Three-dimensional structure of Fab R19.9, a monoclonal murine antibody specific for the p-azobenzenearsonate group.

Abstract

The crystal structure of Fab R19.9, derived from an anti-p-azobenzenearsonate monoclonal antibody, has been determined and refined to 2.8-A resolution by x-ray crystallographic techniques. Monoclonal antibody R19.9 (IgG2bK) shares some idiotopes with a major idiotype (CRIA) associated with A/J anti-p-azobenzenearsonate antibodies.The amino acid sequences of the variable (V) parts of the heavy (VH) and light (VL) polypeptide chains of monoclonal antibody R19.9 were determined through nucleotide sequencing of their mRNAs. The VL region is very similar to that of CRIA-positive anti-p-azobenzenearsonate antibodies as is VH, except for its third complementarity-determining region, which is three amino acids longer; it makes a loop, unique to R19.9, that protrudes into the solvent. A large number of tyrosine residues in the complementarity-determining region of VH and VL, with their side chains pointing towards the solvent, may have an important function in antigen binding. Murine antibodies to model antigens have provided valuable experimental systems to study the molecular bases of the specificity, diversity, and genetic control of immune responses. The hapten, p-azobenzenearsonate (Ar), has been used in several laboratories as a suitable probe for such studies (1-5), which have been facilitated by the presence of an intrastrain cross-reactive idiotype, designated CRIA, among the anti-Ar antibodies of A/J mice or ofclosely related strains. The expression of CRIA is linked to genetic loci encoding heavy (H) chains (6) and light (L) chains (7). On the average, about half of the anti-Ar antibodies induced by keyhole limpet hemocyanin-Ar in A/J mice share this idiotype. The variable (V) regions, VH and VL, of CRIA antibodies appear to be encoded by single germ-line genes (8,9), and the diversity (D) region is encoded by a variant of the DFL16.1 gene (10). CRIA molecules also utilize the VK10, K chain joining (J) 1, and, almost invariably, JH2 gene segments (4, 5, 11). Idiotypeexpressing antibodies from hyperimmunized mice display somatic variants ofamino acid sequences in each ofthese gene segments (4, 5, 12), whereas the antibodies from an early primary response reflect few if any mutations (5). The VH region appears to be somewhat more susceptible to somatic variation than VL (4). A disproportionate number of mutations in VH and VL occurs in their complementarity-determining regions (CDR); this probably reflects selection by antigen of variants with higher affinity (5).Among the serum anti-Ar antibodies of immunized A/J mice are molecules that carry some but not all ofthe idiotopes associated with CRIA (13,14). Such antibodies are bound by anti-CRIA antibodies, but they are unable to completely displace labeled CRI+ antibodies from such anti-idiotype antibodies. Antibodies of this type were designated "minor idiotypes" (13,14). The subject of the present investigation, monoclonal antibody (mAb) R19.9 (IgG2bK), has these serological properties and is thus a member of a minor idiotypic anti-Ar family. The ...

A computer screening approach to immunoglobulin superfamily structures and interactions: Discovery of small non-peptidic CD4 inhibitors as novel immunotherapeutics

“…6). For example, a similar pocket consisting of the FG and CCЈ loops is commonly observed to be involved in dimerization of Ig superfamily molecules, such as homodimers of CD8␣ (35) and antibody V H V L (40). On the other hand, this pocket also mediates heterophilic interactions as seen in FcRI␣ 2 , which is the domain of the IgE high-affinity receptor that binds IgE (36), CD2 that binds LFA-3 (37), and CD28 that binds CD80͞86 (38).…”

Structural correlates of high antibody affinity: three engineered amino acid substitutions can increase the affinity of an anti-p-azophenylarsonate antibody 200-fold.

“…1), and therefore their effect on affinity is not surprising. The x-ray crystallographic structures of two anti-Ars antibodies, R19.9 and 36-71, have been reported (32,33). From the structure of 36-71, Ile-57, Thr-58, Glu-96, and Lys-lOOc do not seem to be involved in binding to Ars and probably affect affinity indirectly: Glu-96 and Lys-lOOc by influencing the positions of two H-chain aromatic side chains (Tyr-50 and Tyr-lOOb) that stack against the phenyl group of Ars, and Ile-57 and Thr-58 by "anchoring" that part of CDR2 and possibly influencing the position of Tyr-50 (R. K. Strong, G. A. Petsko, J.S., and M. N. Margolies, unpublished data).…”

Three‐dimensional structure of the Fab fragment of a neutralizing antibody to human rhinovirus serotype 2

“…2; Kinemage 1). This elbow angle, defined as the angle between the pseudodyad axes that relate the light chain and heavy chain domains in both the variable and constant modules, was found to vary from about 132" in New and McPC603 Fab fragments (review in Sheriff et al, 1988) to about 179" in the R19.9 and 36-71 Fab structures (Lascombe et al, 1989;Strong et al, 1991). The rotation-translation operations that optimize the superposition of the backbone atoms of the VH and VL domains are 173.59", 1.5 A; whereas for the CL onto the CH1 domains they are 169.54", -1.47 A.…”

“…seen to vary from 132" to 179" in known Fab structures (Sheriff et al, 1988;Lascombe et al, 1989;Strong et al, 1991). Because changes in the relative orientation of these two modules greatly influence the Patterson function, we have calculated independently the rotation and translation functions for each module following the example of Cygler and Anderson (1988a,b) for the structure solution of HED10 (Cygler et al, 1987).…”

“…Because changes in the relative orientation of these two modules greatly influence the Patterson function, we have calculated independently the rotation and translation functions for each module following the example of Cygler and Anderson (1988a,b) for the structure solution of HED10 (Cygler et al, 1987). The molecular models that proved useful in the analysis were the Fv module (V,:V, domains) of antibody R19.9 (Lascombe et al, 1989) (PDB file lF19), and the constant module, CL:CHl domains, of 1719 (Rini et al, 1992). The fast rotation function (Crowther, 1972) was used with 10.0-4.0-A resolution data and a radius of integration of 23.9 A ( Table 2).…”