Owen Darerca, Campbell Louise J, Littlefield Keily, Evetts Katrina A, Li Zhigang, Sacks David B, Lowe Peter N, Mott Helen R
Department of Biochemistry, University of Cambridge, 80 Tennis Court Rd., Cambridge CB2 1GA, United Kingdom.
Department of Biochemistry, University of Cambridge, 80 Tennis Court Rd., Cambridge CB2 1GA, United Kingdom.
J Biol Chem. 2008 Jan 18;283(3):1692-1704. doi: 10.1074/jbc.M707257200. Epub 2007 Nov 5.
IQGAP1 contains a domain related to the catalytic portion of the GTPase-activating proteins (GAPs) for the Ras small G proteins, yet it has no RasGAP activity and binds to the Rho family small G proteins Cdc42 and Rac1. It is thought that IQGAP1 is an effector of Rac1 and Cdc42, regulating cell-cell adhesion through the E-cadherin-catenin complex, which controls formation and maintenance of adherens junctions. This study investigates the binding interfaces of the Rac1-IQGAP1 and Cdc42-IQGAP1 complexes. We mutated Rac1 and Cdc42 and measured the effects of mutations on their affinity for IQGAP1. We have identified similarities and differences in the relative importance of residues used by Rac1 and Cdc42 to bind IQGAP1. Furthermore, the residues involved in the complexes formed with IQGAP1 differ from those formed with other effector proteins and GAPs. Relatively few mutations in switch I of Cdc42 or Rac1 affect IQGAP1 binding; only mutations in residues 32 and 36 significantly decrease affinity for IQGAP1. Switch II mutations also affect binding to IQGAP1 although the effects differ between Rac1 and Cdc42; mutation of either Asp-63, Arg-68, or Leu-70 abrogate Rac1 binding, whereas no switch II mutations affect Cdc42 binding to IQGAP1. The Rho family "insert loop" does not contribute to the binding affinity of Rac1/Cdc42 for IQGAP1. We also present thermodynamic data pertaining to the Rac1/Cdc42-RhoGAP complexes. Switch II contributes a large portion of the total binding energy to these complexes, whereas switch I mutations also affect binding. In addition we identify "cold spots" in the Rac1/Cdc42-RhoGAP/IQGAP1 interfaces. Competition data reveal that the binding sites for IQGAP1 and RhoGAP on the small G proteins overlap only partially. Overall, the data presented here suggest that, despite their 71% identity, Cdc42 and Rac1 appear to have only partially overlapping binding sites on IQGAP1, and each uses different determinants to achieve high affinity binding.
IQGAP1含有一个与Ras小G蛋白的GTP酶激活蛋白（GAPs）催化部分相关的结构域，但它没有RasGAP活性，而是与Rho家族小G蛋白Cdc42和Rac1结合。据认为，IQGAP1是Rac1和Cdc42的效应器，通过E-钙黏蛋白-连环蛋白复合体调节细胞间黏附，该复合体控制着黏附连接的形成和维持。本研究调查了Rac1-IQGAP1和Cdc42-IQGAP1复合体的结合界面。我们对Rac1和Cdc42进行了突变，并测量了突变对它们与IQGAP1亲和力的影响。我们确定了Rac1和Cdc42用于结合IQGAP1的残基相对重要性方面的异同。此外，与IQGAP1形成复合体所涉及的残基与与其他效应蛋白和GAPs形成复合体所涉及的残基不同。Cdc42或Rac1的开关I中相对较少的突变会影响IQGAP1的结合；只有32位和36位残基的突变会显著降低对IQGAP1的亲和力。开关II突变也会影响与IQGAP1的结合，尽管Rac1和Cdc42的影响有所不同；Asp-63、Arg-68或Leu-70中的任何一个突变都会消除Rac1的结合，而开关II的突变均不影响Cdc42与IQGAP1的结合。Rho家族的“插入环”对Rac1/Cdc42与IQGAP1的结合亲和力没有贡献。我们还提供了与Rac1/Cdc42-RhoGAP复合体相关的热力学数据。开关II对这些复合体的总结合能贡献很大一部分，而开关I的突变也会影响结合。此外，我们确定了Rac1/Cdc42-RhoGAP/IQGAP1界面中的“冷点”。竞争数据表明，小G蛋白上IQGAP1和RhoGAP的结合位点仅部分重叠。总体而言，此处提供的数据表明，尽管Cdc42和Rac1有71%的同一性，但它们在IQGAP1上的结合位点似乎仅部分重叠，且各自使用不同的决定因素来实现高亲和力结合。
