Broadly neutralizing antibodies (bNAbs) isolated from HIV-1-infected individuals inform HIV-1 vaccine design efforts. Developing bNAbs with increased efficacy requires understanding how antibodies interact with the native oligomannose and complex-type N-glycan shield that hides most protein epitopes on HIV-1 envelope (Env). Here we present crystal structures, including a 3.8-Å X-ray free electron laser dataset, of natively glycosylated Env trimers complexed with BG18, the most potent V3/N332gp120 glycan-targeting bNAb reported to date. Our structures show conserved contacts mediated by common D gene-encoded residues with the N332gp120 glycan and the gp120 GDIR peptide motif, but a distinct Env-binding orientation relative to PGT121/10-1074 bNAbs. BG18’s binding orientation provides additional contacts with N392gp120 and N386gp120 glycans near the V3-loop base and engages protein components of the V1-loop. The BG18-natively-glycosylated Env structures facilitate understanding of bNAb–glycan interactions critical for using V3/N332gp120 bNAbs therapeutically and targeting their epitope for immunogen design.
In the ongoing fight against the HIV-1 pandemic, the discovery and characterization of broadly neutralizing antibodies (bNAbs) against HIV-1 envelope (Env) fuel new efforts in treatment and management of infection. Next-generation bNAbs protected against and reduced viral loads in humanized mouse1,2 and non-human primate3,4 models of infection and exhibited anti-viral activity in human trials5,6,7,8,9. Therefore, a vaccine that elicits such antibodies is likely to be efficacious. Despite their promise, unusual properties of HIV-1 bNAbs—such as a high degree of somatic hypermutations10, long heavy chain complementarity determining region 3 (CDRH3) loops11, and/or short light chain complementarity determining region 3 (CDRL3) loops12—have made it difficult to elicit bNAbs by immunization. In addition, innate features of the viral Env spike that interfere with broad-based immunity include the diversity of Env sequences that arise by mutation13, a low Env density on the virion surface that interferes with bivalent antibody binding14,15, and host glycans that shield most of the Env surface16.
The glycan shield comprises ~50% of the mass of HIV-1 Env, a (gp120-gp41)3 trimer, and consists of N-glycans attached to an average of 30 ± 3 potential N-linked glycosylation sites (PNGSs) per gp120-gp41 protomer17. The carbohydrates decorating the surface of Env reduce access to protein epitopes and are generally non-immunogenic because they are assembled by host cell machinery16. Although under-processed oligomannose N-glycans cover parts of HIV-1 Env such as the N332gp120 glycan region of gp120, processed complex-type N-glycans predominate in other regions of Env18 and protect the CD4 binding site (CD4bs) and the variable loop 3 (V3-loop) of gp12019. The production of soluble native-like Env trimers (SOSIPs)20 has enabled structure determinations of glycosylated Env–bNAb complexes and a better understanding of bNAb epitopes11,21,22,23,24. However, structural information pertaining to bNAb recognition of highly glycosylated HIV-1 Env trimers in the context of native glycan shields has been difficult to obtain due to chemical and conformational heterogeneity of N-glycans that usually precludes crystallization required for an X-ray structure determination. Thus with one exception25, all monomeric and trimeric Env crystal structures were solved using glycoproteins produced with exclusively high mannose-only forms of N-glycans, which were usually enzymatically trimmed after being complexed with antibody Fabs22,26,27,28,29,30,31,32,33,34,35,36,37. A single-particle cryo-electron microscopy (cryo-EM) structure of a natively glycosylated HIV-1 Env showed complex-type N-glycans at the gp120-gp41 interface, but many of the remaining glycans were not ordered in the EM map38,39.
The V3/N332gp120 class of HIV-1 bNAbs, exemplified by the PGT121/10-1074 family40,41, evolved to interact with both glycan and protein components on HIV-1 Env to effect the neutralization of a majority of HIV-1 strains31,42,43,44. These bNAbs possess long CDRH3s that interact with the N332gp120 glycan and penetrate the glycan shield to contact the conserved 324GD/NIR327 peptide motif at the base of the gp120 V3-loop25,32,42,45. bNAbs against the V3/N332gp120 site isolated from several HIV-1-infected donors can adopt different Env-binding orientations to engage the conserved epitope29,44 and display a wide array of interactions with surrounding glycans, including glycans at positions N301gp120 (the PGT128 bNAb), N137gp120/N156gp120/N301gp120 (PGT121/10-1074 family), and N386gp120/N392gp120 (PGT135). While under-processed N-glycans presenting as a oligomannose patch predominate in the V3/N332gp120 epitope, the recent structure of 10-1074 bound to a natively glycosylated BG505 SOSIP trimer included complex-type N-glycans at positions N156gp120 and N301gp12025, and sialic acid-bearing complex-type glycans at the N156gp120 position were shown to be critical for maturation of the CAP256 V2 apex bNAb lineage46. Thus, providing structural information of bNAbs bound to Env trimers that include both complex-type and high mannose glycans will facilitate developing strategies for improving bNAb breadth and for design of high-affinity germline-binding immunogens.
BG18 exhibits the highest potency among the V3/N332gp120 bNAbs described to date47. Isolated from an elite controller infected with clade B HIV-1, BG18 displays a similar breadth of coverage (~64%) across a panel of HIV-1 strains to the PGT121/10-1074 family bNAbs (Supplementary Figure 1), but BG18 neutralizes these strains with a geometric mean IC50 value of 0.03 µg/mL, a higher potency than 10-1074 and other bNAbs in human clinical trials8. The structure of unliganded BG18 Fab47 exhibited a cleft between the CDRH2 and CDRH3 loops and a long CDRH3 loop that forms a two-stranded β-sheet as previously observed for PGT121 and 10-1074 Fab structures solved in the absence of HIV-1 Env40. However, comparison of the BG18 Fab structure (PDB 5UD9 (https://doi.org/10.2210/pdb5UD9/pdb)) with structures of unbound PGT121 and 10-1074 Fabs (4FQ1 (https://doi.org/10.2210/pdb4FQ1/pdb) and 4FQ2 (https://doi.org/10.2210/pdb4FQ2/pdb)) demonstrated structural differences including: (1) a displaced, shorter, and more compact CDRH3 stabilized by a network of hydrogen bonds, (2) differences in variable light (VL) domain orientation relative to the variable heavy (VH) domain, and (3) a second cleft in the antigen-binding site between CDRH3 and CDRL1/CDRL3. In addition, single-particle electron microscopy showed that BG18 exhibited a different orientation compared with PGT121/10-1074 and other V3/N332gp120 bNAbs for binding HIV-1 Env47.
To better understand the molecular mechanism underlying BG18’s interactions with Env glycans and increased potency compared with other V3/N332gp120 bNAbs, we determined the crystal structures of natively glycosylated clade A (BG505) and clade B (B41) Envs in complex with BG18 Fab. We used the increased brightness of an X-ray free electron laser48 (XFEL) to circumvent the limitations of crystal size and improve the resolution to 3.8 Å for our BG18-BG505-35O22 complex. We found that BG18 binds the V3/N332gp120 glycan site in a distinct manner relative to PGT121-like bNAbs, primarily through rearrangements in its VH and VL domains. Analysis of BG18 interactions bound to a natively glycosylated Env showed engagement with oligomannose glycans near the base of the V3-loop and the conserved GDIR peptide motif. Moreover, BG18’s CDRL1 and CDRL2 formed part of a cleft within 8 Å of gp120’s variable loop 1 (V1-loop), increasing BG18’s protein surface contact with Env. These structures provide valuable information for understanding the promiscuity of V3/N332gp120 glycan-directed bNAbs that will facilitate current efforts to evaluate them for therapeutic use in HIV-1-infected humans8 and to target their epitope for immunogen design49,50.
Structures of natively glycosylated Env-BG18 complexes
Structural insight into BG18 binding in the context of a natively glycosylated Env was achieved by crystallizing bNAb Fabs complexed with Env trimers expressed and purified from Chinese hamster ovary (CHO) cells51, which include similar glycoforms as Env trimers expressed in human cell lines52. Our crystals comprised natively glycosylated Env SOSIP.664 trimers from clade A (BG505) or clade B (B41) strains bound to Fabs from BG18 and either 35O22, a gp120–gp41 interface-spanning bNAb53, or IOMA, a VH1-2 CD4bs bNAb25. We solved the structures of BG18-BG505-35O22, BG18-B41-35O22, and BG18-BG505-IOMA complexes to resolutions of 4.1, 4.9, and 6.7 Å, respectively (Fig. 1 and Table 1) by molecular replacement (Methods) using synchrotron radiation datasets. Subsequently, XFEL diffraction data collected by manually targeting individual crystals (~75 µm × 75 µm × 50 µm dimensions) improved intensities of high-angle Bragg reflections (Supplementary Figure 2) as previously observed for XFEL datasets48,54, resulting in a 3.8 Å resolution structure of the BG18-BG505-35O22 complex from 520 still diffraction images (Fig. 1a). The use of B-factor sharpening55 in our BG18-BG505-35O222 structures permitted visualization and refinement of most amino acid side chains, particularly at the Fab–Env interface.
The BG18-BG505-35O22, BG18-B41-35O22, and BG18-BG505-IOMA structures each comprised an Env trimer bound to three BG18 Fabs and three 35O22 or IOMA Fabs (Fig. 1a–c), with ordered electron density corresponding to native glycans at the Fab interfaces (Fig. 1d and Supplementary Figure 3a). The orientations of BG18 Fab with respect to gp120 were preserved across the three crystal structures and a low-resolution EM structure of a BG18-BG505 complex47 (Supplementary Figure 3b–d), indicating that the orientation was conserved across different Env strains and was not an artifact of crystal packing (Supplementary Figure 3e,f). In addition, BG18 did not alter binding modes at the gp41/gp120 interface and CD4bs by 35O22 or IOMA, respectively, as these Fabs adopted similar conformations as previously observed on trimeric Envs25,27.
Superimposition of the BG18 VH-VL coordinates in the BG18-BG505-35O22 structure with VH-VL in unbound BG18 Fab (PDB 5UD9 (https://doi.org/10.2210/pdb5UD9/pdb)) resulted in a 1.3-Å root mean square deviation (rmsd) (240 Cα atoms), demonstrating that BG18 CDR loops (except for CDRL2, which was disordered in unbound BG1847) did not undergo large conformational rearrangements upon binding Env, and thereby maintained the previously observed clefts (Fig. 2a, b). Most notably, the conformation and location of CDRH3 in unbound BG18, which differs from CDRH3s in unbound PGT121 and 10-107440,47, were preserved in the Env-bound BG18 structure (Fig. 2c, d). Additionally, interactions with gp120 and glycans at the base of the V3-loop resulted in stabilization of CDRL2.
BG18 adopts a distinct Env-binding orientation
A low-resolution Env-bound BG18 structure derived by negative stain EM showed an orientation for BG18 distinct from the orientations of 10-1074 and other V3/N332gp120 bNAbs47. Despite crystallization contacts involving Fabs (Supplementary Figure 3e,f), BG18 maintained this distinct orientation in our crystal structures compared with orientations in structures of HIV-1 Env trimers with Fabs from 10-1074, PGT122, a PGT121 intermediate, and PGT12425,27,31,50 (Fig. 3 and Supplementary Figure 4). To analyze these differences, we calculated the rotation and translation for VH-VL domains of Env-bound BG18 Fab when compared to Env-bound 10-1074 Fab structures. We found that the mature BG18 VH-VL domains differed by ~78˚ relative to the orientations of 10-1074 Fab VH-VL domains (Fig. 3d, e), which contrasts with the ~5˚ difference between Env-bound PGT122 and 10-1074 Fab orientations. Notwithstanding its different orientation, BG18, 10-1074, and PGT121-like bNAbs share a common mode of interaction with the N332gp120 glycan through their CDRH3 loops (Fig. 3c). This interaction includes a structural motif with a consensus R-I-Y-G-V/I-I sequence (BG18 residue numbers 101-106 and 10-1074/PGT122 residue numbers 100-100E; Fig. 2d) encoded by the same antibody D3-3 gene segment, likely explaining the nearly identical N332gp120 glycan recognition. Given that CDRH3 is a primary determinant of the interactions of V3/N332gp120 bNAbs with Env trimer29,32,40,44, its displacement in both unbound and Env-bound BG18 (Fig. 2c) rationalizes its orientation of Env binding relative to other V3/N332gp120 bNAbs. Consistent with these observations, BG18’s footprint at the V3/N332gp120 epitope differs from PGT121/10-1074-like bNAbs, such that its interactions with Env glycans and protein components are mediated by different CDR loops relative to the CDR loops used by 10-1074 (Fig. 4a, b and Supplementary Figure 5).
N-linked glycan interpretation and interactions with BG18
Modeling of glycans was achieved in our structures by interpreting electron density at PNGSs using 2Fo−Fc maps calculated with model phases and in composite annealed omit maps to reduce model bias56. In the BG18-BG505-35O22 structures, 17 N-linked glycans were modeled into ordered electron density, forming glycan arrays that extended ~22 Å from the Env surface (Fig. 1a and Supplementary Figure 6). N-glycans near the BG18 and 35O22 interfaces were modeled as mostly oligomannose (Man5-9GlcNAc2) in our structures to avoid over-interpretation of the electron density, except for complex-type glycans at positions N88gp120 and N156gp120, which were assigned as complex-type based on density for a core fucose in our 3.8 Å XFEL structure (Supplementary Figure 7). Although complex-type N-glycans were modeled at positions N160gp120, N276gp120, N301gp120, and N392gp120 in a previous natively glycosylated 10-1074-Env-IOMA structure25 and observed at positions N160gp120, N197gp120, and N276gp120 by mass spectrometry18,52,57, our datasets did not show characteristic densities for complex glycans at these positions. Modeling of oligomannose N-linked glycans into ordered electron density was also possible in the 4.9 Å BG18-B41-35O22 structure, and for the N332gp120 glycan in the 6.7 Å BG18-BG505-IOMA structure (Fig. 1b,c and Supplementary Figure 6). Overlay of BG505 and B41 Env structures showed conservation of the glycan shield surrounding the BG18 binding site (Supplementary Figure 6). Despite resolution limitations that necessitated glycan modeling as predominantly oligomannose at PNGSs shown to attach complex glycoforms, the use of natively glycosylated Env trimers in the crystallization complexes ensured accuracy of bNAb binding orientations and interfaces with Env trimer.
As seen in other PGT121-like bNAb-Env SOSIP structures25,27,31,50, BG18’s primary interaction was with the N332gp120 glycan (Man9GlcNAc2), which interfaced with CDRH3, CDRH1, and CDRL2 (Fig. 4a,c; 1170 Å2 total buried surface area (BSA)). This contrasts with PGT121-like bNAb interactions that pack the N332gp120 glycan into a groove between CDRH3 and CDRL1/225,29,31 (Fig. 4b). Indeed, the observed differences in the orientation of the BG18 Fab light chain on Env influenced the N332gp120 glycan conformation, as the glycan D1 arm reached in close proximity to CDRL2 and the gp120 GDIR motif in the BG18-BG505 complex (Fig. 4c), which differs from the interactions of the N332gp120 glycan D1 arm with light chain framework region 3 (FWRL3) in the 10-1074-BG505 complex (Fig. 4b). Modeling of Env-bound 10-1074 Fab conformation onto the gp120 subunit from the BG18-BG505-35O22 structure showed clashes between the N332gp120 glycan and the CDRL1/2 loops and FWRL3 of 10-1074 (Fig. 4d), suggesting that the distinct BG18 interaction with Env stabilizes a N332gp120 glycan conformation that is not possible when binding PGT121/10-1074-like bNAbs29,31.
In addition to the BG18 interaction with the N332gp120 glycan, BG18 makes secondary interactions with the N392gp120, N386gp120, and N156gp120 glycans (Fig. 4a and Supplementary Data 1). These interactions are in contrast with PGT121-like bNAbs that interact with the N137gp120, N156gp120 glycans in the V1-loop, and N301gp120 glycans at the base of the V3-loop (Fig. 4b and Supplementary Data 1)25,27,32. When comparing the BG18–Env interaction with the only other structure of a V3/N332gp120 bNAb bound to natively glycosylated Env (10-1074–BG505)25, the rearrangement of BG18’s VL domain reduced BG18’s contact with the N301gp120 glycan (modeled as a core pentasaccharide in the BG18-BG505-35O22 structure) compared to 10-1074’s contacts with the N301gp120 glycan (modeled as complex-type biantennary in the 10-1074-BG505-IOMA structure; PDB 5T3X (https://doi.org/10.2210/pdb5T3X/pdb)) (Fig. 4a, b). Interestingly, VL domain rearrangements allowed BG18 engagement of the N392gp120 glycan through its CDR loops (Fig. 4a). The N392gp120 glycan threads between the BG18 cleft that is located between CDRH3 and CDRL1/L347 (Fig. 2b), burying ~425 Å2 of Fab surface area against the glycan. This interaction resembles the PGT135 interaction with monomeric gp120 in which ~550 Å2 of gp120 surface area is buried against the N392gp120 glycan44. However, in contrast to PGT135, which does not neutralize strains lacking the N392gp120 or N386gp120 glycans, BG18 remains potent against strains lacking glycans at these positions, showing only a ~4-fold reduction in potency (Table 2). Moreover, binding affinities determined by surface plasmon resonance (SPR) showed no effect when the N392gp120 glycan was removed from BG505 SOSIP (Supplementary Figure 8). These results are comparable to PGT121-like bNAbs, which equivalently neutralize strains +/− the N392gp120 and N386gp120 glycans (Table 2), suggesting that BG18 combines binding and neutralization properties of both PGT121-like and PGT135-like bNAbs.
Molecular details of BG18–Env interactions
BG18 conserves interactions with the gp120 GDIR peptide motif at the base of the V3-loop that are seen in other V3/N332gp120 bNAbs (Fig. 5a, b and Supplementary Figure 5). However, rotation of BG18’s light chain relative to other V3/N332gp120 bNAbs places only CDRL2 in close proximity to the gp120 GDIR peptide motif, compared to engagement of GDIR by multiple CDR loops and framework regions observed in 10-1074/PGT121-like bNAb recognition (Fig. 5b, c)25,29,31. These differences in light chain interactions reflect the germline origins of the BG18 and PGT121/10-1074 light chains, which derive from different VL gene segments (Fig. 5d). However, in an example of convergence toward a common chemical binding mechanism, light chain residues involved in GDIR recognition by BG18 CDRL2 derive from hypermutation from the germline LV3-25*02 gene segment, whereas serines in the CDRL3s of PGT121 and 10-1074 derive from J regions chosen during V–J joining.
B41 Env harbors a GNIR sequence instead of GDIR, allowing us to analyze the D325Ngp120 substitution in our BG18-B41-35O22 structure. Side chain placement was not possible due to low resolution (4.9 Å), but the BG18 interaction with B41 resembled its interaction with BG505 in the BG18-BG505-35O22 structure (Supplementary Figure 9), suggesting that BG18 recognizes the GNIR motif analogously to how it recognizes GDIR. Consistent with this result, analysis of BG18 neutralization of HIV-1 isolates containing GNIR motifs showed only a 2-fold loss in potency, by contrast to PGT121 or PGT122, which showed ~6-fold and ~44-fold losses, respectively (Table 2). However, while BG18 S53LC potentially engages N325gp120 (Supplementary Figure 8b), the loss of BG18 Q54LC contacts resulted in the disorder of CDRL2 residues 54-60 in our BG18-B41 structure.
Further comparisons between BG18-Env and 10-1074-Env bound structures showed that BG18 CDR loops and light chain framework region 3 are located in close proximity to the gp120 V1-loop (residues 126–158, Supplementary Figure 5), of which residues 140–150 were disordered in the BG18–Env structures (Fig. 6a) and other Env structures22,25,27,31. Despite variability in V1 across HIV-1 Envs, we observed engagement of V1-loop protein components with BG18 CDRL2 and FWRL3 residues (Fig. 6b), accommodating the V1-loop into the nearby positively charged cleft formed by CDRH3, CDRL1/L2, and FWRL3 loops (Fig. 6b, c). The electronegativity of this cleft potentially increases protein–protein interactions with BG505 and other HIV-1 strains harboring polar or acidic residues in this region (Fig. 6c). Although PNGSs are commonly found in the V1-loop, the presence of glycans in this region do not notably affect BG18 neutralization (e.g., N133gp120, N137gp120, and N156gp120 glycans; Fig. 6a and Table 2), despite the roof of the cleft being only ~6–8 Å above the V1-loop site. In comparison, the surface of 10-1074 in the comparable region is neutral and does not exhibit a cleft architecture, and only minimally contacts the V1-loop backbone through R94LC (Supplementary Figure 10). Taken together, these observations may explain BG18’s increased potency, as BG18 can engage multiple glycan and protein component regions of gp120 (Supplementary Data 1).
Structures of bNAbs complexed with HIV-1 Env trimers have helped elucidate the molecular correlates for anti-HIV-1 antibody breadth and potency. Here we report four crystal structures of the highly potent V3/N332gp120 bNAb BG18 bound to natively glycosylated clade A and clade B Envs (Fig. 1a–c). Our structures of clade A BG505 and clade B B41 HIV-1 strains represent only the second example of fully and natively glycosylated Env crystal structures, with the first being BG505 complexed with another V3/N332gp120 bNAb, 10-1074, and with the CD4bs bNAb IOMA25. Given the crucial role the Env glycan shield plays in HIV-1’s immune evasion strategies13,58, the prevalence of bNAbs that interact with complex glycans18,25,58, and the importance of complex glycans in bNAb maturation46, solving structures containing both complex and high-mannose glycans provides a more complete picture of bNAb recognition of Env epitopes. The newly identified BG18-Env-35O22 crystal lattice system packs solely through Fab interactions (Table 1 and Supplementary Figure 3e), similar to crystals of previously described Env-bNAb complexes25,27, thus providing an additional system to study HIV-1 Env diversity. Moreover, the improvement in resolution from 4.1 Å using conventional crystals to 3.8 Å resolution by exposing smaller crystal volumes using an XFEL (Supplementary Figure 2) offers the potential to examine natively glycosylated Env trimer–Fab structures to higher resolutions. The demonstration that two HIV-1 Env trimers (BG505 and B41) can be crystallized in different crystal packing lattices without converting their glycans to exclusively high-mannose forms provides an impetus for further crystallization efforts using natively glycosylated HIV-1 Envs. Resulting crystal structures can be compared to natively glycosylated Env structures determined by cryo-EM38, a method that does not require crystallization and can therefore be used for heterogeneously glycosylated samples.
Our structures were consistent with previous evidence that BG18 binds with a distinct orientation compared to the prototype PGT121/10-1074 bNAbs in the V3/N332gp120 glycan-targeting family47 and showed extensive interactions with both protein and glycan components of gp120 (Fig. 1d). BG18’s CDRH3 interactions with GDIR and the N332gp120 glycan are conserved with other V3/N332gp120 bNAbs (Supplementary Figure 5), serving as the main driver for epitope recognition (Figs 3c and 5b). Strikingly, the D3-3 gene for BG18/10-1074/PGT121 bNAbs that encodes a CDRH3 consensus structural motif responsible for N332gp120 glycan interactions plays a role analogous to the VH1-2 gene for VRC01-like bNAbs24,59,60 for epitope targeting. This common feature, which unites the PGT121 and BG18 bNAb families, suggests that it provides the key interaction in the initial binding of their unmutated ancestor precursors to Env. Interestingly, this common interaction occurs despite different orientations for the rest of the VH-VL domains (Fig. 4a, b). For example, unlike other V3/N332gp120 bNAbs, BG18’s light chain CDR loops straddle the V1-loop of gp120 (Fig. 6), increasing surface contact with gp120 protein components. In addition, BG18’s interactions with gp120 N-glycans differ from the PGT121-like family in both engagement and ability to retain potent neutralization properties. For instance, despite extensive interactions with glycans at positions N392gp120 and N386gp120 (Fig. 4a, e and Supplementary Data 1), BG18 can potently neutralize HIV-1 strains lacking these glycans, by contrast to the more weakly neutralizing V3/N332gp120 bNAb, PGT135, which relies on N332gp120, N392gp120, and N386gp120 glycans for its anti-HIV-1 activity44 (Table 2). Consistent with this observation, analysis of neutralization by BG18 and PGT121/10-1074 family bNAbs of HIV-1 strains including the N332gp120 glycan, but with and without glycans known to interact with bNAbs targeting the V3/N332gp120 epitope, showed that BG18 and 10-1074 retained their anti-HIV-1 potency (mean IC50s < 0.6 µg/mL), whereas PGT121/PGT122 potency was abrogated upon removal of the N301gp120 glycan (Table 2). Furthermore, PGT122 showed sensitivity to D325Ngp120 mutations in the GDIR peptide motif. Taken together, these differences illustrate the divergence of solutions evolved by bNAbs to target this epitope, and BG18’s successful strategies to accommodate Env sequence diversity.
Recent studies showed that priming with designed SOSIP Env trimers that bind weakly to the common inferred germline sequence (iGL) of PGT121 and 10-107450 could elicit bNAbs resembling PGT121 in PGT121 iGL Ig knock-in mice49. However, the iGL of BG18 did not bind detectably to the tightest-binding designed priming immunogen 11MUTB (Supplementary Figure 11a), which can be rationalized by one of several differences in BG18 and PGT121/10-1074 recognition of Env. First, mature BG18 CDRL2 residues are involved in GDIR recognition with S53LC and Q54LC engaging D325gp120 of GDIR (Fig. 5b). Comparison of iGL and mature BG18 CDRL2 amino acid sequences showed that four of five residues were mutated (50YKDSE54 vs. 50SRSSQ54, respectively) (Fig. 5d). Not only does the germline CDRL2 have increased bulk, but also acidic residues flank S53LC, which interacts with D325gp120. In contrast, iGL sequences for CDRL1/3 in PGT121/10-1074 show conservation of serines responsible for interacting with D325gp120 in GDIR and flanking residues (Fig. 5d). Thus, since the 11MUTB priming immunogen contains no substitutions in GDIR, BG18 iGL likely shows a reduced ability to interact with this region compared with PGT121/10-1074 iGLs. An additional predicted impediment to iGL BG18 binding to 11MUTB is the relative proximity of light chain CDRs to the gp120 V1-loop (Fig. 6). Since the CDRs are the most heavily substituted between iGL and mature sequences, altering the chemical properties of this cleft could negatively affect binding. Moreover, 11MUTB harbors seven mutations in the V1-loop necessary for iGL PGT121 binding50, which likely clash with iGL BG18 given its V1-loop interactions (Supplementary Figure 11b). Finally, mature BG18 is heavily substituted compared to iGL sequences (35 and 26 heavy chain and light chain amino acid mutations, respectively). Previous structural studies of a PGT121 intermediate bound to BG505 Env showed that orientations are defined early during maturation and that differences in amino acid composition can alter antibody footprints on gp12031. Thus, it is possible that the conformation of iGL BG18 is incompatible with 11MUTB binding.
Although eliciting BG18-like bNAbs would require a different set of designed immunogens than being used for PGT121/10-1074 bNAbs49,50, they might be easier to elicit because of a shorter CDRH3 than PGT121/10-1074 bNAbs. By elucidating the molecular details of BG18’s distinct interaction with the V3/N332gp120 epitope, the structural information reported here will facilitate future immunogen design efforts.
Protein expression and purification
Fabs from BG18 (including a N26QHC substitution47), 35O22, and IOMA IgGs were produced as previously described37. Briefly, Fabs were expressed by transiently transfecting HEK293-6E cells (National Research Council of Canada) with vectors encoding the appropriate light chain and C-terminal 6x-His tagged heavy chain genes. Secreted Fabs were purified from cell supernatants using Ni2+-NTA affinity chromatography (GE Healthcare), followed by size exclusion chromatography (SEC) with a Superdex200 16/60 column (GE Healthcare). Purified Fabs were concentrated and maintained at 4 °C in storage buffer (20 mM Tris pH 8.0, 150 mM NaCl, and 0.02% sodium azide).
Genes encoding BG505 SOSIP.664 gp14020 or B41 SOSIP.664 gp14061 trimers were stably expressed in CHO Flp-InTM cells (Invitrogen) as described51 using cell lines kindly provided by John Moore (Weill Cornell Medical College). Plasmids encoding the BG505 SOSIP.664 gp140 N392 gene variant (see Supplementary Table 1 for primer sequences) was transiently expressed in HEK293-6E cells (National Research Council of Canada) as previously described22. In both cases, secreted SOSIP trimers expressed in the absence of kifunensine were isolated from cellular supernatants using 2G12 immunoaffinity chromatography by covalently coupling 2G12 IgG monomer to an activated-NHS Sepharaose column (GE Healthcare). Trimers were eluted using 3 M MgCl2 and immediately dialyzed into storage buffer before SEC purification with a Superdex200 16/60 column (GE Healthcare) against the same buffer. Peak fractions pertaining to SOSIP trimers were pooled and repurified over the same column and buffer conditions. Twelve 1.0-mL fractions were stored separately at 4 °C.
Crystallization of BG18–Env complexes
Complexes for crystallization were produced by an overnight incubation of SOSIP with BG18 and 35O22 or IOMA Fabs at a 1:1:1 molar ratio, and subsequently concentrated to 5–8 mg/mL by centrifugation with a 30-kDa concentrator (Amicon). Initial matrix crystallization trials were performed at room temperature using the sitting drop vapor diffusion method by mixing equal volumes of protein sample and reservoir using a TTP LabTech Mosquito robot and commercially available screens (Hampton Research and Qiagen). Initial hits were optimized and crystals were obtained for BG18-BG505-35O22 and BG18-B41-35O22 in 0.1 M Tris pH 8.0, 5% Tacismate pH 8.0, and 14% polyethylene glycol (PEG) 3350 at 20 °C. BG18-BG505-IOMA crystals were obtained in 0.1 M citric acid pH 3.7, 16% PEG 3350 at 20 °C. Crystals were cryo-protected stepwise with reservoir and a final 20% v/v glycerol concentration before being cryopreserved in liquid nitrogen.
Structure determination and refinement
Conventional X-ray diffraction data were collected for BG18-Env complexes at the Stanford Synchroton Radiation Lightsource (SSRL) beamline 12-2 on a Pilatus 6M pixel detector (Dectris). Data from a single crystal for each complex were indexed and integrated in XDS62, and merged with AIMLESS in the CCP4 software suite63. Structures were determined by molecular replacement in PHASER64 using a single search with coordinates of an a glycosylated gp120-4 protomer (PDB 5T3Z (https://doi.org/10.2210/pdb5T3Z/pdb)), BG18 Fab (PDB 5UD9 (https://doi.org/10.2210/pdb5UD9/pdb)), and 35O22 Fab (PDB 4TOY (https://doi.org/10.2210/pdb4TOY/pdb)) or IOMA Fab (PDB 5T3Z (https://doi.org/10.2210/pdb5T3Z/pdb)). Models were refined using B-factor refinement in CNS65 and Phenix56, followed by several cycles of manual building with B factor sharpening in Coot55,66. Glycans were initially interpreted and modeled using Fo−Fc maps calculated with model phases contoured at 2σ, followed by 2Fo−Fc simulated annealing composite omit maps in which modeled glycans were omitted to remove model bias56. N-linked glycans identified at individual PNGSs in our crystallographic studies on both BG505.664 and B41.664 SOSIP trimers were generally consistent with the mixture of glycans observed by mass spectroscopy18,57 and previous crystallographic studies of a natively and fully glycosylated Env trimer25. Therefore, modeling of complex-type glycans at positions N88gp120 and N156gp120 were primarily determined by the presence of electron density characteristic of a core fucose, which when modeled, slightly lowered Rfree values. Additional details of glycan modeling and coordinate refinement were followed as previously described25,67. Inclusion of higher resolution data with weak intensities improved refinement behavior and stereochemistry as described68.
XFEL diffraction experiments were performed at the MFX endstation of LCLS using a standard goniometer setup48, 9.5 keV X-ray pulses with 40 fs duration and a 5-µm beam focus at the crystal interaction point. Diffraction images were recorded on a Rayonix MX325 detector data and were integrated with IOTA69 using the data reduction algorithms implemented in cctbx.xfel70. Of the 627 collected images, 589 contained discernible diffraction; of these, 570 were successfully integrated and yielded correct crystal symmetry and unit cell values. Scaling, post-refinement, and merging was carried out using PRIME71. Of the 570 integrated diffraction images, 526 were included in the final merged dataset, which was complete (99.1%) to 3.8 Å, and exhibited good multiplicity (9.2-fold) and reasonable merging statistics (Table 1). Phases were generated by molecular replacement, using our refined 4.1 Å synchrotron structure with glycans omitted as a search model.
Superposition and figures were rendered using PyMOL (Version 126.96.36.199 Schrodinger, LLC), and protein electrostatic calculations were achieved using APBS and PDB2PQR webservers72. BSAs were determined with PDBePISA using a 1.4-Å probe73. Potential hydrogen bonds were assigned using a distance of <3.6 Å and an A-D-H angle of >90°, while the maximum distance allowed for a van der Waals interaction was 4.0 Å. Putative H-bonds, van der Waals assignments, and total BSA should be considered tentative, owing to the relatively low structure resolutions.
SPR experiments were carried out on a Biacore T100 (Biacore) using a standard single-cycle kinetics method as previously described22,37. Briefly, a CM5 chip, primarily amine coupled with Protein A, was used to immobilize 8ANC195 IgG, a gp120-gp41 interface bNAb37. Remaining Protein A sites were blocked with 1 µM human Fc. BG505 HIV-1 SOSIP trimers were captured by injecting 10 µM solutions at a flow rate of 30 µL/s for 180 s. Mature BG18 Fab was injected at decreasing concentrations (3-fold dilution series with a starting top concentration of 110 nM) at 30 µL/s for 60 s and allowed to dissociated for 300 s. Kinetic analyses were done after subtraction of reference curves to obtain ka, kd, and KD values for a 1:1 binding model with or without a bulk refractive index change correction as appropriate (Biacore T200 Evaluation software).
An ELISA to evaluate binding of mature and iGL BG18 IgG to 11MUTB SOSIP50 was performed by coating of High-Bind 96-well plates (Corning #9018) with 50 µL per well of a 2-µg/mL solution of purified 11MUTB in PBS overnight at 4 °C. Plates were washed six times with washing buffer (1× PBS with 0.05% Tween 20 (Sigma-Aldrich)) and incubated in blocking buffer (1× PBS with 1% non-fat milk) for 1 h at room temperature (RT). Immediately after blocking, IgGs were added in blocking buffer and incubated for 2 h at RT. Antibodies were assayed at a 5-µg/mL starting dilution and seven additional 3-fold serial dilutions. Plates were washed six times with washing buffer and then incubated with anti-human IgG secondary antibody conjugated to horseradish peroxidase (HRP) (Jackson Laboratories) in washing buffer at a 1:5000 dilution. Plates were developed by addition of the HRP substrate, ABTS (Life Technologies), and absorbance was measured at 405 nm with an ELISA microplate reader (FluoStar Omega, BMG Labtech).
Coordinates and structure factors reported in this manuscript have been deposited in the Protein Data Bank with accession codes 6CH7, 6CH8, 6CH9, and 6CHB. Other data are available from the corresponding author upon reasonable request.
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We thank J. Vielmetter, S. Khomandiak, and the Caltech Protein Expression Center for producing proteins, J. Kaiser and the beamline staff at SSRL for data collection assistance, and members of the Bjorkman and Nussenzweig laboratories for critical reading of the manuscript. We also thank Silvia Russi, Clyde Smith, Jinhu Song, and Alexander Batyuk for user support during our MFX experiment and the lab of Guillermo Calero (University of Pittsburgh) for providing loop supports to mount multiple crystals during XFEL experiments. C.O.B holds a Hanna Gray Fellowship from the Howard Hughes Medical Institute and a Postdoctoral Enrichment Program Award from the Burroughs Wellcome Fund. This research was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health Grant HIVRAD P01 AI100148 (P.J.B.); (the content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health), the Bill and Melinda Gates Foundation (Collaboration for AIDS Vaccine Discovery Grant OPP1124068 (M.C.N./P.J.B.)), and the Molecular Observatory at Caltech supported by the Gordon and Betty Moore Foundation. Use of the Linac Coherent Light Source (LCLS) and use of the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the US Department of Energy Office of Biological and Environmental Research, and by the National Institutes of Health (NIH), National Institute of General Medical Sciences (including P41GM103393).
The authors declare no competing interests.
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