Figures and data
Cryo-EM structure of ABCG5/G8 showing a potential sterol-binding site buried between the TMDs of ABCG5 and ABCG8.
A and B. Cryo-EM map and structure of ABCG5/G8 in the nucleotide-free (ATP-apo) and sterol-bound states. (a and b) The zoomed-in pictures of sterol-like density observed in between the TMDs of ABCG5/G8, adjacent to ABCG5Y432 and Alanine 429 (ABCG5A429). C. Ergosterol (blue) modeled into the observed cryo-EM density. D. RMSD profiles of ergosterol (blue), cholesterol (yellow), and CHS (orange) within the ABCG5/G8 binding site during 200 ns MD simulations across independent replicates.
Structural features of the ABCG5/G8 sterol-binding pocket and molecular determinants of sterol recognition.
A. Electrostatic surface map of ABCG5/G8. The zoomed-in view highlights the electrostatic potential within the binding pocket surrounding the observed bound ligand (ergosterol shown in blue). B. Alignment of the cryo-EM structure of ABCG5/G8 bound to ergosterol (ergosterol shown in blue; ABCG5 in pink and ABCG8 in purple) with the crystal structure (PDB ID: 8CUB; shown in pale yellow), which is apo at this binding site. (a) The alignment highlights conformational changes in residues forming the potential sterol-binding pocket in ABCG5. Serin 538 (highlighted in the blue box) is positioned 3.5 Å from the hydroxyl group of ergosterol. (b) The same structural alignment focusing on ABCG8, showing residue movements within the corresponding binding pocket. C. Per-residue binding free energy decomposition from the MD simulations of ABCG5/G8 in the presence of ergosterol (blue), cholesterol (yellow), or CHS (orange) over 200 ns MD simulations across independent replicates.
ATP- and sterol-dependent modulation of ABCG5/G8 ATPase activity.
A. ATPase activity of ABCG5WT/G8WT measured over the 2–8 min interval in the presence of ergosterol, cholesterol, or CHS, compared with the sterol-free condition and the catalytically inactive ABCG5WT/G8G216D mutant. B. ATPase activity of ABCG5Y432F/G8WT measured over the 2–8 min interval in the presence of ergosterol or cholesterol, compared with the corresponding ergosterol- and cholesterol-stimulated ATPase activities of ABCG5WT/G8WT shown in panel A. Statistical significance in panels A and B was determined using one-way ANOVA with multiple comparisons. ns, not significant; *P < 0.05; ****P < 0.0001. C. ATP concentration-dependent ATPase activity of ABCG5WT/G8WT measured in the presence of 0.25 mM ergosterol, cholesterol, CHS, or under sterol-free conditions. D. Sterol concentration-dependent ATPase activity of ABCG5WT/G8WT measured in the presence of 5 mM ATP and increasing concentrations of ergosterol, cholesterol, or CHS. Ergosterol was insoluble above 0.4 mM under the current assay conditions.
Proposed functional model of ABCG5/G8 highlighting sterol-binding sites identified in the crystal structure (PDB ID: 8CUB) (17) and the cryo-EM structure determined in this study.
A. The model depicts sterol efflux through a binding site observed in the cryo-EM structure, with the sterol located between the TMDs indicated in the red box corresponding to ergosterol. Sterols are proposed to enter from the membrane into the binding cavity, where ATP binding and hydrolysis drive conformational changes that enable substrate release. B. Model of cholesterol translocation along the protein surface, illustrating a proposed flip-flop mechanism between membrane leaflets. The cholesterol molecule shown in the green box corresponds to the bound cholesterol observed in the crystal structure (PDB ID: 8CUB) (17). C. Surface representation of ABCG5/G8 (top view) showing sterol-binding sites identified in both structures. The site highlighted in red corresponds to that shown in panel A, while the site in green corresponds to panel B. Key residues, including ABCG5I539 and ABCG8L458, partition the transmembrane cavity into asymmetric subcavities. The ABCG5-ABCG8-facing cavity is spacious and accessible to sterols, whereas the ABCG8-ABCG5-facing cavity is more restricted and is not predicted to accommodate sterol entry.
