Organization of FliN subunits in the flagellar motor of Escherichia coli.
FliN is a major constituent of the C ring in the flagellar basal body of many bacteria. It is present in >100 copies per flagellum and together with FliM and FliG forms the switch complex that functions in flagellar assembly, rotation, and clockwise-counterclockwise switching. FliN is essential for flagellar assembly and switching, but its precise functions are unknown. The C-terminal part of the protein is best conserved and most important for function; a crystal structure of this C-terminal domain of FliN from Thermotoga maritima revealed a saddle-shaped dimer formed mainly from beta strands (P. N. Brown, M. A. A. Mathews, L. A. Joss, C. P. Hill, and D. F. Blair, J. Bacteriol. 187:2890-2902, 2005). Equilibrium sedimentation studies showed that FliN can form stable tetramers and that a FliM1FliN4 complex is also stable. Here, we have examined the organization of FliN subunits by using targeted cross-linking. Cys residues were introduced at various positions in FliN, singly or in pairs, and disulfide cross-linking was induced by oxidation. Efficient cross-linking was observed for certain positions near the ends of the dimer and for some positions in the structurally uncharacterized N-terminal domain. Certain combinations of two Cys replacements gave a high yield of cross-linked tetramer. The results support a model in which FliN is organized in doughnut-shaped tetramers, stabilized in part by contacts involving the N-terminal domain. Electron microscopic reconstructions show a bulge at the bottom of the C-ring whose size and shape are a close match for the hypothesized FliN tetramer.
Paul
Koushik
K
Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.
Blair
David F
DF
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry* / Q000166:cytology / Q000235:genetics; D005407:Flagella / Q000737:chemistry* / Q000378:metabolism*; D017353:Gene Deletion; D008958:Models, Molecular; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; C077389:FliN protein, Bacteria; D021122:Protein Subunits