| Abstract
| - Staphylococcal protein A (SpA) is a cell-surface component of Staphylococcus aureus. Inaddition to the well-characterized interaction between SpA and the Fc-region of human IgG, an alternativebinding interaction between SpA and the Fab-region of immunoglobulin domains encoded by the VH3gene family has been described. To characterize structurally the interface formed by SpA repeats andtype-3 VH-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 NMRspectroscopy. Protocols were established for efficient incorporation of 15N, 13C, and 2H into EZ4 and theVH- and VL-domains of the Fv, allowing backbone resonances to be assigned sequentially for EZ4 andthe VH-domain in both free and complexed states. Broadening of certain VH-resonances in the free andbound Fv-fragment suggests microsecond to millisecond time-scale motion in CDR3. Residues experiencingsignificant chemical shift changes of backbone 1HN, 15N, and 13CO resonances upon complex formationdelineate contiguous surfaces on EZ4 and the VH-domain that define the binding interfaces of the twoproteins. The interaction surfaces identified by chemical shift mapping are comprised of predominantlyhydrophilic residues. This is in contrast to the SpA−Fc interface which is predominantly hydrophobic innature. Further analysis of the surface properties suggests a probable binding orientation for SpA- andVH3-domains.
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