Transmembrane Polar Relay Drives the Allosteric Regulation for ABCG5/G8 Sterol Transporter

Abstract

The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely, a polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.

Keywords: ABCG5; ABCG8; ATP-binding cassette transporter; cholesterol; polar relay; sitosterolemia.

Figure 1

Disease-causing mutations and single-nucleotide polymorphisms (SNPs) in ATP-binding cassette (ABC) sterol transporters (ABCG5/G8). (A) Localization of ABCG5/G8 residues carrying missense mutations. The positions of disorder-related polymorphisms or mutations are highlighted in black spheres on the structures of ABCG5 (Protein Data Bank (PDB) identifier (ID): 5D07, chain C) and ABCG8 (PDB ID 5D07, chain D). Structural motifs are indicated in dashed ovals: triple-helical bundle (black), transmembrane domain (TMD) polar relay (yellow), and extracellular domain with re-entry helices (green). (B) Microenvironment of G5-E146, G5-A540, and G8-R543. (Middle) The transmembrane domains (white) and the triple-helical bundle (gray) are plotted in tube-styled cartoon presentation, showing the α-carbons (spheres) of G5-E146 (orange), G8-R543 (blue), and G5-A540 (black). The polar relays are plotted in dotted yellow spheres. (Top left) Slapped top view shows G5-A540 situated more than 10 Å away from the polar relay of either subunit (red dot-ended lines). (Top right) Near G5-A540 shows a cholesterol-shaped electron density (mesh) in the crystal structure of ABCG5/G8. The Fo−Fc difference electron density map was contoured at 3.0 σ. (Bottom left) At the triple helical bundle of ABCG5, E146 interacts with R377 through their side-chain termini in a distance of hydrogen bonding, 3.5 Å (black dashed line). (Bottom right) In the ABCG8 polar relay, R543 interacts E503 through their side-chain termini in a distance of hydrogen bonding, 3.1 Å (black dashed line).

Figure 2

(A) Chemical structures of cholesterol, cholesteryl hemisuccinate (CHS), and cholic acid (cholate). Source: PubChem. (B) Schematic illustration of sterol-coupled ATPase activity of ABCG5/G8. Dodecyl-maltoside (DDM)-purified ABCG5/G8 (light/dark-gray surface) is preincubated with phospholipids and cholate. Addition of CHS (four-ringed steroid structure) stimulates hydrolysis of ATP to ADP and inorganic phosphate (Pi) in the presence of the divalent magnesium ions (Mg²⁺). Using the colorimetric and bismuth citrate-based assay, the liberated Pi is then captured by ammonium molybdate in the presence ascorbic acid. The color is developed upon mixing with bismuth citrate and sodium citrate, and the absorbance was measured at 695 nm. See details in Section 4.

Figure 3

ATPase activity of ABCG5/G8. The ATP hydrolysis was used as a measure of ABCG5/G8 ATPase activity at 37 °C in conditions with 5 mM ATP and 4.1 mM CHS. The protocol is entailed in Section 4. (A) Data points are presented as the means ± standard deviations from 4–8 independent experiments using 2–4 independently purified proteins; where not visible, the error bars are covered by the plot symbols. A linear regression, plotted from the first 12 min, is used to calculate the specific activities. (B) Bar graphs show the specific activities of ATP hydrolysis by wild type (WT) in the presence and absence of CHS and the catalytically deficient mutant G8-G216D in the presence of CHS. The specific activity of WT in the absence of CHS is regarded as the basal ABCG5/G8 ATPase activity. (C) Bar graphs represent the percentage inhibition of ABCG5/G8 ATPase activity by 0.015 mM orthovanadate, where a p-value of <0.0001 (marked as ****) was obtained using ordinary one-way ANOVA (Prism 8).

Figure 4

Lipid dependence of ABCG5/G8 ATPase activity. (A) Purified ABCG5/G8 was assayed in the presence of either Escherichia coli or bovine liver polar lipids, and the specific activities of ATP hydrolysis were obtained by the ATP concentration-dependent experiments (0–5 mM ATP). Both curves are fitted to the Michaelis–Menten equation (Prism 8), and, using two independently purified proteins, the means of at least three independent experiments along with standard deviations are plotted here. The kinetic parameters are listed in Table 1. (B) In conditions of 5 mM ATP and 4.1 mM CHS, ATP hydrolysis of purified ABCG5/G8 was assayed in the presence of egg phosphatidylethanolamine (PE), soy phosphatidylcholine (PC), or egg phosphatidylglycerol (PG), where p-values of 0.0006 and 0.0003 (marked as ***), respectively, were obtained using ordinary one-way ANOVA (Prism 8).

Figure 5

ATP dependence of ABCG5/G8 ATPase activity. Purified proteins were assayed in the presence of E. coli polar lipids, and the specific activities of ATP hydrolysis were obtained from the ATP concentration-dependent experiments (0–5 mM ATP). The curves are fitted to the Michaelis–Menten equation (Prism 8), and, using two-to-four independently purified proteins, the means of at least three independent experiments along with standard deviations are plotted here. The kinetic parameters are listed in Table 1.

Figure 6

CHS dependence of ABCG5/G8 ATPase activity. Purified proteins were assayed in the presence of E. coli polar lipids, and the specific activities of ATP hydrolysis were obtained by the CHS concentration-dependent experiments (0–4.1 mM CHS). The curves are fitted to the Michaelis–Menten equation (Prism 8), and, using two independently purified proteins, the means of at least two independent experiments along with standard deviations are plotted here. The kinetic parameters are listed in Table 2.

Figure 7

Fluctuation of root-mean-square deviations (RMSDs) along molecular dynamics (MD) simulation time course. RMSDs were calculated using the main-chain atoms of all residues (black lines) or secondary structures only (red lines): (A) wild type; (B) E146Q mutant in ABCG5; (C) A540F mutant in ABCG5; (D) R543S mutant in ABCG8. G5G8: ABCG5/G8; SS: secondary structure.

Figure 8

Representative structures of the WT ABCG5/G8 and its three missense mutants. The representative structures (shown as blue cartoons and bluish sticks) were aligned to the crystal structure (green cartoons, and greenish lines). The three mutation residues, E146Q, A540F, and R543S, are shown as spheres. The hypothetical surrounding residues of ATP are shown as dashed rectangles. (A,E) Wild type; (B,F) E146Q; (C,G) A540F; (D,H) R543S. G5: ABCG5; G8: ABCG8. Residues in G5 and G8 are separately colored in black and red. Root-mean-square deviations (RMSDs) for the main-chain atoms (rmsd_MC) and all heavy atoms (rmsd_HEV) are shown in the lower panels. If R211 is omitted from RMSD calculations, RMSDs of the main-chain atoms are 0.69, 1.30, 0.88, and 0.78 Å for WT, E146Q, A540, and R543S, respectively; the corresponding RMSDs of heavy atoms are 0.85, 1.42, 1.13, and 0.96 Å.

Figure 9

Proposed mechanism of sterol-coupled ATP catalysis by ABCG5/G8. (Mode 1) A sequential pathway is derived from experiments on the disease mutants, G5-E146Q and G8-R543S. ABCG5/G8 first recruits ATP and Mg²⁺ ions, likely causing a conformational change of the nucleotide-binding domain (NBD) for ATP binding. CHS/sterol then binds the transporter and triggers ATP hydrolysis that may result in its dissociation. (Mode 2) A concerted pathway is derived from experiments on the putative sterol-binding mutant, G5-A540F. ABCG5/G8 simultaneously recruits CHS, ATP, and Mg²⁺ ions, induces a transient conformational change of the NBD, and activates ATP hydrolysis and CHS/sterol dissociation from the transporter. G5: ABCG5; G8: ABCG8; E: ABCG5/G8 heterodimer; Pi: inorganic phosphate.