Mapping of synergistic components of weakly interacting protein-protein motifs using arrays of paired peptides
Mapping of synergistic components of weakly interacting protein-protein motifs using arrays of paired peptides
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DOI：
10.1074/jbc.m211887200
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发表时间：
2003-04-25
影响因子：
4.8
通讯作者：
van Huijsduijnen, RH
中科院分区：
文献类型：
--
作者：
Espanel, X;Wälchli, S;van Huijsduijnen, RH
蛋白质-蛋白质识别通常涉及分散在蛋白质表面的不同基序之间的多种相互作用。为了识别这种弱相互作用，我们开发了一种新型双肽合成（DS）方法。该方法使我们能够通过使用空间可寻址的合成肽协同对来绘制涉及多肽中两个线性不连续成分的蛋白质-蛋白质相互作用图。 DS 程序基于“SPOT”膜结合肽合成技术，但为了合成两种肽的混合物，它在第一个循环中同时使用 Fmoc (N-(9-芴基)甲氧基羰基))-丙氨酸和 Alloc-丙氨酸。这使得它们可以分别通过哌啶或三丁基锡/钯处理来选择性脱保护。使用 SPOT DS，我们证实了 ERK-2 激酶对 Elk-1 Ser(383) 磷酸化的原理证明，Elk-1 对接结构域的存在会刺激 Elk-1 Ser(383) 磷酸化。 SPOT DS 还可用于剖析定义磷酸酶底物亲和力的蛋白质-蛋白质基序。利用这项技术，我们确定了胰岛素受体中的三个新区域，它们通过蛋白酪氨酸磷酸酶 (PTP) 1B 刺激受体去磷酸化，并可能增加 PTP 对这种底物的选择性。这些数据表明，SPOT DS 技术可以识别非线性弱相互作用的蛋白质基序，这些基序是蛋白质、激酶和磷酸酶底物特异性以及一般蛋白质-蛋白质相互作用的重要决定因素。
Protein-protein recognition usually involves multiple interactions among different motifs that are scattered over protein surfaces. To identify such weak interactions, we have developed a novel double peptide synthesis (DS) method. This method allows us to map protein-protein interactions that involve two linear discontinuous components from a polypeptide by the use of spatially addressable synergistic pairs of synthetic peptides. The DS procedure is based on the "SPOT" membrane-bound peptide synthesis technique, but to synthesize a mixture of two peptides, it uses both Fmoc (N-(9-fluorenyl)methoxycarbonyl))-alanine and Alloc-alanine at the first cycle. This allows their selective deprotection by either piperidine or tributyltin/palladium treatment, respectively. Using SPOT DS, we confirmed as a proof of principle that Elk-1 Ser(383) phosphorylation by ERK-2 kinase is stimulated by the presence of the Elk-1-docking domain. SPOT DS can also be used to dissect protein-protein motifs that define phosphatase substrate affinity. Using this technique, we identified three new regions in the insulin receptor that stimulate the dephosphorylation of the receptor by protein-tyrosine phosphatase (PTP) 1B and presumably increase the selectivity of PTP for this substrate. These data demonstrate that the SPOT DS technique allows the identification of non-linear weakly interacting protein motifs, which are an important determinant of protein, kinase and phosphatase substrate specificity and of protein-protein interactions in general.