Structural and biophysical insights into the role of the insert region in Rac1 function.
Structural and biophysical insights into the role of the insert region in Rac1 function.
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关于插入区域在 Rac1 功能中的作用的结构和生物物理学见解。
DOI：
10.1021/bi0120087
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Campbell,SharonL
中科院分区：
文献类型：
--
作者：
Thapar,Roopa;Karnoub,AntoineE;Campbell,SharonL
在 β 链 5 和 α 螺旋 4 之间形成一个短的 310 个螺旋的 13 个氨基酸插入是 GTP 酶 Rho 家族大多数成员的一个显着特征，但该区域在信号转导中的确切作用尚不清楚。先前的体内功能研究表明，RhoA、Rac1 和 Cdc42 的插入区域对于细胞转化、肌动蛋白细胞骨架的调节、控制 DNA 合成以及下游靶标的激活非常重要。在 Rac1 中，我们最近的生物学研究表明，插入片段对于 SRF 激活和板状伪足的形成很重要，但对于该蛋白质的所有其他细胞功能来说是可有可无的。在本文报道的研究中，我们描述了插入缺失对 Rac1 结构、热力学稳定性和核苷酸缔合动力学的影响。这些体外研究有助于澄清生物学数据，并提供关于这 13 个氨基酸区域在调节 Rac1 功能中的作用的进一步见解。研究表明，删除插入片段对 Rac1 结构没有影响，仅导致 Rac1·GDP·Mg2+ 的 ΔGfold 略有下降（∼0.8 kcal/mol）。与野生型相比，Rac1·Δinsis 的核苷酸解离的内在速率降低了约1.5倍，并且观察到GEF(Vav2)介导的交换速率增加了3倍。此外，插入片段的删除不会改变 Rac1 与 GDI 相互作用的 KD，并且与之前在 Cdc42 中观察到的类似，相对于天然蛋白，没有观察到删除突变体对 GDP 解离的抑制。总的来说，这里报告的结构和生化研究与我们之前报告的生物学数据一致，并表明插入区最可能的作用必须是充当下游效应器的结合界面，特别是那些对于肌动蛋白调节重要的效应器。
A 13 amino acid insertion that forms a short 310helix between β-strand 5 and α-helix 4 is a distinguishing feature among most members of the Rho family of GTPases, yet the precise role of this region in signal transduction is poorly understood. Previous in vivo functional studies have implicated the insert region of RhoA, Rac1, and Cdc42 to be important for cell transformation, regulation of the actin cytoskeleton, controlling DNA synthesis, and in the activation of downstream targets. In Rac1, our recent biological studies have suggested that the insert is important for SRF activation and the formation of lamellipodia but is dispensable for all other cellular functions of this protein. In the studies reported herein, we have characterized the effect of the insert deletion on Rac1 structure, thermodynamic stability, and the kinetics of nucleotide association. These in vitro studies help clarify biological data and provide further insights as to the role of this 13 amino acid region in modulating Rac1 function. The studies reveal that deletion of the insert has no effect on Rac1 structure and causes only a marginal (∼0.8 kcal/mol) decrease in the ΔGfoldof Rac1·GDP·Mg2+. The intrinsic rate of nucleotide dissociation of Rac1·Δinsis decreased by about 1.5-fold compared to that of wild type, and a 3-fold increase in the GEF (Vav2)-mediated exchange rate is observed. In addition, deletion of the insert does not change theKDfor the interaction of Rac1 with GDI, and similar to that previously observed for Cdc42, no inhibition of GDP dissociation is observed for the deletion mutant relative to that for the native protein. Taken together, the structural and biochemical studies reported here are consistent with our biological data reported previously and suggest that the most likely role of the insert region must be to serve as a binding interface for downstream effectors, particularly those important for actin regulation.
Ras 信号转导的复杂性不断增加：Rho 家族蛋白的参与。
影响因子：
--
作者：
Khosravi-Far,R;Campbell,S;Rossman,KL;Der,CJ
通讯作者：
Der,CJ