title: Dimeric plant RhoGAPs are regulated by its CRIB effector motif to stimulate a sequential GTP hydrolysis.
abstract: RopGAPs are GTPase-activating proteins (GAPs) for plant Rho proteins (ROPs). The largest RopGAP family is characterized by the plant-specific combination of a classical RhoGAP domain and a Cdc42/Rac interactive binding (CRIB) motif, which, in animal and fungi, has never been found in GAPs but in effectors for Cdc42 and Rac1. Very little is known about the molecular mechanism of the RopGAP activity including the regulatory role of the CRIB motif. Previously, we have shown that they are dimeric and form a 2:2 complex with ROPs. Here, we analyze the kinetics of the GAP-mediated GTP hydrolysis of ROPs using wild-type and mutant RopGAP2 from Arabidopsis thaliana. For an efficient GAP activity, RopGAP2 requires both the catalytic Arg159 in its GAP domain indicating a similar catalytic machinery as in animal RhoGAPs and the CRIB motif, which mediates high affinity and specificity in binding. The dimeric RopGAP2 is unique in that it stimulates ROP·GTP hydrolysis in a sequential manner with a 10-fold difference between the hydrolysis rates of the two active sites. Using particular CRIB point and deletion mutants lead us to conclude that the sequential mechanism is likely due to steric hindrance induced by the Arg fingers and/or the CRIB motifs after binding of two ROP molecules.
authors: Department of Structural Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn Str. 11, 44227 Dortmund, Germany.
authors: Antje
authors: A
authors: Schaefer
authors: Mandy
authors: M
authors: Miertzschke
authors: Antje
authors: A
authors: Berken
authors: Alfred
authors: A
authors: Wittinghofer
mesh_terms: D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D020690:GTPase-Activating Proteins; C085379:rho GTPase-activating protein; D006160:Guanosine Triphosphate; D020559:Monomeric GTP-Binding Proteins; C505651:RAC2 protein, Arabidopsis; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins