Structure of an integral membrane sterol reductase from Methylomicrobium alcaliphilum

Abstract

Sterols are essential biological molecules in the majority of life forms. Sterol reductases1 including Delta-14 sterol reductase (C14SR), 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 Delta-14 sterol reductase (maSR1) from the methanotrophic bacterium Methylomicrobium alcaliphilum 20Z, a homolog of human C14SR, LBR, and DHCR7, with the cofactor NADPH. The enzyme contains 10 transmembrane segments (TM). Its catalytic domain comprises the C-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 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.

Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT

“…The dimensions of the cavity in MaMTase and its exposure to the membrane suggest that the substrates of MaMTase are hydrophobic molecules with smaller dimensions than a farnesyl or geranylgeranyl prenyl group. c , Overall structure of the prokaryotic integral membrane sterol reductase MaSR1 (PDB ID: 4QUV) 18 . The orientation is based on superposition of the cofactor binding domain with ICMT, with corresponding colouring.…”

“…The carbon atoms of a bound NADPH cofactor are coloured cyan. A crevice between the magenta- and dark blue-coloured helices may serve as access for lipophilic sterol substrates (arrow) 18 .…”

“…Nevertheless, the binding site for AdoHcy is analogous (Extended Data Fig. 7) and the cofactor-binding domain, that is, the portion spanning from M6 of ICMT through the cap helix, shares a recognizable fold not only with regions of MaMTase but also with regions of a prokaryotic integral membrane sterol reductase that utilizes a nicotinamide adenine dinucleotide phosphate (NADP + ) cofactor 16,18 . Evidently, this domain represents a structural motif for soluble cofactor binding to integral membrane enzymes (Extended Data Fig.…”

Cholesterol-mediated Degradation of 7-Dehydrocholesterol Reductase Switches the Balance from Cholesterol to Vitamin D Synthesis

“…Cholesterol-mediated Degradation of DHCR7 Does Not Involve Putative Cholesterol-binding Sites nor the Insig Retention Protein-There are at least two ways that DHCR7 may respond to increased cholesterol: first, via a predicted sterolsensing domain (32,33), and second, by interacting with putative cholesterol-binding sites (34). In HMGCR, crucial residues were identified in the enzyme's sterol-sensing domain, which must bind to the endoplasmic reticulum retention protein Insig to induce HMGCR degradation (35).…”

“…Two cholesterol-binding sites (tyrosine 280 and aspartic acid 411) were recently predicted through solving the crystal structure of a ⌬(14)-sterol reductase that is a homologue of human DHCR7 found in the methanotrophic bacterium Methylomicrobium alcaliphilum 20Z (34). Using site-directed mutagenesis, we mutated these residues both singly and together to determine their importance in the degradation of DHCR7.…”