Research Article

Crystal Structure of a Soluble Cleaved HIV-1 Envelope Trimer

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Science  20 Dec 2013:
Vol. 342, Issue 6165, pp. 1477-1483
DOI: 10.1126/science.1245625

Knowing the Enemy

Infection of host cells by HIV-1 is mediated by an envelope glycoprotein (Env) trimeric spike on the surface of the virus. Proteins comprising the Env trimer must be cleaved for infectivity, and thus viral fusion involves three Env conformations. The flexibility of the Env trimer has made it a challenge to determine a high-resolution structure, although such a structure is key both for understanding trimer function and for guiding vaccine design. Lyumkis et al. (p. 1484) and Julien et al. (p. 1477) studied soluble cleaved trimers stabilized by specific mutations but that have kept a near-native antigenicity profile. Lyumkis et al. present a high-resolution structure of the trimer in complex with a broadly neutralizing antibody, and Julien et al. present a crystal structure of the trimer in complex with another broadly neutralizing antibody.

Abstract

HIV-1 entry into CD4+ target cells is mediated by cleaved envelope glycoprotein (Env) trimers that have been challenging to characterize structurally. Here, we describe the crystal structure at 4.7 angstroms of a soluble, cleaved Env trimer that is stabilized and antigenically near-native (termed the BG505 SOSIP.664 gp140 trimer) in complex with a potent broadly neutralizing antibody, PGT122. The structure shows a prefusion state of gp41, the interaction between the component gp120 and gp41 subunits, and how a close association between the gp120 V1/V2/V3 loops stabilizes the trimer apex around the threefold axis. The complete epitope of PGT122 on the trimer involves gp120 V1, V3, and several surrounding glycans. This trimer structure advances our understanding of how Env functions and is presented to the immune system, and provides a blueprint for structure-based vaccine design.

The envelope glycoprotein (Env) trimer is the only virally encoded antigen on the surface of HIV-1, the pathogen that causes AIDS, and is responsible for viral entry into host cells. The trimer is composed of gp120/gp41 heterodimers and is the target for neutralizing antibodies. Various structures of components of gp120 and gp41, alone and in complex with different ligands, have been determined (110). Cryogenic electron microscopy (cryo-EM) and tomography have been integrated with core gp120 x-ray structures to visualize the Env trimer at resolutions that extend from 30 Å to below 10 Å and, thereby, provide insights into its overall conformation before and after receptor binding (11, 12). However, determining an atomic-level structure of the Env trimer has been difficult. A higher-resolution structure would not only help to explain how the trimer functions during virus-cell fusion, but also guide HIV-1 vaccine design by delineating the key antigenic sites recognized by the humoral immune system and the defenses evolved by the virus as a countermeasure.

During Env synthesis, gp160 precursors trimerize and are subsequently cleaved by proteases of the furin family into gp120 and gp41 subunits, which associate noncovalently before the native complex reaches the surface of infected cells and is then packaged onto virions (13). Cleavage is obligatory for Env trimers to function in viral infection of target cells (14). Virus-cell fusion is a multistep process involving three major Env conformations, each with distinct roles: (i) prefusion (interacts with CD4 receptor); (ii) extended gp41 intermediate (interacts with CCR5 or CXCR4 co-receptors); and (iii) gp41 six-helix bundle (hemi-fusion of viral and cell membranes) (15).

The requirement for the cleaved, native Env trimer to undergo conformational changes during receptor binding and fusion makes it metastable, which has substantially hindered both structure determination and vaccine development. The extensive N-linked glycosylation (on average, 81 sites per trimer) creates additional complications for x-ray structural studies. Moreover, membrane-associated forms of Env are more difficult to express and purify in appropriate quantities and qualities than soluble versions. Our approach to these various problems has been to express soluble (that is, truncated before the gp41 transmembrane domain), cleaved forms of trimeric Env (SOSIP gp140) that are engineered to improve their stability and homogeneity. Specifically, a disulfide bond (termed SOS) between gp120 residue 501 (HXB2 numbering) and gp41 residue 605 covalently links these subunits, whereas an Ile-to-Pro change at position 559 (termed IP) strengthens gp41-gp41 associations (16). A recent version of the SOSIP gp140 trimer, based on a tier-2 subtype A virus (BG505) (17), was further engineered to delete all but four residues of the hydrophobic membrane proximal external region (MPER) of gp41 (1720). Together, these various modifications allow the expression of a thermostable, nonaggregating, and homogeneous soluble Env trimer, BG505 SOSIP.664 gp140, suitable for structural characterization by x-ray crystallography (Fig. 1A). These trimers are reactive with a large panel of diverse broadly neutralizing antibodies (bnAbs), including those to quaternary epitopes, while being minimally reactive with non-neutralizing antibodies that preferentially recognize individual gp120/gp41 subunits and/or uncleaved, non-native trimer forms (17, 18). The near-native antigenic properties of the BG505 SOSIP.664 gp140 trimer suggest that its structure resembles the native viral spike, although we cannot completely rule out slight conformational differences resulting from engineered features, such as truncation of the gp41 MPER and transmembrane domain (19). Here, we show that the BG505 SOSIP.664 gp140 trimers could be successfully crystallized with a highly potent bnAb, PGT122, that targets the glycan-dependent Asn332 (N332) supersite of vulnerability on gp120 (21). These crystals allowed the structure of an Env trimer to be determined at a resolution of 4.7 Å.

Fig. 1 Overall architecture of a soluble, cleaved, recombinant HIV-1 Env trimer in complex with bnAb PGT 122.

(A) Schematic of the HIV-1 Env BG505 SOSIP.664 construct in comparison to full-length gp160. N-linked glycans are shown and numbered on their respective Asn residues. The constant (C1 to C5) and variable (V1 to V5) regions in gp120 and the FP, HR1 and HR2 helices, MPER, transmembrane (TM), and cytoplasmic (CT) elements in gp41 are indicated. The SOSIP mutations are shown in red, as well as the added N332 glycan site. The color coding is preserved in (B) to (D). (B) Side view of the soluble Env trimer complex with PGT122 showing two of the three Env gp140 protomers associated with PGT122 Fab (blue). A 2Fo-Fc electron-density map contoured at 1.0σ is shown as a gray mesh around the leftmost gp140 protomer. The membrane to which gp41 is attached would be at the bottom of the figure. (C) Side view of the Env trimer. For one of the three protomers on Env, core gp120 is shown in yellow, whereas V1/V2 and V3 regions are highlighted in orange and red, respectively. The main gp41 helical elements are colored in different shades of green. Protein components are rendered according to their secondary structure, and glycans are depicted as spheres. (D) View of Env down the trimer axis. Loops of high variability in gp120 (V1 to V5) all map to the periphery of the trimer and are labeled. Glycans have been omitted for clarity. Dashed lines indicate the location of gp120 V2 and V4 loops for which electron density was absent or ambiguous. The figure was generated with PyMOL (63).

Structure Determination

The BG505 SOSIP.664 construct was expressed in human embryonic kidney 293S GnTI−/− cells, yielding trimers enriched for oligomannose (Man5-Man9) glycans [see supplementary materials (22)]. After incubation of the purified trimers with a sixfold molar excess (twofold for the binding sites) of PGT122 Fab, the complex was treated with EndoH glycosidase (fig. S1, A and B) to truncate any accessible N-linked glycans (that is, not buried or occluded by PGT122) to a single N-acetyl glucosamine (NAG) moiety that remains covalently attached to the Asn side chain. Crystals of the purified complex diffracted well, albeit anisotropically, to 3.7 Å along the c axis, but to lower resolution along the other two axes (fig. S1, C and D). Merging diffraction data from two crystals resulted in a complete data set to a maximum resolution of 4.7 Å (table S1). Phases were obtained by molecular replacement using integrative approaches in which a search model was generated from crystal structures of the unliganded PGT122 Fab variable region [Protein Data Bank identification number (PDB ID): 4JY5 (23)] and CD4-bound gp120 core [PDB ID: 3JWD (5)], docked into our previous ~14 Å EM reconstruction of the same complex [Electron Microscopy Data Bank identification number: 5624 (23)]. Only one complex was present in the asymmetric unit with 82% solvent, which simplified structure determination (fig. S2). Initial phases resulted in a well-defined electron-density map that enabled subsequent placement of the high-resolution PGT122 Fab constant domains and the gp120 V1/V2 [PDB ID: 3U4E (4)] and V3 [PDB ID: 2ESX (24)] structures. Previously uncharacterized elements in the trimer—such as the gp41 helices, the V1/V2/V3 loops, and various Man5-Man9 glycans—were visible in the electron-density maps, as were residual NAG moieties attached to their respective Asn residues (fig. S3). Together, these identifiable features, along with aromatic side chains, aided in model building and refinement at this moderate resolution. In addition, the prominent new features for the Env trimer that we observed in the electron-density maps are the same as those visualized in the accompanying 5.8 Å cryo-EM reconstruction of the same trimer in complex with bnAb PGV04 (fig. S4) (25).

Architecture of the Env Trimer

The soluble BG505 SOSIP.664 trimer adopts a compact mushroom shape reminiscent of the Env trimer prefusion closed conformation determined at 20 Å resolution by EM (11, 26). Three PGT122 Fabs protrude vertically from the membrane-distal gp120 subunits, whereas the gp41 components are membrane-proximal and interspersed with the gp120 C1 and C5 elements (Fig. 1, B to D). Previous high-resolution crystal structures of core gp120 [PDB ID: 3JWD (5)], scaffolded V1/V2 [PDB ID: 3U4E (4)], and the variable domains of PGT122 Fab [PDB ID: 4JY5 (23)] fit well into the electron density of the trimer complex (Figs. 1 and 2 and fig. S5) and have Cα root mean square deviations of 1.3, 2.9, and 0.8 Å with the final trimer model, respectively. Thus, the core gp120 elements in the trimer adopt conformations similar to those observed in unliganded gp120 (2) or gp120 in complex with various ligands [specifically CD4 (3, 5), b12 (9), VRC01 (8), PGT128 (7), PGT135 (1), and PG9/PG16 (4, 6)]. In addition, all previously described disulfide bonds in gp120 (3) are present, and all of the variable loops (V1 to V5) are on the outside of the structure (Fig. 1D).

Fig. 2 Comparison of gp120 and components, as observed in high-resolution crystal structures and in the soluble HIV-1 Env trimer.

(A) High-resolution crystal structure of core gp120 [PDB ID: 3JWD (5), pale green] superimposed on the gp120 component of the soluble, cleaved SOSIP.664 trimer crystal structure (yellow). A longer α1 helix (gp120 residues 99 to 117) likely contributes to rearrangement in the bridging sheet, particularly in β2 and β3, which extend into V1/V2 atop the trimer. The gp120 β2-proximal residues 115 to 125 are highlighted in blue and brown in core gp120 and trimeric gp140, respectively. A 2Fo-Fc electron-density map contoured at 1.0σ is shown as a gray mesh around the α1 helix. (B) Superimposition of the scaffolded gp120 V1/V2 crystal structure (PDB ID: 3U4E, pale green) on V1/V2 in the trimer crystal structure (orange). There are differences in V1 and in the β2 and β3 connecting strands. Electron density for carbohydrates at gp120 N156 and N160 is shown as a 2Fo-Fc gray mesh contoured at 1.0σ. (C) Structural arrangement of gp120 V1/V2 (orange) and V3 (red) in the context of the trimer. A 2Fo-Fc electron-density map contoured at 1.0σ is shown as a gray mesh around V1/V2 and V3 elements. All structures are depicted according to secondary structure elements, with glycans depicted as yellow spheres and the Cys126-Cys196 disulfide bond shown in blue. The figure was generated with PyMOL (63).

Our Env trimer structure contrasts markedly with one recently described for an uncleaved, membrane-bound JR-FL Env trimer (27). Compared with our soluble cleaved structure, the uncleaved trimer EM structure differs substantially even in the gp120 core, as well as in the arrangement of the gp41 helices. Our structure also does not contain the large hole that has been reported to be present in the uncleaved Env trimer (2729). It remains to be determined whether these differences are attributable to the use of different forms of trimer (soluble, truncated, cleaved versus detergent-solubilized, almost full-length, uncleaved) or reflect concerns about the cryo-EM methodology used to derive the uncleaved trimer structure (3034).

Comparison with Component Crystal Structures

High-resolution crystal structures of monomeric gp120 have largely been obtained with the use of a core construct stabilized in the CD4-bound conformation and with the V1/V2/V3 elements truncated (1, 3, 810). We observe only a few small deviations from these gp120 crystal structures, mostly in elements leading to and from V1/V2 (Fig. 2A and fig. S6). The differences are expected because V1/V2 undergoes major conformational changes when gp120 binds CD4 (2, 11, 12). In the trimer structure, clear density for an additional helical turn (residues 114 to 117) is observed in the gp120 α1 helix (residues 99 to 113), which leads into V1/V2 (Fig. 2A). In addition, the β2 (119 to 123) and β3 (199 to 201) strands are only partially involved in forming the bridging sheet with β20 and β21; instead, compared with gp120 monomeric cores, β2 and β3 flip and translocate slightly toward the trimer apex to connect with strands A and D of V1/V2, respectively (Fig. 2A). This elongated α1 helix-bridging sheet arrangement is also observed in the accompanying cryo-EM structure of the SOSIP gp140 trimer in complex with bnAb PGV04 (25). Whether this β-strand inversion at the V1/V2 base in the trimer structures—compared with the CD4-bound (3, 5), antibody-bound (810, 35), and unliganded core monomeric crystal structures (2)—can be attributed to oligomer-monomer differences, changes induced by CD4-binding, or the truncation of V1/V2 in core gp120 is not yet known.

Electron density at the trimer apex indicates that V1/V2 adopts a four-stranded Greek-key β-sheet arrangement similar to that observed in the high-resolution crystal structures of V1/V2 scaffolds in complex with bnAbs PG9 [(4), Fig. 2B)] and PG16 (6). The V1 loop (residues 132 to 140) connecting strands A and B adopts a more flattened structure that is parallel to the four-stranded β-sheet topology, differing slightly from its more vertical conformation in the V1/V2 scaffolded structure (4) and in the accompanying high-resolution cryo-EM trimer structure in complex with a receptor binding site antibody (25). It is unclear whether this difference in V1 orientation is a consequence of PGT122 binding or arises because V1 is a flexible loop. In the trimer crystal structure, the Cys126-Thr128 and Leu193-Cys196 segments diverge from one another, while still maintaining the Cys126-Cys196 disulfide bond, and these segments no longer contribute to V1/V2 strands A and D (Fig. 2B). Instead, they interact atop the Env spike with V1/V2 strands B and C and the conserved crown of the V3 loop from a neighboring protomer (Fig. 2C). We observe no clear electron density for V2 loop residues 178 to 190, which were also disordered in the V1/V2 scaffolded structures (4, 6).

The gp120 V3 loop completes the trimer apex and forms a β-hairpin with its two antiparallel β strands nestling directly below V1/V2 strands B and C of the same protomer (Fig. 2C). V3 peptides complexed with Abs adopt similar β-hairpin structures (24, 36, 37), as does the V3 region of monomeric gp120 (38) (fig. S5). The V3 crown is buried under a NAG moiety from the N197 glycan at the C-terminal end of V2 strand D from an adjacent protomer (Fig. 2C). Removing this glycan from other viruses increases nAb sensitivity (39) and can confer CD4-independent entry into CCR5-expressing cells (40). Our structure suggests that the N197 glycan helps to stabilize native Env by occluding V3 and inhibiting its premature release before CD4 binding. Any heterogeneity in the presence or composition of the N197 glycan may contribute to the reactivity of non-neutralizing V3 antibodies with BG505 SOSIP.664 trimers in some binding assays (17). Overall, the trimer structure is compatible with both intra- and intertrimeric (cis-trans) V3 shielding by V1/V2 (41, 42).

gp41 Architecture

Strong electron density for helices attributable to gp41 is clearly visible in the trimer structure (Fig. 3A). Three central helices (six turns) extend ~30 Å along the trimer axis, perpendicular to the viral membrane, and are ascribed to gp41 heptad-repeat 1 (HR1). Similar helices were recently observed in an ~9 Å cryo-EM reconstruction of the KNH1144 SOSIP gp140 trimer (12). An additional short helix (two and a half turns) extends from the central helix bundle but is kinked away from the trimer axis. These two HR1 helices pack against hydrophobic gp120 residues in C1 and C5, as well as loop A (Fig. 3B), and are capped by the gp120 α1 helix (Fig. 3B). Overall, these topological features are consistent with previous mutagenesis and structural studies that suggest gp41-gp120 interactions may propagate long-range effects upon receptor binding (5, 43).

Fig. 3 Structural organization of gp41 in the soluble cleaved HIV-1 Env trimer.

(A) Overall arrangement of gp41 elements from one protomer is shown in a gray 2Fo-Fc electron-density map contoured at 1.0σ. Carbohydrates are shown as spheres. Dashed lines delineate connecting electron density for which a chain trace and secondary structure determination was ambiguous at this moderate resolution. (B) Regions of contact between the gp120 inner domain and the gp41 central helix. The inset shows hydrophobic residues in the gp120 high-resolution crystal structure from C1, α0, and loop A that line the interface with gp41 HR1. (C) A surface rendering of two of the three HIV-1 Env and influenza hemagglutinin (HA) (PDB ID: 4FNK) protomers (back protomer omitted) emphasizes the similarity in position and size of a small, central interprotomer opening that presumably facilitates conformational changes during the fusion process. (D) Comparison between the structures of HIV-1 Env and influenza HA (PDB ID: 4FNK), the prototype type I fusion protein. There are notable similarities in the position of structural elements in the two glycoproteins. (E) The trimeric arrangement of gp41 HR1 in the postfusion conformation [PDB ID: 2X7R (50), beige] superimposes closely with the central HR1 in the soluble HIV-1 Env trimer, which is similar to the retention of the three-helix bundle at the top of the long HA2 helix in influenza HA. All structures are depicted according to secondary structure elements. The figure was generated with PyMOL (63).

The weak and diffuse electron density corresponding to the fusion peptide (FP) and the residues that connect it to the HR1 central helix makes model building of this region particularly difficult at this resolution (fig. S7A), but this is likely indicative of a lack of regular secondary structure (44). Nonetheless, residues adjacent to the N termini of the HR1 helices initially wrap around the gp120 α0 helix, forming a metastable loop-helix structure akin to similar elements in influenza HA2 (45) (Fig. 3, C and D) that lead to an extension of the central helix in the postfusion form (46). Overall, the central trimeric coiled-coil arrangement of the fusion protein (gp41) surrounded by three receptor subunits (gp120) is a characteristic of type-1 membrane-fusion proteins (4749). We now extend that hallmark to the capping of the central helices by another helix (α1 and α105-115) in both gp120 and HA. A small interprotomer central opening is created at the top of the central helix in both fusion proteins (Fig. 3C), as well as in Ebola and PIV5/RSV fusion proteins, that presumably facilitates conformational rearrangements of the head domains (e.g., gp120, HA1) during the fusion process. The Env trimer structure also agrees well with what is known about the transition from a pre- to postfusion conformation, in that the three HR1 central helices adopt a similar arrangement in the postfusion six-helix bundle (Fig. 3E) (50).

A second well-defined helical element that is ~40 Å long at an ~60° angle relative to the HR1 central helix is located at the bottom of the trimer, proximal to the membrane, where it wraps around the trimer base (Fig. 3A). We interpret this long helix (seven turns) as the C-terminal portion of the HR2 sequence, which is consistent with a cryo-EM reconstruction in the accompanying paper (25), where deletion of residues 651 to 664 from the BG505 SOSIP.664 trimer eliminated the density corresponding to the end of this helix (25, 51). Strong electron density between the bottom of HR1 and the middle of the HR2 helix is likely indicative of substantial intrasubunit gp41-stabilizing interactions. The residues that are N-terminal to the long HR2 helix appear to adopt a horseshoe conformation for the polypeptide backbone (Fig. 3A and fig. S4B); the NAG moieties at glycosylation sites 625 and 637 are clearly visible in the electron-density maps and define the location of turns. Strong electron density, predominantly in a flat-extended shape characteristic of β strands, is present between the C terminus of HR1 and the N terminus of HR2, where the gp41 disulfide loop [(DSL), residues 585 to 609] is thought to be located (fig. S7B). However, we cannot determine the arrangement of these gp41 components with confidence at this resolution, and a further complication is the close proximity of gp120 C1 and C5 elements that most likely participate in tertiary structure interactions with residues connecting HR1 to HR2, including the DSL (5, 52). As such, we attribute the approximate location of the gp41 DSL and, hence, the engineered intersubunit disulfide bond to the base of the Env trimer. Finally, no conclusion can be drawn about the arrangement of the gp41 MPER because it is not present in the BG505 SOSIP.664 gp140 trimer (19, 20). The gp41 HR2 may continue to extend in the same orientation as it enters the MPER, or it could change direction near residue 664 (53, 54) so that the MPER could align parallel to the trimer axis.

Structural Definition of the PGT122 Epitope

The PGT121 family of antibodies potently neutralizes ~70% of circulating HIV-1 viruses (21, 55), and PGT121 is highly effective in protecting against mucosal challenge in a macaque model of HIV-1 infection (56). The crystal structure presented here agrees well with our lower-resolution negative-stain EM reconstruction of the same PGT122-trimer complex (23) (fig. S8) but now allows a more complete description of the epitope. As initially predicted (21), the N332 Man8/9 glycan that is a central feature of the PGT122 epitope sits directly at the junction between the light-chain complementarity-determining region 2 (LCDR2) and the 26-residue heavy-chain CDR3 (HCDR3) (Fig. 4A and fig. S9). Residues in the LCDR1, LCDR3 and light-chain framework 3 (LFR3) regions of PGT122, which were predicted by alanine-scanning mutagenesis to be important for HIV-1 neutralization (23), interact directly with the gp120 V3 base near Ile323-Arg327, a key part of the epitope (21, 57). The trimer structure also helps explain why PGT121-class bnAbs depend on V1/V2 residues for locking gp120 into the pre-CD4 conformation (23); in fact, gp120 V1 residues 135 to 139, including the glycan at N137, come into close proximity to HCDR1, HCDR2, HCDR3, and LCDR3 of PGT122 (Fig. 4A and fig. S9). The N137 oligomannose glycan in the trimer structure superimposes almost perfectly with the biantennary complex glycan that occupies the groove formed by the PGT121 heavy-chain CDRs in previous crystal structures (23, 55, 58) (Fig. 4, A and B). Alanine scanning mutagenesis of gp120 glycans clearly indicates that bnAbs of the PGT121 family depend on N137 for neutralizing the BG505 virus (Fig. 4C and fig. S10). Locating this key residue in the crystal structure has allowed us not only to delineate the full PGT122 epitope, but also to confidently determine the relative position of V1/V2 elements at the trimer apex. In addition to N137 and N332, the N156 and N301 glycans are also protected from EndoH deglycosylation by PGT122. Although the composition and nature of the glycoforms on native Env are still uncertain, PGT122 recognition is compatible with the presence of a complex or hybrid carbohydrate at position N156/N173, as observed in the crystal structure of a V1/V2 scaffold with bnAb PG16 (6) (Fig. 4B). The N156 and N301 glycans do have a clear involvement, albeit a minor one, in neutralization by PGT121-family bnAbs (Fig. 4C and fig. S10). Thus, the Env trimer structure reveals the complexity of the PGT122 epitope, which involves the V1 and V3 base and four glycans (N137, N156/N173, N301, and N332).

Fig. 4 Complete structural definition of the PGT122 epitope.

(A) In addition to the N332 glycan (yellow), the PGT122 bnAb recognizes both protein and glycan elements near the base of gp120 V1 (orange) and V3 (red) to mediate broad and potent HIV-1 neutralization. Heavy- and light-chain CDRs are shown as dark and light blue tubes, respectively. Electron density for oligomannose glycans (spheres) surrounding the PGT122 epitope is shown as a 2Fo-Fc gray mesh contoured at 1.0σ. (B) Superimposition of the PGT122 epitope with glycans from PGT121 [PDB ID: 4JY4 and 4FQC (23, 55)] and PG16 [PDB ID: 4DQO (6)] liganded crystal structures. PGT122 binding is compatible with the involvement of complex or hybrid glycans at gp120 N137 and N156/173 (green) when the trimer is expressed in human cells capable of making this type of glycan. The figure was generated with PyMOL (63). (C) The use of glycan knock-out mutants of HIV-1 BG505 pseudoviruses reveals the importance of glycans at N137, N156, and N301 for PGT122 recognition and neutralization. These glycans are part of the PGT122 epitope in the trimer crystal structure to varying extents. A, Ala; T, Thr; WT, wild type. Error bars indicate SEM obtained from duplicate measurements.

Revisiting Glycan-Dependent bnAb Epitopes in the Context of the HIV-1 Env Trimer

The recent isolation (21, 59) and structural characterization (1, 4, 6, 7) of potent glycan-reactive bnAbs has emphasized the need to precisely define the composition of various glycans that are sits of vulnerability on Env. The trimer structure allows us to reevaluate previous crystal structures of glycan-dependent bnAbs that were obtained with monomeric core gp120 or epitope scaffolds. When viewed in the context of the trimer, the gp120 epitopes for the N160-dependent (PG9/PG16) and N332-dependent (PGT122, PGT128, and PGT135) epitopes are in close proximity (Fig. 5A). Once considered separate sites of vulnerability (Fig. 5B), these two epitope clusters are larger than were first thought and overlap to some extent, helping to explain why some of these antibodies compete with one another in certain binding assays (21) (Fig. 5C). All of these glycan-dependent bnAbs use their six CDR loops, in addition to framework regions, when recognizing their epitopes. As previously suggested (18), PG9 and PG16 would interact with two N160 glycans across different protomers in the trimer apex (Fig. 5D). In addition, whereas one side of the elongated HCDR3 hammerhead inserts into the trimer apex, the other is available to interact with elements of V3 (Fig. 5D) and may help explain why V3 residues influence neutralization by PG16 (59). The trimer crystal structure also suggests that HCDR1 and HFR3 of PG9/PG16 may interact with the V3-protecting N197 glycan on an adjacent protomer (Fig. 5D). PGT128 would also make contacts with Env elements additional to those previously reported in the crystal structure with a gp120 outer domain lacking V1/V2 (7) (Fig. 5E). Thus, LCDR1, LCDR2, and LFR3 of PGT128, for which no previous role could be assigned, are now predicted to be in close proximity to V1 elements, including N137 and N156 (60), whereas a change in the V1 loop orientation is required to allow PGT135 binding (Fig. 5F). Indeed, a V1 orientation closer to that observed in the V1/V2 scaffold or in the high-resolution cryo-EM trimer structure (25) would remove a clash with N137 and facilitate PGT135 interaction with V1. A flexible role for V1 might help to explain the plasticity of PGT135-Env interactions seen in previous EM studies (1).

Fig. 5 Revisiting glycan-dependent epitopes of bnAbs in the context of the soluble Env trimer structure.

(A) Superimposition of the cocrystal structures of PG16 (green), PGT128 (red), and PGT135 (light blue) on the PGT122 (dark blue)–Env trimer crystal structure reveals that these glycan-dependent epitopes overlap. (B) Cocrystal structures of glycan-dependent bnAbs in complex with monomeric gp120 or scaffolded gp120 elements provide crucial, but incomplete details of their epitopes and, hence, how they recognize Env. The various bnAb epitopes are colored individually on the surfaces, with overlapping elements in purple. (C) Superimposition of the PG9/16, PGT128, and PGT135 cocrystal structures on the soluble trimer-PGT122 cocrystal structure reveals that these glycan-dependent bnAbs have expanded epitopes, which creates overlapping sites of vulnerability on HIV-1 Env. (D) Model of the expanded PG16 epitope on Env. Antibody interactions additional to those previously described in the PG9/PG16-V1/V2–scaffold cocrystal structures (4, 6) are predicted to occur with gp120 V3, as well as with residues N160 and N197 of the adjacent protomer. Protomers are denoted with P1, P2, and P3 subscripts, and the positions of the hypervariable loops 1 and 2 (attached to the V1/V2 framework) are denoted by V1/V2. The view is slightly offset from looking down the trimer axis. (E). Model of the expanded PGT128 epitope includes additional interactions with glycans at N137 and N156 mediated via the PGT128 light chain (light blue). (F) PGT135 would clash (yellow star) with the gp120 V1 conformation recognized by PGT122, based on superimposition of the PGT135-core gp120 cocrystal structure (1). A slight reorientation of gp120 V1 (green) would allow PGT135 to interact, consistent with the hypothesis that the V1 loop is flexible enough to permit different modes of bnAb interaction. Heavy and light chains are shown as dark and light blue tubes, respectively. The figure was generated with PyMOL (63).

Conclusion

The crystal structure of the soluble BG505 SOSIP.664 trimer is in excellent agreement (fig. S4) with the accompanying cryo-EM structure of the same Env construct as a complex with PGV04 Fab (25). Structures determined by these two independent techniques show complete concordance in the major new features that are visualized at the present level of 5 to 6 Å resolution (fig. S4). We have defined the overall architecture of the soluble trimer, as well as the secondary, tertiary, and quaternary interactions between gp120 and gp41 that are involved in its assembly. The trimer is relatively tightly packed, especially in gp41, but with a small opening between the Env apex and the top of the central gp41 helices. The gp120 subunits are held together, at least in part, by association of the V1/V2/V3 regions at the apex of the trimer (11, 61, 62). The gp41 central helices provide the main stabilizing contact between gp41 and gp120. Finally, the complete definition of how neutralization epitopes are presented in the context of the trimer, here and in the accompanying manuscript (25), should help the design of next-generation immunogens as candidate vaccines.

Supplementary Materials

www.sciencemag.org/content/342/6165/1477/suppl/DC1

Materials and Methods

Figs. S1 to S10

Tables S1 and S2

References (6477)

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
  2. The BG505 SOSIP.664 trimer possesses most, if not all, of the characteristics associated with the prefusion closed Env trimer conformation: the presence of a V1V2/V3 cap assembled at the membrane-distal apex; antigenic reactivity with antibodies to quaternary epitopes; the likely inability to gp41 to bind to free HR2 peptides; a coherent binding site for residues associated with binding of the small-molecule inhibitor of HIV-1 entry, BMS806; and many residues of the inner domain previously analyzed by mutational substitution for which we can now explain their mutational phenotypes.
  3. A hole was also reported in cleaved trimer structures determined by EM (11) but seems to result from the low resolution of the reconstructions [see discussion in (25)].
  4. Stronger electron density reappears for a loop before the N terminus of the gp41 HR1 central helix, near the gp120/gp41 interface. We assign this electron density to the gp41 FP proximal region elements. gp41 and gp120 elements in this region (gp120 residues 73 to 84, glycans at gp120 N88 and gp41 N625, and the C-terminal HR2 helix from an adjacent protomer) are particularly hydrophobic and could easily interact with and occlude the hydrophobic gp41 FP. In addition, this ascribed FP region is in close relative proximity to the C terminus of gp120 C5, to which it was connected in the uncleaved gp160 precursor before furin cleavage.
  5. A slight difference in HR2 helix orientation is observed between the trimer crystal structure and the cryo-EM structure reported in the accompanying manuscript (25). Crystal packing interactions at the gp41 C terminus probably account for this small shift (fig. S2).
  6. The complex biantennary glycan observed in the PGT121 paratope in crystal structures (23, 55) is fortuitous, as the glycan comes from a symmetry-related PGT121 Fab molecule in the crystal. Fab PGT121 is glycosylated and was expressed in mammalian cells. PGT121 also reacts on the glycan array with complex biantennary glycans possessing α1-6 sialylated ends (23), which make substantial contacts with the paratope in the crystal structures. Although the N137 glycoform remains uncharacterized on virus-derived HIV-1 Env, PGT121 reactivity with the glycan array, and the liganded crystal structures, suggest that N137 is probably a biantennary complex glycan. The relatively weaker electron density for the oligomannose N137 glycan in the trimer structure also suggests that, when it is not an α1-6 sialylated complex carbohydrate, the N137 glycan lacks major putative sites of interaction with PGT122. Hence, it might not be fully protected from glycosidase treatment, or it may be slightly disordered.
  7. The PGT122 and PGT128 bnAbs appear to recognize a closely related N332-dependent epitope, but they approach gp120 with an inverted disposition of their light and heavy chains. Whereas the PGT128 heavy chain predominantly interacts with the gp120 V3 base, PGT122 recognizes this region mainly through its light chain. Conversely, N137 is recognized by the PGT122 heavy chain but is putatively contacted by the PGT128 light chain.
  8. Acknowledgments: We thank Y. Hua, L. Kong, P. S. Lee, X. Zhu, R. Pejchal, H. Tien, T. Clayton, K. Saye, and J. Korzun, for technical assistance, previous contributions to the Env trimer project, and insightful discussions and C. R. King and W. Koff for support and encouragement. This work was supported by NIH grant P01 AI82362 (J.P.M., I.A.W.), as well as the International AIDS Vaccine Initiative Neutralizing Antibody Consortium and Center (D.R.B., I.A.W., J.P.M., A.B.W.), CHAVI-ID UM1 AI100663 (D.R.B., I.A.W., AB.W.), NIH grant R01 AI084817 (I.A.W.), NIH grant R37 AI36082 (J.P.M.), NIH grant R01 AI33292 (D.R.B.), a Vidi grant from the Netherlands Organization for Scientific Research (R.W.S.), a Starting Investigator Grant from the European Research Council (R.W.S.), Canadian Institutes of Health Research fellowship (J.-P.J.), and the Ragon Institute. D.L. is supported by the U.S. NIH National Institute of General Medical Sciences (NIGMS) Biomedical Technology Research Center program (grant GM103310). The Joint Center of Structural Genomics is supported by NIH NIGMS via a Protein Structure Initiative grant U54 GM094586 (I.A.W.). Use of the Advanced Photon Source for data collection was supported by the U.S. Department of Energy (DOE), Basic Energy Sciences, Office of Science, under contract no. DE-AC02-06CH11357. GM/CA CAT has been funded in whole or in part with federal funds from the National Cancer Institute (NCI) (grant Y1-CO-1020) and NIGMS (grant Y1-GM-1104). Extensive crystal screening was also carried out at the Stanford Synchrotron Radiation Lightsource (SSRL) and at the Canadian Light Source (CLS). SSRL, a Directorate of the SLAC National Accelerator Laboratory and an Office of Science User Facility, is operated for the U.S. DOE Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research; NIH’s National Center for Research Resources, Biomedical Technology Program (P41RR001209); and the NIGMS. CLS is supported by the Natural Sciences and Engineering Research Council of Canada, the National Research Council Canada, the Canadian Institutes of Health Research, the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan. The content is the responsibility of the authors and does not necessarily reflect the official views of the NIGMS, NCI, or NIH. Coordinates and structure factors have been deposited with the Protein Data Bank under accession code 4NCO. The International AIDS Vaccine Initiative has previously filed a patent relating to the BG505 SOSIP.664 trimer: U.S. Provisional Application no. 61/772,739, titled “HIV-1 envelope glycoprotein,” with inventors M. Caulfield, A.C., H. Dean, S. Hoffenberg, C. R. King, P.J.K., A. Marozsan, J.P.M., R.S., A.B.W., I.A.W., J.-P.J., but no patents have been filed on any work described here. Materials and information will be provided under a Materials Transfer Agreement. This is manuscript 25054 from The Scripps Research Institute.
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