Characterization of the binding interface between the E-domain of Staphylococcal protein A and an antibody Fv-fragment.
Staphylococcal protein A (SpA) is a cell-surface component of Staphylococcus aureus. In addition to the well-characterized interaction between SpA and the Fc-region of human IgG, an alternative binding interaction between SpA and the Fab-region of immunoglobulin domains encoded by the V(H)3 gene family has been described. To characterize structurally the interface formed by SpA repeats and type-3 V(H)-domains, we have studied the 32-kDa complex formed between an E-domain mutant (EZ4) and the Fv-fragment of the humanized anti-HER2 antibody (Hu4D5-8) using heteronuclear NMR spectroscopy. Protocols were established for efficient incorporation of (15)N, (13)C, and (2)H into EZ4 and the V(H)- and V(L)-domains of the Fv, allowing backbone resonances to be assigned sequentially for EZ4 and the V(H)-domain in both free and complexed states. Broadening of certain V(H)-resonances in the free and bound Fv-fragment suggests microsecond to millisecond time-scale motion in CDR3. Residues experiencing significant chemical shift changes of backbone (1)H(N), (15)N, and (13)CO resonances upon complex formation delineate contiguous surfaces on EZ4 and the V(H)-domain that define the binding interfaces of the two proteins. The interaction surfaces identified by chemical shift mapping are comprised of predominantly hydrophilic residues. This is in contrast to the SpA-Fc interface which is predominantly hydrophobic in nature. Further analysis of the surface properties suggests a probable binding orientation for SpA- and V(H)3-domains.
Meininger
D P
DP
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.
Rance
M
M
Starovasnik
M A
MA
Fairbrother
W J
WJ
Skelton
N J
NJ
D000595:Amino Acid Sequence; D001666:Binding Sites, Antibody / Q000235:genetics; D005822:Genetic Vectors / Q000138:chemical synthesis; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010449:Peptide Mapping; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D013816:Thermodynamics
D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D018719:Erb-b2 Receptor Tyrosine Kinases; D013205:Staphylococcal Protein A; C055966:immunoglobulin Fv