Refolding Dynamics of gp41 from Pre-fusion to Pre-hairpin States during HIV-1 Entry.

The HIV-1 infection is triggered by the binding of the viral envelope glycoprotein (Env) gp120-gp41 trimer to host-cell receptor CD4 and co-receptor CCR5/CXCR4, which leads to substantial conformational changes of Env, that is, structural transition of gp120 from a closed to an open state followed by gp41 refolding from pre-fusion to post-fusion states. The latter finally promotes membrane fusion, likely via visiting a critical pre-hairpin state of gp41. The complete conformational dynamics of the pre-hairpin formation at atomic resolution, however, is still unknown. Here, by constructing a Markov state model based on the all-atom molecular dynamics (MD) with an aggregated simulation time of ∼24 μs, we reveal the gp41 refolding dynamics from pre-fusion to pre-hairpin state and the key metastable states involved. Moreover, we further explored the drug resistance mechanism of two C-terminal heptad repeat-derived gp41 inhibitors, T20 and sifuvirtide, based on the constructed inhibitor-bound gp41 pre-hairpin complexes. The results indicate that these two inhibitors have distinct binding sites on gp41 but share a common drug resistance region that usually exhibits a helical structure in the pre-hairpin state yet adopts various secondary structures in other metastable states. Moreover, we conducted several mutant MD simulations to further investigate the mechanisms of how some drug-resistant mutations might affect the pre-hairpin formation, which in turn prevent the inhibitor recognition. Our findings provide deep structural insights into the molecular mechanisms of the pre-hairpin formation for gp41, which helps to guide future anti-HIV drug design.