Structural modeling of the flagellum MS ring protein FliF reveals similarities to the type III secretion system and sporulation complex

Abstract

The flagellum is a large proteinaceous organelle found at the surface of many bacteria, whose primary role is to allow motility through the rotation of a long extracellular filament. It is an essential virulence factor in many pathogenic species, and is also a priming component in the formation of antibiotic-resistant biofilms. The flagellum consists of the export apparatus on the cytosolic side; the basal body and rotor, spanning the bacterial membrane(s) and periplasm; and the hook-filament, that protrudes away from the bacterial surface. Formation of the basal body MS ring region, constituted of multiple copies of the protein FliF, is one of the initial steps of flagellum assembly. However, the precise architecture of FliF is poorly understood. Here, I report a bioinformatics analysis of the FliF sequence from various bacterial species, suggesting that its periplasmic region is composed of three globular domains. The first two are homologous to that of the type III secretion system injectisome proteins SctJ, and the third possesses a similar fold to that of the sporulation complex component SpoIIIAG. I also describe that Chlamydia possesses an unusual FliF protein, lacking part of the SctJ homology domain and the SpoIIIAG-like domain, and fused to the rotor component FliG at its C-terminus. Finally, I have combined the sequence analysis of FliF with the EM map of the MS ring, to propose the first atomic model for the FliF oligomer, suggesting that FliF is structurally akin to a fusion of the two injectisome components SctJ and SctD. These results further define the relationship between the flagellum, injectisome and sporulation complex, and will facilitate future structural characterization of the flagellum basal body.

Keywords: Chlamydia; Flagellum; Homology modeling; Salmonella; Secretion system; Sporulation.

Figure 1. The flagellar, T3SS, and sporulation complexes.

Schematic representation of the bacterial flagellum (A), the T3SS (B), and the sporulation complex (C). SctJ-like components are in blue, SctD-like components in green, and outer-membrane components in yellow. The EM maps are shown in grey for (A) and (B). The ring structures identified in the flagellum are also indicated. IM, inner membrane; OM, outer membrane; IFM, inner forespore membrane; OFM, outer forespore membrane.

Figure 2. Domain organization of FliF.

Multiple sequence alignment of FliF sequences from various human pathogens (S. typhimurium, Escherichia coli, Yersinia pestis, Bordetella pertussis, Pseudomonas aeruginosa, Legionella pneumophilia, Helicobacter pylori, Campylobacter jejuni, Listeria monocytogenes, Streptococcus pneumonia, Vibrio cholerae, Bacillus subtilis, Clostridium difficile, Treponema palladium). Conserved residues are in red box, similar residues are in red characters. Identified domains are highlighted in colored boxes, with the TM helices in yellow, the FliG-binding domain in green, the signal sequence in purple, the SctJ homology domain in blue and the FliF-specific domain in orange. The predicted secondary structure elements for the S. typhimurium FliF are in blue at the top.

Figure 3. The Chlamydia FliF orthologue has unusual domain architecture.

(A) Multiple sequence alignment of FliF sequences from the Chlamydiacae family (C. trachomatis, C. muridarum, C. suis from the genus Chlamydia, and C. psittaci, C. abortus, C. felis, C. caviae, C. ibidis, C. pneumonia, and C. pecorum from the genus Chlamydophila. Labeling is as in figure A, with the secondary structure prediction of the C. trachomatis orthologue shown at the top. (B) Schematic representation of FliF and its interaction with FliG (top), and of the Chlamydia FliF-FliG fusion (bottom).

Figure 4. Modeling of the S. typhimurium FliF SctJ homology domain.

(A) Sequence alignment of the periplasmid domains from the EPEC and S. typhimurium SctJ homologues, EscJ and PrgK, with that of the SctJ homology domain of FliF (residues 50–221). Secondary structure elements for EscJ (PDB ID: 1YJ7) and PrgK (PDB ID: 3J6D) are shown at the top, in blue and green respectively. (B) and (C) Energy plot for the refinement of the FliF RBM1 and RBM2. The RMSD values are computed for all atoms, relative to the lowest-energy model. (D) and (E) Cartoon representation of the lowest-energy models for the FliF RBM1 and RBM2, with rainbow coloring indicating N- to C-termini.

Figure 5. Modeling of the FliF-specific domain.

(A) Sequence alignment of the EscJ RBM2, PrgK RBM2, SpoIIIAG and SpoIIIAH, with that of the FliF-specific domain (residues 228–439). Secondary structure elements for EscJ (PDB ID: 1YJ7) and SpoIIIAH (PDB ID: 3UZ0) are shown at the top, in blue and green respectively. The location of the insert ion FliF and SpoIIIAG is indicated. (B) Energy plot for the refinement of the FliF RBM3. The RMSD values are computed for all atoms, relative to the lowest-energy model. (C) Cartoon representation of the lowest-energy model for the FliF RBM3, with rainbow coloring indicating N- to C-termini. The location of the insert is indicated.

Figure 6. Comparison of the flagellar and T3SS basal body.

(A) EM maps of the S. typhimurium T3SS basal body (EMBD ID: 1875) in black, overlaid on that of the S. typhimurium flagellum basal body (EMBD ID: 1887) in yellow. A close-up view of the S-ring region of the flagellum is shown on the left, with the SctJ/SctD ring model (PDB ID 3J6D) docked in the T3SS EM map. The SctJ RBM1 is in cyan and the RBM2 in blue, and the three SctD RBMs are in green. (B) Schematic representation of SctJ, SctD and FliF, with the TMs in yellow, and the RBMs colored as in (A) for SctJ and SctD. Corresponding domains are indicated in FliF.

Figure 7. Modeling of the FliF oligomer.

(A) Docking of the three FliF RBM models in the FliF EM map. The domains are colored as in figure 5B. (B) Energy plot for the EM-guided symmetry docking procedure of the FliF RBM3. The RMSDs are computed for backbone atoms of the entire modeled 24mer complex, relative to the lowest-energy model, and color-coded depending on the fit to EM map. Three clusters of low-energy models were identified (Cl-1, Cl-2 and Cl-3), with two adjacent molecules for each cluster shown in (C). The 25-mer radius axis is represented by a dotted line. (D) 25-mer model of the FliF periplasmic region, viewed from the top.