Biochemical and Biophysical Research Communications 529 (2020) 1005e1010

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

The crystal structure of ORP3 reveals the conservative PI4P binding

pattern
Xue Dong 1, Zhiming Wang 1, Sheng Ye, Rongguang Zhang*
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: Oxysterol-binding protein (OSBP) and its related protein (ORP) constitute a conserved family of lipid
Received 18 June 2020 transfer proteins (LTPs). ORPs have been implicated as intracellular lipid exchanger and sensor in recent
Accepted 18 June 2020 years, which regulate the lipid homeostasis and signal pathway. OSBP-related protein 3 plays key role in
controlling cell adhesion and migration and could be developed as the drug target for cancer therapy.
Here, we report the crystal structures of human ORP3 ORD to 2.1 Å and ORD-PI4P complex to 3.2 Å. The
Keywords:
binding assay in vitro confirms the ORP3 has the capability of PI4P binding. This study further verifies
Oxysterol-binding protein
that the PI4P is the common ligand of all ORPs and ORPs should be the lipid exchanger in membrane
ORP3
OSBP-Related domain
contact sites(MCS).
PI4P © 2020 Elsevier Inc. All rights reserved.

1. Introduction also phosphatidylinositol-4-phosphate (PI4P) and phosphati-

The unevenly distribution of lipid between cell membrane
define the organelle identity, which base on the lipid movement
across membrane and between organelles [1,2]. The intracellular
transport of lipid via three major mechanisms, including flip-flop,
vesicular/tubular transport and non-vesicular transport. The non-
vesicular transport play key roles in lipid fluxes between indi-
rectly connected organelles, which could be mediated by either
spontaneous lipid diffusion or by lipid-transfer proteins(LTPs)
[3e5]. Based on the lipid binding domains, the LTPs could be
classified in six distance families (SEC14, PITP, START, GLTP, SCP-2
and OSBP/ORP), which are widely distributed in eukaryotic
kingdom [6].
Oxysterol-binding protein(OSBP) and OSBP-related protei-
n(ORPs) constitute a evolutionarily conserved family in eukaryote,
which are characterized by the C-terminal OSBP-related domai-
n(ORD). Due to the initial discovery of binding oxysterol, the pro-
totype member is named as OSBP, while they have been shown to
accommodate a variety of oxysterols, cholesterol, ergosterol, but
Abbreviations: OSBP, Oxysterol-binding protein; ORP, OSBP related protein; LTP,
lipid transfer protein; ORD, OSBP-related domain; PI4P, phosphatidylinositol-4-
phosphate.
* Corresponding author.
E-mail addresses: dongxuedejia@hotmail.com (X. Dong), rzhang@ibp.ac.cn
(R. Zhang).
1
These authors contributed equally to this work.
dylserine(PS) in subsequent research [7e10]. Recent researches
reveal some ORPs are lipid exchangers, which exchange phos-
phoinositide for the second lipid such as sterol or phosphati-
dylserine, and they explore the differences in the phosphoinositide
concentration between two membranes to drive the exchange
[11e14].
OSBP-related protein 3 interacts with R-Ras and play roles in
controlling cell adhesion and migration [15,16]. The phosphoryla-
tion regulates its interaction with ER membrane anchors-VAPs, the
ORP3-VAP complex simulate the R-Ras signaling and regulate the
b-integrin activity [17]. The abnormal high expression of ORP3 have
been reported in specific types of leukemia and solid tumors
[18e20], indicating ORP3 may control cell signaling and adhesion
that facilitates the cancer cell malignant growth. Nowadays, several
structures of ORPs have been confirmed including Osh3 PH domain
and ORD, Osh4 ORD, Osh6 ORD and ORP1-ORD, however our un-
derstanding of human ORP structure is very limited. In order to
verify the mechanism of PI4P-coupled lipid transport in ORPs, more
evidence should be collected. In our study, the structures of ORD of
ORP3 were determined, one of the long ORP members in human.
We confirmed ORP3 could bind the PI4P via its ORD by binding
assay in vitro and structural information. The study provide the
farther evidence for the conserved mode of phosphoinositide
binding in all ORPs.

https://doi.org/10.1016/j.bbrc.2020.06.090
0006-291X/© 2020 Elsevier Inc. All rights reserved.
1006 X. Dong et al. / Biochemical and Biophysical Research Communications 529 (2020) 1005e1010
2. Materials and methods
2.1. Protein preparation
DNA sequence of the ORD domain (residues 489e887) of ORP3,
was subcloned into pET28a expression vector (Novagen, USA).
Escherichia coli strain BL21(DE3) cells transformed with the plasmid
encoding ORD were cultured in LB medium to express native ORP3
ORD protein after the addition of 0.5 mM IPTG.
After centrifugation, collected cells were resuspended in Tris
buffer (50 mM Tris-HCl pH 8.0 and 500 mM NaCl) and lysed by
sonication. Target proteins were isolated from the clarified lysate
via nickel affinity chromatography followed by overnight treatment
with Ulp1 protease at 4  C to remove the fusion tag. then decreased
imidazole concentration to lower than 1 mM. The protein was then
loaded onto a Hitrap Q HP column (GE Healthcare, USA) for anion-
exchange chromatography and a Superdex 200 column (GE
Healthcare, USA) for subsequent size-exclusion chromatography in
a solution consisting of 20 mM TriseHCl pH 8.0, 5 mM MgCl2,
100 mM NaCl.
2.2. Crystallization
Preliminary crystallization screening was using the hanging-
drop vapour diffusion method at 16  C in 96-well crystallization
plates automatically by a Mosquito robot (TTP Labtech, England).
Hanging drops consisting of 0.1 ml protein solution (10 mg ml 1)
and 0.1 ml reservoir solution were equilibrated against 100 ml
reservoir solution. Screening of 600 commercially available crys-
tallization conditions gave several hits, which were further opti-
mized manually in 2 ml drops against 500 ml reservoir solution.
Diffraction-quality crystals of ORP3 ORD were obtained 2 d after
setup in a solution consisting of 0.08 M Cacodylate sodium pH 6.5,
16% PEG 8000, 0.16 M Magnesium acetate, 20%glycerol. Native
ORP3 ORD was then co-crystallized with PI(4)P diC8 (Avanti Polar
Lipids, USA). Following a 7 h incubation at 16  C, complex crystals
were obtained in a condition consisting of 0.16 M Calcium Acetate,
0.08 M Sodium Cacodylate:HCl pH 6.5, 14.4% PEG 8000, 20% Glyc-
erol. Both native and complex crystals were soaked in cryoprotec-
tant consisting of reservoir solution plus 30%(v/v) glycerol and
flash-cooled in liquid nitrogen prior to data collection.
2.3. Data collection, phasing and model refinement
Crystallographic diffraction data sets for crystals of ORP3 ORD
and its complexes were collected on the BL-19U and BL-17U
beamlines of Shanghai Synchrotron Radiation Facility, People’s
Republic of China, respectively. The phasing problem was solved for
the apo ORP3 ORD structure by the molecular-replacement method
with phenix. phaser (McCoy et al., 2007) using the structure of
Osh3 ORD (PDB entry 4IC4) as a search model. The final model was
manually adjusted using Coot (Emsley & Cowtan, 2004) and refined
with phenix.refine (Adams et al., 2010; Afonine et al., 2012). The
refined model of ORP3 ORD was then used as a search model in the
molecular-replacement method to solve the structures of the ORP3
ORD-PI4P complexes.
2.4. Isothermal titration calorimetry
Calorimetric titrations were performed using an Auto-ITC200
microcalorimeter from MicroCal (Northampton, MA). The acquisi-
tion and analysis of data were performed using the Origin software
supplied by MicroCal. ORP ORD and PI4P diC8 were dialyzed into a
buffer containing 50 mM Tris(pH8) and 150 mM NaCl for
isothermal titration calorimetry (ITC) experiments. A typical
titration was performed by injections of 180 mM ORP3 ORD into the
calorimeter cell containing 23 mM PI4P solution. 19 successive in-
jections were separated by 120s intervals to allow the endothermic
peak resulting from the reaction to return to the baseline. A con-
stant stirring speed of 800 rpm and a temperature of 25  C were
maintained throughout the titration. Dilution heats of CaM were
measured by titrating 180 mM ORP3 ORD from the syringe into the
cell containing only buffer. The resulting ORP3 ORD dilution heat
changes were subtracted from the measured heats of ORP3 ORD
and PI4P binding.
3. Results
3.1. Overall structure of ORP3 ORD
ORP3 contains PH domain (residues 51e146) in the N terminal
region, FFAT motif (residues 450e456) in the middle region and
ORD in the C terminal region (Fig. 1). The structure of human ORP3
ORD (residues 489e887) was determined at 2.1 Å resolution by
molecular replacement using Osh3 ORD as a search model. The
space group of crystal belongs to P3121 (Table 1). Two monomers
(monomer A and monomer B) in one asymmetric unit exhibit
pseudo twofold symmetry (Supp. Fig.1). In the dimeric form of
hORP3 ORD, only 1 salt bridge and 8 hydrogen bonds were found
between the monomers, besides the interface area (750 Å2) is only
4.2% of surface area of monomer (17800 Å2) based on the calcula-
tion by PISA. Meanwhile, the ORP3 ORD reveals the monomeric
state in solution (Supp. Fig.2). So that the crystallographic dimer
appears to form merely due to the crystal packing restraints.
The electron densities of the major part are clear, while the
densities of N-terminal flexible loop (residues 489e511) is invisible
in the Chain B and the densities of residues(residues 489e516,
522e543) is invisible in Chain A. In addition, seven chemically
modified cysteine residues are found in the crystal structures
(Chain A- Cys515, Cys615, Cys689, Cys760 and ChainB-Cys615,
Cys689, Cys760). Considering the influence of the crystallographic
reagent sodium cacodylate, the side chain of cysteine could not well
fit the electron density. The thiol group of cysteine should be
esterified by cacodylate group and they are named as CAF. All the
CAF residues locate at the surface of protein.
The ORP3 ORD comprises 9 a-helices and 19 b-strands. Like
Osh3 ORD, it can be divided into three major regions(Fig. 2). The N-
terminal extended loop and lid region consist of residues 512e546
(residues 525e533 a1). The ligand-binding tunnel contains resi-
dues 547e772 (residues 550e554 b1, 555e563 a2, 567e572 a3,
578e591 a4, 596e600 a5, 604e606 b2, 612e617 b3, 622e631 b4,
634e642 b5, 645e656 b6, 662e667 b7, 669e674 b8, 679e684 b9,
687e691 b10, 699e703 b11, 705e710 b12, 717e722 b13, 735e741
b14, 746e753 b15, 757e761 b16, 766e771 b17). The C-terminal
region includes residues 773e877 (residues 787e792 a6, 812e818
a7, 823e845 a8, 855e857 b18, 863-835 b19, 869e876 a9).
The ORD core is the ligand-binding tunnel which is comprised of
an incomplete b-barrel of 19 b-strands and an 50 Å a-helix bundle
of two-stranded b-sheet and three a-helix. The tunnel is located in
the center of the b barrel. A short a-helix of three turns and the U-
shape loop formed the lid (residues 525e546) cover the entrance of
tunnel. The C terminal region followed the b-barrel contains four a-
helix and two b-strand forming an hairpin. The N terminal
extended loop (residues 512e524) locates on the concave surface of
the C-terminal region.
3.2. Comparison of ORP3 ORD with other proteins of this family
ORPs could be divided into two subtypes by molecular structure,
the short ORPs and long ORPs. Most human ORPs are long ORPs
 X. Dong et al. / Biochemical and Biophysical Research Communications 529 (2020) 1005e1010 1007

Fig. 1. Schematic representation of the domain structures of ORP.

Table 1 PIPs binding (Supp. Fig.4A) [8]. Meanwhile, the Osh4/Osh6 lack the
Diffraction data collection and structure refinement statistics. N terminal extended loop due to their independent ORD (Supp.
Apo ORD ORD (PI4P diC8 Complex) Fig.4B). In addition, the structural arrangement of C terminal region
PDB Accession Code is different and the length of C terminal region in ORP3/Osh3 is
Data Collection obviously shorter than that in Osh4/Osh6 (Supp. Fig.4C). Although
Resolution limit (Å) 41.3-2.10 (2.14-2.10) 37.9-3.17 (3.22-3.17)
Wavelength (Å) 0.97852 0.97852
the detailed reason is unknown, the long ORPs have the multiple
Space group P3121 P3121 domains (PH domain/FFAT motif) which are involved in different
Cell parameters membranes recognizing and binding, whereas the short ORPs have
a, b, c (Å) 95.3, 95.3, 185.3 95.5, 95.5, 188.7 the sole ORD, it could be speculated that the ORD of short ORPs
a, b, g ( ) 90, 90, 120 90, 90, 120
have multiple functions not only involved in ligands transporting
Measured reflections 567936 173319
Multiplicity 9.8(9.2) 9.7(8.2) but also participate in membrane recognizing and binding. The
Mean I/s(I) 31.7(3.2) 16.4(2.6) RMSD between ORP3 ORD and Osh3 ORD is merely 2.1 Å which
Completeness (%) 100.0 (100.0) 99.9(100.0) indicate the structures are high conserved in evolution. The major
Ramerge 0.074(0.717) 0.129(0.727) differences are potential PIP binding loops and the lid region.
Refinement statistics
Resolution range (Å) 41.3e2.1 37.9e3.2
3.3. The N terminal lid of ORP3 ORD
Reflections
Working set 57616 17815
Test set 2920 908 The ORD lid region is deemed to be the molecular switch for
Rbcryst (%) 16.6 22.3 ligand uptake and release [8,14], it suggests the lid exhibits the
c
Rfree (%) 21.1 26.5 flexible state which allows the transition between open and closed
r.m.s.d.
Bonds (Å) 0.014 0.002
conformations and the ligand binding stabilize the conformation of
Angles ( ) 1.511 0.616 lid. In solved ORD structures, only the Osh3 has the electron density
Average B-factors (Å2) 41.61 93.4 of lid when no ligand binds. However, the protruding tip of the lid is
Protein (Å2) 41.62 93.4 partially disordered with the high B-factors in the Osh3 ORD ligand
Waters (Å2) 41.58
free structure, so that the interaction between the lid and the
Ramachandran plot
Favored regions (%) 96.4 94.4 entrance of the hydrophobic tunnel is hard to analyze. In our ORP3
Outliers (%) 0 0.27 ORD ligand free structure, chain B has the completely closed lid
based on the low B factor (Fig. 3A), which provide the details of
interaction between the lid and the entrance of the hydrophobic
except ORP1s and ORP4s, while only Osh1-3 are long ORPs in yeast. tunnel. The conformation of lid is stabilized via the hydrogen bonds
The short ORPs contain the sole ORD and the long ORPs include including internal hydrogen bonds and lid-b barrel hydrogen bonds
other N terminal variable region, such as two phenylalanines in an (Fig. 3B). Among of these, Asn525 can form hydrogen bond with
acidic tract (FFAT) motif, Ankyrin motif, GOLD domain and pleck- Ser527 via the water to link the lid and N terminal extension of lid.
strin homology (PH) domain. ORP3 ORD shares low sequence Two hydrogen bonds fix the U sharp conformation of lid, Ser527
similarity with homologous members (23% identity with Osh4, 25% form hydrogen bond with Glu548 and Asn534 form hydrogen bond
identity with Osh6, 37% identity with Osh3) (Supp. Fig.3), and the with Ala544 via two waters. In addition, several hydrogen bonds
overall structure of ORP3 ORD is similar to that of Osh3 ORD (PDB stabilize the closed conformation of lid. Asn535/Gly537/Lys538
code 4IC4; Z-score 44.9; RMSD 2.1 Å), Osh4 (PDB code 1ZHZ; Z- form hydrogen bonds with Phe657 (b6-b7) via the water, Gly537
score 30.8; RMSD 2.9 Å) and Osh6 (PDB code 4PH7; Z-score 33.9; form hydrogen bond with Lys656 (b6), Asp539 can form hydrogen
RMSD 2.9 Å). The structural homology search using Dalisever. Su- bonds with Trp653/Asn655 via water, Leu540 from hydrogen bond
perposition of the ORP3 ORD structure with Osh3 ORD, Osh4 and with Asn655 and Met545 form hydrogen bond with Asn550 located
Osh6 structures show that the ligand-binding tunnel are highly in b1 via water. The abundant hydrogen bonds between the lid and
conserved in these ORDs, whereas the loops surrounding the b- b-barrel stabilize the conformation of the long loop located in lid.
barrel are variant and they are likely to be associated with different The lid upstream 13 residues form an extended loop and make
tight interaction with the surface of the C terminus of ORD. The
1008 X. Dong et al. / Biochemical and Biophysical Research Communications 529 (2020) 1005e1010

Fig. 2. The overall structure of ORP3 ORD. The N-terminal lid and the N-terminal upstream residues of lid (residues 512e546) is shown in magenta. The central helices (residues
547e603) in yellow, the bbarrel (residues 604e772) in limon, and C-terminal region (residues 773e877) in cyan. CAF615, CAF689 and CAF760 are shown as stick mode. (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3. The N terminal Lid of ORP3 ORD. (A) B-factor representation of apo ORP3 ORD (top view). (B) Cartoon representation of apo ORP3 ORD lid, the residues which participated in
interaction are show in stick model. (C) The N-terminal upstream residues of the lid, the residues which participated in interaction are show in stick model.
 X. Dong et al. / Biochemical and Biophysical Research Communications 529 (2020) 1005e1010
Fig. 4. PI4P binding to the ORP3 ORD. (A) ITC titration curves showing the binding affinities of ORP3 ORD with PI4P diC8 (1:1 and 1:0.8). (B) Binding of diC8 in the ORP3 tunnel. (C)
The hydrophobic binding tunnel.
upstream residues well match the shallow pocket which is
comprised of b17-a6, a6-a7 and a7-a8 loops (Fig. 3C). Meanwhile,
the tight interaction could not be disrupted by the conformational
changes of lid, the Arg513 (Arg630 in Osh3) form a salt bridge with
the Glu818 (Glu930 in Osh3) and Leu516 (Ile633) is fixed in the
hydrophobic pocket formed by the b17-a6 loop and a6.
3.4. PI4P binding to the ORP3 ORD
OSBP was named as the oxysterol binding protein since it was
first discovered as a receptor for oxysterol, in addition, many ORPs
have the capability of binding sterol. However, it was thought as a
misnomer due to the specific ligands of ORPs. So far, the ligands of
several ORPs has been identified, the photo-cholesterol and photo-
25OH-cholesterol were identified as the ligands of ORP3 by photo-
cross-linking method in vitro [21], whereas t further verification is
required. ITC is thought to be one of the most reliable and high-
precision methods to quantitate noncovalent, equilibrium protein
and ligands interactions. By using ITC, we obtained thermodynamic
parameters for the interaction between ORP3 ORD and PI4P diC8
(Fig. 4A) The calorimetric data were best fit to a model assuming a
single set of identical sites. Our ITC assay clearly indicated that
ORP3 ORD and PI4P diC8 formed a 1:1 complex with a dissociation
constant around 29 nM.
1009
On account of these evidence, we try to obtain the ORP3 ORD
and ligands complex by cocrystallization method. At last, we only
obtain the ORP3 ORD-PI4P complex, extensive trials to obtain
cholesterol/25-hydroxycholesterol and ORP3 ORD complex were
failed. The structure models of the crystals grown in the presence of
5 mM cholesterol/25-hydroxycholesterol and the crystals of ORP3
ORD soaked sterol for 10 h showed empty binding pockets. We also
modeled the structures of ORP3-cholesterol complex and ORP3-25-
hydroxycholesterol complex based on the complex structures of
Osh4 (Supp. Fig. 5Ae5B), however, the ligand binding tunnel is too
narrow to accommodate the tetracyclic rings of sterol, several
residues clashed with the ligands. In the model of ORP3 and
cholesterol complex, the side chain of Glu562 (a2) clashed with the
hydroxyl of cholesterol, the side chain of Tyr653 (b6) clashed with
the tetracyclic rings of cholesterol, the main chain of Gly601 and
Ser602(a5-b2) clashed with the side chain of cholesterol. The
clashed situation in the ORP3 and 25-hydroxycholesterol is similar
to ORP3 and cholesterol except for the a5-b2 loop. In addition, its
orthologous Osh3 does not bind sterols as well [22]. Therefore, we
speculate that ORP3 ORD may not bind cholesterol or 25-
hydroxyoxycholesterol.
The structure of ORP3 ORD-PI4P diC8 complex was determined
at 3.2 Å resolution by molecular replacement using the apo form.
The phosphorylated inositol head is recognized by the shallow
1010 X. Dong et al. / Biochemical and Biophysical Research Communications 529 (2020) 1005e1010
pocket around the entrance of the tunnel and the short acyl chain of
PI4P loosely interact with the hydrophobic tunnel (Fig. 4B and C).
The cluster of hydrogen bonds hold the conformation of PI4P head
and it is high conserved in Osh3/Osh4/Osh6. More specifically, the
most hydrogen bonds could be divided into three parts, Lys603/
Asn606 (b2) directly bind the inositol ring and 1-phosphate group,
His631/His632 (b4-b5) directly bind 4-phosphate group and
Lys829/Glu833/Arg837/Arg840 (a8) bind the polar head. Besides
these, the Asn655 (b6) also directly bind the carbonyl oxygen of acyl
chain. We aligned the amino sequences of ORPs, the key residues
binding PI4P are highly conservative except Arg840 (Supp. Fig.6A),
among them, the OSBP fingerprint motif EQVSHHPP could be
defined as the PI4P binding motif, which indicates the PI4P is a
common ligand to ORPs. Meanwhile, the binding pattern of PI4P
acyl chain is not conservative in all ORPs. Structural comparison of
apo- and PI4P bound ORP3 ORD reveals that there is no obviously
conformational change during ligand binding with the rmsd of 1 Å
for all Ca atoms. The conformational change of b-barrel part is
limited, whereas the changes are concentrated on the orientation of
side chain in several PI4P-binding residues (Supp. Fig.6A).
4. Conclusions
The crystal structure of ORP3 ORD provide the structural infor-
mation of human ORPs. The structural similarity of human ORP3
ORD and three yeast Oshs ORD (Osh3/Osh4/Osh6) indicates the
overall structure of ORD is well conserved among ORPs. Meanwhile,
the structure of ORP3-PI4P, together with biophysical result and
reported Osh ORD-PI4P structures, further confirm that the PI4P is
the common ligand to ORPs. In our structures, the upstream resi-
dues of lid tightly bind to the surface of b-barrel, which may serve
as the fixed point between the N terminal domains and ORD lid.
And together with the structural information of Osh3, we suggest it
is the common character in all long ORPs. We also find the C ter-
minal regions of ORP3 and Osh3 are smaller than Osh4/Osh6, the
ORPs always has the smaller C terminal region and the large C
terminal region of short ORPs may participate in other unknown
function.
Although we have ascertained the PI4P is the first ligand of
ORP3, ORP3 may transfer PI4P and another ligand bidirectionally.
Transport of PI4P followed by its hydrolysis would provide the
energy for transfer of the other ligand against its concentration
gradient. So that seeking the second ligand is foremost in future.
The atomic coordinates of ORP3 ORD and ORD/PI4P complex pro-
vide a structural basis for seeking second ligand to investigate the
diverse roles of ORP3 and other ORPs in cell signaling.
Declaration of competing interest
We declare that we do not have any commercial or associative
interest that represents a conflict of interest in connection with the
work submitted.
Acknowledgement
This work was supported by the National Natural Science
Foundation of China (Project Nos. 31271491 and 31470792).The
members of Zhang’ laboratory are highly appreciated.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2020.06.090.
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