THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 16, pp.
11161–11166, April 21, 2006
Structure of the ATP Binding Domain from the
Archaeoglobus fulgidus Cuⴙ-ATPase*□
Received for publication, September 30, 2005, and in revised form, December 27, 2005 Published, JBC Papers in Press, February 22, 2006, DOI 10.1074/jbc.M510708200
Matthew H. Sazinsky‡1, Atin K. Mandal§, José M. Argüello§2, and Amy C. Rosenzweig‡3
From the ‡Departments of Biochemistry, Molecular Biology, and Cell Biology and of Chemistry, Northwestern University,
Evanston, Illinois 60208 and the §Department of Chemistry and Biochemistry, Worcester Polytechnic Institute,
Worcester, Massachusetts 01609
The P-type ATPases translocate cations across membranes using
the energy provided by ATP hydrolysis. CopA from Archaeoglobus
fulgidus is a hyperthermophilic ATPase responsible for the cellular
export of Cuⴙ and is a member of the heavy metal P1B-type ATPase
subfamily, which includes the related Wilson and Menkes diseases
proteins. The Cuⴙ-ATPases are distinct from their P-type counter-
parts in ion binding sequences, membrane topology, and the pres-
ence of cytoplasmic metal binding domains, suggesting that they
employ alternate forms of regulation and novel mechanisms of ion
transport. To gain insight into Cuⴙ-ATPase function, the structure
of the CopA ATP binding domain (ATPBD) was determined to 2.3
Å resolution. Similar to other P-type ATPases, the ATPBD includes
nucleotide binding (N-domain) and phosphorylation (P-domain)
domains. The ATPBD adopts a closed conformation similar to the
nucleotide-bound forms of the Ca2ⴙ-ATPase. The CopA ATPBD is
much smaller and more compact, however, revealing the minimal
elements required for ATP binding, hydrolysis, and enzyme phos-
phorylation. Structural comparisons to the AMP-PMP-bound
form of the Escherichia coli Kⴙ-transporting Kdp-ATPase and to
the Wilson disease protein N-domain indicate that the five con-
served N-domain residues found in P1B-type ATPases, but not in the
other families, most likely participate in ATP binding. By contrast,
the P-domain includes several residues conserved among all P-type
ATPases. Finally, the CopA ATPBD structure provides a basis for
understanding the likely structural and functional effects of various
mutations that lead to Wilson and Menkes diseases.
The P-type ATPases encompass a large family of integral membrane
proteins that couple ATP hydrolysis with the transport of cations across
cell membranes (1, 2). Within this protein family, members of the P1B
subgroup specifically transport transition metal ions including Cu⫹/
Ag⫹ or Zn2⫹/Cd2⫹/Pb2⫹ (3, 4). Widely distributed in nature, the P1B-
type ATPases confer heavy metal tolerance to microorganisms (5, 6)
and are essential for the absorption, distribution, and bioaccumulation
of metal micronutrients by multicellular eukaryotes (7, 8). The impor-
* This work was supported in part by NIGMS National Institutes of Health Grant GM58518
(to A. C. R.) and National Science Foundation Grant MCM-0235165 (to J. M. A.). The
costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
□S
The on-line version of this article (available at http://www.jbc.org) contains supple-
mental Figs. S1 and S2.
The atomic coordinates and structure factors (code 2B8E) have been deposited in the Protein
Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New
Brunswick, NJ (http://www.rcsb.org/).
1 4
Supported by National Research Service Award Fellowship GM073457.
2
To whom correspondence may be addressed. Tel.: 508-831-5326; Fax: 508-831-5933;
E-mail: arguello@wpi.edu.
3
To whom correspondence may be addressed. Tel.: 847-467-5301; Fax: 847-467-6489;
E-mail: amyr@northwestern.edu.
APRIL 21, 2006 • VOLUME 281 • NUMBER 16
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
S
tance of these enzymes for metal homeostasis is underscored by the two
Cu⫹-ATPases present in humans, ATP7A (MNK) and ATP7B (WND)
(7, 9). Mutations in the genes encoding the ATP7A and ATP7B proteins
lead to Menkes and Wilson diseases, respectively. Menkes disease is
characterized by impaired transport of dietary copper across the small
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intestine, resulting in a copper deficiency in peripheral tissues.
Improper function of WND leads to toxic copper accumulation in the
liver because of reduced biliary excretion (10, 11).
Although all P-type ATPases share essential structural elements, the
subgroups differ in the number of predicted transmembrane helices and
the arrangement of these segments with respect to the cytoplasmic ATP
binding domain (ATPBD).4 In addition, the soluble regions vary in
length and composition (4, 5, 12). The P1B-type ATPases comprise eight
transmembrane helices, a large ATPBD, an actuator domain (A-do-
main), and N- or C-terminal soluble metal binding domains (MBDs).
Binding sites responsible for metal coordination during transport are
located within the membrane and are believed to involve a CPX
sequence motif as well as other key residues that confer metal ion selec-
tivity (3, 13). The N-terminal MBDs (N-MBDs), ranging from one to six
depending on the organism, receive copper ions from metallochaper-
ones (14), participate in ATPase regulation (15), and facilitate intracel-
lular relocalization (16). In the absence of copper, the six WND
N-MBDs interact with the ATPBD (17), suggesting distinct mecha-
nisms of regulation by substrates in P1B-type ATPases.
The P1B-type ATPase transport mechanism follows the classical Post-
Albers E1/E2 model (7, 18, 19). The central element of this catalytic
cycle is the coupling of metal transport to enzyme phosphorylation by
ATP. Phosphorylation of the aspartic acid residue from the signature
sequence DKTGT is the unifying characteristic of all P-type ATPases (1,
2). The ATP binding, hydrolysis, and enzyme phosphorylation steps
occur within the ATPBD, which consists of a phosphorylation domain
(P-domain) and a nucleotide binding domain (N-domain). The ATPBD
from WND (17), as well as those from P2 and P1A-ATPases (20 –22),
have been isolated in soluble form. These cytoplasmic fragments are
able to bind nucleotides and hydrolyze ATP, albeit at a low rate, and thus
contain the key components needed for energy dependent ion transport
by these ATPases.
With the exception of solution structures of N-MBDs (23, 24), struc-
tural information for P1B-type ATPases is lacking. Several structures of
the sarcoplasmic reticulum (SERCA1) Ca2⫹-ATPase have been
reported recently (25–29) and provide insight into the conformational
changes that P2-type ATPases such as the Na⫹,K⫹-ATPase or the
H⫹,K⫹-ATPase undergo during catalysis. Because of significant archi-
The abbreviations used are: ATPBD, ATP binding domain; A-domain, actuator domain;
AMP-PCP, adenosine 5⬘-(␤,␥-methylene)triphosphate; N-MBD, N-terminal metal
binding domain; KdpB, potassium-transporting ATPase B chain; P-domain, phospho-
rylation domain; SERCA1, sarcoplasmic reticulum Ca2⫹-ATPase; MOPS, 3-[N-morpho-
lino]propanesulfonic acid.
JOURNAL OF BIOLOGICAL CHEMISTRY 11161
Structure of the CopA ATP Binding Domain
tectural differences between heavy metal transporting and P2-type TABLE 1
ATPases, homology modeling based on the SERCA1 structures is of Data collection, phasing, and refinement statistics
limited utility in understanding P1B-type ATPase function and transport r.m.s.d., root mean square deviation.
mechanisms at the molecular level. We have determined the 2.3 Å res- Selenomethionine
ATPBD
olution crystal structure of the ATPBD from CopA from the hyperther-
mophile Archaeoglobus fulgidus, a well characterized Cu⫹-ATPase that Data collection
Wavelength (Å) 0.979
contains all of the key elements present in eukaryotic Cu⫹-ATPases (13, Resolution (Å)a 30.0–2.3
15, 30). The CopA ATPBD structure, because of its small size and com- Unique observations 33,406
Total observations 366,735
pactness, constitutes a minimal ATP binding-phosphorylation unit. As Completeness (%) 99.6 (100.0)
such, the structure allows for the identification of the central catalytic Redundancy 10.7 (10.7)
I/␴ 29.3 (8.4)
features independently of the secondary structural elements likely asso- Rsym (%)b 6.5 (24.2)
ciated with trafficking, targeting, and regulatory functions. Selenium sites 9
FOM 0.93
EXPERIMENTAL PROCEDURES Refinement
Rwork (%)c 24.0
407 Rfree (%)d 29.4
Cloning of the CopA ATPBD—The CopA ATPBD (residues Lys -
Molecules in ASU 3
Lys671) was PCR-amplified from CopA cDNA (30) by using the Number of protein-nonhydrogen atoms 5,463
primers 5⬘-GCCCTTGGTCTCTAATGAAAAATGCCGACGCT- Number of solvent atoms 289

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CTCGAA-3⬘ and 5⬘-CGGGAAGGTCTCTGCGCTTTTGCTCAT-
GGTCTTTCTGCT-3⬘, which introduce a Bsa1 restriction site at the
5⬘- and 3⬘-ends of the PCR product. The PCR product was digested with
Bsa1 and cloned into the pPR-IBA1vector (IBA), which introduces an
8-amino-acid (WSHPQFEK) streptactin tag at the C terminus of the pro-
tein. The accuracy of the insert was confirmed by DNA sequencing.
BL21Star(DE3)pLysS Escherichia coli cells carrying the plasmid pSJS1240
encoding for rare tRNAs (tRNAargAGA/AGG and tRNAileAUA) (31) were
transformed with the pATPBD construct.
Expression and Purification of the CopA ATPBD—E. coli cells con-
taining the pATPBD plasmid were grown at 37 °C in Luria-Bertani
medium containing 100 mg/liter ampicillin and 30 mg/liter chloram-
phenicol. ATPBD expression was induced with 250 ␮M isopropyl ␤-D-
thiogalactopyranoside at an A600 of 0.7. The cells were harvested by
centrifugation at 6000 ⫻ g for 5 min 3– 4 h after induction. The pellet
was frozen in liquid nitrogen and stored at ⫺80 °C.
For purification, the frozen cells were resuspended in 100 mM Tris,
pH 7.5, 150 mM NaCl (Buffer W), to which DNase, 5 mM MgCl2, and 1
mM phenylmethylsulfonyl fluoride were added. The cell suspension was
sonicated for 10 min (30-s pulses with a 30-s rest) and centrifuged at
163,000 ⫻ g for 1 h. The supernatant was then loaded onto a 10-ml
streptactin column (IBA) conditioned in buffer W, and the ATPBD was
purified according to the manufacturer’s protocol. SDS-PAGE of the
eluted protein indicated it was greater than 95% pure. The ATPBD was
exchanged into 20 mM MOPS, pH 7.0, 20 mM NaCl, and 5% glycerol by
several concentration and dilution steps using an Amicon Ultra YM-10
concentrator, frozen at 30 mg/ml in liquid nitrogen, and stored at
⫺80 °C. This protocol yielded ⬃15 mg of protein/liter of medium. The
protein concentration was estimated by using the theoretical extinction
coefficient, ⑀280 ⫽ 11,400 cm⫺1 M⫺1, and a molecular mass of 29.6 kDa.
A selenomethionine derivative of the CopA-ATPBD was prepared
using LeMaster’s medium (32) and purified as described above.
ATPase Activity—Activity assays were carried out at 70 °C for 20 min
in a 500-␮l solution containing 50 mM Tris, pH 7.5, 25 mM NaCl, 1 mM
MgCl2, 5 mM ATP, and ⫾ 20 ␮M ATPBD. The reaction was stopped by
quickly cooling the solution to 4 °C. The free phosphate concentration
was measured at room temperature by using the EnzChek phosphate
assay kit (Molecular Probes). The ATPBD had an activity of 4.2 nmol
Pi/mg/min. Background activity (ATP hydrolysis in the absence of pro-
tein) was less than 50% in all of our determinations.
Crystallization—Both wild-type and selenomethionine CopA-
ATPBD were diluted to 10 mg/ml with a 20 mM MOPS, pH 7.0, 20 mM
NaCl, and 5% glycerol solution and crystallized by using the hanging
R.m.s.d. bond length (Å) 0.007
R.m.s.d. bond angle (°) 1.3
Average B-value (Å2) 40.3
a
Values in parentheses are for the highest resolution shell.
b
Rsym ⫽ 兺i 兺hkl 兩Ii(hkl) ⫺ 具I(hkl)典兩/兺hkl 具I(hkl)典, where Ii(hkl) is the ith measured
diffraction intensity and 具I(hkl)典 is the mean of the intensity for the Miller index
(hkl).
c
Rwork ⫽ 兺hkl 储Fo(hkl)兩 ⫺ 兩Fc(hkl)储/兺hkl兩Fo(hkl)兩.
d
Rfree ⫽ Rwork for a test set of reflections (5%).
drop method at room temperature (22–24 °C). Equal volumes of pro-
tein and a precipitant solution containing 100 mM sodium acetate, pH
4.6, 5– 8% polyethylene glycol 4000, and 10 –20% glycerol were com-
bined. Large rectangular crystals grew within 2–3 days. Prior to data
collection, the ATPBD crystals were quickly exchanged into a cryosol-
vent comprising 50 mM sodium acetate, pH 4.6, 5% polyethylene glycol
4000, and 25% glycerol and flash frozen in liquid nitrogen.
Data Collection and Structure Determination—A SAD data set was
collected at 100 K at the sector 32 beamline at the Advanced Photon
Source (see Table 1). The crystals diffracted to 2.3 Å resolution, belong
to the space group P31, and have unit cell dimensions of a ⫽ b ⫽ 80.78
Å and c ⫽ 105.96 Å. The data sets were integrated by using MOSFLM
(33) and scaled with SCALA (34). Both SOLVE (35) and CNS (36) were
used to find nine heavy atom sites corresponding to three selenium
positions in each of the three ATPBD molecules in the asymmetric unit.
CNS yielded the most interpretable electron density maps after density
modification. The three ATPBDs comprising the asymmetric unit were
built using XtalView (37), and the model was refined with CNS (Table
1). Non-crystallographic symmetry restraints were imposed throughout
the refinement. Most of the residues in the three asymmetric units were
observed in the electron density maps except for residues 407– 410 at
the N terminus, 670 – 671 at the C terminus, and 637– 645 in the P-do-
main. A Ramachandran plot calculation with PROCHECK (38) indi-
cated that 91% of the residues had the most favored geometry with the
rest in additionally allowed regions. All figures were generated with
PyMOL (39).
RESULTS AND DISCUSSION
Overall Structure—The A. fulgidus CopA ATPBD spanning residues
407– 671 was expressed and purified to homogeneity. This isolated
domain was able to bind and hydrolyze ATP at a measurable rate (4.2
nmol Pi/mg/min) when incubated at 70 °C, the growth temperature of
the source organism. The A. fulgidus ATPBD exhibits a kidney bean-like
topology with a cleft for ATP binding at the interface between the N-
and P-domains (Fig. 1a). Two 5– 6-amino-acid loops (residues 429 –

11162 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 16 • APRIL 21, 2006
 Structure of the CopA ATP Binding Domain

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FIGURE 1. Ribbon diagrams of the CopA and SERCA1 ATPBDs. a, CopA ATPBD. The ␣-helices and ␤-sheets are labeled, and the location of a disordered loop between residues
637– 645 is marked by an asterisk (*). b, closed form of the SERCA1 ATPBD with bound AMP-PCP and Mg2⫹ (PDB accession code 1VFP) depicted as spheres. Carbon atoms are colored
orange, oxygen atoms are colored red, nitrogen atoms are colored blue, and the Mg2⫹ ion is colored green. c, open form of the SERCA1 ATPBD (PDB accession code 1SU4).

FIGURE 2. Sequence alignment of the ATPBDs from WND, MNK, and CopA. The alignment was generated with ClustalW. Residues highlighted in green are conserved among the
different P-type ATPase families, whereas residues highlighted in blue are conserved only among P1B-type ATPases (alignment not shown). Residues comprising the P-domain are
highlighted with a gray background, whereas the light pink background denotes the N-domain. Asterisks (*) represent the locations of point mutations in WND linked to disease.

434 between ␤1 and ␤2 and residues 547–552 between ␤7 and ␣6) form
a hinge region connecting the N- and P-domains. Notably, the CopA
ATPBD adopts a closed conformation similar to the SERCA1 structures
with bound AMP-PCP or AlF4⫺ and ADP, which both mimic the E1P-
ADP state of the protein (27, 29) (Fig. 1b). This closed conformation is
characterized by a quasiparallel orientation of helices ␣5 and ␣7, similar
to helices ␣7 and ␣10 in the nucleotide-bound SERCA1 structures (Fig.
1b). By contrast, these helices adopt a perpendicular orientation in the
open forms of P2-type ATPases (25) (Fig. 1c). Among the different
P-type ATPases, and within the specific P1, P1B, and P2 subfamilies,
there is little sequence conservation in these helices (Fig. 2 and supple-
mental Fig. S1). Nevertheless, mutations in the corresponding MNK
and WND helices are known to lead to Wilson and Menkes diseases
(Fig. 2) (40). A crystal structure with a nucleotide analogue bound to the
CopA ATPBD could not be obtained despite several cocrystallization
and soaking efforts. The ATPBD may be locked in this closed state
either by the crystallization conditions or by intrinsic interactions that
confer thermostability to CopA.
Hinge Region—The two short loops linking the N- and P-domains
likely function as a hinge, allowing the two domains to undergo struc-
tural rearrangements concomitant with nucleotide binding and phos-
phoester intermediate formation (25, 27, 29). Several highly conserved
residues are present in the hinge region. These include Thr430 and
Asp548, which appear to be universally conserved among all ATPases,
and Gly432, which is conserved only among the P1B family members. The
Thr430 and Asp548 side chains interact via hydrogen bonding. In addition,
the Gly432 amide nitrogen interacts weakly with the Asp548 side chain. A
similar pattern is observed in SERCA1 (supplemental Fig. S2) (25–29).
These interactions may be critical for stabilizing the conformation of the
phosphorylation loop (D424KTGT), which is just upstream of Thr430 in the
P-domain, and are the focal point about which the hinge flexes. In support
of a key role for these residues, mutations in Thr430 and Gly432 in WND are
linked to disease states (Fig. 2) (40).
The N-domain—The N-domain consists of a six stranded anti-
parallel ␤-sheet sandwiched between two ␣-helices on each side of the
sheet (Fig. 3a). Structures of N-domains from SERCA1 (25), the
Na⫹,K⫹-ATPase (41), the Kdp-ATPase (KdpB) (42) and more recently
WND,5 all exhibit this overall fold despite a lack of sequence homology
(Fig. 3 and supplemental Fig. S1). Moreover, SERCA1, the Na⫹/K⫹-
ATPase, and WND differ significantly from CopA in that their
sequences include several insertions ranging from 10 to 60 residues in
5
O. Dmitriev, R. Tsivkovskii, F. Abildgaard, C. T. Morgan, J. L. Markley, and S. Lutsenko, in
press.

APRIL 21, 2006 • VOLUME 281 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 11163
Structure of the CopA ATP Binding Domain
FIGURE 3. Comparison of the CopA N-domain to structural homologs. a, CopA N-
domain. b, WND N-domain (PDB accession code 2ARF). c, superposition of CopA (gray)
and WND (red). d, superposition of CopA (gray) and KdpB (red) (PDB accession code
1X6K). e, surface representation of the CopA N-domain (light gray) with docked AMP-PNP
(sticks) from the superimposed KdpB structure. Surfaces of conserved residues predicted
to bind to AMP-PNP are colored by atom type with oxygen red, nitrogen blue, and carbon
orange. For AMP-PNP, carbon is colored green, and phosphate is colored pink.
length. The WND N-domain has an unstructured 39-amino-acid loop
between ␤4 and ␤5 that extends into the cytosol away from the N-do-
main (Fig. 3, b and c). This loop, which spans 56 amino acids in MNK, is
not essential for catalysis5 and comprises just four residues in CopA.
The MNK and WND loops do not bear any sequence resemblance to
each other, suggesting that these elements may specifically target the
two proteins to different cellular compartments. Like WND, the Ca2⫹
and Na⫹,K⫹-ATPases have additional loops and structural elements,
such as a seventh strand on the ␤-sheet (Fig. 1 and supplemental Fig.
S1). Some of these inserts in SERCA1 participate in interactions with
the A-domain (25–29). The additional structural elements found in
the various P-type ATPases may therefore be specific for their func-
tion and help to promote domain-domain interactions as well as
proper cellular localization.
The CopA N-domain is quite similar to the KdpB N-domain with
bound AMP-PNP. There are no extra insertions in the KdpB sequence,
and the root mean square deviation for C␣ atoms between KdpB and
CopA is 1.54 Å (Fig. 3d). When KdpB is superimposed onto CopA, the
adenine and ribose rings of the ATP analogue fit well within a conserved
pocket (Fig. 3e). Sequence analysis of the different P1B-type ATPase
N-domains reveals that five amino acids, Glu457, His462, Gly490, Gly492,
11164 JOURNAL OF BIOLOGICAL CHEMISTRY
and Gly501, are universally conserved (Fig. 2).5 All of these residues are
predicted to interact with the docked nucleotide in CopA on the basis of
the superimposed KdpB structure. Residues Glu457, His462, and Gly492
may function to orient and bind the adenine ring through hydrogen
bonding, whereas Gly490 and Gly501 likely interact primarily with the
ribose moiety and ␣-phosphate. Mutagenesis and NMR studies on
WND (44)5 and the E. coli Zn2⫹-transporting P1B-type ATPase ZntA
(45) are consistent with a role for these residues in ATP binding. Inter-
estingly, none of these residues are conserved in the other ATPase
families, and each family appears to preserve its own set of residues
specific for ATP binding. For example, in KdpB (42) and SERCA1 (27,
29), a phenylalanine residue on the strand equivalent to ␤3 forms
␲-stacking interactions with the adenine ring.
In the model of nucleotide-bound CopA, His462 is not positioned
properly for strong contacts with the adenine ring (Fig. 3e). Given the
morphology of the adenine-binding cleft, the adenine may form better
interactions with His462 and Glu457 by rotating 90° with respect to its
position in KdpB. Further movement of the S460EHP loop between ␣2
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and ␣3 may also facilitate interaction. The WND N-domain structure,
which was determined with bound ATP, differs from CopA in that ␣2
has rotated by ⬃30° to form a different set of interactions with the
␤-sheet (Fig. 3c). As a result, the position of the SEHP loop changes and
␣3 rotates by ⬃10°. These differences may be related to interdomain
regulatory interactions associated with the multiple MBDs present in
eukaryotic proteins. It has been demonstrated that the first four MBDs
of WND interact with the N-domain and that this interaction modu-
lates ATP affinity (17, 44). CopA, which requires only one MBD for
maximum activity (15), may function in a slightly different fashion.
Because the N-domain in the full-length ATPase is likely to participate
in domain-domain interactions required for catalysis, movement of the
SEHP loop is likely to be important for protein function.
The P-domain—The P-domain comprises a six-stranded parallel
␤-sheet sandwiched between six ␣-helices, three on each side of the
sheet (Fig. 4a). The SERCA1 P-domain, which is the only other known
structure of a P-domain, has a similar fold (root mean square deviation
for C␣ atoms ⫽ 1.7 Å) with the exception of a 50-amino-acid insert
between ␣10 and ␤14 (Fig. 4, b and c). In CopA, both the invariant
D424KTGT and T572GD loops are positioned in the back of the ATPBD
crevice near the hinge region (Fig. 1a). These loops, which are critical for
enzyme phosphorylation during catalysis, adopt similar conformations
to those observed in the different SERCA1 structures (Fig. 4, c and d)
(25–29). The loop between ␤10 and ␣9 containing the sequence
A614XXGDGXND rests at the edge of the crevice marking the interface
between the N- and P-domains. In the SERCA1 structures, the equiva-
lent loop interacts with the A-domain and adopts different orientations.
Finally, residues 637– 645, for which no electron density was observed,
would be located on a loop between ␤11 and ␤12 far from the phospho-
rylation site on the protein exterior (Figs. 1a and 4a). The analogous
region in SERCA1 forms a small helix (Fig. 1b) and interacts with the
A-domain in several different ways depending upon conformation of
SERCA1. This loop in CopA would likely become more ordered in the
presence of other cytosolic domains.
The similarities in the core fold of the P-domain are not surprising
considering that the majority of the conserved residues among the dif-
ferent P-type ATPase ATPBDs are located in this domain (Figs. 2, 4d,
and supplemental Fig. S1). Conserved residues are clustered in the loops
facing the ATP binding site of the N-domain. A superimposition of the
CopA P-domain with the AlF4⫺ and ADP-bound form of SERCA1 (Fig.
4d) suggests that CopA residues Asp424, Thr426, Thr572, Gly573, Asn621
probably interact with the ␤- and ␥-phosphates, Asp424 and Asp618
VOLUME 281 • NUMBER 16 • APRIL 21, 2006

FIGURE 4. Comparison of the CopA (a) and SERCA1
P-domains (b). c, stereo view of superimposed
structures of CopA (red) and SERCA (gray). d, stereo
view of conserved P-domain residues participat-
ing in ATP binding. The SERCA1 (gold) (PDB acces-
sion code 1T5T) residues involved in binding ADP,
AlF4, and Mg2⫹ and their CopA (light blue) coun-
terparts are superimposed. The ligands are col-
ored green, Mg2⫹ ions are shown as green spheres,
nitrogen atoms are colored blue, and oxygen
atoms are colored red.
likely coordinate Mg2⫹, and Asp574 hydrogen bonds to the nucleotide
ribose moiety. The remaining conserved residues likely participate in
second coordination sphere hydrogen bonding or serve a specific struc-
tural role to preserve the active site geometry.
Domain Interactions—In P-type ATPases, transmembrane ion trans-
port is coupled to ATP hydrolysis via key structural and conformational
changes (1, 2). These various movements include transient domain-
domain interactions such as those described for the P- and N-domains
of P2-type ATPases (25–28). Electrostatic surface maps of the CopA
ATPBD (Fig. 5a) do not reveal any obvious patches of positive or
negative charge suggestive of a domain interaction site. Compari-
sons to the SERCA1 structures provide some insight into potential
interaction surfaces, however. Mapping the known A-domain inter-
action sites in SERCA1 (28) onto the surface of the CopA ATPBD
identifies specific parts of the P-domain as likely to interact with the
APRIL 21, 2006 • VOLUME 281 • NUMBER 16
Structure of the CopA ATP Binding Domain
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A-domain (Fig. 5, b and c). These sites correspond to the slightly
positively charged surface at the crest of the P-domain ATP-binding
cleft (Fig. 5a). Notably, interactions between the A-domain and the
N-domain in the SERCA1 structures (26 –29) involve regions of sec-
ondary structure not present in P1B-ATPases. This observation does
not preclude possible interactions between the A- and N-domains in
CopA but could be related to the additional requirement for metal-
dependant interactions between the N-MBDs and the ATPBD (17,
44) and between the N-MBDs and metallochaperones (46).
Structural Significance of WND and MNK Mutations—A number of
mutations in the ATPBDs of WND and MNK have been identified in
Wilson and Menkes diseases patients (Fig. 2) (40). The structure of the
CopA ATPBD provides new insight into the probable effects of these
mutations on enzyme function. For example, mutations T1031S,
T1033A, G1035V, and R1038K occur in the WND hinge region and are
JOURNAL OF BIOLOGICAL CHEMISTRY 11165
Structure of the CopA ATP Binding Domain
FIGURE 5. Surfaces on the P-domain predicted to
interact with the A-domain. a, electrostatic surface
of the CopA ATPBD. Positively charged surfaces are
colored blue, and negatively charged surfaces are
colored red. b, the A-domain interaction sites from
SERCA1 mapped onto CopA (left). Magenta surfaces
correspond to predicted interactions in the E1 and
E1P-ADP states, and green surfaces correspond to
additional interactions occurring upon formation of
the E2 state. Residue His462 is colored orange to
denote the nucleotide binding site. A ribbon repre-
sentation of the structure with the same color cod-
ing is shown on the right. The electrostatic surfaces
were generated by using APBS (43).
predicted to alter P- and N-domain movement. Other mutations
might affect ligand binding by direct participation in ligand-protein
interactions or by proximity to interacting residues. These muta-
tions not only include the well characterized mutations in the SEHP
region like H1069Q but also the I1102T, C1104F, I1148T, and
R1151H WND mutations that probably affect the positions of gly-
cines interacting with the nucleotide. A similar case can be made for
mutations that involve the phosphate binding site in the P-domain,
like T1220M, D1222N, D1267A, and N1270S in WND. Finally, sev-
eral mutations occur in regions not directly involved in ligand inter-
action or conformational changes. In WND, these mutations include
L1043P, L1083P, T1232P, V1252I, and D1296N. Such mutations may
affect the folding and stability of the protein.
In sum, the CopA ATPBD structure reveals the key components
required for ATP binding and hydrolysis by P1B-type ATPases. The
overall fold, which resembles that observed in P2-type ATPases, repre-
sents the basic scaffold capable of performing key steps in the classical
P-type ATPase E1/E2 mechanism. The structure additionally serves as a
basis to explore domain-domain interactions specific to heavy metal
transport. Finally, the CopA ATPBD structure provides a new frame-
work for the structural analysis of the many mutations leading to Wilson
and Menkes diseases.
Acknowledgments—We thank Joe Brunzelle at LS-CAT and Liliya Yatsunyk
for help with data collection and processing. We also thank Svetlana Lutsenko
and Oleg Dmitriev for providing coordinates of the WND N-domain and a
preprint of their manuscript.
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VOLUME 281 • NUMBER 16 • APRIL 21, 2006
 Structure of the ATP Binding Domain from the Archaeoglobus fulgidus Cu+
-ATPase
Matthew H. Sazinsky, Atin K. Mandal, José M. Argüello and Amy C. Rosenzweig
J. Biol. Chem. 2006, 281:11161-11166.
doi: 10.1074/jbc.M510708200 originally published online February 22, 2006

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 Structure of the ATP Binding Domain from the Archaeoglobus fulgidus Cu+-

ATPase*

Supplemental Data

Matthew H. Sazinsky‡, Atin K. Mandal§, José M. Argüello§, and Amy C. Rosenzweig‡

From the ‡Departments of Biochemistry, Molecular Biology, and Cell Biology and of

Chemistry, Northwestern University, Evanston IL 60208, §Department of Chemistry and

Biochemistry, Worcester Polytechnic Institute, Worcester MA 01609

Address correspondence to: Amy C. Rosenzweig, Departments of Biochemistry, Molecular

Biology, and Cell Biology and of Chemistry, Northwestern University, Evanston IL 60208, Tel.:

847-467-5301; Fax. 847-467-6489; E-mail: amyr@northwestern.edu and José M. Argüello,

Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester MA

01609, Tel. (508)-831-5326; Fax. (508)-831-5933; E-mail: arguello@wpi.edu

 α1 β1 β2 β3

SERCA1 LGTRRMAKKNAIVRSLPSVETLGCTSVICSDKTGTLTTNQMSVCKMFIIDKVDGDVCSLN 380

Na/K-ATPase LTAKRMARKNCLVKNLEAVETLGSTSTICSDKTGTLTQNRMTVAHMWSDNQIHEADTTEN 403
KdpB AGMSRMLGANVIATSGRAVEAAGDVDVLLLDKTGTITLGNRQASEFIPAQGVD------- 329
CopA VGMGKGAELGILIKNADALEVAEKVTAVIFDKTGTLTKGKPEVTDLVPLNG--------- 444

β3 α2 α3 β4 β4

SERCA1 EFSITGSTYAPEGEVLKNDKPVRAGQYDGLVELATICALCNDSSLDFNETKGVYEKVGEA 440

Na/K-ATPase QSGVSFDKTS-----------------ATWLALSRIAGLCNRAVFQANQENLPILKRAVA 446
KdpB -----------------------EKTLADAAQLASLADETPEGRSIVILAKQRFNLRER- 365
CopA -------------------------DERELLRLAAIAERRSEHPIAEAIVKKALEHGIEL 480
α4 α5 β6 β7

SERCA1 TETALTTLVEKMNVFNTEVRSLSKVERANACNSVIRQLMKKEFTLEFSRDRKSMSVYCSP 500

Na/K-ATPase GDASESALLKCIELC---------------CGSVKEMRERYTKIVEIPFNSTNKYQLSIH 491
KdpB ---------------------------------------------DVQSLHATFVPFTAQ 380
CopA ----------------------------------------------------------GE 481

β8 α6 β9 α7

SERCA1 AKSSRAAVGNKMFVKGAPEGVIDRCNYVRVGTTRVPLTGPVKEKIMSVIKEWGTGRDTLR 560

Na/K-ATPase KNPNTAEPRHLLVMKGAPERILDRCSSILIHGKEQPLDEELKDAFQNAYLELGGLGERVL 551
KdpB SRMSGINIDNRMIRKGSVDAIRRHVEAN-----GGHFPTDVDQKVDQVARQG-------- 427
CopA PEKVEVIAGEGVVADGILVGNKRLMEDF-----GVAVSNEVELALEKLEREA-------- 528

β10 α8 β11 α9

SERCA1 CLALATRDTPPKREEMVLDDSAKFMEYEMDLTFVGVVGMLDPPRKEVTGSIQLCRDAGIR 620

Na/K-ATPase GFCHLFLPDEQFPEGFQFDTDDVNFPLDN-LCFVGLISMIDPPRAAVPDAVGKCRSAGIK 610
KdpB ATPLVVVE---------------------GSRVLGVIALKDIVKGGIKERFAQLRKMGIK 466
CopA KTAVIVAR---------------------NGRVEGIIAVSDTLKESAKPAVQELKRMGIK 567

β12 α10 β13 α11

SERCA1 VIMITGDNKGTAIAICRRIGIFSENEEVTDRAYT--------------------GREFDD 660

Na/K-ATPase VIMVTGDHPITAKAIAKGVGIISEGNETVEDIAARLNIPVSQVNPRDAKACVVHGSDLKD 670
KdpB TVMITGDNRLTAAAIAAEAGVDD------------------------------------- 489
CopA VGMITGDNWRSAEAISRELNLDL------------------------------------- 590
α12 β14 α13 β15 α14 β16
SERCA1 LPLAEQREACR--RACCFARVEPSHKSKIVEYLQSYDEITAMTGDGVNDAPALKKAEIGI 718
Na/K-ATPase MTSEQLDDILKYHTEIVFARTSPQQKLIIVEGCQRQGAIVAVTGDGVNDSPASKKADIGV 730
KdpB ----------------FLAEATPEAKLALIRQYQAEGRLVAMTGDGTNDAPALAQADVAV 533
CopA ----------------VIAEVLPHQKSEEVKKLQAK-EVVAFVGDGINDAPALAQADLGI 633

β16 α15 β17 α16

SERCA1 AMGSG-TAVAKTASEMVLADDNFSTIVAAVEEGRAIYNNMKQFIRYLISSNVGEVVCIFL 777
Na/K-ATPase AMGIAGSDVSKQAADMILLDDNFASIVTGVEEGRLIFDNLKKSIAYTLTSNIPEITPFLI 790
KdpB AMNSG-TQAAKEAGNMVDLDSNPTKLIEVVHIGKQMLMTRGSLTTFSIANDVAKYFAIIP 592
CopA AVGSG-SDVAVESGDIVLIRDDLRDVVAAIQLSRKTMSKIKQNIFWALIYNVILIPAAAG 692

Fig. S1 Sequence alignment of CopA, SERCA1, Na+/K+ ATPase, and KdpB ATPBDs. The

alignment was generated by using ClustalW. Blue shading denotes the beginning and end of the

N-domain. Conserved residues are highlighted in green, and the SERCA1 secondary structure

assignment is depicted above the sequences.

Fig. S2 Conserved hydrogen bonding patterns in the a, CopA and b, SERCA1 ATPBD hinge

region. Carbon atoms are colored green, oxygen atoms are colored red, and nitrogen atoms are

colored blue.