Protein expression, purification, and site-specific labeling

All bMRP1 constructs were expressed and purified as described previously (Johnson and Chen, 2017). Briefly, baculovirus with each bMRP1 construct was generated and used to infect HEK293S GntI^- suspension cells. All constructs contained a C-terminal PreScission-Protease-cleavable GFP tag. Cell pellets were solubilized by adding 2% lauryl maltose neopentyl glycol (LMNG) supplemented with 0.2% cholesteryl hemisuccinate (CHS). After removal of the insoluble fraction by centrifugation, supernatants were batch bound to GFP-nanobody-conjugated Sepharose 4 Fast Flow resin (GE Healthcare). The resin was then packed into a column, washed with buffer containing 0.06% digitonin, and protein was eluted by digestion with PreScission Protease. After elution, protein was concentrated and applied to a Superose 6 10/300 GL column (GE Healthcare) equilibrated in 0.06% digitonin, 150 mM KCl, 50 mM Tris-HCl pH 8.0, 2 mM MgCl₂, and 2 mM DTT. Peak fractions were pooled and concentrated, and either used immediately to prepare cryo-EM samples or flash-frozen in liquid nitrogen and stored at −80°C for future FRET and ATPase assays.

For site-specific fluorescence labeling, the 12-residue S6 peptide (GDSLSWLLRLLN) was substituted at linker residues 868–879, and the 12-residue A1 peptide (GDSLDMLEWSLM) was added to the C-terminus immediately following V1530 (Yin et al., 2006; Zhou et al., 2007). A His₁₀-tag was also added at the N-terminus of bMRP1 for surface immobilization. Fluorescent labeling of the A1 site was carried out after elution off the GFP nanobody column by adding 5 µM AcpS, 25 µM LD655-CoA, and 10 mM MgCl₂ and incubating for 1 hr at room temperature. Excess dye was removed by size-exclusion chromatography as described above. The S6 site was then labeled by adding 5 µM Sfp, 25 µM Cy3-CoA, and 10 mM MgCl₂ and incubating for 1 hr at room temperature. The doubly labeled protein was cleaned up by size-exclusion chromatography. The labeling efficiencies were estimated to be ~30% for Cy3 and ~80% for LD655 from the respective extinction coefficients of the protein and fluorophores. Sfp and AcpS synthases, as well as dye-CoA conjugates, were purified as described previously (Yin et al., 2006; Zhou et al., 2007).