Volume 159, Issue 1, Pages (September 2014)

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Volume 159, Issue 1, Pages 69-79 (September 2014) Structural Evolution of Glycan Recognition by a Family of Potent HIV Antibodies  Fernando Garces, Devin Sok, Leopold Kong, Ryan McBride, Helen J. Kim, Karen F. Saye-Francisco, Jean-Philippe Julien, Yuanzi Hua, Albert Cupo, John P. Moore, James C. Paulson, Andrew B. Ward, Dennis R. Burton, Ian A. Wilson  Cell  Volume 159, Issue 1, Pages 69-79 (September 2014) DOI: 10.1016/j.cell.2014.09.009 Copyright © 2014 Elsevier Inc. Terms and Conditions

Cell 2014 159, 69-79DOI: (10.1016/j.cell.2014.09.009) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Crystal Structure of PGT124 in Complex with HIV-1 gp120 and CD4 (A) The ternary complex of PGT124 (purple and brown for heavy and light chains, respectively) and two-domain CD4 (gray) bound to gp120 (light blue) are shown as a ribbon representation. PGT124 contacts the N332 glycan and the base of V3. The glycan is represented in ball and stick with green carbons, red oxygens, and blue nitrogens and covered with its molecular surface. (B) CDR H3, L1, L2, and L3 loops and the light-chain FR3 make extensive contacts with both the Man8 N332 glycan and V3 (in red) of gp120. (C) The surface areas (Å2) buried on the gp120 protein and N332 glycan by individual CDR loops and framework regions on PGT124 are presented on the lower right. (D) Fitting of the PGT122-BG505 SOSIP.664 gp140 crystal structure into the PGT124:BG505 SOSIP EM reconstruction at 17.8 Å resolution. The PGT122 fitting is excellent with a correlation of 0.9, indicating that PGT122 and PGT124 share a similar angle of approach for interaction with Env (see also Figures S1 and S2 and Tables S1 and S6). Cell 2014 159, 69-79DOI: (10.1016/j.cell.2014.09.009) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Interaction between PGT124 and the Base of the V3 Loop (A) The interaction between PGT124 CDR loops (purple ribbons) and the base of the V3 loop (red ribbon). Red dots represent residues at the tip of the mini-V3 loop that are not visible in the electron density. The interaction with contacting residues is displayed in ball-and-stick representation below. Contacts between side chains of the 324GDIR327 motif on the V3 loop and the CDR loops of PGT124 are indicated with solid lines for van der Waals’ interactions and dotted lines for hydrogen bonds. (B) For comparison, the interaction between PGT128 CDR loops (pale green) and the base of the V3 (gray) (PDB 3TYG) for comparison with (A). The PGT128 antibody-antigen interactions with the V3 base are all H bonds of the Fab main chain with V3 main chain. (C) The V3 loop structures bound to PGT124 or PGT128 are superimposed for comparison. (D) The effect on the neutralization activity of PGT124 and PGT128 by single and double mutations of the V3 loop GDIR motif. Values are shown as IC50 in μg/ml (see also Figures S3, S4, and S5). Cell 2014 159, 69-79DOI: (10.1016/j.cell.2014.09.009) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Interaction between PGT124 and the N332 Glycan (A) Interaction of the CDR loops of PGT124 in purple with the oligomannose glycan at the N332 position in red ball-and-stick representation with the D1, D2, and D3 arms labeled. (B) For comparison, the interaction of the PGT128 CDR loops (light green) is shown with the N332 glycan (gray ball-and-stick representation). (C) The N332 glycan structures bound to PGT128 or PGT124 are shown after superposition of the gp120 protein components. Conformational differences are highlighted by comparative distances between equivalent mannose moieties. (D) Schematic diagram of the Man8GlcNAc2 glycan with the glycan moiety nomenclature. (E) The effect on PGT124 neutralization activity of differences in gp120 glycosylation arising from different cell expression systems and removal of the N332 glycan. Values are shown as IC50 in μg/ml (see also Figures S3, S5, and S6 and Tables S2 and S3). Cell 2014 159, 69-79DOI: (10.1016/j.cell.2014.09.009) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Epitope Core of the PGT121 Germline Lineage (A) Cartoon representation of the interaction between mV3-gp120 core and the PGT124 variable region (left) and close-up views of the PGT124 epitope core with the 324GDIR327 motif at the V3 base in blue spheres and the N332 glycan as pale green ball and stick (middle and right). The PGT124 residues that contact the N332 glycan and the V3 protein motif are represented in red and orange as a ball and stick, respectively, and covered with its molecular surface. (B) Sequence alignment of heavy and light chains of PGT121–124 Abs and germline precursors (asterisk indicates that the germline CDR L3 and CDR H3 sequences are not used for alignment because of ambiguity associated with D gene assignment). Red and orange boxes indicate that residues that contact the N332 glycan and the GDIR motif, respectively, on the epitope core of PGT124 are also largely conserved in PGT121–123 and, importantly, in the germline sequence (see also Figure S1). Cell 2014 159, 69-79DOI: (10.1016/j.cell.2014.09.009) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 PGT122 Contacts Glycans at N137 and N301, but PGT124 Accommodates These Glycans without Forming Contacts (A) The PGT122 epitope is composed of V1 and V3 regions from gp120, as well as four glycans at N137, N156, N301, and N332. The CDR loops and FR3 are shown in cartoon representation (in gray) (PDB ID: 4NCO). (B) Superposition of PGT124 (in purple) on the PGT122 epitope. (C) Although PGT124 seems capable of accommodating a glycan in the open face formed by H2 and H3, those loops appear to be shifted toward the center of the epitope when compared to the PGT122 paratope, thereby avoiding contacts with the N137 glycan. (D) However, PGT124 is predicted to clash with the 301 glycan and presumably pushes this glycan away to access the protein surface below. Cell 2014 159, 69-79DOI: (10.1016/j.cell.2014.09.009) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Evolution of Recognition of Complex Glycans by the PGT121 Family (A) Key residues on PGT121 (Kabat numbering) that contact the complex glycan attached at N137 of gp120 V1 are represented in red as a stick and ball and are covered with its molecular surface on the ribbon representation of the antibody variable regions (left). Residues at equivalent positions on PGT124 are highlighted in orange (right). (B) Sequence alignment for heavy-chain variable region for the germline precursor (GL) (asterisk indicates that the germline CDR H3 sequence is not used for the alignment because of ambiguity associated with D gene assignment), intermediate precursors (3H, 32H, and 9H), and mature antibodies of the PGT121 family (PGT121–124). Red boxes indicate residues involved in N137 glycan binding by PGT121, whereas orange boxes indicate the residues at equivalent positions on PGT124. (C) Glycan arrays illustrate that PGT124, in contrast to PGT121, PGT128, or 2G12, does not bind either complex or high-mannose glycans with any demonstrable affinity (see also Figures S1, S3, and S7). Error bars, SD; n = 6; RFU, relative fluorescence units. Cell 2014 159, 69-79DOI: (10.1016/j.cell.2014.09.009) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 Summary of the Contributions of the N137, N156, N301, and N332 Glycans to Recognition of the BG505 Isolate by Antibodies of the PGT121 Lineage Early precursors are highly dependent on the N301 and N332 glycans for neutralization and are negatively impacted by the presence of the N137 glycan (asterisk indicates that N137 glycan can play a double role in glycan recognition—first it can inhibit antibody binding by shielding the epitope, here represented in blue, and second, although it can still inhibit antibody binding to some extent, the N137 glycan can also be used by PGT121–123 to neutralize BG505 in the absence of N332, here represented in blue/red). Affinity maturation appears to create two distinct lineages that evolve separate dependencies on glycans that surround N332. N332 is the only critical glycan in the PGT124 epitope, whereas the N137 glycan contributes negatively to recognition by this antibody. Fold changes in neutralization IC50 for single and double glycan mutants (Table S4) were used to determine whether a glycan positively contributes to the epitope (decrease in neutralization IC50 upon removal) or contributes to shielding of the epitope (increase in neutralization IC50 upon removal). A relative scale for both positive and negative glycan contributions to the epitope was determined based on the extent of the change in neutralization IC50. Interestingly, for PGT121–123, the N137 glycan contributes to shielding in the presence of N332 but is a critical part of the epitope in the absence of N332 (see also Table S4 and S5). Cell 2014 159, 69-79DOI: (10.1016/j.cell.2014.09.009) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure S1 Superposition of the Variable Regions of PGT121 Family Antibodies (A) Analysis of PGT124 binding to gp120. The variable regions of gp120-bound (yellow) and unbound (purple) structures have an all-atom r.m.s.d of 0.6 Å, which indicates that PGT124 does not undergo major conformational changes when it binds to its epitope. (B) Superposition of PGT124 (in purple), PGT122 (in blue), 10-1074 (in pale green) and germline (in gray) Fab structures indicate a similar overall architecture featuring an open “U”-shaped depression in the binding site (top surface). (C) Sequence alignment of the variable regions of PGT124 and 10-1074 antibodies with the non-conserved residues shown below. The three heavy chain (HC) and three light chain (LC) CDRs are indicated based on Kabat alignment. Red boxes indicate the residues that contact the N332 glycan and GDIR motif from the crystal structure of PGT124 in complex with gp120. Orange boxes indicate the positions of residues involved in N137 glycan binding by the PGT121–123 antibody branch but that are not conserved at equivalent positions on the PGT124/10-1074 branch (related to Figures 1, 4, and 6). Cell 2014 159, 69-79DOI: (10.1016/j.cell.2014.09.009) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure S2 PGT124 Binding to the Env Trimer (A) EM density map for the PGT124-BG505 SOSIP.664 gp140 trimer complex. The EM reconstruction clearly shows density for the SOSIP gp140 trimer and for three PGT124 Fabs binding to each gp120 in the three protomers of the trimer. (B) Reference-free 2D class averages of the PGT124-BG505 SOSIP.664 gp140 trimer samples stained with 2% uranyl formate. (C) 2D projections of the 3D models and the corresponding reference-free 2D class averages of PGT124-BG505 SOSIP.664 gp140 trimer complex. (D) The 17.8 Å resolution of the final EM map is calculated from the Fourier shell correlation (FCS) (related to Figure 1). Cell 2014 159, 69-79DOI: (10.1016/j.cell.2014.09.009) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure S3 Binding of PGT124 to gp120 by ITC (A–D) Binding affinity of PGT124 Fab to (A) JRCSF WT (wild-type) gp120 (full-length), (B) JRCSF gp120 with a GDIR to GAIA double mutation, (C) JRCSF gp120 with a glycan knockout mutation N332D and (D) JRCSF gp120 lacking the V1/V2 loop. (E) Competition experiments between PGT124 and sCD4 for binding to full-length JRCSF. ITC plots of PGT124 and CD4 binding to gp120 individually are shown on the left. ITC plots of CD4 binding to the PGT124-gp120 complex and PGT124 binding to the CD4-gp120 complex are shown on the right. The sequential binding experiments clearly show that binding affinity is not affected when PGT124 and CD4 are titrated into a solution previously saturated with either PGT124 or CD4. The dissociation constant (Kd) and stoichiometry of binding (N) as determined by ITC are shown (related to Figures 2, 3, and 6). Cell 2014 159, 69-79DOI: (10.1016/j.cell.2014.09.009) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure S4 Superposition of V3 Loops when Either PGT122 or PGT124 Are Bound to gp120 Superposition of gp120-core miniV3 (gray) bound to PGT124 onto one protomer of the SOSIP.664 gp140 (gray) bound to PGT122 (PDB ID:4NCO). The black box shows that the GDIR motif located at the base of the V3 loop has a similar but not identical conformation in gp120 monomer and gp140 BG505 SOSIP trimer. The antibodies are not shown (related to Figure 2). Cell 2014 159, 69-79DOI: (10.1016/j.cell.2014.09.009) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure S5 Paratope Mapping of PGT124 (A) Single alanine-scanning mutagenesis identifies key residues on the Ab paratope for binding to the N332 glycan and GDIR motif. The effect of mutations on neutralization activity is color-coded from black (no effect) to red (most effect). (B) Mapping of the residues on the paratope surface using the same color code (related to Figures 2 and 3). Cell 2014 159, 69-79DOI: (10.1016/j.cell.2014.09.009) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure S6 N332 Glycan Conformation in Different Structures (A) Superposition of the N332 glycan in gp120 from various complexes of gp120 and the gp120 outer domain with PGT128 (PDB ID:3TYG) (gray), PGT135 (PDB ID:4JM2) (wheat), and PGT124 (red). The gp120 is represented as a light blue cartoon while the V3 loop is in dark blue. Although the N332 Man8/9 structures have some differences particularly at their distal mannose ends, they all seem to share the same general overall orientation. (B) The angles between the major axis of inertia of each N332-glycan structure after alignment at the gp120 protein are indicated. The angles were determined by calculating the principal vectors using all atoms of the N332 glycans. (C) Superposition of the same N332 structures only on the carbohydrate to calculate the all-atom RMSD (related to Figure 3). Cell 2014 159, 69-79DOI: (10.1016/j.cell.2014.09.009) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure S7 PGT124 Utilizes High-Mannose Glycoforms for Neutralization The isolate WEAU was tested for neutralization as removal of the N332 glycan site does not result in complete loss of neutralization potency. WT and N332A variants of WEAU were produced in 293T cells (solid lines) and in 293T cells with kifunensine (dashed lines) and tested for neutralization by PGT124. The results show that the neutralization potency for PGT124 seems to increase when only high mannose glycans are present on the virus surface. As a control, the ability of PG9 to neutralize is greatly diminished in the presence of only high mannose glycans on the Env trimer as PG9 relies on a complex glycan for productive binding. Altogether, these data strongly suggest that PGT124 does not bind complex glycans even in the absence of a glycan at N332 (related to Figure 6). Error bars, SD. Experiments were performed in duplicate and repeated three times. Cell 2014 159, 69-79DOI: (10.1016/j.cell.2014.09.009) Copyright © 2014 Elsevier Inc. Terms and Conditions