Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9

Abstract

Variable regions 1 and 2 (V1/V2) of human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein are critical for viral evasion of antibody neutralization, and are themselves protected by extraordinary sequence diversity and N-linked glycosylation. Human antibodies such as PG9 nonetheless engage V1/V2 and neutralize 80% of HIV-1 isolates. Here we report the structure of V1/V2 in complex with PG9. V1/V2 forms a four-stranded β-sheet domain, in which sequence diversity and glycosylation are largely segregated to strand-connecting loops. PG9 recognition involves electrostatic, sequence-independent and glycan interactions: the latter account for over half the interactive surface but are of sufficiently weak affinity to avoid autoreactivity. The structures of V1/V2-directed antibodies CH04 and PGT145 indicate that they share a common mode of glycan penetration by extended anionic loops. In addition to structurally defining V1/V2, the results thus identify a paradigm of antibody recognition for highly glycosylated antigens, which-with PG9-involves a site of vulnerability comprising just two glycans and a strand.

Figure 1. Overall structure of the V1/V2 domain of HIV-1 gp120 in complex with PG9

V1/V2 from the CAP45 strain of HIV-1 is shown, in magenta ribbons, in complex with Fab of antibody PG9. The PG9 heavy and light chains are shown as yellow and blue ribbons, respectively, with CDRs in different shades. Although the rest of HIV-1 gp120 has been replaced by the 1FD6 scaffold (shown in white ribbons), the positions of V1/V2, PG9 and the scaffold are consistent with the proposal that the viral spike, and hence the viral membrane, is positioned towards the top of the page. The extended CDR H3 of PG9 is able to penetrate the glycan shield that covers the V1/V2 cap on the spike and to reach conserved elements of polypeptide, while residues in heavy- and light-chain-combining regions recognize N-linked glycans. The disordered region of the V2 loop is represented by a dashed line. Perpendicular views of V1/V2 are shown in Figs 2 and 6, and the structure of PG9 in complex with V1/V2 from HIV-1 strain ZM109 is shown in Supplementary Fig. 7.

Figure 2. Structure of the V1/V2 domain of HIV-1 gp120

a–i, The four anti-parallel strands that define V1/V2 fold as a single domain, in a topology known as ‘Greek key’ that is observed in many proteins. a, Schematic of V1/V2 topology. V1/V2 resides between strands β2 and β3 of core gp120, and its structure completes the crystallographic determination of all portions of HIV-1 gp120. Strands are depicted as arrows and disulphide bonds as yellow lines. b, c, Ribbon diagram of V1/V2 residues 126–196 from HIV-1 strains CAP45 (magenta) and ZM109 (green). Conserved disulphide bonds are represented as ball and stick, and the beginning and terminating residues of each strand are labelled. d, Superposition of the structures shown in b and c. e, Amino acid conservation of V1/V2. The backbone is shown as a tube of variable thickness, coloured as a rainbow from cold (blue) to hot (red), corresponding to conserved (thin) and to variable (thick), respectively, based on an alignment of 166 HIV-1 sequences. Aliphatic and aromatic side chains are shown as sticks with semi-transparent molecular surface, coloured by conservation as in i. f, Electrostatic surface potentials of CAP45 V1/V2 coloured blue to red, corresponding to positive and negative surface potentials, respectively. g, Molecular surfaces corresponding to main-chain atoms including C_β are coloured orange, with other surfaces coloured white. h, Superposition of ZM109 and CAP45 models containing V1 and V2 loops and associated glycans. For each glycosylated asparagine, only the first N-acetylglucosamine attached to the asparagine is shown and represented as sticks with a transparent molecular surface. Modelled amino acids and glycans that are disordered in the crystal structures are shown in grey. i, Sequence alignment of nine HIV-1 strains that are potently neutralized by PG9. Glycosylated asparagine residues are boxed and in bold. Identical residues have a dark green background with white characters, whereas conserved residues have white backgrounds with dark green characters. Above the alignment, β-strands are shown as arrows, coloured magenta and green for CAP45 and ZM109, respectively. Residues and attached glycans that make hydrogen bonds to PG9 are denoted with symbols above the alignment (side-chain hydrogen bonds are indicated by open circles with dashes, main-chain hydrogen bonds are indicated by closed circles, or both).

Figure 3. PG9 V1/V2 interactions

a–f, Glycan, electrostatic and sequence-independent interactions of antibody PG9 facilitate recognition of V1/V2 from the ZM109 strain of HIV-1 gp120. a, PG9 is shown as a grey molecular surface, and strands B and C of V1/V2 are shown as green ribbons. Mannose and N-acetylglucosamine residues are shown in stick representation, as are the side chains of Asn 160 and 173. Electron density (2F_o−F_c) is contoured at 1σ and shown as a blue mesh. b, Ribbon representations of strands B and C of ZM109 V1/V2 (green), PG9 heavy chain (yellow) and PG9 light chain (blue). V1/V2 glycans and PG9 residues that hydrogen bond are shown as sticks. Nitrogen atoms are coloured blue, oxygen atoms are coloured red, and dotted lines represent hydrogen bonds. c, Schematic of the Man₅GlcNac₂ moiety attached to Asn 160. GlcNacs are shown as blue squares, and mannoses as green circles. Hydrogen bonds to PG9 are listed to the right of the symbols, as is the total surface area buried at the interface between PG9 and each sugar. d, Schematic of the PG9–main-chain interaction with V1/V2. Disulphide bonds in V1/V2 are shown as yellow sticks. e, f, Ribbon representation of V1/V2 (green) and PG9 CDR H3 (yellow). Hydrogen bonds are represented by dotted lines. Main-chain interactions are shown in e, and side chain interactions in f (with the two images related by a 90° rotation about a vertical axis). Details of the PG9 interaction with V1/V2 from the CAP45 strain of HIV-1 are shown in Supplementary Fig. 8.

Figure 4. PG9 and PG16 recognition of the HIV-1 viral spike, monomeric gp120 and scaffolded V1/V2

a–c,Quaternary-structure-preferring antibodies show different affinities for oligomeric, monomeric and scaffolded V1/V2. Both structural and arginine-scanning mapping, however, suggest that the epitopes of PG9 and PG16 are mostly present in scaffolded V1/V2. a, Affinities of PG9 (filled symbols) and PG16 (open symbols) are shown for the functional viral spike (gp120/gp41)₃ (circles), monomeric gp120 (triangles), and scaffold-V1/V2 (squares), based on neutralization (black), ELISA (blue) and surface plasmon resonance (red). b, Negative stained images are shown for ternary complexes of wild-type gp120 (HIV-1 strain 16055) in complex with antibody PG9 and the CD4-binding-site antibody T13. Six different classifications were observed, and are superimposed in the top left panel and labelled PG9-1 to PG9-6. Individual fitting for classes PG9-1, PG9-3 and PG9-5 are shown after rigid-body alignment of Fab PG9–scaffold-V1/V2, Fab T13 and core gp120 (in the conformation bound by the CD4-binding site antibody F105; ref. 20). c, Comparison of crystallographically defined PG9 paratope with neutralization-defined PG16 paratope. Scaffold-V1/V2 interactive surface of PG9 in ZM109 (left) and CAP45 (middle) contexts is shown along with the PG16 paratope (right) as defined by arginine-scanning mutagenesis (orange-highlighted residue is Trp 64 in the CDR H2). Perpendicular views of the paratope, rotated by 90° about a horizontal axis, are shown in top and bottom rows.

Figure 5. CDR H3 features of V1/V2-directed broadly neutralizing antibodies

a, b, A protruding anionic CDR H3 is preserved in members of this broadly neutralizing class of antibodies. a, CDR H3 sequence alignment. Cohort, donor information and sequences in the CDR H3 (Kabat definition and numbering) are shown for V1/V2-directed antibodies. Positively charged residues are boxed in blue and negatively charged residues in red. Residues that make hydrogen bonds to CAP45 residues (magenta) or glycans (purple) are denoted with symbols above the alignment (side-chain hydrogen bonds are indicated by open circles with dashes, main-chain hydrogen bonds by closed circles, or both). Similar contacts are shown for ZM109 residues (green) or glycans (grey). Sulphated tyrosines are circled or squared if the post-translational modification has been confirmed crystallographically or by mass spectrometry, respectively. The sequence for the V1/V2-directed strain-specific antibody, 2909, is also included. b, Protruding CDR H3, displayed as ribbon diagrams with sulphated tyrosines shown in spheres and paired with electrostatic surface potentials coloured blue to red, corresponding to positive and negative surface potentials, respectively. All CDR H3s are aligned so that the light chain would be on the left and the heavy chain on the right (as in Supplementary Fig. 17). Average electrostatic potentials are shown for specified CDR H3 and V1V2 surfaces.

Figure 6. Two glycans and a strand comprise a V1/V2 site of vulnerability

a, b, Glycan, electrostatic and sequence-independent interactions allow PG9 to recognize a glycopeptide site on V1/V2. a, Site characteristics in CAP45 strain of HIV-1. Glycans 160 and 156 (173 with ZM109) are highlighted in green, and strands B and C are highlighted in magenta, with the rest of V1/V2 in semi-transparent white. The interactive surface of V1/V2 with PG9 is shown, coloured according to the local electrostatic potential as in Fig. 5b. The contribution of each structural element to that surface is provided as a percentage of the total. Although the V1/V2 scaffolds used here do not allow a comprehensive analysis of the overall antibody response to this region of gp120, in addition to assisting with structural definition of effective V1/V2-directed neutralization, the V1/V2 scaffolds may have utility in attempts to direct the V1/V2-elicited response away from the hypervariable loops to the conserved strands—especially the site of vulnerability highlighted here. b, Saturation transfer difference (STD) NMR for Man₅GlcNAc₂-Asn binding to PG9. Top, STD spectrum of 1.5 mM Man₅GlcNAc₂-Asn in the presence of 15 μM Fab PG9 (lower spectrum) is paired with the corresponding reference spectrum (upper spectrum). Bottom left, Langmuir binding curve as a function of glycan concentration, used to obtain the K_D (A_STD signals correspond to N-acetyl protons, which are shown in the boxed area of the top panel). Bottom right, stacked STD NMR spectra as a function of Man₅GlcNAc₂-Asn concentration.