Rossman Kent L, Worthylake David K, Snyder Jason T, Cheng Li, Whitehead Ian P, Sondek John
Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 2002 Dec 27;277(52):50893-8. doi: 10.1074/jbc.M208580200. Epub 2002 Oct 24.
Guanine nucleotide exchange factors (GEFs) directly engage small GTPases to facilitate the exchange of bound GDP for GTP, leading to GTPase activation. Several recent crystal structures of GEFs in complex with Rho family GTPases highlight the conserved interactions and conformational alterations necessary for catalyzing exchange. In the present study, functional roles were defined for specific residues within Cdc42 implicated by the crystal structures as important for physiological exchange of guanine nucleotides within Rho GTPases. In particular, this study highlights the paramount importance of the phosphate-binding loop and interactions with the magnesium co-factor as critical for proper regulation of RhoGEF-catalyzed exchange. Other conformational alterations of the GTPases affecting interactions with the sugar and base of guanine nucleotides are also important but are secondary. Of particular note, substitution of alanine for cysteine at position 18 of Cdc42 leads to a fast cycling phenotype for Cdc42 with heightened affinity for RhoGEFs and produces a dominant negative form of Cdc42 capable of inhibiting RhoGEFs both in vitro and in vivo.
鸟嘌呤核苷酸交换因子（GEFs）直接作用于小GTP酶，促进结合的GDP与GTP的交换，从而导致GTP酶激活。最近一些GEF与Rho家族GTP酶复合物的晶体结构突出了催化交换所需的保守相互作用和构象改变。在本研究中，确定了Cdc42内特定残基的功能作用，晶体结构表明这些残基对于Rho GTP酶内鸟嘌呤核苷酸的生理交换很重要。特别是，本研究强调了磷酸结合环以及与镁辅助因子的相互作用对于正确调节RhoGEF催化的交换至关重要。GTP酶的其他构象改变影响与鸟嘌呤核苷酸的糖和碱基的相互作用也很重要，但属于次要因素。特别值得注意的是，将Cdc42第18位的半胱氨酸替换为丙氨酸会导致Cdc42出现快速循环表型，对RhoGEF的亲和力增强，并产生一种在体外和体内均能抑制RhoGEF的Cdc42显性负性形式。
