The hen (chicken) egg-white lysozyme (HEWL) epitope for the monoclonal antibody HyHEL-IO Fab (Fab-IO) was investigated by alanine scan mutagenesis. The association rate constants (/con) for the HEWL.Fab-10 complexes were obtained from the homogenous solution method described in the preceding paper (Taylor et al., 1998). A new method for determining the dissociation rate constant (kOff) for the complex, by trapping nascent free antibody with an inactive HEWL mutant is described. The values of k,, fall within a factor of 2 of the wild-type (WT) HEWL value (1.43 f 0.13 X lo6 M" s-'), while the increases in kc,w more nearly reflect the total change in free energies of the complex (AAG"). The dissociation constants (KO) were measured directly in those cases where satisfactory kinetic data could not be obtained. The Y20A, K96A, and K97A HEWL.Fab-IO complexes are destabilized by more than 4 kcal/mol compared to the WT complex. The R21A, L75A, and DlOlA antibody complexes are moderately destabilized (0.7 < AAGo 5 1 .O kcal/mol). Additional mutations of the "hotspot" residues (TYRO, Lys96, Lys97) were constructed to probe, more precisely, the nature of their contributions to complex formation. The results show that the entire hydrocarbon side chains of Tyr20 and Lys97, and only the s-amino group of Lys96, contribute to the stability of the complex. The value of AAG,] for the R21A mutant complex is a distinct outlier in the Arg2l replacement series demonstrating the importance of supplementing alanine scan mutagenesis with additional mutations.

The rate constants, k,,, for the formation of hen (chicken)

The complex formed between hen egg white lysozyme (HEL) and the monoclonal antibody HyHEL-10 Fab fragment has an interface composed of van der Waals interactions, hydrogen bonds, and a single ion pair. The antibody overlaps part of the active site cleft. Putative critical residues within the epitope region of HEL, identified from the x-ray crystallographic structure of the complex, were replaced by site-directed mutagenesis to probe their relative importance in determining affinity of the antibody for HEL. Twenty single mutations of HEL at three contact residues (Arg-21HEL, Asp-101HEL, and Gly-102HEL) and at a partially buried residue (Asn-19HEL) in the epitope were made, and the effects on the free energies of dissociation were measured. A correlation between increased amino acid side-chain volume and reduced affinity for HELs with mutations at position 101 was observed. The D101GHEL mutant is bound to HyHEL-10 as tightly as wild-type enzyme, but the AAGdi. is increased by about 2.2 kcal (9.2 kJ)/mol for the larger residues in this position. HEL variants with lysine or histidine replacements for arginine at position 21 are bound 1.4-2.7 times more tightly than those with neutral or negatively charged amino acids in this position.These exhibit 1/40 the affinity for HyHEL-10 Fab compared with wild type. There is no side-chain volume correlation with AAGdbwc at position 21. Although are in the epitope, replacements at these positions have no effect on the affinity of HEL for the antibody.A major goal of immunochemistry has been to provide a quantitative understanding of the specificity of immune receptors for antigens. Such knowledge requires the elucidation of both the structural and the functional bases of complementarity. We have focused on monoclonal antibodies (mAbs) specific for hen egg-white lysozyme (HEL) as a model for understanding protein-protein interactions in general. The detailed topography of the interaction between a mAb and a macromolecular antigen became clear when high-resolution x-ray structures for three HEL-mAb complexes were published (1-3). The three antibodies, D1.3, HyHEL-5, and HyHEL-10, recognize different sites (epitopes) on the HEL molecule. There is a small overlap of the D1.3 and HyHEL-10

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