Symmetry mismatch in the MS-ring of the bacterial flagellar rotor explains the structural coordination of secretion and rotation

Abstract

The bacterial flagellum is a complex self-assembling nanomachine that confers motility to the cell. Despite great variation across species, all flagella are ultimately constructed from a helical propeller that is attached to a motor embedded in the inner membrane. The motor consists of a series of stator units surrounding a central rotor made up of two ring complexes, the MS-ring and the C-ring. Despite many studies, high-resolution structural information is still lacking for the MS-ring of the rotor, and proposed mismatches in stoichiometry between the two rings have long provided a source of confusion for the field. Here, we present structures of the Salmonella MS-ring, revealing a high level of variation in inter- and intrachain symmetry that provides a structural explanation for the ability of the MS-ring to function as a complex and elegant interface between the two main functions of the flagellum-protein secretion and rotation.

Extended Data Fig. 1. Structure determination of 33mer

a. SDS-PAGE of samples taken throughout purification of FliF in DDM. Lanes contain (1, 16) PageRuler Markers (2) whole cell lysate (3) supernatant from low speed spin (4) supernatant from high speed spin (5) solubilised membranes (6) supernatant from second low speed spin (7) supernatant from second high speed spin (8) resuspended pellet from second high speed spin (9-15 and 17-20) fractions from top to bottom of sucrose gradient post-high speed equilibration. Note – this gel shows samples from sucrose gradient without glutaraldehyde run in parallel with tubes containing glutaraldehyde. The fractions equivalent to those indicated (red box) were selected from the cross-linked gradients and used for structural analysis. b. Example micrograph (1.5 μm defocus) of cross-linked FliF on a graphene oxide surface. Scale bar 500Å c. FSC curves from PostProcessing in RELIONv3.0 for volumes calculated in (i) C33, (ii) C21 and (iii) C3 respectively. d. Slab through C3 volume coloured by local resolution as estimated using RELIONv3.0.

Extended Data Fig. 2. 33-fold symmetry does not resolve lower ring detail

a. Volume generated by refinement in C33 shows lack of detail in RBM2_inner region below, later explained by C21 symmetry in this region. b. Slab through central section of composite C33/C21/C3 volume reveals layered density derived from the detergent micelle at the periphery of the RBM3 ring and a central column of density below the C21 ring that presumably results from density associated with the 24 copies of RBM1 that are not located elsewhere in the map, the N-terminal trans-membrane helices attached to these and associated detergent.

Extended Data Fig. 3. Proteomic analysis reveals limited clipping at extreme C-terminus of FliF

a. Purified, non-crosslinked S. Typhimurium FliF was run on blue native PAGE, then the gel band corresponding to the FliF complex was cut out and run on SDS-PAGE (lane marked X). The SDS-PAGE bands were cut out and mass spectrometry was used to identify the protein. The identity of each band is indicated on the gel. b. The three S Typhimurium FliF bands all produced peptides spread throughout the FliF amino acid sequence (i.e. from residues 2 to 560/552), however the two lighter bands had a lower intensity of peptides from the sequence post the folded RBM3 domain suggesting these bands differ in trimming of the extreme C-terminus beyond the structured region.

Extended Data Fig. 4. Comparisons of individual RBM domains

a. Overlay of RBM2 and RBM3 domains of FliF (chain A), rmsd of 2.3 Å over 78 Cα. b. Superposition of the RBM2 domain of FliF on the closest structural homologue – the RBM2 of SctJ, rmsd of 1.1 Å over 79 Cα c. Superposition of the RBM3 domain of FliF on the SpoIIIAG RBM domain, rmsd of 2.3 Å over 80 Cα. The beta-insertions are not used to derive the superposition and the different relationship between the RBM domains and these inserts can therefore be appreciated. d. The N-terminal (blue) and C-terminal strands (red) of the beta-insert cross at the transition between tilted and vertically oriented strands.

Extended Data Fig. 5. Structural Observations

a. The Electrostatic potential is mapped onto the surface of the FliF assembly (upper panel) and monomer using APBS within PyMol, revealing that the overall object is highly charged whilst the monomer interfaces are largely hydrophobic. b. A putative glutaraldehyde cross-link (marked with an asterisk) is observed in the beta-collar region of FliF.

Extended Data Fig. 6. Building the C21 portion of the structure

a. Superposing a pair of neighbouring RBM3 domains on to a pair of neighbouring RBM2_inner by aligning the first domain shows the rearrangements driven by the C33 versus C21 packing. b. The RBM2_outer domains provide the major contact between the RBM2_inner and RBM3 rings adapting between the C21 and C33 symmetries.

Extended Data Fig. 7. Subtle differences in RBM domain packing drives different assemblies

a. Superposing a pair of neighbouring RBM3 domains on to a pair of neighbouring SpoIIIAG RBM domains by aligning the first domain shows the subtle alteration in packing needed to form the C33 rather than C30 assemblies. b. Superposing a pair of neighbouring RBM2_inner domains on to a pair of neighbouring SctJ RBM2 domains by aligning the first domain shows the subtle alteration in packing needed to form the C21 rather than C24 assemblies.

Extended Data Fig. 8. Structure determination of 34mer

a. FSC curves from PostProcessing in RELIONv3.0 for volumes calculated in (i) C34, (ii) C22 and (iii) C2 respectively. b. Slab through C2 volume coloured by local resolution as estimated using RELIONv3.0.

Extended Data Fig. 9. Supervised 3D classification reveals assembly diversity

Distribution of particles between different symmetries in the RBM3 ring/β-collar region following supervised 3D-classification. 20% of particles were allocated to a C36 class, but the volume was uninterpretable from this class and presumably reflected damaged particles / particles with a variety of other symmetries.

Extended Data Fig. 10. Putative location of missing RBM1 domain in tomogram

A slab through the centre of the P. shigelloides flagellar tomogram (grey surface; EMDB 10057) with the FliF C34 volume (blue surface) placed. The density of appropriate volume for the currently unresolved RBM1 domains is highlighted with red circles.

Figure 1. Overall structure of the flagellar MS ring

a, Schematic showing the location of the MS ring (blue) within the bacterial flagellum. b, Cartoon representation of the domain structure of FliF. c, Composite 3D cryo-EM reconstruction with different symmetries applied within masks (see methods). Regions occupied by RBM2 and RBM3 for each chain are similarly coloured in an alternating scheme, with the exception of chains for which only RBM3 can be seen (yellow). Regions assigned to 9 RBM1 domains are indicated in red as connectivity cannot be definitively assigned. d, Representative 2D class of cross-linked FliF complexes on graphene oxide surface. Scale bar, 100Å. e, Representative density for regions where de-novo building of protein domains was possible in (i, ii) the C33 averaged RBM3-region map and (iii) the C21 averaged RBM2_inner-region map. f, Final model for the 33mer FliF, coloured as in (b). g, Representative density for docking of the RBM2_outer and RBM1 domains.

Figure 2. Domain rearrangements required to build the full assembly

a, The locations of conformationally equivalent subunits within the assembly are highlighted; blue RBM3/collar/RBM2_inner; green RBM3/collar/RBM2_outer; maroon RBM3/collar/RBM2_{not observed}. b, The three domain arrangements observed for RBM3/β-collar/RBM2 domains within the complex are shown with the RBM3/collar region aligned, coloured as in (Fig 1f).

Figure 3. Conserved or co-varying residues map to the inter-subunit interfaces

a, Conservation of FliF between bacterial species is plotted on the surface of the monomer (ConSurf http://consurf.tau.ac.il) revealing that the monomer-monomer interfaces are the most conserved regions. b, Strongly co-varying residue pairs which do not form contacts within a single subunit (yellow) can all be explained by close contacts (green dotted lines) with neighbouring subunits (shades of orange).

Figure 4. Comparison to structurally or functionally homologous assemblies

a, The closest structural homologue to the RBM3 portion of the FliF ring is the 30-mer B. subtilis protein SpoIIIAG (PDB-5wc3). A view from the outer-membrane side is shown above and from the side below, with a cartoon representation of a single, extracted, monomer also shown. A small beta-insertion structure is indicated (arrow on monomer structures). b, The virulence T3SS basal body is constructed from two protein chains in the MS-ring equivalent region, which both form 24-mer rings consisting of multiple RBM domains. Central sections of the Salmonella SPI-1 injectisome basal body (PDB-5tcr, LH panel) and the FliF ring (RH panel) show the striking similarity in overall shape despite fundamental differences in the chains and domain types used. They also show the 21-fold RBM2_inner domains are very similarly arranged to the PrgK/SctJ-RBM2 24-fold ring, whilst the FliF-RBM1 and PrgK/SctJ-RBM1 domains are very differently arranged with respect to these.

Figure 5. The flagellar MS ring is structurally heterogenous

a, Composite 3D cryo-EM reconstruction from a 34-fold stoichiometric subset of particles. C34 symmetry is applied within the RBM3 region, C22 within the RBM2_inner region and C2 symmetry applied elsewhere. The colour scheme mimics that of Figure 1c. b, Comparison of the C33/C34 and C21/C22 regions by overlaying the complete rings using a single chain reveals the subtle differences in the sizes of the respective ring-like assemblies built. c, Despite assembling to form rings of different symmetries, the specific interactions from which they are built are entirely conserved, including potential salt bridges, in both the C33 (cyan and blue)/C34 (grey) rings (left hand panel) and the C21 (cyan and blue)/C22 (grey)rings (right hand panel).

Figure 6. The MS-ring as a structural adapter

a, A model for the 34-mer MS-ring, coloured to highlight the different structural regions, is placed in the single particle reconstruction of the S. Typhimurium flagellar basal body (grey) (EMD-1887), showing the good match in overall shape and links to the 34-fold symmetric C-ring. The 34-mer FliF was built in the map shown in Figure 5a and extended to the C-terminus using a continuous helix of the correct length, ending in a homology model based on the crystal structure of residues 523-559 of Helicobacter pylori FliF (PDB: 5wuj). b, The FliF model (coloured as in (a)) is shown placed in a P. shigelloides tomographic volume (EMD-10057) and a model for the export gate complex (blue) (PDB-6r69) is then docked within FliF. The panel on the right is an update of the cartoon from Figure 1a, using this colour scheme. c, Exploded diagram of FliF coloured to emphasise the roles the different symmetries play in adapting between components within the flagellar assembly.