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. 2019 Nov 5;116(45):22556-22566.
doi: 10.1073/pnas.1912427116. Epub 2019 Oct 17.

Topological analysis of the gp41 MPER on lipid bilayers relevant to the metastable HIV-1 envelope prefusion state

Yi Wang  1   2 , Pavanjeet Kaur  3   4 , Zhen-Yu J Sun  5 , Mostafa A Elbahnasawy  1   2 , Zahra Hayati  3   4 , Zhi-Song Qiao  1   2 , Nhat N Bui  3 , Camila Chile  1   2 , Mahmoud L Nasr  5 , Gerhard Wagner  5 , Jia-Huai Wang  1   6 , Likai Song  3 , Ellis L Reinherz  7   2 , Mikyung Kim  7   8
Affiliations

Topological analysis of the gp41 MPER on lipid bilayers relevant to the metastable HIV-1 envelope prefusion state

Yi Wang et al. Proc Natl Acad Sci U S A. .

Abstract

The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein (gp) 41 is an attractive vaccine target for elicitation of broadly neutralizing antibodies (bNAbs) by vaccination. However, current details regarding the quaternary structural organization of the MPER within the native prefusion trimer [(gp120/41)3] are elusive and even contradictory, hindering rational MPER immunogen design. To better understand the structural topology of the MPER on the lipid bilayer, the adjacent transmembrane domain (TMD) was appended (MPER-TMD) and studied. Membrane insertion of the MPER-TMD was sensitive both to the TMD sequence and cytoplasmic residues. Antigen binding of MPER-specific bNAbs, in particular 10E8 and DH511.2_K3, was significantly impacted by the presence of the TMD. Furthermore, MPER-TMD assembly into 10-nm diameter nanodiscs revealed a heterogeneous membrane array comprised largely of monomers and dimers, as enumerated by bNAb Fab binding using single-particle electron microscopy analysis, arguing against preferential trimeric association of native MPER and TMD protein segments. Moreover, introduction of isoleucine mutations in the C-terminal heptad repeat to induce an extended MPER α-helical bundle structure yielded an antigenicity profile of cell surface-arrayed Env variants inconsistent with that found in the native prefusion state. In line with these observations, electron paramagnetic resonance analysis suggested that 10E8 inhibits viral membrane fusion by lifting the MPER N-terminal region out of the viral membrane, mandating the exposure of residues that would be occluded by MPER trimerization. Collectively, our data suggest that the MPER is not a stable trimer, but rather a dynamic segment adapted for structural changes accompanying fusion.

Keywords: Env; HIV-1; MPER; nanodiscs; transmembrane domains.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Effect of cytoplasmic and TM sequences on the immersion depth of MPER-TMD in a membrane environment. (A) Domain architecture of gp41. Cyto, cytoplasmic domain; FP, fusion peptide. The peptide sequence used in this study comprised a L692C mutation for R1 spin labeling circled in red. (B) NMR structure of the HxB2 MPER-TMD segment simulated in the liposome (43). The colored region representing the lipid membrane is for illustration only. Lipid headgroup is represented in light blue and aliphatic region is shaded in darker blue. R1 spin labels are placed at the position of green L692 and V705. R696 side-chain position is shown in red relative to the lipid headgroup. (C) Immersion depths of L692 residue in various MPER-TMD segments from EPR measurements. Mutations are color-coded on the wild-type sequence. Cysteine mutations for R1 spin labeling in the MPER-TMD chimeras are highlighted in blue. (D) A model structure of TMD from integrin α-IIb (green) and from integrin β-3 (cyan) used for chimeric MPER-TMD peptide. The models were adapted from 2K9J of the Orientations of Proteins in Membranes (OPM) database.
Fig. 2.
Fig. 2.
(A–C) Modulation of MPER-specific bNAb recognition linked to the TMD and its sequence. Antigenicity analysis of indicated antibodies to the denoted MPER segments arrayed on liposomes as measured by BIAcore. DOPC:DOPG liposomes were prepared at a peptide-to-lipid ratio of 1:50. Then, 30 μL of 20 μg/mL antibodies was injected to monitor the binding. See Materials and Methods for details. As shown in SI Appendix, Fig. S2, bNAbs binding to bare liposome and an irrelevant mAb 1A3 (anti-ADA gp120 V3 loop) binding to various MPER-TMD/liposomes were used as a negative control.
Fig. 3.
Fig. 3.
MPER-TMD ND assembly. (A) Models of MPER-TMD in NDs in monomeric (Left) versus trimeric (Right) configurations. The MPER-TMD segment is simplified for illustration purposes and does not imply any precise structural and membrane-immersion information. (B and C) MPER-TMD was assembled into NDs as 1:1 (B) and 3:1 (C) MPER-TMD segment to ND ratios. In both, peptides were first codried with POPC:POPG (3:2), and then the mixture was solubilized and incubated with MSP. After dialysis, the assembled mixture was purified by size-exclusion column and peak corresponding fractions were analyzed on tricine SDS/PAGE gels with positions of hMSP and MPER-TMD relative to molecular weight markers given. (D) Minor peak at fraction 12 and major peak at fraction 15, both from C, were visualized by negative-stain EM. (Scale bar, 20 nm.)
Fig. 4.
Fig. 4.
Characterization of MPER-TMD/NDs. (A) The binding of MPER-specific bNAbs to MPER-TMD/NDs was measured by ELISA. NDs with MPER-TMD at 1:1 (Left) and 3:1 (Center) ratio was coated onto plates at a ND concentration of 50 nM. The lipid polyreactivity of bNAbs was analyzed by examining bNAb binding to empty ND (peptide-to-MSP ratio at 0:1) (Right) and served as a negative control. (B) 2F5 Fab binding to the ND was visualized by negative-stain EM. Then, 2 μM of MPER-TMD/ND at 1:1 ratio was incubated with 10 μM Fab at 4 °C for 1 h and then diluted accordingly for grid preparation. The field images (Left) and the selected gallery (Right) of ND images represent NDs with different numbers of associated Fab molecules. (Scale bars, 20 nm.) (C) Heterogeneity of MPER-TMD incorporation into ND was visualized by binding of bNAb Fabs, quantitated and graphically represented. Fab fragments of each bNAbs were complexed with MPER-TMD/ND samples at both peptide assembly ratios and the complexes were imaged by negative-stain EM, as above. The particles were categorized based on the number of bound Fabs (1 to 3). Over 3,000 particles were characterized in each Fab-MPER-TMD/ND combination.
Fig. 5.
Fig. 5.
Impact of Ile mutations in the gp41 CHR region on Env trimer antigenicity and fusogenicity. (A) CHR-MPER junction sequence comparison of ADA Env (ΔCT) cell expression constructs for the Ile mutants. Introduced mutations are highlighted in blue and the CHR region is shaded. (B) Schematic diagram of the hypothesized conformation for mutant Env trimers on the membrane with gp140 shown in blue, MPER in red, and TMD in green. (C) Antigenicity of 293T cell expressing ADA Env (ΔCT) variants by flow cytometry based on gp140 reactive bNAbs. Cells were incubated without (blue) or with (red) sCD4 and subsequently incubated with the indicated primary antibody followed by staining with fluorochrome-labeled anti-IgG secondary antibody. Geometric mean fluorescence intensity (gMFI) of the single live cell population of each mutant was measured. Normalized gMFI was the percentage of net gMFI for each antibody relative to that of pre-CD4 1A3, where net gMFI was calculated by subtracting MFI of untransfected cells from that of Env-expressing cells. Four replicates were included, and SEMs are shown as error bars. (D) Comparable antigenicity analysis as in C but probed with the designated anti-MPER bNAbs. (E) Fluorescence microscopy analysis of cell–cell fusion. Env-expressing 293T effector cells (green, Calcein-AM) and 3T3.CD4.CCR5 target cells (red, CMTMR) were stained and coincubated. Bright field (Left) and fluorescence images (Right) were collected 4 h (for wild-type) or 18 h postcoincubation for Ile mutants. Fused effector targets are indicated by yellow arrows. (Magnification, 40×.)
Fig. 6.
Fig. 6.
10E8-induced conformational change of the MPER. (A) Membrane immersion depth analysis of the MPER in the absence and presence of 10E8 Fab by EPR. A cysteine mutation was introduced at the site of measurement where an R1 spin label was coupled. (B) Membrane immersion depth measurement of MPER-nTM (amino acids 662 to 693) in the absence and presence of 10E8 Fab. The residues of interest were 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid (TOAC)-labeled for EPR analysis. Residues measured in both MPER and MPER-nTM analysis are highlighted. Depth values between −5 Å and 0 Å and larger than 0 Å correspond to lipid headgroup region and acyl-chain region, respectively. The precise depths of residues exposed to aqueous phase (depth < −5 Å) cannot be determined experimentally and are thus indicated by the striped bars. (C) MPER peptide docked to lipid bilayer surface based on EPR membrane immersion depth data. (D) 10E8-bound MPER peptide docked to lipid bilayer based on EPR data. The dark blue-shaded area represents lipid aliphatic region and the light blue-shaded area represents lipid head-group region.
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