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

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.

Structure of an HIV-1–neutralizing antibody target, the lipid-bound gp41 envelope membrane proximal region trimer

“…Others are consistent with a more compact structure having a width of ∼35 Å where the Env stalk enters the membrane (6,7,10). The crystal structure of the postfusion six-helix bundle shows that the MPER packs onto the outside of the bundle, leading to significant separation of the C termini of the MPER domain (13). In our structure, the C termini of the MPER trimer segments are not self-associated and, instead, are associated with the detergent micelle.…”

“…The short peptides were either solubilized in detergent micelles (39,44,45) or in bound forms cocrystallized with MPER-recognizing antibodies (31,32). Of the trimeric structures, one was not membrane-associated and does not bind 2F5 or 4E10 antibodies (38), and the other was part of a six-helix bundle representing the postfusion state of gp41 (13).…”

“…The formation of this conformation is thought to drive membrane fusion. This conformation is stable, and its structure has been well studied using X-ray crystallography techniques (13). Binding studies have shown that the broadly neutralizing antibodies 2F5 and 4E10 do not bind with high affinity to either the postfusion six-helix bundle or the prefusion resting state, suggesting that a prefusion intermediate state is the target for these antibodies (12).…”

Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

“…GeneJuice (Millipore) was used for all transient transfection reactions in adherent cells. Stably transfected dual-split protein (DSP)1 and DSP2 cells expressing DSP [1][2][3][4][5][6][7] or DSP [8][9][10][11] , respectively, are derived from the human glioma cell line NP-2/CD4 (29). DSP cell lines were maintained in MEM with 7.5% (vol/vol) FBS at 37°C and 5% CO 2 .…”

“…One, designated "HR-A or HR-1," is located in close proximity to the fusion peptide [in the case of HIV gp41, HR-1 is separated through a 13-residue fusion peptide proximal region from the fusion peptide (9); in the case of paramyxovirus F, HR-A is directly linked to the fusion peptide]. The other, designated "HR-B or HR-2," is in close proximity to the transmembrane domain [in the case of gp41, HR-2 is separated through an 18-residue membrane proximal external region from the transmembrane domain (9); in the case of paramyxovirus F, HR-B is separated through a seven-to nine-residue region from the transmembrane domain (10)]. In the final 6HB fusion core, the fusion peptides and transmembrane domains, and thus the target and donor membranes, are brought into immediate proximity.…”