Two-Ball Structure of the Flagellar Hook-Length Control Protein FliK as Revealed by High-Speed Atomic Force Microscopy

Kodera, Noriyuki; Uchida, Kaoru; Ando, Toshio; Aizawa, Shin‐Ichi

doi:10.1016/j.jmb.2014.11.007

Cited by 27 publications

(20 citation statements)

References 32 publications

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“…Interestingly, FliK C decreases its volume as the distance between FliK N and FliK C increases whereas the volume of FliK N does not change at all. This suggests that the N‐terminal portion of the FliK T3S4 domain is less stable and tends to become unstructured (Kodera et al ., ). Since helix α1 and strand β1 are not directly involved in the substrate specificity switch (Minamino et al ., ), we propose that conformational rearrangements of the N‐terminal potion of FliK T3S4 may allow Val‐302 and Ile‐304 to be exposed to the solvent, thereby promoting the interaction between the hydrophobic core domain of FliK T3S4 and FlhB CC .…”

Section: Discussionmentioning

confidence: 97%

The role of intrinsically disordered C‐terminal region of FliK in substrate specificity switching of the bacterial flagellar type III export apparatus

Kinoshita

Aizawa

Inoue

et al. 2017

Molecular Microbiology

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The bacterial flagellar export switching machinery consists of a ruler protein, FliK, and an export switch protein, FlhB and switches substrate specificity of the flagellar type III export apparatus upon completion of hook assembly. An interaction between the C-terminal domain of FliK (FliK ) and the C-terminal cytoplasmic domain of FlhB (FlhB ) is postulated to be responsible for this switch. FliK has a compactly folded domain termed FliK (residues 268-352) and an intrinsically disordered region composed of the last 53 residues, FliK (residues 353-405). Residues 301-350 of FliK and the last five residues of FliK are critical for the switching function of FliK. FliK is postulated to regulate the interaction of FliK with FlhB , but it remains unknown how. Here we report the role of FliK in the export switching mechanism. Systematic deletion analyses of FliK revealed that residues of 351-370 are responsible for efficient switching of substrate specificity of the export apparatus. Suppressor mutant analyses showed that FliK coordinates FliK action with the switching. Site-directed photo-cross-linking experiments showed that Val-302 and Ile-304 in the hydrophobic core of FliK bind to FlhB . We propose that FliK may induce conformational rearrangements of FliK to bind to FlhB .

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Section: Discussionmentioning

confidence: 97%

The role of intrinsically disordered C‐terminal region of FliK in substrate specificity switching of the bacterial flagellar type III export apparatus

Kinoshita

Aizawa

Inoue

et al. 2017

Molecular Microbiology

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“…An alignment of these FliK proteins revealed a low similarity between these proteins (data not shown); this result was not unexpected since it has been observed that FliK proteins show a low similarity degree, even in closely related species (36). This low similarity is more pronounced in the N-terminal region, which has been suggested to have an intrinsically disordered structure (37,38).…”

Section: Am1 Cells Display Severalmentioning

confidence: 59%

Structural Characterization of the Fla2 Flagellum of Rhodobacter sphaeroides

et al. 2015

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Rhodobacter sphaeroides is a free-living alphaproteobacterium that contains two clusters of functional flagellar genes in its genome: one acquired by horizontal gene transfer (fla1) and one that is endogenous (fla2). We have shown that the Fla2 system is normally quiescent and under certain conditions produces polar flagella, while the Fla1 system is always active and produces a single flagellum at a nonpolar position. In this work we purified and characterized the structure and analyzed the composition of the Fla2 flagellum. The number of polar filaments per cell is 4.6 on average. By comparison with the Fla1 flagellum, the prominent features of the ultra structure of the Fla2 HBB are the absence of an H ring, thick and long hooks, and a smoother zone at the hook-filament junction. The Fla2 helical filaments have a pitch of 2.64 m and a diameter of 1.4 m, which are smaller than those of the Fla1 filaments. Fla2 filaments undergo polymorphic transitions in vitro and showed two polymorphs: curly (righthanded) and coiled. However, in vivo in free-swimming cells, we observed only a bundle of filaments, which should probably be left-handed. Together, our results indicate that Fla2 cell produces multiple right-handed polar flagella, which are not conventional but exceptional. ؊ mutant grown phototrophically and in the absence of organic acids. The Fla1 system produces a single lateral or subpolar flagellum, and the Fla2 system produces multiple polar flagella. The two kinds of flagella are never expressed simultaneously, and both are used for swimming in liquid media. The two sets of genes are certainly ready for responding to specific environmental conditions. The characterization of the Fla2 system will help us to understand its role in the physiology of this microorganism. Motility provides microorganisms with a fundamental survival advantage. Flagella are one of the most complex and effective organelles of locomotion, capable of propelling bacteria through liquids (swimming) and through viscous environments or over surfaces (swarming), and are widely used among Bacteria and Archaea. In addition, these organelles play an important role in adhesion to substrates and biofilm formation, and they contribute to the virulence process in pathogenic bacteria (1, 2).The bacterial flagellum is a rotary motor powered by the electrochemical proton or sodium potential. The morphology of the flagellum is similar among different bacterial species. Nevertheless, there are differences in their substructures, which are not yet clearly understood. The improvement in powerful microscopy techniques has revealed variations in the architecture of the bacterial flagellar motors (3, 4). The bacterial flagellum is a supramolecular complex made of about 30 different proteins with copy numbers that range from a few to thousands. The structure has been divided into three parts: filament, hook, and basal body. The basal body spans the bacterial cell envelope and comprises a rod and a series of rings. In the cytoplasm the basal body for...

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“…That is, FliK C always interacted with the secretion gate when the hook was longer than 55 nm. We concluded that FliK uses the minimal length as a signal to switch secretion modes but does not measure hook length per se (17). In other words, FliK acts to prevent hook polymerization beyond an optimal functional length (55 nm).…”

mentioning

confidence: 90%

“…In other words, FliK acts to prevent hook polymerization beyond an optimal functional length (55 nm). High-speed atomic force microscopy revealed that the molecular shape of intact FliK in solution was composed of two folded domains of differing sizes, linked by a flexible linker (17). The larger domain corresponded to FliK N , and the smaller domain corresponded to FliK C .…”

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confidence: 99%

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Identification of the Key Sequence in the FliK C-Terminal Domain for Substrate Specificity Switching in the Flagellar Protein Secretion

2016

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The flagellar hook is a short tubular structure located between the external filament and the membrane-bound basal body. The average hook length is 55 nm and is determined by the soluble protein FliK and the integral membrane protein FlhB. Hook elongation is terminated by FliK-mediated cessation of hook protein secretion, followed by the secretion of filamentous proteins. This process is referred to as the substrate specificity switch. Switching of the secretion modes results from a direct interaction between the FliK C-terminal domain (FliK C ) and the secretion gate in FlhB. FliK C consists of two ␣-helices and four ␤-strands. Loop 2 connects the first two ␤-sheets and contains a conserved sequence of 9 residues. Genetic and physiological analyses of various fliK partial deletion mutants pointed to loop 2 as essential for induction of a conformational change in the FlhB gate. We constructed single-amino-acid substitutions in the conserved region of loop 2 of FliK and discovered that the loop sequence LRL is essential for the timely switching of secretion modes. IMPORTANCEFlagellar protein secretion is controlled by the soluble protein FliK. We discovered that the loop 2 sequence LRL in the FliK C terminus was essential for timely switching of secretion modes. This mechanism is applicable to type three secretions systems that secrete virulence factors in bacterial pathogens. One of the most important and mysterious regulatory processes is the control of size and length determination of biological structures. A few examples exist in which the molecular mechanism for length determination is known, including the tobacco mosaic virus, the bacteriophage lambda tail, and muscle thin filaments (1, 2). In recent years, the bacterial flagellum has been regarded as another example for which molecular elucidation of length control is possible (3-6).The flagellum is a supramolecular structure for motility consisting of three substructures with distinctive functions. The basal body acts as a rotary motor, and the external filament works as a screw. The hook conveys torque generated at the basal body toward the filament (7). Hook length is controlled and averages approximately 55 nm (8, 9). The flagellar protein FliK controls hook length (3,8,10). FliK consists of 405 amino acids and is structurally divided into two distinct N-and C-terminal regions, FliK N and FliK C (11). The molecular length of FliK N is linearly correlated with hook length, while FliK C is necessary for switching the substrate specificity of protein secretion at FlhB (10, 12). Mutations in FliK C often result in hooks without filaments and uncontrolled lengths, called polyhooks (8,11,13). Although defects in FliK N lead to uncontrolled hook length, substrate switching eventually occurs, as long as FliK C is intact. This results in filament formation on polyhooks, referred to as polyhook-filaments (11,14). Minamino et al. (13) performed site-directed mutagenesis and reported that 302Val, 304Ile, 335Leu, 401Val, and 405Ala of FliK C were critical f...

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Two-Ball Structure of the Flagellar Hook-Length Control Protein FliK as Revealed by High-Speed Atomic Force Microscopy

Cited by 27 publications

References 32 publications

The role of intrinsically disordered C‐terminal region of FliK in substrate specificity switching of the bacterial flagellar type III export apparatus

The role of intrinsically disordered C‐terminal region of FliK in substrate specificity switching of the bacterial flagellar type III export apparatus

Structural Characterization of the Fla2 Flagellum of Rhodobacter sphaeroides

Identification of the Key Sequence in the FliK C-Terminal Domain for Substrate Specificity Switching in the Flagellar Protein Secretion

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