Membrane fusion between HIV and host cells requires interaction between the N-terminal and C-terminal repeat regions (NHR and CHR) of the gp41 envelope subunit. A deep hydrophobic pocket (HP) on the surface of NHR is considered crucial in this interaction. Targeting the viral gp41 CHR with stabilized trimeric NHR peptides or chimeric proteins effectively inhibits HIV infection. However, the contribution of each specific structural element, particularly the HP, in this mode of inhibition remains unclear. Here, we describe three chimeric proteins that structurally mimic the full NHR region interacting intramolecularly with CHR segments of varying lengths, either covering or exposing the HP. The intramolecular NHR-CHR interaction strongly stabilized all the chimeras. Binding analysis using a CHR-derived peptide and the hydrophobic probe 8-anilino-1-naphthalene sulfonate (ANS) combined with X-ray crystallography assessed the degree of exposure of the HP in the chimeras. Despite differences in HP accessibility, none of the chimeras displayed relevant inhibitory activity against several HIV-1 strains, suggesting that an exposed HP alone is insufficient to disrupt the NHR-CHR interaction in a viral context. The crystal structure of the ANS-chimera complex revealed the binding pose of ANS within the HP, while the overall CHR-NHR interaction closely resembled the canonical post-fusion six-helix-bundle structure. To our knowledge, this is the first crystallographic structure of a small molecule ligand independently bound to the HP. These findings provide insight into the role of the HP in NHR-based fusion inhibitors and guide the design of new, more focused and effective HIV inhibitors.
Polo-Megías et al. (Mon,) studied this question.