Glycine Substitution at Helix-to-Coil Transitions Facilitates the Structural Determination of a Stabilized Subtype C HIV Envelope Glycoprotein.

Advances in HIV-1 envelope glycoprotein (Env) design generate native-like trimers and high-resolution clade A， B， and G structures and elicit neutralizing antibodies. However， a high-resolution clade C structure is critical， as this subtype accounts for the majority of HIV infections worldwide， but well-ordered clade C Env trimers are more challenging to produce due to their instability. Based on targeted glycine substitutions in the Env fusion machinery， we defined a general approach that disfavors helical transitions leading to post-fusion conformations， thereby favoring the pre-fusion state. We generated a stabilized， soluble clade C Env (16055 NFL) and determined its crystal structure at 3.9 Å. Its overall conformation is similar to SOSIP.664 and native Env trimers but includes a covalent linker between gp120 and gp41， an engineered 201-433 disulfide bond， and density corresponding to 22 N-glycans. Env-structure-guided design strategies resulted in multiple homogeneous cross-clade immunogens with the potential to advance HIV vaccine development.