Merck
CN
  • Direct metal recognition by guanine nucleotide-exchange factor in the initial step of the exchange reaction.

Direct metal recognition by guanine nucleotide-exchange factor in the initial step of the exchange reaction.

Acta crystallographica. Section D, Biological crystallography (2013-03-23)
Tamami Uejima, Kentaro Ihara, Mariko Sunada, Masato Kawasaki, Takashi Ueda, Ryuichi Kato, Akihiko Nakano, Soichi Wakatsuki
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.

MATERIALS
Product Number
Brand
Product Description

Supelco
L-Aspartic acid, certified reference material, TraceCERT®, Manufactured by: Sigma-Aldrich Production GmbH, Switzerland
Sigma-Aldrich
L-Aspartic acid, BioUltra, ≥99.5% (T)
Sigma-Aldrich
L-Aspartic acid hemimagnesium salt dihydrate, ≥97.0% (KT)
SAFC
L-Aspartic acid
Sigma-Aldrich
L-Aspartic acid, from non-animal source, meets EP, USP testing specifications, suitable for cell culture, 98.5-101.0%
Sigma-Aldrich
L-Aspartic acid, BioXtra, ≥99% (HPLC)
Sigma-Aldrich
L-Aspartic acid, reagent grade, ≥98% (HPLC)
Sigma-Aldrich
DL-Aspartic acid, ≥99% (TLC)
Supelco
L-Aspartic acid, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
L-Aspartic acid potassium salt, ≥98% (HPLC)