Insight Into Distinct Functional Roles of the Flagellar ATPase Complex for Flagellar Assembly in Salmonella

Abstract

Most motile bacteria utilize the flagellar type III secretion system (fT3SS) to construct the flagellum, which is a supramolecular motility machine consisting of basal body rings and an axial structure. Each axial protein is translocated via the fT3SS across the cytoplasmic membrane, diffuses down the central channel of the growing flagellar structure and assembles at the distal end. The fT3SS consists of a transmembrane export complex and a cytoplasmic ATPase ring complex with a stoichiometry of 12 FliH, 6 FliI and 1 FliJ. This complex is structurally similar to the cytoplasmic part of the F_OF₁ ATP synthase. The export complex requires the FliH₁₂-FliI₆-FliJ₁ ring complex to serve as an active protein transporter. The FliI₆ ring has six catalytic sites and hydrolyzes ATP at an interface between FliI subunits. FliJ binds to the center of the FliI₆ ring and acts as the central stalk to activate the export complex. The FliH dimer binds to the N-terminal domain of each of the six FliI subunits and anchors the FliI₆-FliJ₁ ring to the base of the flagellum. In addition, FliI exists as a hetero-trimer with the FliH dimer in the cytoplasm. The rapid association-dissociation cycle of this hetero-trimer with the docking platform of the export complex promotes sequential transfer of export substrates from the cytoplasm to the export gate for high-speed protein transport. In this article, we review our current understanding of multiple roles played by the flagellar cytoplasmic ATPase complex during efficient flagellar assembly.

Keywords: ATPase; F0F1 ATP synthase; bacterial flagella; flagellar assembly; protein translocation; proton motive force (pmf); type III secretion system (T3SS).

FIGURE 1

Schematic diagrams of the flagellar type III export apparatus and F_OF ATP synthase. The flagellar type III secretion system (fT3SS) is composed of five membrane proteins, FlhA, FlhB, FliP, FliQ, and FliR and three cytoplasmic proteins, FliH, FliI, and FliJ. FlhA, FlhB, FliP, FliQ and FliR assembles into a transmembrane export complex within the MS-ring of the basal body of the flagellum. FliH, FliI, and FliJ form a cytoplasmic ATPase ring. The FliI₆-FliJ₁ ring complex is structurally similar to the α₃β₃γ₁ ring complex of the F_OF₁ ATP synthase. The N-terminal and C-terminal domains of FliH structurally are similar in structure to the b and δ subunits, respectively, of the F_OF₁ ATP synthase. The FliH dimer acts as a peripheral stalk that anchors the FliI₆-FliJ ring complex to the base of the flagellum in a similar manner as the b and δ subunits of the F_OF1 ATP synthase connect the α₃β₃γ ring complex to membrane-embedded F_O. The stoichiometry of the c-ring varies dramatically from c₈ up to at least c₁₅. CM, cytoplasmic membrane.

FIGURE 2

Schematic diagram of the bacterial flagellum. The bacterial flagellum is composed of basal body rings, namely the C-ring, MS-ring, L-ring, and P-ring, and an axial structure consisting of the rod, the hook, the hook-filament junction, the filament, and the filament cap. To construct the axial structure beyond the cytoplasmic membrane, flagellar axial proteins are translocated through the fT3SS, diffuse down a narrow central channel, and assemble at the tip of the growing structure. OM, outer membrane; PG, peptidoglycan layer; CM, cytoplasmic membrane.

FIGURE 3

CryoEM structure of the FlhB₁-FliP₅-FliQ₄-FliR₁ complex (PDB ID: 6S3L). FliP and FliR assemble into the FliP₅-FliR₁ complex with the help of the flagellum-specific transmembrane protein, FliO. Four copies of FliQ associates with the outside of the FliP₅-FliR₁ complex. The central pore of the FliP₅-FliR₁ complex is thought to be a polypeptide channel. The FliP₅-FliQ₄-FliR₁ complex adopts a right-handed helix similar to that of the flagellar axial structure. The transmembrane domain of FlhB (FlhB_TM) associates with the FliP₅-FliQ₄-FliR₁ complex. The highly conserved M-loop formed by Met-209, Met-210, and Met-211 of FliP (FliP_M–loop) and the plug loop composed of residues 106–122 of FliR (FliR_plug) block leakage of any small molecules during protein translocation. The cytoplasmic loop of FlhB FlhB_Loop) interacts with all four FliQ subunits. Because the entrance gate of the FlhB₁-FliP₅-FliQ₄-FliR₁ complex is closed, FlhB is proposed to regulate opening of the gate to the polypeptide channel. Cyan, FliP; green, FliQ; magenta, FliR; yellow, FlhB_TM.

FIGURE 4

Atomic model of the cytoplasmic domain of FlhA (PDB ID: 3A5I). (A) Topological model of FlhA. FlhA is composed of an N-terminal transmembrane region with eight transmembrane helices (FlhA_TM) and a large C-terminal cytoplasmic domain (FlhA_C). FlhA_TM acts as a dual-ion channel that can conduct both H⁺ and Na⁺. The highly conserved charged residues R94, K203, D208 and D249 are involved in H⁺-coupled protein export. The highly conserved residues D456, F459 and T490 of FlhA_C are critical for substrate recognition. A flexible linker region of FlhA (FlhA_L), which connects FlhA_C with FlhA_TM, is involved in the interaction with FliJ. The interaction between FlhA_L and FliJ activates the FlhA ion channel. (B) Model of the FlhA_C-ring. FlhA_C forms a homo-nonameric ring structure. The C-terminal part of FlhA_L binds to the neighboring FlhA_C subunit to stabilize the open conformation of FlhA_C, allowing flagellar export chaperones in complex with their cognate substrates to bind to a chaperone-binding site of FlhA_C, which includes the D456, F459, and T490 residues.

FIGURE 5

Atomic model of the FliI₆-FliJ₁ ATPase ring complex. (A) Cα ribbon representation of FliI (PDB ID: 5B0O). FliI consists of an N-terminal (FliI_N), an ATPase (FliI_CAT), and a C-terminal (FliI_C) domain. FliI_N is involved in formation of the FliI₆ ring. FliI_CAT contains the highly conserved P-loop, the catalytic glutamate (E211), and an arginine finger (R374), which are all involved in ATP hydrolysis. The ATP catalytic cycle induces sequential and cooperative conformational changes of FliI_C, which interacts with FliJ. (B) Electron micrograph of negatively stained FliI ring-like structures in complex with Mg²⁺-ADP-AlF₄. The inset shows a 2D class average of the FliI ring structure. (C) Model of the FliI₆-FliJ₁ ring model. R33, N73, and R76 of FliI_N regulate FliI ring formation. FliJ binds to the center of the FliI₆ ring. (D) Cα ribbon representation of FliJ (PDB ID: 3AJW). FliJ forms a two stranded coiled-coil structure. The highly conserved Q38, L42, Y45, Y49, F72, L76, A79 and H83 residues of FliJ extends out of the FliI₆ ring and are interact with FlhA_L.

FIGURE 6

Atomic model of the FliH_C12-FliI₆ ring complex. Cα ribbon representation of the FliH_C2-FliI₁ complex (PDB ID: 5B0O) is shown. The C-terminal domain of FliH (residues 141–235, FliH_C) forms a dimer via an interaction of residues 101–140, which adopt a coiled-coil structure. The FliH_C dimer binds to each N-terminal domain (FliI_N) of the FliI₆ ring. Interestingly, one FliH_C domain (blue) binds to the N-terminal α-helix consisting of residues 2–21 of FliI (brown, FliI_EN), and the other (cyan) binds to a positively charged region formed by R26, R27, R30, R33, R76, and R93 of FliI. These two domains adopt conformations that are completely different from each other.

FIGURE 7

Energy coupling mechanism of the fT3SS. The transmembrane export complex remains inactive until the cytoplasmic ATPase ring complex is formed at the base of the flagellum; the polypeptide and proton channels of the export complex remain closed (step 1). When ATP hydrolysis by FliI induces the rotation of FliJ in the FliI₆ ring, interactions between FliJ and FlhA_L induces conformational rearrangements of the export complex. As a result, the complex becomes an active protein transporter (step 2). The cytoplasmic FliH₂-FliI₁ complex acts as a dynamic carrier to deliver export substrates from the cytoplasm to the export complex (step 3). Upon docking of an export substrate to the entrance gate of the polypeptide channel, the gates of both polypeptide and proton channels are opened. The export complex can now act as a H⁺/protein antiporter that couples H⁺ flow through the ion channel with protein translocation into the polypeptide channel (step 4).