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Determination of the sequence specificity of XIAP BIR domains by screening a combinatorial peptide library

机译：通过筛选组合肽库确定XIAP BIR域的序列特异性

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摘要

Inhibitor of apoptosis (IAP) proteins regulate programmed cell death by inhibiting members of the caspase family of proteases. The X-chromosome-linked IAP (XIAP) contains three baculovirus IAP repeat (BIR) domains, which bind directly to the N-termini of target proteins including those of caspases-3, -7, and -9. In the present study, we defined the consensus sequences of the motifs that interact with the three BIR domains in an unbiased manner. A combinatorial peptide library containing four random residues at the N-terminus was constructed and screened using BIR domains as probes. We found that the BIR3 domain binds a highly specific motif containing an alanine or valine at the N-terminus (P1 position), an arginine or proline at the P3 position, and a hydrophobic residue (Phe, Ile, and Tyr) at the P4 position. The BIR2-binding motif is less stringent. Although it still requires an N-terminal alanine, it tolerates a wide variety of amino acids at P2-P4 positions. The BIR1 failed to bind to any peptides in the library. SPR analysis of individually synthesized peptides confirmed the library screening results. Database searches with the BIR2- and BIR3-binding consensus sequences revealed a large number of potential target proteins. The combinatorial library method should be readily applicable to other BIR domains or other types of protein modular domains.
机译：凋亡抑制剂（IAP）蛋白通过抑制蛋白酶caspase家族的成员来调节程序性细胞死亡。 X染色体连接的IAP（XIAP）包含三个杆状病毒IAP重复（BIR）域，它们直接与目标蛋白的N末端结合，包括caspases-3，-7和-9的蛋白质。在本研究中，我们定义了与三个BIR结构域以无偏方式相互作用的基序的共有序列。构建并在N端包含四个随机残基的组合肽库，并使用BIR域作为探针进行筛选。我们发现，BIR3域结合了一个高度特异性的基序，该基序在N端（P1位置）包含丙氨酸或缬氨酸，在P3位置包含精氨酸或脯氨酸，在P4处包含疏水残基（Phe，Ile和Tyr）。位置。 BIR2结合基序不太严格。尽管它仍然需要N端丙氨酸，但它可以耐受P2-P4位置上的多种氨基酸。 BIR1未能与文库中的任何肽结合。单独合成的肽的SPR分析证实了文库筛选结果。数据库搜索与BIR2和BIR3结合的共识序列揭示了大量潜在的目标蛋白。组合文库方法应易于应用于其他BIR域或其他类型的蛋白质模块化域。

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