Electrostatic Forces Mediate the Specificity of RHO GTPase-GDI Interactions

Abstract

Three decades of research have documented the spatiotemporal dynamics of RHO family GTPase membrane extraction regulated by guanine nucleotide dissociation inhibitors (GDIs), but the interplay of the kinetic mechanism and structural specificity of these interactions is as yet unresolved. To address this, we reconstituted the GDI-controlled spatial segregation of geranylgeranylated RHO protein RAC1 in vitro. Various biochemical and biophysical measurements provided unprecedented mechanistic details for GDI function with respect to RHO protein dynamics. We determined that membrane extraction of RHO GTPases by GDI occurs via a 3-step mechanism: (1) GDI non-specifically associates with the switch regions of the RHO GTPases; (2) an electrostatic switch determines the interaction specificity between the C-terminal polybasic region of RHO GTPases and two distinct negatively-charged clusters of GDI1; (3) a non-specific displacement of geranylgeranyl moiety from the membrane sequesters it into a hydrophobic cleft, effectively shielding it from the aqueous milieu. This study substantially extends the model for the mechanism of GDI-regulated RHO GTPase extraction from the membrane, and could have implications for clinical studies and drug development.

Differential role for rapid proteostasis in Rho GTPase-mediated control of quiescent endothelial integrity

“…In marked contrast to RhoA and Rac1, RhoB has a very short half-life of 1 to 2 h ( 33 ). This is likely due to the fact that RhoB does not bind the ubiquitously expressed chaperone RhoGDI1, which is known to protect GTPases from activation and degradation ( 38 , 39 , 40 , 41 ). Consequently and because of the abundance of GTP in the cytosol, newly synthesized RhoB rapidly becomes GTP-bound, signaling competent, and sensitive to degradation.…”

Rapid proteostasis controls monolayer integrity of quiescent endothelium

“…In marked contrast to RhoA and Rac1, RhoB has a very short half-life of 1-2 h (Lebowitz, Davide, and Prendergast 1995). This is likely due to the fact that RhoB does not bind the ubiquitously expressed chaperone RhoGDI1, which is known to protect GTPases from activation and degradation (Garcia-Mata, Boulter, and Burridge 2011; Michaelson et al 2001; Mosaddeghzadeh et al 2021; Boulter et al 2010). Consequently and because of the abundance of GTP in the cytosol, newly synthesized RhoB rapidly becomes GTP-bound, signaling competent and sensitive to degradation.…”

The Dual Function of RhoGDI2 in Immunity and Cancer

“…[ 17 ]. The N-terminal domain of the RhoGDIs interacts with the switch 1 and switch 2 regions of GDP-bound Rho GTPases which prevents the exchange of GDP for GTP and therefore keeps them in their inactive form [ 18 , 19 ], whereas the C-terminal domain also contributes towards their inhibition by extracting Rho GTPases from the membrane [ 17 , 20 ].…”

“…Although their extreme N-terminal domain (25 and 22 amino acids for RhoGDI 1 and 2, respectively) are completely divergent, RhoGDI 1 and 2 show 73.6% identity for the remaining C-terminal sequence ( Figure 2 ). RhoGDI1 and RhoGDI2 interact with and form complexes with the classical Rho GTPases, i.e., RhoA, RhoC, Rac1, Rac2, Rac3, RhoG and Cdc42 [ 19 , 54 , 55 , 56 ]. However, the interaction potency of RhoGDI2 with Cdc42 is 10–20 folds lower than that of RhoGDI1.…”