**1. Introduction**

All living cells depend on the ability to translocate nutrients, metabolites, and other molecules across their membranes. One major way to achieve this is through membrane-anchored transporter proteins. The evolutionarily conserved ATP-binding cassette (ABC) transporter superfamily, for example, carries out ATP-dependent and active transport of a wide range of substances across cellular membranes, including both hydrophilic and hydrophobic molecules such as sugars, peptides, antibiotics, or cholesterol [1–4]. As a key component of cellular membranes, cholesterol constitutes ~50% of cellular lipid content; it is also the precursor of steroid hormones that modulate gene regulation and bile acids that enable nutrient absorption. Translocation of cholesterol molecules on biological

membranes plays an essential role in maintaining cellular and whole-body cholesterol homeostasis. Thus, excess cholesterol needs to be eliminated from cells and tissues through either sterol acceptors in the circulation or direct excretion into the bile or the gut [5,6]. A large body of evidence indicates that ABC sterol transporters regulate cholesterol metabolism, and their defects are associated with dysregulation of whole-body cholesterol homeostasis, a major risk factor for cardiovascular diseases [7,8]. Yet, we have almost no understanding of how these transporters actually translocate cholesterol molecules and how the sterol-transport process is controlled by ATP catalysis. Given the dysregulation of cholesterol metabolism as a major risk factor for cardiovascular disease, there is a pressing need to elucidate of mechanism of these transporters in moving molecules across the cell membranes.

Recent progress in solving a heterodimeric crystal structure of human ABCG5 and ABCG8 established a new molecular framework toward such a mechanistic understanding of ABC sterol transporters. ABCG5 and ABCG8 are half-sized ABC sterol transporters and co-expressed on the apical surface of the hepatocytes along the bile ducts and the enterocytes from the intestinal brush-border membranes [9,10]. ABCG5 and ABCG8 function as obligate heterodimers (ABCG5/G8) and serve as the primary and indispensable sterol-efflux pump that effectively exports excess cholesterol, non-cholesterol sterols, and dietary plant sterols into the bile and the intestinal lumen. In mammals, most cholesterol is eliminated via its metabolism into bile acids or via biliary secretion as free cholesterol. The latter is considered as the last step of reverse cholesterol transport (RCT), where ABCG5/G8 accounts for more than 75% biliary cholesterol secretion [11–14]. Recent studies have shown that, in human subjects and animal models, ABCG5/G8 is also responsible for eliminating neutral sterols via the transintestinal cholesterol efflux (TICE), a cholesterol-lowering process independent of RCT [15]. Thus, physiologically, ABCG5/G8 plays an essential role in controlling cholesterol homeostasis in our bodies.

In general, the smallest functional unit of an ABC transporter consists of two transmembrane domains (TMD1 and TMD2) and two nucleotide-binding domains (NBD1 and NBD2), and both NBDs concertedly bind and hydrolyze ATP to provide the energy and drive substrate transport. The TMDs, on the other hand, have been shown to share low sequence similarity in the amino-acid sequences and three-dimensional structural folds, suggesting substrate-specific mechanisms for individual transporters [16]. Mechanistic analyses of ABC cholesterol transporters have largely centered on sequence requirement at the canonical ATP-binding sites [17–20], whereas little is known about the sterol–protein interaction and its relationship with ATP catalysis. Recent progress solving a crystal structure of human ABCG5/G8 revealed a unique TMD fold and several structural motifs [21]. In particular, for each subunit, a network of polar and charged amino acids is present in the core of the TMD, namely, a polar relay, whose role remains to be characterized. A triple-helical bundle is located at the transmission interface between the NBD and the TMD and consists of an elbow connecting helix, a hotspot helix (also known as an E-helix), and an intracellular loop-1 (ICL1) coupling helix. However, on the triple-helical bundle or the transmembrane polar relay, several residues have been shown to bear disease-causing missense mutations from sitosterolemia or other metabolic disorders with lipid phenotypes (Figure 1A). Notably, several disease-causing mutations are clustered in the membrane-spanning region or at the NBD–TMD interface [8,22]. This suggests the unique roles of these structural motifs in regulating the ABCG5/G8 function, yet no prior knowledge was available to explain the role of these structural motifs in the sterol-transport function.

Loss-of-function (LOF) mutations in *ABCG5* or *ABCG8* are linked to sitosterolemia, a rare autosomal recessive disease, while several other missense mutations are also associated with other lipid disorders, such as gallstone formation or elevated low-density lipoprotein (LDL) cholesterol [23–28]. At the cellular level, many of the missense mutations lead to defects in post-translational trafficking of ABCG5/G8 from the endoplasmic reticulum (ER), an abnormality commonly observed in other ABC transporters with missense mutations, e.g., ∆F508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR or ABCC7) [29,30]. However, specific missense mutants of ABCG5/G8 heterodimers have shown no defect in protein maturation [29], suggesting alternative disease-causing mechanisms. Therefore, studies of these mutants will not only show how they alter the transporter activity, but also provide mechanistic insights into the function of wild-type (WT) ABCG5/G8 sterol transporter.

**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).

Disease mutations are instrumental in studying the mechanisms of affected proteins in vitro, e.g., familial hypercholesterolemia mutations for proteins involved in low-density lipoprotein metabolism [31]. Guided by the structural framework of ABCG5/G8, we can now investigate its mechanisms using enzymological approaches with purified proteins. For this, we first need to establish at least one robust and consistent in vitro functional assay. Using ATPase activity as the functional benchmark in this study, we optimized an in vitro colorimetric ATPase assay that allows high-throughput activity assessment of detergent-purified ABCG5/G8. Using a soluble cholesterol memetic, cholesteryl hemisuccinate (CHS), we report here the CHS-stimulated ATP hydrolysis by ABCG5/G8 proteo-micelles, consisting of phospholipids, cholate, and dodecyl-maltoside (DDM), and we present an enzymatic analysis for the sterol-coupled ATPase activity on ABCG5/G8 sterol

transporter. Using ATPase activity as functional readout of ABCG5/G8, we show differentially inhibition of the CHS-stimulated ATPase activity by three LOF missense mutants, two sitosterolemia mutations, and one sterol-binding mutation, where residues bearing the two disease mutations are located along the polar relay. Our data hereby demonstrate the mechanistic basis on regulating ABCG5/G8 function by the transmembrane polar relay (Figure 1B).
