IDENTIFICATION OF SRC, FYN, LYN, PI3K AND ABL SH3 DOMAIN LIGANDS USING PHAGE DISPLAY LIBRARIES
IDENTIFICATION OF SRC, FYN, LYN, PI3K AND ABL SH3 DOMAIN LIGANDS USING PHAGE DISPLAY LIBRARIES
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DOI：
10.1002/j.1460-2075.1994.tb06897.x
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发表时间：
1994-12-01
期刊：
影响因子：
11.4
通讯作者：
ZOLLER, MJ
中科院分区：
文献类型：
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作者：
RICKLES, RJ;BOTFIELD, MC;ZOLLER, MJ
许多参与细胞内信号转导的蛋白质含有小的50-60个氨基酸结构域，称为Src同源3（SH 3）结构域。该结构域似乎介导参与对细胞外信号的响应的关键蛋白质-蛋白质相互作用。先前的研究表明，来自几种蛋白质的SH 3结构域识别富含脯氨酸残基的短的连续氨基酸序列。虽然迄今为止鉴定的所有SH 3识别序列都具有保守的P-X-X-P基序，但对单个SH 3结构域的序列识别特异性知之甚少。我们已经采用了一种新的修改噬菌体展示涉及偏见库，以确定肽配体的Src，Fyn，林恩，PI 3 K和Abl SH 3结构域。利用有偏向的文库，我们在12个氨基酸窗口上探测SH 3识别。Src SH 3结构域优选序列XXXRPLPPLPXP，Fyn优选XXXRPLPP（I/L）PXX，林恩优选RXXRPLPPLPXP，PI 3 K优选RXXRPLPPLPPP，而Abl SH 3结构域选择含有序列PPPYPPPP（I/V）PXX的噬菌体。我们还分析了Abl和Src SH 3配体的结合性质。我们发现，尽管噬菌体展示的Abl和Src SH 3配体富含脯氨酸，但它们是不同的。在表面等离子体共振结合测定中，当序列展示在噬菌体表面上或作为合成肽时，这些SH 3结构域显示出与其同源配体的高度选择性结合。这些高亲和力SH 3肽配体的选择提供了关于SH 3结构域的识别基序的有价值的信息，作为干扰SH 3结构域介导的过程的细胞功能的新工具，并形成疾病途径的SH 3特异性抑制剂的设计基础。
Many proteins involved in intracellular signal transduction contain a small, 50-60 amino acid domain, termed the Src homology 3 (SH3) domain. This domain appears to mediate critical protein-protein interactions that are involved in responses to extracellular signals. Previous studies have shown that the SH3 domains from several proteins recognize short, contiguous amino acid sequences that are rich in proline residues. While all SH3 recognition sequences identified to date share a conserved P-X-X-P motif, the sequence recognition specificity of individual SH3 domains is poorly understood. We have employed a novel modification of phage display involving biased libraries to identify peptide ligands of the Src, Fyn, Lyn, PI3K and Abl SH3 domains. With biased libraries, we probed SH3 recognition over a 12 amino acid window. The Src SH3 domain prefers the sequence XXXRPLPPLPXP, Fyn prefers XXXRPLPP(I/L)PXX, Lyn prefers RXXRPLPPLPXP, PI3K prefers RXXRPLPPLPPP while the Abl SH3 domain selects phage containing the sequence PPPYPPPP(I/V)PXX. We have also analysed the binding properties of Abl and Src SH3 ligands. We find that although the phage-displayed Abl and Src SH3 ligands are proline rich, they are distinct. In surface plasmon resonance binding assays, these SH3 domains displayed highly selective binding to their cognate ligands when the sequences were displayed on the surface of the phage or as synthetic peptides. The selection of these high affinity SH3 peptide ligands provides valuable information on the recognition motifs of SH3 domains, serve as new tools to interfere with the cellular functions of SH3 domain-mediated processes and form the basis for the design of SH3-specific inhibitors of disease pathways.