Nucleotide Dependent Switching in Rho GTPase: Conformational Heterogeneity and Competing Molecular Interactions

Abstract

Ras superfamily of GTPases regulate myriad cellular processes through a conserved nucleotide (GTP/GDP) dependent switching mechanism. Unlike Ras family of GTPases, for the Rho GTPases, there is no clear evidence for the existence of “sub-states” such as state 1 & state 2 in the GTP bound form. To explore the nucleotide dependent conformational space of the Switch I loop and also to look for existence of state 1 like conformations in Rho GTPases, atomistic molecular dynamics and metadynamics simulations on RhoA were performed. These studies demonstrate that both the nucleotide-free state and the GDP bound “OFF” state have very similar conformations, whereas the GTP bound “ON” state has unique conformations with signatures of two intermediate states. The conformational free energy landscape for these systems suggests the presence of multiple intermediate states. Interestingly, the energetic penalty of exposing the non-polar residues in the GTP bound form is counter balanced by the favourable hydrogen bonded interactions between the γ-phosphate group of GTP with the highly conserved Tyr34 and Thr37 residues. These competing molecular interactions lead to a tuneable energy landscape of the Switch I conformation, which can undergo significant changes based on the local environment including changes upon binding to effectors.

Conformational regulation of AP1 and AP2 clathrin adaptor complexes

“…Each Arf1 simultaneously engages each AP1 via two interfaces: the central region of one AP1's γ subunit and the N‐terminus of the other AP1's β1 subunit. This second interface buries the switch I and II regions of Arf1 that are known to engage effectors upon GTP binding . Modeling of the dimeric AP1‐Arf1 structure on a virtual membrane suggests that all of the membrane‐ and cargo‐binding sites could simultaneously engage a single surface (Figure A).…”

“…This second interface buries the switch I and II regions of Arf1 that are known to engage effectors upon GTP binding. 45 Modeling of the dimeric AP1-Arf1 structure on a virtual membrane suggests that all of the membraneand cargo-binding sites could simultaneously engage a single surface ( Figure 4A). This structure further suggests that initiation of clathrincoated structures on intracellular membranes may require two membrane-associated, GTP-bound Arf1 proteins that cooperate to convert two AP1 complexes to the active state.…”

SmgGDS-607 Regulation of RhoA GTPase Prenylation Is Nucleotide-Dependent

“…As a GTPase, RhoA undergoes conformational changes depending on whether it is in the GTP- or GDP-bound state and these conformational changes can affect the affinity for GEFs, GAPs, and effectors. 23,24 Thus, we hypothesized that the nucleotide status of RhoA could regulate the affinity with SmgGDS-607 and this in turn, would regulate prenylation by GGTase-I. To begin to test this model, we first measured the binding affinities of SmgGDS-607 for RhoA-GDP and RhoA-GTP using a bio-layer interferometry (BLI) assay.…”

RHO Guanine Nucleotide Exchange Factors Predict Prognosis of Non-Small Cell Lung Cancer: A Comprehensive Bioinformatics Analysis

“…RHO guanosine triphosphatases (GTPases) are essential molecular switches involved in the regulation of numerous downstream pathways in various types of cancer [5,6]. The cycling between guanosine triphosphate (GTP)-bound (active) state and guanosine diphosphate (GDP)-bound (inactive) state contributes to the activation or inactivation of downstream effectors [7]. In the "on" state (GTPbound) of RHO GTPases, target proteins are recognized and subsequently a response is generated until the RHO GTPases are turned to "off" state (GDP-bound) [5].…”