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

Abstract

The 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 T 35 with bound Mg 2C 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.

SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits

“…We refer to references2223242526 for recent reviews on the mechanisms of C9ORF72 -mediated neurotoxicity.…”

“…The pathophysiology mediated by C9ORF72 repeat expansions potentially involves three extensively-studied mechanisms which may all contribute to neuronal injury and disease progression: (i) RNA toxic gain-of-function by sequestration of RNA-binding factors 8 9 10 11 12 ; (ii) protein toxic gain-of-function due to repeat-associated non-ATG (RAN) translation that occurs in all sense and antisense reading frames to produce five dipeptide-repeat proteins (DPRs) 6 13 14 15 16 ; (iii) haploinsufficiency due to decreased expression of the C9ORF72 protein 2 17 18 which has recently been shown to play a key role in the Rab GTPase-dependent regulation of autophagy 19 20 21 . We refer to references 22 23 24 25 26 for recent reviews on the mechanisms of C9ORF72 -mediated neurotoxicity.…”

Characterizing Protein Conformational Spaces using Dimensionality Reduction and Algebraic Topology

“…To visualize the topology of the conformational The Switch-I region (residues 25-37) in blue, the Switch-II region (residues 57-75) in orange and the insert region (residues 122-135) in red. It can be shown that these three regions represent the highest variability, consistent with the fact that they are involved with binding and activation [62], [63]. These two clusters are then compared to each other to deduce conformational information.…”

Two Small Molecules, ZCL278 and AZA197 Show Promise in Influencing Protein Interactions Involving the Ras-Related Protein Cell division cycle 42 [Cdc42] to Modulate Its Oncogenic Potential

“…While experimental validation is still needed, these findings highlight that the Switch 1 binding region may serve as a prime region for small molecules to bind and possibly alter Cdc42-effector interactions. Studies from this laboratory have characterized how backbone dynamics changes in the Switch 1 region of a Cdc42 variant can alter a protein interaction with a peptide derivative of a GTPase inhibitor and restore normal GTP hydrolytic activity [10]. Therefore, it should be possible for a small molecule such as ZCL278 to bind to the Switch 1 region in Cdc42 and change its structure or alter backbone dynamics of the protein to disturb or block the Cdc42-effector PPI.…”

“…Furthermore, these residues might not need to occupy large surfaces on the protein to influence the PPI. Recent biophysical and biochemical studies, using Cdc42, have characterized the structurally dynamic Switch 1 region that serves as the binding interface for a GTPase inhibitor protein that fosters Cdc42 hyperactive state [10] [11]. These studies suggested that Switch 1 could serve as a prime region for small molecule targeting to modulate Cdc42 structure and conformational dynamics that may influence PPIs.…”