Abstract
Guanine nucleotide exchange factors (GEFs) catalyze exchange of GDP for GTP by stabilizing the nucleotide-free state of the small GTPases through their Dbl homology/pleckstrin homology (DH.PH) domains. Unconventionally, PDZ-RhoGEF (PRG), a member of the RGS-RhoGEFs, binds tightly to both nucleotide-free and activated RhoA (RhoA.GTP). We have characterized the interaction between PRG and activated RhoA and determined the structure of the PRG-DH.PH-RhoA.GTPgammaS (guanosine 5'-O-[gamma-thio]triphosphate) complex. The interface bears striking similarity to a GTPase-effector interface and involves the switch regions in RhoA and a hydrophobic patch in PRG-PH that is conserved among all Lbc RhoGEFs. The two surfaces that bind activated and nucleotide-free RhoA on PRG-DH.PH do not overlap, and a ternary complex of PRG-DH.PH bound to both forms of RhoA can be isolated by size-exclusion chromatography. This novel interaction between activated RhoA and PH could play a key role in regulation of RhoGEF activity in vivo.
Highlights
The Rho family of monomeric GTPases belongs to the Ras superfamily and is composed of several proteins including the well characterized Rho (A, B, and C), Rac (1 and 2), and Cdc42 proteins
PDZ-RhoGEF Recognizes GTP-activated RhoA via Its pleckstrin homology (PH) Domain—Full-length PRG and p115RhoGEF were each expressed in bacteria with an N-terminal cleavable GST tag and a C-terminal His6 tag for purification
Mutations of key residues of the Dbl homology (DH) domain, which lead to diminished guanine nucleotide exchange factors (GEFs) activity and weak binding to nucleotide-free RhoA, have very little effect on binding of DH1⁄7PH to activated RhoA
Summary
Expression Construct—Coding regions of human PRG were subcloned into a pGEX-KG vector containing the protease recognition site for the tobacco etch virus (pGEX-KG-TEV) for proteolytic cleavage of the expressed domains from glutathione S-transferase. The resin was washed with incubation buffer, and His6-tagged RhoGEF proteins (12 pmol) were added to the immobilized GST-RhoA in incubation buffer (100 l) containing no additional guanine nucleotide, 10 M GDP, or 10 M GTP␥S. Equal moles of RhoA(⌬C)1⁄7GTP␥S and PRG-DH1⁄7PH (residues 712–1085) were mixed and filtered through the gel filtration columns preequilibrated with Buffer B (25 mM Tris-Cl, pH 8.5, 1 mM dithiothreitol, 100 mM NaCl, and 1 mM EDTA) and 2 mM MgCl2. The binary complex between DH1⁄7PH and nucleotide-free RhoA (full length) was formed by mixing equal moles of nucleotidefree RhoA (full length) and PRG-DH1⁄7PH and filtered through gel filtration columns at 4 °C pre-equilibrated with Buffer B. RhoA(⌬C)1⁄7GTP␥S was added to the binary complex and incubated for 15 min at room temperature, and the mixture was filtered through gel filtration columns pre-equilibrated with Buffer B and 2 mM MgCl2. Electrostatic potential maps were created with PyMOL and APBS [24]
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