The hydrophobic core of FliG domain II is the stabilizer in the flagellar motor Free

Abstract

The flagellar protein FliG is the major component of the flagellar torque generator, and consists of two separate domains, I and II. Domain I is essential for flagellar assembly, while domain II in the C-terminal region is not essential for flagellar assembly but is dedicated to torque generation. Previously, we found that some mutants were temperature-hypersensitive (hyper-TS) and identified three residues (F236V, D244Y and K273E) on domain II responsible for the temperature-sensitive (TS) phenotype. In this study, we substituted the three residues with all 20 amino acids (X) and analysed the behaviour of the variants at various temperatures. Each group of F236X, D244X and K273X variants gave rise to several hyper-TS mutants. In F236X, only substitution with F and W gave rise to wild-type, while other hydrophobic residues resulted in hyper-TS mutants and hydrophilic residues resulted in non-motile variants. The atomic arrangement around the F236 residue indicated that F236 together with neighbouring residues forms a hydrophobic core in the centre of domain II, which is well conserved among many species. These data suggest that the hydrophobic core may play an essential role in stabilizing the whole structure of domain II, so that changes of physiological conditions in the microenvironment of domain II do not perturb torque generation.

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2012-10-01
2024-03-28
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References

  1. Aizawa S.-I. ( 2009). Flagella. Desk Encyclopedia of Microbiology.393–403 Schaechter M. Oxford: Elsevier; [View Article]
    [Google Scholar]
  2. Blair K. M., Turner L., Winkelman J. T., Berg H. C., Kearns D. B. ( 2008). A molecular clutch disables flagella in the Bacillus subtilis biofilm. Science 320:1636–1638 [View Article][PubMed]
    [Google Scholar]
  3. Bochner B. R., Huang H.-C., Schieven G. L., Ames B. N. ( 1980). Positive selection for loss of tetracycline resistance. J Bacteriol 143:926–933[PubMed]
    [Google Scholar]
  4. Brown P. N., Hill C. P., Blair D. F. ( 2002). Crystal structure of the middle and C-terminal domains of the flagellar rotor protein FliG. EMBO J 21:3225–3234 [View Article][PubMed]
    [Google Scholar]
  5. Datsenko K. A., Wanner B. L. ( 2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645 [View Article][PubMed]
    [Google Scholar]
  6. Dundas J., Ouyang Z., Tseng J., Binkowski A., Turpaz Y., Liang J. ( 2006). CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. Nucleic Acids Res 34:Web Server issueW116–W118 [View Article][PubMed]
    [Google Scholar]
  7. Eswar N., Webb B., Marti-Renom M. A., Madhusudhan M. S., Eramian D., Shen M.-Y., Pieper U., Sali A. ( 2006). Comparative protein structure modeling using Modeller. Current Protocols in Bioinformatics 15:5.6.1–5.6.30 [View Article]
    [Google Scholar]
  8. Francis N. R., Sosinsky G. E., Thomas D., DeRosier D. J. ( 1994). Isolation, characterization and structure of bacterial flagellar motors containing the switch complex. J Mol Biol 235:1261–1270 [View Article][PubMed]
    [Google Scholar]
  9. Fukuoka H., Yakushi T., Homma M. ( 2004). Concerted effects of amino acid substitutions in conserved charged residues and other residues in the cytoplasmic domain of PomA, a stator component of Na+-driven flagella. J Bacteriol 186:6749–6758 [View Article][PubMed]
    [Google Scholar]
  10. Garza A. G., Harris-Haller L. W., Stoebner R. A., Manson M. D. ( 1995). Motility protein interactions in the bacterial flagellar motor. Proc Natl Acad Sci U S A 92:1970–1974 [View Article][PubMed]
    [Google Scholar]
  11. Irikura V. M., Kihara M., Yamaguchi S., Sockett H., Macnab R. M. ( 1993). Salmonella typhimurium fliG and fliN mutations causing defects in assembly, rotation, and switching of the flagellar motor. J Bacteriol 175:802–810[PubMed]
    [Google Scholar]
  12. Katayama E., Shiraishi T., Oosawa K., Baba N., Aizawa S.-I. ( 1996). Geometry of the flagellar motor in the cytoplasmic membrane of Salmonella typhimurium as determined by stereo-photogrammetry of quick-freeze deep-etch replica images. J Mol Biol 255:458–475 [View Article][PubMed]
    [Google Scholar]
  13. Khan S., Khan I. H., Reese T. S. ( 1991). New structural features of the flagellar base in Salmonella typhimurium revealed by rapid-freeze electron microscopy. J Bacteriol 173:2888–2896[PubMed]
    [Google Scholar]
  14. Kojima S., Nonoyama N., Takekawa N., Fukuoka H., Homma M. ( 2011). Mutations targeting the C-terminal domain of FliG can disrupt motor assembly in the Na+-driven flagella of Vibrio alginolyticus . J Mol Biol 414:62–74 [View Article][PubMed]
    [Google Scholar]
  15. Konishi M., Kanbe M., McMurry J. L., Aizawa S.-I. ( 2009). Flagellar formation in C-ring-defective mutants by overproduction of FliI, the ATPase specific for flagellar type III secretion. J Bacteriol 191:6186–6191 [View Article][PubMed]
    [Google Scholar]
  16. Lee L. K., Ginsburg M. A., Crovace C., Donohoe M., Stock D. ( 2010). Structure of the torque ring of the flagellar motor and the molecular basis for rotational switching. Nature 466:996–1000 [View Article][PubMed]
    [Google Scholar]
  17. Lloyd S. A., Tang H., Wang X., Billings S., Blair D. F. ( 1996). Torque generation in the flagellar motor of Escherichia coli: evidence of a direct role for FliG but not for FliM or FliN. J Bacteriol 178:223–231[PubMed]
    [Google Scholar]
  18. Lloyd S. A., Whitby F. G., Blair D. F., Hill C. P. ( 1999). Structure of the C-terminal domain of FliG, a component of the rotor in the bacterial flagellar motor. Nature 400:472–475 [View Article][PubMed]
    [Google Scholar]
  19. Macnab R. M. ( 1996). Flagella. Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn.123–145 Neidhardt F. C., Curtiss R. III, Ingraham J. L., Lin E. C. C., Low K. B., Magasanik B., Reznikoff W. S., Riley M., Schaechter M., Umbarger H. E. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  20. Makishima S., Komoriya K., Yamaguchi S., Aizawa S.-I. ( 2001). Length of the flagellar hook and the capacity of the type III export apparatus. Science 291:2411–2413 [View Article][PubMed]
    [Google Scholar]
  21. Maloy S. R., Nunn W. D. ( 1981). Selection for loss of tetracycline resistance by Escherichia coli . J Bacteriol 145:1110–1111[PubMed]
    [Google Scholar]
  22. Martí-Renom M. A., Stuart A. C., Fiser A., Sánchez R., Melo F., Sali A. ( 2000). Comparative protein structure modeling of genes and genomes. Annu Rev Biophys Biomol Struct 29:291–325[PubMed] [CrossRef]
    [Google Scholar]
  23. Mashimo T., Hashimoto M., Yamaguchi S., Aizawa S.-I. ( 2007). Temperature-hypersensitive sites of the flagellar switch component FliG in Salmonella enterica serovar Typhimurium. J Bacteriol 189:5153–5160 [View Article][PubMed]
    [Google Scholar]
  24. Sockett H., Yamaguchi S., Kihara M., Irikura V. M., Macnab R. M. ( 1992). Molecular analysis of the flagellar switch protein FliM of Salmonella typhimurium . J Bacteriol 174:793–806[PubMed]
    [Google Scholar]
  25. Thomas D. R., Morgan D. G., DeRosier D. J. ( 1999). Rotational symmetry of the C ring and a mechanism for the flagellar rotary motor. Proc Natl Acad Sci U S A 96:10134–10139 [View Article][PubMed]
    [Google Scholar]
  26. Togashi F., Yamaguchi S., Kihara M., Aizawa S.-I., Macnab R. M. ( 1997). An extreme clockwise switch bias mutation in fliG of Salmonella typhimurium and its suppression by slow-motile mutations in motA and motB . J Bacteriol 179:2994–3003[PubMed]
    [Google Scholar]
  27. Wallace A. C., Laskowski R. A., Thornton J. M. ( 1996). ligplot: a program to generate schematic diagrams of protein–ligand interactions. Protein Eng 8:127–134[PubMed] [CrossRef]
    [Google Scholar]
  28. Williams A. W., Yamaguchi S., Togashi F., Aizawa S.-I., Kawagishi I., Macnab R. M. ( 1996). Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium . J Bacteriol 178:2960–2970[PubMed]
    [Google Scholar]
  29. Yakushi T., Yang J.-H., Fukuoka H., Homma M., Blair D. F. ( 2006). Roles of charged residues of rotor and stator in flagellar rotation: comparative study using H+-driven and Na+-driven motors in Escherichia coli . J Bacteriol 188:1466–1472 [View Article][PubMed]
    [Google Scholar]
  30. Yamaguchi S., Fujita H., Ishihara A., Aizawa S.-I., Macnab R. M. ( 1986a). Subdivision of flagellar genes of Salmonella typhimurium into regions responsible for assembly, rotation, and switching. J Bacteriol 166:187–193[PubMed]
    [Google Scholar]
  31. Yamaguchi S., Aizawa S.-I., Kihara M., Isomura M., Jones C. J., Macnab R. M. ( 1986b). Genetic evidence for a switching and energy-transducing complex in the flagellar motor of Salmonella typhimurium . J Bacteriol 168:1172–1179[PubMed]
    [Google Scholar]
  32. Yorimitsu T., Sowa Y., Ishijima A., Yakushi T., Homma M. ( 2002). The systematic substitutions around the conserved charged residues of the cytoplasmic loop of Na+-driven flagellar motor component PomA. J Mol Biol 320:403–413 [View Article][PubMed]
    [Google Scholar]
  33. Yorimitsu T., Mimaki A., Yakushi T., Homma M. ( 2003). The conserved charged residues of the C-terminal region of FliG, a rotor component of the Na+-driven flagellar motor. J Mol Biol 334:567–583 [View Article][PubMed]
    [Google Scholar]
  34. Zhou J., Lloyd S. A., Blair D. F. ( 1998a). Electrostatic interactions between rotor and stator in the bacterial flagellar motor. Proc Natl Acad Sci U S A 95:6436–6441 [View Article][PubMed]
    [Google Scholar]
  35. Zhou J., Sharp L. L., Tang H. L., Lloyd S. A., Billings S., Braun T. F., Blair D. F. ( 1998b). Function of protonatable residues in the flagellar motor of Escherichia coli: a critical role for Asp 32 of MotB. J Bacteriol 180:2729–2735[PubMed]
    [Google Scholar]
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