Structure-activity relationships in flexible protein domains: Regulation of rho GTPases by RhoGDI and D4 GDI
Structure-activity relationships in flexible protein domains: Regulation of rho GTPases by RhoGDI and D4 GDI
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DOI：
10.1006/jmbi.2000.4262
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发表时间：
2001-01-05
影响因子：
5.6
通讯作者：
Roberts, GCK
中科院分区：
文献类型：
--
作者：
Golovanov, AP;Chuang, TH;Roberts, GCK
鸟嘌呤解离抑制剂 RhoGDI 和 D4GDI 抑制鸟苷 5'-二磷酸从 Rho GTPases 解离，使这些小 GTPase 保持非活性状态。 GDI 由两个结构域组成：约 70 个氨基酸残基的柔性 N 端结构域和折叠的 134 个残基 C 端结构域。在这里，我们使用一系列 NMR 实验（包括 N-15 弛豫和酰胺溶剂可及性测量）来表征 RhoGDI 和 D4GDI N 端区域的构象。在每种蛋白质中，鉴定出两个具有形成螺旋倾向的区域：RhoGDI 中的残基 36 至 58 和 9 至 20，以及 D4GDI 中的残基 36 至 57 和 20 至 25。为了检查 RhoGDI N 端结构域的功能作用，使用 N 端截短的蛋白质进行了体外和体内功能测定。这些研究表明，前 30 个氨基酸残基不是抑制 GDP 解离所必需的，但似乎对 GTP 水解很重要，而去除前 41 个残基则完全消除了 RhoGDI 抑制 GDP 解离的能力。结构和功能研究的结合使我们能够解释为什么 RhoGDI 和 D4GDI 能够以类似的方式与鸟苷 5'-二磷酸结合的 GTPase 相互作用，但它们调节 GTP 结合形式的能力不同；这些功能差异归因于鸟苷 5'-二磷酸解离抑制剂 N 端结构域的构象差异。因此，这两个瞬时螺旋似乎与 RhoGDI 的不同生物效应相关，为柔性蛋白质结构域中的结构-活性关系提供了清晰的例子。 (C) 2001 年学术出版社。
The guanine dissociation inhibitors RhoGDI and D4GDI inhibit guanosine 5'-diphosphate dissociation from Rho GTPases, keeping these small GTPases in an inactive state. The GDIs are made up of two domains: a flexible N-terminal domain of about 70 amino acid residues and a folded 134-residue C-terminal domain. Here, we characterize the conformation of the N-terminal regions of both RhoGDI and D4GDI using a series of NMR experiments which include N-15 relaxation and amide solvent accessibility measurements. In each protein, two regions with tendencies to form helices are identified: residues 36 to 58 and 9 to 20 in RhoGDI, and residues 36 to 57 and 20 to 25 in D4GDI. To examine the functional roles of the N-terminal domain of RhoGDI, in vitro and in vivo functional assays have been carried out with N-terminally truncated proteins. These studies show that the first 30 amino acid residues are not required for inhibition of GDP dissociation but appear to be important for GTP hydrolysis, whilst removal of the first 41 residues completely abolish the ability of RhoGDI to inhibit GDP dissociation. The combination of structural and functional studies allows us to explain why RhoGDI and D4GDI are able to interact in similar ways with the guanosine 5'-diphosphate-bound GTPase, but differ in their ability to regulate GTP-bound forms; these functional differences are attributed to the conformational differences of the N-terminal domains of the guanosine 5'-diphosphate dissociation inhibitors. Therefore, the two transient helices, appear to be associated with different biological effects of RhoGDI, providing a clear example of structure-activity relationships in a flexible protein domain. (C) 2001 Academic Press.