$995,227
National Institute of Diabetes and Digestive and Kidney Diseases
Maryland
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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 and bicelles consisting of DMPC and dihexanoyl phosphatidylcholine suggested the importance of trimerization of gp41s transmembrane helix to prevent complete dissociation of the trimer during the course of fusion. The membrane proximal external region (MPER) of HIV-1 gp41 contains epitopes for at least four broadly neutralizing antibodies. Depending on solution conditions and construct design, different structures have been reported for this segment. We have found that in aqueous solution the MPER fragment (gp160 residues 660-674) exists in a monomer-trimer equilibrium with an association constant in the micro-molar range. Thermodynamic analysis revealed that the association is exothermic, more favorable in D2O than H2O, and increased with ionic strength, indicating hydrophobically driven intermolecular interactions. Circular dichroism, 13C chemical shifts, NOE, and hydrogen exchange rates revealed that MPER undergoes a structural transition from predominately unfolded monomer at low concentrations to an alpha-helical trimer at high concentrations. This result has implications for antibody recognition of MPER prior to and during the process where gp41 switches from a pre-hairpin intermediate to its post-fusion 6-helical bundle state. Preliminary experiments applied to the ecto-domain of the Spike protein of SARS-CoV-2 indicate this protein contains a three-helical N-terminal heptad repeat (NHR) that is considerably more stable than the corresponding region of gp41, and the propensity for the C-terminal heptad (CHR) repeat albeit lower than that of NHR, is also substantially higher than for the CHR of gp41, whereas the propensity to trimerize for its membrane proximal region is substantially lower. Work has been submitted for publication but remains under review.