Crystal Structure of HIV-1 gp41 Including Both Fusion Peptide and Membrane Proximal External Regions

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Abstract
The HIV-1 envelope glycoprotein (Env) composed of the receptor binding domain gp120 and the fusion protein subunit gp41 catalyzes virus entry and is a major target for therapeutic intervention and for neutralizing antibodies. Env interactions with cellular receptors trigger refolding of gp41, which induces close apposition of viral and cellular membranes leading to membrane fusion. The energy released during refolding is used to overcome the kinetic barrier and drives the fusion reaction. Here, we report the crystal structure at 2 Å resolution of the complete extracellular domain of gp41 lacking the fusion peptide and the cystein-linked loop. Both the fusion peptide proximal region (FPPR) and the membrane proximal external region (MPER) form helical extensions from the gp41 six-helical bundle core structure. The lack of regular coiled-coil interactions within FPPR and MPER splay this end of the structure apart while positioning the fusion peptide towards the outside of the six-helical bundle and exposing conserved hydrophobic MPER residues. Unexpectedly, the section of the MPER, which is juxtaposed to the transmembrane region (TMR), bends in a 90°-angle sideward positioning three aromatic side chains per monomer for membrane insertion. We calculate that this structural motif might facilitate the generation of membrane curvature on the viral membrane. The presence of FPPR and MPER increases the melting temperature of gp41 significantly in comparison to the core structure of gp41. Thus, our data indicate that the ordered assembly of FPPR and MPER beyond the core contributes energy to the membrane fusion reaction. Furthermore, we provide the first structural evidence that part of MPER will be membrane inserted within trimeric gp41. We propose that this framework has important implications for membrane bending on the viral membrane, which is required for fusion and could provide a platform for epitope and lipid bilayer recognition for broadly neutralizing gp41 antibodies. HIV-1 employs its envelope glycoprotein complex (Env) composed of gp120 and gp41 to catalyze cell entry. Both Env subunits undergo conformational changes triggered by the gp120-mediated interactions with cellular receptors. Notably, gp41 refolds into a core six-helical bundle structure which is central to the fusion process. Here we report the structural basis for the folding of the linker regions connecting to the membrane anchors of gp41, namely to the transmembrane region (MPER) and to the fusion peptide (FPPR). Our structural analysis shows helical assemblies of FPPR and MPER which increase the melting temperature of gp41 and position the fusion peptide towards the outside of the six-helix bundle structure at this stage of gp41 refolding. It suggests that part of MPER must be inserted into the viral membrane, which would induce membrane curvature as postulated to be required for the fusion reaction. Thus our findings shed new light on the refolding of gp41, which contributes energy to the fusion reaction and reveals for the first time the structural principles of MPER membrane interaction within trimeric gp41. We propose that the structure presents a late fusion intermediate state that provides a new framework for fusion inhibitor development and MPER immunogen design.