HIGH-RESOLUTION MAPPING OF THE HYHEL-10 EPITOPE OF CHICKEN LYSOZYME BY SITE-DIRECTED MUTAGENESIS

The complex formed between hen egg white lysozyme (HEL) and the monocl onal antibody HyHEL-10 Fab fragment has an interface composed of van d er Waals interactions, hydrogen bonds, and a single ion pair. The anti body overlaps part of the active site cleft. Putative critical residue s within the epitope region of HEL, identified from the x-ray crystall ographic structure of the complex, were replaced by site-directed muta genesis 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 bu ried 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 D101G(HEL) mutant is bound to HyHEL-10 as tightly as wild-type enzyme, but the DELTADELTAG( dissoc) is increased by about 2.2 kcal (9.2 kJ)/mol for the larger res idues in this position. HEL variants with lysine or histidine replacem ents 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 posi tion. These exhibit 1/40 the affinity for HyHEL-10 Fab compared with w ild type. There is no side-chain volume correlation with DELTADELTAG(d issoc) at position 21. Although Gly-102HEL and Asn-19HEL are in the ep itope, replacements at these positions have no effect on the affinity of HEL for the antibody.