Ligand binding induces conformational changes in human cellular retinol-binding protein 1 (CRBP1) revealed by atomic resolution crystal structures

Article abstract of DOI:10.1074/jbc.M116.714535

Important in regulating the uptake, storage, and metabolism of retinoids, cellular retinol-binding protein 1 (CRBP1) is essential for trafficking Vitamin A through the cytoplasm. However, the molecular details of ligand uptake and targeted release by CRBP

Full text of DOI:10.1074/jbc.M116.714535

JBC Papers in Press. Published on February 21, 2016 as Manuscript M116.714535
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.714535
Ligand Binding Induces Conformational Changes in Human Cellular Retinol-Binding Protein 1 (CRBP1)
revealed by atomic resolution crystal structures§
Josie A. Silvaroli1#, Jason M. Arne1#, Sylwia Chelstowska1,2, Philip D. Kiser1,3, Surajit Banerjee4,5,
Marcin Golczak1,6
From the 1Department of Pharmacology, School of Medicine, Case Western Reserve University,
Cleveland, OH
2Laboratory of Hematology and Flow Cytometry, Department of Hematology, Military Institute of
Medicine, Warsaw, Poland
3Research Service, Louis Stokes Cleveland VA Medical Center, Cleveland, OH
4Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY
5Northeastern Collaborative Access Team, Argonne National Laboratory, Argonne, IL
6Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve
University, Cleveland, OH
Running title: Crystal structure of apo-CRBP1
To whom the correspondence should be addressed: Marcin Golczak, Ph.D., Department of Pharmacology,
School of Medicine, Case Western Reserve University, 10900 Euclid Ave, Cleveland, Ohio 44106, USA;
Phone: 216–368–0302; Fax: 216–368–1300; E–mail: mxg149@case.edu.
Keywords: cellular retinol-binding protein, vitamin A, retinylamine, retinol, lipid transport
ABSTRACT
Important in regulating the uptake,
storage, and metabolism of retinoids, cellular
retinol-binding protein 1 (CRBP1) is essential
backbone mobility in the apo-protein that
became more rigid upon retinoid binding. This
conformational flexibility of human apo-
CRBP1 facilitates interaction with the ligands,
whereas the more rigid holo-protein structure
protects the labile retinoid moiety during
vitamin A transport. These findings suggest a
mechanism of induced fit upon ligand binding
for trafficking vitamin
A
through the
cytoplasm. However, the molecular details of
ligand uptake and targeted release by CRBP1
remain unclear. Here we report the first
structure of CRBP1 in a ligand-free form as
well as ultra-high-resolution structures of this
protein bound to either all-trans-retinol or
retinylamine, the latter a therapeutic retinoid
by
mammalian
cellular
retinol-binding
proteins.
Effective distribution of vitamin A (all-
trans-retinol) and its derivatives (called
‘retinoids’) throughout the body and cellular
compartments determines their essential functions
in embryonic development, growth, immunology,
reproduction, and vision (1-4). However, because
their lipophilic nature, free retinoids exist only at
extremely low concentrations in plasma or cytosol,
making diffusion kinetics between sites of action
inefficient. This solubility limitation is overcome
by specialized retinoid-binding proteins that
regulate retinoid metabolism and physiological
functions (5). Thus, an appreciation of the
molecular basis for ligand uptake and targeted
release by retinoid-binding proteins is critical for
that
prevents
light-induced
retinal
degeneration. Superpositioning of human apo-
and holo-CRBP1 revealed major differences
within segments surrounding the entrance to
the retinoid-binding site. These included α-helix
II and hairpin turns between β-strands βC-βD
and βE-βF as well as several side chains such as
Phe57, Tyr60, and Ile77 that change their
orientations to accommodate the ligand.
Additionally, we mapped hydrogen-bond
networks inside the retinoid-binding cavity and
demonstrated their significance for the ligand
affinity. Analyses of the crystallographic B-
factors indicated several regions with higher
1
Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

understanding retinoid homeostasis. Yet, current
knowledge of this process is very limited.
potentially useful in delivering therapeutic agents.
An example of this approach is retinylamine, an
inhibitor of the visual cycle that provides
Three classes of retinoid-binding proteins
named for their selectivity, i.e. cellular retinol-
binding protein (CRBP)1, cellular retinoic acid-
binding protein (CRABP), and -cis retinoid
protection
against
light-induced
retinal
degeneration (24,25). The high efficacy of
retinylamine is attributed to its efficient LRAT-
driven uptake into the RPE, where it exerts its
therapeutic effects (26-28). Delivery of
retinylamine to LRAT by CRBP1 can facilitate
formation of retinylamides. Thus, as with vitamin
A, acylation pulls retinylamine into the cells via
specific
cellular
retinal-binding
proteins
(CRALBP), carry vitamin A and its metabolites
within cells (5-7). All representatives of CRBPs
and CRABPs are members of intracellular lipid-
binding proteins (iLBPs) (8), whereas CRALBP
belongs to the Sec14 protein family (9). Four
CRBPs (CRBP1, 2, 3, and 4) are encoded in the
human genome (10,11). Among them, CRBP1 is
the most widely expressed in numerous tissues
with the highest abundance in the liver, kidney,
lung, and retinal pigment epithelium (RPE) cells
of the eye (12-14). In contrast, expression of
CRBP2 is limited to the small intestine (15),
human CRBP3 to kidney and liver (16), and
CRBP4 to kidney, heart, and colon (11). CRBPs
also differ in their vitamin A binding affinities.
The lowest Kd values were reported for CRBP1
and CRBP2 (10 – 50 nM, current study) (17,18),
whereas affinity of CRBP3 and 4 for all-trans-
retinol is lower (Kd ≈60 and 200 nM, respectively)
(10,11). These differences in expression pattern
and binding affinity are consistent with the diverse
physiological functions of CRBPs. In addition to
serving as chaperones for the labile retinoids,
accumulating evidence indicates that CRBPs are
critical for the regulation of vitamin A uptake and
metabolism. CRBP1 is an acceptor of retinol
transported from the plasma to cytosol via a cell-
surface receptor named STRA6, which interacts
with serum retinol-binding protein (19,20).
CRBP1 also facilitates the synthesis of retinyl
esters by lecitihin:retinol acyltransferase (LRAT)
that preferentially utilizes substrate delivered by
this binding protein (21). These findings are
consistent with the phenotype of CRBP1-deficient
mice characterized by dramatically decreased
levels of retinyl esters in hepatic stellate cells and
the RPE. This decrease in ocular retinyl ester
levels correlated with a two-fold reduction in
visual chromophore regeneration rate after light
exposure due to slower diffusion of vitamin A
from photoreceptors to the RPE where it is
esterified (22,23).
mass-action and provides
intracellular source of this drug (27,29).
a
long lasting
Transport of retinoids requires a precise
mechanism of ligand uptake and targeted release.
Despite numerous structural studies on iLBP
proteins, neither process is yet understood. Crystal
structures of mammalian fatty acid-binding protein
(I-FABP) (30,31) and CRBP2 (32) revealed only
minimal changes upon ligand binding. This is
quite surprising owing to the nearly completely
encapsulated retinol-binding cavities in CRBP1
(33), CRBP2 (34), and CRBP3 (10) that are rather
inaccessible. Thus retinol entry and exit would
require significant alterations in the positions of
selected regions of the protein. This puzzle was
partially resolved by NMR studies that revealed
conformational flexibility at the entrance to the
ligand binding pocket in apo-iLBPs (35-38). These
findings led to the formulation of the “dynamic
portal hypothesis” in which transient loss of
structure provides a passageway for a ligand to
enter an internal binding site, which would
otherwise be inaccessible in the occluded
conformation of the protein.
One of the main obstacles to better
understanding of the role of conformational
changes upon ligand binding and release by
CRBPs is the lack of an apo-CRBP1 crystal
structure. Therefore, to clarify this mechanistic
aspect of CRBPs function, we obtained the first
bona fide X-ray structure of CRBP1 in the ligand-
free form and compared it to atomic resolution
structures of the protein bound to either all-trans-
retinol or retinylamine. These analyses indicate the
occurrence of well-defined conformational
changes involved in the binding and release of
these ligands and provide evidence for
conformational flexibility of the “portal” region.
Thus, we provide compelling crystallographic
verification of a general mechanism facilitating
The ability of CRBPs to bind and
transport hydrophobic compounds makes them
2

increased access to the binding cavity in the apo-
form of mammalian CRBPs.
dropwise to 200 mL of ethanol containing a
stoichiometric excess of sodium borohydride. The
reduction reaction was carried out for 2 h on ice
with vigorous stirring. Retinoids were extracted
with ethyl acetate, the organic solvent was washed
twice with a saturated solution of NaCl and loaded
onto a silica gel (Sigma-Aldrich). After an
extensive wash with ethyl acetate, retinylamine
was eluted with a mixture of methanol, ethyl
EXPERIMENTAL PROCEDURES
Expression and purification of human
apo-CRBP1 – The synthetic cDNA sequence of
human CRBP1 (hCRBP1) (GI:132387) with the
addition of C-terminal 6 His residues subcloned
into pD441 vector was purchased from DNA2.0.
The protein was expressed in the BL21 (DE3)
E.coli strain (New England Biolabs). Bacteria
were grown in a shaker incubator at 37 °C in the
presence of 50 µM kanamycin. Protein expression
was induced with 0.5 mM isopropyl β-D-1
thiogalactopyranoside (Roche). After 4 h of
incubation, cells were harvested by centrifugation
(6,000 × g, 15 min, 4 °C). Bacteria were disrupted
by osmotic shock (39) and the lysate was
centrifuged at 36,000 × g for 30 min at 4 °C.
Buffer composition of the supernatant was
adjusted to 50 mM Tris-HCl, pH 8.0, 250 mM
NaCl prior to loading onto a column filled with
Ni-NTA agarose resin (Qiagen) and equilibrated
with loading buffer composed of 50 mM Tris-HCl,
pH 8.0, 250 mM NaCl. The column was washed
with 100 mL of loading buffer containing 5 mM
imidazole and the protein was eluted by raising the
imidazole concentration to 250 mM. Fractions of
the eluted protein were combined and concentrated
to 5 mL in an Amicon Ultra-4 centrifugal filter,
cutoff 10,000 Da (Millipore). The protein solution
then was diluted 40 fold to a final volume of 200
ml with 10 mM Tris-HCl buffer, pH 8.0 and
loaded onto a Mono-Q ion exchanger column (GE
Healthcare). hCRBP1 was eluted with a gradient
of NaCl (0 – 0.5 M) over 30 min at a flow rate 0.5
mL/min. Collected fractions were examined by
SDS-PAGE and those containing purified
hCRBP1 were pooled together (Fig. 1A). The
protein was concentrated to 3 mg/mL and 0.2 mL
aliquots were stored at -80 °C.
acetate, and
7
N
ammonia in methanol
(49.5%/50%/0.5%, v/v, respectively). The purity
of retinylamine was verified by HPLC using a
normal phase Luna 10 μm PREP silica 100 Å, 250
x 4.6 mm column (Phenomenex) and ethyl acetate
with 0.5% ammonia solution as the mobile phase
at a flow rate of 2.0 mL/min.
Retinoid fluorescence binding assays –
The affinity of all-trans-retinol or retinylamine for
hCRBP-I was evaluated by monitoring the
quenching of protein fluorescence at increasing
concentrations of the ligand. All measurements
were performed on a PerkinElmer Life Sciences
LS55 model fluorometer. After the excitation
wavelength was set at 285 nm, emission spectra
were recorded between 300 and 420 nm with
bandwidths for excitation and emission fixed at 10
nm. Titrations were carried out at 25 °C in 20 mM
Tris/HCl buffer, pH 7.6, containing 50 mm NaCl
and 5% glycerol (v/v). Concentrations of all-trans-
retinol and retinylamine stock solutions were
determined spectrophotometrically by using the
molar absorbance coefficient ε = 52,770 for the
maximum absorbance at 325 nm in ethanol (40).
Retinoids were delivered in dimethyl sulfoxide
(DMSO). Final concentrations of the solvent did
not exceed 0.2% of the sample's total volume. All
binding data were corrected for background and
self-absorption of excitation and emission light
(41). Apparent Kd values were calculated by
nonlinear regression of the experimental data
using saturation ligand-binding models available
in the SigmaPlot software package (Systat
Software).
Synthesis of all-trans-retinylamine – all-
trans-Retinylamine
(retinylamine)
was
synthesized by reacting 0.5 g of all-trans-retinal
(Toronto Research Company) dissolved in 5 mL
of ethanol with ammonia (7 N in methanol
(Sigma-Aldrich)) (24). After 2 h of stirring at
room temperature, the resulting retinyl imine was
reduced by sodium borohydride (Sigma-Aldrich).
To avoid formation of side products and increase
product yield, retinyl imine solution was added
Formation of holo-hCRBP1 with all-trans-
retinol or retinylamine – To obtain holo-hCRBP1,
3 mg of the apo-protein was incubated for 20 min
on ice in 10 mM Tris-HCl, pH 8.0, 10% glycerol
(v/v) with either all-trans-retinol (Toronto
Research Company) or retinylamine delivered in
DMSO (2%, v/v) to a final concentration of ~0.2
mM (~2 molar excess over the protein). To
3

remove unbound retinoids, the protein solution
was diluted 10 fold with 10 mM Tris-HCl, pH 8.0,
and centrifuged to remove potential precipitates
(36,000 x g, 20 min, 4 °C). Holo-hCRBP1 was re-
purified on a Mono-Q column as described above.
The effectiveness of holo-hCRBP1 formation was
verified spectrophotometrically. The complex of
hCRBP1 with all-trans-retinol or retinylamine
revealed an A350/A280 ratio of 1.6 as reported
previously for holo-CRBP1 purified from rat liver
(Fig. 1B) (42).
and a 300 mm crystal-to-detector distance. Data
for the hCRBP1 complex with retinylamine (PDB
# 5HA1) were obtained at NE-CAT beamline 24-
ID-E with ADSC Quantum 315 CCD detector at
the Advanced Photon Source at Argonne National
Laboratory. The detector distance was 150 mm,
oscillation range 1.0°, and wavelength 0.9792 Å.
An ultra-high-resolution data set for all-trans-
retinol holo-hCRBP1 (PDB # 5HBS) was
collected at the BL12-2 beamline at the Stanford
Synchrotron Radiation Lightsource equipped with
Pilatus 6M PAD detector. Images were measured
at 0.2° increments by using an X-ray wavelength
of 0.7293 Å at a 200 mm crystal-to-detector
distance.
Crystallization conditions – hCRBP1 in
complex with all-trans-retinol or retinylamine as
well as the apo-protein were crystallized with the
sitting drop vapor diffusion method. Protein
samples at 3 mg/mL concentration were mixed in
1:1 ratio with 1.3 µL of 0.1 M Bis-Tris, pH 5.5,
and 25% PEG 6000 or PEG 3350. Plates were
sealed and incubated at room temperature. Rod-
like crystals with dimensions of 0.2 – 1.0 mm in
length and 10 – 25 µm in width grew within 2-5
days. Mature crystals were collected and flash
cooled in liquid nitrogen in preparation for X-ray
diffraction experiments.
Data processing, model building, and
refinement – Data from single crystals of apo- and
holo-hCRBP1 were integrated and scaled with
Mosflm (43). The 0.89 Å resolution data set for
hCRBP1 bound to all-trans-retinol was processed
with XDS (44). The structure hCRBP1 was solved
by molecular replacement with PHASER_MR
(45) using the atomic coordinates of rat CRBP1
(rCRBP1) in complex with all-trans-retinol (PDB
# 1CRB). Models were built and manually
adjusted in COOT (46,47), including adding
alternative conformations where appropriate and
refined with PHENIX (48), using a riding
Verification of complex stability between
hCRBP1 and retinylamine – Fifty µg of the
protein was extracted with ethyl acetate. The
organic phase was collected and the solvent was
dried down in a stream of nitrogen. Retinoids were
re-solubilized in 100 µL of acetonitrile, loaded
onto a reverse phase Gemini 5 μm, 100 Å, 250 x
4.6 mm C18 column (Phenomenex), and eluted
with a gradient of 20% to 100% of acetonitrile in
water developed over 20 min at a flow rate of 1.5
mL/min. Both solvents contained 0.1% (v/v)
formic acid. Similar analyses were performed
using retinylamine holo-hCRBP1 crystals. Ten
protein crystals were harvested and washed 3
times by transferring them to fresh crystallization
solutions. Finally, resulting crystals were
dissolved in 50 µL of 50% methanol/water (v/v),
retinoids were extracted with ethyl acetate and
analyzed by HPLC as described above.
hydrogen
model,
individual
anisotropic
temperature factors, and occupancy refinement for
alternative conformers and waters. Allowing
individual hydrogen refinement did not result in
better geometry statistics or R factors. Geometry
of the refined models was assessed with the
MolProbity server (49). The final atomic
coordinates and structure factors for all four
structures described in this manuscript were
deposited in the RCSB Protein Data Bank. The
accession codes as well as the data-collection and
refinement statistics are summarized in Table 1.
Visualization of the macromolecules and figure
preparation were carried out with the CHIMERA
software package version 1.10.1 (50).
X-ray data collection – Diffraction data
Calculations of average crystallographic
for apo-hCRBP-I crystals (PDB # 5H9A) and all-
trans-retinol holo-hCRBP1 crystals (PDB # 5H8T)
were collected at NE-CAT beamline 24-ID-C at
the Advanced Photon Source at Argonne National
Laboratory. Data were collected in the shutterless
mode by a Dectris Pilatus 6MF pixel array
detector with 0.5° images at 0.9792 Å wavelength
B-factors and distribution of anisotropy within
refined models – To compare residue mobility, the
averages
of
the
equivalent
isotropic
crystallographic B-factors (Beq) for the main chain
of each residue were calculated. They represent
isotropic displacement of atoms that were
described by anisotropic displacement parameters
4

during refinement. Then the residue Beq were
normalized with the “z-score normalization”
methodology (51),
composed of ten β-strands organized in two anti-
parallel β-sheets (Fig. 2A). These form a flattened
β-barrel that surrounds the retinoid-binding site.
One of the two potential entrances to this site is
completely enclosed by the side chains of the β-
sheets and the N-terminus, whereas the other is
protected by two short α-helices (I and II) and the
hairpin turns between β-strands βC-βD and βE-βF.
These α-helices and turns constitute a so-called
‘portal region’. Holo-hCRBP1 and holo-rCRBP1
exhibit high structural similarity with an overall
RMSD of 0.89 Å between equivalent main-chain
atoms (Table 2).
B
eqx-zscore(i) = [Beqx(i) – ۦBeq(i) ۧ]/s(i)
where Beqx-zscore(i) is the normalized z-score for
residue x in structure i, Beqx(i) is the equivalent
isotropic B-factors for residue x, ۦBeqۧ(i) is the
average residue equivalent isotropic B-factor for
structure i, and s(i) is the corresponding standard
deviation among atoms in the structure.
The anisotropy of individual atoms, defined as the
ratio of the minimum and maximum eigenvectors
of the anisotropic displacement parameters matrix
(52) as well as the anisotropy mean values for the
protein structures, were calculated using
PARVATI (53) and ANISOANL (47) validation
tools.
Electron density for all-trans-retinol was
well-defined for the entire molecule thereby
allowing all of the ligand atoms to be refined at
100% occupancy. The retinoid moiety is almost
planar. The β-ionone ring is embedded in a
hydrophobic cleft between α-helices I and II, and
it turns connecting β-strands βC-βD and βE-βF.
The orientation of the β-ionone ring is essentially
identical to those found in other CRBPs and
adopts the 6-s-trans configuration (Fig. 2B). The
dihedral angles defined by atoms C5-C6/C7-C8 in
PDB # 5HBS and PDB # 5H8T are nearly
RESULTS
High resolution structure of hCRBP1 in
the
retinol-bound
state
–
Nearly
all
crystallographic information about CRBPs
originates from structures of CRBP2. CRBP1 is
represented by only a single set of crystallographic
coordinates for a holo-protein purified from rat
liver refined at a modest resolution of 2.1 Å (PDB
# 1CRB) (33). Attempts to replicate the previously
reported rCRBP1 crystallization conditions that
included cadmium (II) chloride as a precipitant
(54), were unsuccessful in case of hCRBP1.
Crystals instead were obtained from PEG 6000 or
PEG 3350-containing solutions. Despite the
differing crystallization conditions, hCRBP1
crystals grew in space group P212121 with unit cell
constants similar to those of rCRBP1 with a single
protein molecule in the asymmetric unit.
identical, with
ψ
=
-152.3° and -153.9°,
respectively. Free rotation of the β-ionone ring is
restricted by hydrophobic side chains of Phe16,
Phe57, Leu36, Val25, Leu29, and Leu20 (Fig.
2C). The first residue seems to be particularly
important for defining the ring position. An
alternative 6-s-cis configuration would cause steric
clashes between the gem-dimethyls and the Phe16
side chain benzyl ring (Fig. 2C). The rest of ligand
binding cavity is lined with well-conserved
hydrophobic residues that provide non-polar
interactions with the retinoid moiety.
Several structures of CRBP2 and CRBP
from zebrafish contained degradation products of
the labile retinoid moiety caused by X-ray
radiation exposure (34,55,56). These were
characterized by the alteration of torsion angles
measured between carbons C12-C13/C14-C15
along the polyene chain that were not consistent
with the geometry of a trans double bond. This
aberration caused spatial relocation of the
hydroxyl group and formation of hydrogen bonds
with the side chain of Lys40 in addition to the
canonical Gln108. We found no evidence for
radiation damage of all-trans-retinol in the
hCRBP1 structures. The torsion angles for C12-
The 0.89 Å and 1.21 Å structures of hCRBP1
(PDB # 5HBS and PDB # 5H8T, respectively)
reported here represent a significant improvement
over rCRBP1 reported previously (33). Their
higher resolution facilitated the building of a more
accurate model of the structure, including residues
directly involved in retinoid binding. In fact, the
electron density maps are well-defined for all the
residues including the C-terminal histidine
residues of a 6xHis tag with the exception of
Lys68 side chain. The overall structure of
hCRBP1 closely resembles those of other iLBPs
with
a
compact single-domain architecture
5

C13/C14-C15 were ψ = -169.6° and -169.1° in the
0.89 Å and 1.21 Å structures, respectively. These
values are nearly identical to those found in retinol
bound to human CRBP2 (PDB # 4QZT), a model
structure for which crystallographic data were
collected at low X-ray energy (34) (Fig. 2B).
Interestingly, optimal geometry of all-trans-retinol
in hCRBP1 does not prevent the hydroxyl group
from hydrogen bonding with side chains of both
Gln108 and Lys40 (Fig. 3A). This finding
represents a major dissimilarity between previous
models of rCRBP1 or CRBP2 and current high-
resolution hCRBP1 structures. The key difference
that permits direct formation of a hydrogen bond
with Lys40 is the position of its side chain that
adopts a conformation distinct from those
previously reported by bringing the ζ-nitrogen
atom about ~1.0 Å closer to the hydroxyl group of
vitamin A (Fig. 3A). Importantly, the electron
density for the entire Lys40 including every atom
of the side chain is very well defined and the
geometric parameters for this residue are well
within allowed ranges. Also, there is no difference
in the position of Lys40 between the 0.89 Å and
1.21 Å data sets. The side chain conformation of
Lys40 is stabilized by a network of hydrogen
bonds between the hydroxyl group of Thr53 and
the water molecule, W1 (Fig. 3B). W1 further
interacts with ε1-carbonyl oxygen of Gln128 and
with water W2, which in turn is hydrogen bonded
to ε2-amino group of Gln128. Additionally, Thr53
interacts with W3 to complete the functional
network of the hydrogen bonds. The high-
resolution structure reveals a total of 7 well-
defined water molecules buried in the binding site,
all of which interact with internal polar side
chains. Waters W4, W5, and W6 form yet another
network, in which W4 interacts with ε1-nitrogen
atom of the Trp106 indole ring, W6 with the
hydroxyl group of Tyr19, whereas W5 bridges
these two water molecules together. Finally, W7
interacts with side chains of Tyr19 and Gln97.
hCRBP1 in retinylamine-bound state – To
hCRBP1 after 2 days incubation at room
temperature. As shown in Fig. 4A, an HPLC
chromatogram revealed a single retinoid peak,
with an elution time and a UV/Vis absorbance
spectrum which perfectly matched that of a
synthetic retinylamine standard. The amount of
extracted retinylamine and number of the protein
molecules were closely correlated, revealing ~1.1
stoichiometry. The same analyses performed on
retinylamine holo-hCRBP1 crystals indicated the
presence of retinylamine in the crystalline form of
the protein.
Comparison of the aligned structures of hCRBP1
in complex with all-trans-retinol and retinylamine
indicated a similar positioning of the two ligands
within the binding pocket (Fig. 4B). Thus, the
amino group of retinylamine is perfectly located to
form a hydrogen bond with ε1-carbonyl oxygen of
Gln108. In contrast, an interaction between
protonated amino groups of the ligand (pKa ≈7.2)
(27) and Lys40 would be unfavorable owing to
electrostatic repulsion. Thus only a single
hydrogen bond could be made to stabilize
retinylamine binding. To investigate whether the
alteration of the hydrogen bonding affects ligand
binding, we determined the dissociation constant
for all-trans-retinol and retinylamine with a
fluorescence quenching assay. As shown in Fig.
4C, incubation of these retinoids with hCRBP1 led
to an exponential decay of protein fluorescence
that displayed a saturable binding isotherm.
Subsequent calculation of the apparent
dissociation constants revealed a Kd = 18.5 ± 1.1
nM for all-trans-retinol and a Kd = 64.1 ± 16.9 nM
for retinylamine. This result identifies hCRBP’s
ability to form hydrogen bonds with side chains of
both Lys40 and Gln108 as a major factor
responsible for its nearly 4 times higher affinity
for all-trans-retinol than retinylamine.
Comparison of apo- and holo-structures
of hCRBP1 – The enclosed retinoid-binding cavity
observed for holo-hCRBP1 raises the question of
whether the apo-protein structure would reveal any
conformational differences that could allow ligand
binding. Comparison of both all-trans-retinol- and
retinylamine-bound structures to the ligand-free
model revealed a high level of conservation of the
overall structure. The RMSD values calculated for
the main-chain atoms of superimposed holo- and
apo-structures ranged between 0.92 – 1.07 Å
(Table 2). However, they are significantly larger
shed light on the role of hydrogen bonds within
the retinoid-binding pocket, we co-crystallized
hCRBP1 with retinylamine, a retinol derivative in
which a hydroxyl is replaced by an amino group.
Prior to crystallization, we verified that labile
retinylamine was stable in its hCRBP1 bound state
by determining the composition and quantity of
retinoids extracted from retinylamine holo-
6

(1.7 – 1.8 Å) in discrete regions including α-helix
II (residues 24 – 36) as well as the hairpin βE-βF
and βC-βD turns (residues 53 – 60 and 73 – 81,
respectively) (Fig. 5A). These differences reflect
conformational rearrangements of hCRBP1 in the
absence of a ligand. The most significant transition
is repositioning of the main chains of βC-βD and
particularly the βE-βF loops towards the interior of
the retinoid-binding cavity, accompanied by α-
helix II bending away from the rest of the protein.
Rearrangement of the main chains is followed by
displacement of the side-chains of several residues
that surround vitamin A. The most striking
changes are noted for Phe57, Tyr60, and Ile77
(Fig. 5B). Their side chains adopt orientations that
protrude into the binding cavity, occupying space
reserved for the β-ionone ring of retinoid in the
holo-structures. Concomitantly, the side chains of
Phe16, Leu20, Val25, and Leu29 that belong to α-
helix II are pushed backwards to avoid close
contact with Phe57 and Ile77. Interestingly, the
remaining amino acids that line the interior of the
binding pocket do not change their orientations
upon the holo to apo transition. This observation
could be explained by a preserved hydrogen-
bonding network in which a hydroxyl group of
retinol is substituted by a water molecule in the
could exhibit significantly greater conformational
flexibility. To further evaluate the dynamics of the
portal region in a more quantitative manner, we
analyzed the equivalent isotropic crystallographic
B-factors (Beq). Because Beq values could be
affected by refinement strategies or differences in
data resolution, they were scaled by the z-score
normalization method (51) prior to the analysis.
To further ensure proper interpretation of any
differences, we compared Beq values for the main
chain atoms of the apo-protein to both the 0.89 Å
and 1.21 Å structures of all-trans-retinol-bound
hCRBP1 as well as to its retinylamine-bound
form. Also as an internal control, we calculated the
corresponding differences between all-trans-
retinol and retinylamine holo-proteins. All
comparisons of the normalized crystallographic B-
factors between apo- and folo-forms of hCRBP1
were very consistent with each other leading to the
conclusion that the higher back-bone mobility
occurs at the well-defined portal region of the
protein (Fig. 6). Importantly, these differences
were not seen among the holo-structures. Thus, the
ligand-free crystal structure most likely represents
a dominant, but only one of many possible
conformations of the apo-protein.
In addition to the magnitude of atomic
apo-structure,
structural role of waters in hCRBP1 (Fig. 5C).
Backbone dynamics of apo- and holo-
hCRBP1 – The conformational changes described
above seem counterintuitive because they provide
evidence for a significant steric hindrance that
prevents entrance of vitamin A into the apo-
protein’s binding pocket. Although crystals of
apo- and holo-hCRBP1 are not isomorphous,
underscoring
the
important
displacement, experimental anisotropic B-factors
provide information about preferred directions of
atomic movement. As analyzed by PARVATI
software, the mean anisotropy for all non-
hydrogen protein atoms in hCRBP1 structures are
0.37 (σ 0.11) for apo-, 0.35 (σ 0.10) for all-trans-
retinol holo-, and 0.40 (σ 0.12) for retinylamine-
bound proteins. Slightly lower anisotropy, 0.54 (σ
0.13) is observed for 0.89 Å holo-hCRBP1, which
correlates with exceptionally low thermal B-
factors for this structure. Nevertheless, these
values deviate significantly from 1, which refers to
perfect isotropy. Contrary to the Beq, the
anisotropy parameters revealed low variability
along the polypeptide chains and were not affected
by the ligand binding. Although analysis of the
Rosenfield plots (57) indicated that the quasi-rigid
body model cannot be applied to the parts of
hCRBP1 that form the portal region, it is
interesting to note significant changes in the
directional preference of individual atom
fluctuations in the turn connecting β-strands βE-βF
(Fig. 7). In addition to an increase in the level of
motion, movement of the main chain atoms in
dissimilarities
in
the
crystallographic
intermolecular contacts do not affect the protein
regions that reveal conformational changes. Thus,
the differences observed can be attributed to the
presence or absence of a ligand rather than crystal
packing. In an attempt to explain this
phenomenon, we closely examined the atomic
positions in the refined models. Contrary to the
structures of holo-hCRBP1 where the electron
density was very well defined throughout the
model, we found only satisfactory electron
densities for the main chain and poor electron
densities for part of the side chain in the portal
region of apo-protein (Fig. 6). This observation
suggests that some regions of the apo-hCRBP1
7

apo-hCRBP1 is characterized by the major
vibrational axes oriented towards and being almost
parallel to the anisotropy axes of loop βC-βD. This
dynamic flexibility in combination with positions
of the side chains of Phe57, Arg58, Tyr60, and
Ile77, can further contribute to narrowing the
aperture of the portal. Binding of vitamin A
greatly affects the conformation of the βE-βF turn
and induces a change in the orientation of the
major vibrational axes. They become nearly
perpendicular to that observed in the apo-structure
and are directed towards the retinoid molecule.
Overall, the above data provide evidence
suggests a pivotal role for local structural
dynamics in providing retinoid access to the
binding site and fosters a tempting speculation
about the putative mechanism for ligand
recognition. How then can retinoids find their way
to the binding cavity? In vivo, the interaction of
vitamin A with CRBP1 occurs in the vicinity of
lipid membranes. However experiments done in
vitro clearly indicate that the apo-protein binds all-
trans-retinol spontaneously without assistance of
specific micro-environments, lipid membranes or
other proteins. Thus, it seems plausible that the
initial interaction of the retinoid polyene chain
with the mobile portal region could shift the
equilibrium towards the latter’s more open
conformation and allow the ligand to slip deeper
into the binding cavity. Once inside, hydrophobic
interactions with the ligand’s β-ionone ring could
stabilize the conformation of the protein’s βC-βD
and βE-βF turns as well as α-helix II locking
vitamin A inside. These interactions appear to be
critical for discrimination between retinoids and
other hydrophobic ligands and thus could provide
binding specificity.
that binding of all-trans-retinol or retinylamine
increases local backbone stability, locking
hCRBP1 in a well-defined conformation.
DISCUSSION
The mechanistic basis for retinoid
interactions with CRBPs has been the subject of
extensive studies that have not yet converged into
a general model for ligand binding or release. The
major difficulty in formulating such a model was
due to the relatively stable conformation of the
apo-forms of CRBP2, 3, and 4 that are virtually
identical except for the side chains of certain
amino acids (10,11,55,58). Thus, these data did
not provide obvious evidence for the dynamic
portal entrance leading to the binding cavity.
However, the solution structures of rCRBP1 and
CRBP2 determined by multidimensional NMR
spectroscopy drew a different picture revealing
high local flexibility of the polypeptide main chain
that predominantly affected the βE-βF turn and to
a lesser degree the βC-βD loop (37,59). This
observation was further confirmed by mass
spectrometry methods indicating that a localized
backbone disorder resulted in increased rates of
hydrogen/deuterium exchange in these regions of
rCRBP1 (60).
Interestingly, similar conformational
changes were observed in zebrafish CRBP, an
ortholog of mammalian CRBP2 (56). Although the
exact positions of the side chain atoms of Phe57,
Arg58, Tyr60, and Ile77 (Leu77 in zebrafish)
differ from those found in apo-hCRBP1, they all
occupy some of the space reserved for retinol in
the holo-protein and thus had to be displaced upon
ligand binding. This correlation could indicate a
common evolutionarily conserved interaction
mechanism with retinoids in the CRBP family.
More intriguing than binding is the
question of targeted release of vitamin A from
CRBP1. It is tempting to speculate that ligand
liberation requires a transition back to the dynamic
state of the portal region or its alternative
conformation. Previous studies showed that
spontaneous all-trans-retinol dissociation could be
achieved at a relatively low pH (<4.0) or in a high
concentration of alcohols (>20%) (61).
Additionally, lipid membranes did not stimulate an
effective release. If we consider that vitamin A
needs to be transported from the plasma
membrane or its storage sites to specific
compartments within the cell, it becomes clear that
this cannot be a stochastic process. Thus, targeted
release of retinoids could be facilitated instead by
In contrast to previous crystallographic
data, the high resolution X-ray structures of
hCRBP1 reported herein revealed an unanticipated
conformational transformation in response to
ligand binding, that affected the side and main
chains which frame the ligand entry portal.
Surprisingly, comparison of the apo- and holo-
structures indicated that the dominant orientations
of the side chains spatially close to the internal
cavity blocked access to this site by narrowing the
aperture of the entrance. This scenario strongly
8

specific interactions of CRBP1 with its protein
binding partners, which are still unknown.
pocket, we attempted to crystalize hCRBP1 in
complex with all-trans-retinal. Unfortunately, the
protein crystals obtained revealed a very low
occupancy for this ligand. Thus, it might be that
the efficiency of all-trans-retinal binding is limited
by a residual network of water molecules in the
apo-protein that mediated hydrogen bonds
between Gln108 and Lys40, thereby limiting the
ability of the Gln108 side chain to rotate favorably
for aldehyde binding.
High-resolution structures of hCRBP1
allowed us to map hydrogen bond networks in the
binding site and investigate their role in ligand
affinity. The higher affinity for all-trans-retinol in
CRBP1 and 2 can be attributed to the formation of
a hydrogen bond between the retinol hydroxyl
group and ε1-carbonyl oxygen of Gln108. The
significance of this residue is underscored by the
fact that its substitution to histidine in CRBP3 and
4 lowered ligand affinity by a factor of ten (10,11).
By analyzing the structure of hCRBP1, we provide
convincing evidence that the canonical interaction
of all-trans-retinol with Gln108 is supplemented
by an additional hydrogen bond formed with the
amino group of Lys40 (Fig. 3B). The Lys40
interaction clearly contributes to the binding as
illustrated by the retinylamine example. The
inability for hydrogen bonding with Lys40
increased Kd values for retinylamine to 64.1 nM
from the 18.6 nM determined for all-trans-retinol
under identical experimental conditions (Fig. 4B).
Importantly, the affinity measured for retinylamine
corresponds closely to that calculated for all-trans-
retinal (Kd = 50 nM) (62). Because of the
potentially free rotation of the Gln108 side chain
in hCRBP1, the retinal carbonyl could form a
hydrogen bond with its ε2-amine group. However,
similar to retinylamine, the retinal carbonyl cannot
interact with Lys40. The above finding provided
additional evidence for the role of Lys40 in
hCRBP1 that ensures the highest affinity for
vitamin A among CRBPs. To shed more light on
the interaction of retinoids inside the binding
In conclusion, crystal structures of
hCRBP1 offer valuable new insights into the
structure-function relationships of the retinol-
binding protein family. The outcome of this study
explains importance of the entry portal region and
its involvement in conformational changes
associated with ligand binding and provides an
alternative view about the interactions of the
retinoid moiety in the hCRBP1 binding site.
Importantly, our data clarify some of the
controversies that have arisen about different
findings obtained with crystal and in-solution
structures of CRBPs.
ACKNOWLEDGMENTS
We thank L.T. Webster, Jr. for help in editing of this manuscript.
This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines,
which are funded by the National Institute of General Medical Sciences from the National Institutes of
Health (P41 GM103403). The Pilatus 6M detector on 24-ID-C beam line is funded by a NIH-ORIP HEI
grant (S10 RR029205). This research used resources of the Advanced Photon Source, a U.S. Department
of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne
National Laboratory under Contract No. DE-AC02-06CH11357.
We also thank staff of SLAC National Accelerator Laboratory, Menlo Park, CA, operated by Stanford
University for the U.S. Department of Energy Office of Science. Use of the Stanford Synchrotron
Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of
Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515.
The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and
9

Environmental Research, and by the National Institutes of Health, National Institute of General Medical
Sciences (including P41GM103393).
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest with the contents of this article.
AUTHOR CONTRIBUTIONS
M. G. conceived and designed the study. J. A. S., J. M. A., S. C., P. D. K., S. B., and M. G. performed
experiments and analyzed the data. M. G. wrote the manuscript with valuable input from J. A. S. and J.
M. A. All authors reviewed the results as well as edited and approved the final version of the manuscript.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
Alvarez, R., Vaz, B., Gronemeyer, H., and de Lera, A. R. (2014) Functions, therapeutic
applications, and synthesis of retinoids and carotenoids. Chemical reviews 114, 1‐125
Blomhoff, R., and Blomhoff, H. K. (2006) Overview of retinoid metabolism and function. Journal
of neurobiology 66, 606‐630
O'Byrne, S. M., and Blaner, W. S. (2013) Retinol and retinyl esters: biochemistry and physiology.
Journal of lipid research 54, 1731‐1743
Al Tanoury, Z., Piskunov, A., and Rochette‐Egly, C. (2013) Vitamin A and retinoid signaling:
genomic and nongenomic effects. Journal of lipid research 54, 1761‐1775
Noy, N. (2000) Retinoid‐binding proteins: mediators of retinoid action. The Biochemical journal
348 Pt 3, 481‐495
Kono, N., and Arai, H. (2015) Intracellular transport of fat‐soluble vitamins A and E. Traffic 16,
19‐34
Chytil, F., and Ong, D. E. (1983) Cellular Retinol‐Binding and Retinoic Acid‐Binding Proteins. Adv
Nutr Res 5, 13‐29
Schaap, F. G., van der Vusse, G. J., and Glatz, J. F. (2002) Evolution of the family of intracellular
lipid binding proteins in vertebrates. Molecular and cellular biochemistry 239, 69‐77
Bankaitis, V. A., Mousley, C. J., and Schaaf, G. (2010) The Sec14 superfamily and mechanisms for
crosstalk between lipid metabolism and lipid signaling. Trends in biochemical sciences 35, 150‐
160
10.
11.
12.
13.
14.
15.
Folli, C., Calderone, V., Ottonello, S., Bolchi, A., Zanotti, G., Stoppini, M., and Berni, R. (2001)
Identification, retinoid binding, and x‐ray analysis of a human retinol‐binding protein.
Proceedings of the National Academy of Sciences of the United States of America 98, 3710‐3715
Folli, C., Calderone, V., Ramazzina, I., Zanotti, G., and Berni, R. (2002) Ligand binding and
structural analysis of a human putative cellular retinol‐binding protein. The Journal of biological
chemistry 277, 41970‐41977
Eriksson, U., Das, K., Busch, C., Nordlinder, H., Rask, L., Sundelin, J., Sallstrom, J., and Peterson,
P. A. (1984) Cellular retinol‐binding protein. Quantitation and distribution. The Journal of
biological chemistry 259, 13464‐13470
Kato, M., Kato, K., and Goodman, D. S. (1984) Immunocytochemical studies on the localization
of plasma and of cellular retinol‐binding proteins and of transthyretin (prealbumin) in rat liver
and kidney. The Journal of cell biology 98, 1696‐1704
Saari, J. C., Buntmilam, A. H., Bredberg, D. L., and Garwin, G. G. (1984) Properties and
Immunocytochemical Localization of 3 Retinoid‐Binding Proteins from Bovine Retina. Vision
research 24, 1595‐&
Li, E., Demmer, L. A., Sweetser, D. A., Ong, D. E., and Gordon, J. I. (1986) Rat Cellular Retinol‐
Binding Protein‐Ii ‐ Use of a Cloned Cdna to Define Its Primary Structure, Tissue‐Specific
10

Expression, and Developmental Regulation. Proceedings of the National Academy of Sciences of
the United States of America 83, 5779‐5783
16.
17.
18.
19.
20.
Vogel, S., Mendelsohn, C. L., Mertz, J. R., Piantedosi, R., Waldburger, C., Gottesman, M. E., and
Blaner, W. S. (2001) Characterization of a new member of the fatty acid‐binding protein family
that binds all‐trans‐retinol. The Journal of biological chemistry 276, 1353‐1360
MacDonald, P. N., and Ong, D. E. (1987) Binding specificities of cellular retinol‐binding protein
and cellular retinol‐binding protein, type II. The Journal of biological chemistry 262, 10550‐
10556
Levin, M. S., Locke, B., Yang, N. C., Li, E., and Gordon, J. I. (1988) Comparison of the ligand
binding properties of two homologous rat apocellular retinol‐binding proteins expressed in
Escherichia coli. The Journal of biological chemistry 263, 17715‐17723
Kawaguchi, R., Yu, J., Ter‐Stepanian, M., Zhong, M., Cheng, G., Yuan, Q., Jin, M., Travis, G. H.,
Ong, D., and Sun, H. (2011) Receptor‐mediated cellular uptake mechanism that couples to
intracellular storage. ACS chemical biology 6, 1041‐1051
Kawaguchi, R., Yu, J., Honda, J., Hu, J., Whitelegge, J., Ping, P., Wiita, P., Bok, D., and Sun, H.
(2007) A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A.
Science 315, 820‐825
21.
22.
Herr, F. M., and Ong, D. E. (1992) Differential interaction of lecithin‐retinol acyltransferase with
cellular retinol binding proteins. Biochemistry 31, 6748‐6755
Ghyselinck, N. B., Bavik, C., Sapin, V., Mark, M., Bonnier, D., Hindelang, C., Dierich, A., Nilsson, C.
B., Hakansson, H., Sauvant, P., Azais‐Braesco, V., Frasson, M., Picaud, S., and Chambon, P. (1999)
Cellular retinol‐binding protein I is essential for vitamin A homeostasis. The EMBO journal 18,
4903‐4914
23.
24.
Saari, J. C., Nawrot, M., Garwin, G. G., Kennedy, M. J., Hurley, J. B., Ghyselinck, N. B., and
Chambon, P. (2002) Analysis of the visual cycle in cellular retinol‐binding protein type I (CRBPI)
knockout mice. Investigative ophthalmology & visual science 43, 1730‐1735
Golczak, M., Kuksa, V., Maeda, T., Moise, A. R., and Palczewski, K. (2005) Positively charged
retinoids are potent and selective inhibitors of the trans‐cis isomerization in the retinoid (visual)
cycle. Proceedings of the National Academy of Sciences of the United States of America 102,
8162‐8167
25.
26.
Maeda, A., Maeda, T., Golczak, M., and Palczewski, K. (2008) Retinopathy in mice induced by
disrupted all‐trans‐retinal clearance. The Journal of biological chemistry 283, 26684‐26693
Zhang, J., Dong, Z., Mundla, S. R., Hu, X. E., Seibel, W., Papoian, R., Palczewski, K., and Golczak,
M. (2015) Expansion of first‐in‐class drug candidates that sequester toxic all‐trans‐retinal and
prevent light‐induced retinal degeneration. Molecular pharmacology 87, 477‐491
Golczak, M., Imanishi, Y., Kuksa, V., Maeda, T., Kubota, R., and Palczewski, K. (2005)
Lecithin:retinol acyltransferase is responsible for amidation of retinylamine, a potent inhibitor of
the retinoid cycle. The Journal of biological chemistry 280, 42263‐42273
27.
28.
29.
Golczak, M., Sears, A. E., Kiser, P. D., and Palczewski, K. (2015) LRAT‐specific domain facilitates
vitamin A metabolism by domain swapping in HRASLS3. Nature chemical biology 11, 26‐32
Maeda, A., Maeda, T., Golczak, M., Imanishi, Y., Leahy, P., Kubota, R., and Palczewski, K. (2006)
Effects of potent inhibitors of the retinoid cycle on visual function and photoreceptor protection
from light damage in mice. Molecular pharmacology 70, 1220‐1229
30.
Sacchettini, J. C., Gordon, J. I., and Banaszak, L. J. (1989) Crystal structure of rat intestinal fatty‐
acid‐binding protein. Refinement and analysis of the Escherichia coli‐derived protein with bound
palmitate. Journal of molecular biology 208, 327‐339
11

31.
Sacchettini, J. C., Gordon, J. I., and Banaszak, L. J. (1989) Refined apoprotein structure of rat
intestinal fatty acid binding protein produced in Escherichia coli. Proceedings of the National
Academy of Sciences of the United States of America 86, 7736‐7740
32.
33.
Winter, N. S., Bratt, J. M., and Banaszak, L. J. (1993) Crystal structures of holo and apo‐cellular
retinol‐binding protein II. Journal of molecular biology 230, 1247‐1259
Cowan, S. W., Newcomer, M. E., and Jones, T. A. (1993) Crystallographic studies on a family of
cellular lipophilic transport proteins. Refinement of P2 myelin protein and the structure
determination and refinement of cellular retinol‐binding protein in complex with all‐trans‐
retinol. Journal of molecular biology 230, 1225‐1246
34.
Nossoni, Z., Assar, Z., Yapici, I., Nosrati, M., Wang, W., Berbasova, T., Vasileiou, C., Borhan, B.,
and Geiger, J. (2014) Structures of holo wild‐type human cellular retinol‐binding protein II
(hCRBPII) bound to retinol and retinal. Acta crystallographica. Section D, Biological
crystallography 70, 3226‐3232
35.
36.
37.
38.
Hodsdon, M. E., and Cistola, D. P. (1997) Ligand binding alters the backbone mobility of
intestinal fatty acid‐binding protein as monitored by 15N NMR relaxation and 1H exchange.
Biochemistry 36, 2278‐2290
Lu, J., Lin, C. L., Tang, C., Ponder, J. W., Kao, J. L., Cistola, D. P., and Li, E. (1999) The structure and
dynamics of rat apo‐cellular retinol‐binding protein II in solution: comparison with the X‐ray
structure. Journal of molecular biology 286, 1179‐1195
Franzoni, L., Lucke, C., Perez, C., Cavazzini, D., Rademacher, M., Ludwig, C., Spisni, A., Rossi, G. L.,
and Ruterjans, H. (2002) Structure and backbone dynamics of Apo‐ and holo‐cellular retinol‐
binding protein in solution. The Journal of biological chemistry 277, 21983‐21997
Xiao, H., and Kaltashov, I. A. (2005) Transient structural disorder as a facilitator of protein‐ligand
binding: native H/D exchange‐mass spectrometry study of cellular retinoic acid binding protein I.
J Am Soc Mass Spectrom 16, 869‐879
39.
40.
41.
42.
43.
44.
45.
Burger, A., Berendes, R., Voges, D., Huber, R., and Demange, P. (1993) A rapid and efficient
purification method for recombinant annexin V for biophysical studies. FEBS letters 329, 25‐28
Bridges, C. D. (1975) Storage, distribution and utilization of vitamins A in the eyes of adult
amphibians and their tadpoles. Vision research 15, 1311‐1323
Lakowicz, J. R. (1999) Principles of fluorescence spectroscopy, 2nd ed., Kluwer Academic/Plenum,
New York
Ong, D. E., and Chytil, F. (1978) Cellular retinol‐binding protein from rat liver. Purification and
characterization. The Journal of biological chemistry 253, 828‐832
Leslie, A. G. W., and Powell, H. R. (2007) Processing diffraction data with MOSFLM. Nato Sci Ser Ii
Math 245, 41‐51
Kabsch, W. (2010) Integration, scaling, space‐group assignment and post‐refinement. Acta
Crystallogr D 66, 133‐144
Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M.,
Krissinel, E. B., Leslie, A. G. W., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S.,
Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A., and Wilson, K. S. (2011) Overview of the
CCP4 suite and current developments. Acta Crystallogr D 67, 235‐242
46.
47.
48.
Emsley, P., and Cowtan, K. (2004) Coot: model‐building tools for molecular graphics. Acta
Crystallogr D 60, 2126‐2132
Bailey, S. (1994) The Ccp4 Suite ‐ Programs for Protein Crystallography. Acta Crystallogr D 50,
760‐763
Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung,
L. W., Kapral, G. J., Grosse‐Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R.
J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a
12

comprehensive Python‐based system for macromolecular structure solution. Acta Crystallogr D
66, 213‐221
49.
50.
51.
52.
Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L.
W., Richardson, J. S., and Richardson, D. C. (2010) MolProbity: all‐atom structure validation for
macromolecular crystallography. Acta Crystallogr D 66, 12‐21
Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., and
Ferrin, T. E. (2004) UCSF Chimera‐‐a visualization system for exploratory research and analysis.
Journal of computational chemistry 25, 1605‐1612
Schneider, B., Gelly, J. C., de Brevern, A. G., and Cerny, J. (2014) Local dynamics of proteins and
DNA evaluated from crystallographic B factors. Acta crystallographica. Section D, Biological
crystallography 70, 2413‐2419
Trueblood, K. N., Burgi, H. B., Burzlaff, H., Dunitz, J. D., Gramaccioli, C. M., Schulz, H. H., Shmueli,
U., and Abrahams, S. C. (1996) Atomic displacement parameter nomenclature ‐ Report of a
subcommittee on atomic displacement parameter nomenclature. Acta Crystallogr A 52, 770‐781
Merritt, E. A. (1999) Comparing anisotropic displacement parameters in protein structures. Acta
Crystallogr D 55, 1997‐2004
53.
54.
Newcomer, M. E., Liljas, A., Eriksson, U., Sundelin, J., Rask, L., and Peterson, P. A. (1981)
Crystallization of and preliminary X‐ray data for an intracellular vitamin A‐binding protein from
rat liver. The Journal of biological chemistry 256, 8162‐8163
55.
56.
Tarter, M., Capaldi, S., Carrizo, M. E., Ambrosi, E., Perduca, M., and Monaco, H. L. (2008) Crystal
structure of human cellular retinol‐binding protein II to 1.2 A resolution. Proteins 70, 1626‐1630
Calderone, V., Folli, C., Marchesani, A., Berni, R., and Zanotti, G. (2002) Identification and
structural analysis of a zebrafish apo and holo cellular retinol‐binding protein. Journal of
molecular biology 321, 527‐535
57.
58.
59.
60.
Rosenfield, R. E., Trueblood, K. N., and Dunitz, J. D. (1978) Test for Rigid‐Body Vibrations, Based
on a Generalization of Hirshfeld Rigid‐Bond Postulate. Acta Crystallogr A 34, 828‐829
Winter, N. S., Bratt, J. M., and Banaszak, L. J. (1993) Crystal‐Structures of Holo and Apo‐Cellular
Retinol‐Binding Protein‐Ii. Journal of molecular biology 230, 1247‐1259
Lu, J., Cistola, D. P., and Li, E. (2003) Two homologous rat cellular retinol‐binding proteins differ
in local conformational flexibility. Journal of molecular biology 330, 799‐812
Careri, M., Elviri, L., Mangia, A., Zagnoni, I., Torta, F., Cavazzini, D., and Rossi, G. L. (2006) Mass
spectrometry techniques for detection of ligand‐dependent changes in the conformational
flexibility of cellular retinol‐binding protein type I localized by hydrogen/deuterium exchange.
Rapid communications in mass spectrometry : RCM 20, 1973‐1980
61.
62.
Torta, F., Dyuysekina, A. E., Cavazzini, D., Fantuzzi, A., Bychkova, V. E., and Rossi, G. L. (2004)
Solvent‐induced ligand dissociation and conformational states of Cellular Retinol‐Binding Protein
type I. Biochimica et biophysica acta 1703, 21‐29
Li, E., Qian, S. J., Yang, N. C., d'Avignon, A., and Gordon, J. I. (1990) 19F nuclear magnetic
resonance studies of 6‐fluorotryptophan‐substituted rat cellular retinol binding protein II
produced in Escherichia coli. An analysis of four tryptophan substitution mutants and their
interactions with all‐trans‐retinol. The Journal of biological chemistry 265, 11549‐11554
FOOTNOTES
§This work was supported by grants EY023948 from the National Eye Institute of the National Institutes
of Health (NIH) (to M.G.) and IK2BX002683 Career Development Award from the Department of
Veterans Affairs (to P.D.K.).
#equal contributions
13

1The abbreviations used are: Beq, equivalent isotropic crystallographic B-factors; CRABP, cellular retinoic
acid-binding protein; CRALBP, cellular retinaldehyde-binding protein; CRBPs, family of cellular retinol-
binding proteins; DMSO, dimethyl sulfoxide; hCRBP1, human cellular retinol-binding protein 1; HPLC,
high-performance liquid chromatography; iLBPs, intracellular lipid-binding proteins; LRAT,
lecithin:retinol acyltransferase; rCRBP1, rat cellular retinol-binding protein 1; RMSD, root-mean-square
deviation; RPE, retinal pigment epithelium.
14

FIGURE LEGENDS
Figure 1 – Purification of recombinant hCRBP1 and formation of complexes with all-trans-retinol and
retinylamine. A, Elution profile of apo- (a), all-trans-retinol- (b), and retinylamine-bound (c) proteins
from a Mono-Q ion exchanger column. Solid and dashed traces represent absorbance at 280 nm and 325
nm, respectively. Inset shows SDS-PAGE of purified hCRBP1 after Coomassie R-250 staining. The
arrowhead indicates the position of expressed protein in a 12% polyacrylamide gel. B, the absorbance
spectra of hCRBP1 in complex with all-trans-retinol (solid line) and retinylamine (dashed line) collected
in 10 mM Tris/HCl buffer, pH 8.0.
Figure 2 – Crystal structure of all-trans-retinol holo-hCRBP1. A, Ribbon diagrams of overlay structures
of hCRBP1 depicted in light blue color (PDB # 5HBS) and rCRBP1 colored in gray (PDB # 1CRB). B,
Electron density maps for all-trans-retinol and the ligand geometry. In the top panel, the green mesh
represents an unbiased 0.89 Å resolution σA-weighted Fo – Fc omit electron density map contoured at
6.0σ. The appearance of a strong positive electron density clearly indicates the location of the retinoid
moiety. The gray mesh corresponds to the 2Fo – Fc electron density map contoured at 2.0σ. The middle
panel signifies a refined 2Fo – Fc electron density map for the ligand at 1.6σ shown in blue. The bottom
panel shows torsion angles between carbons C12-C13/C14-C15 of the retinoid moiety found in the X-ray
structures of hCRBP1 (orange) and human CRBP2 (gray), PDB # 5HBS and PDB # 4QZT, respectively.
C, Molecular organization of the retinoid-binding pocket with selected residues proposed to be involved
in formation of the internal cavity.
Figure 3 – Interaction of the hydroxyl group of vitamin A in the binding pocket. A, Orientation of the side
chains and local interactions of Lys40 and Gln108. The side chain of Lys40 in rCRBP1 is shown in
salmon color, whereas the side chains of the human protein are tinted in light blue. Blue mesh represents
the 2Fo – Fc electron density map for the side chain of Lys40 in hCRBP1 contoured at 2.0σ. Hydrogen
bonds are indicated by dashed lines. Distances are shown in Å. B, The hydrogen bond network in the
retinoid-binding pocket of hCRBP1. Water molecules are shown as red spheres.
Figure 4 – Binding of retinylamine to hCRBP1. A, To determine the retinoid composition carried by
hCRBP1 pre-incubated with retinylamine, the protein was extracted with ethyl acetate and the retinoids
were analyzed by reverse phase HPLC. The chromatograms revealed a single peak (black trace) with an
elution time and UV/Vis absorbance spectrum (inset) that perfectly match that of a synthetic retinylamine
standard (gray trace). Similar analyses were performed on the protein crystals also revealed a robust peak
corresponding to retinylamine (dashed trace). B, An overlay of the side chains at the binding site of
hCRBP1 in complex with all-trans-retinol (light blue, PDB # 5HBS) and retinylamine (light orange, PDB
# 5HA1). Blue mesh represents an 2Fo – Fc electron density map for retinylamine contoured at 1.6σ. C,
Determination of the apparent dissociation constants for all-trans-retinol and retinylamine. Quenching of
the maximum fluorescence emissions upon increasing concentrations of all-trans-retinol (▲) or
retinylamine (●) were monitored at 337 nm, corrected for background and inner filter effects, and used to
calculate the apparent Kd values, which equal 18.5 ± 1.1 nM for all-trans-retinol and 64.1 ± 16.9 nM for
retinylamine. The inset represents an expanded titration curve for all-trans-retinol. All experiments were
repeated three times in duplicate. Data are presented as mean values ± standard deviations.
Figure 5 – Overview of the structure of apo-hCRBP1 and its comparison to the holo-form. A, Ribbon
representation of superimposed structures of the all-trans-retinol holo- (PDB # 5HBS) and apo-protein
(PDB # 5H9A) colored in light blue and orange, respectively. Hairpin turns between β-strands βE-βF and
α-helix II reveal the highest level of conformational alterations. B, α-Carbon RMSD deviations calculated
between apo- and all-trans-retinol holo-protein as a function of residue position. RMSDs for amino acids
that belong to the βE-βF turn and α-helix II are marked. C, Differences in conformation of the side chains
in hCRBP1 induced by ligand binding (holo-protein, light blue; apo-form, light orange). Residues that
15

show the most significant changes are labeled. The side chains of Phe57, Arg 58, Tyr60, and Ile77 that
contribute to steric hindrance in the apo-protein are labeled with darker colors. Their shifts in position are
marked by arrows. D, The hydrogen bond network in apo-hCRBP1. All waters are represented by red
spheres. The water molecule that is located at the position of a hydroxyl group of all-trans-retinol in holo-
protein is shown with a larger diameter.
Figure 6 – Differences in the equivalent isotropic B-factors between apo- and holo-hCRBP1. A, Graphic
representation of the residue average Beq in all-trans-retinol holo-protein (left, PDB # 5HBS), its
retinylamine-bound state (middle, PDB # 5HA1), and apo-form (right, PDB # 5H9A) colored from low to
high (blue to red). Regions of the apo-hCRBP1 with the highest conformational flexibility are identified
by labels. B, 2Fo – Fc electron density maps (blue mesh) of α-helix II and hairpin turns βC-βD and βE-βF
for a 1.21 Å structure of all-trans-retinol holo-hCRBP1 (left) (PDB # 5H8T) and apo-protein (right).
Purple mesh represents electron density for the ligand. All maps were contoured at 1.2σ. C, Comparison
of the normalized Beq. The plot represents relative differences and indicates regions with increased
conformational dynamics. The color scheme corresponds to the following paired structures: green – apo-
(PDB # 5H9A) vs. vitamin A holo- (0.89 Å resolution structure, PDB # 5HBS), blue – apo- (PDB #
5H9A) vs. vitamin A holo- (1.21 Å, PDB # 5H8T), red – apo- (PDB # 5H9A) vs. retinylamine holo-
(PDB # 5HA1), and gray – vitamin A holo- (PDB # 5H8T) vs. retinylamine holo-hCRBP1 (PDB #
5HA1).
Figure 7 – Changes in the directional mobility of the portal region upon the ligand binding. Individual
atom anisotropic thermal ellipsoids drown at 25% probability level for the backbone atoms of all-trans-
retinol-bond hCRBP1, PDB # 5H8T (A) and its apo-form, PDB # 5H9A (B). The orientations of the major
vibrational axis for selected atoms in the βE-βF loop are ma rked with arrows. The length of arrows is
arbitrarily exaggerated for clarity. The color scheme is the following: red – oxygen, blue – nitrogen, grey
– carbon. Vitamin A molecule is colored orange.
16

Table 1 – X-ray data collection and refinement statistics.
CRBP1
(all-trans-retinol)
CRBP1
(all-trans-retinol)
CRBP1
(retinylamine)
CRBP1
(APO)
PDB accession code
5HBS
5H8T
5HA1
5H9A
Beam line
Wavelength (Å)
BL12-2
0.7293
24-ID-C
0.9792
24-ID-E
0.9792
24-ID-C
0.9792
Data collection
Space group
Cell dimensions
a, b, c (Å)
()
Resolution (Å)
P212121
P212121
P212121
P212121
37.22, 49.77, 73.48
90, 90, 90
37.50, 44.99, 84.97 37.15, 49.57, 73.30 37.35, 37.96, 94.96
90, 90, 90
37.55-1.21
(1.23-1.21)1,2
7.4 (50.4)2
10.6 (1.9)2
99.4 (98.5)2
5.0 (4.2)2
90, 90, 90
27.55-1.35
(1.32-1.35)1,2
10.3 (60.4)2
9.4 (3.2)2
90, 90, 90
47.48-1.38
(1.41-1.38)1,2
8.2 (59.9)2
11.0 (2.8)2
98.9 (98.9)2
5.3 (4.6)2
33.20-0.89
(0.94-0.89)1,2
8.8 (89.7)2
Rsym (%)
I / I
Completeness (%)
Redundancy
24.5 (3.2)2
98.7 (92.7)2
12.4 (10.4)2
100 (100)2
5.9 (5.9)2
Refinement
Resolution (Å)
No. reflections
33.20-0.89
104073
12.5/13.3
1580
34.35-1.21
44760
11.8/14.7
1529
27.55-1.35
30442
12.2/15.6
1502
35.25-1.38
28067
13.3/17.1
1362
Rwork / Rfree (%)
No. atoms
Protein
Ligand
1223
1198
1196
1180
21 (RTL)3
336
21 (RTL)3
310
21 (RNE)3
285
14 (BTB)3
168
Water
Mean B-factor (Å2)
Protein
6.2
9.6
8.3
18.8
Ligand
Water
6.9 (RTL)3
14.9
10.1 (RTL)3
25.5
15.1 (RNE)3
17.2
20.5 (BTB)3
30.5
R.m.s. deviations
Bond lengths (Å)
Bond angles ()
0.005
0.989
0.009
1.194
0.010
1.131
0.011
1.123
Validation
Ramachandran plot
Favored/outliers (%)
98.0/04
97.9/04
98.6/04
99.3/04
1Reported data set was collected on a single crystal
2Highest-resolution shell is shown in parentheses
3Ligand’s accession code
4As defined by MolProbity
17

Table 2 – Root mean square deviations (RMSD) between various holo-and apo-forms of CRBP1.
holo
holo
holo
(retinylamine)
PDB # 5HA1
apo
rat holo
(retinol)
PDB # 1CRB
(retinol) 0.89 Å (retinol) 1.21 Å
PDB # 5HBS
RMSD (Å)
PDB # 5H8T
PDB # 5H9A
holo (retinol) 0.89 Å
PDB # 5HBS
holo (retinol) 1.21 Å
PDB # 5H8T
holo (retinylamine)
PDB # 5HA1
0.42
0.15
0.33
1.84
0.83
0.70
0.77
1.79
0.38
0.27
1.07
0.89
1.71
1.76
0.29
0.92
0.65
apo
0.97
PDB # 5H9A
rat holo (retinol)
PDB # 1CRB
0.83
1.08
RMSD values were calculated for the main-chain atoms of entire polypeptide chains (black) and the
portal regions (residues 24 – 36, 53 – 60, and 73 – 81) (italic) of available CRBP1 structures. RMSD
values for apo- vs. holo- pairs are shown in bold. All values were calculated using Baverage software
available in CCP4 package.
18

Figure 1
19

Figure 2
20

Figure 3
21

Figure 4
22

Figure 5
23

Figure 6
24

Figure 7
25

Ligand Binding Induces Conformational Changes in Human Cellular Retinol-Binding
Protein 1 (CRBP1) revealed by atomic resolution crystal structures
Josie A. Silvaroli, Jason M. Arne, Sylwia Chelstowska, Philip D. Kiser, Surajit Banerjee
and Marcin Golczak
J. Biol. Chem. published online February 21, 2016
Access the most updated version of this article at doi: 10.1074/jbc.M116.714535
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