Contribution of domain interface residues to the stability of antibody CH3 domain homodimers.
Dimers of CH3 domains from human IgG1 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 CH3 dimer in which the carboxyl terminus of one domain was joined to the amino terminus of the second domain via a (G4S)4 linker. Single-chain variants were expressed in Escherichia coli grown in a fermentor 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 CH3 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 K409) 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 DeltaG of 1.1-1.3 kcal mol-1) or very small (seven have a Delta DeltaG of </=0.7 kcal mol-1). Thus, at the center of the CH3 structural interface there is a small "functional interface" of residues that make significant contributions to complex stability.
Dall'Acqua
W
W
Department of Molecular Oncology, Genentech Inc., South San Francisco, California 94080, USA.
Simon
A L
AL
Mulkerrin
M G
MG
Carter
P
P
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D019281:Dimerization; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D011994:Recombinant Proteins; D000409:Alanine