ARTICLE https://doi.org/10.1038/s41467-019-10279-w OPEN Structural basis for human sterol isomerase in cholesterol biosynthesis and multidrug recognition Tao Long 1, Abdirahman Hassan 1, Bonne M Thompson2, Jeffrey G McDonald1,2, Jiawei Wang3 & Xiaochun Li 1,4 3-β-hydroxysteroid-Δ8, Δ7-isomerase, known as Emopamil-Binding Protein (EBP), is an endoplasmic reticulum membrane protein involved in cholesterol biosynthesis, autophagy, 1234567890():,; oligodendrocyte formation. The mutation on EBP can cause Conradi-Hunermann syndrome, an inborn error. Interestingly, EBP binds an abundance of structurally diverse pharmacolo- gically active compounds, causing drug resistance. Here, we report two crystal structures of human EBP, one in complex with the anti-breast cancer drug tamoxifen and the other in complex with the cholesterol biosynthesis inhibitor U18666A. EBP adopts an unreported fold involving five transmembrane-helices (TMs) that creates a membrane cavity presenting a pharmacological binding site that accommodates multiple different ligands. The compounds exploit their positively-charged amine group to mimic the carbocationic sterol intermediate. Mutagenesis studies on specific residues abolish the isomerase activity and decrease the multidrug binding capacity. This work reveals the catalytic mechanism of EBP-mediated isomerization in cholesterol biosynthesis and how this protein may act as a multi-drug binder. 1 Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. 2 Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. 3 State Key Laboratory of Membrane Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China. 4 Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Correspondence and requests for materials should be addressed to X.L. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:2452 | https://doi.org/10.1038/s41467-019-10279-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10279-w holesterol maintains membrane structure, is a precursor shown to cause the death of cancer cells by influencing cholesterol Cin the biosynthesis of steroid hormones and bile acid, metabolism17,18. and has various roles in cell signaling. Acetyl-CoA, the Bioinformatics analysis shows that EBP shares structural fea- precursor of cholesterol biosynthesis, is converted into lanos- tures with both the membrane protein TM6SF2, which is asso- 19 terol, the first sterol-like intermediate, through a series of ciated with nonalcoholic fatty liver disease , and the σ2 receptor, reactions in the endoplasmic reticulum1–3. Due to its low which is highly expressed in multiple types of cancer cells20,21. solubility, lanosterol is handled by a number of membrane Here we present two structures of human EBP protein each in enzymes and is eventually converted to cholesterol4–6 (Sup- complex with a different pharmacologically active compound, plementary Fig. 1). To date, among those membrane enzymes revealing its mechanism of action in cholesterol biosynthesis and only the structure of sterol C14-demethylaase7 (CYP51) and the multidrug recognition. homolog of Δ14-sterol reductase4 have been determined. EBP catalyzes the conversion of Δ8-sterols (e.g zymosterol and Results zymostenol) to their corresponding Δ7-isomers (Fig. 1a). Functional characterization. To validate the function of EBP, the Mutations in EBP can lead to Conradi–Hunermann syndrome, human EBP-encoding plasmid was transferred to a yeast sterol which commonly causes growth deficiency, short stature, and isomerase erg2 knockout strain22 (Fig. 1b). The expression of curvature of the spine8. human EBP, but not the vector alone, allowed the yeast to survive Inhibition of EBP causes an accumulation of its sub- under exposure to 50 ng/ml cycloheximide, suggesting that strates zymosterol and zymostenol, contributing to autophagy in human EBP functions as a sterol isomerase in this system tumor cells9,10 and oligodendrocyte formation in the central (Fig. 1b). However, when we supplemented the medium with nervous system11. Notably, EBP binds an abundance of structu- either U18666A (an inhibitor of cholesterol biosynthesis and rally diverse pharmacologically active compounds, including Niemann-Pick C1 protein)23,24 or tamoxifen (Fig. 1c), growth of antidepressants, antipsychotics, opioid analgesics, sterol bio- – the yeast was inhibited (Fig. 1b). Our competition binding assay synthesis inhibitors and anti-tumor reagents12 14 (Supplementary shows that either U18666A or tamoxifen can compete with the Fig. 2). This type of broad specificity is similar to the σ1 receptor [3H]-Ifenprodil binding of purified EBP in vitro12 (Fig. 1d). This that has been linked to a wide variety of signal transduction observation is consistent with a previous ligand-binding study in pathways15, although the sequence analysis shows that EBP and the yeast microsome12, suggesting that these compounds may σ1 receptor share no structural similarity. Remarkably, as a bind the catalytic site of EBP to block enzymatic activity. component of the microsomal anti-estrogen-binding site (AEBS), which is involved in estrogen receptor-independent effects of tamoxifen, EBP can lower the availability of intracellular The overall structure. The purified EBP protein presented a tamoxifen, causing resistance16. Some EBP ligands have been monodisperse peak on gel filtration encouraging us to continue a c O R R R 9 8 + 8 N 7 ON U18666AU18 HO HO HO Δ8-sterol Carbocationic intermediate Δ7-isomer R= R= N Zymosterol Zymostenol Dehydrolathosterol Lathosterol ON Tamoxifen b d HO erg2 N H3 [H3]-Ifenprodil 9 8 3 7 HO H HO HO 7000 Tamoxifen IC = 165 ± 13 nM Fecosterol Episterol 50 ± 6000 U18666A IC50 = 351 12 nM Input 5000 EV 4000 WT 3000 EV + 50 μM Tamoxifen WT 2000 ]-Ifenprodil binding (CPM) 3 H EV [ 1000 +100 μM U18666A WT 0 Without +50 ng/ml –10 –9 –8 –7 –6 –5 –4 –3 cycloheximide cycloheximide Log[ligand](M) Fig. 1 Functional characterization of human EBP protein. a Reaction catalyzed by human EBP in which zymosterol (Δ8-sterol) is converted to dehydrolathosterol (Δ7-sterol). b Yeast complementation assay. EBP can rescue the growth of a Saccharomyces cerevisiae sterol isomerase Erg2 (yeast EBP homologue) deletion strain (ΔErg2). Growth of yeast expressing human EBP in the presence of sub-inhibitory concentrations of cycloheximide for 24 to 48 h with or without pharmacological compound. c The structures of U18666A and tamoxifen. d The binding of EBP to different ligands. Inhibition of [3H]- Ifenprodil binding to the purified EBP protein by U18666A (red) and tamoxifen (black). Data shown are the mean ± SD of three determinations. Source data are provided as a Source Data file. EV empty vector, WT wild type 2 NATURE COMMUNICATIONS | (2019) 10:2452 | https://doi.org/10.1038/s41467-019-10279-w | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10279-w ARTICLE a b TM5 Cytosol TM4 TM3 TM5 U18666A TM2 TM1 90° TM4 H1 U18666A TM2 TM1 H1 Lumen TM3 65 Å c d TM5 Cytosol TM4 TM3 Tamoxifen TM1 TM4 TM2 90° H1 TM2 Tamoxifen TM1 H1 TM3 Lumen e f U18666A Tamoxifen Tamoxifen –5 kT/e 5 kT/e Fig. 2 Molecular architecture of human EBP. a Overall structure of EBP with U18666A viewed parallel to the membrane. Each molecule of the dimer is indicated in the different colors. The two lines show the approximate location of the lipid bilayer. b The bottom view of EBP with U18666A from the lumen. c Overall structure of EBP with tamoxifen viewed parallel to the membrane. d The bottom view of EBP with tamoxifen from the lumen. e Electrostatic surface representation of EBP and the ligand-biding site with tamoxifen. f, 2Fo – Fc map for U18666A and tamoxifen (blue mesh) contoured at 1σ with our structural investigation (Supplementary Fig. 3). Crystals Tactin sepharose (Supplementary Fig. 7a), implying that EBP can were only obtained with U18666A or tamoxifen in space group form a dimer as shown by our structural observation. Mutations Δ P21212, owing to these two compounds presenting a high binding in the dimer interface lead to slower growth of the Erg2 affinity to EBP (Supplementary Fig. 2). The structure was deter- Saccharomyces cerevisiae at exposure of 50 ng/ml cycloheximide mined by selenium-based single-wavelength anomalous disper- (Supplementary Fig. 7b, c), implying that the dimerization may sion (SAD) and refined at 3.2 Å resolution with U18666A and play a role in fully activating EBP. Notably, sequence analysis 3.5 Å resolution with tamoxifen (Supplementary Figs. 4, 5 and suggests that the TM6SF2 protein contains two repeat units, each Supplementary Table 1). This protein contains five transmem- of which shares sequence homology with EBP21. brane helices (TMs 1–5) (Fig. 2a–e). Based on the previous immunoblotting study25 and positive inside rule26, we assigned the N-terminal face to the lumen (Fig. 2a, b). In the density map, The horizontal helix linker. A linker between TM2 and TM3 the ligand (U18666A or tamoxifen) was determined unambigu- forms a membrane horizontal helix (H1) that blocks a hydro- ously (Fig. 2f). Comparison between U18666A-bound and phobic cavity created by the residues of TMs 2–5(Fig.2a–d). tamoxifen-bound structures revealed a similar conformation The residues of H1, which face the cavity, are hydrophobic and (Supplementary Fig. 6). aromatic (Fig. 3a, b and Supplementary Fig. 8), while the A DALI search for structural homologues failed to identify a residues facing the lumen are hydrophilic. Importantly, Ala similar entry for the entire structure, implying that EBP presents substitutions of Trp101, Tyr104 and Tyr111, which face the an unreported fold. EBP forms a homodimer in the crystal, as hydrophobic cavity, abolish over 90% of the isomerase activity reported in previous solution studies27, with dimensions of 65 × by gas chromatography analysis28. Conversion of Δ8-sterols to 30 × 55 Å. The area of the dimer interface including TMs 3–5of Δ7-isomers occurs through the uptake of solvent hydrogen from each monomer is 1460 Å2 (Fig.