Structure of an integral membrane sterol reductase from Methylomicrobium alcaliphilum

Article abstract of DOI:10.1038/nature13797

Sterols are essential biologicalmolecules in themajority 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 cofactorNADPH. The enzymecontains ten transmembrane segments (TM1-10). Its catalytic domain comprises the carboxy-terminal half (containingTM6-10) 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 humanC14SR. Therefore, our structure as a prototype of integral membrane sterol reductases providesmolecular insight intomutations inDHCR7 and LBR for inborn human diseases.

Full text of DOI:10.1038/nature13797

LETTER
doi:10.1038/nature13797
Structure of an integral membrane sterol reductase
from Methylomicrobium alcaliphilum
1
2
1
Xiaochun Li , Rita Roberti & G u¨ nter Blobel
Sterolsareessentialbiologicalmoleculesinthemajorityoflifeforms. enzymes is lacking, and therefore a mechanistic understanding cannot be
1
14
Sterolreductases includingD -sterolreductase(C14SR, alsoknown developed.
as TM7SF2), 7-dehydrocholesterol reductase (DHCR7) and 24-
To gain more insight into sterol reductases, we set out to determine the
dehydrocholesterol reductase (DHCR24) reduce specific carbon– crystal structure of one of its family members. In screening for crystal-
carbon double bonds of the sterol moiety using a reducing cofactor forming representatives of integral membrane sterol reductases, we
2
during sterol biosynthesis. Lamin B receptor (LBR), an integral found a homologue from the methanotrophic bacterium Methylomi-
inner nuclear membrane protein, also contains a functional C14SR crobium alcaliphilum 20Z, MaSR1, which shares 38–45% sequence
14
domain. Here we report the crystal structure of a D -sterol reduc- identity and 51–62% similarity to human C14SR, DHCR7 and the C-
tase (MaSR1) from the methanotrophic bacterium Methylomicro- terminal portion of LBR (Extended Data Fig. 2). Methylomicrobium
bium alcaliphilum 20Z (a homologue of human C14SR, LBR and alcaliphilum 20Z is an aerobic methanotroph, the cell membrane of
1
4
DHCR7)withthecofactor NADPH. Theenzymecontainstentrans- which contains significant levels of sterols and hopanoids .
1
4
membrane segments (TM1–10). Its catalytic domain comprises the
Expression of MaSR1 complements the deletion of the D -sterol re-
carboxy-terminal half(containingTM6–10) and envelopstwo inter- ductase gene (ERG24) in yeast, indicating thatMaSR1isabona fide sterol
connected pockets, one of which faces the cytoplasm and houses reductase(ExtendedDataFig. 3, lanes1–3). Totestwhether MaSR1 can
NADPH, while the other oneisaccessible fromthe lipid bilayer. Com- function inhumancholesterolbiosynthesis, weperformed thesterolre-
3
parison with a soluble steroid 5b-reductase structure suggests that ductaseactivityassayofMaSR1afterexpressioninhumanHEK293cells
8,14
the reducing end of NADPH meets the sterol substrate at the junc- and employed 5a-cholesta-8,14-dien-3b-ol (C27D ), a human cho-
1
5–17
ture of the two pockets. A sterol reductase activity assay proves that lesterol biosynthetic intermediate analogue
of 4,4-dimethylcholesta-
MaSR1 can reduce the double bond of a cholesterol biosynthetic in- 8,14-dien-3b-ol (C29D , Extended Data Fig. 1), asthe substrate (Fig. 1a).
8,14
1
5
termediate, demonstrating functional conservation to human C14SR. This assay has been used for initial identification and further invest-
1
6,17
Therefore, our structure as a prototype of integral membrane sterol igation
reductasesprovidesmolecularinsight intomutationsinDHCR7and MaSR1 is about 75% of that of human C14SR (Fig. 1b, c). We conclude
LBR for inborn human diseases.
that MaSR1canfunctionlike humanC14SR and specificallyreducethe
Sterols, amphipathic molecules, are widespread in animals, plants, double bond of the approximate cholesterol biosynthetic intermediate.
of mammalian sterol reductases. The catalytic efficiency of
4
fungi and some prokaryotes and a large variety exist , including ergo-
WecrystallizedMaSR1inspacegroupP1 withNADPH. Thediffrac-
sterol, hopanoids, phytosteroland cholesterol. Themost abundant sterol tion of the crystal is anisotropic (Methods and Extended Data Fig. 4).
in animals is cholesterol, which not only has a vital role in the main- The structure was determined by selenium-based single-wavelength
˚
tenance of membrane strength and permeability, but also serves as a anomalousdispersionandrefined at2.74 Aresolution(Extended Data
5,6
precursor for the biosynthesis of steroid hormones . Endogenous bio- Tables 1 and 2). Introduction of additional selenium anomalous scat-
synthesis is the major source of sterols, and the biosynthetic pathway terers by selective mutation and preparation of platinum derivatives con-
from water-soluble small metabolites via intermediates of increasing firmed that the atomic model was correct (Extended Data Fig. 5 and
complexity up to water-insoluble sterols encompasses numerous distinct Extended Data Table 3).
enzymes , manyofwhichcontain multipletransmembrane segments.
TwoMaSR1molecules(rotatedby180u)packintoacrystallographic
Todate,nostructureoftheintegralmembraneenzymesinvolvingsterol dimer that forms the asymmetric unit. The dimensions of the MaSR1
1
,7
1
˚
biosynthesis has been determined. Sterol reductases , integral mem- monomer are 50 3 45 3 58 A. The enzyme contains ten transmem-
brane enzymes including C14SR, DHCR7 and DHCR24, can reduce brane helices (TM1–10). On the basis of the ‘positive inside rule’ for
1
8
specific carbon–carbon double bonds of the sterol moiety using a re- membrane proteins , we assigned the amino and carboxy termini to
ducing cofactor at distinct steps in sterol and cholesterol biosynthesis facethecytoplasm, consistentwiththe biochemicallydeterminedtopo-
(
Extended Data Fig. 1).
Curiously, the multifunctional lamin B receptor (LBR), located in parallel b-sheet regions (b1–4) interspersed in the cytoplasm-exposed
the inner nuclear envelope membrane, also contains a domain in its C- loops as well as two short a-helices, designated a1 and a2 (Fig. 2a and
logy of the yeast homologue Erg24 (ref. 19). There are two short anti-
2
8
terminal portion that is highly homologous to human sterol reductase
ExtendedDataFig. 2).Residues1–23and162–176(partoftheloopbet-
(
Extended Data Fig. 2). Indeed, complementary DNA of human LBR ween TM4 and TM5) are not visible in the electron density map and
complements reductase gene (ERG24) deletion in yeast, supporting the therefore presumed to be disordered. The binding pocket for NADPH
9
idea that LBR can substitute for sterol reductase activity . Mutations in was localizedtothe C-terminaldomain(TM6–10)and acavitywithan
1
LBR and DHCR7 leadtovarioushumangeneticdiseases (Pelger–Hu e¨ t unidentified ligand density facing the lipid bilayer is surrounded by
1
0
11
anomaly (PHA) and Greenberg skeletal dysplasia (also known as TM7 and TM10 (Fig. 2b, c).
hydrops-ectopic calcification-moth-eaten skeletal dysplasia, HEM) related
to LBR; and Smith–Lemli–Opitz syndrome (SLOS)related to DHCR7). NADPH molecule could be clearly identified (Extended Data Fig. 6a, b).
However, structuralknowledgeof theseimportant membrane-embedded Thenicotinamideringisthehydrogendonorinthetransientinteraction
Except for the nicotinamide-ribose moiety, the remainder of the
12,13
1
2
Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10065, USA. Department of Experimental Medicine, University of Perugia, Perugia 06132,
Italy.
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RESEARCH LETTER
a
c
5
4
0
0
Untransfected
cells
3
MaSR1
C14SR
30
1
20
2
4
C27Δ8,14
C27Δ8
10
15
b
0
0
.4
.3
16
17
18
19
20
21
22
Time (min)
30
MaSR1
3
2
0
0
0
.2
.1
0
1
2
4
10
15
16
17
18
19
20
21
22
Time (min)
3
6
5
4
0
0
0
Flag–MaSR1
1
2
0.35
d
30
4
20
10
0
0
0
.25
.15
1
5
16
17
18
19
20
21
22
Time (min)
4
3
0
0
Human C14SR
2
3
1
2
0
4
.05
0
1
0
15
16
17
18
19
20
21
22
Time (min)
1
. m/z 372, 5α-cholestane (internal standard)
2
3
4
. m/z 458, endogenous cholesterol
. m/z 456, C27Δ
. m/z 458, C27Δ
WB:
anti-Flag
8,14
8
1
4
Figure 1
|
D -reductase activity of MaSR1. a, Reaction catalysed by
assay. Peak 1, mass to charge ratio (m/z) 372, 5a-cholestane (an internal
standard); peak 2, m/z 458, endogenous cholesterol; peak 3, m/z 456, C27D
peak 4, m/z 458, C27D . The reduced products (peak 4) were detected in
8
,14
transfected human C14SR or MaSR1 in which 5a-cholesta-8,14-dien-3b-ol
C27D ) is converted to 5a-cholesta-8-en-3b-ol (C27D ). b, The average
;
8,14
8
8
(
catalytic efficiency in two different experiments (calculated as the integrated
gas chromatography–mass spectrometry (GC–MS) chromatogram peak area
ratio C27D /(C27D 1 C27D )) of untransfected HEK293 cells and cells
MaSR1, Flag–MaSR1 and C14SR but little in untransfected cells. d, The
catalytic efficiency of Flag–MaSR1 mutants. All mutants expressed by anti-Flag
western blot (WB) detection. The plotted values in the grey bars are the
8
8,14
8
transfected with MaSR1, Flag–MaSR1 and human C14SR is 3.6%, 29.7%, 28.9% average of two different experiments. The thin black lines show the spread
and 38.4%, respectively. The thin black lines show the spread between the two between the measurements.
14
individual measurements c, GC–MS chromatograms of D -sterol reductase
withthesubstrate.Inourstructure, theabsenceofa sterolsubstratepro- double bond reduction. Analogous to the catalytic pocket of soluble
3
bably fails to coordinate the nicotinamide ring and hence causes it to steroid 5b-reductase (AKR1D1, Protein Data Bank (PDB) accession
be disordered. By contrast, the other half of the NADPH molecule is number 3COT), bound to progesterone (Fig. 3b), the lipid-bilayer-
well defined in the electron density and stabilized by a hydrogen bond facingcavity ofMaSR1 isthe likely candidateforthe sterolbindingpocket
from the centrally located Tyr 414 of a2 (Fig. 3a). The remainder of the (Fig. 3c). Notably, the sterol binding pockets of both enzymes contain
NADPH pocket is lined with residues from TM9 and TM10 as well as a ‘signature’ motif forming triangular hydrogen bonds that coordinate
two residues from TM8 (Fig. 3a). The NADPH pocket extends further the b3 hydroxyl of either sterol or steroid; for MaSR1, this signature
around TM10 providing enough space to house the nicotinamide-ribose motif includes Tyr 241 bonded to Asp 363 (Fig. 3c) and for steroid 5b-
moiety of NADPH (Extended Data Fig. 6c). We mutated several residues reductase, Tyr 58 bonded to Glu 120 (Fig. 3b). The distance between
˚
intheNADPHbinding pocketofMaSR1 (Extended Data Fig. 2). These Tyr 58 and Glu 120 (4.1 A) in steroid 5b-reductase is similar to that of
˚
mutations led to the loss of catalytic activity, as judged by the human Tyr 241 and Asp 363 (3.9 A) in MaSR1. The MaSR1double mutant Y241F/
1
4
14
D -sterol reductase activity assay (Fig. 1d) and by the Erg24 comple- D363A loses sterol reductase function in the human D -sterol reduc-
mentation assay in yeast (Extended Data Fig. 3, lanes 4, 6 and 7).
tase activity assay (Fig. 1d) and the yeast Erg24 complementation assay
The working principle of reductases is to bring the nicotinamide of (Extended Data Fig. 3, lane 5). For the putative sterol binding pocket,
NADPHintocloseproximitytothesubstrate, leadingtocarbon–carbon extensive hydrophobic contacts between the highly conserved Trp 274
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Figure 2
|
The molecular
a
b
architecture of MaSR1. a, Overall
structure of MaSR1 viewed parallel
to the membrane. The ten
5
5
α1
transmembrane segments are
divided into an N-terminal half
(TM1–5 and a1, grey) and a
C-terminal half (TM6–10 and a2,
yellow). The two black lines show the
approximate location of the lipid
bilayer. b, MaSR1 structure cartoon
with NADPH shown by stick
8
8
α1
2
2
7
9
7
NADPH
3
9
1
1
3
3
5 Å
10
4
6
4
1
0
o c
representation. 2F – F map for an
6
unidentified molecule (blue mesh)
and simulated annealing (SA)-omit
map (F 2 F densities) for NADPH
(magenta mesh) both contoured
at 2s. c, The cavity is indicated by
an orange circle in an electrostatic
surface representation. The right
and left panels represent two
perpendicular views.
Cytoplasm
α2
α2
Unidentified
molecule
o
c
c
Acidic
Basic
90°
Cavity
Cytoplasm
and Tyr 387 residues enforce the interaction of TM7 and TM10 (Ex- purification), it may represent a substrate for the putative sterol bind-
tendedDataFig. 6d). Notably, ineachmonomerofthecrystallographic ing pocket. We modelled that two binding pockets bring the reducing
dimer there is the extra electron density of an unidentified molecule end of NADPH into close proximity to the sterol (steroid) carbon–
in front of the cavity (Figs 2b and 3c). Although the molecular identity carbon double bondto be reduced (Fig. 3d), similar to the aldo-keto re-
2
0
of this density could not be unambiguously determined (maybe an ductase (AKR) family of enzymes involved in human steroidgenesis
endogenous molecule from Escherichia coli or the detergent used for (for example, AKR1C3 and AKR1C2).
Owing to the high sequence homology with human LBR and human
DHCR7, we generated structural models based on MaSR1 to highlight
disease-relatedmutations(Fig. 4). ThePHA/HEM-relatedmutationsof
LBRand theSLOS-related mutationsofDHCR7couldbealmostentirely
mapped to the sterol reductase catalytic domain affecting the cofactor
binding or sterol entry/binding sites. The similarities in pathogenesis
between PHA/HEM and SLOS could therefore arise from a defect in
sterol reduction.
a
b
N359
W352
Y360
Y58
E120
K319
Y414
NADP+
D399
Intriguingly, substrate recognition for sterol reductases is not very
8
,14
R323
Progesterone
specific.MaSR1couldreducethedoublebondofbothC27D (Fig.1b)
and of the yeast sterol substrate ergosta-8,14-dien-ol (Extended Data
Fig. 3). This is consistent with previous observations for LBR: it can
1
0
α2
Y407
Steroid 5β-reductase
8
,14
2/2
K406
reduce C27D (ref. 16); complement C14SR function in C14SR
21
d
2/2
(
alsoknownasTm7sf2 )mice ;andalsoreducedifferentyeaststerol
9
c
substrates . Finally, our structure also provides insight into the func-
tionofLBR. A DALIsearchforstructuralhomologuesofMaSR1 shows
6
8
D363
6
9
Y241
Y241
Figure 3
|
NADPH, putative sterol binding pockets and homology
modellingwithsteroid 5b-reductase. a, Close-upview oftheNADPH binding
pocket. NADPH is shown in stick representation with the phosphates in red.
The interactions between NADPH and MaSR1 are indicated by dashed
lines. b, Close-up view of the active pockets of steroid 5b-reductase (PDB
accession number 3COT). Tyr58 and Glu120 clamp the b3 carbonyl oxygen of
progesterone. c, The putative sterol binding site is accessible from the lipid
8
D363
9
7
7
o c
bilayer. An unidentified ligand density is shown with 2F – F map (blue mesh)
10
at a 2.5s. d, Modelling of MaSR1 putative sterol binding pocket. On the basis
of the active sites of steroid 5b-reductase, the missing nicotinamide-ribose
Unidentified
molecule
Docked C29Δ8,14
Docked nicotinamide ribose
8
,14
moiety (purple) of NADPH was docked into the NADPH pocket and C29D
light green) modelled into the pocket of the MaSR1 structure.
(
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RESEARCH LETTER
a
Figure 4
|
Models of human LBR
b
and human DHCR7. a, Human
LBR model and the distribution of
PHA and HEM mutations in green
and purple, respectively. b, Human
DHCR7 model and the distribution
of SLOS mutations in blue. The
cofactor NADPH (grey) is shown in
stick representation.
5
5
α1
α1
7
8
8
7
9
1
2
10
9
6
10
6
3
1
3
4
4
2
α2
α2
Human LBR C14SR domain model
Human DHCR7 model
SLOS mutation
HEM mutation
PHA mutation
PHA and HEM mutation
no similar entry for the entire MaSR1 structure. However, it identified
the membrane-embedded isoprenylcysteine carboxyl methyltransfer-
methanotroph Methylomicrobium alcaliphilum 20Z. Microbiology 80, 595–605
2011).
(
22
15. Paik, Y. K., Trzaskos, J. M., Shafiee, A. & Gaylor, J. L. Microsomal enzymes of
cholesterol biosynthesis from lanosterol. Characterization, solubilization, and
ase (ICMT, PDB accession number 4A2N) as the closest entry for the
TM6–10 segments of MaSR1 (Extended Data Fig. 7). The function of
ICMT, which recognizes and then carboxymethylates the farnesylated
cysteine of its substrate, points towards a similar role of the C14SR
domain ofLBR, which may recognizethe farnesylatedcysteine ofeither
8,14
partialpurification of NADPH-dependent D
-steroid 14-reductase. J. Biol. Chem.
2
59, 13413–13423 (1984).
1
6. Roberti, R. et al. Cloning and expression of sterol D14-reductase from bovine liver.
Eur. J. Biochem. 269, 283–290 (2002).
1
7. Bennati, A. M. et al. Sterol dependent regulation of human TM7SF2 gene
23
14
prelamin A or lamin B as the ligand .
expression: role of the encoded 3b-hydroxysterol D -reductase in human
cholesterol biosynthesis. Biochim. Biophys. Acta 1761, 677–685 (2006).
Online Content Methods, along with any additional Extended Data display items
andSource Data, are available in the onlineversion of the paper; references unique
to these sections appear only in the online paper.
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).
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).
Received 15 June; accepted 26 August 2014.
Published online 12 October 2014.
2
2
2
2
0. Miller, W. L. & Auchus, R. J. The molecular biology, biochemistry, and
physiology of human steroidogenesis and its disorders. Endocr. Rev. 32, 81–151
(
2011).
1. Bennati, A. M. et al. Disruption of the gene encoding 3b-hydroxysterol D
reductase (Tm7sf2) in mice does not impair cholesterol biosynthesis. FEBS J. 275,
034–5047 (2008).
1.
2.
3.
Porter, F. D. & Herman, G. E. Malformation syndromes caused by disorders of
cholesterol synthesis. J. Lipid Res. 52, 6–34 (2011).
14
-
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).
Di Costanzo, L., Drury, J. E., Penning, T. M. & Christianson, D. W. Crystal structure of
5
2. Yang, J. et al. Mechanism of isoprenylcysteine carboxyl methylation from the
crystal structure of the integral membrane methyltransferase ICMT. Mol. Cell 44,
4
human liver D -3-ketosteroid 5b-reductase (AKR1D1) and implications for
9
97–1004 (2011).
substrate binding and catalysis. J. Biol. Chem. 283, 16830–16839 (2008).
Fahy, E. et al. A comprehensive classification system for lipids. J. Lipid Res. 46,
3. 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,
4.
5.
6.
7.
8.
9.
8
39–862 (2005).
Miller, W. L. & Bose, H. S. Early steps in steroidogenesis: intracellular cholesterol
trafficking. J. Lipid Res. 52, 2111–2135 (2011).
1
019–1029 (1994).
Miller, W. L. Steroid hormone synthesis in mitochondria. Mol. Cell. Endocrinol. 379,
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
6
2–73 (2013).
Goldstein, J. L., DeBose-Boyd, R. A. & Brown, M. S. Protein sensors for membrane
sterols. Cell 124, 35–46 (2006).
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).
Silve, S., Dupuy, P. H., Ferrara, P. & Loison, G. Human lamin B receptor exhibits
sterol C14-reductase activity in Saccharomyces cerevisiae. Biochim. Biophys. Acta
sterol reductase assays, and E. Coutavas, E. Debler and H. Shi for constructive
8,14
comments in manuscript preparation. The C27D
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.
1
392, 233–244 (1998).
1
0. Hoffmann, K. et al. Mutations in the gene encoding the lamin B receptor produce
an altered nuclear morphology in granulocytes (Pelger–Hu e¨ t anomaly). Nature
Genet. 31, 410–414 (2002).
Author 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.
1
1. Waterham, H. R. et al. Autosomal recessive HEM/Greenberg skeletal dysplasia is
14
caused by 3b-hydroxysterol D -reductase deficiency due to mutations in the
lamin B receptor gene. Am. J. Hum. Genet. 72, 1013–1017 (2003).
2. Porter, F. D. Smith–Lemli–Opitz syndrome: pathogenesis, diagnosis and
management. Eur. J. Hum. Genet. 16, 535–541 (2008).
1
1
1
Author Information Coordinates and structure factors for MaSR1 are deposited in the
Protein Data Bank under accession code 4QUV. Reprints and permissions information
is available at www.nature.com/reprints. The authors declare no competing financial
interests. Readers are welcome to comment on the online version of the paper.
Correspondence and requests for materials should be addressed to X.L.
(xli05@rockefeller.edu) or G.B. (blobel@rockefeller.edu).
3. Herman, G. E. & Kratz, L. Disorders of sterol synthesis: beyond Smith–Lemli–Opitz
syndrome. Am. J. Med. Genet. C 160, 301–321 (2012).
4. Shchukin, V. N., Khmelenina, V. N., Eshinimaev, B., Suzina, N. E. & Trotsenko, Yu. A.
Primary characterization of dominant cell surface proteins of halotolerant
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LETTER RESEARCH
were subcloned into pEGFP-N1 (Clontech) without the EGFP tag. HEK293 cells
METHODS
were grown in a 5% CO incubator at 37 uC in DMEM supplemented with 10%
2
Protein expression and purification. We expressed a homologue of eukaryotic
sterol reductases from the methanotrophic bacterium Methylomicrobium alcali-
philum20Z(MaSR1, NCBI GI number: 503913803). Its cDNAwas clonedinto pET-
21 21
fetalbovine serum, 2 mM L-glutamine, 100 U ml penicillinand 0.1 mg ml strep-
tomycin. Cells were cultured in 10-cm Petri dishes to 80% confluence and trans-
fectedwith the plasmids, usingLipofectamine2000(LifeTechnologies). After48 h,
transfected cells were recovered and the washed cell pellet was resuspended in PBS
containing complete protease inhibitor cocktail (Sigma-Aldrich). Cells were lysed
by sonication three times for 10 s on ice. After low-speed centrifugation, the result-
ingsupernatantwasultracentrifugedtosedimentamembranefraction. Theisolated
21b (Novagen) with an N-terminal 8-His tag and expressed in E. coli C43(DE3)
(
Lucigen). The transformed cells were grown to an optical density of 1.0 at OD600
andinducedwith0.2 mMisopropyl-b-D-thiogalactopyranoside(IPTG).Cellswere
disrupted using a French press with two passes at 15,000 p.s.i., in a buffer contain-
ing 25 mM Tris-Cl, pH 8.0, and 150 mM NaCl (buffer A). After low-speed centri-
fugation, the resulting supernatant was centrifuged at high speed to sediment a
4
membranefraction wasresuspended in 10 mMKPO /0.5 mMEDTA (pH7.4) and
membrane fraction, which then was incubated in buffer A with 2% (w/v) N-dodecyl- frozen in aliquots for further analyses. Protein concentration was determined by
b-D-maltopyranoside (DDM, Anatrace) for 1 h at 4 uC. The lysate was centrifuged Bradfordmethod, usingbovineserumalbuminasastandard.Proteinsofthemem-
21
again and the supernatant was loaded onto Ni -NTA affinity column (Qiagen). brane fraction were separated by SDS–PAGE, blotted on PVDF and probed with
After washing twice, the protein was elutedwith25mMTris-Cl,pH8.0,150mMNaCl, mousemonoclonal anti-FlagM2(Sigma-Aldrich) and peroxidase-conjugatedgoat
3
00mM imidazole, and 0.1% DDM, and concentratedby centricon forsubsequent anti-mouse (Santa Cruz). The protein was detected using Super Signal West Pico
gel filtration (Superdex-200, GE Healthcare) in buffer A with 0.4% (w/v) N-nonyl- Chemiluminescent Substrate (Pierce).
14
b-D-glucopyranoside (b-NG, Anatrace). The peak fraction was collected for crys-
D -reductase activity was assayed in the membrane fractions obtained from trans-
8
,14
tallization. All mutations were generated using two-step PCR. Selenomethionine fected cells (0.25 mg protein per assay) using 5a-cholesta-8,14-dien-3b-ol (C27D
(
[
)
1
5–17
SeMet)-labelled protein was purified similarly with the exception that 1 mM Tris asasubstrate . Aftertheadditionof 5a-cholestane(5mg)asaninternalstandard,
2-carboxyethyl] phosphine(TCEP)was included during the purificationprocess. sterolswere extracted withpetroleum ether, desiccated undernitrogen stream and
Crystallization. Beforecrystallization, theproteinsolutionwas incubatedwith2 mM converted to trimethylsilyl derivatives using N,O-Bis (trimethylsilyl) trifluoroace-
NADPH(Sigma-Aldrich). Crystalswere grownat20 uCbythehanging-dropvapour- tamide (BSTFA) and pyridine (1:1, v/v). Gas chromatography–mass spectrometry
diffusion method. The crystals appeared after 5 days in the well buffer containing (GC–MS) analysis was performed in multiple ion detection mode using a Varian
0
4
.1 M Tris-Cl pH 7.0, 0.2 M NH Ac, 30% (v/v) pentaerythritol ethoxylate (15/4 GC-MS 2000 apparatus with a Varian CP-Sil8 CB low bleed capillary column. The
EO/OH). DDMwasaddedintocrystallizationbufferat1%(v/v)finalconcentration
to improve diffraction. SeMet-labelled protein was crystallized in the same buffer
supplementedwith6 mMdithiothreitol(DTT).Platinumderivativeswereobtained
trimethylsilylation of sterol products yields a molecular mass increase of 72 Da. Sterol
retention times were: 15.31 min, 5a-cholestane (m/z 372); 19.90 min, cholesterol
8
,14
(
5
m/z 458); 20.16 min, 5a-cholesta-8,14-dien-3b-ol (C27D , m/z 456); 20.34 min,
21
by soaking native crystals for 12 h in mother liquor plus 10 mg ml
K
2
Pt(NO2)
4
.
8
14
a-cholesta-8-en-3b-ol (C27D , m/z 458). D -reductase catalytic efficiency is cal-
All crystals were directly flash-frozen in a cold nitrogen stream at 100 K.
8
8,14
8
8
culated as the peak area ratio C27D /(C27D 1 C27D ). The C27D sterol was
Data collection andstructure determination. Thedata were collected at National
SynchrotronLightSource(NSLS) beamline X29. Alldatasets were processedusing
HKL2000 (ref. 24). Owing to the anisotropic diffraction properties, the outlier re-
flections were rejected based on extreme-value Wilson statistics using the program
XTRIAGE in the PHENIX package . The anomalous signal in the SeMet-derivative
data was further magnified with the local-scaling algorithm using the program
undetectable at zero incubation time.
24. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in
oscillation mode. Methods Enzymol. 276, 307–326 (1997).
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).
25
26
2
7
28
SOLVE . Then, theseleniumsiteswere determinedusingtheprogramSHELXD .
The identified sites were refined and the initial phases were generated in the pro-
2
6. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for
macromolecular structure solution. Acta Crystallogr. D 66, 213–221
(2010).
29
gram PHASER with the single-wavelength anomalous dispersion experimental phas-
ing module. Twofold NCS averaging along with solvent flattening and histogram
matching was performed using DM . The initial model was built in COOT man-
27. Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta
Crystallogr. D 55, 849–861 (1999).
28. Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta
Crystallogr. D 58, 1772–1779 (2002).
30
31
26
ually. Thestructurewas refined withphenix.refine . Modelvalidationwas performed
32
with MolProbity . Introduction of additional selenium anomalous scatterers by
selective mutation and preparation of platinum derivatives confirmed the correct-
ness of the atomic model.
2
9. McCoy, A. J. etal.Phasercrystallographicsoftware. J. Appl. Crystallogr. 40, 658–674
2007).
(
3
0. 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).
The homology models of human LBR and human DHCR7 were generated by
33
the program MODELLER on the basis of the structure of MaSR1 in which the
N-terminal regions (1–200 of LBR and 1–58 of DHCR7) were excluded because of
lowsequenceconservation(ExtendedDataFig. 2). Allfiguresweregeneratedusing
the program PyMOL (http://www.pymol.org/).
3
3
3
1. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta
Crystallogr. D 60, 2126–2132 (2004).
2. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular
crystallography. Acta Crystallogr. D 66, 12–21 (2010).
3. Fiser, A. & Sali, A. Modeller: generation and refinement of homology-based protein
structure models. Methods Enzymol. 374, 461–491 (2003).
Yeast reductase complementation assay. Wild-typeandmutantMaSR1and ScErg24
were subcloned into the URA3 shuttle vector pCM190 (Euroscarf, Germany). The
plasmids were introduced in Erg24-deficient Saccharomycescerevisiaestrain Y11164
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,
2
(
Euroscarf) by electroporation. A single colony was picked from a URA selective
2
plate. For the yeast rescue assay, the yeast was grown on URA plates either in
the absence or the presence of sub-inhibitory concentrations of cycloheximide
(
4
673–4680 (1994).
2
1
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).
36. Holm, L. & Rosenstrom, P. Dali server: conservation mapping in 3D. Nucleic Acids
Res. 38, W545–W549 (2010).
20 ng ml ) at 30 uC for 24 to 48 h. The results were confirmed by three indepen-
dent experiments with different colonies.
D -reductaseassay. The cDNAencoding human D -sterol reductase (C14SR) was
14
14
subclonedintopCMV-SPORT6(OpenBiosystems).Wild-typeandmutantMaSR1
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RESEARCH LETTER
1
Extended Data Figure 1
|
Cholesterol biosynthesis pathway and sterol
which are catalysed by C14SR, LBR and DHCR7 (MaSR1 homologues, in red).
C14SR, LBR and DHCR7 are homologues of NADPH-dependent reductases
reductase family. Acetyl-CoA is the precursor for cholesterol biosynthesis.
After several reactions, the intermediate lanosterol is synthesized. Conversion that catalyse the reduction of the sterol double bonds indicated in the
of lanosterol to cholesterol (Bloch pathway) involves many reactions, some of green circles.
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2014 Macmillan Publishers Limited. All rights reserved

LETTER RESEARCH
Extended Data Figure 2
|
Sequence alignment of MaSR1 with human
proteins). Putative cholesterol hydroxyl group binding sites are highlighted
in red, NADPH binding sites arehighlightedin cyan. Humandisease mutations
are also highlighted by different symbols. Sequence alignment was carried
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
3
4
out using ClustalW .
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RESEARCH LETTER
2
Yeast complementation assay. MaSR1 can rescue concentrations of cycloheximide (20 ng ml ) for 24 to 48 h (lower panel).
1
Extended Data Figure 3
|
14
the growth of a Saccharomyces cerevisiae D -sterol reductase Erg24 (yeast
MaSR1 homologue) deletion strain (DErg24). DErg24 yeast expressing wild-
type MaSR1, ScErg24 and mutated MaSR1 from a URA3 shuttle vector can
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 yeast . Results are representative of
3
5
2
grow under URA selection (upper panel). Growth of yeast expressing MaSR1, three independent experiments.
ScErg24 and various mutated MaSR1 versions in the presence of sub-inhibitory
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LETTER RESEARCH
Extended Data Figure 4
crystals with various resolution rings indicated by the circles.
|
MaSR1 crystal and X-ray diffraction image. a, Photograph of MaSR1 crystal. b, A representative X-ray diffraction image of MaSR1
©
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RESEARCH LETTER
Extended Data Figure 5
|
Anomalous difference Fourier electron density.
anomalous difference signals, right panel shows mutated SeMet anomalous
a, Overview of the anomalous difference Fourier map for selenium atoms in an difference signals at 3s (blue mesh). d, A view of the anomalous difference
asymmetric unit. The electron density is contoured at 4.5s (purple mesh). Fourier map for platinum atoms in an asymmetric unit. The electron density is
Two molecules (MolA, molecule A; MolB, molecule B) were observed in each contoured at 3s (purple mesh). There are four platinum atoms binding to
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 3s (purple mesh). c, Examination of the atomic model in TM8 by
selenium anomalous difference signals. Left panel shows wild-type SeMet
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 2F 2 F electron density, contoured at 2s, in one
o c
asymmetric unit.
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LETTER RESEARCH
Extended Data Figure 6
|
NADPH binding pocket and interaction between panel (same orientation as Fig. 2a), rotated by 180u. c, The rebuilt missing
Trp274 and Tyr 387 of MaSR1. a, The structure of NADPH with the missing moiety (purple) of NADPH in MaSR1. d, The surface representation shows
moiety in the MaSR1 structure indicated in the black circles. b, Overview of Trp274 (orange) and Tyr387 (blue) located in the back of the sterol
the NADPH-bound MaSR1. SA-omit map (F 2 F densities, magenta mesh) binding pocket. 2F – F map for an unidentified ligand (blue mesh)
for NADPH contoured at 2s. The right panel is an enlargement of the left contoured at 2s.
o
c
o
c
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RESEARCH LETTER
Extended Data Figure 7
structure. a, A comparison of MaSR1 (grey and yellow) and ICMT (cyan)
structure with S-adenosyl-L-homocysteine (SAH) bound (PDB accession
|
Comparison of MaSR1 structure with ICMT
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
2
2
36
number 4A2N). DALI search shows the closest entry (Z-score of 7.5) to
MaSR1 is the structure of ICMT, consisting of 5 transmembrane helices, which coordinating tyrosine residues in the cofactor pockets of these two enzymes.
˚
had 193 Ca atoms aligned to MaSR1 (TM6–10 and a2) with r.m.s.d. of 2.8A.
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LETTER RESEARCH
Extended Data Table 1
|
Data collection and refinement statistics
Values in parentheses are for the highest resolution shell. Rfree was calculated with 5% of the reflections selected in the thin shell. ASU, asymmetric unit.
See Extended Data Table 2 of the native data completeness of each shell.
*
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Extended Data Table 2
|
Native data completeness of each shell
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LETTER RESEARCH
Extended Data Table 3
|
Data collection statistics for the MaSR1 mutants I151M, L304M and Pt-derivatives
Values in parentheses are for the highest resolution shell.
©
2014 Macmillan Publishers Limited. All rights reserved