An Experimentally Based Computer Search Identifies Unstructured Membrane-binding Sites in Proteins APPLICATION TO CLASS I MYOSINS, PAKS, AND CARMIL
An Experimentally Based Computer Search Identifies Unstructured Membrane-binding Sites in Proteins APPLICATION TO CLASS I MYOSINS, PAKS, AND CARMIL
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DOI：
10.1074/jbc.m109.066910
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发表时间：
2010-02-19
影响因子：
4.8
通讯作者：
Korn, Edward D.
中科院分区：
文献类型：
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作者：
Brzeska, Hanna;Guag, Jake;Korn, Edward D.
存在用于搜索潜在的膜穿透片段(疏水区)和具有高度定义的三级结构的脂结合位点的蛋白质序列的程序，例如PH、FERM、C2、THEN和其他结构域。然而，越来越多的膜相关蛋白(包括细胞骨架蛋白、激酶、GTP结合蛋白及其效应物)通过结构较差的区域与脂类结合。在这里，我们描述了一种简单的计算机搜索程序的开发和测试，该程序可以识别非结构化潜在的膜结合位点。最初，我们发现碱性氨基酸和疏水性氨基酸都有助于与合成多肽的酸性磷脂小泡结合，这些多肽与棘阿米巴I类肌球蛋白推测的膜结合结构域相对应。基于这些结果，我们修改了疏水性标度，给出了Arg和Lys-的正值，而不是负值。使用这个基本的疏水尺度和标准的搜索算法，我们成功地在所有测试的16个蛋白质中确定了先前确定的非结构膜结合位点。重要的是，基本和疏水搜索在I类肌球蛋白、PAK和CARMIL(封闭蛋白，Arp2/3，肌球蛋白I连接物；膜相关细胞骨架蛋白)中发现了先前未知的潜在膜结合位点，以及包含这些新发现的体外酸性磷脂结合位点的合成肽和蛋白结构域。
Programs exist for searching protein sequences for potential membrane-penetrating segments (hydrophobic regions) and for lipid-binding sites with highly defined tertiary structures, such as PH, FERM, C2, ENTH, and other domains. However, a rapidly growing number of membrane-associated proteins (including cytoskeletal proteins, kinases, GTP-binding proteins, and their effectors) bind lipids through less structured regions. Here, we describe the development and testing of a simple computer search program that identifies unstructured potential membrane-binding sites. Initially, we found that both basic and hydrophobic amino acids, irrespective of sequence, contribute to the binding to acidic phospholipid vesicles of synthetic peptides that correspond to the putative membrane-binding domains of Acanthamoeba class I myosins. Based on these results, we modified a hydrophobicity scale giving Arg- and Lys-positive, rather than negative, values. Using this basic and hydrophobic scale with a standard search algorithm, we successfully identified previously determined unstructured membrane-binding sites in all 16 proteins tested. Importantly, basic and hydrophobic searches identified previously unknown potential membrane-binding sites in class I myosins, PAKs and CARMIL (capping protein, Arp2/3, myosin I linker; a membrane-associated cytoskeletal scaffold protein), and synthetic peptides and protein domains containing these newly identified sites bound to acidic phospholipids in vitro.