Structural similarity between the flagellar type III ATPase FliI and F1-ATPase subunits

Abstract

Construction of the bacterial flagellum in the cell exterior proceeds at its distal end by highly ordered self-assembly of many different component proteins, which are selectively exported through the central channel of the growing flagellum by the flagellar type III export apparatus. FliI is the ATPase of the export apparatus that drives the export process. Here we report the 2.4 A resolution crystal structure of FliI in the ADP-bound form. FliI consists of three domains, and the whole structure shows extensive similarities to the alpha and beta subunits of F0F1-ATPsynthase, a rotary motor that drives the chemical reaction of ATP synthesis. A hexamer model of FliI has been constructed based on the F1-ATPase structure composed of the alpha3beta3gamma subunits. Although the regions that differ in conformation between FliI and the F1-alpha/beta subunits are all located on the outer surface of the hexamer ring, the main chain structures at the subunit interface and those surrounding the central channel of the ring are well conserved. These results imply an evolutionary relation between the flagellum and F0F1-ATPsynthase and a similarity in the mechanism between FliI and F1-ATPase despite the apparently different functions of these proteins.

Fig. 1.

Structure-based sequence alignment of FliI and the F₁-α/β subunits from bovine mitochondria (1BMFA and 1BMFB) (21) and the thermophilic Bacillus PS3 (1SKYA and 1SKYB) (28). The regions of secondary structural elements are shown below each sequence: blue line, α helix; green line, β structure. The secondary structural elements are labeled with initials of three domains (N, A, and C for α helix; n, a, and c for β structure) and numbers. The P loop is shown by the yellow box. The residues conserved between FliI and any of the F₁ subunits are highlighted in red. Red and blue boxes indicate the residues forming a hydrophobic pocket for nucleotide binding and basic residues located on the top surface of the crown-like structure, respectively. The residues included in the molecular model of FliI are shown in bold characters.

Fig. 2.

Structure of FliI(Δ1–18). (A) C^α ribbon drawing of FliI(Δ1–18). All of the secondary structure elements are labeled as in Fig. 1. The linker connecting the N-terminal and ATPase domains, which is missing in the model, is indicated by a dashed line. (B) Close-up stereoview of the nucleotide-binding site. The bound ADP is colored green, and the residues interacting with ADP are shown in cyan. Conserved residues involved in catalysis are indicated by yellow. (C–F) Comparison of the relative domain orientation. FliI(Δ1–18) (cyan) is superimposed onto the F₁-β subunits in various states, for which only corresponding atoms in the ATPase domain were used for fitting: (C) β_E (green), (D) β_TP (magenta), (E) β_DP (yellow) in 1BMF (21), and (F) β_ADP+Pi (red) in 1H8E (22).

Fig. 3.

FliI hexamer model. (A) Stereoview of the ribbon diagram. (B–D) Superposition of FliI (blue and yellow) onto the α (blue green) and β (orange) subunits of F₁-ATPase [1BMF (ref. 21)]. (B) N-terminal domain. (C) ATPase domain. (D) C-terminal domain. The N and C termini of the model are labeled for one subunit in B and D, respectively. (E–H) Electrostatic surface potential of the FliI hexamer. (E) Side view of two opposite subunits. (F) End-on view from the C-terminal side. (G) End-on view of a cross-section from the C-terminal side. (H) End-on view from the N-terminal side. Black and gray arrows indicate the hydrophobic and acidic sleeves, respectively. The surface potential is color coded as blue (positive) or red (negative).