Biophysics in Africa: Cryptic No More: Characterising a Novel Allosteric Site in the HIV-1 Protease Cantilever Region

HIV-1 protease is a critical enzyme for viral maturation and one of the most well-studied proteins in structural biology. While much attention has focused on its active site, there is interest in how the enzyme might interact with small molecules at other regions on its surface. In particular, some of these potential sites, called cryptic pockets, are not visible in the unbound structure and only emerge under certain conditions. Until now, no cryptic sites had been identified in HIV-1 protease. We used mixed-solvent molecular dynamics simulations to uncover a previously unrecognised cryptic pocket in the cantilever region of the enzyme. Interestingly, this region shows conserved structural behaviour across related retroviral proteases, hinting at a broader functional role. When we locked this region in place using disulfide cross-linking, the protease adopted a distinct semi-open conformation, suggesting that the cantilever region influences flap movement. We then performed fragment-based screening which revealed that this cryptic pocket can bind small molecules. Simulations with one such fragment showed that binding alters the dynamics of the protease, particularly in the flap region. Overall, our findings highlight the cantilever region as a key regulatory element in HIV-1 protease function and a promising target for future drug discovery efforts.