Structure of 2G12 Fab2 in complex with soluble and fully glycosylated HIV-1 Env by negative-stain single-particle electron microscopy

Abstract

The neutralizing anti-HIV-1 antibody 2G12 is of particular interest due to the sterilizing protection it provides from viral challenge in animal models. 2G12 is a unique, domain-exchanged antibody that binds exclusively to conserved N-linked glycans that form the high-mannose patch on the gp120 outer domain centered on a glycan at position N332. Several glycans in and around the 2G12 epitope have been shown to interact with other potent, broadly neutralizing antibodies; therefore, this region constitutes a supersite of vulnerability on gp120. While crystal structures of 2G12 and 2G12 bound to high-mannose glycans have been solved, no structural information that describes the interaction of 2G12 with gp120 or the Env trimer is available. Here, we present a negative-stain single-particle electron microscopy reconstruction of 2G12 Fab2 in complex with a soluble, trimeric Env at ∼17-Å resolution that reveals the antibody's interaction with its native and fully glycosylated epitope. We also mapped relevant glycans in this epitope by fitting high-resolution crystal structures and by performing neutralization assays of glycan knockouts. In addition, a reconstruction at ∼26 Å of the ternary complex formed by 2G12 Fab2, soluble CD4, and Env indicates that 2G12 may block membrane fusion by induced steric hindrance upon primary receptor binding, thereby abrogating Env's interaction with coreceptor(s). These structures provide a basis for understanding 2G12 binding and neutralization, and our low-resolution model and glycan assignments provide a basis for higher-resolution studies to determine the molecular nature of the 2G12 epitope.

Importance: HIV-1 is a human virus that results in the deaths of millions of people around the world each year. While there are several effective therapeutics available to prolong life, a vaccine is the best long-term solution for curbing this global epidemic. Here, we present structural data that reveal the viral binding site of one of the first HIV-1-neutralizing antibodies isolated, 2G12, and provide a rationale for its effectiveness. These structures provide a basis for higher-resolution studies to determine the molecular nature of the 2G12 epitope, which will aid in vaccine design and antibody-based therapies.

FIG 1

Negative-stain single-particle EM reconstruction of BG505 SOSIP.664 Env bound to 2G12 Fab₂. (A) Top (left) and side (right) views of the reconstruction are rendered with the segmented densities corresponding to 2G12 Fab₂ in blue and BG505 SOSIP.664 Env in white. (B) Crystal structures of 2G12 Fab₂ (PDB ID 1OP5) (23) and Env (PDB ID 4NCO) (20) were fit into the EM density map. All glycans in the Env crystal structure were removed for clarity. Top (left) and side (right) views of the fitted maps are shown with gp120 rendered in yellow, V1/V2 loops in orange, V3 loops in red, gp41 in green, and 2G12 Fab₂ in dark blue, with bound glycans in purple.

FIG 2

Interaction of 2G12 with Env BG505 SOSIP.664. (A) Distance measurements between pairs of atoms were made between the closest protein residues in the docked 2G12 Fab₂ (PDB ID 1OP5) and the crystal structure of Env BG505 SOSIP.664 trimer (PDB ID 4NCO), which were then averaged for each Fab. The closest protein interaction with 2G12 occurs within the V4 loop, which is not completely modeled in the docked crystal structure because of disorder (dashed red line). All glycans were removed for clarity. (B) Although the Env structures in complex with antibodies against different sites of vulnerability as determined by X-ray crystallography and cryo-EM are overall very similar, their V1 loop positions are different. The V1/V2 motif from the cryo-EM structure (red) (PDB ID 3J5M) was overlaid on the X-ray crystallography structure (cyan and gray) (PDB ID 4NCO). Residue N137 is highlighted in each structure, and the dashed arrows indicate the direction that the residue is pointing relative to 2G12. No glycan is modeled in the cryo-EM structure, but there is one in the X-ray crystallography structure (which is bound by PGT122 Fab). The V1 loop in the X-ray crystal structure points toward 2G12 and into the density of the negative-stain EM structure. The V1 loop in the cryo-EM structure, however, is pointed away from 2G12. These differences demonstrate that antibody interaction with Env variable loops, especially with glycans, may alter the position of these loops and influence binding/neutralization.

FIG 3

Details of the glycan epitope of 2G12. (A) A closeup view of the interface between BG505 SOSIP.664 Env (PDB ID 4NCO) and 2G12 Fab₂ (PDB ID 1OP5) (contact interface highlighted with dotted black line) showing the 2G12-relevant glycans. Glycans from the Env crystal structure that fall within the 2G12 epitope are rendered in space-filling mode and highlighted by different colors as indicated in the key. gp120 is rendered in gray ribbon. Our 2G12-Env EM reconstruction was fit into a model of unliganded membrane-anchored HIV-1 trimer (gray surface) (EMDB accession numbers EMD-5019 and EMD-5021) (60) in order to fit the Fab₂ crystal structures. The BG505 SOSIP.664 crystal structure was subsequently fit into our EM density map in order to determine which glycans fell within the 2G12 epitope (20). The crystal structures used in the docking were solved after partial deglycosylation, such that some of the N-linked glycans only harbor an N-acetylglucosamine (NAG), while others contain high-mannose glycans (20). (B) On the left, we show a superimposition of glycan-dependent monoclonal antibody (MAb) cocrystal structures of PGT135 (PDB ID 4JM2) (13) and PGT122 (PDB ID 4NCO) fit onto the Env trimer crystal structure along with our own EM fitting of 2G12 to expand on the mapping of this glycan supersite of vulnerability on Env, showing the overlap of these three antibodies. The extents of these epitopes are detailed on the right, further emphasizing the large size of the 2G12 epitope on the trimeric surface of Env and where it overlaps with other glycan-dependent MAbs.

FIG 4

Assignment of glycan positions. Crystal structures of 2G12 bound to four Man₉GlcNAc sugars and Env were fit into the 2G12-Env EM density. Using UCSF Chimera, distance measurements were made from the OH of C-1 on the terminal GlcNAc of 2G12-bound sugars and the OH of C-4 from the terminal GlcNAcs of N295, N332, N339, and N392 that were modeled in the crystal structure (PDB ID 4NCO). There was no sugar modeled at position N339 in the crystal structure; therefore, the structure of PGT135 (PDB ID 4JM2), which has a terminal GlcNAc modeled at this position, was overlaid onto Env. Glycan assignments bound to 2G12 were assigned on the surface of Env according to the shortest distance, with the exception of N332. Three independent crystal structures with antibodies bound to N332 were overlaid onto Env, and in each case, N332 aligned nearly exactly with glycan 2 and not glycan 4. The structure of PGT135 also contains glycan N392, which overlays with glycan 1, in agreement with our distance measurements. Distances in angstroms are tabulated below the structure.

FIG 5

Model of the 2G12 epitope in the context of trimeric Env. Our model was constructed using the hybrid methods of docking separate crystal structures of Env and glycan-bound 2G12 Fab₂ into our low-resolution EM reconstruction, distance measurements (Fig. 3), and previous biochemical data (24, 25). We place glycans N392 and N295 within the primary combining sites of 2G12, while N332 and N339 interact with the secondary combining sites, which are formed by the V_H-V_H interactions arising from the V_H/V_H′ domain swap. Glycans linked to positions N392 and N332 both bind to the upper Fab in the Fab₂ structure. The upper Fab is also closer to the variable loops and the gp120 outer domain than the lower Fab. The glycan at N339 likely stabilizes these interactions, although it is not required for 2G12 to recognize Env (as shown by the N339A glycan knockout below), while the glycan at N295 may be important for glycan processing.

FIG 6

Glycan knockout analysis of BG505 SOSIP.664. (A) Individual glycan sites on BG505 pseudovirus were removed by alanine mutagenesis and tested for neutralization in a TZM-bl assay (46). Each glycan site was then removed and tested for binding to WT BG505 virus. Here, we define wild-type (WT) virus as that with an Asn residue at position 332. Tabulated neutralization IC₅₀ are presented. Glycan knockouts with an IC₅₀ less than 1 were not considered important for 2G12 binding, those with an IC₅₀ greater than 1 and less than 50 were considered important but not critical to binding, and those with an IC₅₀ greater than 50 were considered critical for binding. (B) Glycans at positions N332, N295, and N392 are critical for 2G12-based neutralization of HIV, with N339 also playing an important role.

FIG 7

Negative-stain single-particle EM reconstruction of BG505 SOSIP.664 Env bound to 2G12 Fab₂ and soluble 2-domain CD4 (sCD4). (A) Coomassie-stained, nonreducing SDS-PAGE gel analysis of the complex components and the complex purified by size exclusion chromatography. BG505 SOSIP.664, 2G12 Fab, and sCD4 were stained similarly with equal amounts of protein. Note that some 2G12 F(ab′)2 remained with 2G12 Fab₂ during purification and subsequently bound to SOSIP; because 2G12 is domain exchanged, these two products are virtually identical except on a nonreducing SDS-PAGE gel. (B) SEC-MALS was used to determine that the observed shift in elution volume and molar mass for the SOSIP-2G12 Fab-sCD4 (MM_Complex, red) complex in comparison to those of the unliganded trimer (MM_SOSIP, blue) correspond to one trimer binding three Fab₂ and three sCD4 molecules simultaneously. (C) Structural characterization of BG505 SOSIP.664 Env bound to three 2G12 Fab₂ and three sCD4 by negative-stain single-particle EM. Top (left) and side (right) views of the reconstruction are rendered with the segmented densities corresponding to 2G12 Fab₂ in blue and BG505 SOSIP.664 Env in white. (D) Crystal structures of 2G12 Fab₂ (PDB ID 1OP5) and gp120 bound to sCD4 (PDB ID 2B4C) (53) were fit into the EM density map. All glycans were removed for clarity. Top (left) and side (right) views of the fitted maps are shown with gp120 rendered in yellow, V3 loops in red, sCD4 in gray, and 2G12 Fab₂ in blue.

FIG 8

Proposed model of 2G12 steric blockade of gp120-coreceptor interaction. (A) Unliganded HIV-1 Env contacts CD4 from a target cell (left), which causes conformational changes in Env, exposes the V3 loops (red), and makes Env competent to bind its coreceptor and initiate viral fusion (right). (B) Env is bound by 2G12 at an initially shallow angle relative to the plane of the membrane and is still capable of binding CD4 (left); however, conformational changes and gp120 rotation brought about by CD4 binding cause the angle of 2G12 in relation to the viral membrane to increase significantly. This increase in angle may lead to steric clashes with the target cell membrane, such that the V3 loop is prevented from contacting the coreceptor. Env is in white, the V3 loop in red, CD4 in green, the membrane proximal external region in gray, and 2G12 in blue. Unliganded Env was solved previously by negative-stain EM (12). The CD4-bound Env reconstruction is deposited in EMDB under accession number EMD-5708.