Anti-HIV Antibodies

IUCr Abstract

Crystallization

Fab cleavage
Crystallization of Fab fragments

anti V3-loop f50.1

27. Stura E.A., Stanfield R.L., Rini J.M., Fieser G.G., Silver S., Roguska M., Hincapie L.M., Simmerman H.K.B., Profy A.T., Wilson I.A. (1992) Crystallization, Sequence and Preliminary Crystallographic data for an anti-peptide Fab 50.1 and peptide complexes with the principal neutralizing determinant of HIV-1. Proteins, 14, 499-508.

Abstract: X-ray quality crystals of an Fab fragment from an antipeptide monoclonal antibody (R/V3-50.1) that recognizes the principal neutralizing determinant (PND) of the gp120 glycoprotein of human immunodeficiency virus type 1 (HIV-1) (MN isolate) were grown as uncomplexed and peptide complexed forms. Crystals of the free Fab grew from high salt in orthorhombic space groups P212121 and I222 and from polyethylene glycol in space groups P1 and P221. Seeds from either the P1 and P221 native (uncomplexed) Fab crystals induced nucleation of crystals of the Fab complexed to a 16-residue synthetic peptide corresponding to the PND when streak seeded into pre-equilibrated solutions of this complex. Data were collected from these complex crystals and from each of the four native Fab forms to at least 2.8 Å resolution. The genes for the variable domain of the Fab were cloned and sequenced and the primary amino acid sequence was deduced from this information. Knowledge of the were collected from these complex crystals and from each of the four native Fab forms to at least 2.8 Å resolution. The genes for the variable domain of the Fab were cloned and sequenced and the primary amino acid sequence was deduced from this information. Knowledge of the three-dimensional structure of this Fab-peptide complex will be important in the understanding of the PND of HIV-1 and its recognition by neutralizing monoclonal antibodies.

28. Rini, J.M., Stanfield, R.L., Stura, E.A., Salinas, P.A., Profy, A.T., Wilson, I.A. Crystal structure of Neutralizing antibody 50.1 in complex with its V3 loop peptide antigen. Proceedings of the National Academy of Sciences of the United States of America. 90, 6325-6329.

Abstract: The crystal structure of the Fab fragment of a human immunodeficiency virus type 1 (HIV-1) neutralizing monoclonal antibody Fab has been determined at 2.8 Å resolution in complex with a linear 16-residue peptide from the third hypervariable region (V3) of gp120. The first 9 residues of the peptide are ordered in the electron density maps, and their conformation is in partial agreement with the beta-strand-type II beta-turn structure predicted for this portion of the V3 loop. Notably, several of the peptide residues that are well conserved among different HIV-1 isolates contact a nonpolar 25-Å-long groove in the antibody-combining site. The largely extended structure of the peptide differs from the b-turns seen as the primary determinants in other published anti-peptide Fab structures. Analysis of the specific Fab-peptide interactions only partially explains the MN isolate specificity shown by this antibody.

Stanfield, R.L., Takimoto-Kamimura M., Rini J.M., Profy A.T., Wilson I.A. Major antigen-induced domain rearrangements in an antibody. Structure. 1, 83-93, 1993 Abstract: BACKGROUND: Recent structural results have shown that antibodies use an induced fit mechanism to recognize and bind their antigens. Here we present the crystallographically determined structure of an Fab directed against an HIV-1 peptide (Fab 50.1) in the unliganded state and compare it with the peptide-bound structure. We perform a detailed analysis of the components that contribute to enhanced antigen binding and recognition. RESULTS: Induced fit of Fab 50.1 to its peptide antigen involves a substantial rearrangement of the third complementarity determining region loop of the heavy chain (H3), as well as a large rotation of the variable heavy (VH) chain relative to the variable light (VL) chain. Analysis of other Fab structures suggests that the extent of the surface area buried at the VL-VH interface correlates with the ability to alter antibody quaternary structure by reorientation of the VL-VH domains. CONCLUSION: Fab 50.1 exhibits the largest conformational changes yet observed in a single antibody. These can be attributed to the flexibility of the variable region. Comparisons of new data with previous examples lend to the general conclusion that a small VL-VH interface, due in part to a short H3 loop, permits substantial alterations to the antigen-binding pocket. This has major implications for the prediction, engineering and design of antibody-combining sites.

anti V3-loop f59.1

44. Ghiara J.B., Stura E.A., Stanfield R.L. Profy A.T., Wilson I.A. (1994) Crystal Structure of the Principle Neutralizing Site of HIV-1. Science 264: 82-85.

Abstract: The crystal structure of a complex between a 24-amino acid peptide from the third variable (V3) loop of human immunodeficiency virus-type 1 (HIV-1) gp 120 and the Fab fragment of a broadly neutralizing antibody (59.1) was determined to 3 angstrom resolution. The tip of the V3 loop containing the Gly-Pro-Gly-Arg-Ala-Phe sequence adopts a double-turn conformation, which may be the basis of its conservation in many HIV-1 isolates. A complete map of the HIV-1 principal neutralizing determinant was constructed by stitching together structures of V3 loop peptides bound to 59.1 and to an isolate-specific (MN) neutralizing antibody (50.1). Structural conservation of the overlapping epitopes suggests that this biologically relevant conformation could be of use in the design of isolates. A complete map of the HIV-1 principal neutralizing determinant was constructed by stitching together structures of V3 loop peptides bound to 59.1 and to an isolate-specific (MN) neutralizing antibody (50.1). Structural conservation of the overlapping epitopes suggests that this biologically relevant conformation could be of use in the design of synthetic vaccines and drugs to inhibit HIV-1 entry and virus-related cellular fusion.

anti V3-loop f58.2 with linear and cyclic peptides

Paper in preparation: See IUCr Abstract