Contribution of domain interface residues to the stability of antibody CH3 domain homodimers

Dimers of C(H)3 domains from human IgG(1) were used to study the effect of mutations constructed at a domain-domain interface upon domain dissociation and unfolding, "complex stability". Alanine replacement mutants were constructed on one side of the interface for each of the sixteen interdomain contact residues by using a single-chain C(H)3 dimer 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 variants were expressed in Escherichia coli grown in a fermenter and recovered in yields of 6-90 mg L-1 by immobilized metal affinity chromatography. Guanidine hydrochloride-induced denaturation was used to follow domain dissociation and unfolding. Surprisingly, the linker did not perturb the complex stability for either the wild type or two destabilizing mutants. The C(H)3 domain dissociation and unfolding energetics are dominated by six contact residues where corresponding alanine mutations each destabilize the complex by >2.0 kcal mol(-1). Five of these residues (T366, L368, F405, Y407, and K-409) form a patch at the center of the interface and are located on the two internal antiparallel beta-strands. These energetically key residues are surrounded by 10 residues 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 interface there is a small "functional interface" of residues that make significant contributions to complex stability.