与Src SH3结构域结合的FN3单结构域抗体的分子识别特性。

Molecular recognition properties of FN3 monobodies that bind the Src SH3 domain.

作者信息

Karatan Ece, Merguerian Matthew, Han Zhaozhong, Scholle Michael D, Koide Shohei, Kay Brian K

机构信息

Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439, USA.

出版信息

Chem Biol. 2004 Jun;11(6):835-44. doi: 10.1016/j.chembiol.2004.04.009.

DOI:10.1016/j.chembiol.2004.04.009

PMID:15217616

Abstract

We have constructed a phage-displayed library based on the human fibronectin tenth type III domain (FN3) scaffold by randomizing residues in its FG and BC loops. Screening against the SH3 domain of human c-Src yielded six different clones. Five of these contained proline-rich sequences in their FG loop that resembled class I (i.e., +xxPxxP) peptide ligands for the Src SH3 domain. The sixth clone lacked the proline-rich sequence and showed particularly high binding specificity to the Src SH3 domain among various SH3 domains tested. Competitive binding, loop replacement, and NMR perturbation experiments were conducted to analyze the recognition properties of selected binders. The strongest binder was able to pull down full-length c-Src from murine fibroblast cell extracts, further demonstrating the potential of this scaffold for use as an antibody mimetic.

摘要

我们通过随机化人纤连蛋白第十个III型结构域（FN3）支架的FG和BC环中的残基，构建了一个噬菌体展示文库。用人c-Src的SH3结构域进行筛选，得到了六个不同的克隆。其中五个在其FG环中含有富含脯氨酸的序列，类似于Src SH3结构域的I类（即+xxPxxP）肽配体。第六个克隆缺乏富含脯氨酸的序列，并且在测试的各种SH3结构域中对Src SH3结构域表现出特别高的结合特异性。进行了竞争性结合、环置换和NMR扰动实验，以分析所选结合物的识别特性。最强的结合物能够从小鼠成纤维细胞提取物中拉下全长c-Src，进一步证明了该支架用作抗体模拟物的潜力。