Closed prefusion structure of B41 SOSIP.664

We chose the soluble SOSIP.664 Env of the T/F virus B41 for investigation of structural flexibility within the Env trimer sub-domains, as it was reported to contain mixed conformational populations28. Partially open conformations, distinct from the CD4-induced fully-open state, were attributed to loop and/or domain movements that equilibrate between closed and partially open states on HIV-1 Envs31,32,38,39,40,41. We found that B41 SOSIP.664 trimers expressed in either HEK293S cells (thermal denaturation midpoint, T m = 57.2 °C) or HEK293F cells (T m = 57.6 °C) cells are stable (Supplementary Figure 1A), but less so than BG505 SOSIP.66410. For crystallographic studies, the B41 SOSIP.664 trimers were further stabilized by binding to Fabs PGT124 and 35O22, which increase the melting temperatures by ~5 °C (to 62.8 °C) and an additional ~1.5 °C (to 64.5 °C), respectively (Supplementary Fig. 1b). After complex formation, the unprotected glycans were trimmed with EndoH glycosidase and the trimer-Fab complexes were then purified using SEC (Supplementary Fig. 1d). The trimer-Fab complex crystallized in two different conditions, and x-ray structures were determined at 3.50 Å and at 3.80 Å resolutions in cubic (P23) and hexagonal (P6 3 ) crystal lattices (Fig. 1a, b, Supplementary Fig. 1e and f) with data complete to ~100% in the highest resolution shell (Supplementary Table 1). PGT124 recognizes the N332 glycan and the 324GDIR327 motif at the V3 base on B41 SOSIP.664 trimers with an angle of approach similar to ones seen with other trimers42. We detect conserved interactions (N325 gp120 to S30 in CDRL3, S93 in CDRL1 and Y100b in CDRH3) despite a D325N mutation in the 324GDIR327 motif compared to BG505 (Supplementary Fig. 2). The Asp/Asn duo appears at an 80%/17% frequency at position 325 across 5451 aligned HIV-1 Env sequences (Los Alamos HIV database). B41 SOSIP.664 (3.50 Å) adopts overall protomer/trimer conformations, similar to previously determined trimer structures from other isolates/clades25,43,44,45, with Cα root mean square deviations (r.m.s.d) ranging between 0.6–1.0 Å (protomer) and 1.0–1.3 Å (trimer) (Fig. 1c). Thus, despite no significant gp120 conformational differences, the same B41 SOSIP.664 complex could be crystallized in two different crystal lattices (Supplementary Fig. 3). However, gp41 exhibits substantial conformational heterogeneity at its N-terminus (Fig. 1e, f), although the HR1N regions in both crystal lattices are disordered. These features may explain in part the ~10 °C lower thermal stability compared to BG505 SOSIP.664 (T m = 68.1 °C)10. Mutations in FPPR (T538F) and HR1N (I548F) of B41 trimer showed improvement in thermal stability46. We observed electron density at 24 of the 29 potential N-glycosylation sites (PNGS) present on B41 SOSIP.664 trimers (Fig. 1d)47, whereas the other five sites are mainly located in disordered loops. The N289 glycan is 72% conserved between HIV-1 isolates, but is not present in B41 (n.b. the sequence around 289 is 289NEA291, which does not code for a glycan). The glycan hole on the wild-type (WT) B41SOSIP.664 trimer is similar to that on BG505, which lacks both N241 and N28929,48. N-glycosylation sites at positions 241, 295, 339, and 448 surround the B41 N289 glycan hole (Supplementary Fig. 4). Elicited antibodies targeting this immunogenic glycan hole, common to both BG505 and B41 viruses, could possibly be exploited for heterologous cross-neutralization in sequential boosting strategies with HIV-1 trimers29.

Fig. 1 Crystal structures of closed prefusion structure of B41 SOSIP.664 Env trimer. a, b Views of the Fab complex down the trimer axis with gp120 in pink and gp41 in cyan. The cartoon representation is overlaid with a transparent molecular surface. Fabs PGT124 (dark gray) and 35O22 (green) are from the 3.50 and 3.80 Å structures in space groups P23 (a) and P6 3 (b), respectively. c Comparison of the protomer/trimer backbones from crystal structures of trimers of clade A (BG505 SOSIP.664, bound and unbound), B (JRFL SOSIP.664), G (X1193.c1 SOSIP.664), and the new B41 SOSIP.664 structure at 3.50 Å. d Glycosylation observed in the electron density maps in the two different B41 SOSIP.664 crystal structures. The blue, gray, and green bars along the Y-axis represent the number of N-glycan moieties of N-acetylglucosamine (NAG/GlcNAc) (maximum possible, two), β-d-mannopyranose (BMA) (maximum, one), and α-d-mannopyranose (MAN) (maximum, eight), respectively. The X-axis represents N-glycosylation sites on B41 SOSIP.664. e, f Side views of B41 SOSIP.664 protomer (gp120: light blue, gp41: light orange) in cartoon representation, overlaid with a transparent molecular surface showing two conformational states of the free N-terminus FP (pink) going away from (3.50 Å) and towards (3.80 Å) the C-terminus (i.e. proximal to the membrane) Full size image

Flexibility in FP and its proximal region

To gauge the flexibility of the hydrophobic FP in the prefusion state, we compared the gp41 sub-domains in both crystal structures comprising of B41 SOSIP.664 bound to bNAbs PGT124 and 35O22 (Fig. 2). The FP, which is fully resolved to A512 at the N-terminus in the P23 lattice, is in a conformation that points away from the C-terminus of gp120 (Fig. 2a) and is sandwiched between HR2 of the neighboring protomer in the trimer and the light-chain (LC) variable region (CDRL1 and LFR3) of 35O22 (from another ternary complex in the crystal lattice) (Supplementary Fig. 5a). The FP turns upwards and is stabilized through polar (H641 and T644) and hydrophobic (L645 and V648) interactions with HR2 of the neighboring protomer in the same Env trimer (Supplementary Fig. 5b). In contrast, the fully resolved FP in B41PGT12 4+35O22 complex in the hexagonal lattice P6 3 (Fig. 2b) points toward the C-terminus of gp120. Strikingly, on superimposing the two crystal structures, we observe large rearrangements (up to ~11.6 Å) in the FPPR without altering the M530 position in the tryptophan clasp (W623, W628, W631) that anchors the gp41 sub-domain in the prefusion conformation (Fig. 2c and Supplementary Fig. 6). Despite rearrangements in FP and its proximal region (FPPR), the overall Env conformation in both structures remain unchanged. Comparing the B41 SOSIP.664 structure in P6 3 to structures of PGT151-bound WT JRFL ΔCT (PDB 5FUU)44, VRC34.01-bound BG505 SOSIP.664 (PDB 5I8H)35,44, and vFP16-bound and vFP20-bound BG505 DS-SOSIP (PDB 6CDI, 6CDE)36, we observe different conformations that suggests the FP is dynamic and flexible (Fig. 2d). In the FP-antibody bound or unbound trimer structures, FPPR and HR1N adopt overall similar conformations up to the hydrophobic F522 residue, which appears to be the pivot point for the FP structural plasticity37 and is buried inside a pocket formed at the gp120/gp41 interface (inset, Fig. 2d). In the P23 structure, F522 exits the interface pocket and the FP and FPPR regions rotate with M530 acting as the anchor (Fig. 2e). This altered conformation due to loss of the F522 interface interaction does not otherwise perturb the gp41 prefusion state. Such flexibility in the FP may aid the Env trimer in accessing the conformations observed in the pre-fusion state, antibody-bound states (e.g. PGT151), and the transition to intermediate states. These include the fully closed native-like state and those in equilibrium between the closed and more open conformations observed by NS-EM in different strains and clades28. It has been reported that optimizing FPPR (L543N) and HR1N in B41 SOSIPv3.2 improves PGT145 binding and stability, respectively23.

Fig. 2 Different conformational states of the fusion peptide in B41 SOSIP.664. a The FP (red) of gp41 at 3.50 Å points away from HR2. Inset shows the 2Fo–Fc electron density composite omit maps (1.0σ) for the fusion peptide (residues 512–527, red), and FPPR (residues 528–540, green). b The FP from the 3.80 Å structure extends towards HR2. Inset shows the 2Fo–Fc electron density map (1.0σ) for the FP and FPPR. c Overlay of FP and FPPR regions of gp41 from the two crystal structures (3.50 Å: light pink, 3.80 Å: light blue). Inset illustrates the large rearrangement of FPPR and major Cα displacement for example of S534 (~11.6 Å) in B41 SOSIP.664 following the same resolution-based color-code. d Different conformational states of FP illustrated in the overlay of the FP, FPPR, and HR1N regions of PGT151-bound WT JRFL ΔCT (PDB ID: 5FUU; cyan), vFP16-bound BG505 DS-SOSIP (PDB ID: 6CDI; green), vFP20-bound BG505 DS-SOSIP (PDB ID: 6CDE; blue), and VRC34.01-bound BG505 SOSIP.664 (PDB ID: 5I8H; orange), recognized by the respective bNAbs. Inset shows the structural plasticity of the FP around F522. e F522 in FP (pink) of B41 SOSIP.664 in the 3.80 Å structure is tucked inside the gp120/gp41 interface and acts as a pivot for the remaining FP residues. Another conformational state of FP is captured in the 3.50 Å structure where F522 exits its hydrophobic-binding pocket, causing the FP and FPPR to rotate in opposite directions without affecting the location and conformation of M530 (green), which acts an anchor in the gp41 prefusion state Full size image

The current arsenal of crystal and cryo-EM structures illustrate that the FP has a dynamic range of conformations, independent of crystal contact formation, which facilitate bNAb (VRC34.01, ACS202, vFP16, vFP20, and PGT151) engagement from various angles of approach. These results illustrate two representative conformational states where the FP is fully solvent-exposed and accessible to FP-specific antibodies. This FP and FPPR conformational flexibility may be contributing factors that affect the stability46 and expression of B41 versus BG505 SOSIP trimers, despite eliciting comparable titers of autologous antibody responses8. However, only a single conformation of FP in the cleavage-independent (NFL) trimers17 has been observed to date that points towards the C-terminus18,19. The N-terminus of the FP is not free on the cleavage-independent sc-gp14016 and NFL trimers, as the furin-cleavage site has been deleted. This design feature limits the presentation and/or accessibility of the epitopes for the various FP-directed antibodies. As a result, the FP-directed human bNAbs VRC34.01, ACS202, and PGT151 bind poorly, with high off-rates, to cleavage-independent trimers34,35. Further introducing an enterokinase cleavage site, for post-expression enzymatic cleavage, upstream of the FP in NFL improved binding for VRC34.01 and PGT15149, highlighting the requirement for a charged and free N-terminus for this epitope.

Restoration of the VRC34.01 epitope on the B41

To test our hypothesis that the amino-acid sequence and register of certain FP residues have an important role in antibody binding, we analyzed the FP sequence conservation in HIV-1 group M, including recombinants, and compared the sequences to BG505 SOSIP.664 (Fig. 3a). We found that positions 515, 518, and 519 are relatively poorly conserved in the B41 sequence compared to group M as a whole (Supplementary Fig. 7a). Thus, residue L515 on B41 is 41% conserved whereas I515 on BG505 is 53% conserved, showing that both Leu and Ile can be tolerated at FP position 515. However, an I515L mutation to BG505-Env pseudovirus did not affect VRC34.01 neutralization35. Much less conservation is seen for B41 residues F518 (~1.5%) and I519 (~13.0%), implying that antibodies with epitopes that include these adjacent residues may be particularly susceptible to antigenic variation in the FP. Although we captured a FP conformation that highly resembles the VRC34.01-bound state on BG505 (PDB 5I8H) (Fig. 2d), this bNAb did not bind to B41 SOSIP.664 trimers (Fig. 3c). To restore the VRC34.01 epitope to the B41 trimer, we aligned the FPs (residues 512–527) of BG505 and B41 and then genetically engineered insertion of Val at position 518 and deletion of Ile at position 519 in B41 SOSIP.664 trimer. In the modified sequence, Phe518 then moved to position 519. Thus, the modified B41 FP sequence is aligned with BG505 except for position 515, which is Leu in B41 but Ile in BG505 (Fig. 3b). We used isothermal titration calorimetry (ITC) to test the binding of VRC34.01 to the FP mutant, designated B41mut1 SOSIP.664, and observed a large increase in affinity compared to the parental B41 SOSIP.664 trimer (Fig. 3c). We conclude that residue 518 is critical for the binding of VRC34.01 to the FP of the B41 trimer.

Fig. 3 Restoration of the VRC34.01 epitope on B41 and delineation of Env breathing. a Amino acid conservation of the FP (X-axis: residues 512–527, Y-axis: percent conservation) in BG505 SOSIP.664 (black) and B41 SOSIP.664 (gray). The difference in conservation is shown in rectangles for BG505 SOSIP.664 and ovals for B41 SOSIP.664. For assessing conservation of FP residues, 5451 HIV-1 sequences from Los Alamos National Laboratory (LANL) HIV-1 database were analyzed. b The FP residues of both isolates are aligned to highlight the differences at positions 515, 518, and 519. Construction of the B41mut1 SOSIP.664 is illustrated in the lower panel. c No binding is observed between FP-directed Fab VRC34.01 with wild-type FP of B41 SOSIP.664 (left panel). Restoration of VRC34.01 binding to the mutated B41mut1 SOSIP.664 FP (K d = 12 nM, three Fabs bind per trimer) is illustrated (right panel). The enthalpy and entropy are measured in kcal per mol and cal per mol per deg, respectively. Data points not included in the fit are indicated by an asterisk. All binding experiments are measured by ITC and reported values are averages from two independent measurements. d Binding kinetics of B41 FP and FP (mut1) peptide variants of VRC34.01, as determined by BLI. e Crystal structure of His-tagged B41mut1FP peptide bound to VRC34.01. f View down the three-fold trimer apex of B41 SOSIP.664 showing opening and closing of the trimer at 3.50 Å (left) and 3.80 Å (right) resolutions, respectively. Inter-V2 distances (Å; dashed lines) are measured between the Cα residues of R166 (cyan). Top views of both crystal structures showing center of mass of each protomer (gp120: green, gp41: red) overlaid on the Env cartoon (light gray) for orientation. Inter-gp41 and inter-gp120 distances (measured in Å; lines) are measured between the center of masses. The insets of the two B41 structures are shown in the side view (right). The angle is measured between center of mass of gp41 and gp120 of a protomer to reflect the movement between domains that leads to a slight opening of the trimer Full size image

To understand the underlying structural basis for this epitope restoration, we used biolayer interferometry (BLI) to measure the binding of the VRC34.01 Fab to a C-terminally His-tagged B41mut1 FP (residues 512–521) (Fig. 3d), and then determined its x-ray structure at 1.98 Å resolution (Fig. 3e and Supplementary Table 2). Clear electron density was observed for the entire B41mut1 FP, including the His 6 -tag (Supplementary Fig. 7a). The paratope, comprising of four shallow cavities formed by all CDR loops, except L2, engages this restored FP (Supplementary Fig. 7b) similar to that observed for VRC34.01 bound to the FP of BG505 SOSIP.664 (Supplementary Fig. 7c)35. We found that, when F518 is present as in the WT FP of B41, it would clash with VRC34.01 CDRs and abrogate binding (Supplementary Fig. 7d). Replacing Val for Phe at position 518 in the FP B41mut1 variant restores the geometry of the epitope and prevents a clash. Thus, the nature of the residue at position 518, but not 519, is critical for VRC34.01-like antibody engagement; it is also potentially relevant to HIV-1 escape from neutralization as Phe is present in ~2% of group M isolates.

We detected no binding of PGT151 and ACS202 to either the B41 SOSIP.664 trimer or its mut1 variant (Supplementary Fig. 8), although both bNAbs are known to bind the BG505 SOSIP.664 FP34,50. PGT151 neutralizes the B41 Env-pseudotyped virus, but is not able to bind the B41 SOSIP.664 trimer28,50,51. A T538F mutation to the FPPR stabilizes this flexible epitope at the gp120/gp41 interface of the B41 SOSIP.664 trimer and enhances PGT151 binding46. The major difference between VRC34.01, vFP16, vFP20, and other FP-directed antibodies is the presence of a hydrophobic YYYY motif in CDRH3 of PGT15144 that interacts with the FP and is predictably similar in ACS20234. However, VRC34.01, vFP16, and vFP20 bind to soluble BG505 SOSIP despite not having this YYYY motif in their CDRH3. Thus, we can categorize these FP-directed antibodies into two separate classes: the first, which includes ACS202, is dependent on a hydrophobic patch in CDRH3 for interaction while, in contrast, VRC34.01-like antibodies, lack this requirement. This variety in angles of approach results from multiple FP conformations that also enable residues to be shared at this epitope, but in completely different recognition modes (Supplementary Fig. 9). Notably, ACS202 had slightly stronger binding to its autologous AMC011 SOSIP.v4.2 trimer, compared to BG505 SOSIP.664, despite an identical FP; there may therefore be some (albeit minor) dependence on position 229 (K229 in BG505 but N229 in AMC011), while glutamate remains identical at positions 83 and 8734. In AMC011, an E87A change abrogates ACS202 binding completely. In HIV-1 group M, glutamate is at position 87 in ~55% of isolates, and glycine in ~15%. We deduce that G87 in B41, as found in clone 2D7 of AMC011 (consensus of the infectious molecular clones 2D6, 2D7, and 2G9 of the individual viruses from month 8 that elicited the ACS202 lineage), hampers ACS202 binding and aids viral escape34. Despite sharing partially overlapping epitopes, the binding stoichiometries of these FP-directed bNAbs on the trimer is distinct; ACS202, VRC34.01, vFP16, and vFP20 bind in a stoichiometry of three Fabs per trimer, while only 2 PGT151 Fabs bind per trimer due to an antibody-induced asymmetry in the trimer44.

Our analysis suggests that FP flexibility allows for different modes of antibody recognition of the epitopes in which the FP is involved. This same flexibility also creates challenges for epitope stabilization, which is critical for effective germline antibody engagement and vaccine design when targeting this region of the virus.

gp41 plays a role in Env breathing

Conformational changes induce opening of soluble Env in response to receptor and/or co-receptor binding31. However, structural movements, such as relaxation of variable regions and movement between and within sub-domains, induce partial opening/closing of the trimer termed breathing. Previous studies have shown that CD4-binding opens Env, and that some bNAbs can also induce trimer opening21,31,32,33,40,41,52. To understand the structural implications of induced opening versus breathing in the same isolate, we compared both structures of the B41–Fab complexes. We observed breathing at the trimer apex when identical antibodies were bound to B41 SOSIP.664 (Fig. 3f and Supplementary Fig. 10). We next examined the cause of apical opening. On measuring the angle between the center of masses of Cα atoms of gp120 and gp41 sub-domains (either excluding or including the FP) of the two structures, we detected a small outward tilt (~1° and ~0.5°, respectively) in the gp120 subunits of the B41 SOSIP.664 trimer in the 3.5 Å structure compared to the 3.8 Å structure (Fig. 3f) relative to gp41. Although small, this tilt angle is sufficient to induce an ~1.6 Å opening at the trimer apex. We also observed movement within both sub-domains and twisting of the trimer (Supplementary Movies 1 and 2). However, rearrangements within gp41 alone are not enough to trigger full trimer opening when compared to that induced by receptor (CD4) binding. Thus, from these results, we observe that conformational isomerism and changes/rearrangements in the gp41 FP and FPPR can have distal effects on the trimer apex.

Comparison of open and closed states of prefusion B41

We next evaluated conformational rearrangements that occur in B41 trimers upon binding of CD4. We compared B41 SOSIP.664 bound by b12 [CD4-binding site antibody, PDB 5VN831] and by sCD4 and Fab 17b [CD4-induced antibody, PDB 5VN331], as observed by cryo-EM, and compared them with the 3.5 Å crystal structure of the closed B41 trimers bound by PGT124 and 35O22. The overall Cα r.m.s.d between the open and closed B41 protomer/trimer structures varies from 2.6 Å/21.7 Å (5VN8) to 8.3 Å/18.7 Å (5VN3). This divergence is a reflection of the changes in conformation of both subunits, as well as relative movements between sub-domains and rotations within the trimer (Supplementary Fig. 11). The b12-bound B41 trimer shows a large outward movement of gp120 (22.7 Å between the center of masses of the closed structure and 5VN8, with their gp41s aligned), that is sufficient to open up the trimer apex. In the sCD4-bound (5VN3) structure, the apical opening (21 Å, calculated as described above) results from more localized V1/V2 rearrangements compared to B41 PGT124+35O22 (Fig. 4a, d and Supplementary Movie 3). In the closed prefusion B41 PGT124+35O22 structure, the FP is largely solvent exposed (and stabilized by crystal contacts) and extends away from the trimer core, whereas the FPPR faces inward as seen in all soluble Env trimers (Fig. 4e). The engagement of CD4 induces major movements in gp120 relative to gp41, thus forming a new pocket to accommodate the FP in a conformation that points towards the trimer core, while the FPPR is dislodged from its usual closed pre-fusion position (Fig. 4b). The closed prefusion B41 structure also lacks the helical α0 (residues 63–72) secondary structure in the C1 region that is observed in the open B41 sCD4+17b structure (Fig. 4c, f)31, while this region is disordered in B41 b12 , indicative of high local flexibility. Our results suggest that the transformation in the FP and FPPR gp41 sub-domains regulates the transition between partially open and fully closed prefusion conformational states of the SOSIP.664 trimer. This inherent conformational flexibility of the FP (Fig. 4g) illustrates that various distinct stable conformational geometries and locations of the FP region can be observed for the prefusion state of Env. These findings highlight and help to explain the differential recognition modes by different FP-directed antibodies37.