Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Structure of an integral membrane sterol reductase from Methylomicrobium alcaliphilum

Nature volume 517, pages 104–107 (2015)Cite this article

Subjects

Abstract

Sterols are essential biological molecules in the majority of life forms. Sterol reductases1 including Δ14 -sterol reductase (C14SR, also known as TM7SF2), 7-dehydrocholesterol reductase (DHCR7) and 24-dehydrocholesterol reductase (DHCR24) reduce specific carbon–carbon double bonds of the sterol moiety using a reducing cofactor during sterol biosynthesis. Lamin B receptor2 (LBR), an integral inner nuclear membrane protein, also contains a functional C14SR domain. Here we report the crystal structure of a Δ14-sterol reductase (MaSR1) from the methanotrophic bacterium Methylomicrobium alcaliphilum 20Z (a homologue of human C14SR, LBR and DHCR7) with the cofactor NADPH. The enzyme contains ten transmembrane segments (TM1–10). Its catalytic domain comprises the carboxy-terminal half (containing TM6–10) and envelops two interconnected pockets, one of which faces the cytoplasm and houses NADPH, while the other one is accessible from the lipid bilayer. Comparison with a soluble steroid 5β-reductase structure3 suggests that the reducing end of NADPH meets the sterol substrate at the juncture of the two pockets. A sterol reductase activity assay proves that MaSR1 can reduce the double bond of a cholesterol biosynthetic intermediate, demonstrating functional conservation to human C14SR. Therefore, our structure as a prototype of integral membrane sterol reductases provides molecular insight into mutations in DHCR7 and LBR for inborn human diseases.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Δ14-reductase activity of MaSR1.
Figure 2: The molecular architecture of MaSR1.
Figure 3: NADPH, putative sterol binding pockets and homology modelling with steroid 5β-reductase.
Figure 4: Models of human LBR and human DHCR7.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors for MaSR1 are deposited in the Protein Data Bank under accession code 4QUV.

References

  1. Porter, F. D. & Herman, G. E. Malformation syndromes caused by disorders of cholesterol synthesis. J. Lipid Res. 52, 6–34 (2011)

    Article  CAS  Google Scholar 

  2. Worman, H. J., Yuan, J., Blobel, G. & Georgatos, S. D. A lamin B receptor in the nuclear envelope. Proc. Natl Acad. Sci. USA 85, 8531–8534 (1988)

    Article  ADS  CAS  Google Scholar 

  3. Di Costanzo, L., Drury, J. E., Penning, T. M. & Christianson, D. W. Crystal structure of human liver Δ4-3-ketosteroid 5β-reductase (AKR1D1) and implications for substrate binding and catalysis. J. Biol. Chem. 283, 16830–16839 (2008)

    Article  CAS  Google Scholar 

  4. Fahy, E. et al. A comprehensive classification system for lipids. J. Lipid Res. 46, 839–862 (2005)

    Article  CAS  Google Scholar 

  5. Miller, W. L. & Bose, H. S. Early steps in steroidogenesis: intracellular cholesterol trafficking. J. Lipid Res. 52, 2111–2135 (2011)

    Article  CAS  Google Scholar 

  6. Miller, W. L. Steroid hormone synthesis in mitochondria. Mol. Cell. Endocrinol. 379, 62–73 (2013)

    Article  CAS  Google Scholar 

  7. Goldstein, J. L., DeBose-Boyd, R. A. & Brown, M. S. Protein sensors for membrane sterols. Cell 124, 35–46 (2006)

    Article  CAS  Google Scholar 

  8. Olins, A. L., Rhodes, G., Welch, D. B., Zwerger, M. & Olins, D. E. Lamin B receptor: multi-tasking at the nuclear envelope. Nucleus 1, 53–70 (2010)

    Article  Google Scholar 

  9. Silve, S., Dupuy, P. H., Ferrara, P. & Loison, G. Human lamin B receptor exhibits sterol C14-reductase activity in Saccharomyces cerevisiae . Biochim. Biophys. Acta 1392, 233–244 (1998)

    Article  CAS  Google Scholar 

  10. Hoffmann, K. et al. Mutations in the gene encoding the lamin B receptor produce an altered nuclear morphology in granulocytes (Pelger–Huët anomaly). Nature Genet. 31, 410–414 (2002)

    Article  CAS  Google Scholar 

  11. Waterham, H. R. et al. Autosomal recessive HEM/Greenberg skeletal dysplasia is caused by 3β-hydroxysterol Δ14-reductase deficiency due to mutations in the lamin B receptor gene. Am. J. Hum. Genet. 72, 1013–1017 (2003)

    Article  CAS  Google Scholar 

  12. Porter, F. D. Smith–Lemli–Opitz syndrome: pathogenesis, diagnosis and management. Eur. J. Hum. Genet. 16, 535–541 (2008)

    Article  CAS  Google Scholar 

  13. Herman, G. E. & Kratz, L. Disorders of sterol synthesis: beyond Smith–Lemli–Opitz syndrome. Am. J. Med. Genet. C 160, 301–321 (2012)

    Article  CAS  Google Scholar 

  14. Shchukin, V. N., Khmelenina, V. N., Eshinimaev, B., Suzina, N. E. & Trotsenko, Yu. A. Primary characterization of dominant cell surface proteins of halotolerant methanotroph Methylomicrobium alcaliphilum 20Z. Microbiology 80, 595–605 (2011)

    Article  CAS  Google Scholar 

  15. Paik, Y. K., Trzaskos, J. M., Shafiee, A. & Gaylor, J. L. Microsomal enzymes of cholesterol biosynthesis from lanosterol. Characterization, solubilization, and partial purification of NADPH-dependent Δ8,14-steroid 14-reductase. J. Biol. Chem. 259, 13413–13423 (1984)

    CAS  PubMed  Google Scholar 

  16. Roberti, R. et al. Cloning and expression of sterol Δ14-reductase from bovine liver. Eur. J. Biochem. 269, 283–290 (2002)

    Article  CAS  Google Scholar 

  17. Bennati, A. M. et al. Sterol dependent regulation of human TM7SF2 gene expression: role of the encoded 3β-hydroxysterol Δ14-reductase in human cholesterol biosynthesis. Biochim. Biophys. Acta 1761, 677–685 (2006)

    Article  CAS  Google Scholar 

  18. von Heijne, G. Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues. Nature 341, 456–458 (1989)

    Article  ADS  CAS  Google Scholar 

  19. Kim, H., Melen, K., Osterberg, M. & von Heijne, G. A global topology map of the Saccharomyces cerevisiae membrane proteome. Proc. Natl Acad. Sci. USA 103, 11142–11147 (2006)

    Article  ADS  CAS  Google Scholar 

  20. Miller, W. L. & Auchus, R. J. The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocr. Rev. 32, 81–151 (2011)

    Article  CAS  Google Scholar 

  21. Bennati, A. M. et al. Disruption of the gene encoding 3β-hydroxysterol Δ14-reductase (Tm7sf2) in mice does not impair cholesterol biosynthesis. FEBS J. 275, 5034–5047 (2008)

    Article  CAS  Google Scholar 

  22. Yang, J. et al. Mechanism of isoprenylcysteine carboxyl methylation from the crystal structure of the integral membrane methyltransferase ICMT. Mol. Cell 44, 997–1004 (2011)

    Article  CAS  Google Scholar 

  23. Hennekes, H. & Nigg, E. A. The role of isoprenylation in membrane attachment of nuclear lamins. A single point mutation prevents proteolytic cleavage of the lamin A precursor and confers membrane binding properties. J. Cell Sci. 107, 1019–1029 (1994)

    CAS  PubMed  Google Scholar 

  24. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  25. Zwart, P. H., Grosse-Kunstleve, R. W. & Adams, P. D. Xtriage and Fest: automatic assessment of X-ray data and substructure structure factor estimation. CCP4 Newsletter 43, 27 (2005)

    Google Scholar 

  26. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    Article  CAS  Google Scholar 

  27. Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999)

    Article  CAS  Google Scholar 

  28. Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D 58, 1772–1779 (2002)

    Article  Google Scholar 

  29. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  30. Cowtan, K. dm: An automated procedure for phase improvement by density modification. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallogr. 31, 34–38 (1994)

    Google Scholar 

  31. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  32. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    Article  CAS  Google Scholar 

  33. Fiser, A. & Sali, A. Modeller: generation and refinement of homology-based protein structure models. Methods Enzymol. 374, 461–491 (2003)

    Article  CAS  Google Scholar 

  34. Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994)

    Article  CAS  Google Scholar 

  35. Clayton, P. et al. Mutations causing Greenberg dysplasia but not Pelger anomaly uncouple enzymatic from structural functions of a nuclear membrane protein. Nucleus 1, 354–366 (2010)

    Article  Google Scholar 

  36. Holm, L. & Rosenstrom, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank W. Shi and H. Robinson at National Synchrotron Light Source (NSLS) beamline X29 and N. Sukumar at Advanced Photon Source (APS) beamline 24-ID-E for on-site assistance and J. Wang for support with the structure determination. We also thank L. Gatticchi and B. Sebastiani for assistance with the sterol reductase assays, and E. Coutavas, E. Debler and H. Shi for constructive comments in manuscript preparation. The C27Δ8,14 substrate was a gift to R.R. by G. Galli, University of Milano, Italy. This work was supported by funds from the Rockefeller University and the Howard Hughes Medical Institute. X.L. is supported by C.H. Li Memorial Scholar Fund fellowship of the Rockefeller University.

Author information

Authors and Affiliations

  1. Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, 10065, New York, USA

    Xiaochun Li & Günter Blobel

  2. Department of Experimental Medicine, University of Perugia, Perugia 06132, Italy,

    Rita Roberti

Authors
  1. Xiaochun Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rita Roberti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Günter Blobel
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.L. designed the research and performed structural biological studies; X.L. and R.R. performed sterol reductase activity assays; X.L., R.R. and G.B. contributed to data analysis and manuscript preparation; X.L. and G.B. wrote the manuscript.

Corresponding authors

Correspondence to Xiaochun Li or Günter Blobel.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Cholesterol biosynthesis pathway1 and sterol reductase family.

Acetyl-CoA is the precursor for cholesterol biosynthesis. After several reactions, the intermediate lanosterol is synthesized. Conversion of lanosterol to cholesterol (Bloch pathway) involves many reactions, some of which are catalysed by C14SR, LBR and DHCR7 (MaSR1 homologues, in red). C14SR, LBR and DHCR7 are homologues of NADPH-dependent reductases that catalyse the reduction of the sterol double bonds indicated in the green circles.

Extended Data Figure 2 Sequence alignment of MaSR1 with human C14SR, DHCR7 and C-terminal domain of LBR.

Secondary structural elements of MaSR1 are indicated above the sequences. Disordered regions in the MaSR1 structure are shown by a dashed line. Invariant amino acids are highlighted in blue (invariant in 3 of 4 proteins) and purple (invariant in all proteins). Putative cholesterol hydroxyl group binding sites are highlighted in red, NADPH binding sites are highlighted in cyan. Human disease mutations are also highlighted by different symbols. Sequence alignment was carried out using ClustalW34.

Extended Data Figure 3 Yeast complementation assay.

MaSR1 can rescue the growth of a Saccharomyces cerevisiae Δ14-sterol reductase Erg24 (yeast MaSR1 homologue) deletion strain (ΔErg24). ΔErg24 yeast expressing wild-type MaSR1, ScErg24 and mutated MaSR1 from a URA3 shuttle vector can grow under URA− selection (upper panel). Growth of yeast expressing MaSR1, ScErg24 and various mutated MaSR1 versions in the presence of sub-inhibitory concentrations of cycloheximide (20 ng ml−1) for 24 to 48 h (lower panel). The yeast expressing MaSR1 or ScErg24 is able to grow in the presence of cycloheximide. R395A (lane 8) corresponds to R583Q in LBR which has been reported to lead to loss of activity in yeast35. Results are representative of three independent experiments.

Extended Data Figure 4 MaSR1 crystal and X-ray diffraction image.

a, Photograph of MaSR1 crystal. b, A representative X-ray diffraction image of MaSR1 crystals with various resolution rings indicated by the circles.

Extended Data Figure 5 Anomalous difference Fourier electron density.

a, Overview of the anomalous difference Fourier map for selenium atoms in an asymmetric unit. The electron density is contoured at 4.5σ (purple mesh). Two molecules (MolA, molecule A; MolB, molecule B) were observed in each asymmetric unit. b, Examination of the atomic model in TM4 by selenium anomalous difference signals. Left panel shows wild-type SeMet anomalous difference signals; right panel shows mutated SeMet anomalous difference signals at 3σ (purple mesh). c, Examination of the atomic model in TM8 by selenium anomalous difference signals. Left panel shows wild-type SeMet anomalous difference signals, right panel shows mutated SeMet anomalous difference signals at 3σ (blue mesh). d, A view of the anomalous difference Fourier map for platinum atoms in an asymmetric unit. The electron density is contoured at 3σ (purple mesh). There are four platinum atoms binding to histidine residues in molecule A (yellow), but there are eight platinum atoms binding to six histidine and two methionine residues in molecule B (red). e, An overall view of the 2Fo − Fc electron density, contoured at 2σ, in one asymmetric unit.

Extended Data Figure 6 NADPH binding pocket and interaction between Trp 274 and Tyr 387 of MaSR1.

a, The structure of NADPH with the missing moiety in the MaSR1 structure indicated in the black circles. b, Overview of the NADPH-bound MaSR1. SA-omit map (Fo − Fc densities, magenta mesh) for NADPH contoured at 2σ. The right panel is an enlargement of the left panel (same orientation as Fig. 2a), rotated by 180°. c, The rebuilt missing moiety (purple) of NADPH in MaSR1. d, The surface representation shows Trp 274 (orange) and Tyr 387 (blue) located in the back of the sterol binding pocket. 2Fo – Fc map for an unidentified ligand (blue mesh) contoured at 2σ.

Extended Data Figure 7 Comparison of MaSR1 structure with ICMT structure.

a, A comparison of MaSR1 (grey and yellow) and ICMT22 (cyan) structure with S-adenosyl-l-homocysteine (SAH) bound (PDB accession number 4A2N). DALI search36 shows the closest entry (Z-score of 7.5) to MaSR1 is the structure of ICMT, consisting of 5 transmembrane helices, which had 193 Cα atoms aligned to MaSR1 (TM6–10 and α2) with r.m.s.d. of 2.8 Å. Both proteins have a similar cofactor binding pocket (magenta circle), although the sequence conservation is low. b, Comparison of NADPH and SAH binding pockets of MaSR1 (grey) and ICMT (cyan). The orientation of adenine–ribose moiety of SAH and NAPDH is similar with respect to the coordinating tyrosine residues in the cofactor pockets of these two enzymes.

Extended Data Table 1 Data collection and refinement statistics
Full size table
Extended Data Table 2 Native data completeness of each shell
Full size table
Extended Data Table 3 Data collection statistics for the MaSR1 mutants I151M, L304M and Pt-derivatives
Full size table

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, X., Roberti, R. & Blobel, G. Structure of an integral membrane sterol reductase from Methylomicrobium alcaliphilum . Nature 517, 104–107 (2015). https://doi.org/10.1038/nature13797

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature13797

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing