title: Direct metal recognition by guanine nucleotide-exchange factor in the initial step of the exchange reaction.
abstract: Rab small GTPases regulate vesicle transport in eukaryotes by interacting with various effectors. Guanine nucleotide-exchange factor (GEF) catalyzes the transition from inactive GDP-bound Rab to active GTP-bound Rab. The existence of several GDP-bound intermediates containing the Arabidopsis thaliana Rab5 homologue ARA7 and the GEF VPS9a prior to the formation of a nucleotide-free binary complex has been proposed [Uejima et al. (2010), J. Biol. Chem. 285, 36689-36697]. During this process, VPS9a directly interacts with the β-phosphate of GDP and the P-loop lysine of ARA7 via a catalytically important aspartate finger, which promotes the release of GDP from ARA7. However, it is unclear how VPS9a removes Mg2+ from ARA7 before forming the GDP-bound ternary complex. Here, the structure of the ARA7-GDP-Ca2+-VPS9a complex is reported, in which the aspartate finger directly coordinates the divalent metal ion. Ca2+ is bound to the canonical Mg2+-binding site, coordinated by the β-phosphate of GDP and the P-loop serine of ARA7. Unexpectedly, Ca2+ is further coordinated by the aspartate finger and the main chain of VPS9a. This structure may represent the earliest intermediate step in the GEF-catalyzed nucleotide-exchange reaction of ARA7 before the metal-free GDP-bound intermediates are created.
authors.affiliation: Structural Biology Research Center, Institute of Materials 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: Mariko
authors.initials: M
authors.lastname: Sunada
authors.forename: Masato
authors.initials: M
authors.lastname: Kawasaki
authors.forename: Takashi
authors.initials: T
authors.lastname: Ueda
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Kato
authors.forename: Akihiko
authors.initials: A
authors.lastname: Nakano
authors.forename: Soichi
authors.initials: S
authors.lastname: Wakatsuki
mesh_terms: D017360:Arabidopsis / Q000201:enzymology; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D020691:rab GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D020662:Guanine Nucleotide Exchange Factors; C556142:Vps9a protein, Arabidopsis; D001224:Aspartic Acid; C495672:Ara7 protein, Arabidopsis; D020691:rab GTP-Binding Proteins; D002118:Calcium
keywords: ARA7; guanine nucleotide-exchange factors