74 and exhibits closer interactions with Val32 and Val56. The bound DRV lacks one hydrogen bond with the main chain of Asp30 in PRL76V relative to PR, possibly accounting for the resistance to DRV. SQV shows slightly improved polar interactions with PRL76V compared to those with PR. Although the L76V mutation significantly slows the N-terminal autoprocessing of the precursor TFR-PRL76V to give rise to the mature PRL76V, the coselected M46I mutation counteracts the effect by enhancing this rate but renders the TFR-PRM46I/L76V precursor less responsive to inhibition by 6 μM LPV while preserving inhibition by SQV and DRV. The correlation of lowered stability, higher Kd, and impaired autoprocessing with reduced internal hydrophobic contacts suggests a novel molecular mechanism for drug resistance. [ABSTRACT FROM AUTHOR]