Insight into structural remodeling of the FlhA ring responsible for bacterial flagellar type III protein export

Abstract

The bacterial flagellum is a supramolecular motility machine. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. A carboxyl-terminal cytoplasmic domain of FlhA (FlhA_C) forms a nonameric ring structure in the flagellar type III protein export apparatus and coordinates flagellar protein export with assembly. However, the mechanism of this process remains unknown. We report that a flexible linker of FlhA_C (FlhA_L) is required not only for FlhA_C ring formation but also for substrate specificity switching of the protein export apparatus from the hook protein to the filament protein upon completion of the hook structure. FlhA_L was required for cooperative ring formation of FlhA_C. Alanine substitutions of residues involved in FlhA_C ring formation interfered with the substrate specificity switching, thereby inhibiting filament assembly at the hook tip. These observations lead us to propose a mechanistic model for export switching involving structural remodeling of FlhA_C.

Fig. 1. FlhA_C forms a ring-shaped structure.

(A) Typical HS-AFM image of FlhA_C placed on mica at a protein concentration of 2.4 μM. The image was recorded at 200 ms per frame in a scanning area of 100 × 100 nm² with 100 × 100 pixels. (B) Comparison of a simulated HS-AFM image (middle panel) constructed from the atomic model of the FlhA_C9 ring structure (left panel) with an experimental HS-AFM image of the FlhA_C ring (right panel) obtained at a concentration of 0.71 μM. The HS-AFM image was recorded at 200 ms per frame in a scanning area of 50 × 50 nm² with 100 × 100 pixels. Color bar shows a range of particle height (nanometers). (C to E) Histograms of the stoichiometry (C), diameter (peak-to-peak distance), (D) and height (E) of the FlhA_C ring. (F) Effect of protein concentrations on FlhA_C ring formation. Ring particles per frame were counted under each condition. The number of the ring formed at the highest protein concentration is set to 1. Relative ring particle numbers versus protein concentrations (micromolar) are plotted and fitted by Hill equation.

Fig. 2. Effect of deletion of residues 328 to 351 in FlhA_L on FlhA_C ring formation.

(A) Crystal structure of the FlhA_C dimer [PDB (Protein Data Base) ID: 3A5I]. FlhA_C contains four domains, D1, D2, D3, and D4, and a flexible linker termed FlhA_L. The asymmetric unit of the crystal contains two FlhA_C molecules. Residues 347 to 361 of FlhA_L bind to the D1 and D3 domains of its neighboring subunit. Residues involved in this interaction are shown. The Cα backbone is color-coded from blue to red, going through the rainbow colors from the N terminus to the C terminus. (B) Typical HS-AFM images of wild-type FlhA_C (indicated as WT) and FlhA_C38K (indicated as 38K) lacking residues 328 to 351 of FlhA_L placed either on mica or mica-supported planar phospholipid bilayer (mica-SLB). The protein concentrations were ca. 2.4 and 11.8 μM when they were placed on the mica and phospholipid surfaces, respectively. The HS-AFM images on the mica surface were recorded at 200 ms per frame in a scanning area of 100 × 100 nm² with 150 × 150 pixels. The images on phospholipids were recorded at 500 ms per frame in a scanning area of 100 × 100 nm² with 200 × 200 pixels. Scale bars, 10 nm. (C) Effect of protein concentrations on FlhA_C ring formation on phospholipid bilayer. Ring particles per frame were counted under each condition. Relative ring particle numbers versus protein concentrations (micromolar) are plotted and fitted by Hill equation. (D) Typical HS-AFM images of wild-type FlhA_C and its mutant variants, W354A, E351A/D356A, E351A/W354A/D356A, R391A/K392A/K393A, and L401A placed on a mica surface at a concentration of 2.4 μM. All images were recorded at 200 ms per frame in a scanning area of 100 × 100 nm² with 150 × 150 pixels. Scale bars, 10 nm.

Fig. 3. Effect of alanine substitutions of residues involved in interactions of FlhA_L with its neighboring subunit on FlhA function.

(A) Secretion assays of flagellar proteins. Immunoblotting using polyclonal anti-FlgD (first row), anti-FlgE (second row), anti-FlgM (third row), anti-FlgK (fourth row), anti-FlgL (fifth row), or anti-FliC (sixth row) antibody, of whole-cell proteins (Cell) and culture supernatants (Sup). Lanes 1 and 9, ΔflhA; lanes 2 and 10, wild-type (WT); lanes 3 and 11, flhA(W354A) (W354A); lanes 4 and 12, flhA(E351A/D356A) (E351A/D356A); lanes 5 and 13, flhA(E351A/W354A/D356A) (E351A/W354A/D356A); lanes 6 and 14, flhA(R391A/K392A/K393A) (R391A/K392A/K393A); lanes 7 and 15, flhA(L401A) (L401A); and lanes 8 and 16, flhA(Q511A) (Q511A). The positions of FlgD, FlgE, FlgM, FlgK, FlgL, and FliC are indicated by arrows. (B) Electron micrograms of HBB isolated from the above stains. The average hook length and SDs are shown. Scale bars, 100 nm. (C) Interaction between FlhA_C and FlgN-FlgK complex. Purified His-FlhA_C (WT) or His-FlhA_C(W354A) (W354A) was mixed with GST-FlgN in complex with FlgK (first and second rows), or GST alone (third row) and dialyzed overnight against phosphate-buffered saline (PBS) and dialyzed overnight against PBS. The mixture (L) was subjected to GST affinity chromatography. Flow through fraction (F.T.), wash fractions (W), and elution fractions (E) were analyzed by CBB (Coomassie brilliant blue) staining.

Fig. 4. Effect of depletions of FliH and FliI on the export function of FlhA(L401A).

(A) Interaction between FliJ and FlhA_C. The mixture of the soluble fractions (Input) prepared from Salmonella ΔflhDC-cheW cells expressing GST-FliJ with those from a Salmonella ΔflhA mutant carrying pTrc99AFF4 (indicated as ΔflhA), pMM130 (WT), pYI001 (W354A), pYI002 (E351A/D356A), pYI003 (E351A/W354A/D356A), pYI004 (R391A/K392A/K393A), pYI005 (L401A), or pYI006 (Q511A) was loaded onto a GST column. After extensive washing, proteins were eluted with a buffer containing 10 mM reduced glutathione. The eluted fractions were analyzed by immunoblotting with polyclonal anti-FlhA antibody (first and second rows) and CBB staining for GST-FliJ (third row). (B) Motility of and flagellar protein export by NH004 (ΔfliH-fliI flhB* ΔflhA) (left panel) and NH002 (flhB* ΔflhA) (right panel) harboring pTrc99AFF4 (V), pMM130 (WT), pYI005 (L401A), or pYI006 (Q511A). Upper panels: Motility assays in soft agar at 30°C. Lower panels: Immunoblotting, using polyclonal anti-FlgD and anti-FlgK antibodies, of whole-cell proteins (Cell) and culture supernatant fractions (Sup).

Fig. 5. Model for cooperative remodeling of the FlhA_C ring structure.

Partial atomic models of the MxiA_C (left; PDB ID: 4A5P) and FlhA_C (right; PDB ID: 3A5I) ring models are shown. Both MxiA_C and FlhA_C consist of domains D1 (cyan), D2 (blue), D3 (light green), and D4 (red). FlhA_C forms a nonameric ring structure mainly through interactions between D3 domains and between domains D1 and D3. Only an N-terminal short stretch forming part (magenta) of a flexible linker is visible, whereas most of residues forming the linker are invisible because of their conformational flexibility. In contrast, about a C-terminal half of FlhA_L binds to its neighboring subunit in the FlhA_C crystal. During hook assembly, FlhA_L does not bind to its neighboring subunit, allowing the flagellar type III protein export apparatus to transport the hook protein. Upon completion of the hook structure, FlhA_L binds to the D1 and D3 domains of its neighboring subunit, inducing conformational changes of the FlhA_C ring in a highly cooperative manner. As a result, the protein export apparatus terminates the export of the hook protein and initiates the export of filament-type proteins responsible for filament formation at the hook tip.