RSS

PDB:2efd

Protein Name

ARA7 complexed with VPS9a

Species

Mouse-ear cress (Arabidopsis thaliana)

Biological Context

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.

Structure Description

2efd2efd_x2efd_y

1. Asp finger

Asp finger conserved perfectly in the Vps9 domain has been named analogous to Glu finger, which has the similar function in the Sec 7 domain, GEF of Arf. GEF with the acidic residues at the active center, such as Asp finger and Glu finger, was thought to convert nucleotides using the electrostatic repulsions between the negative charges. However, from the ARA7 complex structures with VPS9a including the nucleotide, the intermediate form is found, where Asp finger directly recognizes GDP with hydrogen bonds, and the nucleotide release by GEF is not simply explained by the electrostatic repulsion.

2. Mechanism of GDP release

If Asp finger does not release GDP by the electrostatic repulsion, what mechanism should work for the GDP release? A hydrogen bond is confirmed between Asp finger and the beta-phosphate of GDP, and an ion-pair is also observed between Asp finger and the conserved Lys residue in P-loop of small GTPases. In the monomer of the small GTPase, the Lys residue in P-loop forms an ion pair, constructing the active center, together with Ser/Thr residues conserved in P-loop, and with Mg2+ ion ligated with GDP. Asp finger competes with GDP for binding P-loop. A GDP release model is shown, where Asp finger interacts with GDP by hydrogen bonds, attracts Lys in P-loop against GDP, and lowers the affinity of GDP to the small GTPase. The positive charges in Mg2+ and Lys can lower the pKa of GDP protonation, and can increase the GDP acidity. However, GDP protonation may occur by a mechanism, where GEF may sequentially release positive charges from GDP. This protonation may decrease the electrostatic repulsion between Asp finger and GDP. Namely, the interaction between GEF and its ligand, the small GTPase in its GDP-bound form, may increase. Consequently, Lys in P-loop, GDP, and Asp finger can be co-localized, and the nucleotide conversion may be effectively proceeded. In the current crystal structures, Mg2+ ions are not included. Thus, the next step to understand completely the nucleotide conversion by GEF is to study the mechanism of Mg2+ ion release from the small GTPase in its GDP-bound form.

Protein Data Bank (PDB)

References

Source

  • Uejima, T. Ihara, K. Goh, T. Ito, E. Sunada, M. Ueda, T. Nakano, A. Wakatsuki, S.; "Recognition of Rab5-GDP by guanine nucleotide exchange factor of plant Rab5 prior to GDP release"; To be Published; () :-.

Others

Author: Tamami Uejima and Kentaro Ihara Translator: Haruki Nakamura


Japanese version:PDB:2efd