Fig. 3: Functional analysis of OmrDE.

a, b Comparison protein structures between OmrDE (predicted by TmrAB as a template, PDB: 6RAF) and TmrAB (only TMDs and NBDs are indicated). The protein structures are colored: OmrD, cyan; OmrE, yellow; TmrA, orange; TmrB, navy. b The functional important motifs in OmrDE and TmrAB are indicated by black arrows with labels. Walker A, ABC signature, and D-loop are grouped with irregular shapes indicated as dotted lines. c Purified membrane proteins subjected to SDS-PAGE. d Western blotting after the His-Tag pull-down assay. The molecular weights of the purified membrane protein containing hexahistidine (His) and trihemagglutinin (HA) tags are calculated using protein sequences: OmrD-His, 62.86 kDa; OmrE-His, 72.09 kDa; OmrE-HA, 75 kDa. c, d These experiments were performed twice, and the representative data are shown. e MIC tests for E. coli harboring plasmids treated with various drugs, as shown in Supplementary Fig. 10a‒f, Supplementary Table 1. Measurements were conducted in an independent experimental group (n = 3), in which the data were presented as mean value ± SD. Cm, chloramphenicol; Km, kanamycin; Tet, tetracycline; DAPI, 4′,6-diamidino-2-phenylindole; HO342, Hoechst 33342. f DAPI translocation in IMVs containing no protein or membrane proteins was initiated by adding Mg-ATP, followed by measuring DAPI fluorescence; measurements were conducted in an independent experimental group (n = 6). g ATP hydrolysis of the NBDs of purified membrane proteins was performed using colorimetric determination of P_i. Non-linear fits were evaluated using the Michaelis–Menten equation, and measurements were conducted in an independent experimental group (n = 3), in which the data were presented as mean value ± SD. h Suggested model of OcHeR function associated with OmrDE. Membrane proteins are embedded in the membrane. Marker, protein marker: Null, empty vector; CP cytoplasmic side, EC extracellular side.