Mutational analysis of the interaction between staphylococcal protein A and human IgG1.
The interactions have been studied between an IgG-binding domain derivative based on domain B of staphylococcal protein A (designated Z) and human immunoglobulin G class 1 (IgG1) and its Fc fragment (Fc1) respectively. Five single amino acid substituted mutant forms of Z were constructed at the gene level, produced intracellularly in Escherichia coli, purified to homogeneity and characterized. Four of these Z variants, designated Z(L17D), Z(N28A), Z(I31A) and Z(K35A), were mutated in residues suggested to be involved in binding, based on the three-dimensional structure of the complex between a one domain protein A molecule and Fc1 [Deisenhofer, J. (1981) Biochemistry, 20, 2361-2370]. The fifth mutant protein, Z(F30A), had a mutation in a phenylalanine residue which was not expected to be involved in the interaction. Analysis by far UV circular dichroism spectroscopy suggests that all Z mutant proteins have similar folds. Their respective binding to human monoclonal IgG1 and to human recombinant Fc1 were studied in a competitive binding assay using radioactively labeled Z as a tracer, demonstrating that the mutant proteins with a substitution in the postulated binding surface showed a weakened binding to both the full-length antibody and the recombinant Fc1. The affinity constants of the interactions as well as relative binding free energies from the parent Z molecule were calculated. These values were similar for each Z variant to both IgG1 and Fc1, suggesting that Fc and not Fab binding was measured also for IgG1. However, the binding strengths differ significantly, and these binding properties were used to compare the contribution of each mutated amino acid residue in the Fc interaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Cedergren
L
L
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Andersson
R
R
Jansson
B
B
Uhlén
M
M
Nilsson
B
B
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001483:Base Sequence; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D014169:Transformation, Bacterial
D000911:Antibodies, Monoclonal; D006146:Guanidines; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D019791:Guanidine