Organization of FliN subunits in the flagellar motor of Escherichia coli.

Many species of bacteria are propelled by rotating flagella (2) that obtain energy from the membrane ion gradient (16, 23; for reviews, see references 1 and 21) The flagella are built from about 25 different proteins, most of which have roles in forming the flagellar basal body, hook, and filament (22, 26). Only a few proteins function in rotation and switching. The membrane proteins MotA and MotB form ion-conducting complexes that function as the stator (3, 8-11, 33). The proteins FliN, FliM, and FliG form the rotor-mounted switch complex that functions in flagellar assembly, rotation, and clockwise-counterclockwise (CW/CCW) direction control (44, 45). The switch complex contains >100 copies of FliN (47), about 34 copies of FliM (47), and 25 copies of FliG (13, 18, 35, 36, 47). Together, these form the C ring, a 45-nm-diameter, drum-shaped structure at the bottom (the cytoplasm-proximal end) of the basal body (Fig. ​(Fig.1)1) (14, 19, 40). FIG. 1. Cartoon of the flagellar basal body indicating the approximate locations of the proteins that form the stator (MotA and MotB) and the switch complex (FliG, FliM, and FliN). OM, outer membrane; PG, peptidoglycan; IM, inner membrane. FliG functions directly in rotation of the flagellum (17, 24). It interacts with the FliF protein that forms the MS ring (13, 15, 20, 30) and with the membrane-associated stator protein MotA (48) and must presumably be located in the upper (membrane-proximal) part of the C ring (Fig. ​(Fig.1).1). FliM and FliN have not been located precisely but are probably more distant from the membrane because they do not function as directly in rotation (24) and are not believed to interact directly with FliF or MotA. FliM is closely involved in direction switching (34) and contains a binding site for the CW-signaling protein CheY (28, 41, 43). FliN has been implicated in CW/CCW switching (17) and in the export process that occurs during flagellar assembly (42), but its exact functions are unknown. The FliN protein of Escherichia coli or Salmonella contains 137 residues (27). The C-terminal part of the protein, from about residue 58 to the end, is best conserved and most important for function (37). A FliN protein lacking 57 N-terminal residues is sufficient for flagellar assembly and rotation, but the cells swarm at about one-third of the wild-type rate in soft agar (37) and show relatively slow, erratic motility in liquid (K. Paul and D. Blair, unpublished data). In the flagellum, FliN interacts with the C-terminal domain of FliM (FliMC) (28, 41). Portions of FliMC show sequence homology to FliN (28) and may therefore show some similarity in folding. FliN might also bind to FliG, but the evidence for this is less strong (39). Two crystal structures were recently solved for most of the FliN protein from Thermotoga maritima (Protein Data Bank [PDB] entries 1o6a and 1yab) (5). Both structures show a saddle-shaped dimer formed mainly from β strands, with short α helices at the ends (Fig. ​(Fig.2).2). The two structures differ somewhat in the position of α-helix 1, which packs near the body of the dimer in one crystal (1o6a) but extends to contact an adjacent dimer in the other (1yab). The FliN dimer exhibits a sizable surface hydrophobic patch, formed from the side chains of 5 residues (10 residues in the dimer) with a strongly conserved nonpolar character. A mutation in the hydropobic patch (Val113→Asp) affects both flagellar assembly and switching (5). FIG. 2. (A) Crystal structure of FliN (PDB accession no. 1yab), with the hydrophobic patch indicated by gray coloring. (B) Differing positions of helix 1 (colored gray) in the two FliN crystal structures. In the 1o6a structure, the two helices of the dimer have ... Equilibrium sedimentation experiments showed that the FliN protein of E. coli forms a stable tetramer in solution and that the FliN and FliM proteins of T. maritima form a stable complex with the composition FliM1FliN4 (5). (Sedimentation experiments with FliM used the T. maritima proteins because E. coli FliM is prone to aggregation.) Accordingly, we proposed that FliM1FliN4 complexes might be structural units of the C ring (5). The subunit composition of the C ring is not yet firmly established, however; an estimate of subunit stoichiometry in purified basal bodies of Salmonella enterica serovar Typhimurium gave a FliM:FliN ratio closer to 1:3 (47). Because FliMC shows sequence similarity to FliN, it might be capable of replacing a FliN subunit to form a FliM-FliN heterodimer, and such heterodimers in combination with FliN dimers could account for a 1:3 subunit composition. The location and arrangement of the FliN subunits in the C ring are also unknown. The related protein HrcQBC crystallized as an elongated tetramer in which the dimers are joined end to end (12). Patterns of residue conservation indicate that FliN dimers might associate in a similar end-to-end fashion (5), but the subunit associations seen in FliN crystals indicate that other arrangements are also possible, including some with the shape of a ring (Fig. 2C to E). Here, we have used targeted cross-linking to test various models for the organization of FliN. Cys residues were introduced in various positions on the surface of FliN, singly or in pairs; disulfide cross-linking was induced; and the products were analyzed on immunoblots. The subunit replacement model was tested by making Cys replacements at predicted interfacial positions in FliN and FliMC. The results indicate that FliN is organized in tetramers and does not form subunit-exchanged heterodimers with FliMC. The FliN tetramer is not organized like the tetramer seen in the HrcQBC crystal structure but has a doughnut shape similar to the subunit organization seen in one of the FliN crystal structures. The FliN doughnut seen in the crystal structure is puckered and as a result is predicted to be bistable (to exist in two equally stable conformations). In the flagellum, the two conformations would be nonequivalent and might be relevant for CW/CCW switching. Some Cys replacements were also made in the structurally uncharacterized N-terminal domain of FliN. Occurrences of efficient cross-linking show that the N-terminal segments of two FliN subunits are in proximity to each other and are near the hydrophobic patch of another dimer. These interactions appear to stabilize the tetramer, because a protein lacking the N-terminal domain formed mainly dimers. Electron microscopic reconstructions (40, 46) show a bulge at the bottom of the C ring of the appropriate size and shape to fit the FliN tetramers.