Abstract
The binding of the soluble cytoplasmic protein FliG to the transmembrane protein FliF is one of the first interactions in the assembly of the bacterial flagellum. Once established, this interaction is integral in keeping the flagellar cytoplasmic ring, responsible for both transmission of torque and control of the rotational direction of the flagellum, anchored to the central transmembrane ring on which the flagellum is assembled. Here we isolate and characterize the interaction between the N-terminal domain of Thermotoga maritima FliG (FliG(N)) and peptides corresponding to the conserved C-terminal portion of T. maritima FliF. Using nuclear magnetic resonance (NMR) and other techniques, we show that the last ~40 amino acids of FliF (FliF(C)) interact strongly (upper bound K(d) in the low nanomolar range) with FliG(N). The formation of this complex causes extensive conformational changes in FliG(N). We find that T. maritima FliG(N) is homodimeric in the absence of the FliF(C) peptide but forms a heterodimeric complex with the peptide, and we show that this same change in oligomeric state occurs in full-length T. maritima FliG, as well. We relate previously observed phenotypic effects of FliF(C) mutations to our direct observation of binding. Lastly, on the basis of NMR data, we propose that the primary interaction site for FliF(C) is located on a conserved hydrophobic patch centered along helix 1 of FliG(N). These results provide new detailed information about the bacterial flagellar motor and support efforts to understand the cytoplasmic ring's precise molecular structure and mechanism of rotational switching.