Structural basis for human sterol isomerase in cholesterol biosynthesis and multidrug recognition

The N-terminal face of EBP is on the lumen.

“Based on the previous immunoblotting study25 and positive inside rule26, we assigned the N-terminal face to the lumen (Fig. 2a, b).”

• cites [25] Dussossoy, D. et al. Colocalization of sterol isomerase and sigma(1) receptor at endoplasmic reticulum and nuclear envelope level. Eur. J. Biochem. 263, 377–386 (1999). sourceSupported
• cites [26] von Heijne, G. Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues. Nature 341, 456–458 (1989). sourceNot checked — no full text of the source

Conversion of Δ8-sterols to Δ7-isomers occurs through uptake of solvent hydrogen from the membrane.

“Conversion of Δ8-sterols to Δ7-isomers occurs through the uptake of solvent hydrogen from the membrane29, implying that a proton or water molecule can enter the catalytic cavity with helix H1 possibly serving as the gate.”

• cites [29] Wilton, D. C., Rahimtula, A. D. & Akhtar, M. The reversibility of the delta8-cholestenol-delta7-cholestenol isomerase reaction in cholesterol biosynthesis. Biochem. J. 114, 71–73 (1969). sourcePartly supported

  The Results and Discussion report incorporation of hydrogen from tritiated water at C-9 of Δ7-cholestenol, consistent with a reversible Δ8-to-Δ7 isomerization. The Mechanism section proposes proton addition during that conversion, but describes the proton as coming from an enzyme group; it does not establish uptake of solvent hydrogen specifically from the membrane. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Conradi-Hunermann syndrome patients have increased amounts of 8,9-unsaturated sterols in the plasma and tissues due to mutations in the EBP gene.

“Biochemical studies on Conradi–Hunermann syndrome patients demonstrated increased amounts of 8,9-unsaturated sterols in the plasma and tissues due to mutations in the EBP gene8,32.”

• cites [8] Braverman, N. et al. Mutations in the gene encoding 3 beta-hydroxysteroid-delta 8, delta 7-isomerase cause X-linked dominant Conradi-Hunermann syndrome. Nat. Genet. 22, 291–294 (1999). sourcePartly supported

  The Abstract (page not provided in the index) reports increased 8(9)-cholestenol and 8-dehydrocholesterol in tissue samples from seven female probands and EBP mutations in all probands. The sterol-analysis section describes fibroblast and liver samples, but our copy does not report elevated sterols in patients’ plasma; that part of the claim is unsupported. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [32] Kelley, R. I. et al. Abnormal sterol metabolism in patients with Conradi-Hunermann-Happle syndrome and sporadic lethal chondrodysplasia punctata. Am. J. Med. Genet. 83, 213–219 (1999). sourceNot checked — no full text of the source

EBP shares a large cavity in the transmembrane domain for ligand binding with P-glycoprotein, MRP1, and ABCG2.

“Comparison with ligand structures of other known multidrug binding proteins (P-glycoprotein (P-gp)35, multidrug resistance-associated protein 1 (MRP1)36, ABCG237 and σ1 receptor38) reveals that EBP shares a large cavity in the transmembrane domain for ligand-binding with P-gp, MRP1, and ABCG2 (Fig. 6a–c).”

• cites [35] Aller, S. G. et al. Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science 323, 1718–1722 (2009). sourceSupported
• cites [36] Johnson, Z. L. & Chen, J. Structural basis of substrate recognition by the multidrug resistance protein MRP1. Cell 168, 1075–1085 e1079 (2017). sourceCould not verify

  The supplied passages have no section or page labels and contain only reference and software URLs, not structural findings. Searches for a transmembrane ligand-binding cavity, MRP1 substrate binding, and EBP returned no substantive passage, so our copy cannot verify the comparison. (LLM, conf=0.72)

  Experimental — accuracy not yet measured
• cites [37] Manolaridis, I. et al. Cryo-EM structures of a human ABCG2 mutant trapped in ATP-bound and substrate-bound states. Nature 563, 426–430 (2018). sourceSupported

The σ1 receptor includes only one TM that recruits its extracellular domain to bind ligand.

“By contrast, the σ1 receptor includes only one TM that recruits its extracellular domain to bind ligand (Fig. 6d).”

• cites [38] Schmidt, H. R. et al. Crystal structure of the human sigma1 receptor. Nature 532, 527–530 (2016). sourcePartly supported

  The paper’s structure description (Abstract; page unavailable) confirms one transmembrane domain per protomer. But it places the ligand-binding β-barrel in the cytosolic domain, not an extracellular domain, and does not say the transmembrane domain recruits it to bind ligand. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

Blocking EBP activity contributes to the death of tumor cells.

“Blocking EBP activity has been shown to contribute to the death of tumor cells9,10,17,18.”

• cites [9] de Medina, P. et al. Ligands of the antiestrogen-binding site induce active cell death and autophagy in human breast cancer cells through the modulation of cholesterol metabolism. Cell Death Differ. 16, 1372–1384 (2009). sourceNot checked — no full text of the source
• cites [10] Segala, G. et al. Dendrogenin A drives LXR to trigger lethal autophagy in cancers. Nat. Commun. 8, 1903 (2017). sourceSupported
• cites [17] de Medina, P., Silvente-Poirot, S. & Poirot, M. Tamoxifen and AEBS ligands induced apoptosis and autophagy in breast cancer cells through the stimulation of sterol accumulation. Autophagy 5, 1066–1067 (2009). sourcePartly supported

  The section “AEBS Ligands Induce Apoptosis through the Accumulation of Sterols and Oxysterols” reports that AEBS ligands induce apoptosis in MCF-7 breast cancer cells. Our copy does not identify EBP or establish that blocking EBP activity specifically contributes to that death; it attributes the observed death to sterol accumulation and oxidation following treatment with AEBS ligands. Page numbers are unavailable in the indexed copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Human EBP functions as a sterol isomerase in a yeast complementation system.

“The expression of human EBP, but not the vector alone, allowed the yeast to survive under exposure to 50 ng/ml cycloheximide, suggesting that human EBP functions as a sterol isomerase in this system (Fig. 1b).”

• Also stated in: § Introduction · plain

  “EBP can rescue the growth of a Saccharomyces cerevisiae sterol isomerase Erg2 (yeast EBP homologue) deletion strain (ΔErg2).”

Human EBP adopts an unreported fold involving five transmembrane helices (TMs 1-5).

“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.”

• Also stated in: § The overall structure · plain

  “This protein contains five transmembrane helices (TMs 1–5) (Fig. 2a–e).”

• Also stated in: § The overall structure · hedged

  “A DALI search for structural homologues failed to identify a similar entry for the entire structure, implying that EBP presents an unreported fold.”

The five-TM fold of EBP creates a membrane cavity that presents a pharmacological binding site accommodating multiple different ligands.

“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.”

• Also stated in: § The membrane cavity and the putative catalytic mechanism · hedged

  “Structural analysis indicates that the cavity is created by the TMs that provide the hydrophobic environment to host the compound (Fig. 3c).”

• Also stated in: § Discussion · plain

  “The binding pocket in EBP consists of several conserved aromatic residues that provide a hydrophobic environment directly facing the membrane, allowing the cavity to host diversely structured ligands (Fig. 3c).”

Mutations in the EBP dimer interface slow the growth of ΔErg2 Saccharomyces cerevisiae at 50 ng/ml cycloheximide.

“Mutations in the dimer interface lead to slower growth of the ΔErg2 Saccharomyces cerevisiae at exposure of 50 ng/ml cycloheximide (Supplementary Fig. 7b, c), implying that the dimerization may play a role in fully activating EBP.”

H1 residues facing the cavity are hydrophobic and aromatic.

“The residues of H1, which face the cavity, are hydrophobic and aromatic (Fig. 3a, b and Supplementary Fig. 8), while the residues facing the lumen are hydrophilic.”

EBP has a membrane horizontal helix (H1) between TM2 and TM3 that blocks a hydrophobic cavity created by residues of TMs 2-5.

“A linker between TM2 and TM3 forms a membrane horizontal helix (H1) that blocks a hydrophobic cavity created by the residues of TMs 2–5 (Fig. 2a–d).”

The EBP dimer interface, including TMs 3-5 of each monomer, has an area of 1460 Å2.

“The area of the dimer interface including TMs 3–5 of each monomer is 1460 Å2 (Fig. 2a–d).”

The U18666A-bound and tamoxifen-bound EBP structures have a similar conformation.

“Comparison between U18666A-bound and tamoxifen-bound structures revealed a similar conformation (Supplementary Fig. 6).”

H1 residues facing the lumen are hydrophilic.

“The residues of H1, which face the cavity, are hydrophobic and aromatic (Fig. 3a, b and Supplementary Fig. 8), while the residues facing the lumen are hydrophilic.”

EBP forms a homodimer.

“EBP forms a homodimer in the crystal, as reported in previous solution studies27, with dimensions of 65 × 30 × 55 Å.”

• Also stated in: § The overall structure · hedged

  “Western blot showed that His-EBP can be pulled-down by Strep-Tactin sepharose (Supplementary Fig. 7a), implying that EBP can form a dimer as shown by our structural observation.”

• cites [27] Moebius, F. F. et al. Purification and amino-terminal sequencing of the high affinity phenylalkylamine Ca2+ antagonist binding protein from guinea pig liver endoplasmic reticulum. J. Biol. Chem. 269, 29314–29320 (1994). sourceNot checked — no full text of the source

The side-chain distances between Glu80 and Glu122 (2.7 Å) and between Glu80 and His76 (3.2 Å) allow a proton to transfer through a hydrogen-bonding network as a catalytic triad.

“The 2.7 Å distance between the side chains of Glu80 and Glu122 and the 3.2 Å distance between the side chains of Glu80 and His76 allow a proton to transfer through the hydrogen-bonding network as a catalytic triad.”

EBP mutants His76Ala and Glu80Ala completely lose enzymatic activity.

“The results show that mutants with His76Ala or Glu80Ala completely lose the enzymatic activity, while mutants with Glu122Ala or Trp196Ala retain a much lower activity than the wild-type protein (Fig. 4c and Supplementary Fig. 10).”

Trp196 stabilizes EBP ligands through a π-cation interaction.

“Trp196 is capable of stabilizing the compound through a π-cation interaction, while His76, Glu80, Glu122, and Asn193 may form electrostatic interactions and hydrogen bonds with the amine group, further enhancing the binding (Fig. 3a, b).”

Asn193 and Glu122 recognize the amine group of the ligand.

“Asn193 and Glu122 recognize the amine group of the ligand, which is stabilized through a π-cation interaction with Trp196 (Fig. 3a, b).”

The ligand amine group is stabilized through a π-cation interaction with Trp196.

“Asn193 and Glu122 recognize the amine group of the ligand, which is stabilized through a π-cation interaction with Trp196 (Fig. 3a, b).”

Five Conradi-Hunermann syndrome mutations (L18P, E103K, A105D, G107E, R110Q) are found in H1 and the luminal domain.

“Five mutations are found in H1 and the luminal domain, including L18P, E103K, A105D, G107E, R110Q.”

EBP mutants Glu122Ala and Trp196Ala retain much lower enzymatic activity than wild-type EBP.

“The results show that mutants with His76Ala or Glu80Ala completely lose the enzymatic activity, while mutants with Glu122Ala or Trp196Ala retain a much lower activity than the wild-type protein (Fig. 4c and Supplementary Fig. 10).”

U18666A and tamoxifen bind to EBP in the same manner.

“We also showed that U18666A and tamoxifen bind to EBP in the same manner, implying the binding mode of EBP ligands (Fig. 3a, b).”

The EBP membrane cavity contains four polar residues.

“There are four polar residues in this cavity suggesting a special role.”

Ala substitutions of Trp101, Tyr104, and Tyr111 abolish over 90% of EBP isomerase activity.

“Importantly, Ala substitutions of Trp101, Tyr104 and Tyr111, which face the hydrophobic cavity, abolish over 90% of the isomerase activity by gas chromatography analysis28.”

• cites [28] Moebius, F. F. et al. Histidine77, glutamic acid81, glutamic acid123, threonine126, asparagine194, and tryptophan197 of the human emopamil binding protein are required for in vivo sterol delta 8-delta 7 isomerization. Biochemistry 38, 1119–1127 (1999). sourceNot checked — no full text of the source

The ligand (U18666A or tamoxifen) was determined unambiguously in the density map.

“In the density map, the ligand (U18666A or tamoxifen) was determined unambiguously (Fig. 2f).”

Dimerization may play a role in fully activating EBP.

“Mutations in the dimer interface lead to slower growth of the ΔErg2 Saccharomyces cerevisiae at exposure of 50 ng/ml cycloheximide (Supplementary Fig. 7b, c), implying that the dimerization may play a role in fully activating EBP.”

The resulting carbocationic sterol intermediate would be stabilized by Trp196 through a π-cation interaction.

“The resulting carbocationic sterol intermediate would be stabilized by Trp196 through a π-cation interaction (Fig. 4a).”

Helix H1 can allow a proton or water molecule to access the EBP cavity from the ER lumen.

“Helix H1 can allow a proton or water molecule to access the cavity from the ER lumen.”

The produced Δ7-isomers would be released into the membrane from the membrane gate between TM1 and TM5.

“The produced Δ7-isomers would be released into the membrane from the membrane gate between TM1 and TM5 (Fig. 3c).”

His76 may act as a proton donor, protonating Δ8-sterol at C9α and generating a carbocation at C8.

“His76 may play the role of a proton donor residue, protonating Δ8-sterol at C9α with the subsequent generation of a carbonium ion at C8.”

These enzymatic activity results are consistent with previous gas chromatography analysis showing that EBP variants with any of these mutations essentially affected its isomerase activity.

“These results are consistent with the previous gas chromatography analysis, which demonstrated that EBP variants with any of these mutations essentially affected its isomerase activity28.”

• cites [28] Moebius, F. F. et al. Histidine77, glutamic acid81, glutamic acid123, threonine126, asparagine194, and tryptophan197 of the human emopamil binding protein are required for in vivo sterol delta 8-delta 7 isomerization. Biochemistry 38, 1119–1127 (1999). sourceNot checked — no full text of the source

The proton would be returned to His76 through the hydrogen-bonding network.

“After this reaction, the proton would be returned to His76 through the hydrogen-bonding network.”

EBP's putative catalytic mechanism is analogous to the enzymatic action of ketosteroid isomerases, in which acidic residues act as a proton donor or acceptor.

“This putative mechanism is analogous to the enzymatic action of ketosteroid isomerases, for which acidic residues act as a proton donor or acceptor31, although the ketosteroid isomerase catalyzes the reaction in an aqueous environment.”

• cites [31] Pollack, R. M. Enzymatic mechanisms for catalysis of enolization: ketosteroid isomerase. Bioorg. Chem. 32, 341–353 (2004). sourceNot checked — no full text of the source

These five mutations may block solvent entry.

“These mutations may block the solvent entry.”

The hydrophobicity of EBP-binding compounds enables them to partition into the membrane.

“Besides the amine group, the hydrophobicity of compounds enables them to partition into the membrane.”

Compounds that bind to EBP have a positively-charged amine group, mimicking the carbocationic reaction sterol intermediate.

“The compounds that bind to EBP have a positively-charged amine group (Fig. 1c and Supplementary Fig. 2), mimicking the carbocationic reaction sterol intermediate (Fig. 4a).”

Disease-related mutations localized to the EBP membrane cavity may affect sterol binding/entry and catalytic reaction.

“Mutations, which are localized to the membrane cavity, may affect sterol binding/entry and catalytic reaction.”

Glu80, Glu122, and His76 are likely involved in the catalytic mechanism of EBP during cholesterol biosynthesis.

“As these three residues have a 3.5–5 Å distance to the compound, they are likely involved in the catalytic mechanism of EBP during cholesterol biosynthesis (Fig. 4a).”

EBP in the ER is not able to directly export tamoxifen to the extracellular space.

“It is clear that EBP in the ER is not able to directly export tamoxifen to the extracellular space.”