Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding

Abstract

The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6HB trimer and the membrane affinity of gp41's ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of trimerization of gp41’s transmembrane helix to prevent complete dissociation of the trimer during the course of fusion.

HIV‐1 gp41 transmembrane oligomerization monitored by FRET and FCS

“…Roche et al [21], who used a truncated gp41 ectodomain construct where the immunodominant loop (IL) was replaced by a flexible linker, found that the protein was monomeric in the detergent dodecyl phosphocholine (DPC). The same behavior was observed also in the presence of the membrane proximal external region (MPER) [22].…”

The C34 Peptide Fusion Inhibitor Binds to the Six-Helix Bundle Core Domain of HIV-1 gp41 by Displacement of the C-Terminal Helical Repeat Region

“…After isolating the insoluble fraction, the constructs were fractionated on size-exclusion Superdex-200 or -75 columns (GE Healthcare, Piscataway, NJ) under denaturing conditions followed by reverse-phase high-performance liquid chromatography as described previously. 49 6-helix, 5-helix, core S , and core SP were folded by dialysis against 50 mM sodium formate (pH 3) followed by buffer exchange with 10 mM Tris-HCl (pH 7.6) and 150 mM NaCl (buffer A), concentrated to ~ 2 mg/mL, and stored.…”

How HIV-1 entry mechanism and broadly neutralizing antibodies guide structure-based vaccine design

“…Ground-breaking developments – involving diverse technologies including single molecule fluorescence resonance energy transfer (smFRET) [10], cryo-electron microscopy (cryo-EM) [11••,12••], X-ray crystallography [13••,14••] and nuclear magnetic resonance (NMR) [15,16] – are revealing the structures and conformations of the HIV-1 Env, a type 1 fusion machine that uses conformational change to drive fusion of viral and cellular membranes. These studies provide the context in which to situate bNAb sites of vulnerability.…”