Model structure of a fully glycosylated full-length HIV Env trimer embedded in a membrane.

(A) The model structure built by combining the cryo-EM structure of the ectodomain (yellow, PDB ID: 6B0N) with the NMR structure of the MPER, TMD, and CT (purple, PDB ID: 7LOI). The full-length model includes residues A31 to L856, while the CT-truncated (ΔCT) model includes residues A31 to S716. The missing loops in the PDB structures are highlighted in red, and the glycosylation sites are marked by cyan spheres. (B) Left: assignment of functional domains with boundary residue numbers, including signal peptide (SP), variable regions (V1-V5), fusion peptide (FP), heptad repeats (HR1 and HR2), membrane-proximal external region (MPER), transmembrane domain (TMD), and cytoplasmic tail (CT). Right: missing residues (red) and glycosylation sites (blue). The shaded region at the bottom marks CT residues excluded in the ΔCT model. (C) N-linked glycans shown as high-mannose (green) and complex (magenta) types. The full-length model is shown on the left and the ΔCT model on the right. (D) Env trimer embedded in a membrane. Lipid headgroups are highlighted by green spheres and glycans are omitted for visual clarity. The palmitoyl groups covalently attached to C764 and C837 are shown in cyan. Molecular illustrations were prepared using Visual Molecular Dynamics (VMD) (Humphrey et al., 1996).

Tilting motions of the ectodomain and TMD are independent.

(A) Representative structures illustrating different ectodomain tilt angles and the schematic showing how tilt angles are calculated. (B) Probability densities of ectodomain and TMD tilt angles, calculated from CT-truncated systems with various initial configurations.

Ectodomain is rigid, whereas the MPER is highly flexible and adopts diverse conformations.

(A) Top and side views of the ectodomain and MPER in the cleaved system, with RMSF indicated by color. (B) Schematic illustrating the calculation of interchain distances and their distributions at the Cα atoms of G644, E654, D664, and F673. For each residue, the distribution from cleaved systems is shown in dark color (left), and that from uncleaved systems is shown in light color (right). represented by solid and transparent colors, respectively. The initial values of interchain distances are marked by purple stars. (C–F) Local structures of the ectodomain C-terminus and MPER. The HR2 helix and MPER in one protomer are highlighted in dark yellow, with the Cα atoms of four selected residues marked by blue, orange, green and red spheres. (C) The initial conformation and (D) representative snapshot from simulations of the cleaved system. (E) The initial conformation and (F) representative snapshot from simulations of the uncleaved system.

R696 interacts with lipid headgroups and disrupts membrane integrity.

(A–C) MPER and TMD in the CT-truncated system with the “high” TMD configuration. MPER-N, MPER-C, and TMD are shown in magenta, cyan, and white, respectively. Lipid headgroups, R696, and the residues anchored in the lipid headgroups (R683, R707 and R709) are shown in green, blue, and purple, respectively. Lipid headgroups and ions interacting with R696 are highlighted in orange and red, respectively. (A) Initial conformation. (B, C) Representative snapshots from different trajectories. (D–F) MPER and TMD in the CT-truncated system with the “low” TMD configuration. (G) Two side views of the same snapshot where R696 of one protomer interacts with lipid headgroups in the exoplasmic leaflet and R696 of two protomers interact with lipid headgroups in the cytoplasmic leaflet. Lipid headgroups and tails are shown in green and gray, and water molecules in magenta. TMD of three protomers (i.e., chains A, B and C) are shown in light yellow, dark yellow and orange, respectively. (H) Frequency of TMD residues interacting with lipid headgroups, lipid tails, and water. For each TMD residue–interacting component pair, the frequency represents the fraction of snapshots in which the heavy atoms of the TMD residue and the corresponding component are within 5 Å. Bar shading reflects this fraction, with fully filled bars indicating 100% and empty bars indicating 0%.

MPER exhibits diverse conformations, and its exposure depends on both MPER and TMD.

(A) The initial structure of the CHΔCT system, where dF673 of two promoters equals 8.5 Å and 9.2 Å. Lipid headgroups are shown in green and R696 in blue. dF673 is defined as the distance from the Cɑ of F673 (red) to the highest among the adjacent lipid headgroups (orange and purple). (B) Distribution of dF673 in the CLΔCT and CHΔCT systems. The cyan dashed line indicates the mean dF673 of three protomers in the initial structure, and the blue solid line indicates the mean across all data sampled from simulations. (C, D) Representative snapshots illustrating the buried (C) and exposed (D) MPER. (E, F) The entire trimer structures corresponding to (C) and (D), respectively.

Antibody epitope accessibility.

(A) The frequency of accessibility. Each marker represents the epitope on one of the three protomers across all trajectories. For 35O22, red indicates the accessibility frequency without considering steric clashes with the membrane, while purple indicates the frequency accounting for clashes with the membrane. (B–D) Representative snapshots showing conformations with the epitope exposed (upper) and shielded (lower) for antibodies PGT128, 35O22, and 4E10, respectively. The antibody VH and VL domains are shown in surface representation, with lipid head groups in green spheres and glycans that may interfere with the antibody in distinct colors.

Ectodomain tilt versus TMD tilt, grouped by time intervals (cleaved CT-truncated systems).

(A) Three trajectories starting from the “high” TMD configuration. (B) Three trajectories starting from the “low” TMD configuration. The 1-μs trajectory was divided into four intervals, with values from each interval shown in light gray, dark gray, black, and red, respectively.

Ectodomain tilt versus TMD tilt, grouped by time intervals (uncleaved CT-truncated systems).

Labeling and color coding are the same as in Figure 2—figure supplement 1.

Ectodomain tilt versus TMD tilt, grouped by time intervals (cleaved full-length systems).

Labeling and color coding are the same as in Figure 2—figure supplement 1.

Ectodomain tilt versus TMD tilt, grouped by time intervals (uncleaved full-length systems).

Labeling and color coding are the same as in Figure 2—figure supplement 1.

Temporal evolution of Ectodomain and TMD tilt angles (cleaved CT-truncated systems).

Temporal evolution of Ectodomain and TMD tilt angles (uncleaved CT-truncated systems).

Temporal evolution of Ectodomain and TMD tilt angles (cleaved full-length systems).

Temporal evolution of Ectodomain and TMD tilt angles (uncleaved full-length systems).

Probability densities of ectodomain and TMD tilt angles (full-length systems).

Dynamic cross-correlation matrix of Cα atoms (cleaved CT-truncated systems).

The x-axis (left to right) and the y-axis (top to bottom) correspond to residue indices ranging from 31 to 716 for each of the three protomers. TMD residues are enclosed within the dashed lines. Positive and negative correlations are color-coded from red to blue.

Dynamic cross-correlation matrix of Cα atoms (uncleaved CT-truncated systems).

The x-axis (left to right) and the y-axis (top to bottom) correspond to residue indices ranging from 31 to 716 for each of the three protomers. TMD residues are enclosed within the dashed lines. Positive and negative correlations are color-coded from red to blue.

Dynamic cross-correlation matrix of Cα atoms (cleaved full-length systems).

The x-axis (left to right) and the y-axis (top to bottom) correspond to residue indices ranging from 31 to 856 for each of the three protomers. TMD residues are enclosed within the dashed lines. Positive and negative correlations are color-coded from red to blue.

Dynamic cross-correlation matrix of Cα atoms (uncleaved full-length systems).

The x-axis (left to right) and the y-axis (top to bottom) correspond to residue indices ranging from 31 to 856 for each of the three protomers. TMD residues are enclosed within the dashed lines. Positive and negative correlations are color-coded from red to blue.

RMSF and RMSD of the ectodomain.

(A) Top and side views of the ectodomain and MPER in the uncleaved system, with RMSF indicated by color. (B) RMSD relative to the initial model as a function of time, calculated from the trajectories of CT-truncated systems.

Local conformations of the TMD and global conformations of protein and membrane (CHΔCT systems).

Three protomers of the TMD are shown in light yellow, purple, and pink; three protomers of the ectodomain in dark yellow, gray, and white; MPER-N and MPER-C in magenta and cyan, respectively; and lipid headgroups in green. Lipid headgroups interacting with R696 are highlighted in orange, and the ions interacting with R696 in red.

Local conformations of the TMD and global conformations of protein and membrane (CLΔCT systems).

Labeling and color coding are the same as in Figure 4—figure supplement 1.

Local conformations of the TMD and global conformations of protein and membrane (UHΔCT systems).

Labeling and color coding are the same as in Figure 4—figure supplement 1.

Local conformations of the TMD and global conformations of protein and membrane (ULΔCT systems).

Labeling and color coding are the same as in Figure 4—figure supplement 1.

Local conformations of the TMD and global conformations of protein and membrane (CHCT systems).

Labeling and color coding are the same as in Figure 4—figure supplement 1, with the CT additionally shown in red.

Local conformation of the TMD and global conformation of protein and membrane (CLCT systems).

Labeling and color coding are the same as in Figure 4—figure supplement 5.

Local conformations of the TMD and global conformations of protein and membrane (UHCT systems).

Labeling and color coding are the same as in Figure 4—figure supplement 5.

Local conformations of the TMD and global conformations of protein and membrane (ULCT systems).

Labeling and color coding are the same as in Figure 4—figure supplement 5.

Temporal evolution of the distance from the MPER midpoint to the membrane surface.

The distance dF673, defined in Figure 5A, is shown as a function of simulation time for the CHΔCT and CLΔCT systems.

Shielding of antibody PGT128 epitope.

(Left) Variable domains of the heavy and light chains aligned onto our modeled structure. (Middle) Top view of the glycosylated trimeric protein, with the orange arrow indicating the epitope location. (Right) Part of the epitope in the antibody-epitope complex PDB structure was used for structural alignment and is highlighted in purple. Glycans capable of hindering antibody binding are shown in various colors.

Shielding of antibody PG9 epitope.

Labeling and color coding are the same as in Figure 6—figure supplement 1.

Shielding of antibody VRC01 epitope.

Labeling and color coding are the same as in Figure 6—figure supplement 1. (Middle) The glycosylated trimeric protein is shown in a side view instead of the top view.

Shielding of antibody 35O22 epitope.

Labeling and color coding are the same as in Figure 6—figure supplement 3. (Left) The dashed greens indicate the approximate location of the lipid headgroups.

Shielding of antibody 4E10 epitope.

Labeling and color coding are the same as in Figure 6—figure supplement 4.

Shielding of antibody 10E8 epitope.

Labeling and color coding are the same as in Figure 6—figure supplement 4.

Snapshots showing the MPER of one protomer accessible to either 4E10 or 10E8.

(A) Snapshot in which the MPER of the white protomer is accessible to 4E10 (cyan transparent surface) but not to 10E8. (B) Snapshot in which the MPER of the white protomer is accessible to 10E8 (green transparent surface) but not to 4E10.

Snapshots showing the MPER of two protomers are accessible to 4E10.

The MPER is accessible to 4E10, (A) exclusively in the yellow protomer, and (B) exclusively in the gray protomer.

Snapshots showing the MPER of two protomers are accessible to 10E8.

The MPER is accessible to 4E10, (A) exclusively in the yellow protomer, and (B) exclusively in the white protomer.

Snapshots showing the MPER of three protomers are accessible to 10E8.

The MPER is accessible to 4E10, (A) exclusively in the yellow protomer,(B) exclusively in the gray protomer, and (C) exclusively in the white protomer.