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 A 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 degrees-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.

Figure 1. FPPR and MPER increase the melting temperature of gp41.

A) Schematic overview of gp41; FP, fusion peptide; FPPR, fusion peptide proximal region; HR1, heptad repeat 1; HR2, heptad repeat 2; MPER, membrane proximal external region; TMR, transmembrane region. B) Unfolding of gp41_528–683 and gp41_541–665 monitored by circular dichroism spectroscopy at 222 nm.

Figure 2. Crystal structure of gp41_528–683 reveals a 90 Å long rod-like structure.

A) Ribbon representation of gp41. The previously determined core is colored dark blue (HR1) and marine blue (HR2). The flag sequence present at the N-terminus of HR2 is shown in black. FPPR is colored in light blue and MPER in grey. Note that the N-terminus of FPPR (residue 531) points towards the outside of the rod. B) Close up of the MPER and FPPR region shows the exposure of aromatic side chains Trp 678, Trp 680 and Tyr 681 towards the membrane.

Figure 3. The FPPR-MPER regions are splayed apart.

A) Close-up view from the bottom showing residue Leu 545 as the last coiled coil interacting residue of the HR1 core of gp41. The preceding potential heptad positions are Ala 541 and Thr 538. B) Close up view revealing mostly hydrophobic interactions between FPPR and MPER and only one hydrogen bond between the carbonyl of Ala and NE1 of Trp 670. C) Close-up of solvent exposed hydrophobic MPER residues.

Figure 4. Comparison of MPER conformations.

MPER conformations as determined in complex with broadly neutralizing antibodies (A) 2F5 , (B) Z13e1 and (C) 4E10 are shown in comparison to MPER within trimeric gp41. The corresponding MPER segments are colored equally and residues contacting the 4E10 Fab are shown as sticks. (blue, HR2).