Architecture of the flagellar rotor

Abstract

Rotation and switching of the bacterial flagellum depends on a large rotor-mounted protein assembly composed of the proteins FliG, FliM and FliN, with FliG most directly involved in rotation. The crystal structure of a complex between the central domains of FliG and FliM, in conjunction with several biochemical and molecular-genetic experiments, reveals the arrangement of the FliG and FliM proteins in the rotor. A stoichiometric mismatch between FliG (26 subunits) and FliM (34 subunits) is explained in terms of two distinct positions for FliM: one where it binds the FliG central domain and another where it binds the FliG C-terminal domain. This architecture provides a structural framework for addressing the mechanisms of motor rotation and direction switching and for unifying the large body of data on motor performance. Recently proposed alternative models of rotor assembly, based on a subunit contact observed in crystals, are not supported by experiment.

Figure 1

Hypotheses for FliG organization in the flagellar rotor. (A) The flagellar basal body of wild-type Salmonella (Thomas et al, 2001). The dashed box indicates the region shown magnified in the other panels. (B) FliG-domain arrangement discussed by Thomas et al (2006). (C) Hypothesis of Brown et al (2007). (D) Arrangement based on a FliG_M–FliG_C contact observed in crystals. The contact is postulated to involve either two different FliG subunits (Lee et al, 2010) or a single FliG subunit (Minamino et al, 2011).

Figure 2

Structure of the T. maritima FliM_M:FliG_M complex. (A) Overall shape of the complex. The N- and C-termini of FliM_M are oriented towards the bottom in this view; these parts of FliM are directed towards the bottom of the C-ring in the flagellar basal body (Park et al, 2006; Sarkar et al, 2010b). (A stereo version of this figure is provided in Supplementary data.) (B) The FliG_M:FliM_M interface. The EHPQR residues of FliG and the GGXG motif of FliM are indicated. Orange circles mark positions where Cys residues were introduced to confirm the interaction by crosslinking. Numbers are for the E. coli protein. Unbiased electron density for the EHPQR and GGXG motifs is shown in Supplementary data. (C) Crosslinking through the introduced Cys residues. Crosslinking was induced using Cu-phenanthroline. (D) Packing of the helix near the C-terminus of FliG_M against the body of the domain. The helix is shown in lighter colour. In the previous crystal structure of FliG_MC (Brown et al, 2002), this helix is detached from the domain and makes extensive inter-subunit crystal contacts instead. (E) Hydrophobic contacts between the helix and FliM_M that stabilize the close-packed conformation of the helix.

Figure 3

Interaction between FliM and FliG_C detected in GST pull-down assays. (Representative results are shown; see Supplementary Figure S2 for additional data.) Blots were probed with anti-FliM antibody. (A) Effects of FliG_C mutations on the binding to FliM. Positions where mutations eliminated binding are coloured red; black indicates positions where mutations had no effect. (B) Effects of FliM mutations on binding to FliG_C. Colouring as in panel (A), plus orange to indicate positions where binding was weakened.

Figure 4

Crosslinking experiments to probe the FliM_M–FliG_C relationship. (A) Positions of Cys replacements and summary of the crosslinking results. Dotted blue lines connect Cys pairs of residues that formed disulphide crosslinks, with the thickness of the line indicating relative yield. Representative gels are shown below; blots were probed with anti-HA antibody. The red dashed line connects a Cys pair that, in addition to crosslinking, showed mutational suppression (see the text and Supplementary Figure S3A). (B) Model for the FliM_M–FliG_C assembly based on the crosslinking results. The highest yield Cys pairs are indicated. (C) Tests of the crystal contact-based model for FliG organization. The 117/166 Cys pair in FliG_M was shown previously to crosslink efficiently (Lowder et al, 2005) and is included as a positive control. The 159/218 and 162/196 Cys pairs are in close proximity in the crystal contact model (Lee et al, 2010; see Supplementary Figure S4 for an illustration). These failed to crosslink, using either Cu-phenanthroline (shown) or iodine (data not shown).

Figure 5

Proximity of FliG_N to FliG_M. (A) Crosslinking of position 43 in FliG_N to position 147 in FliG_M by bis-maleimidohexane. Crosslinking was carried out at 23°C for 10 min. (B) A hypothetical arrangement of the FliG_N and FliG_M domains that could account for the observed FliG_N–FliG_M and FliG_M–FliG_M crosslinking. The FliG_N domain (residues 5–89) is pale-cyan and FliG_M (residues 104–184) is cyan. The segment linking the domains (residues 90–103) is yellow and the positions to which it would connect (carboxy-terminus of FliG_N and amino-terminus of FliG_M) are red and blue. The relative orientation of the FliG_M domains is based on a previous study (Lowder et al, 2005), which identified positions giving efficient FliG_M–FliG_M crosslinking; one such pair (117–166) is shown. The orientation of FliG_N, which is intended to be approximate only, is based on the observed FliG_N–FliG_M crosslink (A) and constraints imposed by the inter-domain connection (the length of the connecting helix and the positions it must connect). Spheres indicate positions of Cβ positions, (grey in FliG_N and black in FliG_M). Residue numbers are for the E. coli protein. For previously identified instances of crosslinking, including the indicated 117/166 Cys pair, see Lowder et al (2005).

Figure 6

Structural model for the upper part of the C-ring. (A) Overall plan of FliG and FliM organizations. The arrangement is similar to that proposed by Brown et al (2007), with adjustments to reflect more-current information on FliG structure. FliM is light brown and FliG is cyan. (B) More detailed view of a section of the rotor. Colouring is as in (A), but with the three parts of FliG (FliG_NM, linking helix and FliG_C) coloured with increasing intensity, and the active-site ridge shown in atom colours to highlight the conserved charged residues that interact with the stator (Zhou et al, 1998a). The dashed line indicates the hypothesized path of the stator (relative to the rotor) as the motor turns (see the text). (C) Stereo-view (crossed-eye) of a section of the rotor. The view is in a roughly radial direction (out-to-in). The active-site ridge on FliG_C is coloured white.