Crystallization and preliminary X-ray analysis of the periplasmic domain of FliP, an integral membrane component of the bacterial flagellar type III protein-export apparatus

Fukumura, Takuma; Furukawa, Yoshiko; Kawaguchi, Tatsuya; Saijo-Hamano, Yumiko; Namba, Keiichi; Imada, Katsumi; Minamino, Tetsuo

doi:10.1107/s2053230x14014678

Cited by 5 publications

(8 citation statements)

References 36 publications

(34 reference statements)

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“…Ala-186 and Phe-187 make hydrophobic interactions with Met-127, Val-131, and the side chain arm of Arg-130. There is no direct contact between Mol A and Mol D. Since sedimentation equilibrium analytical ultracentrifugation measurements have revealed that Tm- FliP P forms a homotetramer in solution [ 37 ], we conclude that the tetramer structure observed in the crystal appears to be equivalent to that in solution.…”

Section: Resultsmentioning

confidence: 76%

“…To clarify the role of FliP P in FliP 6 ring formation, we determined the crystal structure of FliP P . Although no St- FliP P crystal was obtained, the Tm- FliP P crystals were grown [ 37 ], and its structure was solved at 2.4 Å resolution. Tm- FliP P formed a homotetramer in the crystal (Mol A, Mol B, Mol C, and Mol D) related by pseudo D2 symmetry (Protein Data Bank [PDB] ID: 5H72) ( Fig 3A ).…”

Section: Resultsmentioning

confidence: 99%

“…Details of the expression, purification, and crystallization of Tm- FliP P have been described previously [ 37 ].…”

Section: Methodsmentioning

confidence: 99%

“…FliP P of T . maritia ( Tm -FliP P ) forms a homotetramer in solution [ 37 ], raising the possibility that Salmonella FliP ( St- FliP) forms an oligomer through interactions between FliP P domains. To study the oligomeric structure of FliP, we purified St- FliP from the membrane fraction by solubilizing it with 1% n-dodecyl β-D-maltoside (DDM) and analyzed it by electron microscopy (EM) and image analysis.…”

Section: Introductionmentioning

confidence: 99%

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Assembly and stoichiometry of the core structure of the bacterial flagellar type III export gate complex

et al. 2017

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The bacterial flagellar type III export apparatus, which is required for flagellar assembly beyond the cell membranes, consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. FlhA, FlhB, FliP, FliQ, and FliR form the gate complex inside the basal body MS ring, although FliO is required for efficient export gate formation in Salmonella enterica. However, it remains unknown how they form the gate complex. Here we report that FliP forms a homohexameric ring with a diameter of 10 nm. Alanine substitutions of conserved Phe-137, Phe-150, and Glu-178 residues in the periplasmic domain of FliP (FliPP) inhibited FliP6 ring formation, suppressing flagellar protein export. FliO formed a 5-nm ring structure with 3 clamp-like structures that bind to the FliP6 ring. The crystal structure of FliPP derived from Thermotoga maritia, and structure-based photo-crosslinking experiments revealed that Phe-150 and Ser-156 of FliPP are involved in the FliP–FliP interactions and that Phe-150, Arg-152, Ser-156, and Pro-158 are responsible for the FliP–FliO interactions. Overexpression of FliP restored motility of a ∆fliO mutant to the wild-type level, suggesting that the FliP6 ring is a functional unit in the export gate complex and that FliO is not part of the final gate structure. Copurification assays revealed that FlhA, FlhB, FliQ, and FliR are associated with the FliO/FliP complex. We propose that the assembly of the export gate complex begins with FliP6 ring formation with the help of the FliO scaffold, followed by FliQ, FliR, and FlhB and finally FlhA during MS ring formation.

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Section: Resultsmentioning

confidence: 76%

Section: Resultsmentioning

confidence: 99%

“…Details of the expression, purification, and crystallization of Tm- FliP P have been described previously [ 37 ].…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Assembly and stoichiometry of the core structure of the bacterial flagellar type III export gate complex

et al. 2017

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“…Previous estimates of FliP stoichiometry suggested the presence of 4-5 copies per basal body (Fan et al, 1997), and structural studies of a sub-domain of FliP similarly indicate the likelihood of FliP multimers (Fukumura et al, 2014). To further test the proposal that the apparatus contains multiple, jointly functioning copies of FliP, we examined inter-subunit complementation between FliP mutations that were previously found to cause fairly severe motility impairments individually (Erhardt et al, 2017).…”

Section: Resultsmentioning

confidence: 99%

Type‐III secretion pore formed by flagellar protein FliP

Ward

Renault

Kim

et al. 2017

Molecular Microbiology

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SummaryDuring assembly of the bacterial flagellum, protein subunits that form the exterior structures are exported through a specialized secretion apparatus energized by the proton gradient. This category of protein transport, together with the similar process that occurs in the injectisomes of gram-negative pathogens, is termed type-III secretion. The membrane-embedded part of the flagellar export apparatus contains five essential proteins: FlhA, FlhB, FliP, FliQ and FliR. Here, we have undertaken a variety of experiments that together support the proposal that the proteinconducting conduit is formed primarily, and possibly entirely, by FliP. Chemical modification experiments demonstrate that positions near the center of certain FliP trans-membrane (TM) segments are accessible to polar reagents. FliP expression sensitizes cells to a number of chemical agents, and mutations at predicted channel-facing positions modulate this effect. Multiple assays are used to show that FliP suffices to form a channel that can conduct a variety of mediumsized, polar molecules. Conductance properties are strongly modulated by mutations in a methionine-rich loop that is predicted to lie at the inner mouth of the channel, which might form a gasket around cargo molecules undergoing export. The results are discussed in the framework of an hypothesis for the architecture and action of the cargo-conducting part of the type-III secretion apparatus.

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