FliG is a component of the switch complex on the rotor of the bacterial flagellum. Each flagellar motor contains about 25 FliG molecules. The protein of Escherichia coli has 331 amino acid residues and comprises at least two discrete domains. A C-terminal domain of about 100 residues functions in rotation and includes charged residues that interact with the stator protein MotA. Other parts of the FliG protein are essential for flagellar assembly and interact with the MS ring protein FliF and the switch complex protein FliM. The crystal structure of the middle and C-terminal parts of FliG shows two globular domains joined by an alpha-helix and a short extended segment that contains two well-conserved glycine residues. Here, we describe targeted cross-linking studies of FliG that reveal features of its organization in the flagellum. Cys residues were introduced at various positions, singly or in pairs, and cross-linking by a maleimide or disulfide-inducing oxidant was examined. FliG molecules with pairs of Cys residues at certain positions in the middle domain formed disulfide-linked dimers and larger multimers with a high yield, showing that the middle domains of adjacent subunits are in fairly close proximity and putting constraints on the relative orientation of the domains. Certain proteins with single Cys replacements in the C-terminal domain formed dimers with moderate yields but not larger multimers. On the basis of the cross-linking results and the data available from mutational and electron microscopic studies, we propose a model for the organization of FliG subunits in the flagellum.
