Cryo-EM structure of a native, fully glycosylated, cleaved HIV-1 envelope trimer

Abstract

The envelope glycoprotein trimer (Env) on the surface of HIV-1 recognizes CD4(+) T cells and mediates viral entry. During this process, Env undergoes substantial conformational rearrangements, making it difficult to study in its native state. Soluble stabilized trimers have provided valuable insights into the Env structure, but they lack the hydrophobic membrane proximal external region (MPER, an important target of broadly neutralizing antibodies), the transmembrane domain, and the cytoplasmic tail. Here we present (i) a cryogenic electron microscopy (cryo-EM) structure of a clade B virus Env, which lacks only the cytoplasmic tail and is stabilized by the broadly neutralizing antibody PGT151, at a resolution of 4.2 angstroms and (ii) a reconstruction of this form of Env in complex with PGT151 and MPER-targeting antibody 10E8 at a resolution of 8.8 angstroms. These structures provide new insights into the wild-type Env structure.

Fig. 1. CryoEM reconstruction of JR-FL EnvΔCT

(A) Reconstruction of JR-FL EnvΔCT in complex with PGT151 Fab at 4.2 Å resolution, segmented to highlight densities corresponding to gp120 (yellow), gp41 (blue), PGT151 Fab (pink), and the micelle surrounding the MPER and TM domain (gray). The three possible PGT151 binding sites are labeled as interface 1 (unliganded), interface 2, and interface 3. (B) Model of the EnvΔCT ectodomain, colored as in (A). The Fab LC and HC are colored in pink and magenta, respectively. Glycans are shown as spheres, with the gp120 and gp41 glycans shown in light and dark green, respectively. (C) Simplified cartoon rendering of gp41. Most of HR1 (residues 534–593) that is missing in the x-ray structure (residues 548–568) is here revealed to be an α-helix. To distinguish this region from the central HR1 helix (residues 571–593), we call these two helices HR1_N and HR1_C, respectively. The complete HR1 spans residues 534–593. The cartoon cylinder and loops are colored according to the sequence shown at the bottom.

Figure 2. Conformational changes induced by PGT151 binding

(A) Compared to interface 1 (gray), HR1_N in interface 2 (blue) and 3 (teal) are shifted about 24° outwards towards the Env surface, and 26° towards gp120 of the same protomer (yellow surface). The position of I559 residue is shown in yellow. (B) The FP (teal) is inserted into a hydrophobic pocket formed by the PGT151 CDR loops (CDRH2: magenta, CDRH3: purple, CDRL3: orange). The hydrophobic aromatic residues are shown as sticks.

Figure 3. Glycan structures on the Env trimer

(A) The glycan at position N611 makes extensive contacts with PGT151 Fab. The glycan residues are colored according to the diagram in (C). (B) As in (A) but for the N637 glycan. The glycans modeled at N611 (C) and N637 (D) The dark shades represent sugar moieties resolved in the current structure while the light shades represent inferred sugars that are disordered. (E) The N241 and N448 glycans (different shades of green) are in close proximity to CDRL3 and FWRH3 of PGT151.

Figure 4. The complete PGT151 epitope

(A) A model of PGT151 and glycan interactions. Glycans from up to four different subunits (two gp120, two gp41) from two protomers of the trimer can lock the Fab in its bound form (left). Some of the glycans bind PGT151 with high affinity (black arrows), but there are numerous steric barriers that need to be overcome (red arrows). Glycans N241 and N448 likely have an inhibitory effect on PGT151 binding by influencing the conformations of each other. Lack of the N241 glycan (right) alleviates steric pressure by N448 (blue arrow). Different gp120 subunits are shown in shades of yellow, gp41 subunits in shades of blue, Fab LC and HC in pink and magenta, gp120 glycans in green, and gp41 glycans in dark green. (B) When PGT151 is bound to glycans at N611 and N637, HR2 is locked in a bent conformation, and therefore cannot undergo conformational changes into the extended post-fusion form. (C) PGT151 CDRL1 (yellow) and the N637 glycan fucose (dark green) interact with a Glu/Asn rich region of HR1_N on the adjacent gp41 (blue). The CDRH3 (purple) inserts between HR1_N and the FP, and these interactions cap HR1_N to lock gp41 in the prefusion conformation. The interacting residues in the Fab and HR1_N are shown in orange. Only the core Man(Fuc)GlcNAc₂ residues are shown for the N637 glycan for clarity. (D) A measurement of the inter-gp41 distances in PGT151 bound JR-FL (left) compared to the unliganded BG505 trimer (right). Relative to the unliganded BG505 trimer which measures ~37 Å between Cα of N628 and N637 on the adjacent protomer, the distance between the same two residues measures ~35 Å at the PGT151 liganded interfaces. On the other hand, the inter-gp41 distance at interface 1 (~44 Å) is ~9 Å further apart in comparison to the liganded interfaces, indicating that the trimer becomes asymmetric in the PGT151-bound form.

Figure 5. JR-FL EnvΔCT bound to PGT151 and 10E8

(A) CryoEM reconstruction of JR-FL EnvΔCT in complex with both PGT151 and the MPER binding antibody 10E8 at 8.8 Å resolution (left). The JR-FL EnvΔCT-PGT151 reconstruction low-pass filtered to 8.8 Å is shown on the right for comparison. The reconstructions indicate that when 10E8 is bound, the trimer is lifted off the membrane (red arrow), suggesting a conformational change in the MPER/TM. (B) The Env HR2-MPER connectivity in 10E8-bound form is modeled into the EM density. (C) A comparison of the position of residues 659–670 in the two asymmetric units (ASU) of the 10E8 bound MPER peptide x-ray model (dark and light gray), superimposed on the complete Env model (blue), in which the primary MPER epitope (residues 671–685) is shown in yellow. This N-terminal segment exhibits different conformations in the two ASUs. D664 is colored in red as a point of reference. 10E8 is shown as the white surface.

Figure 6. 10E8 contact analysis in the context of the Env ectodomain

(A) and (B) A model of the 10E8 epitope in the context of the intact Env trimer. The Fab constant region and the nearby Env gp120 is also shown. In blue is the gp41 that the 10E8 Fab makes primary interactions with the MPER residues 671–685 (yellow). Additional contacts could be made with the HR2 and C-terminal region of the FP in the adjacent gp41 (teal), as well as regions in gp120 (white). These additional contacts to Env within a 4 Å radius of 10E8 are shown in red. Many of these interactions are likely FRWH3 (orange) mediated. The model also demonstrates that the N88 (A), and N625 (B) glycans could sterically obstruct 10E8 binding. The glycans modeled here are ManGlcNAc₂ for N88 and GlcNAc for N625 (Table S1), but are expected to be larger in native Env. (C) Glycans at N88 and N625 sterically hinder 10E8 binding to the trimer (left, red arrows). Binding of 10E8 (left) or CD4 (center right) lifts the MPER up from the membrane, relative to the ground state (center left). In the CD4-bound conformation, the opening of the trimers results in rotation of the gp120s, moving N88 away from the 10E8 binding site relieving some steric hindrance (right, blue arrow).