Structural basis of tyrosine sulfation and VH-gene usage in antibodies that recognize the HIV type 1 coreceptor-binding site on gp120

Abstract

The conserved surface of the HIV-1 gp120 envelope glycoprotein that binds to the HIV-1 coreceptor is protected from humoral recognition by multiple layers of camouflage. Here we present sequence and genomic analyses for 12 antibodies that pierce these defenses and determine the crystal structures of 5. The data reveal mechanisms and atomic-level details for three unusual immune features: posttranslational mimicry of coreceptor by tyrosine sulfation of antibody, an alternative molecular mechanism controlling such sulfation, and highly selective V(H)-gene usage. When confronted by extraordinary viral defenses, the immune system unveils novel adaptive capabilities, with tyrosine sulfation enhancing the vocabulary of antigen recognition.

Fig. 1.

Structure of the archetype CD4i antibody, 17b. (A) Complexed versus free structure of 17b. The Left two structures show the rerefined YU2 and HXBc2 ternary complexes after superposition of the 17b V_H framework, with the two complexed Fab 17b in black C^α worm, interacting 17b side chains in green, the N-terminal domain of CD4 in yellow, and the molecular surface of YU2 core gp120 in red, except for the surface within 3.5 Å of 17b, which is blue. In this orientation, the viral membrane would be positioned toward the top of the page. The Right two structures show the two independent copies of free 17b from the P2₁2₁2₁ crystals superimposed on the complexed structures. The color and orientation for the complexed structures are the same as in Left, with the free 17b structures shown in blue with magenta interactive residues. The Far Right shows the entire Fab, including the constant portion. Whereas the variable domains are quite similar, considerable differences are seen in the constant portions, especially between the two free structures. (B) Details of gp120–17b interaction at CDR H2 and CDR H3. The electrostatic potential of gp120 is shown at the molecular surface colored blue for electropositive, red for acidic, and white for apolar. The Left two structures show 17b in the same orientation as A. The portion corresponding to the V_H gene, VH1-69, has been colored green, except for residues altered by somatic mutation, which are colored magenta. The five side chains of the CDR H2 that interact with gp120 are shown: I52, I53, L54, V56, and H58. The Right two structures show an ≈90° view, adjusted so that the pseudo twofold axes of the Fab are aligned with the edges of the page. In this view, the acidic CDR H3 loop (yellow C^α worm) can be seen reaching up to contact a basic gp120 surface. Side chains of VH1-69 that interact with the CDR H3 loop are shown.

Fig. 2.

CD4i antibody variable domain sequences. A multiple sequence alignment of CD4i antibodies is shown with CDRs labeled. Both Kabat CDR definitions and numbering are used. Sequences have been ordered according to CDR H3 length, which varies from 10 for 48d to 25 for E51. Antibodies isolated from phage display are labeled with an asterisk. N-terminal sequences influenced by sequencing primers are shown in lowercase; those influenced by phage library construction are shown in italics. Somatic mutations are highlighted with yellow background, acidic CDR residues with red, and CDR tyrosines with green. Boxed residues of 17b contact gp120. Because of allelic differences, somatic mutations were determined by using the closest genomic progenitor. The N termini of CCR5 and CXCR4 are shown for reference.

Fig. 3.

Tyrosine sulfation and HIV-1 gp120 recognition for m16 and X5. (A) Modulation of antibody sulfation with sulfotransferase and small hairpin RNA (shRNA). Initially 293T cells were transfected with plasmid encoding shRNA complementary to the message of the two known tyrosyl protein sulfotransferases (ST-shRNA) or to an irrelevant message. Two days later, cells were transfected with the indicated antibody in the presence (TPST2) or absence of tyrosine sulfotransferases. Cells were divided and labeled with [³⁵S]cysteine and [³⁵S]methionine or [³⁵S]sulfate. Cell supernatants were immunoprecipitated with staphylococcal protein A-Sepharose and analyzed by SDS/PAGE. CD4 binding-site antibody b12 is shown as a control. (B) Sulfation does not contribute to gp120 association. Antibody from assay shown in A was incubated with radiolabeled gp120 of the CCR5-using isolate ADA and an excess of unlabeled CD4. Antibody and gp120/CD4 complexes were immunoprecipitated with protein A-Sepharose and analyzed by SDS/PAGE. With the functionally sulfated E51, such ST-shRNA treatment reduces binding by 6- to 8-fold (19).

Fig. 4.

Structure and electrostatic surface of 48d, 17b, 47e, 412d, and E51. CD4i antibodies were aligned by superposition of their V_H framework. The two independent copies of both 17b and 412d as well as the eight independent copies of 47e are shown. The disordered portions of the CDR H3 loops for 47e, 412d, and E51 were modeled and subjected to molecular dynamics. Shown for the disordered regions are 8 models of 47e and 10 for 412d and E51. (A) Fab structures. Blue C^α worms are shown with CDR H2 green and CDR H3 yellow. The modeled sections, subjected to molecular dynamics, are shown with C^α worm in gray and sulfated tyrosines in red. The orientation of this figure is the same as the Right two structures of Fig. 1B.(B) Perpendicular view of A, showing only the variable fragment (Fv) portion of the Fab for clarity. (C) Electrostatic surface. The electrostatic potential is displayed at the molecular surface, with electropositive surface in blue, electronegative surface in red, and apolar surface in white. The Fabs are in the same orientation as in B. Although diluted somewhat by the disorder of 47e, 412d, and E51, an acidic surface can be seen on all of the CD4i antibodies. The alterations in shape of the overall surface are due primarily to variations in the positions of the constant domains.

Fig. 5.

Atomic-level details of antibody sulfation. The sulfated tyrosine at position H100 of 412d is shown. Two of the five coordinating ligands (Lys-145 and Gln-147) are from the light chain of a symmetry-related molecule. Electron density (2F_o - F_c) is shown at 0.5σ.