The flagellum is a long filamentous organelle used for bacterial motility. It consists of a basal body, a short curved segment called the hook and a long helical filament. The basal body is a reversible rotary motor and generates torque. The hook is a universal joint that transmits the motor torque to the long helical propeller in its different orientations. The filament is a thin helical structure that typically grows to around 15 µm in length and rapidly rotates as a propeller for cell locomotion in viscous environments.
The basal body spans the inner and outer cell membranes, while the hook and the filament are external to the cell. Therefore, most of the flagellar component proteins have to be exported from the cytoplasm for flagellar assembly. These proteins are exported across the cell membranes by the flagellar protein-export apparatus, which is a member of the type III export system. The export apparatus is believed to be located within the C ring, a large cytoplasmic structure of the flagellar basal body. Previous genetic and biochemical studies have demonstrated that the export apparatus consists of six integral membrane components (FlhA, FlhB, FliO, FliP, FliQ and FliR) and three cytoplasmic components (FliH, FliI and FliJ; Minamino & Macnab, 1999
). FliI is an ATPase and presumably pushes the export-substrate proteins out through the central pore of the basal body by utilizing the energy of ATP hydrolysis (Dreyfus et al.
; Fan & Macnab, 1996
). FliH regulates the ATPase activity of FliI and contributes to the interaction between FliI and FlhA or FliI and FlhB (Auvray et al.
; González-Pedrajo et al.
; Minamino & Macnab, 2000b
; Minamino et al.
). FliJ acts as a general chaperone for the export-substrate proteins (Minamino et al.
). FlhB functions as the substrate-specificity switch for the export apparatus during the flagellar assembly (Minamino & Macnab, 2000a
; Zhu et al.
; Fraser et al.
FlhA consists of 692 amino-acid residues with a molecular weight of 75 kDa. flhA
-deletion mutants cannot produce the flagellar axial structure including the rod, the hook and the filament (Kubori et al.
). There are also mutations in flhA
that allow FliI to bind to the export apparatus in the absence of FliH (Minamino et al.
). These reports indicate that FlhA is an essential component of the export mechanism. FlhA can be divided into two major domains: a hydrophobic N-terminal transmembrane domain (FlhATM
, 34.5 kDa) and a hydrophilic C-terminal cytoplasmic domain (FlhAC
, 40.5 kDa). FlhAC
consists of two distinct portions: a compact 38 kDa fragment, FlhAC
38K, and a linker region connecting FlhATM
38K, which contains amino-acid residues 352–629 of FlhA, is directly involved in the export of flagellar proteins, because temperature-sensitive flhA
mutations and the bypass mutation described above lie within FlhAC
. The linker region is thought to be important for stabilizing the interactions between FlhAC
38K and the cytoplasmic components of the export apparatus, such as FliI, FliH, FliJ and the cytoplasmic domain of FlhB (Saijo-Hamano et al.
Genetic and biochemical studies have revealed the properties of these proteins (Minamino & Namba, 2004
). However, the export mechanism is not clear except that energy released by ATP hydrolysis by FliI is used for the export process. To elucidate the protein-export mechanism, the atomic structures of the component proteins are essential. Here, we report an X-ray crystallographic study of FlhAC