Albert T. Profy

Major antigen-induced domain rearrangements in an antibody

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.

Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing fabs.

BACKGROUND The third hypervariable (V3) loop of HIV-1 gp120 has been termed the principal neutralizing determinant (PND) of the virus and is involved in many aspects of virus infectivity. The V3 loop is required for viral entry into the cell via membrane fusion and is believed to interact with cell surface chemokine receptors on T cells and macrophages. Sequence changes in V3 can affect chemokine receptor usage, and can, therefore, modulate which types of cells are infected. Antibodies raised against peptides with V3 sequences can neutralize laboratory-adapted strains of the virus and inhibit syncytia formation. Fab fragments of these neutralizing antibodies in complex with V3 loop peptides have been studied by X-ray crystallography to determine the conformation of the V3 loop. RESULTS We have determined three crystal structures of Fab 58.2, a broadly neutralizing antibody, in complex with one linear and two cyclic peptides the amino acid sequence of which comes from the MN isolate of the gp120 V3 loop. Although the peptide conformations are very similar for the linear and cyclic forms, they differ from that seen for the identical peptide bound to a different broadly neutralizing antibody, Fab 59.1, and for a similar peptide bound to the MN-specific Fab 50.1. The conformational difference in the peptide is localized around residues Gly-Pro-Gly-Arg, which are highly conserved in different HIV-1 isolates and are predicted to adopt a type II beta turn. CONCLUSIONS The V3 loop can adopt at least two different conformations for the highly conserved Gly-Pro-Gly-Arg sequence at the tip of the loop. Thus, the HIV-1 V3 loop has some inherent conformational flexibility that may relate to its biological function.

Recurring conformation of the human immunodeficiency virus type 1 gp120 V3 loop

The crystal structure of the human immunodeficiency virus type 1 (HIV-1) neutralizing, murine Fab 83.1 in complex with an HIV-1 gp120 V3 peptide has been determined to 2.57 A resolution. The conformation of the V3 loop peptide in complex with Fab 83.1 is very similar to V3 conformations seen previously with two other neutralizing Fabs, 50.1 and 59.1. The repeated identification of this same V3 conformation in complex with three very different, neutralizing antibodies indicates that it is a highly preferred structure for V3 loops on some strains of the HIV-1 virus.

Complementary use of chemical modification and site-directed mutagenesis to probe structure-activity relationships in enzymes.

Publisher Summary This chapter uses chemical modification and site-directed mutagenesis to probe structure-activity relationships in enzymes. Site-directed mutagenesis provides a useful test of validity of conclusions based on chemical modification experiments. A number of putative essential residues can be replaced without a substantial activity loss. This was particularly true when the initial identifications were based on labeling by common, side-chain-selective modification reagents. Thus, His-291 in ribulose-bisphosphate carboxylase, Cys-395 in glycyl-tRNA synthetase, Cys-148 in lac permease, and even Tyr-248 in carboxypeptidase A are expendable. The surprising result in the carboxypeptidase Tyr-248 case demonstrates the power of site-directed mutagenesis as a structure-function probe. It was also expected that replacement of some “essential” amino acids would cause a dramatic loss in enzymatic activity, thereby confirming their importance. This was generally observed when the initial assignment was based on affinity labeling, such as in the case of Ser-70 in p-lactamase, Lys-166 and Lys-329 in ribulose-bisphosphate carboxylase, Lys-84 in aspartate transcarbamoylase, and Glu-165 of triosephosphate isomerase. These results confirm that affinity labeling is a much more reliable way to identify active-site amino acids than is modification by common side-chain-selective reagents.

Immunoassay to measure staphylococcal protein A in the presence of murine immunoglobulins

Immunoassays designed to measure low concentrations of staphylococcal protein A are subject to varying degrees of interference by excess IgG. We have developed an enzyme-linked immunosorbent assay (ELISA) that overcomes this problem by analyzing IgG-containing protein A samples in solutions buffered at pH 3.5. Under these carefully selected conditions, protein A is absorbed efficiently by solid-phase chicken anti-protein A antibodies, but protein A-IgG complexes are largely dissociated. The assay has a protein A detection limit of 0.1 ng/ml, and the response is unaffected by 0.25 mg/ml murine IgG. The method should be useful for determining protein A contamination levels in antibodies purified by affinity chromatography on immobilized protein A resins.