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Structure of an integral membrane sterol reductase from Methylomicrobium alcaliphilum.

Li X, Roberti R, Blobel G - Nature (2014)

Bottom Line: Comparison with a soluble steroid 5β-reductase structure 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.

View Article: PubMed Central - PubMed

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

ABSTRACT
Sterols are essential biological molecules in the majority of life forms. Sterol reductases including Δ(14)-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 receptor (LBR), an integral inner nuclear membrane protein, also contains a functional C14SR domain. Here we report the crystal structure of a Δ(14)-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 structure 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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Yeast complementation assay. maSR1 can rescue the growth of a Saccharomyces cerevisiae Delta-14 sterol reductase ERG24 (yeast maSR1 homolog) 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 under in the presence of sub-inhibitory concentrations of cycloheximide (20ng/ml) for 24 to 48 hours (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.
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Figure 3: Yeast complementation assay. maSR1 can rescue the growth of a Saccharomyces cerevisiae Delta-14 sterol reductase ERG24 (yeast maSR1 homolog) 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 under in the presence of sub-inhibitory concentrations of cycloheximide (20ng/ml) for 24 to 48 hours (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.

Mentions: Expression of maSR1 complements the deletion of the Delta-14 sterol reductase gene (ERG24) in yeast, indicating that maSR1 is a bona fide sterol reductase (Extended Data Fig. 3, lanes 1-3). To test whether maSR1 can function in human cholesterol biosynthesis, we preformed the sterol reductase activity assay of maSR1 after expression in human HEK293 cells and employed 5α-cholesta-8,14-dien-3β-ol (C27Δ8,14), a human cholesterol biosynthetic intermediate analogue15-17 of 4,4-dimethylcholesta-8,14-dien-3β-ol (C29Δ8,14, Extended Data Fig. 1) as the substrate (Fig. 1a). This assay has been used for initial identification15 and further investigations16,17 of mammalian sterol reductases. The catalytic efficiency of maSR1 is about 75% of that of human C14SR (Fig. 1b, c). We conclude that maSR1 can function like human C14SR and specifically reduce the double bond of the approximate cholesterol biosynthetic intermediate.


Structure of an integral membrane sterol reductase from Methylomicrobium alcaliphilum.

Li X, Roberti R, Blobel G - Nature (2014)

Yeast complementation assay. maSR1 can rescue the growth of a Saccharomyces cerevisiae Delta-14 sterol reductase ERG24 (yeast maSR1 homolog) 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 under in the presence of sub-inhibitory concentrations of cycloheximide (20ng/ml) for 24 to 48 hours (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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4285568&req=5

Figure 3: Yeast complementation assay. maSR1 can rescue the growth of a Saccharomyces cerevisiae Delta-14 sterol reductase ERG24 (yeast maSR1 homolog) 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 under in the presence of sub-inhibitory concentrations of cycloheximide (20ng/ml) for 24 to 48 hours (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.
Mentions: Expression of maSR1 complements the deletion of the Delta-14 sterol reductase gene (ERG24) in yeast, indicating that maSR1 is a bona fide sterol reductase (Extended Data Fig. 3, lanes 1-3). To test whether maSR1 can function in human cholesterol biosynthesis, we preformed the sterol reductase activity assay of maSR1 after expression in human HEK293 cells and employed 5α-cholesta-8,14-dien-3β-ol (C27Δ8,14), a human cholesterol biosynthetic intermediate analogue15-17 of 4,4-dimethylcholesta-8,14-dien-3β-ol (C29Δ8,14, Extended Data Fig. 1) as the substrate (Fig. 1a). This assay has been used for initial identification15 and further investigations16,17 of mammalian sterol reductases. The catalytic efficiency of maSR1 is about 75% of that of human C14SR (Fig. 1b, c). We conclude that maSR1 can function like human C14SR and specifically reduce the double bond of the approximate cholesterol biosynthetic intermediate.

Bottom Line: Comparison with a soluble steroid 5β-reductase structure 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.

View Article: PubMed Central - PubMed

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

ABSTRACT
Sterols are essential biological molecules in the majority of life forms. Sterol reductases including Δ(14)-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 receptor (LBR), an integral inner nuclear membrane protein, also contains a functional C14SR domain. Here we report the crystal structure of a Δ(14)-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 structure 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.

Show MeSH
Related in: MedlinePlus