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. To gain further insight into the role of the transmembrane domain, we followed up on a recent study (Science 353: 172-175, 2016) which reported the trimeric structure of this segment. Even while our spectra were indistinguishable from those reported, analysis of residual dipolar couplings (RDCs) reported with both paramagnetic tagging and with using anisotropically compressed gels, indicated this trimeric structure is incompatible with the RDCs. The absence of a strong trimerization affinity is confirmed by density-matched sedimentation equilibrium ultracentrifuge measurements and by the absence of intermolecular paramagnetic relaxation effects in samples that employed mixed labeling. Remarkably, when extending the TM construct by 20 residues in the N-terminal direction, such that it includes the MPER regio, a weak trimerization tendency can be observed, even though the MPER region is found to be embedded on the surface of the membrane, separated from the TM domain by a dynamic 2-residue kink. The TM helix extends over 30 residues and adopts a geometry that is remarkably close to that of an ideal helix and, based on hydrophobic mismatch, must be oriented at a large angle relative to the bilayer normal.
| Chiliveri, Sai Chaitanya; Louis, John M; Ghirlando, Rodolfo et al. (2018) Tilted, Uninterrupted, Monomeric HIV-1 gp41 Transmembrane Helix from Residual Dipolar Couplings. J Am Chem Soc 140:34-37 |
| Roche, Julien; Louis, John M; Aniana, Annie et al. (2015) Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding. J Biomol NMR 61:235-48 |
| Roche, Julien; Louis, John M; Grishaev, Alexander et al. (2014) Dissociation of the trimeric gp41 ectodomain at the lipid-water interface suggests an active role in HIV-1 Env-mediated membrane fusion. Proc Natl Acad Sci U S A 111:3425-30 |
| Lakomek, Nils-Alexander; Kaufman, Joshua D; Stahl, Stephen J et al. (2013) Internal dynamics of the homotrimeric HIV-1 viral coat protein gp41 on multiple time scales. Angew Chem Int Ed Engl 52:3911-5 |
| Lakomek, Nils-Alexander; Ying, Jinfa; Bax, Ad (2012) Measurement of ยน?N relaxation rates in perdeuterated proteins by TROSY-based methods. J Biomol NMR 53:209-21 |
