ARA7 complexed with VPS9a
Mouse-ear cress (Arabidopsis thaliana)
Small GTPases are molecular switches, using the conformation differences between their GTP-bound forms and the GDP-bound forms. This is mainly regulated by 4 factors. Change from the inactive GDP-bound form to the active GTP-bound form is mediated by GEF (Guanine nucleotide exchange factor). The active small GTPase functions through the interaction with the target protein called "effecter". Conversion from the active form to the inactive form is made by its own GTPase function with GAP (GTPase activating protein). The resulted inactive small GTPase forms a complex with GDI (Guanosine nucleotide dissociation inhibitor), and keeps the state, which does not interact with the effector. Once the GDI is released with the aid of GDF (GDI-displacement factor), GEF becomes inactive, and the above cycle returns to the initial state.
Several different Rab small GTPases are engaged in the endoplasmic reticulum transportation, and Rab5 is, in particular, involved in endocytosis. In Arabidopsis thaliana, there are 3 known Rab5s: ARA6, ARA7, and RHA1. VPS9a is GEF of Rab5 from Arabidopsis thaliana, and the function and structure have been studied. These days, very many structure of the small GTPases in their GTP- and GDP-bound forms have been determined, and their complex structures with GEF have also been revealed. However, the reaction mechanism with GEF has not been known well. Here, the crystal structure for the binary complex of ARA7 complexed with VPS9a in its nucleotide free form and that for the ternary complex with GDP or GDPNH2 are determined.
The perfectly conserved Asp residue (Asp finger), which is essential for GEF activity, was thought to be involved in the nucleotide conversion, using the electrostatic repulsion. However, in the ternary complex structure, the nucleotide is recognized with the hydrogen bonds, in contrast to the electrostatic repulsion. On the contrary, Lys side-chain in the P-loop, which is perfectly conserved and recognizes the beta-phosphate in the nucleotide, interacts with the Asp finger. Thus, the electrostatic competitions among Lys, Asp finger, and GDP are suggested. The above binary and ternary complex structures of ARA7 with VPS9a are expected to contribute to reveal the conversion mechanism of nucleotide by GEF.
Protein Data Bank (PDB)
Author: Tamami Uejima and Kentaro Ihara Translator: Haruki Nakamura