Figures and data
Biochemical analysis of B. subtilis MinD ATPase cycle.
(A) Phosphate release plotted against different MinD concentrations, fitted with a simple linear regression (R² = 0.97). Phosphate release was measured using the EnzChek™ phosphate assay kit. Samples contained 0.2 mg ml-1 liposomes and were pre-incubated for 10 minutes before addition of 2 mM Mg2+-ATP; n ≥ 3.
(B) Specific activity of the MinD ATPase from (A) measured as a function of MinD concentration.
(C) Specific activity of MinD measured as a function of ATP concentration, determined as in (B) with fixed MinD concentration of 10 µM. Fitting the Michaelis–Menten equation (black lines) gives kcat = 36.27 h−1, Vmax = 19.50 nmol mg-1 min-1 and KM = 0.173 mM.
(D) Specific activity of MinD measured as a function of liposome concentration, determined as in (B) with fixed MinD concentration of 6 µM. Fitting the Michaelis–Menten equation (black lines) gives kcat = 41.01 h−1, Vmax = 22.05 nmol mg-1 min-1 and KM = 0.0437 mg/ml.
Cartoon of MinD AlphaFold model with highlighted key amino acids and the effects of their mutagenesis.
(A) Left: Rendering of MinD AlphaFold model with ATP binding region highlighted in light blue, membrane targeting sequence (MTS) in turquoise and key residues in orange; Box: explanation of mutagenesis effects. Right: Close-up on hydrophobic region of amphipathic helix.
(B) Representative wide-field microscopy images of B. subtilis expressing indicated Halo-MinD variants as the only MinD copy from the native locus, stained with TMR ligand. Left: Phase contrast; scale bar 5 µm. Right: fluorescence.
BLI analysis of His-MinD and different mutants.
(A) Left: Cartoon representation of the different BLI steps, starting with (1) establishing a baseline in protein buffer, (2) binding of liposomes through the biotinylated DSPE-PEG(2000) phospholipid, (3) establishing a new baseline, (4) binding of MinD and finally (5) dissociation of MinD. Right: Exemplary graph resulting from sensor readouts of steps (1) -(5). Scheme adapted from (70).
(B) MinD binding plotted against time through association and dissociation phase (steps 4-5) at different protein concentrations, including the His-MinD-I260E mutant.
(C) – (E) Same as (B) using the indicated respective mutant of His-MinD (G12V, K16A and D40A).
Kinetic constants of His-MinD and variants obtained via BLI.
Single-molecule localization microscopy analysis of Halo-MinD and mutants expressed in B. subtilis.
Exponentially growing B. subtilis cells expressing Halo-MinD and variants (n ≥ 48 cells, respectively) were stained with TMR ligand and subsequently imaged. Individual protein trajectories were recorded using SMLM and analyzed with Zen blue (Zeiss), Trackmate, the SMTracker 2.0 software package and manual scripts in R. Minimum track-length 4 frames of 24 ms, with at least 2596 trajectories per strain.
(A) Heat map representation of intracellular localization of individual molecules of Halo-MinD and variants, respectively, plotted on normalized cells. Brighter colors indicate higher abundance.
(B) Barplot of stationary localization analysis (SLA), comparing different track types within the protein population. Tracks were considered static when not leaving a circular area of 97 nm diameter within 5+ frames. Mobile populations were further divided into free and mixed tracks, where mixed tracks displayed a switch between free and confined movement.
(C) Plot of the mean-squared displacement of Halo-MinD and variants over time, fitted with a linear fit excluding the last timelag.
(D) Bubble plot displaying single-molecule diffusion rates of the indicated MinD fusions. Populations were determined by fitting the probability distributions of the frame-to-frame displacement (jump distance) data of all respective tracks to a three components model (fast mobile, slow mobile and confined protein populations).
(E) Probability distributions of jump distances of Halo-MinD and variants. Data was fit with a three component model, indicating confined, slow and fast tracks.
