Background: HIV-1 protease is a key antiviral drug target due to its essential function of processing viral polyproteins. Among primary protease mutations, I50V is commonly observed in patients failing therapy with PIs APV and DRV. In addition to conferring resistance to PIs, I50V mutation also impairs substrate processing. Mutations in the p1-p6 cleavage site are statistically associated with I50V protease mutation in the viral sequences retrieved from patients. However, the molecular basis of how compensatory mutations restore substrate recognition in drug resistance is not clear. In this study, we report the structural basis for the co-evolution of I50V/A71V protease with the p1-p6 substrate.
Methods: By applying crystallography method and molecular dynamics simulation, we investigate the structural basis of I50V/A71V protease and p1-p6 substrate co-evolution.
Results: The LP1’F substrate has more vdW contacts with I50V/A71V protease compared to those in either I50V/A71VWT or WTLP1’F. This is also the case for the PP5’L substrate. In addition, the LP1’F mutation causes a distal change at the substrate P5′ proline that is in an alternative position in the WT complex. This change increases the P5′ proline’s vdW contacts.
The RP4’S substrate forms an additional hydrogen bond with both WT and I50V/A71V protease through the P4′ serine side chain. This extra hydrogen bond may compensate for the loss of vdW contacts due to the smaller size of serine in these two complexes
In LP1’F substrate mutation, the peptide bond between Gly51 and Gly52 in the I50V/A71VPP5’L structure is flipped compared to the other structures, and this flipped peptide bond pushes the 50s loop towards the substrate, causing increased vdW contacts.
In the dynamic conformational ensemble of the WTWT structure, the distance between 80-80 loops is around 17.5 Å, and expands to 19–19.5 Å with mutations in either the protease or substrate, the co-evolution brings this distance back to 17.5–18.0 Å, similar to the WT inter-loop distance.
Conclusions: Coevolving mutations in the substrate enhance substrate–protease interactions through a variety of molecular mechanisms. The effects of substrate mutations are not local, but propagate to distal parts of both the substrate and the protease. Drug resistance mutations in the protease or the substrate disturbed the active site dynamics, which was restored in all co-evolved complexes bearing complementary mutations in both the protease and the substrate.