A switch I mutant of Cdc42 exhibits less conformational freedom.
A switch I mutant of Cdc42 exhibits less conformational freedom.
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DOI：
10.1021/bi2004284
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发表时间：
2011-07-19
期刊：
影响因子：
2.9
通讯作者：
Adams, Paul D.
中科院分区：
文献类型：
--
作者：
Chandrashekar, Reena;Salem, Omar;Krizova, Hana;McFeeters, Robert;Adams, Paul D.
Cdc42是一种ras相关的小g蛋白，在与细胞生长和分化相关的信号转导途径中起着分子开关的作用。它通过gtp结合（活性）和gdp结合（非活性）形式之间的循环来控制。核苷酸的结合和水解是通过与效应器和/或调节蛋白的相互作用来调节的。这些相互作用集中在两个相对灵活的“开关”区域，其特征是在多个时间尺度上的内部动态（Loh et al., (2001) Biochemistry 40, 4590-4600），这种灵活性可能对蛋白质相互作用至关重要。在Switch I区域，Thr35似乎对功能至关重要，因为它在ras相关蛋白中是完全不变的。为了研究Cdc42开关I构象柔韧性的重要性，我们将苏氨酸突变为丙氨酸，确定了单点突变蛋白Cdc42（T35A）的溶液结构并表征了其骨架动力学。主干动力学数据表明，突变改变了几个残基内部运动的时间尺度，在Cdc42野生型中出现了几个无法识别的共振（Adams和Oswald（2007）生物分子核磁共振分配1,225 - 227）。该突变似乎不会影响Cdc42的热稳定性，并且胰凝乳酶消化数据进一步表明，相对于野生型，Switch I构象柔韧性的变化减缓了蛋白水解裂解。体外结合实验显示，与野生型相比，Cdc42（T35A）与抑制GTP水解的GTP酶结合蛋白的结合减少。这些结果表明，T35的突变导致开关I的构象自由丧失，从而影响效应蛋白/调节蛋白的相互作用。
Cdc42 is a Ras-related small G-protein, and functions as a molecular switch in signal transduction pathways linked with cell growth and differentiation. It is controlled by cycling between GTP-bound (active) and GDP-bound (inactive) forms. Nucleotide binding and hydrolysis are modulated by interactions with effectors and/or regulatory proteins. These interactions are centralized in two relatively flexible “Switch” regions as characterized by internal dynamics on multiple timescales (Loh et al., (2001) Biochemistry 40, 4590–4600), and this flexibility may be essential for protein interactions. In the Switch I region, Thr35 seems critical for function, as it is completely invariant in Ras-related proteins. To investigate the importance of conformational flexibility in Switch I of Cdc42, we mutated threonine to alanine, determined the solution structure and characterized the backbone dynamics of the single-point mutant protein, Cdc42(T35A). Backbone dynamics data suggests that the mutation changes the timescale of the internal motions of several residues, with several resonances appearing not discernable in Cdc42 wild type (Adams and Oswald (2007) Biomolecular NMR Assignments 1, 225–227). The mutation does not appear to affect the thermal stability of Cdc42, and chymotrypsin digestion data further suggests that changes in conformational flexibility in Switch I slow proteolytic cleavage relative to wild type. In-vitro binding assays show reduced binding of Cdc42(T35A), relative to wild type, to a GTPase binding protein that inhibits GTP hydrolysis in Cdc42. These results suggest that the mutation of T35 leads to the loss of conformational freedom in Switch I that could affect effector/regulatory protein interactions.
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影响因子：
5.7
作者：
Buhrman, Greg;Wink, Glenna;Mattos, Carla
通讯作者：
Mattos, Carla
影响因子：
2.9
作者：
Geyer, M;Schweins, T;Kalbitzer, HR
通讯作者：
Kalbitzer, HR
DOI：
10.1107/s0907444998003254
发表时间：
1998-09-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Brunger, AT;Adams, PD;Warren, GL
通讯作者：
Warren, GL
影响因子：
2.7
作者：
DELAGLIO, F;GRZESIEK, S;BAX, A
通讯作者：
BAX, A
影响因子：
2.9
作者：
Adams, PD;Loh, AP;Oswald, RE
通讯作者：
Oswald, RE