Fig. 2: Mechanistic dissection of the FlhF/FliG interaction.

a Coomassie-stained SDS-PAGE of an in vitro pulldown assay employing different StrepII-tagged variants of the B-domain as bait (the amino acid number are indicated) and full-length FliG as prey. b Structural analysis of the FID domain of FlhF. Upper panel: Revised scheme of the domain architecture of FlhF with the FliG-interacting domain (FID, orange), the structurally uncharacterized linker region (dashed line), followed by the NG domain (green). The domains are drawn to scale. Lower panel, left: X-ray structure of the FID domain of FlhF. Lower panel, right: X-ray structures of the FID domain (this study) and the GDP-bound state of the NG domain (PDB-ID: 8R9R⁴⁹;) from S. putrefaciens FlhF. The structurally uncharacterized linker is indicated by a dashed line, not drawn to scale. c Coomassie-stained SDS-PAGE employing StrepII-tagged FlhF as bait and FliG and its variants (given in the panel above) as prey. d Chromatogram of an analytical size-exclusion chromatography of the FID domain of FlhF (black), the MC-domains of FliG (grey) and their complex (red). Coomassie-stained SDS-PAGE of the peak fraction of each run is shown in the inset. The arrows show the elution volumes of molecular weight markers (kDa). e FlhF-tethered FliG can interact with FliF-C. Coomassie-stained SDS-PAGE employing StrepII-tagged FlhF as bait and FliG or FliG and FliF-C as prey. f FlhF-tethered FliG cannot interact with FliM/N. Coomassie-stained SDS-PAGE employing FliG-bound to StrepII-tagged FlhF as bait in the absence and presence of FliM/N. For Fig. 2a, c–f: Each of the shown experiments were repeated in at least three biological replicates. The molecular weight marker (MW) is in kiloDalton (kDa).