Membrane binding of ScMreB5 is modulated by lipid charge and conformational changes driven by Glu134. (A) A representative 12% SDS-PAGE gel of liposome pelleting assay for determining membrane binding of ScMreB5^WT and the hydrophobic loop mutants with a neutral lipid DOPC and an anionic lipid DOPG. P and S represent the pellet and supernatant fractions of the reaction. Concentrations of DOPG and DOPC liposomes and protein used in the assay are 1 mM and 2 µM, respectively. (B) Plot showing relative intensities of the fraction of protein in the pellet corresponding to ScMreB5^WT and mutant constructs in the SDS-PAGE gels from five independent experiments. The binding is specifically observed for liposome composed of the anionic lipid DOPG for the ScMreB5^WT as well as the mutants. Negligible binding is seen for both ScMreB5^WT and the mutants for the liposome made from neutral lipid DOPC. The error bar denotes the mean with SEM; unpaired two-tailed Student’s t test; ns, P > 0.05; ***, P < 0.0001). (C) Weblogo of C-terminal end, showing the presence of lysine and arginine in Spiroplasma MreB5s. The numbering on the x axis is with respect to the last 10 residues of ScMreB5. (D) A representative 12% SDS-PAGE gel of liposome pelleting assay showing a decrease in binding for ScMreB5^WT and mutants (ScMreB5^ΔC10, ScMreB5^I95A, ScMreB5^W96A, ScMreB5^I95A,W96A, and ScMreB5^E134A, denoted ΔC10, I95A, W96A, IWA, and E134A, respectively) at 20%:80% (DOPC:DOPG) liposome ratio. P and S represent the pellet and supernatant fractions of the reaction. Concentrations of liposomes and protein used in the assay are 600 and 2 µM, respectively. (E) Plot showing relative intensities of the fraction of protein in the pellet calculated from the SDS-PAGE gels from at least four independent experiments (representative image in D). The error bar denotes the mean with SEM; unpaired two-tailed Student’s t test; ns, P > 0.20; ***, P < 0.0001; **, P = 0.001–0.002). (F) Different views of membrane-binding face of double-protofilament ScMreB5. Electrostatic surface potential of the membrane-binding face (IA and IB subdomains; middle and right subpanels) of double protofilament of ScMreB5 is shown corresponding to the ribbon views of the double protofilament (left). Circled regions within the surface show the regions of positive and neutral charge for the membrane-binding face of the filament. The double protofilament of ScMreB5 was modeled using CcMreB double protofilament, PDB accession no. 4CZE. Subdomains IA and IB are colored pink and light green. (G) A representative 12% SDS-PAGE gel of liposome pelleting assay showing binding for ScMreB5^WT in different nucleotide states at 20%:80% (DOPC:DOPG) liposome ratio. P and S represent the pellet and supernatant fractions of the reaction. Concentrations of liposomes and protein used in the assay are 600 and 2 µM, respectively. Concentration of nucleotides and MgCl₂ used are 1 mM each. (H) Plot showing relative intensities of the fraction of protein in the pellet in different nucleotide conditions calculated from the SDS-PAGE gel from at least four independent experiments (representative image in G). The error bar denotes the mean with SEM; unpaired two-tailed Student’s t test; ns, P = 0.09; ***, P ≤ 0.0001). Source data are available for this figure: SourceData F7.