title: A switch I mutant of Cdc42 exhibits less conformational freedom.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, United States.
authors.forename: Reena
authors.initials: R
authors.lastname: Chandrashekar
authors.forename: Omar
authors.initials: O
authors.lastname: Salem
authors.forename: Hana
authors.initials: H
authors.lastname: Krizova
authors.forename: Robert
authors.initials: R
authors.lastname: McFeeters
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D018360:Crystallography, X-Ray; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D015398:Signal Transduction / Q000235:genetics; D013912:Threonine / Q000235:genetics; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry* / Q000235:genetics*
chemical_list: D013912:Threonine; D020764:cdc42 GTP-Binding Protein; D000409:Alanine