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.
Highlights
The RHO family GTPases, most prominently RAC1, CDC42, and RHOA, share two common functional characteristics, membrane anchorage and an on/off switch cycle [1].They typically contain a conserved GDP/GTP binding domain, called G domain, and a C-terminal hypervariable region (HVR) ending with a consensus sequence known as CAAX (C is cysteine, A is any aliphatic amino acid, and X is any amino acid)
That: (1) the geranylgeranyl moiety is dispensable for RAC1-GDI1 interaction, (2) seven out twelve RHO family GTPases did not interact with GDI1, (3) a conserved G domain is not rate-limiting for the GDI1 binding, (4) RAC1 polybasic motif dictates GDI1 binding, (5) electrostatic pincer residues of GDI1 grasp RAC1 HVR, and (5) GDI1 buckles RAC1 into its site
To understand the impact of the isoprenyl moiety of RAC1 on GDI1 binding, we compared the biochemical properties of geranylgeranylated RAC1 (RAC1GG ) from insect cells and non-isoprenylated RAC1 full-length (RAC1FL ) from Escherichia coli
Summary
The RHO family GTPases, most prominently RAC1, CDC42, and RHOA, share two common functional characteristics, membrane anchorage and an on/off switch cycle [1] They typically contain a conserved GDP/GTP binding domain, called G domain, and a C-terminal hypervariable region (HVR) ending with a consensus sequence known as CAAX (C is cysteine, A is any aliphatic amino acid, and X is any amino acid). It has been reported that GDI1 binds the isoprenylated RAC1 and RHOA proteins with extremely high binding affinities 0.4 and 0.005 nanomolar affinity, respectively, as compared to non-isoprenylated proteins [41,42] These studies have clearly demonstrated how GDIs interact with and serve as negative regulators of RHO GTPases, yet the basic mechanisms of how they pull the isoprenoid moiety from the membrane remain elusive. (4) RAC1 polybasic motif dictates GDI1 binding, (5) electrostatic pincer residues of GDI1 grasp RAC1 HVR, and (5) GDI1 buckles RAC1 into its site
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