Dynamic interaction between Arf GAP and PH domains of ASAP1 in the regulation of GAP activity.
Dynamic interaction between Arf GAP and PH domains of ASAP1 in the regulation of GAP activity.
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DOI：
10.1016/j.cellsig.2008.07.007
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发表时间：
2008-11
影响因子：
4.8
通讯作者：
Gruschus, James
中科院分区：
文献类型：
--
作者：
Luo, Ruibai;Jenkins, Lisa M. Miller;Randazzo, Paul A.;Gruschus, James
关键词：
ASAP家族Arf GAPs诱导与Ras超家族蛋白Arf1结合的GTP水解，调节细胞粘附和迁移，并与癌变有关。ASAP蛋白具有PH、Arf、GAP和Ank重复结构域的核心催化结构域。PH结构域对于ASAP1的生物和催化功能都是必需的，并且已经被提出与Arf GAP结构域完整折叠。先前报道的保护性研究和分析性超离心研究表明，这些结构域至少部分折叠在一起。在这里，利用核磁共振波谱和生化分析，我们进一步验证了这一假设，并表征了域间相互作用。由ASAP1分离的PH结构域、Arf GAP和锚蛋白重复结构域或全部三个结构域组成的三种重组蛋白的NMR谱比较表明，PH结构域确实与Arf GAP和Ank重复结构域相互作用；然而，我们发现PH和Arf GAP结构域之间存在大量的动态独立性，这与相互作用是短暂的一致。相反，Arf GAP和Ank重复结构域形成相对刚性的结构。PH-Arf GAP结构域的相互作用部分阻断了可溶性蛋白中的磷酸肌苷结合位点，但结合研究表明，PIP2结合位点在与脂质双分子层表面结合的ASAP1中是可接近的。磷酸肌肽结合改变了PH结构域的构象，但对Arf GAP结构域的结构影响不大。连接Arf GAP结构域的PH结构域环中的突变影响了PIP2结合以及将Arf1•GTP转化为Arf1•GDP的Km和kcat。基于这些结果，我们建立了复合PH/Arf GAP/Ank重复结构域结构的同源性模型。我们提出PH结构域通过结合或定位Arf1•GTP来促进Arf GAP活性，Arf1•GTP同时与Arf GAP结构域结合。
ASAP family Arf GAPs induce the hydrolysis of GTP bound to the Ras superfamily protein Arf1, regulate cell adhesion and migration and have been implicated in carcinogenesis. The ASAP proteins have a core catalytic domain of PH, Arf GAP and Ank repeat domains. The PH domain is necessary for both biological and catalytic functions of ASAP1 and has been proposed to be integrally folded with the Arf GAP domain. Protection studies and analytical ultracentrifugation studies previously reported indicated that the domains are, at least partly, folded together. Here, using NMR spectroscopy and biochemical analysis, we have further tested this hypothesis and characterized the interdomain interaction. A comparison of NMR spectra of three recombinant proteins comprised of either the isolated PH domain of ASAP1, the Arf GAP and ankyrin repeat domain or all three domains indicated that the PH domain did interact with the Arf GAP and Ank repeat domains; however, we found a significant amount of dynamic independence between the PH and Arf GAP domains, consistent with the interactions being transient. In contrast, the Arf GAP and Ank repeat domains form a relatively rigid structure. The PH-Arf GAP domain interaction partially occluded the phosphoinositide binding site in the soluble protein, but binding studies indicated the PIP2 binding site was accessible in ASAP1 bound to a lipid bilayer surface. Phosphoinositide binding altered the conformation of the PH domain, but had little effect on the structure of the Arf GAP domain. Mutations in a loop of the PH domain that contacts the Arf GAP domain affected PIP2 binding and the Km and kcat for converting Arf1•GTP to Arf1•GDP. Based on these results, we generated a homology model of a composite PH/Arf GAP/Ank repeat domain structure. We propose that the PH domain contributes to Arf GAP activity by either binding to or positioning Arf1•GTP that is simultaneously bound to the Arf GAP domain.
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