Characterization of the Binding Interface between the E-Domain of Staphylococcal Protein A and an Antibody Fv-Fragment

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• 作者：David P. Meininger ; Mark Rance ; Melissa A. Starovasnik ; Wayne J. Fairbrother ; and Nicholas J. Skelton
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：January 11, 2000
• 年：2000
• 卷：39
• 期：1
• 页码：26 - 36
• 全文大小：667K
• 年卷期：v.39,no.1(January 11, 2000)
• ISSN：1520-4995

文摘

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 V_H3gene family has been described. To characterize structurally the interface formed by SpA repeats andtype-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 NMRspectroscopy. Protocols were established for efficient incorporation of ¹⁵N, ¹³C, and ²H into EZ4 and theV_H- and V_L-domains of the Fv, allowing backbone resonances to be assigned sequentially for EZ4 andthe V_H-domain in both free and complexed states. Broadening of certain V_H-resonances in the free andbound Fv-fragment suggests microsecond to millisecond time-scale motion in CDR3. Residues experiencingsignificant chemical shift changes of backbone ¹H^N, ¹⁵N, and ¹³CO resonances upon complex formationdelineate contiguous surfaces on EZ4 and the V_H-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- andV_H3-domains.