ScMreB5 binding to DOPG liposome is specific and concentration dependent. (A) A representative 12% SDS-PAGE gel of pelleting assay with WT, ATP hydrolysis mutant, and membrane-binding mutants showing that the protein does not pellet in the absence of liposomes at 100,000 g spin. (B) A representative 12% SDS-PAGE gel of liposome pelleting assay with C-terminal deletion mutant (ΔC10) shows binding with the charged liposome, DOPG. Concentrations of DOPG and DOPC liposomes used in the assay are 1 mM each, and that of protein is 2 µM. (C) Liposome-binding curves showing the increase in the fraction of ScMreB5^WT in the pellet (liposome-bound fraction) at 2-µM protein concentration, with increasing concentration of the liposomes mimicking Spiroplasma lipid composition and 100% DOPG liposomes. The purple dotted line marks the 600-µM liposome concentration chosen for further liposome-binding assays. (D) A representative 12% SDS-PAGE gel of liposome pelleting assay showing the binding specificity of ScMreB5^WT by varying the DOPC and DOPG ratios at 600-µM liposome concentration. Protein in the pellet is observed at the higher DOPG percentages. (E) A representative 12% SDS-PAGE gel of pelleting assay with WT, in the presence of 1 mM ATP/ADP/AMP-PNP, showing that the protein does not pellet in the presence of nucleotides in the absence of liposomes at 100,000 g spin. P and S represent the pellet and supernatant fractions of the protein. Source data are available for this figure: SourceData FS4.