title: The unique plant RhoGAPs are dimeric and contain a CRIB motif required for affinity and specificity towards cognate small G proteins.
abstract: Plant Rho proteins (ROPs) are inactivated by specific GTPase activating proteins, called RopGAPs. Many of these comprise the exclusive combination of a classic, catalytic Arg-containing RhoGAP domain, and a Cdc42/ Rac interactive binding (CRIB) motif which in animal and fungi has been identified in effectors for Cdc42 and Rac1, but never in any GAP protein. Both elements are required for an efficient RopGAP activity. Here, we analyzed the effect of the CRIB motif on the complex formation and the binding reaction with plant and human Rho proteins by using kinetic and equilibrium methods. We show that RopGAP2 from Arabidopsis thaliana dimerizes via its GAP domain and forms a 2:2 complex with ROP. The CRIB effector motif mediates high affinity and specificity in binding. The catalytic Arg in the context of the CRIB motif is inhibitory for binding. The unusually slow association and dissociation reactions suggest a major conformational change whereby the CRIB motif functions as a lid for binding and/or release of ROP. We propose a two-site interaction model where ROP binds to the CRIB motif as described for the human CRIB effectors and to the catalytic GAP domain as described for animal RhoGAPs.
authors.affiliation: Department of Structural Biology, Max Planck Institute of Molecular Physiology, Otto Hahn Str. 11, 44227 Dortmund, Germany.
authors.forename: Antje
authors.initials: A
authors.lastname: Schaefer
authors.forename: Kathrin
authors.initials: K
authors.lastname: Höhner
authors.forename: Antje
authors.initials: A
authors.lastname: Berken
authors.forename: Alfred
authors.initials: A
authors.lastname: Wittinghofer
mesh_terms: D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000737:chemistry; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry*; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary
chemical_list: D029681:Arabidopsis Proteins; D020690:GTPase-Activating Proteins; C085379:rho GTPase-activating protein; D019204:GTP-Binding Proteins; C511948:Rop4 protein, Arabidopsis; D020559:Monomeric GTP-Binding Proteins; C505651:RAC2 protein, Arabidopsis