The Binding of a Glycoprotein 120 V3 Loop Peptide to HIV-1 Neutralizing Antibodies

Abstract

The structural and antigenic properties of a peptide ("CRK") derived from the V3 loop of HIV-1 gp120 protein were studied using NMR and SPR techniques. The sequence of CRK corresponds to the central portion of the V3 loop containing the highly conserved "GPGR" residue sequence. Although the biological significance of this conserved sequence is unknown, the adoption of conserved secondary structure (type II ␤-turn) in this region has been proposed. The tendency of CRK (while free or conjugated to protein), to adopt such structure and the influence of such structure upon CRK antigenicity were investigated by NMR and SPR, respectively. Regardless of conjugation, CRK is conformationally averaged in solution but a weak tendency of the CRK "GPGR" residues to adopt a ␤-turn conformation was observed after conjugation. The influence of GPGR structure upon CRK antigenicity was investigated by measuring the affinities of two cognate antibodies: "5023A" and "5025A," for CRK, protein-conjugated CRK and gp120 protein. Each antibody bound to all the antigens with nearly the same affinity. From these data, it appears that: (a) antibody binding most likely involves an induced fit of the peptide and (b) the gp120 V3 loop is probably conformationally heterogeneous. Since 5023A and 5025A are HIV-1 neutralizing antibodies, neutralization in these cases appears to be independent of adopted GPGR ␤-turn structure. The principal neutralizing determinant (PND)1 of HIV-1 has been mapped to the third hypervariable (V3) loop of the HIV-1 envelope protein, gp120 (1). PND-derived peptides are used as immunogens to elicit antibodies that possess HIV-1 virus neutralization capabilities (2, 3). The binding of neutralizing antibodies blocks virus entry into the host cell but does not prevent binding of HIV-1 to its primary cell receptor protein, CD4 (4, 5). Although the V3 loop represents an important target for the development of vaccines against AIDS, its high sequence variability also makes it a very problematic one (6). Nonetheless, the occurrence of the highly conserved residue sequence, "GPGR," at the tip of the V3 loop has raised the possibility that these residues make up a conserved secondary structural element in gp120. The function of such a structural element, if one exists, is presently unknown, however. The precise predicted secondary structure adopted by the GPGR residues is a type II ␤-turn (6, 7).In order to determine whether the conserved GPGR sequence confers certain secondary structural tendencies upon this region of the V3 loop, numerous NMR studies were conducted upon a variety of V3 loop-derived peptides (8 -21). These peptides were shown to have only low density populations of folded structure and to be conformationally averaged in solution. Despite the report of a "core" gp120 protein complex crystal structure, this complex was prepared using a form of gp120 which lacked most of the hypervariable loops including V3 (22). Information regarding the actual three-dimensional structure of the V3 loop in native gp120 is theref...

Peptide–protein interactions studied by surface plasmon and nuclear magnetic resonances

“…A strong correlation between the structure stability and the a¤nity between protein subdomains has been found from SPR data [2] and a combined use of SPR and NMR measurements has been proposed for investigating peptide^antibody [3,4] and virus^receptor interactions [5].…”

Cross‐Reaction of Anti‐Simian Immunodeficiency Virus Envelope Protein Antibodies with Human Immunoglobulins

“…This paper describes experiments addressing the question: can antibodies elicited against REPs bind hIgs in vitro ? The rationale for asking this question was that previously, structural similarities between REPs and hIgs were discovered [10–12]. Amino acid sequence homologies were found between some portions of HIV‐1, HIV‐2 and SIV envelope proteins and hIg V H regions [13].…”

“…The ability of gp120 and, possibly, other retroviral envelope proteins (REPs) to bind certain antigenic epitopes, including idiotopes, may be related to the fact that REPs have a number of antibody‐like structural properties. REPs are folded into loops that resemble Ig domains, containing three constant (C1–C3) and three variable (V1–V3) domains [10–12]. By computer‐assisted amino acid homology search, we have previously shown that certain oligopeptides are shared between the V3 loop of some HIV‐2 and SIV isolates, and the tetrapeptide sequences between positions 56 and 59 on the V region of human Igs (hIgs) heavy‐chain V regions (V H ) coded by a small group of related Ig V region genes (V H 3–30, V H 3–30.3 and V H 3–33 [13]).…”

Design of immunogens that present the crown of the HIV-1 V3 loop in a conformation competent to generate 447-52D-like antibodies

“…In the present study, we have followed the second approach to generate a V3 loop derivative. NMR studies on free V3 loop peptides report the presence of a relatively unstructured ensemble of V3 molecules in solution [17,[23][24][25][26]. NMR and crystal structures have been solved for V3 peptides in complex with a number of neutralizing antibodies [27][28][29][30], including mAb 447-52D [31].…”