Revealing the affinity of individual and combined FliG domains for FliM in the bacterial flagellar motor switch complex by CG-MALS

Complex interactions between flagellar motor proteins modulate the rotational direction of the bacterial flagella. In particular, the middle and C-terminal domains of FliG (FliGM and FliGC, respectively) bind two different sites on the binding partner FliM, as part of the flagellar motor switch. We extend previous NMR studies of the interactions between FliG domains with FliM via composition gradient multi-angle static light scattering (CG-MALS) to confirm specific binding, quantify affinity and identify the stoichiometry of each complex. Though the equilibrium association constants between the individual FliGC domains and FliGM differ by >25x, both complexes appear to reach equilibrium on time scales faster than that of the CG-MALS measurement (∼5 sec). By contrast, binding of the dual-domain protein FliGMC to FliM exhibited unusually slow association kinetics with reaction time constants on the order of 30-60 minutes. CG-MALS revealed complex self-and hetero-association stoichiometries that could not be identified or quantified by means of NMR. Combined with structural studies, these data could provide insight to the mechanism of flagellar motor switching.