Crystallographic Identification of Lipid as an Integral Component of the Epitope of HIV Broadly Neutralizing Antibody 4E10  Adriana Irimia, Anita Sarkar, Robyn L. Stanfield, Ian A. Wilson  Immunity  Volume 44, Issue 1, Pages 21-31 (January 2016) DOI: 10.1016/j.immuni.2015.12.001 Copyright © 2016 Elsevier Inc. Terms and Conditions

Immunity 2016 44, 21-31DOI: (10.1016/j.immuni.2015.12.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Lipid Binding Site in 4E10 CDRH1 Region as Revealed by Binding of sn-Glycerol-3-PO4 and rac-Glycerol-1-PO4 (A) Crystal structure of 4E10 Fab (blue heavy chain and green light chain) bound to gp41 MPER peptide (brown) and sn-glycerol-3-PO4 (sticks). See also Figure S2. The CDRs (Chothia CDR boundaries) are labeled and colored as follows: H1 (wheat, residues 26–32), H2 (pink, residues 52–56), H3 (yellow, residues 95–102), L1 (cyan, residues 24–34), L2 (green, residues 50–56), and L3 (light green, residues 89–97). The same coloring and numbering scheme was used in all figures. The approximate position of the viral membrane was modeled based on the location of the peptide C-terminal residues 684–686 (replaced by lysines in the peptide used for crystallization), which would correspond to N-terminal residues of the gp41 transmembrane domain. (B) Hydrogen bond interactions (dashed lines) of sn-glycerol-3-PO4 with CDRH1. See also Table S2. Immunity 2016 44, 21-31DOI: (10.1016/j.immuni.2015.12.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Lipid Binding Site in 4E10 CDRH1 Region in Co-crystal Structures with 06:0 PA and 06:0 PG (A) Crystal structure (2.73 Å resolution) of 4E10 Fab with two fragments of 06:0 PA (one with only one ordered acyl tail and the other with only the head moiety) bound in the 4E10 CDRH1 region (see also Figure S4). The approximate position of the viral membrane is indicated considering the location of the 06:0 PA molecules. (B) Superposition of all 4E10 Fabs in the 2.73 Å and 2.9 Å 4E10-06:0 PA asymmetric units containing the modeled fragments of 06:0 PA molecules in CDRH1. CDRH3 tryptophans are shown as yellow sticks. See also Figures S3 and S4. (C) Hydrogen-bond interactions (dashed lines) of the polar heads of the bound 06:0 PA molecules with CDRH1. (D) The 06:0 PG fragment bound in CDRH1 site 1. See also Figure S5 and Table S3. Immunity 2016 44, 21-31DOI: (10.1016/j.immuni.2015.12.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 CDRH3 Is Involved in Lipid Binding (A) The orientation of CDRH3 (yellow, with side chains of Trp100(H) and Trp100B(H) as sticks) in the lipid-bound complex compared to the peptide-glycerol-3-PO4 complex (black) as observed from superimposition of the 4E10 Fab-06:0 PA complex on the 4E10 Fab-peptide-glycerol-3-PO4 (gray Fab, brown peptide). (B) Vesicular organization of the 06:0 PAs (green sticks) and PO4s (red-orange sticks) bound to the six 4E10 Fabs in the asymmetric unit and to their six crystallographic symmetry mates in the 4E10-06:0 PA complex. Only the CDRH3s and the bound PO4 and 06:0 PA fragments are shown here in a cross-section of the vesicle. Dashed lines approximate the positions of the missing regions of the CDRH3 loops. (C) Superposition of 4E10 Fab-06:0 PA complex (yellow CDRH3) with 4E10 Fab-MPER(671-683KKK) (brown peptide, black CDRH3; PDB 2FX7; Cardoso et al., 2007). Side chains are shown for residues in close contact to peptide. (D) Structures of the variable regions of 4E10 Fab obtained by co-crystallization with 06:0 PA, 06:0 PG, 06:0 PC, and 06:0 PE, and unliganded 4E10 scFv (PDB 4LLV; Finton et al., 2013) were superposed with the variable region of 4E10 in its peptide-liganded form (PDB 2FX7; Cardoso et al., 2007), with peptide omitted for clarity. CDRH3 (red) in unliganded 4E10 scFv occupies the peptide binding site. CDRH3 of the 4E10-peptide bound structure (PDB 2FX7) is highlighted in black and CDRH3s of 4E10-lipid co-crystal structures are in orange where the lipid was not observed (4E10-06:0 PE and 4E10-06:0 PC) and in yellow for structures where the respective lipid was bound (4E10-06:0 PA and 4E10-06:0 PG). See also Table S3. Immunity 2016 44, 21-31DOI: (10.1016/j.immuni.2015.12.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Structural Models for Concurrent Binding of 4E10 to gp41 and Lipid and Comparison with 2F5 and 10E8 (A) Structural model for binding of 4E10 to gp41 and membrane lipids (PA modeled here). CDRH3 tryptophans and the gp41 peptide are shown as yellow sticks and brown helix, respectively. Dashed lines were added to complete the acyl chains of the observed 06:0 PA fragments (green ball-and-sticks). (B) Angle of approach of 4E10 (blue and green) with respect to the model of gp41 epitope-viral membrane assembly. Only the MPER (red) for one of the protomers of the trimer model is shown for clarity. The lipids highlighted (stick representation) in the model were observed in our crystal structures. (C) Position of CDRH3 (yellow) and CDRH1 (wheat) in 2F5 (PDB 2P8L) (Julien et al., 2008). The sequence of the displayed CDRH1 residues is shown at the bottom (as in E). (D) Position of CDRH3 (yellow) and CDRH1 (wheat) in 10E8 (PDB 4G6F) (Huang et al., 2012). (E) A model of lipid binding to 10E8, constructed by superposing residues 671-683 of the peptides in 4E10-MPER(671-673KKK) (PDB 2FX7; Cardoso et al., 2007) and 10E8-MPER(RRR656-683RRR) (Huang et al., 2012). The peptides bound to 4E10 and 10E8 are in brown and red, respectively. Only the 10E8 Fab and the position of the modeled PAs (green ball-and–stick) with regard to the MPER(671-683KKK) is shown for clarity. (F) Model of cardiolipin binding to 4E10. A cardiolipin model (green ball-and-stick) was built after superposition of the 4E10-06:0PA and 4E10-glycerol-3-PO4 structures, by connecting the two phosphate moieties at CDRH1 site 1 and 2 by a glycerol bridge (dashes). The most complete fragment of 06:0 PA observed at the CDRH1 site 1 was used for construction of the model and dashed lines were used to complete the acyl chains. The sequence of the CDRH1 residues predicted to interact with the cardiolipin head is shown at the bottom, and color-coded to match the displayed residues. CDRH3 residues are represented as yellow sticks. Immunity 2016 44, 21-31DOI: (10.1016/j.immuni.2015.12.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Non-specific Ligand Binding to 4E10 (A) Location of 03:0 PC binding sites in 4E10 Fab-peptide-03:0 PC complex (cyan light chain, blue heavy chain, brown peptide). Neighboring Fab molecules are shown as gray surfaces. The three 03:0 PCs (green sticks) bound to the variable region of the Fab at sites 3, 4, and 5 are shown, but the fourth 03:0 PC bound in the constant region is omitted. (B and C) illustrate the binding of a ligand (pink sticks; sites 3 and 4) in the 4E10-MPER(671-683KKK) structure cryoprotected with 02:0 SM (d18:1/2:0) and 08:0 PA, respectively. The ligand (either PEG or lipid hydrophobic tails) (Table S2) was assigned as “UNL” for unknown ligand. See also Figure S6 and Table S2. Immunity 2016 44, 21-31DOI: (10.1016/j.immuni.2015.12.001) Copyright © 2016 Elsevier Inc. Terms and Conditions