THE 3RD IGG-BINDING DOMAIN FROM STREPTOCOCCAL PROTEIN-G - AN ANALYSISBY X-RAY CRYSTALLOGRAPHY OF THE STRUCTURE ALONE AND IN A COMPLEX WITHFAB

Protein G is a cell surface-associated protein from Streptococcus that binds to IgG with high affinity. We have determined the X-ray crystal structures of the third IgG-binding domain (domain III) alone to a re solution of 1.1 Angstrom (final R-factor of 18.3%), and in complex wit h an Fab fragment to 2.6 Angstrom (final R-factor of 16.8%). The struc ture of domain III is similar to the lower-resolution crystal structur es of protein G domains determined previously by other investigators b ut shows some minor differences when compared with the equivalent NMR structures. Domain III binds to the immunoglobulin by formation of an antiparallel interaction between the second beta-strand in domain III and the last beta-strand in the C(H)1 domain. There is also a minor si te of interaction between the C-terminal end of the alpha-helix in pro tein G and the first beta-strand in the C(H)1 domain. Previous studies by NMR on the interactions between protein G and IgG have concluded t hat different portions of tie protein G domain are involved in binding to the Fab and Fc portions. The results presented here support these findings and permit a detailed analysis of the recognition of Fab by p rotein G; formation of the complex buries a large cater-accessible are a: of a magnitude comparable with that found in antibody/antigen inter actions. The majority of hydrogen bonds between the two proteins invol ve main-chain atoms from the C(H)1 domain. The C(H)1 domain residues t hat are in contact with protein G are shown to be highly conserved in alignments of mouse and human gamma chain amino acid sequences. We con clude that the binding site for protein G on Fab is relatively invaria nt across different species and gamma chain subclasses, providing an e xplanation for the widespread recognition of Fab fragments from mouse and human antibodies by protein G. The solution of tile crystal struct ures of domain III alone and bound to Fab has demonstrated that there is no major structural change apparent in either protein on formation of the complex.