of a Split Domain Forms the Basis of the MS:C Ring Interface within the Bacterial Flagellar Motor.

SUMMARY The interface between the membrane (MS) and cytoplasmic (C) rings of the bacterial ﬂagellar motor couples torque generation to rotation within the membrane. The structure of the C-terminal helices of the integral membrane protein FliF (FliF C ) bound to the N terminal domain of the switch complex protein FliG (FliG N ) reveals that FliG N folds around FliF C to produce a topology that closely resembles both the middle and C-terminal domains of FliG. The interface is consistent with solution-state nuclear magnetic resonance, small-angle X-ray scattering, in vivo interaction studies, and cellular motility assays. Co-folding with FliF C induces substantial conformational changes in FliG N and suggests that FliF and FliG have the same stoichiometry within the rotor. Modeling the FliF C :FliG N complex into cryo-electron microscopy rotor density updates the architecture of the middle and upper switch complex and shows how domain shufﬂing of a conserved interaction module anchors the cytoplasmic rotor to the membrane. assignments for 93% of residues S2A). shift deviations from random coil both FliF 495-532 and FliG 1-102 within thecomplex to bewell folded, with a helicalstruc-ture (Figure S2B). Overall, the data indicate the FliF C :FliG N com-plexisthesamewhetherproducedasafusionproteinorassepa- rate components and that the presence of a linker does not prevent complex assembly. Double-Cysteine Crosslinking Disulﬁde crosslinking between FliF and FliG was studied in strains expressing cysteine-containing FliG variants from the chromosome and cysteine- containing FliF variants from plasmid pEK16. For these experiments, wild-type protein (FliF) was present along with the Cys-mutant FliF. Levels of the two were roughly equal. The plasmid was induced with 0.2 m M salicylate, which gave FliF levels sufﬁcient to compete with the wild-type (cysteine-less) FliF expressed from the chromosome but not so high as to impair motility. (Motility impairment occurred with induction by 1 m M or higher.) Cells were cultured overnight at 37 (cid:3) C and diluted 100-fold into TB containing antibiotic and 0.2 m M sodium salicylate then grown at 32 (cid:3) C for 5–6 hr (to OD 600 0.7–0.8). OD 600 was measured to adjust the cell density, then equal numbers of cells were pelleted and re-suspended with XL buffer (20 mM Na-phosphate (pH 7.4), 150 mM NaCl). For crosslinking with iodine, 0.1 mL of cell mixed with 4 m L of 25 mM iodine for 3 min at room temperature, then sulfhydryl groups were blocked by addition of 2 m L of 0.5 M NEM and incubation at room temperature for 3 min. Samples were mixed with an equal volume of 2 3 non-reducing loading buffer and heated at 95 (cid:3) C for 10 min before loading on SDS-PAGE gels. Some exper- iments included urea during the crosslinking step at the concentrations indicated in the ﬁgures to test the effects of partially destabilizing the proteins. SDS-PAGE and Immunoblotting Proteins were resolved in 7.5% SDS-PAGE gels and transferred onto nitrocel-luloseusingaTransblotturboapparatus(Bio-Rad). Rabbitpolyclonalantibody against FliG was used at 1:1,000 dilution in a solution containing PBS (pH 7.4), and measurements a previously modiﬁcations. a M M NaH PO M b -mercaptoe-thanol). vor- texedfor10s,followedby 30 C for5min.Reactionwas by adding mL of ortho-nitrophenyl- b -galactoside solution (4 Af- ter measuredtimes of reactionat 30 (cid:3) C,thereaction was stoppedby addition of 0.5 mL of1 M NaHCO 3 Product quantiﬁed by measurement of OD 420 a according to the (1,000 3 420 (cid:4) 3 OD reaction and OD cell