Structure of the membrane proximal external region of HIV-1 envelope glycoprotein

Abstract

The membrane-proximal external region (MPER) of the HIV-1 envelope glycoprotein (Env) bears epitopes of broadly neutralizing antibodies (bnAbs) from infected individuals; it is thus a potential vaccine target. We report an NMR structure of the MPER and its adjacent transmembrane domain in bicelles that mimic a lipid-bilayer membrane. The MPER lies largely outside the lipid bilayer. It folds into a threefold cluster, stabilized mainly by conserved hydrophobic residues and potentially by interaction with phospholipid headgroups. Antigenic analysis and comparison with published images from electron cryotomography of HIV-1 Env on the virion surface suggest that the structure may represent a prefusion conformation of the MPER, distinct from the fusion-intermediate state targeted by several well-studied bnAbs. Very slow bnAb binding indicates that infrequent fluctuations of the MPER structure give these antibodies occasional access to alternative conformations of MPER epitopes. Mutations in the MPER not only impede membrane fusion but also influence presentation of bnAb epitopes in other regions. These results suggest strategies for developing MPER-based vaccine candidates.

Keywords: HIV-1 Env; NMR structure; membrane proximal region; transmembrane region.

Fig. 1.

Structure of the MPER-TMD of HIV-1 Env. (A) Ribbon representation of the average structure of the calculated ensemble. The MPER (residues 660–683) and the TMD (residues 684–710) are shown in yellow and green, respectively. (B) A close-up view of the MPER trimer showing the three protomers in different colors, as well as the characteristic features including the N- and C-helices, the hydrophobic core, the turn connecting the N- and C-helices, and the kink between the C-helix and TMD. (C) The “hydrophobic core” consisting of N-helix hydrophobic residues (W666, W670, L661, and L669). (D) The “turn” region containing residues 671–676. (E) The “kink” at residues 680–683 resulting in a ∼45° change in helix orientation (indicated by the arrows). The O(i)−HN(i + 4) distances of 679–683 and 680–684, indicated by red dashed lines, are >5 Å, much greater than a standard hydrogen bond distance (∼2.5 Å).

Fig. 2.

Transmembrane partition of the MPER-TMD in bicelles. (A) Solvent PRE amplitude is plotted against residue number for the MPER-TMD with the MPER shown in orange and the TMD in green. (B) PRE amplitude is plotted against distance from the bilayer center along the trimer axis, fitted to the sigmoidal function. The fit is shown in red. (C) Position of the MPER-TMD trimer in surface representation relative to the lipid bilayer and the bilayer center with the MPER in yellow and the TMD in green. (D) Fit of the MPER-TMD into EM density of the HIV-1 Env trimer on the surface of virion. (Left) Density of the Env trimer [Electron Microscopy Data Bank (EMDB) ID: EMD-5019] and viral membrane (EMDB ID: EMD-5020), derived from cryo-ET (39), is shown in gray. The backbone trace of a natively glycosylated HIV-1 BG505 SOSIP.664 Env trimer [Protein Data Bank (PDB) ID code 5T3Z] (43), fitted to the cryo-ET density, is in light blue, the MPER in yellow, and the TMD in green. (Right) A view from below of the cryo-ET density within the dashed box, with the membrane density and the SOSIP backbone omitted for clarity.

Fig. 3.

Antibody accessibility of the MPER-TMD in bicelles. (A) The MPER-TMD structure, superposed on crystal structures of the MPER epitope peptides in the complex with their corresponding antibodies, 2F5 [PDB ID code 1TJH (20)], 4E10 [PDB ID code 2FX7 (71)], 10E8 [PDB ID code 4G6F (12)], and DH570 [PDB ID code 5DD0 (44)]. Antibody heavy and light chains are in blue and green, respectively; the epitope peptides are in cyan, the MPER trimer in red, and the TMD in gray. (B) Binding of the MPER-TMD to 2F5 Fab was monitored by loss of NMR signal (due to rapid signal relaxation upon Fab binding). The 1D ¹H-¹⁵N TROSY-HSQC spectrum of the tryptophan indole amines was recorded for the MPER-TMD in bicelles (q = 0.55) at various time points, shown in different colors, after addition of 2F5. The reference spectrum in black was recorded without 2F5. The MPER-TMD:antibody molar ratio was 1.0:0.7. (C–E) Same as in B performed for the 4E10, 10E8, and DH570 Fabs, respectively. (F) Same as in B performed for the anti-6xHis Fab (prepared from antibody MA1-21315) using the MPER-TMD with an N-terminal 6xHis tag. (G) Fraction of Fab not bound to the MPER-TMD at various time points, calculated as (I − 0.3)/(I₀ − 0.3), where I₀ is the reference peak intensity normalized to 1, I is the fraction peak intensity at a particular time relative to I₀, and subtraction of 0.3 corrected for the 30% molar excess of MPER-TMD in the mixture. The y axis intercepts indicate the fraction of the MPER-TMD in a conformation that is incompatible with antibody binding. The essentially flat line for VRC01 shows little or no binding to MPER, as expected.

Fig. 4.

Effect of mutations in the MPER on Env antibody sensitivity. Antibody neutralization of pseudoviruses containing either the wild-type 92UG037.8 Env or one of the MPER mutants shown was determined for the ordinarily nonneutralizing antibodies, b6 (CD4 binding site; blue), 3791 (V3; cyan), and 17b (CD4-induced; purple) and for the trimer-specific bnAbs, PG9 (orange), PG16 (red), and PGT145 (magenta). The CD4 binding site bnAb VRC01 (green) was a control. The experiment was performed in duplicate.