The FlhA linker mediates flagellar protein export switching during flagellar assembly.
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ABSTRACT:
SUBMITTER: Inoue Y
PROVIDER: S-EPMC8166844 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:FlhA and FlhB are transmembrane proteins of the flagellar type III protein export apparatus, and their C-terminal cytoplasmic domains (FlhAC and FlhBC) coordinate flagellar protein export with assembly. FlhBC undergoes autocleavage between Asn-269 and Pro-270 in a well-conserved NPTH loop located between FlhBCN and FlhBCC polypeptides and interacts with the C-terminal domain of the FliK ruler when the length of the hook has reached about 55 nm in Salmonella As a result, the flagellar protein export apparatus switches its substrate specificity, thereby terminating hook assembly and initiating filament assembly. The mechanism of export switching remains unclear. Here, we report the role of FlhBC cleavage in the switching mechanism. Photo-cross-linking experiments revealed that the flhB(N269A) and flhB(P270A) mutations did not affect the binding affinity of FlhBC for FliK. Genetic analysis of the flhB(P270A) mutant revealed that the P270A mutation affects a FliK-dependent conformational change of FlhBC, thereby inhibiting the substrate specificity switching. The flhA(A489E) mutation in FlhAC suppressed the flhB(P270A) mutation, suggesting that an interaction between FlhBC and FlhAC is critical for the export switching. We propose that the interaction between FliKC and a cleaved form of FlhBC promotes a conformational change in FlhBC responsible for the termination of hook-type protein export and a structural remodeling of the FlhAC ring responsible for the initiation of filament-type protein export.IMPORTANCE The flagellar type III protein export apparatus coordinates protein export with assembly, which allows the flagellum to be efficiently built at the cell surface. Hook completion is an important morphological checkpoint for the sequential flagellar assembly process. The protein export apparatus switches its substrate specificity from the hook protein to the filament protein upon hook completion. FliK, FlhB, and FlhA are involved in the export-switching process, but the mechanism remains a mystery. By analyzing a slow-cleaving flhB(P270A) mutant, we provide evidence that an interaction between FliK and FlhB induces conformational rearrangements in FlhB, followed by a structural remodeling of the FlhA ring structure that terminates hook assembly and initiates filament formation.
| S-EPMC6964733 | biostudies-literature
Project description:The hook length of the flagellum is controlled to about 55 nm in Salmonella. The flagellar type III protein export apparatus secretes FliK to determine hook length during hook assembly and changes its substrate specificity from the hook protein to the filament protein when the hook length has reached about 55 nm. Salmonella FliK consists of an N-terminal domain (FliKN, residues 1-207), a C-terminal domain (FliKC, residues 268-405) and a flexible linker (FliKL, residues 208-267) connecting these two domains. FliKN is a ruler to measure hook length. FliKC binds to a transmembrane export gate protein FlhB to undergo the export switching. FliKL not only acts as part of the ruler but also contributes to this switching event, but it remains unknown how. Here we report that FliKL is required for efficient interaction of FliKC with FlhB. Deletions in FliKL not only shortened hook length according to the size of deletions but also caused a loose length control. Deletion of residues 206-265 significantly reduced the binding affinity of FliKC for FlhB, thereby producing much longer hooks. We propose that an appropriate length of FliKL is required for efficient interaction of FliKC with FlhB.
| S-EPMC6972891 | biostudies-literature
Project description:Flagellar structural subunits are transported via the flagellar type III secretion system (fT3SS) and assemble at the distal end of the growing flagellar structure. The C-terminal cytoplasmic domain of FlhA (FlhAC) serves as a docking platform for export substrates and flagellar chaperones and plays an important role in hierarchical protein targeting and export. FlhAC consists of domains D1, D2, D3, and D4 and adopts open and closed conformations. Gly-368 of Salmonella FlhA is located within the highly conserved GYXLI motif and is critical for the dynamic domain motions of FlhAC. However, it remains unclear how it works. Here, we report that periodic conformational changes of the GYXLI motif induce a remodeling of hydrophobic side chain interaction networks in FlhAC and promote the cyclic open-close domain motions of FlhAC. The temperature-sensitive flhA(G368C) mutation stabilized a completely closed conformation at 42°C through strong hydrophobic interactions between Gln-498 of domain D1 and Pro-667 of domain D4 and between Phe-459 of domain D2 and Pro-646 of domain D4, thereby inhibiting flagellar protein export by the fT3SS. Its intragenic suppressor mutations reorganized the hydrophobic interaction networks in the closed FlhAC structure, restoring the protein export activity of the fT3SS to a significant degree. Furthermore, the conformational flexibility of the GYXLI motif was critical for flagellar protein export. We propose that the conserved GYXLI motif acts as a structural switch to induce the dynamic domain motions of FlhAC required for efficient and rapid protein export by the fT3SS. IMPORTANCE Many motile bacteria employ the flagellar type III secretion system (fT3SS) to construct flagella beyond the cytoplasmic membrane. The C-terminal cytoplasmic domain of FlhA (FlhAC), a transmembrane subunit of the fT3SS, provides binding sites for export substrates and flagellar export chaperones to coordinate flagellar protein export with assembly. FlhAC undergoes cyclic open-close domain motions. The highly conserved Gly-368 residue of FlhA is postulated to be critical for dynamic domain motions of FlhAC. However, it remains unknown how it works. Here, we carried out mutational analysis of FlhAC combined with molecular dynamics simulation and provide evidence that the conformational flexibility of FlhAC by Gly-368 is important for remodeling hydrophobic side chain interaction networks in FlhAC to facilitate its cyclic open-close domain motions, allowing the fT3SS to transport flagellar structural subunits for efficient and rapid flagellar assembly.
| S-EPMC9431611 | biostudies-literature
Project description:The flagellar type III secretion system (fT3SS) switches substrate specificity from rod-hook-type to filament-type upon hook completion, terminating hook assembly and initiating filament assembly. The C-terminal cytoplasmic domain of FlhA (FlhAC) forms a homo-nonameric ring and is directly involved in substrate recognition, allowing the fT3SS to coordinate flagellar protein export with assembly. The highly conserved GYXLI motif (residues 368-372) of FlhAC induces dynamic domain motions of FlhAC required for efficient and robust flagellar protein export by the fT3SS, but it remains unknown whether this motif is also important for ordered protein export by the fT3SS. Here we analyzed two GYXLI mutants, flhA(GAAAA) and flhA(GGGGG), and provide evidence suggesting that the GYXLI motif in FlhAC requires the flagellar ATPase complex not only to efficiently remodel the FlhAC ring structure for the substrate specificity switching but also to correct substrate recognition errors that occur during flagellar assembly.
| S-EPMC10965912 | biostudies-literature
Project description:For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal end of the flagellum by the flagellar type III protein export apparatus powered by proton motive force (PMF) across the cytoplasmic membrane. FlhA is an integral membrane protein of the export apparatus and is involved in an early stage of the export process along with three soluble proteins, FliH, FliI, and FliJ, but the energy coupling mechanism remains unknown. Here, we carried out site-directed mutagenesis of eight, highly conserved charged residues in putative juxta- and trans-membrane helices of FlhA. Only Asp-208 was an essential acidic residue. Most of the FlhA substitutions were tolerated, but resulted in loss-of-function in the ?fliH-fliI mutant background, even with the second-site flhB(P28T) mutation that increases the probability of flagellar protein export in the absence of FliH and FliI. The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation. Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR. Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate.
| S-EPMC3139655 | biostudies-literature
Project description: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 (FlhAC) 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 FlhAC (FlhAL) is required not only for FlhAC 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. FlhAL was required for cooperative ring formation of FlhAC. Alanine substitutions of residues involved in FlhAC 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 FlhAC.
| S-EPMC5916509 | biostudies-literature
Project description:A soluble protein, FliJ, along with a membrane protein, FlhA, plays a role in the energy coupling mechanism for bacterial flagellar protein export. The water-soluble FliH(X)-FliI(6) ATPase ring complex allows FliJ to efficiently interact with FlhA. However, the FlhA binding site of FliJ remains unknown. Here, we carried out genetic analysis of a region formed by well-conserved residues-Gln38, Leu42, Tyr45, Tyr49, Phe72, Leu76, Ala79, and His83-of FliJ. A structural model of the FliI(6)-FliJ ring complex suggests that they extend out of the FliI(6) ring. Glutathione S-transferase (GST)-FliJ inhibited the motility of and flagellar protein export by both wild-type cells and a fliH-fliI flhB(P28T) bypass mutant. Pulldown assays revealed that the reduced export activity of the export apparatus results from the binding of GST-FliJ to FlhA. The F72A and L76A mutations of FliJ significantly reduced the binding affinity of FliJ for FlhA, thereby suppressing the inhibitory effect of GST-FliJ on the protein export. The F72A and L76A mutations were tolerated in the presence of FliH and FliI but considerably reduced motility in their absence. These two mutations affected neither the interaction with FliI nor the FliI ATPase activity. These results suggest that FliJ(F72A) and FliJ(L76A) require the support of FliH and FliI to exert their export function. Therefore, we propose that the well-conserved surface of FliJ is involved in the interaction with FlhA.
| S-EPMC3554004 | biostudies-literature
Project description:The flagellar type III protein export apparatus plays an essential role in the formation of the bacterial flagellum. FliH forms a complex along with FliI ATPase and is postulated to provide a link between FliI ring formation and flagellar protein export. Two tryptophan residues of FliH, Trp7 and Trp10, are required for the effective docking of the FliH-FliI complex to the export gate made of six membrane proteins. However, it remains unknown which export gate component interacts with these two tryptophan residues. Here, we performed targeted photo-cross-linking of the extreme N-terminal region of FliH (FliH(EN)) with its binding partners. We replaced Trp7 and Trp10 of FliH with p-benzoyl-phenylalanine (pBPA), a photo-cross-linkable unnatural amino acid, to produce FliH(W7pBPA) and FliH(W10pBPA). They were both functional and were photo-cross-linked with one of the export gate proteins, FlhA, but not with the other gate proteins, indicating that these two tryptophan residues are in close proximity to FlhA. Mutant FlhA proteins that are functional in the presence of FliH and FliI but not in their absence showed a significantly reduced function also by N-terminal FliH mutations even in the presence of FliI. We suggest that the interaction of FliH(EN) with FlhA is required for anchoring the FliI hexamer ring to the export gate for efficient flagellar protein export.
| S-EPMC3457192 | biostudies-other
Project description:FliN is a component of the flagellar switch complex in many bacterial species. The crystal structure is known for most of FliN, and a targeted cross-linking study (K. Paul and D. F. Blair, J. Bacteriol. 188:2502-2511, 2006) showed that it is organized in ring-shaped tetramers at the bottom of the basal body C ring. FliN is essential for flagellar assembly and direction switching, but its precise functions have not been defined. Here, we identify functionally important regions on FliN by systematic mutagenesis. Nonconservative mutations were made at positions sampling the surface of the protein, and the effects on flagellar assembly and function were measured. Flagellar assembly was disrupted by mutations in a conserved hydrophobic patch centered on the dimer twofold axis or by mutations on the surface that forms the dimer-dimer interface in the tetramer. The assembly defect in hydrophobic-patch mutants was partially rescued by overexpression of the flagellar export proteins FliH and FliI, and coprecipitation assays demonstrated a binding interaction between FliN and FliH that was weakened by mutations in the hydrophobic patch. Thus, FliN might contribute to export by providing binding sites for FliH or FliH-containing complexes. The region around the hydrophobic patch is also important for switching; certain mutations in or near the patch caused a smooth-swimming chemotaxis defect that in most cases could be partially rescued by overexpression of the clockwise-signaling protein CheY. The results indicate that FliN is more closely involved in switching than has been supposed, possibly contributing to the binding site for CheY on the switch.
| S-EPMC1539977 | biostudies-literature
Project description:UnlabelledFlagellar biogenesis is a complex process that involves multiple checkpoints to coordinate transcription of flagellar genes with the assembly of the flagellum. In Helicobacter pylori, transcription of the genes needed in the middle stage of flagellar biogenesis is governed by RpoN and the two-component system consisting of the histidine kinase FlgS and response regulator FlgR. In response to an unknown signal, FlgS autophosphorylates and transfers the phosphate to FlgR, initiating transcription from RpoN-dependent promoters. In the present study, export apparatus protein FlhA was examined as a potential signal protein. Deletion of its N-terminal cytoplasmic sequence dramatically decreased expression of two RpoN-dependent genes, flaB and flgE. Optical biosensing demonstrated a high-affinity interaction between FlgS and a peptide consisting of residues 1 to 25 of FlhA (FlhANT). The KD (equilibrium dissociation constant) was 21 nM and was characterized by fast-on (kon = 2.9 × 10(4) M(-1)s(-1)) and slow-off (koff = 6.2 × 10(-4) s(-1)) kinetics. FlgS did not bind peptides consisting of smaller fragments of the FlhANT sequence. Analysis of binding to purified fragments of FlgS demonstrated that the C-terminal portion of the protein containing the kinase domain binds FlhANT. FlhANT binding did not stimulate FlgS autophosphorylation in vitro, suggesting that FlhA facilitates interactions between FlgS and other structures required to stimulate autophosphorylation.ImportanceThe high-affinity binding of FlgS to FlhA characterized in this study points to an additional role for FlhA in flagellar assembly. Beyond its necessity for type III secretion, the N-terminal cytoplasmic sequence of FlhA is required for RpoN-dependent gene expression via interaction with the C-terminal kinase domain of FlgS.
| S-EPMC4420913 | biostudies-literature