CONTRIBUTION OF DOMAIN INTERFACE RESIDUES TO THE STABILITY OF ANTIBODY C(H)3 DOMAIN HOMODIMERS

Dimers of C(H)3 domains from human IgG(1) were used to study the effec t of mutations constructed at a domain-domain interface upon domain di ssociation and unfolding, ''complex stability''. Alanine replacement m utants were constructed on one side of the interface for each of the s ixteen interdomain contact residues by using a single-chain C(H)3 dime r in which the carboxyl terminus of one domain was joined to the amino terminus of the second domain via a (G(4)S)(4) linker. Single-chain v ariants were expressed in Escherichia coli grown in a fermenter and re covered in yields of 6-90 mg L-1 by immobilized metal affinity chromat ography. Guanidine hydrochloride-induced denaturation was used to foll ow domain dissociation and unfolding. Surprisingly, the linker did not perturb the complex stability for either the wild type or two destabi lizing mutants. The C(H)3 domain dissociation and unfolding energetics are dominated by six contact residues where corresponding alanine mut ations each destabilize the complex by >2.0 kcal mol(-1). Five of thes e residues (T366, L368, F405, Y407, and K-409) form a patch at the cen ter of the interface and are located on the two internal antiparallel beta-strands. These energetically key residues are surrounded by 10 re sidues on the two external beta-strands whose contribution to complex stability is small (three have a Delta Delta G of 1.1-1.3 kcal mol(-1) ) or very small (seven have a Delta Delta G of less than or equal to 0 .7 kcal mol(-1)). Thus, at the center of the C(H)3 structural interfac e there is a small ''functional interface'' of residues that make sign ificant contributions to complex stability.