Alternate arrangement of PpL B3 domain and SpA D domain creates synergistic double-site binding to VH3 and Vkappa regions of fab.

Shaohua H Jiang, Jufang F Wang, Rong Xu, Yanjun J Liu, Xiaoning N Wang, Jie Cao, Ping Zhao, Yijun J Shen, Tong Yang, Hua Yang, Jiaan A Jia, Qiuli L Chen, Wei Pan

Department of Microbiology, Second Military Medical University, Shanghai, China.

DNA and cell biology 2008 Aug

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In our previous study, a kind of novel hybrid immunoglobulin (Ig)-binding proteins (IBPs) was obtained with the characteristic structure of alternately arranged Finegoldia magna (formerly Peptostreptococcus magnus) protein L (P. magnus protein L, PpL) B3 domain (B3) and Staphylococcal protein A (SpA) D domain (D). In this study, two representative molecules of these novel proteins, LD3 (B3-D-B3) and LD5 (B3-D-B3-D-B3) (LD3/5), showed substantially higher affinity for IgG-F(ab')2, IgM, and IgA than 4L (B3-B3-B3-B3) or SpA, which were also demonstrated by surface plasmon resonance detection. Further, LD5 showed much stronger binding to single-chain Fv (scFv) KM38 (V(H)3-V(kappa)I) than to KM41 (V(H)1-V(kappa)III) or KM36 (V(H)3-V(kappa)III). Competitive inhibition studies showed that 4L alone or in combination with SpA (4L + SpA) was a weaker inhibitor than LD3/5 in inhibiting LD3/5's binding to IgG-F(ab')2, IgM, or IgA. The computer modeling suggested that the B3-D arrangement in LD3/5 could simultaneously bind to V(H)3 and V(kappa). Thus, our results indicated for the first time that alternate arrangement of B3 and D domains creates synergistic double-site binding to V(H)3 and V(kappa) regions of fragment of antigen binding. The different competitive inhibition pattern of binding of LD5 to scFv KM38 by 4L + SpA suggested strict use of antibody conformation for this simultaneous double-site binding. The demonstration of this novel binding property would promote to achieve the designed hybrid IBPs for useful immunological applications.

Citation

Shaohua H Jiang, Jufang F Wang, Rong Xu, Yanjun J Liu, Xiaoning N Wang, Jie Cao, Ping Zhao, Yijun J Shen, Tong Yang, Hua Yang, Jiaan A Jia, Qiuli L Chen, Wei Pan. Alternate arrangement of PpL B3 domain and SpA D domain creates synergistic double-site binding to VH3 and Vkappa regions of fab. DNA and cell biology. 2008 Aug;27(8):423-31