scholarly journals The FlhA linker mediates flagellar protein export switching during flagellar assembly

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yumi Inoue ◽  
Miki Kinoshita ◽  
Mamoru Kida ◽  
Norihiro Takekawa ◽  
Keiichi Namba ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Binding Site ◽  
Protein Export ◽  
Hook Length ◽  
Filament Assembly ◽  
Type Protein ◽  
Flagellar Assembly ◽  
Chaperone Binding ◽  
Flagellar Protein ◽  
Filament Type

AbstractThe flagellar protein export apparatus switches substrate specificity from hook-type to filament-type upon hook assembly completion, thereby initiating filament assembly at the hook tip. The C-terminal cytoplasmic domain of FlhA (FlhAC) serves as a docking platform for flagellar chaperones in complex with their cognate filament-type substrates. Interactions of the flexible linker of FlhA (FlhAL) with its nearest FlhAC subunit in the FlhAC ring is required for the substrate specificity switching. To address how FlhAL brings the order to flagellar assembly, we analyzed the flhA(E351A/W354A/D356A) ΔflgM mutant and found that this triple mutation in FlhAL increased the secretion level of hook protein by 5-fold, thereby increasing hook length. The crystal structure of FlhAC(E351A/D356A) showed that FlhAL bound to the chaperone-binding site of its neighboring subunit. We propose that the interaction of FlhAL with the chaperon-binding site of FlhAC suppresses filament-type protein export and facilitates hook-type protein export during hook assembly.

scholarly journals The FlhA linker mediates flagellar protein export switching during flagellar assembly

2020 ◽  
Author(s):  
Yumi Inoue ◽  
Mamoru Kida ◽  
Miki Kinoshita ◽  
Norihiro Takekawa ◽  
Keiichi Namba ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Cytoplasmic Domain ◽  
Ring Structure ◽  
Protein Export ◽  
Filament Assembly ◽  
Flagellar Assembly ◽  
Chaperone Binding ◽  
Flagellar Protein ◽  
Filament Type

AbstractThe flagellar protein export apparatus switches export specificity from hook-type to filament-type upon completion of hook assembly, thereby initiating filament assembly at the hook tip. The C-terminal cytoplasmic domain of FlhA (FlhAC) forms a homo-nonameric ring structure that serves as a docking platform for flagellar export chaperones in complex with their cognate filament-type substrates. Interactions of the flexible linker of FlhA (FlhAL) with its nearest FlhAC subunit in the ring allow the chaperones to bind to FlhAC to facilitate filament-type protein export, but it remains unclear how it occurs. Here, we report that FlhAL acts as a switch that brings the order to flagellar assembly. The crystal structure of FlhAC(E351A/D356A) showed that Trp-354 in FlhAL bound to the chaperone-binding site of its neighboring subunit. We propose that FlhAL binds to the chaperon-binding site of FlhAC to suppress the interaction between FlhAC and the chaperones until hook assembly is completed.

scholarly journals FliK-Driven Conformational Rearrangements of FlhA and FlhB Are Required for Export Switching of the Flagellar Protein Export Apparatus

2019 ◽  
Vol 202 (3) ◽  
Author(s):  
Tohru Minamino ◽  
Yumi Inoue ◽  
Miki Kinoshita ◽  
Keiichi Namba
Keyword(s):  
Substrate Specificity ◽  
Conformational Change ◽  
Ring Structure ◽  
Protein Export ◽  
Structural Remodeling ◽  
Type Iii ◽  
Type Protein ◽  
Flagellar Assembly ◽  
Flagellar Protein ◽  
Conformational Rearrangements

ABSTRACT 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.

scholarly journals Helicobacter pylori FlhB processing-deficient variants affect flagellar assembly but not flagellar gene expression

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1170-1180 ◽  
Author(s):  
Todd G. Smith ◽  
Lara Pereira ◽  
Timothy R. Hoover
Keyword(s):  
Helicobacter Pylori ◽  
Substrate Specificity ◽  
Reporter Genes ◽  
Inhibitory Effect ◽  
Protein Export ◽  
Flagellar Gene ◽  
Wild Type ◽  
Flagellar Assembly ◽  
Flagellar Protein ◽  
H Pylori

Regulation of the Helicobacter pylori flagellar gene cascade involves the transcription factors σ 54 (RpoN), employed for expression of genes required midway through flagellar assembly, and σ 28 (FliA), required for expression of late genes. Previous studies revealed that mutations in genes encoding components of the flagellar protein export apparatus block expression of the H. pylori RpoN and FliA regulons. FlhB is a membrane-bound component of the export apparatus that possesses a large cytoplasmic domain (FlhBC). The hook length control protein FliK interacts with FlhBC to modulate the substrate specificity of the export apparatus. FlhBC undergoes autocleavage as part of the switch in substrate specificity. Consistent with previous reports, deletion of flhB in H. pylori interfered with expression of RpoN-dependent reporter genes, while deletion of fliK stimulated expression of these reporter genes. In the ΔflhB mutant, disrupting fliK did not restore expression of RpoN-dependent reporter genes, suggesting that the inhibitory effect of the ΔflhB mutation is not due to the inability to export FliK. Amino acid substitutions (N265A and P266G) at the putative autocleavage site of H. pylori FlhB prevented processing of FlhB and export of filament-type substrates. The FlhB variants supported wild-type expression of RpoN- and FliA-dependent reporter genes. In the strain producing FlhBN265A, expression of RpoN- and FliA-dependent reporter genes was inhibited when fliK was disrupted. In contrast, expression of these reporter genes was unaffected or slightly stimulated when fliK was disrupted in the strain producing FlhBP266G. H. pylori HP1575 (FlhX) shares homology with the C-terminal portion of FlhBC (FlhBCC) and can substitute for FlhBCC in flagellar assembly. Disrupting flhX inhibited expression of a flaB reporter gene in the wild-type but not in the ΔfliK mutant or strains producing FlhB variants, suggesting a role for FlhX or FlhBCC in normal expression of the RpoN regulon. Taken together, these data indicate that the mechanism by which the flagellar protein export apparatus exerts control over the H. pylori RpoN regulon is complex and involves more than simply switching substrate specificity of the flagellar protein export apparatus.

scholarly journals Domain Structure of Salmonella FlhB, a Flagellar Export Component Responsible for Substrate Specificity Switching

2000 ◽  
Vol 182 (17) ◽  
pp. 4906-4914 ◽  
Author(s):  
Tohru Minamino ◽  
Robert M. Macnab
Keyword(s):  
Substrate Specificity ◽  
Transmembrane Domain ◽  
Physical Interaction ◽  
Wild Type ◽  
Binding Properties ◽  
Membrane Bound ◽  
Flagellar Protein ◽  
Mutant Host ◽  
Control Protein ◽  
Filament Type

ABSTRACT We have investigated the properties of the cytoplasmic domain (FlhBC) of the 383-amino-acid Salmonellamembrane protein FlhB, a component of the type III flagellar export apparatus. FlhB, along with the hook-length control protein FliK, mediates the switching of export specificity from rod- and hook-type substrates to filament-type substrates during flagellar morphogenesis. Wild-type FlhBC was unstable (half-life, ca. 5 min), being specifically cleaved at Pro-270 into two polypeptides, FlhBCN and FlhBCC, which retained the ability to interact with each other after cleavage. Full-length wild-type FlhB was also subject to cleavage. Coproduction of the cleavage products, FlhBΔCC (i.e., the N-terminal transmembrane domain FlhBTM plus FlhBCN) and FlhBCC, resulted in restoration of both motility and flagellar protein export to an flhB mutant host, indicating that the two polypeptides were capable of productive association. Mutant FlhB proteins that can undergo switching of substrate specificity even in the absence of FliK were much more resistant to cleavage (half-lives, 20 to 60 min). The cleavage products of wild-type FlhBC, existing as a FlhBCN–FlhBCC complex on an affinity blot membrane, bound the rod- and hook-type substrate FlgD more strongly than the filament-type substrate FliC. In contrast, the intact form of FlhBC (mutant or wild type) or the FlhBCC polypeptide alone bound FlgD and FliC to about the same extent. FlhBCN by itself did not bind substrates appreciably. We propose that FlhBC has two substrate specificity states and that a conformational change, mediated by the interaction between FlhBCN and FlhBCC, is responsible for the specificity switching process. FliK itself is an export substrate; its binding properties for FlhBC resemble those of FlgD and do not provide any evidence for a physical interaction beyond that of the export process.

scholarly journals FlhA Undergoes Cyclic Open-close Domain Motions During Flagellar Protein Export in Salmonella

2021 ◽  
Author(s):  
Tohru Minamino ◽  
Yumi Inoue ◽  
Miki Kinoshita ◽  
Akio Kitao ◽  
Keiichi Namba
Keyword(s):  
Closed Form ◽  
Protein Transport ◽  
Building Blocks ◽  
Protein Export ◽  
Side Chain ◽  
Transport Activity ◽  
Flagellar Assembly ◽  
Flagellar Protein ◽  
Domain Motions

Abstract The flagellar type III secretion system (fT3SS) transports flagellar building blocks from the cytoplasm to the distal end of the growing flagellar structure. The C-terminal cytoplasmic domain of FlhA (FlhAC) serves as a docking platform for flagellar chaperones in complex with their cognate substrates and ensures the strict order of protein export for efficient flagellar assembly. FlhAC adopts open and closed conformations, and the chaperones bind to the open form, allowing the fT3SS to transport the substrates to the cell exterior. To clarify the role of the closed form in flagellar protein export, we isolated pseudorevertants from the flhA(G368C/K549C) mutant, in which the closed conformation is stabilized to inhibit the protein transport activity of the fT3SS. Each of M365I, R370S, A446E and P550S substitutions in FlhAC identified in the pseudorevertants affected hydrophobic side-chain interaction networks in the closed FlhAC structure, thereby restoring the protein transport activity to a considerable degree. We propose that a cyclic open-close domain motion of FlhAC is required for rapid and efficient flagellar protein export where a structural transition from the open to the closed form induces the dissociation of empty chaperones from FlhAC.

scholarly journals Substrate specificity of type III flagellar protein export in Salmonella is controlled by subdomain interactions in FlhB

2003 ◽  
Vol 48 (4) ◽  
pp. 1043-1057 ◽  
Author(s):  
Gillian M. Fraser ◽  
Takanori Hirano ◽  
Hedda U. Ferris ◽  
Lara L. Devgan ◽  
May Kihara ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Protein Export ◽  
Type Iii ◽  
Flagellar Protein

scholarly journals A positive charge region of Salmonella FliI is required for ATPase formation and efficient flagellar protein export

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Miki Kinoshita ◽  
Keiichi Namba ◽  
Tohru Minamino
Keyword(s):  
Positive Charge ◽  
Protein Export ◽  
Active Protein ◽  
Gain Of Function ◽  
Charged Cluster ◽  
Charge Cluster ◽  
Flagellar Assembly ◽  
Flagellar Protein ◽  
Positively Charged

AbstractThe FliH2FliI complex is thought to pilot flagellar subunit proteins from the cytoplasm to the transmembrane export gate complex for flagellar assembly in Salmonella enterica. FliI also forms a homo-hexamer to hydrolyze ATP, thereby activating the export gate complex to become an active protein transporter. However, it remains unknown how this activation occurs. Here we report the role of a positively charged cluster formed by Arg-26, Arg-27, Arg-33, Arg-76 and Arg-93 of FliI in flagellar protein export. We show that Arg-33 and Arg-76 are involved in FliI ring formation and that the fliI(R26A/R27A/R33A/R76A/R93A) mutant requires the presence of FliH to fully exert its export function. We observed that gain-of-function mutations in FlhB increased the probability of substrate entry into the export gate complex, thereby restoring the export function of the ∆fliH fliI(R26A/R27A/R33A/R76A/R93A) mutant. We suggest that the positive charge cluster of FliI is responsible not only for well-regulated hexamer assembly but also for substrate entry into the gate complex.

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