Structure of HIV-1 gp41 with its membrane anchors targeted by neutralizing antibodies
- Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), France
- Instituto Biofisika (CSIC, UPV/EHU) and Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Spain
- Institute of Medical Virology, University of Zurich, Switzerland
- Laboratoire de Physique et Chimie Théoriques (LPCT), University of Lorraine, France
- Laboratoire International Associé, CNRS and University of Illinois at Urbana-Champaign, France
- Department of Physics, University of Illinois at Urbana-Champaign, United States
Figures
Figure 1 with 7 supplements
Crystal structure of gp41FP-TM in complex with 2H10.
(A) Schematic drawing of gp41 and expression constructs of gp41 chains N and C. Sequence numbering is based on the HIV-1-HBX2 envelope gp160 sequence. Color coding is as follows: FP, fusion peptide, …
Figure 1—figure supplement 1
Characterization of gp41 containing FP and TM.
(A) Size exclusion chromatography of the gp41FP-TM complex composed of chains N and C and SDS-PAGE showing the two bands corresponding to gp41 chains N and C. (B) SEC of gp41FP-TM in complex with …
Figure 1—figure supplement 2
Biophysical characterization of gp41FP-TM and MPER Ab interaction.
(A) Circular dichroism of gp41FP-TM shows that FP and TM increase the melting temperature of gp41. Temperature-dependent unfolding of gp41FP-TM monitored by circular dichroism spectroscopy recorded …
Figure 1—figure supplement 3
Close-ups of the model and its corresponding electron density.
2Fo-Fc composite omit maps contoured at 1 σ of a central 6HB core region (A and B) of the kinked MPER conformation of protomer C.
Figure 1—figure supplement 4
Comparison of the gp41FP-TM structure with gp41 core structures.
Ribbon presentation of gp41-MPER (pdb 3k9a), gp41FPPR-MPER (pdb 2x7r) and gp41FP-TM (numbering is shown for chain B). Cα super positioning of all three structures onto chains N-B (residues 546–574) …
Figure 1—figure supplement 5
Positioning of gp41FP-TM-2H10 in a bilayer by MD simulation.
(A) Model of gp41FP-TM-2H10 before simulation and (B) after 1 µs simulation, which repositions the 2H10 CDR3 in the membrane and reveals movement of FP of chain C. The orange spheres represent the …
Figure 1—figure supplement 6
Crystal lattice packing.
Crystal packing of protein 2-D layers arranged in the c direction of the crystal unit cell do not show defined crystal contacts. The inset shows the distances between the protein layers indicating …
Figure 1—figure supplement 7
Membrane interaction of 2H10.
Membrane interaction of nanobodies 2H10, 2H10-RKRF, and bnAb 10E8 was tested using liposomes containing the lipid composition of the HIV-1 envelope. Nanobodies 2H10 and 2H10-RKRF as well as bnAb10E8 …
Figure 2 with 1 supplement
Vesicle-vesicle fusion inhibition by 2H10-RKRF, bi-2H10, 2F5, and 10E8.
(A) Time course of the lipid-mixing assay using fusion-committed vesicles. At time 30 s (‘+N-MPER’), peptide (4 µM) was added to a stirring solution of unlabeled vesicles (90 µM lipid), and, after …
Figure 2—figure supplement 1
Vesicle-vesicle fusion inhibition by 10E8 Fabs.
(A) Time course of the lipid-mixing assay using fusion-committed vesicles. At time 30 s (‘+C-MPER’), peptide (4 µM) was added to a stirring solution of unlabeled vesicles (90 µM lipid), and, after …
Figure 3 with 1 supplement
Gp41FP-TM interaction with bnAbs LN01 and 10E8.
(A) Cα superposition of the MPER peptide structure in complex with LN01 (pdb 6snd) onto chain C-C of the gp41FP-TM-2H10 structure. The lower panel shows a close-up of the interaction oriented with …
Figure 3—figure supplement 1
Pull down of gp41FP-TM by bnAb 10E8 and LN01.
Immunoprecipitation of gp41FP-TM by bNAbs 10E8 and LN01. Input and eluted fractions were analyzed on SDS-gel and stained with Coomassie brilliant blue. Input fractions correspond to gp41FP-TM alone …
Figure 4 with 2 supplements
Interactions within the final post fusion conformation of gp41FP-TM modeled by MD.
(A) Model of gp41FP-TM (Figure 1—figure supplement 7C) after 1µs MD simulation in a bilayer. Phosphate groups of the phospholipids are shown as orange spheres to delineate the membrane boundaries. (B…
Figure 4—figure supplement 1
Modeling a post fusion conformation by MD simulation.
(A) Ribbon of the crystal structure of gp41FP-TM. Numbering of chain B is as shown in Figure 1F. (B) Ribbon of the symmetric trimer model built from chains N-B and C-B of the gp41FP-TM structure. …
Figure 4—figure supplement 2
Positions of the conserved tryptophan residues of MPER in the post fusion model.
Tryptophan residues W666, W670, W672, W678, and W680 and their close-by potential contacts are shown as spheres.
Figure 5
Conformational transitions of gp41 that lead to membrane apposition and membrane fusion.
(A) Representation of the different domains of gp41 with the residue numbers delimiting each domain as indicated. The same color code has been used in all the figures. (B) Ribbon presentation of the …
Author response image 1
MALDI-TOF analysis of gp41FP-TM.
The raw spectrum shows the single and doubly charged ion of chains N and C.
Author response image 2
Neutralization potency of 10E8 Fab variants (cell entry inhibition measured against HIV-1 virions pseudotyped with JR-CSF Env) and their ability to block C-MPER-induced lipid-mixing.
The top diagram (Figure 2—figure supplement 1B) shows the mutated residues and their positions with respect to the putative membrane binding.
Author response image 3
Gp41(512-711) used in this experiment, composed of residues 512-581 and 628-711 (including FP-FPPR and MPER-TM), reveals a Tm of 90°C (green curve, buffer DDM) and gp41(528-683) (including FPPR and MPER) has a Tm of 88°C (black curve, no detergent).
Please note that gp41FP-TM used in the current study is composed of residues 512-594 with a 13 aa longer N-terminal coiled coil and a 5 residue C-terminal extension to residue 716. The two curves …
Tables
Table 1
Crystallographic data collection and refinement statistics.
| Data collection | Gp41FP-TM* |
|---|---|
| Space group | C 2 2 21 |
| Cell dimensions | |
| a, b, c (Å) | 96.75, 101.41, 234.42 |
| α, β, γ (°) | 90, 90, 90 |
| Resolution (Å) | 48.38–3.8 (3.94–3.8) * |
| Unique reflexions | 11179 (631)* |
| Rmerge† | 0.23 (1.508)* |
| Rp.i.m ‡ | 0.081 (0.548) |
| I / σI | 4.75 (1.74) * |
| Completeness (%) | 78.01 (54.69) * |
| Multiplicity | 9.1 (9.6) * |
| CC (1/2) | 0.992 (0.628) * |
| Refinement | |
| Resolution (Å) | 48.38–3.8 (3.936–3.8)* |
| No. reflections | 9154 (630)* |
| Reflections used for Rfree§ | 550 (51)* |
| Rwork§ / Rfree** | 0.265/0.308 |
| No. atoms | |
| Protein | 4440 |
| Ligand/ion | 0 |
| Water | 0 |
| Wilson B (Å2) | 75.8 |
| Average B-factors (Å2) | |
| Overall | 91.76 |
| Protein | 91.76 |
| Ligand/ion | |
| Water | |
| R.m.s deviations | |
| Bond lengths (Å) | 0.003 |
| Bond angles (°) | 0.66 |
| Ramachandran Plot (%) | |
| Favored | 96.65 |
| Outliers | 0.37 |
| PDB ID | 7AEJ |
-
*Data collected from two crystals were used for structure determination.
The statistics are for data that were truncated by STARANISO to remove poorly measured reflections affected by anisotropy. Rmerge, Rp.i.m and multiplicity are calculated on unmerged data prior to STARANISO truncation. For comparison, after STARANISO truncation, Rmerge in the resolution shell 3.97 Å - 3.85 Å is 0.787.
-
† Parentheses refer to outer shell statistics.
‡ Rmerge = Σhkl Σi | Ihkl,i- < Ihkl > | / Σhkl ΣiIhkl,i, where Ihkl,i is the scaled intensity of the ith measurement of reflection h, k, l, and <Ihkl > is the average intensity for that reflection.
-
§ R p.i.m. = Σhkl √1/(n-1) Σi | Ihkl,i- < Ihkl > | / Σhkl ΣiIhkl,i,.
¶ Rwork = Σhkl | Fo - Fc | / Σhkl | Fo | x 100, where Fo and Fc are the observed and calculated structures factors.
-
** Rfree was calculated as for Rwork, but on a test set of 5% of the data excluded from refinement.
-
Table 1—source data 1
Env pseudoviruses.
- https://cdn.elifesciences.org/articles/65005/elife-65005-table1-data1-v2.docx
Table 2
Pseudovirus neutralization by 2H10, 2H10-F, 2H10-RKRF, and bi-2H10 in comparison to 2F5 and VRC01.
IC50s are indicated in µg/ml.
| Tier | 2H10 wt | 2H10-F | 2H10-RKRF | Bi-2H10 | 2F5 | VRC01 | |
|---|---|---|---|---|---|---|---|
| NL4-3 | 1 | 25.20 | 18.68 | 9.15 | 1.84 | 0.16 | 0.20 |
| MN-3 | 1 | >50.00 | 30.38 | 9.36 | 1.39 | 0.03 | 0.06 |
| BaL.26 | 1 | >50.00 | 19.38 | 9.63 | 6.05 | 1.21 | 0.13 |
| SF162 | 1a | >50.00 | >50.00 | 25.19 | 6.14 | 1.22 | 0.39 |
| SF162P3 | 2 | 22.04 | 13.14 | 6.76 | 1.32 | 1.96 | 0.24 |
| SC422661.8 | 2 | >50.00 | >50.00 | 27.93 | 3.79 | 1.00 | 0.27 |
| JR-FL | 2 | 44.65 | 16.93 | 6.95 | 1.49 | 0.97 | 0.11 |
| JR-CSF | 2 | >50.00 | 21.66 | 10.85 | 2.85 | 1.24 | 0.37 |
| QH0692.42 | 2 | >50.00 | >50.00 | >50.00 | >50.00 | 1.20 | 1.21 |
| THRO4156.18 | 2 | >50.00 | >50.00 | >50.00 | >50.00 | >50.00 | 3.84 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Recombinant DNA reagent | Chain N pETM20 (plasmid) | This study | pETM20, PEPcore facility-EMBL | |
| Recombinant DNA reagent | Chain C pETM11 (plasmid) | This study | pETM11, PEPcore facility-EMBL | |
| Recombinant DNA reagent | 2H10 pAX51 (plasmid) | Lutje Hulsik et al., 2013 | PMID:23505368 | |
| Recombinant DNA reagent | 2H10-F pAX51 (plasmid) | This study | S100d to F mutation | |
| Recombinant DNA reagent | 2H10-RKRF pAX51 (plasmid) | This study | S27R, S30K, S74R and S100d to F mutations | |
| Strain, strain background (Escherichia coli) | BL21(DE3) | ThermoFisher | Cat# EC0114 | |
| Strain, strain background (Escherichia coli) | C41(DE3) | Lucigen | Cat#60442 | |
| Cell line (Homo sapiens) | TZM-bl | NIH AIDS Reagent Program | Cat#ARP-8129 RRID:CVCL_B478 | |
| Cell line (Homo sapiens) | HEK293T | ATTC | Cat# CRL-11268 RRID:CVCL_1926 | |
| Antibody (human monoclonal) | α-gp41, LN01 | Pinto et al., 2019 | PMID:31653484 | See Materials and methods |
| Antibody (human monoclonal) | α-gp41, 10E8 | Huang et al., 2012 | PMID:23151583 RRID:AB_2491067 | See Materials and methods |
| Antibody (human monoclonal) | α-gp41, 2F5 | Muster et al., 1993 | PMID:7692082 | See Materials and methods |
| Antibody (human monoclonal) | α-gp140, VRC01 | Zhou et al., 2010 | PMID:20616231 RRID:AB_2491019 | See Materials and methods |
| Antibody (llama nanobody) | α-gp41, 2H10 | Lutje Hulsik et al., 2013 | PMID:23505368 | See Materials and methods |
| Antibody (llama nanobody) | α-gp41, bi-2H10 | Lutje Hulsik et al., 2013 | PMID:23505368 | See Materials and methods |
| Antibody (llama nanobody) | α-gp41, 2H10-F | This study | See Materials and methods | |
| Antibody (llama nanobody) | α-gp41, 2H10-RKRF | This study | See Materials and methods | |
| Chemical compound, drug | Chelating sepharose FF | GE Healthcare | Cat# 17057501 | |
| Chemical compound, drug | Q sepharose FF | GE Healthcare | Cat# 17051010 | |
| Chemical compound, drug | Q sepharose FF | GE Healthcare | Cat# 17051010 | |
| Chemical compound, drug | n-Octyl-β-D-glucosid n-Octyl-β-D-Glucopyranoside | Anatrace | Cat#O311 | |
| Chemical compound, drug | CHAPS (3-[(3-cholamidopropyl) diméthylammonio]−1-propanesulfonate) | Euromedex | Cat#1083E | |
| Chemical compound, drug | 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) | Avanti Polar Lipids | Cat#850457P | |
| Chemical compound, drug | 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) | Avanti Polar Lipids | Cat#850757P | |
| Chemical compound, drug | 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L -serine (POPS) | Avanti Polar Lipids | Cat#840034C | |
| Chemical compound, drug | sphingomyelin | Avanti Polar Lipids | Cat#860062C | |
| Chemical compound, drug | cholesterol | Avanti Polar Lipids | Cat#700000P | |
| Chemical compound, drug | N-(7-nitro-benz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (N-NBD-PE) | Molecular Probes | Cat#N360 | |
| Chemical compound, drug | N-(lissamine Rhodamine B sulfonyl) phosphatidylethanolamine (N-Rh-PE) | Molecular Probes | Cat#L1392 | |
| Chemical compound, drug | Biscinchoninic Acid microassay | Pierce | Cat# 23235 | |
| Chemical compound, drug | Bio-Rad Protein Assay Dye Reagent Concentrate | Biorad | Cat# 5000006 | |
| Software, algorithm | XDS | Kabsch, 2010 | PMID:20124693 | |
| Software, algorithm | Phaser | McCoy et al., 2007 | PMID:19461840 | |
| Software, algorithm | COOT | Emsley et al., 2010 | PMID:20383002 | |
| Software, algorithm | PHENIX | Adams et al., 2010 | PMID:20124702 | |
| Software, algorithm | SBGrid | Morin et al., 2013 | https://sbgrid.org/ | |
| Software, algorithm | Pymol | Warren DeLano | http://www.pymol.org | |
| Software, algorithm | Aimless | Evans and Murshudov, 2013 | PMID:23793146 | |
| Software, algorithm | STARANISO | Tickle et al., 2018 | http://staraniso.globalphasing.org/cgi-bin/staraniso.cgi | |
| Software, algorithm | CHARMM-GUI | Jo et al., 2008 | http://www.charmm-gui.org | |
| Software, algorithm | NAMD (Version 2.13) | Phillips et al., 2005 | https://www.ks.uiuc.edu/Research/namd/ | |
| Software, algorithm | Prism 8 | GraphPad | https://www.graphpad.com/scientific-software/prism/ | |
| Software, algorithm | ForteBio analysis software version 11.1.0.25 | ForteBio | https://www.fortebio.com | |
| Software, algorithm | MicroCal Origin software (origin 7) | Malvern Panalytical (MicroCal) | https://www.malvernpanalytical.com | |
| Peptide, recombinant protein | NEQELLELDKWASLW NWFNITNWLWYIK (N-MPER) | This study | See Materials and methods | |
| Peptide, recombinant protein | KKK-NWFDITNWLWYIKLFIMIVGGLV-KK (C-MPER), | This study | See Materials and methods | |
| Commercial assay or kit | Biscinchoninic Acid microassay | Pierce | Cat# A53225 | |
| Commercial assay or kit | Bright-Glo Luciferase Assay System Streptavidin (SA) biosensors | Promega | Cat# E2610 | |
| Commercial assay or kit | Streptavidin (SA) biosensors | ForteBio | Cat#18–5019 | |
| Commercial assay or kit | EZ-LinkNHS-PEG4-Biotinylation Kit | ThermoFisher | Cat#21455 |
