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 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 delta delta Gdissoc 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 delta delta Gdissoc at position 21. Although Gly-102HEL and Asn-19HEL are in the epitope, replacements at these positions have no effect on the affinity of HEL for the antibody.
Kam-Morgan
L N
LN
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Smith-Gill
S J
SJ
Taylor
M G
MG
Zhang
L
L
Wilson
A C
AC
Kirsch
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002645:Chickens; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009113:Muramidase