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 lefthanded 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.I on-transporting rotary ATPases are divided into three types based on their function and taxonomic origin: F-, V-, and A-type ATPases. F-ATPases function as ATP synthases in mitochondria, chloroplasts, and oxidative bacteria (1). V-ATPases function as proton pumps in the membranes of acidic organelles and plasma membranes of eukaryotic cells (2). A-ATPases in archaea function as ATP synthases similar to the F-ATPases (the "A" designation in A-type refers to archaea), although the structure and subunit composition of A-ATPases are more similar to those of V-ATPases (3). These ATPases possess an overall similar structure that is composed of a hydrophilic catalytic portion (F 1 -, V 1 -, or A 1 -ATPase) and a membrane-embedded ion-transporting portion (F o -, V o -, or A o -ATPase), and they have a similar reaction mechanism that occurs through rotation (2).V-ATPases are found in bacteria, such as Thermus thermophilus and Enterococcus hirae. In T. thermophilus, V-ATPase physiologically functions as an ATP synthase (4). Therefore, it has sometimes been called an A-ATPase, although T. thermophilus is a eubacterium that does not belong to archaea (5). However, the E. hirae V-ATPase acts as a primary ion pump (6), which transports Na þ or Li þ instead of H þ (7). The enzyme is composed of nine subunits (Eh-A, -B, -d, -D, -E, -F, -G, -a, -c; previously designated as NtpA, -B, -C, -D, -E, -G, -F, -I, -K) having amino acid sequences that are homologous to those of the corresponding subunits (A, B, d, D, E, F, G, a, c) of eukaryotic V-ATPases (7) (see Fig. 1). The core of the V 1 domain of this ATPase is composed of a hexameric arrangement of alternating A and B subunits responsible for ATP binding and hydrolysis (8). The V o domain, in which rotational energy is converted to drive Na þ translocation, is composed of oligomers of the 16-kDa c subunits and an a subunit (9, 10). The V 1 and V o domains are connected by a central stalk, which is composed of D, F, and d subunits, and two peripheral stalks, which a...