Fig. 2: Lipid interactions in cleaved Ycf1p monomer and comparison of ATPase activity with uncleaved Ycf1p.

a Atomic models of monomeric Ycf1p in IFwide-α (left) and IFwide-β (right) conformations, coloured as in Fig. 1. Close-up views of the interactions between lipids and each conformer are shown below. A phosphatidylethanolamine molecule (PE1) is located between TMD0 and TM helix bundle 1 on the luminal site of the protein for both the IFwide-α (lower panel) and IFwide-β (lower left panel) conformations and is shown as a stick in CPK colouring with the cryo-EM density in grey. The sidechains of interacting residues, defined as those within 4 Å of the lipid, are also shown as sticks with their cryo-EM densities. A phosphatidylethanolamine (PE2) is bound to the ABC core of the IFwide-β conformation (lower right panel). b The sequence of loop-6 and surrounding residues in TM6 and TM7 is shown on top. Below, map-in-model fit for residues in TM6, TM7, and loop-6. The backbone is shown as a blue ribbon with all modeled sidechains shown as sticks. The cryo-EM density is shown at two different thresholds, denoted by the σ values, to highlight the poor (or missing) density for loop-6. The portion of loop-6 missing in the atomic model is schematically represented by a dashed pink curve. Residues labeled in pink are from the loop-6 insertion, whereas residues labeled in blue are from TM6, loop-6, and TM7, and are found in other ABCC proteins. c Plots of the specific ATPase activities of monomeric cleaved Ycf1p (pink) and monomeric uncleaved Ycf1p (grey) in the presence of increasing concentrations of glutathione disulfide, GSSG (left), and estradiol 17β-(D)-glucuronide, E₂17βG (right). Bars represent the mean value with error bars showing the standard deviation derived from three independent measurements each from two separate preparations of cleaved and uncleaved Ycf1p, with the exception of assays with uncleaved Ycf1p and 0 μM, 25 μM, and 125 μM GSSG. In these cases, values are derived from three independent measurements each from three separate preparations of protein.