Intrinsic GTP hydrolysis is observed for a switch 1 variant of Cdc42 in the presence of a specific GTPase inhibitor

Abstract

ABSTRACTThe Ras-related protein Cell division cycle 42 (Cdc42) is important in cell-signaling processes. Protein interactions involving Cdc42 occur primarily in flexible “Switch” regions that help regulate effector binding. We studied the kinetics of intrinsic GTP hydrolysis reaction in the absence and presence of a biologically active peptide derivative of a p21-activated kinase effector (PBD46) for wt Cdc42 and compared it to the Switch 1 variant Cdc42(T35A). While the binding of PBD46 to wt Cdc42 results in complete inhibition of GTP hydrolysis, this interaction in Cdc42(T35A) does not. Comparison of the crystal structure of wt Cdc42 in the absence of effector (1AN0.pdb), as well as the NMR structure of wt Cdc42 bound to an effector in the Switch 1 region (1CF4.pdb) (www.rcsb.org) suggests that the orientation of T35 with bound Mg2+ changes in the presence of effector, resulting in movement of GTP away from the catalytic box leading to the inhibition of GTP hydrolysis. For Cdc42(T35A), molecular dynamics simulations and structural analyses suggest that the nucleotide does not undergo the conformational shift observed for the wt Cdc42-effector interaction. Our data suggest that change in dynamics in the Switch 1 region of Cdc42 caused by the T35A mutation (Chandrashekar, et al. 2011, Biochemistry, 50, p. 6196) fosters a conformation for this Cdc42 variant that allows hydrolysis of GTP in the presence of PBD46, and that alteration of the conformational dynamics could potentially modulate Ras-related over-activity.