Crystal structure of human antibody 2909 reveals conserved features of quaternary structure-specific antibodies that potently neutralize HIV-1

Abstract

Monoclonal antibody 2909 belongs to a class of potently neutralizing antibodies that recognize quaternary epitopes on HIV-1. Some members of this class, such as 2909, are strain specific, while others, such as antibody PG16, are broadly neutralizing; all, however, recognize a region on the gp120 envelope glycoprotein that includes two loops (V2 and V3) and forms appropriately only in the oligomeric HIV-1 spike (gp120(3)/gp41(3)). Here we present the crystal structure of 2909 and report structure-function analysis with antibody chimeras composed of 2909 and other members of this antibody class. The 2909 structure was dominated by a heavy-chain third-complementarity-determining region (CDR H3) of 21 residues, which comprised 36% of the combining surface and formed a β-hairpin club extending ∼20 Å beyond the rest of the antibody. Sequence analysis and mass spectrometry identified sites of tyrosine sulfation at the middle and top of CDR H3; substitutions with phenylalanine either ablated (middle substitution) or substantially diminished (top substitution) neutralization. Chimeric antibodies composed of heavy and light chains, exchanged between 2909 and other members of the class, indicated a substantial lack of complementation. Comparison of 2909 to PG16 (which is tyrosine sulfated and the only other member of the class for which a structure has previously been reported) showed that both utilize protruding, anionic CDR H3s for recognition. Thus, despite some diversity, members of this class share structural and functional similarities, with conserved features of the CDR H3 subdomain likely reflecting prevalent solutions by the human immune system for recognition of a quaternary site of HIV-1 vulnerability.

FIG. 1.

Overall structure of 2909 Fab. The 2909 crystal structure reveals a combining region dominated by a protruding, acidic CDR H3 loop. (A) Ribbon representation of the 2909 Fab structure is shown, with heavy and light chains colored in blue and green, respectively. The CDR H3 loop is highlighted in red, while other CDR loops (as defined by Kabat [19]) are depicted in yellow. (B) Surface representation of the 2909 Fab is shown in the same orientation as in panel A (left) or rotated 180° about the y axis (right). The surfaces are colored by electrostatic potential (−10 to +10 kT/e), with positively and negatively charged regions shown in blue and red, respectively.

FIG. 2.

Details of 2909 CDR H3. 2909 CDR H3 is a 21-residue loop forming a β-hairpin structure. The loop is stabilized at the base in a bulged conformation by other CDR loop residues. Most of the CDR H3 loop extends out from the combining site and is solvent accessible. (A) 2F_o - F_c electron density contoured at 1 σ is shown around 2909 CDR H3. Heavy and light chains are colored blue and green, respectively, while the CDR H3 loop is shown in pink stick representation. The conformations of side chains at the tip of the CDR H3 loop are not clear due to weak density in this region. (B) Hydrogen bonds stabilizing the 2909 CDR H3 loop are drawn as dotted lines. Coloring is the same as in panel A, with additional stabilizing residues from heavy and light chains shown in gray stick.

FIG. 3.

Structural homology of 2909 CDR H3 region. Sixty-seven percent of the long β-hairpin structure of the 2909 CDR H3 loop is exposed to solvent, making it a rare structural motif among all protein structures. Structural homologs to the CDR H3 show <30% sequence identity. Several anti-HIV-1 antibodies have structurally similar CDR H3 regions, including 447-52D and Z13e1. (A) Two thousand two hundred seventy-two structural homologs were identified from a database of 16,938 proteins and analyzed in a statistical manner. The distribution of solvent accessibility of structural homologs is shown, with the value of 2909 CDR H3 marked by a red arrow. Homologs range in length from 15 to 25 residues. (B) The distribution of sequence identity of structural homologs is shown with respect to the 2909 CDR H3. (C) A structural comparison of the 2909 CDR H3 loop with two structural homologs, 447-52D and Z13e1, is shown by superposition of the heavy chain variable domains using LSQKAB (10). 2909 heavy and light chains are colored in blue and green, respectively, while 447-52D and Z13e1 are gray. The CDR H3 regions of 2909, 447-52D, and Z13e1 are highlighted in red, orange, and yellow, respectively.

FIG. 4.

Electrospray ionization mass spectrometry (ESI-MS) data for 2909 Fab. ESI-MS intact mass measurements of 2909 Fab are consistent with two sites of tyrosine sulfation in the heavy chain. Deconvoluted ESI mass spectra are shown for 2909 Fab heavy and light chains (A) or a hen egg white lysozyme standard (B).

FIG. 5.

Sequence and structural comparison of 2909 with PG16. 2909 and PG16 are both from VH3 gene families. 2909 and PG16 both have critical residues in similar positions in their CDR H3 sequences, and these residues are located at approximately the same distance from the combining surface in both antibodies. Acidic CDR H3 regions for 2909 and PG16 are found on opposing faces of each antibody. (A) Sequence alignment of 2909 and PG16 heavy chains is shown, with identical residues shaded in gray. Tyrosine sulfation in PG16 CDR H3 and predicted sites of sulfation in 2909 are highlighted in magenta. The Kabat numbering scheme is used for CDR loop definition. (B) A comparison of 2909 and PG16 CDR H3 loop structures is shown. The variable domains of 2909 and PG16 were superimposed (over Cα atoms, excluding the CDR H3 loop) and are shown on top. The bottom panel shows the same orientation for PG16 while 2909 is flipped, such that the 2909 V_H is aligned with PG16 V_L and 2909 V_L is aligned with PG16 V_H. The PG16 model is based on PDB ID 3MUG (33). PG16 is colored gray, with its CDR H3 loop depicted in tan. Sulfated Tyr^H100h and Asp^H100i are important residues for PG16 activity and are drawn in gray stick. 2909 is colored as in Fig. 1A, with Tyr^H100a and Tyr^H100c shown in red stick. (C and D) A close-up view of the combining sites from 2909 (C) or PG16 (D). The surfaces are colored by electrostatic potential (−10 to +10 kT/e), with positively and negatively charged regions shown in blue and red, respectively. CDR loops are labeled accordingly.