Anat Zvi

Solid–state NMR evidence for an antibody–dependent conformation of the V3 loop of HIV–1 gp120

Solid–state NMR measurements have been carried out on frozen solutions of the complex of a 24–residue peptide derived from the third variable (V3) loop of the HIV–1 envelope glycoprotein gp120 bound to the Fab fragment of an anti–gp120 antibody. The measurements place strong constraints on the conformation of the conserved central GPGR motif of the V3 loop in the antibody–bound state. In combination with earlier crystal structures of V3 peptide–antibody complexes and existing data on the cross–reactivity of the antibodies, the solid–state NMR measurements suggest that the Gly–Pro–Gly–Arg (GPGR) motif adopts an antibody–dependent conformation in the bound state and may be conformationally heterogeneous in unbound, full–length gp120. These measurements are the first application of solid–state NMR methods in a structural study of a peptide–protein complex.

NMR Structure of an Anti-Gp120 Antibody Complex with a V3 Peptide Reveals a Surface Important for Co-Receptor Binding

BACKGROUND The protein 0.5beta is a potent strain-specific human immunodeficiency virus type 1 (HIV-1) neutralizing antibody raised against the entire envelope glycoprotein (gp120) of the HIV-1(IIIB) strain. The epitope recognized by 0.5beta is located within the third hypervariable region (V3) of gp120. Recently, several HIV-1 V3 residues involved in co-receptor utilization and selection were identified. RESULTS Virtually complete sidechain assignment of the variable fragment (Fv) of 0.5beta in complex with the V3(IIIB) peptide P1053 (RKSIRIQRGPGRAFVTIG, in single-letter amino acid code) was accomplished and the combining site structure of 0.5beta Fv complexed with P1053 was solved using multidimensional nuclear magnetic resonance (NMR). Five of the six complementarity determining regions (CDRs) of the antibody adopt standard canonical conformations, whereas CDR3 of the heavy chain assumes an unexpected fold. The epitope recognized by 0.5beta encompasses 14 of the 18 P1053 residues. The bound peptide assumes a beta-hairpin conformation with a QRGPGR loop located at the very center of the binding pocket. The Fv and peptide surface areas buried upon binding are 601 A and 743 A(2), respectively, in the 0.5beta Fv-P1053 mean structure. The surface of P1053 interacting with the antibody is more extensive and the V3 peptide orientation in the binding site is significantly different compared with those derived from the crystal structures of a V3 peptide of the HIV-1 MN strain (V3(MN)) complexed to three different anti-peptide antibodies. CONCLUSIONS The surface of P1053 that is in contact with the anti-protein antibody 0.5beta is likely to correspond to a solvent-exposed region in the native gp120 molecule. Some residues of this region of gp120 are involved in co-receptor binding, and in discrimination between different chemokine receptors utilized by the protein. Several highly variable residues in the V3 loop limit the specificity of the 0.5beta antibody, helping the virus to escape from the immune system. The highly conserved GPG sequence might have a role in maintaining the beta-hairpin conformation of the V3 loop despite insertions, deletions and mutations in the flanking regions.

A cis proline turn linking two beta-hairpin strands in the solution structure of an antibody-bound HIV-1IIIB V3 peptide.

The refined solution structure of an 18-residue HIV-1IIIB V3 peptide in complex with the Fv fragment of an anti-gp120 antibody reveals an unexpected type VI β-turn comprising residues RGPG at the center of a β-hairpin. The central glycine and proline of this turn are linked by a cis peptide bond. The residues of the turn interact extensively with the antibody Fv. 15N{1H} NOE measurements show that the backbone of the peptide, including the central QRGPGR loop, is well ordered in the complex. The solution structure is significantly different from the X-ray structures of HIV-1MN V3 peptides bound to anti-peptide antibodies. These differences could be due to a two-residue (QR) insertion preceding the GPGR sequence in the HIV-1IIIB strain, and the much longer peptide epitope immobilized by the anti-gp120 antibody.

The principal neutralizing determinant of HIV-1 located in V3 of gp120 forms a 12-residue loop by internal hydrophobic interactions

The interactions of the peptide RP135a (RKSIRIQRGPGRAFVT), corresponding to residues 311–326 of gp120 of HIV‐1IIIB, with the anti‐gp120 HIV‐1IIIB neutralizing antibody 0.5β were studied by NMR. The NOESY difference spectra measured using specifically deuterated derivatives of the peptide show exclusively the interactions of the deuterated residues both within the bound peptide and with the Fab fragment of the antibody. These measurements reveal hydrophobic interactions within the bound peptide between Ile‐4, Ile‐6 and Val‐15 that create a 12‐residue loop with these residues at the base and the conserved GPGR sequence at its top.

Expression, purification, and isotope labeling of the Fv of the human HIV-1 neutralizing antibody 447-52D for NMR studies

The Fv is the smallest antigen binding fragment of the antibody and is made of the variable domains of the light and heavy chains, V(L) and V(H), respectively. The 26-kDa Fv is amenable for structure determination in solution using multi-dimensional hetero-nuclear NMR spectroscopy. The human monoclonal antibody 447-52D neutralizes a broad spectrum of HIV-1 isolates. This anti-HIV-1 antibody elicited in an infected patient is directed against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus. The V3 loop is an immunodominant neutralizing epitope of HIV-1. To obtain the 447-52D Fv for NMR studies, an Escherichia coli bicistronic expression vector for the heterodimeric 447-52D Fv and vectors for single chain Fv and individually expressed V(H) and V(L) were constructed. A pelB signal peptide was linked to the antibody genes to enable secretion of the expressed polypeptides into the periplasm. For easy cloning of any antibody gene without potential modification of the antibody sequence, restriction sites were introduced in the pelB sequence and following the termination codon. A set of oligonucleotides that prime the leader peptide genes of all potential antibody human antibodies were designed as backward primers. The forward primers for the V(L) and V(H) were based on constant region sequences. The 447-52D Fv could not be expressed either by a bicistronic vector or as single chain Fv, probably due to its toxicity to Escherichia coli. High level of expression was obtained by individual expression of the V(H) and the V(L) chains, which were then purified and recombined to generate a soluble and active 447-52D Fv fragment. The V(L) of mAb 447-52D was uniformly labeled with 13C and 15N nuclei (U-13C/15N). Preliminary NMR spectra demonstrate that structure determination of the recombinant 447-52D Fv and its complex with V3 peptides is feasible.

Application of NMR to Conformational Studies of an HIV Peptide Bound to a Neutralizing Antibody

NMR spectroscopy has become an important tool for structural studies of proteins and macro molecular complexes. In case of an antibody-antigen complex, different approaches are taken to alleviate the size limit, which is beyond NMR capabilities. The method of choice depends on the kinetic properties of the system under investigation and on the antibody fragment available. We have recently studied the conformation of RP135, a 24-residue HIV-1 peptide corresponding to the principal neutralizing determinant of the virus envelop glycoprotein gp120, in complex with 0.5β, a neutralizing antibody raised against gp120. The binding of the peptide to the antibody was too strong to observe TRNOE, therefore 2D-NOESY difference spectroscopy was applied using three strategies: (a) deuteration of specific residues of the peptide; (b) Arg→Lys replacement and; (c) truncation of the peptide antigen. The restraints on interproton distances within the bound peptide were used to calculate its conformation. The peptide forms a 10-residue loop, while the two segments flanking this loop interact extensively with each other and possibly form antiparallel β-strands.

The principal neutralizing determinant of HIV-1IIIB: Conformation of the peptide bound to a neutralizing antibody studied by 2D-NMR

RP135 is a 24-residue peptide corresponding to the principal neutralizing determinant of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1. We have studied the conformation of RP135 in complex with a neutralizing antibody 0.5β raised against gp120 by 2D NMR spectroscopy. The antigenic determinant recognized by this antibody was mapped using a combination of HOHAHA and ROESY measurements, in which resonances of the Fab and the tightly bound peptide residues are eliminated and the mobile residues of the bound peptide are sequentially assigned. We found that residues Ser6-Thr19 are part of the epitope, while Lys5 and Ile20 are at its boundaries. Difference spectroscopy was applied to study the conformation of the bound peptide representing the epitope within the 52 kDa of the Fab complex. Specific residues of the peptide were deuterated or replaced and the difference between the NOESY spectrum of the complex with the unlabeled residue and the NOESY spectrum of the complex with the modified residue revealed the interactions of the labeled residue both within the peptide and with the Fab fragment. A total of 122 distance restraints derived from the difference spectra enabled the calculation of the structure of the bound peptide. The peptide forms a 10-residue loop, while the two segments flanking this loop interact extensively with each other and possibly form anti-parallel β-strands. The loop conformation could be observed due to the unusual large size (17 residues) of the antigenic determinant recognized by 0.5β.