Crystal structure of the central axis DF complex of the prokaryotic V-ATPase

Abstract

V-ATPases function as ATP-dependent ion pumps in various membrane systems of living organisms. ATP hydrolysis causes rotation of the central rotor complex, which is composed of the central axis D subunit and a membrane c ring that are connected by F and d subunits. Here we determined the crystal structure of the DF complex of the prokaryotic V-ATPase of Enterococcus hirae at 2.0-Å resolution. The structure of the D subunit comprised a long left-handed coiled coil with a unique short β-hairpin region that is effective in stimulating the ATPase activity of V(1)-ATPase by twofold. The F subunit is bound to the middle portion of the D subunit. The C-terminal helix of the F subunit, which was believed to function as a regulatory region by extending into the catalytic A(3)B(3) complex, contributes to tight binding to the D subunit by forming a three-helix bundle. Both D and F subunits are necessary to bind the d subunit that links to the c ring. From these findings, we modeled the entire rotor complex (DFdc ring) of V-ATPase.

Fig. 1.

Schematic model of E. hirae V-ATPase. Previous names of the corresponding subunits of E. hirae V-ATPase are in parentheses.

Fig. 2.

Structure of the DF complex of the E. hirae V₁-ATPase. (A) Cartoon representation of the Eh-DF complex. Eh-D and Eh-F are shown in green and dark red, respectively. (B) Eh-D is presented in blue to red from N to C terminus. (C) Eh-F is colored in the same manner as in B.

Fig. 3.

Structural similarity of the D subunit. (A) Superimposed structures of Eh-D (green) and Tt-D (gray) of V₁-ATPase from T. thermophilus (PDB ID 3A5D). The adjacent Tt-A is shown in blue. The β-hairpin region (residues 90–108) that was deleted in mutation experiments is shown in the red-dotted box. (B and C) Conserved residues of the D subunit. The figures were generated using ConSurf (35). Residues are colored in accordance with conservation among the aligned amino acid sequences of the D subunit from seven species in Fig. S2.

Fig. 4.

Structural similarity of the F subunit. (A) Superimposed structures of Eh-F (dark pink) and the F subunit (cyan) of V₁-ATPase from T. thermophilus (PDB ID 3A5D). The adjacent Tt-B is shown in violet. (B) Structures of Tt-F (orange) extended form (PDB ID 2D00) and Mm-F (green) are superimposed in A. (C) The sequence alignment of the F subunits from different species (E. hirae, T. thermophilus HB8, Methanosarcina mazei Gö1, Saccharomyces cerevisiae, and Homo sapiens). The secondary structures of Eh-F are shown above the sequence. The deleted regions of the mutations are indicated in the green box.

Fig. 5.

Electrostatic potential of the rotor complex. The electrostatic potential surfaces were calculated using APBS (36) and mapped at contouring levels from -3 kT (blue) to 3 kT (red). (A) Eh-DF (Upper, bottom view; Lower, side view). (B) Homology modeled Eh-d (Upper, top view; Lower, side view). (C) Eh-c ring (Upper, top view; Lower, side view). (D) The model of the entire rotor complex.