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Biochemistry. 2011 Jul 19;50(28):6196-207. doi: 10.1021/bi2004284. Epub 2011 Jun 24.

A switch I mutant of Cdc42 exhibits less conformational freedom.

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  • 1Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, United States.


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 time scales [Loh, A. P., et al. (2001) Biochemistry 40, 4590-4600], and this flexibility may be essential for protein interactions. In the Switch I region, Thr(35) seems to be 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 suggest that the mutation changes the time scale of the internal motions of several residues, with several resonances not being discernible in wild-type Cdc42 [Adams, P. D., and Oswald, R. E. (2007) Biomol. NMR Assignments 1, 225-227]. The mutation does not appear to affect the thermal stability of Cdc42, and chymotrypsin digestion data further suggest that changes in the conformational flexibility of Switch I slow proteolytic cleavage relative to that of the wild type. In vitro binding assays show less binding of Cdc42(T35A), relative to that of wild type, to a GTPase binding protein that inhibits GTP hydrolysis in Cdc42. These results suggest that the mutation of T(35) leads to the loss of conformational freedom in Switch I that could affect effector-regulatory protein interactions.

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