Acta Cryst. (2009). F65, 621–624doi:10.1107/S1744309109017023
Acta Crystallographica Section F
Crystallization and preliminary X-ray studies of the
N-domain of the Wilson disease associated protein
Lili Liu,‡ Christopher O’Grady,‡
Sean A. Dalrymple, Lata Prasad,
Oleg Y. Dmitriev* and Louis T. J.
Department of Biochemistry, University of
Saskatchewan, Saskatoon, Saskatchewan,
S7N 5E5, Canada
‡ These authors contributed equally to this
Received 9 March 2009
Accepted 5 May 2009
Wilson disease associated protein (ATP7B) is essential for copper transport in
human cells. Mutations that affect ATP7B function result in Wilson’s disease, a
chronic copper toxicosis. Disease-causing mutations within the N-domain of
ATP7B (WND) are known to disrupt ATP binding, but a high-resolution X-ray
structure of the ATP-binding site has not been reported. The N-domain was
modified to delete the disordered loop comprising residues His1115–Asp1138
(WND?1115–1138). Unlike the wild-type N-domain, WND?1115–1138 formed
good-quality crystals. Synchrotron diffraction data have been collected from
WND?1115–1138at the Canadian Light Source. A native WND?1115–1138crystal
diffracted to 1.7 A˚resolution and belonged to space group P42212, with unit-cell
parameters a = 39.2, b = 39.2, c = 168.9 A˚. MAD data were collected to 2.7 A˚
resolution from a SeMet-derivative crystal with unit-cell parameters a = 38.4,
b = 38.4, c = 166.7 A˚. The WND?1115–1138structure is likely to be solved by
phasing from multiwavelength anomalous diffraction (MAD) experiments.
Wilson’s disease is an autosomal recessive disorder that results from
mutations in the ATP7B gene. Reduced copper efflux out of cells is
the primary defect (Culotta & Gitlin, 2001). The associated protein
ATP7B is a P-type ATPase that consists of a single polypeptide chain
of 1465 amino-acid residues (Cox & Moore, 2002; Lutsenko et al.,
2007; Pedersen & Carafoli, 1987). P1B-type ATPases such as ATP7B
maintain metal homeostasis in bacteria and eukaryotic cells (Rosen,
2002). ATP7B is composed of eight transmembrane helices and four
cytosolic domains (the N-terminal, A-, P- and N-domains; Arguello et
al., 2007). The isolated N-domain of ATP7B (residues 1032–1196) has
no ATPase activity, but does bind ATP, ADP and AMP specifically
(Morgan et al., 2004). Early attempts to crystallize the complete
N-domain were unsuccessful and the high-resolution structure was
determined by NMR (Dmitriev et al., 2006). The NMR structure of
the N-domain (Fig. 1) revealed a folded core consisting of a six-
stranded ?-sheet and two flanking ?-helical hairpins and a flexible
loop comprising residues Ala1114–Gln1142 (the residue numbering
corresponds to that of the full-length ATP7B protein; Dmitriev et al.,
2006). Several invariant residues in the N-domain of P1B-ATPases,
Glu1064, His1069, Gly1099 and Gly1101, and the highly conserved
Gly1149 were found to be located in the ATP-binding site. The exact
nature of the ATP–protein interactions in the binding site could not
be determined from the NMR data. A high-resolution X-ray struc-
ture of the complex of ATP bound to the N-domain would reveal
these interactions at the atomic level and provide a structural basis
for the design of highly specific modulators of Wilson disease
The long loop comprising residues 1114–1142 is not involved in
ATP binding (Dmitriev et al., 2006) and there are no known Wilson’s
disease mutations in this region (Hsi & Cox, 2004), which implies that
this region is not critical for enzyme function and that the core
N-domain structure would not be altered by deletion of this loop. At
# 2009 International Union of Crystallography
All rights reserved
the same time, the disordered state of this loop may have prevented
successful crystallization of the full-length N-domain. To improve the
chances of crystal formation, we deleted a fragment of the ATP7B
gene corresponding to residues His1115–Asp1138. In the resulting
protein WND?1115–1138, the length of the loop connecting the short
from 29 to five amino-acid residues (Fig. 1). The preparation and
crystallization of WND?1115–1138is described in the present com-
2. Materials and methods
2.1. Protein modification, expression and purification
To generate the His1115–Asp1138 deletion, the pTYB12-NABD
plasmid (Morgan et al., 2004) used for the expression of the full-
length N-domain was amplified by the around-the-plasmid PCR. The
primers GCA GTC CCC CAG ACC TTC TCT (forward) and GGC
CAG GAT GCC TTC CAC GTT (reverse) were designed to anneal
immediately outside the region intended for deletion. Both primers
were 50-phosphorylated. The amplified DNA product produced using
these primers corresponded to the complete sequence of the plasmid
with the exception of the deletion region. A total of 30 PCR cycles
were performed with annealing at 328 K and elongation at 345 K for
150 s using the Phusion DNA polymerase kit (New England Biolabs).
The 7.9 kb PCR product was purified by agarose gel electrophoresis
and ligated overnight at 289 K with T4 ligase (New England Biolabs).
DH5? cells were transformed with the ligation mixture and the
transformants were selected on LB plates supplemented with
100 mg ml?1ampicillin. Plasmid DNA isolated from the selected
clones was checked for the correct restriction pattern (7.1 and 0.8 kb
fragments) with NheI and KpnI enzymes, sequenced and transformed
into Escherichia coli BL21 (DE3) for protein expression. Expression
and purification of the modified N-domain was performed essentially
as described previously (Dmitriev et al., 2006; Morgan et al., 2004).
Briefly, the N-domain was expressed as a fusion protein with a chitin-
binding domain and an intein. The fusion protein was bound to chitin
beads (New England Biolabs) and contaminating proteins were
removed by washing the column. Intein cleavage was then induced by
adding dithiothreitol. Pure N-domain was eluted from the column,
whereas the fragment containing the chitin-binding domain and the
intein remained bound to the beads. To produce SeMet-substituted
protein, the M63 medium used for protein expression was supple-
mented with 50 mg l?1l-selenomethionine (Guerrero et al., 2001).
For protein crystallization, samples of the modified N-domain were
additionally purified by size-exclusion chromatography on a
Superdex-75 HK 16/60 column (Pharmacia) in 50 mM sodium phos-
phate buffer pH 6.0 containing 100 mM NaCl and 5 mM DTT. The
resulting fractions containing modified N-domain protein were
pooled and concentrated to 15 mg ml?1before being stored at 277 K.
2.2. NMR spectroscopy
The1H–15N HSQC spectra of WND?1115–1138were recorded at
300 K on a Bruker 600 MHz spectrometer equipped with a Cryo-
probe and z-axis gradients. The sample contained 0.5 mM protein,
50 mM sodium phosphate buffer pH 6.0, 5%(v/v) D2O, 5 mM DTT,
0.05 mM NaN3 and 0.5 mM 2,2-dimethyl-2-silapentane-5-sulfonic
acid (DSS) for chemical shift referencing.
2.3. Protein crystallization
Initial crystallization conditions for the WND?1115–1138 protein
were identified by high-throughput screening at the Hauptman–
Woodward Medical Research Institute using the microbatch-under-
oil method (Luft et al., 2003). Subsequent optimization of the crys-
tallization conditions resulted in WND?1115–1138 crystals being
obtained from a drop consisting of equal volumes (1 ml) of protein
solution (15 mg ml?1protein, 50 mM NaH2PO4 pH 6.0, 100 mM
NaCl, 0.05 mM NaN3and 5 mM DTT) and precipitant solution (30%
PEG 8000, 200 mM sodium citrate pH 5.0 and 200 mM NH4SCN)
which had been covered in 100% mineral oil at room temperature.
Octahedral shaped crystals appeared within 2 d and continued to
grow over the next few days to approximately 0.43 mm in the largest
dimension (Fig. 2). The crystals were harvested into cryosolution
[220 mM glucose, 525 mM sucrose, 16%(v/v) glycerol and 16%(v/v)
ethylene glycol] prior to flash-cooling in liquid nitrogen. For the
selenomethionine-derivative crystals, drops were formed by mixing
equal volumes (1 ml) of protein solution (12.5 mg ml?1protein,
50 mM NaH2PO4pH 6.0, 100 mM NaCl, 0.05 mM NaN3and 5 mM
Na3C3H5O(COO)3pH 5.0 and 200 mM NH4SCN] and were then
covered with 100% mineral oil at room temperature. Octahedral
[37%PEG8000, 200 mM
Liu et al.
? N-domain of Wilson disease protein
Acta Cryst. (2009). F65, 621–624
Crystals of WND?1115–1138. The largest dimension is approximately 0.43 mm.
The previously reported NMR structure of the N-domain (PDB code 2arf)
modified to show the deletion of the His1115–Asp1138 loop. The invariant residues
involved in ATP binding are shown in magenta (Dmitriev et al., 2006).
shaped crystals appeared after 3 d and continued to grow over the
next few days toapproximately 0.38 mm in the largest dimension. The
crystals were subsequently flash-cooled without the need for addi-
tional cryoprotectant owing to the high concentration of PEG in the
2.4. Data collection and processing
Diffraction of the WND?1115–1138protein crystal took place on
the Canadian Macromolecular Crystallography Facility (CMCF-1)
beamline (08ID-1) at the Canadian Light Source (Saskatoon,
Saskatchewan, Canada). The native data set consisted of 180 images,
each of which was collected with 1 s exposure over a 1?oscillation
range at a crystal-to-detector distance of 160 mm. The intensity data
were indexed, integrated and scaled using XDS (Kabsch, 1993). For
the selenomethionine-derivative crystal, multiwavelength anomalous
diffraction (MAD) data were collected based on wavelengths
selected from analysis of the selenium absorption spectrum. Each of
these data sets consisted of 180 images with 1 s exposure and 1?
oscillation at a crystal-to-detector distance of 180 mm. The intensity
data were indexed, integrated and scaled with the HKL-2000 suite of
programs including DENZO and SCALEPACK (Otwinowski &
Minor, 1997). A summary of the crystallographic statistics for both
the native and SeMet-derivative data sets is given in Table 1.
3. Results and discussion
A photomicrograph of a crystal of WND?1115–1138protein is shown in
Fig. 2 and one image of the diffraction pattern is illustrated in Fig. 3.
showed excellent chemical shift dispersion characteristic of a well
folded protein with a high content of ?-sheet structure. The spectrum
was very similar to the spectrum of the wild-type N-domain, with the
exception of the signals from residues His1115–Asp1138, which were
absent, and the signals of the amino-acid residues in the immediate
proximity of the deletion site, which were shifted. No significant
chemical shift changes were observed for the invariant residues of the
P1B-type ATPases, Glu1064, Glu1068, Gly1099 and Gly1101, which
are involved in ATP binding. These data indicate that the three-
dimensional structure of the folded core is very similar in the wild-
type N-domain and in the WND?1115–1138variant of the protein.
Consistent with this conclusion, the ATP-binding affinity of the
N-domain was unaffected by the loop deletion. The dissociation
constants for ATP measured by chemical shift perturbation assay
1H–15N HSQC spectrum of WND?1115–1138(Fig. 4)
Acta Cryst. (2009). F65, 621–624 Liu et al.
? N-domain of Wilson disease protein
Data-collection statistics for native and SeMet-derivative crystals.
Values in parentheses are for the highest resolution shell.
Unit-cell parameters (A˚)
Solvent content (%)
Unit-cell volume (A˚3)
Molecular weight (Da)
Molecules per ASU
Resolution range (A˚)
ijIiðhklÞ ? hIðhklÞij=P
iIiðhklÞ, where hI(hkl)i is the average
intensity over symmetry-related reflections and Ii(hkl) is the measured intensity.
Diffraction image of a WND?1115–1138crystal.
1H–15N HSQC spectra of wild-type N-domain (red) and the loop-deletion variant
WND?1115–1138(black) recorded in the presence of 5 mM ATP. The signals of the
backbone amides of the invariant amino-acid residues are circled. The signal of
His1069 at 8.45 and 125.4 p.p.m. (wild type) is located in a crowded area of the
spectrum and is not shown.
using Gly1101 as a reporter group (Dmitriev et al., 2006) were found
to be in the range 50–70 mM for both the wild-type N-domain and the
WND?1115–1138variant. This suggested WND?1115–1138as a suitable
model protein for the high-resolution structure determination of the
ATP-binding site in the Wilson disease associated protein.
Unfortunately, initial attempts to solve the native WND?1115–1138
structure via Phaser (Storoni et al., 2004) by molecular replacement
using the NMR model of WND (PDB code 2arf) or with the
N-domain of the Archaeoglobus fulgidus Cu+-ATPase (Sazinsky et al.,
2006), which shares 44% sequence homology with WND?1115–1138,
were both unsuccessful. In an effort to solve the structure through
MAD phasing experiments, selenomethionine-derivatized protein
was produced, purified and crystallized. Once the SeMet-derivative
structure has been solved, the resulting model will be employed to
solve the WND?1115–1138 structure by molecular replacement.
Currently, efforts are also under way to produce additional heavy-
atom derivatives to aid in solving the WND?1115–1138structure.
LTJD is a Tier 1 Canada Research Chair in Structural Biochem-
istry. SAD is the recipient of an NSERC Postdoctoral Fellowship.
This research was funded by a Saskatchewan–CIHR Regional Part-
nership Operating Grant to OYD and a Saskatchewan–CIHR
Regional Partnership Operating Grant as well as an NSERC
Discovery Grant to LTJD and in part by a Saskatchewan Health
Research Foundation Team Grant to the Molecular Design Research
Group at the University of Saskatchewan. The authors would like to
thank the team at the CMCF-1 beamline for technical assistance at
the CLS, which is supported by NSERC, NRC, CIHR and the
University of Saskatchewan, and both Eva-Maria Uhlemann and
Yvonne Leduc for excellent technical support.
Arguello, J. M., Eren, E. & Gonzalez-Guerrero, M. (2007). Biometals, 20,
Cox, D. W. & Moore, S. D. (2002). J. Bioenerg. Biomembr. 34, 333–338.
Culotta, V. C. & Gitlin, J. D. (2001). The Molecular Basis of Inherited Disease,
8th ed., edited by C. R. Scriver, A. L. Beaudet, W. S. Sly & D. Valle, pp.
3205–3126. New York: McGraw–Hill.
Dmitriev, O., Tsivkovskii, R., Abildgaard, F., Morgan, C. T., Markley, J. L. &
Lutsenko, S. (2006). Proc. Natl Acad. Sci. USA, 103, 5302–5307.
Guerrero, S. A., Hecht, H.-J., Hofmann, B., Biebl, H. & Singh, M. (2001). Appl.
Microbiol. Biotechnol. 56, 718–723.
Hsi, G. & Cox, D. W. (2004). Hum. Genet. 114, 165–172.
Kabsch, W. (1993). J. Appl. Cryst. 26, 795–800.
Luft, J. R., Collins, R. J., Fehrman, N. A., Lauricella, A. M., Veatch, C. K. &
DeTitta, G. T. (2003). J. Struct. Biol. 142, 170–179.
Lutsenko, S., Barnes, N. L., Bartee, M. Y. & Dmitriev, O. Y. (2007). Physiol.
Rev. 87, 1011–1046.
Morgan, C. T., Tsivkovskii, R., Kosinsky, Y. A., Efremov, R. G. & Lutsenko, S.
(2004). J. Biol. Chem. 279, 36363–36371.
Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307–326.
Pedersen, P. L. & Carafoli, W. (1987). Trends Biochem. Sci. 12, 146–150.
Rosen, B. P. (2002). Comput. Biochem. Physiol. A Mol. Integr. Physiol. 133,
Sazinsky, M. H., Mandal, H. K., Argu ¨ello, J. M. & Rosenzweig, A. C. (2006). J.
Biol. Chem. 281, 11161–11166.
Storoni, L. C., McCoy, A. J. & Read, R. J. (2004). Acta Cryst. D60, 432–438.
Liu et al.
? N-domain of Wilson disease protein
Acta Cryst. (2009). F65, 621–624