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Structure of an integral membrane sterol reductase from Methylomicrobium alcaliphilum


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.

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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.

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.


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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.

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Authors and Affiliations



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.

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

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Li, X., Roberti, R. & Blobel, G. Structure of an integral membrane sterol reductase from Methylomicrobium alcaliphilum . Nature 517, 104–107 (2015).

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