title: GDP-bound and nucleotide-free intermediates of the guanine nucleotide exchange in the Rab5·Vps9 system.
abstract: Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg(2+) and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP β-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP β-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.
authors.affiliation: Structural Biology Research Center, Institute of Material Structure Science, High Energy Accelerator Research Organization, 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan. tamami.uejima@kek.jp
authors.forename: Tamami
authors.initials: T
authors.lastname: Uejima
authors.forename: Kentaro
authors.initials: K
authors.lastname: Ihara
authors.forename: Tatsuaki
authors.initials: T
authors.lastname: Goh
authors.forename: Emi
authors.initials: E
authors.lastname: Ito
authors.forename: Mariko
authors.initials: M
authors.lastname: Sunada
authors.forename: Takashi
authors.initials: T
authors.lastname: Ueda
authors.forename: Akihiko
authors.initials: A
authors.lastname: Nakano
authors.forename: Soichi
authors.initials: S
authors.lastname: Wakatsuki
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D018360:Crystallography, X-Ray; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D020349:Surface Plasmon Resonance; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D020662:Guanine Nucleotide Exchange Factors; C556142:Vps9a protein, Arabidopsis; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; C495672:Ara7 protein, Arabidopsis; D020691:rab GTP-Binding Proteins