Activated RhoA binds to the pleckstrin homology (PH) domain of PDZ-RhoGEF, a potential site for autoregulation.
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.
Chen
Zhe
Z
Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Zhe.Chen@utsouthwestern.edu
Medina
Frank
F
Liu
Mu-ya
MY
Thomas
Celestine
C
Sprang
Stephen R
SR
Sternweis
Paul C
PC
D001665:Binding Sites; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005822:Genetic Vectors; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D013816:Thermodynamics; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*
C577234:ARHGEF11 protein, human; D020662:Guanine Nucleotide Exchange Factors; D010446:Peptide Fragments; D064067:Rho Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020558:GTP Phosphohydrolases; D020742:rhoA GTP-Binding Protein