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. Author manuscript; available in PMC: 2022 Dec 2.
Published in final edited form as: Structure. 2021 Sep 11:S0969-2126(21)00259-8. doi: 10.1016/j.str.2021.07.009

Molecular Structures of Human ACAT2 Disclose Mechanism for Selective Inhibition

Tao Long 1, Yang Liu 1, Xiaochun Li 1,2,3,*
PMCID: PMC8642284  NIHMSID: NIHMS1729956  PMID: 34520735

Summary

Endoplasmic reticulum-localized acyl-CoA:cholesterol acyltransferase (ACAT), including ACAT1 and ACAT2, converts cholesterol to cholesteryl esters that become incorporated into lipoproteins or stored in cytosolic lipid droplets. Selective inhibition on ACAT2 has been shown to considerably attenuate hypercholesterolemia and atherosclerosis in mice. Here, we report cryogenic electron microscopy (cryo-EM) structures of human ACAT2 bound to its specific inhibitor pyripyropene A (PPPA) or the general ACAT inhibitor nevanimibe. Structural analysis reveals that ACAT2 shares a topology in membranes similar to that of ACAT1. A catalytic core with an entry site occupied by a cholesterol molecule and another site for allosteric activation of ACAT2 is observed in these structures. Enzymatic assays show that mutations within sites of cholesterol entry or allosteric activation attenuate ACAT2 activity in vitro. Together, these results reveal mechanisms for ACAT2 mediated esterification of cholesterol, providing a blueprint to design new ACAT2 inhibitors for use in the prevention of cardiovascular disease.

eTOC Blurb

Long et al. report the structures of human ACAT2 bound to its specific inhibitor pyripyropene A or the general ACAT inhibitor nevanimibe. The structures along with functional validations reveal mechanisms for ACAT2 mediated esterification of cholesterol and facilitate the design of new ACAT2 inhibitors for cardiovascular disease treatment

Graphical Abstract

graphic file with name nihms-1729956-f0001.jpg

Introduction

There are two sources of cholesterol in the Endoplasmic reticulum (ER): de novo cholesterol synthesis in the ER by several different enzymes (Li et al., 2015; Long et al., 2019; Porter and Herman, 2011; Song et al., 2005), or cholesterol transport to the ER via receptor-mediated uptake of plasma low-density-lipoprotein (Brown and Goldstein, 1986; Kwon et al., 2009; Li et al., 2016a; Li et al., 2016b; Long et al., 2020a). ER-localized ACAT1 and ACAT2, which belong to the membrane-bound O-acyltransferase (MBOAT) family of enzymes (Masumoto et al., 2015), play a key role in cholesterol homeostasis by catalyzing transfer of fatty acyl groups to the 3β-hydroxyl group of cholesterol (Brown et al., 2018; Chang et al., 2006; Luo et al., 2019; Rogers et al., 2015). The resulting cholesteryl esters are either stored in cytosolic lipid droplets or incorporated into apolipoprotein B containing lipoproteins such as low-density lipoprotein (LDL) that are secreted from cells (Chang et al., 2009).

The cDNA encoding ACAT1 was isolated by Chang and co-workers in 1993, while the ACAT2 cDNA was isolated in 1998 by three distinct laboratories (Anderson et al., 1998; Cases et al., 1998; Oelkers et al., 1998). Subsequent studies revealed that ACAT1 is ubiquitously expressed in mice, whereas expression of ACAT2 is primarily limited to hepatocytes and intestinal cells (Chang et al., 2000; Lee et al., 2000; Parini et al., 2004). Deficient ACAT2 activity results in reduced assembly and secretion of low-density-lipoproteins that abrogates hypercholesterolemia and atherosclerosis in mouse models (Buhman et al., 2000; Ohshiro et al., 2015; Rudel et al., 2005; Willner et al., 2003).

A causal association exists between levels of circulating LDL and atherosclerosis, a chronic disease characterized by the buildup of plaques composed of cholesterol and other lipids in walls of arteries that restrict blood flow (Goldstein and Brown, 2009). The rupture of these plaques, which causes occlusion of arteries, is the initial pathological event that leads to heart attack and stroke responsible for more than 15 million deaths per year. Several drugs are prescribed to lower plasma LDL and reduce atherosclerosis and associated cardiovascular disease. Statins inhibit the cholesterol biosynthetic enzyme 3-hydroxy-3-methylgluataryl coenzyme A (HMG CoA) reductase and thereby enhance expression of hepatic LDL-receptors that remove LDL from circulation. Ezetimibe blocks absorption of dietary cholesterol in the intestine by inhibiting the function of Niemann Pick Type C1 (NPC1) like protein-1 (NPC1L1). Evolocumab blocks proprotein convertase subtilisin/kexin type 9 (PCSK9)-mediated degradation of hepatic LDL-receptors, leading to their enhanced expression. In many cases, ezetimibe and evolocumab are prescribed together with statins to maximize lowering of plasma LDL. However, the efficacy of statins is limited because the drugs trigger an increase in HMG CoA reductase that permits continued synthesis of cholesterol. In addition, adverse side effects that have been associated with statins, including hepatic dysfunction and myopathy (muscle injury), reduce adherence to therapy (Study of the Effectiveness of Additional Reductions in et al., 2010). Finally, evolocumab is not orally administered, but rather through subcutaneous injection.

Thus, efforts to develop novel alternative therapies for prevention and/or treatment of cardiovascular disease are merited. Pyripyropene A (PPPA) (Das et al., 2008), which specifically inhibits ACAT2, has been shown to considerably attenuate hypercholesterolemia and atherosclerosis in mice (Ohshiro et al., 2011; Ohshiro et al., 2015), while the selective inhibition of ACAT1 causes pernicious side effects and cannot reduce the development of atherosclerotic lesions in mouse models (Accad et al., 2000). These observations, which are consistent with reduced atherosclerosis in ACAT2 deficient mice, indicate ACAT2 is a promising target for new cholesterol-lowering therapies.

Our group and others recently determined the cryo-EM structure of ACAT1 (Guan et al., 2020; Long et al., 2020b; Qian et al., 2020). The analysis of our structure revealed that the soluble acyl-CoA substrate enters the catalytic cavity of ACAT1 from the cytosol, whereas the insoluble cholesterol substrate enters the cavity from the membrane. His460 in the catalytic cavity serves as a base to deprotonate the 3β-hydroxyl group of cholesterol and transfer the fatty acyl chain; these reactions are facilitated by Trp420 and Asn421. Once formed, the ester product is released into the ER membrane and subsequently incorporated into lipid droplets and lipoproteins. A previous study suggested ACAT1 and ACAT2 adopt different topologies in the ER membrane (Lin et al., 2003); however, another study proposed the two enzymes share a similar membrane topology (Das et al., 2008). These disparate findings could be resolved by the determination of the molecular structure of ACAT2, which would also provide further insight into ACAT mediated cholesterol esterification and accelerate development of ACAT2 specific inhibitors that lower blood LDL-cholesterol.

Results

Overall Structures of ACAT2 with distinct inhibitors

To determine the structure of ACAT2 enzyme, we expressed human ACAT2 in HEK293 cells. We co-purified ACAT2 in detergents together with either nevanimibe or PPPA. PPPA had been shown to present much higher selectivity for inhibition of ACAT2 than ACAT1 (Das et al., 2008). Consistent with this, we found in enzymatic assays that the IC50 of PPPA to ACAT2 is 25 μM, whereas its IC50 for inhibiting ACAT1 is 179 μM (Figure 1A). The IC50 of nevanimibe for ACAT2 inhibition is 0.71μM and for ACAT1 inhibition is 0.23 μM (Long et al., 2020b) (Figure 1A). ACAT2 was well behaved in the presence of PPPA or nevanimibe, making them suitable for structural investigation by cryo-EM (Figure S1). We were able to collect cryo-EM images for ACAT2 yielding structures at 3.87~3.93 Å resolution (Figures 1B–F, S2–3 and Table 1). The cryo-EM densities of both inhibitors have been well determined (Figure 1C and E).

Figure 1. Overall structure of human ACAT2 Holoenzyme.

Figure 1.

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A. Either nevanimibe or PPPA inhibits the activity of ACAT1 and ACAT2 in vitro. In the activity assays, ACAT activity was measured by monitoring the released sulfhydryl group of coenzyme A. The IC50 of nevanimibe to ACAT2 is 0.71 μM (the deviation range is 0.58~0.87 μM). The IC50 of PPPA to ACAT2 is 25 μM (the deviation range is 17~38 μM) and the IC50 of PPPA to ACAT1 is 179 μM (the deviation range is 136~238 μM). Data are mean ± s.d. (n=3 independent experiments). B. Structure of human ACAT2 and nevanimibe viewed from the side of the membrane. C. Cryo-EM map of nevanimibe at 5σ level. D. Structure of human ACAT2 and PPPA. E. Cryo-EM map of PPPA at 5σ level. F. The top view of the holoenzyme with PPPA. G. Structural comparison of ACAT2 with ACAT1 (pdb code: 6VUM) from the luminal side.

Table 1.

Cryo-EM data collection, refinement and validation statistics

ACAT2-nevanimibe (EMDB-;24209) (PDB-7N6R) ACAT2-PPPA (EMDB-24208) (PDB-7N6Q)
Data collection and processing
Magnification 60024 60024
Voltage (kV) 300 300
Electron exposure (e−/Å2) 60 60
Defocus range (μm) 1.0 to 2.0 1.0 to 2.0
Pixel size (Å) 0.833 0.833
Symmetry imposed C2 C2
Initial particle images (no.) 1,825,855 1,203,637
Final particle images (no.) 210,603 153,208
Map resolution (Å) 3.93 3.87
FSC threshold 0.143
Refinement
Initial model used (PDB code) 6VUM ACAT2-NEV
Model resolution (Å) 3.97 3.93
FSC threshold 0.5
Map sharpening B factor (Å2) −172.4 −141.3
Model composition
Non-hydrogen atoms 12,708 12,752
Protein residues 1,500 1,500
Ligands 16 16
B factors (Å2)
Protein 140.83 139.51
Ligand 126.58 131.64
R.m.s. deviations
Bond lengths (Å) 0.014 0.014
Bond angles (°) 1.705 1.711
Validation
MolProbity score 1.86 1.97
Clashscore 7.69 7.63
Poor rotamers (%) 1.24 1.55
Ramachandran plot
Favored (%) 94.59 92.53
Allowed (%) 5.14 7.47
Disallowed (%) 0.27 0.00
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Consistent with our observations on ACAT1 (Long et al., 2020b), the ACAT2 holoenzyme contains two dimers (Figure 1B and D), thus C2 symmetry was assumed during the data processing. The four ACAT2 molecules are termed “ACAT2-A and ACAT2-B” in dimer 1 and “ACAT2-C and ACAT2-D” in dimer 2 (Figure 1B and D). The local resolution of the transmembrane helix core of ACAT2-A and ACAT2-C is about 3.5 Å in the cryo-EM maps (Figures S2C, 2E, 3C and 3E). The densities of most amino acids along with the inhibitors in the catalytic core are well defined (Figures S4). Each ACAT2 monomer has nine transmembrane helices (TMs) adopting a similar membrane topology as ACAT1 with an R.M.S.D. of 0.8 Å (Figure 1G). Due to the limited local resolution of the N-terminal cytosolic helices that are involved in tetrameric assembly, the identity of each helix could not be distinguished. Therefore, these helices are not included in the final model.

Figure 2. The substrates in the ACAT2.

Figure 2.

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A. ACAT2 with nevanimibe (dark grey), a fatty acid (FA) chain (light grey) and two cholesterol molecules (yellow) in the hydrophobic cavity. B. ACAT2 with PPPA (dark blue), a fatty acid (FA) chain (light grey) and two cholesterol molecules (yellow) in the hydrophobic cavity. C. Electrostatic surface representation of ACAT2 and a close-up view of the cholesterol entrance. Residues that involved in cholesterol substrate recognition are represented as sticks. D. Functional validation of the cholesterol binding site and catalytic residues. Data are mean ± s.d. (n=3 independent experiments).

Figure 3. The interaction details of inhibitors and ACAT enzymes.

Figure 3.

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A. Interaction of nevanimibe with cavity residues of ACAT1. B. Interaction of nevanimibe with cavity residues of ACAT2. C. Interaction of PPPA with cavity residues of ACAT2. Residues are represented as sticks; dashed line represents hydrophilic interactions. D. Enzymatic activity of wild-type (WT) ACAT2 and its variants with the mutations on the PPPA binding site in the presence of PPPA or nevanimibe. Data are mean ± s.d. (n=3 independent experiments). E. Ratio of enzymatic activity of wild-type (WT) ACAT2 and its variants with the mutations on the PPPA binding site in the presence and absence of PPPA or nevanimibe (the value of red or grey column/the value of black column of panel D).

The acyl-CoA substrate binding pocket and catalytic core

Previously, an endogenous acyl-CoA molecule was found in each ACAT1 protomer. In contrast, there is no molecule observed in the acyl-CoA binding pocket of ACAT2. The structural comparison reveals that the acyl-CoA binding in both of ACAT1 and ACAT2 are conserved suggesting the two enzymes employ the similar cytosolic pocket to engage the acyl-CoA for the reaction (Figure S5). TMs 4–9 form a hydrophobic core for catalyzing cholesterol esterification (Figure 2A and B). TM2, TM4, TM5 and TM6 contribute to create a hydrophobic tunnel open to the lipid bilayer (Figure 2C). The catalytic residue His434 is in the center of this tunnel, accessible from the lipid bilayer in the absence of inhibitors.

The cholesterol substrate entrance

Previously, we predicted that the cholesterol substrate could access the catalytic core of ACAT1 through a tunnel open to the lipid bilayer (Long et al., 2020b). In the cryo-EM maps of ACAT2, we observed a sterol-like molecule at the entrance of a similar tunnel (Figure 2C). Moreover, a snake-like density is also present in the catalytic cavity, which we built as a fatty acid chain according to the morphology of the map (Figure 2C). The position of this putative fatty acid is consistent with that of our previous observation on ACAT1 (Long et al., 2020b), although the function of this molecule remains unclear. The residues Glu235 and Arg238 are responsible for engaging the hydroxyl groups of putative cholesterol and fatty acid (Figure 2C). Residues Phe234 and Trp362 have hydrophobic contacts with the cholesterol molecule, implying that they may assist cholesterol in accessing the catalytic core. To verify our observations, we generated several ACAT2 variants with a single amino acid mutation either on the catalytic core (His434 and Asn395), the charged residues that engage the substrate (Glu235 and Arg238) or the substrate gate (Phe234 and Trp362). These variants were well behaved in solution with detergent (Figure S1C). The functional analysis showed that these variants abolish the enzymatic activity in vitro supporting our structural observation (Figure 2D).

How does cholesterol access the catalytic cavity? Our structural analysis reveals that the ~20 amino acid linker between TM1 and TM2, which is flexible in the lipid bilayer, has several hydrophobic residues (Leu151, Phe153, Leu156 and Phe160) that face the tunnel entrance (Figure 2C) that are conserved between ACAT1 and ACAT2. It is tempting to speculate that this linker serves as a gate to control the movement of cholesterol from the lipid bilayer to the catalytic core.

The nevanimibe binding site

The catalytic His434 of ACAT2 aligns well with His460 of ACAT1, the catalytic residue of ACAT1 (Figure 3A–C). Nevanimibe, which forms polar contacts with the catalytic His434, was stabilized by Trp394 through a π-π interaction and the carbonyl oxygen of nevanimibe forms a hydrogen bond with Asn395 blocking access of substrates to the catalytic His434 residue (Figure 3B). This mechanism is similar to ACAT1 and explains why nevanimibe can target both ACAT1 (Long et al., 2020b) and ACAT2 (Figure 1A).

The PPPA binding site

The structure of the ACAT2 complex with PPPA reveals a distinct inhibitor binding site (Figures 2B and 3C). PPPA binds closer to the luminal side of the catalytic cavity, compared to where nevanimibe binds (Figure 2B). Residues Trp394 and Asn395 are not involved in binding PPPA; in contrast, Phe438 in TM7b, Gln488 and Val489 in TM9 engage the PPPA molecule (Figure 3C). Interactions with these residues are consistent with a previous study that showed Gln488 is required for PPPA-mediated ACAT2 inhibition (Das et al., 2008).

To further validate the PPPA binding site, we mutated Phe438, Gln488 and Val489 to leucine individually, since the residues in a similar position in ACAT1 are leucines (Figure 3A). These variants were well behaved in solution with detergent (Figure S1D). We first measured the enzymatic activity of these variants. With the exception of ACAT2V489L, the ACAT2F438L and ACAT2Q488L present a similar activity as wild type ACAT2 protein. Notably, when the inhibitors were supplemented into the reaction, the activity of these variants was inhibited by nevanimibe but not by PPPA, while both inhibitors can inhibit wild type ACAT2 (Figure 3D). The ratio of ACAT2 variants’ activity in the presence of PPPA to that without PPPA is 2 fold higher compared to that of the wild type protein, supporting the idea that these residues are crucial for recognizing PPPA (Figure 3E). The previous study showed that the half-life of PPPA is ~15 minutes in liver and ~8 hours in plasma, because its three O-acetyl residues can be hydrolyzed quickly in vivo (Ohshiro et al., 2015). The short half-life of this compound restricts any sort of clinical application. Based on our structural observation, the development of PPPA derivatives could generate more potent and stable ACAT2 inhibitors potentially for cardiovascular diseases treatment.

The allosteric site

Several previous studies showed that ACATs enzymes are subject to allosteric regulation and that cholesterol is the most efficient allosteric activator (Liu et al., 2005; Rogers et al., 2012). The iso-octyl side chain of cholesterol is indispensable for allosteric activation, whereas the orientation of the hydroxy group at C-3 or the double bond between C-5 and C-6 are not important (Liu et al., 2005). In addition to the cholesterol molecule in the substrate entrance, we also observed a cholesterol molecule in each protomer, in a hydrophobic pocket flanked by TMs1, 5 and 6 (Figure 4A). Tyr124, Phe127, Leu352 and Phe356 have several interactions with this cholesterol molecule (Figure 4B). The sterol ring makes a hydrophobic contact with Trp382 that might affect the conformation of Trp394 through π-π interactions that are mediated by Trp381 in the catalytic core (Figure 4B). The interactions between cholesterol, Leu352, Phe355 and Phe356 might confine Trp362 in TM6 in a state that allows the cholesterol substrate to enter the catalytic core through the hydrophobic tunnel in the membrane (Figure 4B).

Figure 4. The allosteric site of ACAT2 and the comparison with that of ACAT1.

Figure 4.

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A. The allosteric site in ACAT2 dimer. The TM1, TM5 and TM6 that binds to the cholesterol are indicated. B. The interaction details of cholesterol and ACAT2. Cholesterol (yellow) may have hydrophobic contact with residues to stabilize Trp362 and Trp394. C. The interaction details of cholesterol and ACAT1. Cholesterol (yellow) may have hydrophobic contact with residues to stabilize Trp388 and Trp420. D. Functional validation of the allosteric site. Data are mean ± s.d. (n=3 independent experiments).

The structural comparison with ACAT1 shows that the cholesterol in each allosteric site shares a similar position (Figure 4C); however, the binding environments are slightly different (e.g., Phe120 and Leu352 in the ACAT2 have been changed to Ile138 and Phe378 in the ACAT1). These differences encouraged us to design/screen a specific allosteric inhibitor of ACAT2 in order to block its enzymatic activity. Specifically, when we mutated Phe356 or Trp382 to alanine individually, the activity of these variants was completely abolished suggesting that interference with the allosteric site can inhibit ACAT2 activity (Figures 4D and S1E).

Discussion

When lipid concentration in cells is low, ACAT2 can be ubiquitinated on Cys277 for degradation. This induces cellular reactive oxygen species to maintain the lipid homeostasis (Wang et al., 2017). Our structure reveals that Cys277 is located on the loop between TM4 and TM5 that faces to the cytosolic site (Figure S5D). Interestingly, a short helix functions like an obstacle to prevent Cys277 from being exposed to the cytoplasm. This suggests a special mechanism that can protect Cys277 from excessive oxidation and keep ACAT2 in the ER at an optimal concentration for cholesterol esterification.

Enterocytes employ Niemann-Pick C1-like 1 protein (NPC1L1) to absorb cholesterol (Betters and Yu, 2010), while ABCG5 (G5) and ABCG8 (G8) form a heterodimer to export cholesterol from hepatocytes (Graf et al., 2003). Additionally, NPC1L1 on the apical surface of hepatocytes transport the cholesterol back to cells, avoiding excessive biliary cholesterol loss (Temel et al., 2007). ACAT2 converts cholesterol to its esters, which are then transported in lymph and plasma as chylomicrons in both enterocytes and hepatocytes. However, it remains unclear whether ACAT2 activity affects the transport activity of NPC1L1 and G5G8. A clinical study revealed that the intestinal mRNA level of NPC1L1 and ACAT2 of patients with cholesterol gallstone disease were considerably higher than that of normal people (Jiang et al., 2009). Further study on the network of NPC1L1-G5G8-ACAT2 may facilitate our understanding on cholesterol metabolism in the intestine and liver.

Moreover, specific knockout of ACAT2 in the intestine or liver can prevent accumulation of hepatic cholesterol esters and hypercholesterolemia in mice (Zhang et al., 2012). It would be intriguing to determine whether ezetimibe (which inhibits NPC1L1) together with an ACAT2 specific inhibitor can further decrease the blood cholesterol level. Here, our structural work with functional analysis provides the evidence to develop a class of potent molecules based on PPPA selectively inhibit ACAT2 for prevention and treatment of cardiovascular diseases.

STAR ★METHODS

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Xiaochun Li (xiaochun.li@utsouthwestern.edu).

Materials availability

Any unique reagents/materials used in this study are available from the lead contact with a completed Materials Transfer Agreement.

Data and code availability

  • The 3D cryo-EM density maps of PPPA-bound ACAT2 and nevanimibe-bound ACAT2 have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-24208 and EMD-24209. Atomic coordinates for the atomic model of PPPA-bound ACAT2 and nevanimibe-bound ACAT2 have been deposited in the Protein Databank under the accession number 7N6Q and 7N6R.

  • This paper does not report original code.

  • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Cell line

Spodoptera frugiperda Sf9 and HEK293S GnTI−

Culture conditions for in vitro system

Spodoptera frugiperda Sf9 cells for baculovirus expression were cultured in Sf-900™ III SFM media (Thermo Fisher Scientific) at 27°C with shaking (135 rpm). HEK293S GnTI− cells used for protein expression were cultured in FreeStyle™ 293 media (Thermo Fisher Scientific) supplemented with 2% Fetal Bovine Serum (Corning) and 1% Pen Strep (Thermo Fisher Scientific) at 37°C with shaking (130 rpm).

METHOD DETAILS

Protein expression and purification

The cDNA of human ACAT2 (GenBank: BC096090.1) was cloned into pEG BacMam with a N-terminal Flag-tag. The protein was expressed in HEK-293S GnTI− cells (ATCC) using baculovirus system. For ligand-bound states, 10 μM nevanimibe or 5 μM PPPA was added in purification steps. The cells were harvested after 48 hours of infection, and then the cells were disrupted by sonication in buffer A (20 mM Hepes, pH 7.5, 150 mM NaCl) supplemented with 1 mM PMSF and 5 μg/mL leupeptin. After low-speed centrifugation, the resulting supernatant was incubated in buffer A with 1% (w/v) n-Dodecyl-β-D-Maltopyranoside (DDM, Anatrace) for 1 hour at 4 °C. The lysate was then centrifuged at high-speed to remove non-soluble components, and the supernatant was loaded onto the Flag-M2 affinity resin (Sigma-Aldrich). After two tandem washes, the protein was eluted in buffer A supplemented with 100 μg/mL 3×Flag peptide, 0.06% (w/v) Digitonin (ACROS Organics) and concentrated. The concentrated protein was purified by Superose 6 Increase size-exclusion chromatography column (GE Healthcare) in a buffer containing buffer A and 0.06% (w/v) Digitonin.

Fluorescence-based ACAT Assay

ACAT activity was measured by monitoring released CoA from the acyltransferase-mediated reaction (Cao et al., 2011). Micelles of 2 mM cholesterol/10 mM POPC/18.6 mM taurocholate in reaction buffer (100 mM Hepes pH 7.5 and 150 mM NaCl) were prepared as described previously (Chang et al., 1998). For the functional validations (Figures 2D and 4D), the assay was carried out in a total volume of 10 μL under the following conditions: 7.1 μL mixed micelles, 2.5 μL protein (at 1 μM concentration) and 0.4 μL 2.5 mM oleoyl-CoA. The reaction was initiated by adding protein and incubated at 37 °C for 10 minutes. The reaction was terminated by adding 2.5 μL 10% SDS. Then 100 μL 50 μM 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) in reaction buffer was added to the reaction system and the mixture was transferred to 96 well plate. The plate was incubated at room temperature for 30 minutes, then the fluorescent signal was detected by BioTek Synergy Neo2 Hybrid Multi-Mode Reader (excitation 355 nm; emission 460 nm). Relative fluorescence intensity was obtained by subtracting the fluorescence intensity of oleoyl-CoA free reaction system for the corresponding protein.

To measure the IC50 (Figure 1A) and the inhibition of enzymatic activity (Figure 3D), the assay was carried in a total volume of 20 μL under the following conditions: 14 μL mixed micelle, 1 μL different concentrations of inhibitors as indicated, 2.5 μL protein (at 4 μM concentration) and 2.5 μL 0.8 mM oleoyl-CoA. The protein was added first, and the mixture was incubated at 37 °C for 10 minutes. Then, the reaction was initiated by addition of oleoyl-CoA and incubated at 37 °C for 10 minutes. The reaction was terminated by adding 2.5 μL 10% SDS. The data were collected as described above and IC50 calculations were performed using GraphPad Prism8.

EM Sample Preparation and Imaging

The sample was added to Quantifoil R1.2/1.3 400 mesh Au holey carbon grids (Quantifoil), blotted using a Vitrobot Mark IV (FEI), and frozen in liquid ethane. The grids were imaged in a 300 keV Titan Krios (FEI) with a Gatan K3 Summit direct electron detector (Gatan). Data were collected at 0.833 Å/pixel with a dose rate of 23 electrons per physical pixel per second. Images were recorded for 1.8 s exposures in 60 subframes to give a total dose of 60 electrons per Å2.

Imaging Processing and 3D reconstruction

For nevanimibe-bound ACAT2, the images were collected in three batches (Figure S2). Dark subtracted images were first normalized by gain reference that resulted in a pixel size of 0.833 Å/pixel. Drift correction was performed using MotionCor2 (Li et al., 2013). The contrast transfer function (CTF) was estimated using CTFFIND4 (Rohou and Grigorieff, 2015). To generate ACAT2 templates for automatic picking, around 3000 particles were manually picked and 2D classified in RELION-3 (Zivanov et al., 2018). After auto-picking, the low-quality images and false-positive particles were removed manually. About 0.16/1/0.64 million particles of ACAT2 with nevanimibe were extracted. The 8,000 particles from Dataset 1 were selected to generate the initial model. Then, all the particles were 3D classified. To improve the 3D classification in Dataset 3, 192,697 good particles from Dataset 2 was used as the seeds and then removed in the later 3D auto-refine. The best class, containing 49,942/192,697/54,959 particles, provided a 10.18/8.33/8.33 Å map after 3D auto-refinement without a mask in RELION-3. Applying a soft mask in RELION-3 post-processing yielded cryo-EM maps of 8.87/8.09/7.73 Å. Bayesian polishing of particles were then performed in RELION-3. The 3D refinement using a soft mask and solvent-flattened Fourier shell correlations (FSCs) after combining Dataset 1 and Dataset 2 yielded a reconstruction at 4.66 Å. Then, a 3D Classification without image alignment was performed. The resulting 155,664 particles were refined using a soft mask and solvent-flattened Fourier shell correlations (FSCs) yielded a reconstruction at 4.36 Å. The 3D refinement using a soft mask and solvent-flattened Fourier shell correlations (FSCs) after combining Dataset 1, Dataset 2 and Dataset 3 yielded a reconstruction at 4.10 Å. The resulting particles were further refined with another soft mask yielded a reconstruction at 3.93 Å. Applying a soft mask in RELION-3 post-processing yielded a final cryo-EM map of 3.93 Å. To further improve the quality of our cryo-EM map, a focused refinement with a small mask was attempted. The focused refinements included protomer A and C. After CTF refinement, 3D refinement and postprocess, a final focused cryo-EM map of 3.87 Å was yielded. Both full map and focused map were used to generate a composite map for refinement.

For PPPA-bound ACAT2, 10,334 images were collected (Figure S3). Dark subtracted images were first normalized by gain reference that resulted in a pixel size of 0.833 Å/pixel. Drift correction was performed using MotionCor2 (Li et al., 2013). The contrast transfer function (CTF) was estimated using CTFFIND4 (Rohou and Grigorieff, 2015). After auto-picking, the low-quality images and false-positive particles were removed manually. About 1.2 million particles of ACAT2 with PPPA were extracted. The particles were then subjected to cryoSPARC (Punjani et al., 2017). After 2D classification, heterogenous refinement and NU-Refinement in cryoSPARC, a final cryo-EM map of 4.15 Å was yielded using 350,545 good particles. Then, a 3D Classification was performed in RELION-3. The resulting 153,208 particles were refined using a soft mask and solvent-flattened Fourier shell correlations (FSCs) yielded a reconstruction at 6.11Å. Applying a soft mask in RELION-3 post-processing yielded cryo-EM maps of 4.36 Å. Bayesian polishing were then performed using RELION-3. The 3D refinement using a soft mask and solvent-flattened Fourier shell correlations (FSCs) yielded a reconstruction at 3.98 Å. CTF refinement was performed, and the resulting particles were refined using a soft mask and solvent-flattened Fourier shell correlations (FSCs) yielded a reconstruction at 3.93 Å.

Applying a soft mask in RELION-3 post-processing yielded a final cryo-EM map of 3.87 Å. To further improve the quality of our cryo-EM map, a focused refinement with a small mask was attempted. The focused refinement included protomer A and C. After 3D refinement and postprocess, a final focused cryo-EM map of 3.67 Å was yielded. Both full map and focused map were used to generate a composite map for refinement.

Model Construction

To generate a composite map for refinement, full map and focused map that have been described above were combined and aligned using phenix.combine_focused_maps, which would coalescence the best parts of several maps together. For nevanimibe-bound ACAT2 structure, the structure of nevanimibe-bound ACAT1 (pdb code: 6VUM) was docked to the map and then manually adjusted and refined using COOT (Emsley and Cowtan, 2004). For PPPA-bound ACAT2 structure, the model was manually adjusted and refined using COOT based on the structure of nevanimibe-bound ACAT2. The residues 1–99, 145–149, 192–197, 258–264, 464–471 and 501–522 of human ACAT2 were not built due to the limited resolution.

Model Refinement and Validation

The models were refined in real space using PHENIX (Adams et al., 2010) and in reciprocal space using Refmac with secondary-structure restraints and stereochemical restraints (Murshudov et al., 1997). For cross-validations, the final model vs. map FSC curves were generated in the Comprehensive validation module in PHENIX.PHENIX and MolProbity (Chen et al., 2010) were used to validate the final model. Local resolutions were estimated using RELION-3. Structure figures were generated using PyMOL (http://www.pymol.org) and Chimera (Pettersen et al., 2004)

QUANTIFICTION AND STATISTICAL ANALYSIS

All quantification and statistical analysis were carried out using GraphPad Prism8. Data in all the fluorescence-based ACAT assays are mean ± s.d. (n=3 independent experiments)

Supplementary Material

2

KEY RESOURCES TABLE.

REAGENT or RESOURCE SOURCE IDENTIFIER
Chemicals, peptides, and recombinant proteins
Digitonin Acros Organics Cat#407565000
Lauryl maltose neopentyl glycol Anatrace Cat#NG310
Phenylmethylsulfonyl fluoride Goldbio Cat#P-470-25
Leupeptin Peptides International Cat#ILP-4041
pyripyropene A BioVision Inc Cat# B1910500
Nevanimibe Medchemexpress LLC Cat# HY100399A
POPC Avanti Polar Lipids Cat# 850457
Cholesterol Steraloids Cat#C6760-000
Oleoyl-CoA MP Biomedicals Cat# ICN10090405
CPM Sigma-Aldrich Cat#C1484
Deposited data
Atomic coordinates of PPPA-bound ACAT2 This paper PDB: 7N6Q
3D cryo-EM map of PPPA-bound ACAT2 This paper EMD-24208
Atomic coordinates of nevanimibe-bound ACAT2 This paper PDB: 7N6R
3D cryo-EM map of nevanimibe-bound ACAT2 This paper EMD-24209
Atomic coordinates of nevanimibe-bound ACAT1 Long et al, 2020 PDB: 6VUM
3D cryo-EM map of nevanimibe-bound ACAT1 Long et al, 2020 EMD-21390
Experimental models: Cell lines
HEK293S GnTI- ATCC CRL-3022
Sf9 ATCC CRL-1711
Recombinant DNA
pEG BacMam This paper N/A
pEG BacMam-ACAT2 This paper N/A
pEG BacMam-ACAT1 Long et al, 2020 N/A
Software and algorithms
Serial EM Mastronarde, 2005 http://bio3d.colorado.edu/SerialEM
CTFFIND 4 Rohou and Grigorieff, 2015 http://grigoriefflab.janelia.org/ctffind4
MotionCor 2 Zheng et al., 2017 https://emcore.ucsf.edu/ucsf-software
RELION 3 Zivanov et al., 2018 http://www2.mrclmb.cam.ac.uk/relion
GraphPad Prism8 GraphPad https://www.graphpad.com/scientific-software/prism/
Coot Emsley et al., 2010 http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot
PHENIX Adams et al., 2010 https://www.phenix-online.org
Refmac Brown et al., 2015 Murshudov et al., 1997 https://www.ucl.ac.uk/~rmhasek/refmac.html
MolProbity Chen et al., 2010 http://molprobity.biochem.duke.edu
UCSF Chimera Pettersen et al., 2004 https://www.cgl.ucsf.edu/chimera
PyMOL PyMOL http://www.pymol.org
Other
R1.2/1.3 400 mesh Au holey carbon grids Quantifoil Cat#1210627
Superose 6, 10/300 GL GE Healthcare Cat#17-5172-01
ANTI-FLAG M2 Affinity Gel Millipore Sigma Cat#A2220
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Highlights:

  • Structure of ACAT2 bound PPPA reveals a mechanism for selective inhibition.

  • ACAT2 shares a membrane topology similar to that of ACAT1.

  • The entry site of the catalytic core is occupied by a cholesterol molecule.

  • Mutations within cholesterol entry site or allosteric site abolish ACAT2 activity.

Acknowledgements

The data were collected at the UT Southwestern Medical Center Cryo-EM Facility (funded in part by the CPRIT Core Facility Support Award RP170644). We thank our colleague D. Stoddard for assistance in data collection and R. DeBose-Boyd and P. Schmiege for editing the manuscript. This work was supported by NIH grant P01 HL020948, R01 GM134700 and R01 GM135343, the Endowed Scholars Program in Medical Science of UT Southwestern Medical Center, O’Donnell Junior Faculty Funds, Welch Foundation (I-1957) (to X.L.). X.L. is a Damon Runyon-Rachleff Innovator supported by the Damon Runyon Cancer Research Foundation (DRR-53S-19) and a Rita C. and William P. Clements Jr. Scholar in Biomedical Research at UT Southwestern Medical Center.

Footnotes

Declaration of interests The authors declare no competing interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

2
NIHMS1729956-supplement-2.pdf (2.9MB, pdf)

Data Availability Statement

  • The 3D cryo-EM density maps of PPPA-bound ACAT2 and nevanimibe-bound ACAT2 have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-24208 and EMD-24209. Atomic coordinates for the atomic model of PPPA-bound ACAT2 and nevanimibe-bound ACAT2 have been deposited in the Protein Databank under the accession number 7N6Q and 7N6R.

  • This paper does not report original code.

  • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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