Polar confinement of a macromolecular machine by an SRP-type GTPase

The basal structure of the bacterial flagellum includes a membrane embedded MS-ring (formed by multiple copies of FliF) and a cytoplasmic C-ring (composed of proteins FliG, FliM and FliN). The SRP-type GTPase FlhF is required for directing the initial flagellar protein FliF to the cell pole, but the mechanisms are unclear. Here, we show that FlhF anchors developing flagellar structures to the polar landmark protein HubP/FimV, thereby restricting their formation to the cell pole. Specifically, the GTPase domain of FlhF interacts with HubP, while a structured domain at the N-terminus of FlhF binds to FliG. FlhF-bound FliG subsequently engages with the MS-ring protein FliF. Thus, the interaction of FlhF with HubP and FliG recruits a FliF-FliG complex to the cell pole. In addition, the modulation of FlhF activity by the MinD-type ATPase FlhG controls the interaction of FliG with FliM-FliN, thereby regulating the progression of flagellar assembly at the pole.

Protein extracts from the respective E. coli strains were separated by SDS-PAGE, transferred to membranes and the Shewanella proteins HubP (SpHubP), FlhF(SpFlhF) or FliG (SpFliG) were visualized by western blotting with antibodies against GFP.For each strain the same OD units (10) were loaded for each sample.pBAD and pBBR are loaded as empty vector controls.S1.Data collection and refinement statistics for the structure determination of the FlhF-FID domain.This study Supplementary Table 4.Plasmids used in this study.

Supplementary Figure 1 Supplementary Figure 4 .
. a. Electron density of FID domain of SpFlhF.The 2Fobs-Fcalc electron density map after final refinement is shown as a blue mesh at 1σ.Protein is represented as cartoon with side chains as sticks.b.-c.Alpha-Fold (1) model of HubP.b.Overall Confidence Assessment.The confidence analysis of the HubP model reveals a predominantly low level of confidence.The accompanying inset provides a color-coded reference corresponding to the confidence scores.c.The Alpha-Fold model of HubP is presented in a simplified cartoon format, employing a rainbow color scheme to depict the sequence from N-to C-terminus.Notably, 'N' and 'C' signify the N-terminus and C-terminus, respectively.The code of the model is: AF-A4Y880-F1 (last assessed in January 2024).The deletion of the N-or C-terminal domain of FliG results in a FlhF-mVenus accumulation.a.Quantification of FlhF-mVenus foci intensity (a.u.arbitrary units).Asterisks represent a p-value <0.0001 (Two-way ANOVA).b.Protein levels of FlhF-mVenus.Protein extracts from the respective Shewanella strains were separated by SDS-PAGE, transferred to membranes and the proteins were visualized by western blotting with antibodies against GFP.Supplementary Figure 5.The Shewanella proteins are stable produced in E. coli DH5α.
frame substitution of Thr166 to Cys in the polar major flagellin protein FlaB1 (Sputcn32_2585) and Thr174 to Cys in the polar minor flagellin protein FlaA1 (Sputcn32_2586), deletion of the N-terminal domain (Δ2-85) of FliG(Sputcn32_2575) mVenus tag of FlhF (Sputcn32_2561) linked with Gly and Ser, deletion of the N-terminal domain (frame substitution of Thr166 to Cys in the polar major flagellin protein FlaB1 (Sputcn32_2585) and Thr174 to Cys in the polar minor flagellin protein FlaA1 (Sputcn32_2586),deletion of the C-terminal domain (Δ209-348) of FliG(mVenus tag of FlhF (Sputcn32_2561) linked with Gly and Ser, deletion of the C-terminal domain (Δ209-348) of FliG (Sputcn32_2575).
Expression of N-terminal His-tagged polar FliG-MC domains This study pET24d_FliG-NM_N-His Expression of N-terminal His-tagged polar FliG-NM domains This study pGAT3_FliF-C Expression of N-terminal GST-tagged polar FliF-C This study pET24d_FliM_N-His Expression of N-terminal His-tagged polar FliM This study in frame complementation of flhF (Sputcn32_2561) with FlhF-1xGS-mVenus; Kan r (6) pNPTS138-R6KT flhF KO deletion of the flhF gene (Sputcn32_2561); Kan r (4) pNPTS138-R6KT fliG KO deletion of the fliG gene (Sputcn32_2575); Kan r This study the FlhF bait protein fused to the Gal4 DNA-binding domain This study pGBKT7_FlhF-B pGBKT7 plasmid expressing the FlhF B-domain bait protein fused to the Gal4 DNA-binding domain This study pGADT7_FliF-C pGADT7 plasmid expressing the FliFc prey protein fused to Gal4 activation domain This study pGADT7_FliG_polar pGADT7 plasmid expressing the polar FliG prey protein fused to Gal4 activation domain This study pGADT7_FliG_lateral pGADT7 plasmid expressing the lateral FliG prey protein fused to Gal4 activation domain This study pGADT7_FlhG pGADT7 plasmid expressing the FlhG prey protein fused to Gal4 activation domain This study pGADT7_FliM pGADT7 plasmid expressing the FliM prey protein fused to Gal4 activation domain This study pGADT7_FliN pGADT7 plasmid expressing the FliN prey protein fused to Gal4 activation domain This study pGADT7_FlhF pGADT7 plasmid expressing the FlhF prey protein fused to Gal4 activation domain This study Oligonucleotides used for Escherichia coli work:

Table 2 .
Escherichia coli strains used in this study.