From the Magnetic Resonance Center and
Department of Chemistry, University of Florence, Via Luigi Sacconi 6, Sesto Fiorentino, Florence, 50019, Italy and the Departments of
¶ Chemistry and
Biochemistry, Molecular Biology, and Cell
Biology, Northwestern University, Evanston, Illinois 60208
Received for publication, September 13, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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Ccc2 is an intracellular copper transporter in
Saccharomyces cerevisiae and is a physiological target of
the copper chaperone Atx1. Here we describe the solution structure of
the first N-terminal MTCXXC metal-binding domain,
Ccc2a, both in the presence and absence of Cu(I). For Cu(I)-Ccc2a, 1944 meaningful nuclear Overhauser effects were used to obtain a
family of 35 structures with root mean square deviation to the average
structure of 0.36 ± 0.06 Å for the backbone and 0.79 ± 0.05 Å for the heavy atoms. For apo-Ccc2a, 1970 meaningful nuclear
Overhauser effects have been used with 35 3JHNH Saccharomyces cerevisiae Ccc2 is a member of a class of
proteins that transport heavy metals across vesicular membranes (1). Members of this family, referred to as P-type or CPx-type ATPases, have
been identified in a variety of bacteria, yeast, nematodes, and
mammals. The cytoplasmic N terminus of the copper-transporting ATPases
contains one or more metal-binding domains (2-7) characterized by a
GMXCXXC motif. In humans, this intracellular
copper pump is encoded by the Wilson's and Menkes' disease genes
(2-6), and a functional homologue (Ccc2) has been characterized in
yeast (7). Copper is incorporated into trans-Golgi vesicles via the action of Ccc2 and ultimately into the multicopper oxidase Fet3p, which
translocates to the plasma membrane and works in conjunction with an
iron permease to mediate high affinity iron uptake (8, 9). Both the
Wilson's and Menkes' disease proteins complement the function of Ccc2
in this pathway (10-12). The Menkes CPx-type ATPase contains six
N-terminal GMXCXXC motifs (2-4), is located in
the trans-Golgi network, and translocates copper across intracellular membranes into the secretory pathway (13). Metal binding studies on the
complete N-terminal cytoplasmic region have established that this
region binds Cu(I) selectively (relative to cadmium, cobalt, or zinc)
with a stoichiometry of one copper per metal-binding domain (14).
The yeast metallochaperone Atx1 is a cytosolic Cu(I) receptor that
delivers its metal ion cargo to Ccc2 (15). The thermodynamic gradient
for metal transfer between Atx1 and the first metal-binding domain of
Ccc2 (Ccc2a) is shallow, yet copper transfer is facile, suggesting that
Atx1 works like an enzyme to catalyze the rate of copper transfer
between partners (16). A high resolution (1.02-Å) x-ray
crystallographic structure of Hg(II)-Atx1 reveals that the mercury is
coordinated in a bidentate fashion from two cysteine sulfurs with a
S-Hg-S bond angle of 167° (17). Mutation of several conserved
lysines on the surface of Atx1 greatly reduces the
copper-dependent interaction of Atx1 and Ccc2 in
vivo (15, 18). The Atx1 metallochaperone (17), domain I of the
copper chaperone for superoxide dismutase (CCS) (19), and the fourth metal-binding domain of the Menkes protein (20) all adopt a The mechanism of copper transfer between Atx1 and Ccc2 is proposed to
involve a series of two- and three-coordinate intermediates (15, 16).
The factors responsible for facile and reversible copper transfer
between Atx1 and Ccc2a are probably mediated in part by
metal-dependent conformational changes in Atx1 and Ccc2. In
Atx1, copper release involves a series of structural changes, in which
both cysteines change conformation (46). In apo-Atx1, the loop is not
well defined. We report here the solution structures of Cu(I)-Ccc2a
from S. cerevisiae obtained for a nonlabeled sample and its
reduced apo form, obtained for a 15N-labeled sample.
Relative to Atx1, the first loop is well defined, and Cu(I) binding
induces fewer changes in the conformation of Ccc2a. Unlike Cu(I)-Atx1,
the metal binding residues in Cu(I)-Ccc2a are on the surface of a
helix, and the copper cargo is more accessible to incoming ligands in
the latter. Finally, a negative patch is observed on the surface of
Ccc2a in a site that corresponds to a positive patch on Atx1,
suggesting that a complementary docking interface is employed in the
copper transfer mechanism.
Sample Isolation and Preparation--
Ccc2a was uniformly
labeled with 15N by expressing the protein in
Escherichia coli strain BL21(DE3) (Novagen), transformed
with pDLHV021, in minimal media supplemented with
15NH4Cl. Unlabeled Ccc2a was isolated as
described previously (16). Ccc2a(15N) was purified from the
cell pellet by freeze-thaw extraction with 20 mM
MES1/Na,
pH 6.0, followed by streptomycin sulfate precipitation (5%, w/v) of
the extract. The supernatant was further purified by repetitive runs on
Superdex 75 (Amersham Pharmacia Biotech). The yield of labeled protein
was ~2 mg/liter of culture.
Protein samples were purified and stored in the presence of reducing
agent. NMR samples were prepared in a Vac Atmospheres nitrogen
atmosphere chamber at 12 °C. Protein concentrations were determined
by the Bradford assay and calibrated as described previously (16).
Copper concentration was determined by ICP-AES. The NMR sample of
apo-Ccc2a and apo-Ccc2a(15N) was prepared by exchanging the
reduced form of the protein into 100 mM sodium phosphate,
pH 7, 90% H2O, 10% D2O via ultrafiltration; the final concentrations were 2.7 and 3.3 mM, respectively.
Cu(I)-Ccc2a was prepared as previously described (16), and the buffer
was exchanged by ultrafiltration into 100 mM sodium
phosphate, pH 7, 90% H2O, 10% D2O. The
metal/protein ratio was 1.2 with a protein concentration of 1.2 mM. No exogenous thiols were added to the sample buffers.
Approximately 0.6 ml of sample was loaded into 535-PP 5-mm quartz NMR
tubes (Wilmad), which were capped with a latex serum cap in the
VacAtmospheres chamber.
NMR Spectroscopy--
The NMR spectra were acquired on Avance
800 and 600 Bruker spectrometers operating at a proton nominal
frequency of 800.13 and 600.13 MHz, respectively. A QXI probe has been
used on the Avance 800 spectrometer, and a triple resonance (TXI) 5-mm
probe has been used on the 600 spectrometer. All probes were equipped with pulsed field gradients along the z axis. Total
correlation spectroscopy (25, 26) spectra were recorded on the 600-MHz spectrometer with a spin-lock time of 100 ms, a recycle time of 1 s and a spectral window of 14 ppm. Two-dimensional NOESY maps (27, 28)
were acquired on the 800-MHz spectrometer with a mixing time of 100 ms,
a recycle time of 1 s, and a spectral window of 14 ppm.
On the 15N apo-Ccc2a sample, a two-dimensional
15N-1H heteronuclear single quantum coherence
(29-31) map was obtained at 800 MHz with an INEPT delay of 2.66 ms, a
recycle time of 1 s, and spectral windows of 14 and 33 ppm for the
1H and 15N dimensions, respectively. A
three-dimensional NOESY-15N heteronuclear multiple quantum
coherence experiment (32) was recorded with 290 (1H) × 96 (15N) × 2048 (1H) data points on
the 600-MHz spectrometer. The INEPT delay was set to 5.4 ms, the mixing
time was 100 ms, and the carrier frequency was set in the center of the
amide proton region, at 7.35 ppm. Spectral windows of 14, 14, and 29 ppm were used for the direct 1H dimension and the indirect
1H and 15N dimensions. A HNHA experiment
(33) was performed at 600 MHz to determine
3JHNH
For all the experiments, quadrature detection in the indirect
dimensions was performed in the time-proportional phase incrementation mode (28), and water suppression was achieved through WATERGATE sequence (34). All two-dimensional data consisted of 4K data points in
the acquisition dimension and of 1K experiments in the indirect
dimension. All three- and two-dimensional spectra were collected at 298 K, processed using the standard Bruker software (XWINNMR), and analyzed
on IBM RISC 6000 computers through the XEASY program.
Constraints Used in Structure Calculations--
The peaks used
for the structure calculations were integrated in the two-dimensional
NOESY map acquired at 298 K in H2O. Intensities of dipolar
connectivities were converted into upper distance limits, to be used as
input for structure calculations, by using the approach provided by the
program CALIBA (35). The calibration curves were adjusted iteratively
as the structure calculations proceeded. Stereospecific assignments of
diastereotopic protons were obtained using the program GLOMSA (35).
3JHNH
Hydrogen bond constraints were introduced for backbone amide protons
that were found to be within hydrogen bond distance and to have the
correct orientation with respect to hydrogen bond acceptors in
structural models obtained without inclusion of these constraints. The
distance between the NH proton and the hydrogen bond acceptor was
constrained to be in the 1.8-2.4 Å interval by inclusion of the
corresponding upper and lower distance limits in structure
calculations. In addition, upper and lower distance limits of 3.0 and
2.7 Å between the N and the acceptor atoms were also included.
Structure Calculations--
The structure calculations were
performed using DYANA (36). 200 random conformers were annealed in
10000 steps using NOE and 3J values (when available)
constraints. The 35 conformers with the lowest target function
constitute the final family. The copper ion was included in the
calculations by adding a new residue in the amino acid sequence, formed
by a chain of dummy atoms that have their van der Waals radii set to 0 so that it can freely penetrate into the protein and one atom with a
radius of 1.4 Å, which mimics the copper ion. The sulfur atoms of
Cys13 and Cys16 were linked to the metal ion
through upper distance limits of 2.5 Å. This approach does not impose
any fixed orientation of the ligands with respect to the copper. REM
was then applied within the molecular mechanics and dynamic module of
SANDER (37). The force field parameters for the copper(I) ion were
adapted from similar systems (38). In particular, no constraint on the
S(Cys13)-Cu-S(Cys16) angle was used. The
values of NOE and torsion angle potentials were calculated with force
constants of 50 kcal mol
The program CORMA (39), which is based on relaxation matrix
calculations, was used to back calculate the NOESY cross-peaks from the
calculated structure to check the consistency of the analysis. The
quality of the structure was evaluated in terms of deviations from
ideal bond lengths and bond angles and through Ramachandran plots,
obtained using the programs PROCHECK (40) and PROCHECK-NMR (41).
Structure calculations and analyses were performed on IBM RISC 6000 computers.
Sequence-specific Assignment of Apo-Ccc2a and Cu(I)-Ccc2a--
The
1H NMR spectra of apo-Ccc2a and Cu(I)-Ccc2a are reported in
Fig. 1, A and B,
respectively. The most relevant difference between apo and Cu(I) form
is observed in the HN region. In particular, the HN resonance of
Thr12 is broader in the apo form than in Cu(I)-Ccc2a and
has a change in the shift from 9.72 ppm, in the apo form, to 9.21 ppm,
in the Cu(I) form. In the 1H NMR spectrum of the Cu(I)
form, it is also possible to identify the HN resonance of
Ala15 at 9.50 ppm, a residue very close to the cysteine
binding motif, that cannot be detected in the apo form.
Assignments of the resonances of apo-Ccc2a started from the analysis of
the 15N heteronuclear single quantum coherence map, which
allowed the identification of the 15N and 1HN
resonances. Then through the analysis of the three-dimensional NOESY-heteronuclear multiple quantum coherence and of two-dimensional NOESY and total correlation spectroscopy, the sequence-specific assignment was performed. The assignment of the Cu(I)-Ccc2a derivative was performed through the analysis of two-dimensional NOESY and total
correlation spectroscopy maps only. Resonances for all 72 residues both
for the apo and Cu(I) forms of Ccc2a have been assigned. In the apo and
in the Cu(I) protein, about 97 and 98% of the proton resonances,
respectively, could be located in the maps, and all of the
15N resonances have been assigned, with the exception of
Ser14 and Ala15 in the apo form and with the
exception of Ser14 in the Cu(I) form. The 1H
and 15N resonance assignments of the apo and Cu(I) forms
are reported in Tables I and II of the supplementary materials,
respectively.2
Secondary Structure from NMR Data--
The elements of secondary
structure were identified by analyzing the pattern of assigned NOEs.
Backbone short, medium range NOEs were used to generate Fig.
2, A and B. From
their analysis, it is apparent that the secondary structure is not
significantly affected by the presence or absence of the copper ion. In
Ccc2a, two elements of helical secondary structure can be predicted, which are characterized by a high number of sequential and medium range
connectivities such as dNN(i,
i + 1), dNN(i,
i + 2),
d Solution Structure Calculations and Analysis of Apo-Ccc2a--
A
total of 3785 NOESY cross-peaks were assigned, integrated, and
transformed in upper distance limits with the program CALIBA (35). They
corresponded to 2314 unique upper distance limits, of which 1970 were
found to be meaningful (nonmeaningful distance constraints are those
that cannot be violated in any structure conformation and those
involving proton pairs at fixed distance). The number of NOEs per
residue is reported in Fig.
4A. The average number of NOEs
per residue is 32 for apo-Ccc2a, of which 27 are meaningful. 35 3JHNH
The 35 conformers constituting the final DYANA family had an average
target function of 0.64 ± 0.10 Å2 and average r.m.s.
deviation values over all of the 72 residues with respect to the mean
structure of 0.39 ± 0.05 Å for the backbone and of 0.80 ± 0.07 Å for the heavy atoms. The family of conformers was then
subjected to further refinement through energy minimization (37). The
REM family has an average target function of 0.14 ± 0.02 Å2, to which the NOE contribution is 0.13 Å2,
while the torsional angle one is 0.01 Å2. The average
r.m.s. deviation values for the family with respect to the mean
structure are 0.38 ± 0.06 Å for the backbone and 0.82 ± 0.07 Å for the heavy atoms for all of the amino acids in the sequence.
The r.m.s. deviation values per residue of the final REM family to the
mean structure are shown in Fig. 4B.
The final family of conformers was analyzed with PROCHECK-NMR (41), and
results are reported in Table I.
According to this program, the secondary structure elements in the
energy-minimized mean structure involve residues 2-9 (
The structure of apo-Ccc2a, shown in Fig.
5, is well defined all over its sequence.
All of the Solution Structure Calculations and Analysis of Cu(I)-Ccc2a--
A
total of 3866 NOESY cross-peaks were assigned, integrated, and
transformed in upper distance limits with the program CALIBA (35). They
corresponded to 2338 upper distance limits, of which 1944 were found to
be meaningful. The number of NOEs per residue is reported in Fig.
6A. The average number of NOEs
per residue is 32, of which 27 are meaningful. Hydrogen bond
constraints for 22 amide protons were used at later stages of
structural calculations. A total of 47 proton pairs were
stereospecifically assigned through the program GLOMSA (35). The
constraints used for structure calculations and the stereospecific
assignments are reported in the supplementary
materials.2
The 35 conformers constituting the final DYANA family had an average
target function of 0.48 ± 0.11 Å2 and an average
r.m.s. deviation value over all of the 72 residues with respect to the
mean structure of 0.39 ± 0.06 Å for the backbone and of
0.79 ± 0.04 Å for the heavy atom. The family of conformers was
then subjected to further refinement through energy minimization (37).
The REM family has an average target function of 0.26 ± 0.03 Å2. The average r.m.s. deviation values for the family
with respect to the mean structure are 0.36 ± 0.06 Å for the
backbone and 0.79 ± 0.05 Å for the heavy atoms for all of the
amino acids in the sequence. The r.m.s. deviation values per residue of
the final REM family to the mean structure are shown in Fig.
6B.
The final family of conformers was analyzed with PROCHECK-NMR (41), and
results are reported in Table II. The
secondary structure elements in the energy-minimized mean structure
involve residues 2-9 (
The structure of Cu(I)-Ccc2a, shown in Fig.
7, is also well defined all over its
sequence. All of the Comparison between the Structures of Apo- and Cu-Ccc2a--
The
solution structure of S. cerevisiae Ccc2a exhibits the
The backbone r.m.s. deviation values for each secondary structure
element are obtained either superimposing all residues (global r.m.s.
deviation) or three residues at a time (local r.m.s. deviation) and are
reported in Table III. The highest r.m.s.
deviation values are found for strand
Comparing the copper binding region in apo- and Cu(I)-Ccc2a, helix
Upon copper release, the most important change in the shallow binding
pocket is observed for the side chain of Cys13; the sulfur
flips away from the hydrophobic interior toward the surface (Fig.
9). Calculations of the solvent
accessibility on the apo-Ccc2a structure show that exposure of
Cys13 is remarkably increased (from 23% in the
copper-bound form to 36% accessible surface in the apo form), while
Cys16 is always more buried (with 7% in the copper-bound
form and 11% accessible surface in the apo form).
The changes in HN and H
The methionine in the GMXCXXC metal binding loop
(Met11) is highly conserved between other N-terminal,
membrane-tethered domains of heavy metal ATPases and small
metallochaperones alike. Of all of the residues in the protein,
Met11 shows the greatest change in 1H Comparison between the Solution Structures of the Fourth Metal
Binding Cytosolic Domain from Menkes Copper-transporting ATPase and
Cu(I)-Ccc2a--
The structure of Cu(I)-Ccc2a is similar to the
Ag(I)-bound solution structure of the fourth metal-binding domain
(mbd4) of Menkes ATPase (20), the human homologue of S. cerevisiae Ccc2. A sequence alignment of Ccc2a with this protein
(Fig. 11A) reveals 29%
identity. The structures were superimposed according to the sequence
alignment. Both proteins show the same ferrodoxin-like fold (Fig.
11B). The overall backbone r.m.s. deviation value is 1.15 Å between Cu(I)-Ccc2a and Ag(I)-mbd4. The major structural differences
are represented by simple translations of the secondary structure
elements. All
It is worth noting that in the refinement of the Ag(I)-mbd4 solution
structure, a linear digonal coordination was imposed by
modifying the AMBER force field (20). In the data refinement for the
Cu(I)-Ccc2a family of conformers, no S-Cu-S angle constraints were
included. The resulting value of the S-Cu-S angle is 119 ± 29°.
When the S-Cu-S angle is constrained to linearity, a digonal copper thiolate center can be refined. This suggests that coordination is not rigid but that Cu(I) in this environment may represent a mixture
of coordination numbers of two and higher or that a bent two-coordinate
S-Cu-S geometry is adopted. No other protein atoms appear close enough
to be the third ligand in Ccc2a. A third coordinating atom, if there is
one at all, can come only from an exogenous ligand, such as a buffer
component. While no low molecular weight thiols (e.g. DTT,
GSH) are present in the sample, other buffer components could be
coordinating to the Cu(I). The NMR data here do not allow us to
distinguish between these possibilities.
Comparison between the Solution Structures of Ccc2a and Atx1 for
both Apo and Cu(I) States--
The solution structures of Cu(I)- and
apo-Atx1 from S. cerevisiae have been recently solved (46).
The global folding of Atx1 is very similar to that of Ccc2a. When
superimposing Cu(I)-Ccc2a and Cu(I)-Atx1 structures according to the
sequence alignment (see Fig. 11A), the overall backbone
r.m.s. deviation value is 2.8 Å. Helix
The structures of apo-Ccc2a and apo-Atx1 show an overall backbone
r.m.s. deviation value of 2.93 Å (Fig.
12) and exhibit more differences than
do the two copper-loaded forms (Fig. 11C). The
Another important difference between these copper donor and acceptor
proteins is apparent in Fig. 13,
A and B, namely the accessibility of the copper
to incoming nucleophiles. The solvent-accessible surface of the Cu(I)
center in Cu-Atx1 is 9% but over 18% in Cu-Ccc2a. Access to the Cu(I)
in Atx1 is partially obscured by Lys65, which is highly
conserved among copper chaperones. In Ccc2a, this residue corresponds
to Phe64, and the structure reveals that it extends into
the hydrophobic core, away from the surface. In fact, a Phe (or a Tyr)
is highly conserved among all of the domains of the CPx ATPases, while
Lys65 is conserved among the diffusable Atx1 and CCS copper
chaperone proteins (44). The Phe64 side chain packs
adjacent to the conserved Met11 and is anticipated to
contribute to the stability of the metal binding loop in Ccc2a. In
contrast, the lysine at this position in Atx1 (Lys65) can
access several conformations and may play a role in partner recognition
and control of metal ion access.
Electrostatic Surface and Structural Implications for Interaction
with Physiological Partners--
Partnership between Atx1 and Ccc2
in vivo requires several basic residues, which cluster in
several sites on the surface of Atx1 (18). The structure of Ccc2a
reveals a complementary set of acidic residues. The surface
electrostatic potential distribution was generated with the program
MOLMOL (45) using the refined coordinates of the Cu(I)-Ccc2a structure
(Fig. 13A). This protein has a region comprising several
glutamate and aspartate residues that generate a negatively charged
face on the protein surface in the proximity of the copper binding
region. These residues are Glu67, Glu57,
Glu60, Asp65, Asp61, and
Asp53. In particular, Asp65, Asp61,
and Glu60 are conserved in the metal-binding domain of a
large number of metal-transporting ATPases. The surface electrostatic
potential distribution was also generated for human mbd4 (Fig.
13B). A very similar negative patch, formed by
Asp67, Glu62, and Asp63, which are
conserved in the homologous yeast Ccc2a, and Asp10 is
observed on one surface of the protein. The electrostatic potential
distribution of Cu(I)-Atx1 is almost complementary and shows the
presence of 7 lysines (46), in positions 24, 28, 59, 61, 62, 65, and
71, which generate a positively charged face on the molecule, as shown
in Fig. 13C. It is known that the ATPase Ccc2 is the target
of copper delivery by Atx1 (15, 16, 24). The interaction between Atx1
and Ccc2a could be therefore determined by the complementary
attractions between the positive cluster in Atx1 and the negative
region in Ccc2a.
Finally, a large "nonpolar" area is present for both Ccc2a and mbd4
proteins adjacent to the metal binding site in a region composed of
residues Leu37, Val38, and Ile35.
In fact the side chain of Val38 extends into the solvent,
and in Atx1 this residue is replaced with Glu (Fig. 11A).
These solvent-exposed nonpolar regions may be important after transfer
of copper from Atx1 to Ccc2a. For instance, in the subsequent steps of
the original copper-trafficking mechanism, Cu(I) is transferred to a
cytosolic face of the membrane-spanning ATPase (15). The fate of the
Cu(I) bound by Ccc2a is not known, but it is speculated to be
transferred to a cation translocation site within the membrane portions
of Ccc2, such as the canonical CPC motif. In an extension of this
model, we propose that a hydrophobic patch on mobile N-terminal
domains of Ccc2a or mbd4 can serve as a site for interaction with other
hydrophobic sites essential for copper movement across the membrane.
After transfer and ATP-induced conformational changes, the Cu(I) would
be subsequently released to the interior of the ATPase-containing
vesicle. Tests of this model are under way.
This paper presents the first report of the solution structure of
a copper-transporting ATPase domain bound to the native metal ion
Cu(I). The fold is similar to its physiological partner, the
cytoplasmic copper chaperone Atx1 (17) and is even more similar to the
structure of a metal-binding domain of one of its human orthologs, the
Menkes' disease protein (20). Unlike other structurally characterized
members of this family, both of the metal-binding cysteines of Ccc2a
are located in a helical region, and the metal is more accessible to
solution. Comparisons of the cytosolic domains of the transporters with
the chaperones reveal complementary features that are important in
their respective copper-trafficking functions. First, unlike Atx1, few
conformational changes are observed in Ccc2a upon copper release, and
the metal binding region is well defined in both the apo and holo forms of this domain. In the case of apo-Atx1, the amide NH for residues close to the copper binding, in particular Cys18, are not
detected, and this disappearance could be ascribed to an increased
disorder/mobility of the loop region. In contrast, apo-Ccc2a exhibits a
higher degree of order and both amide HN of the metal-binding cysteines
are detected. Furthermore, NOE signal intensity is significant for both
Cys13 and Cys16 in apo-Ccc2. Together, these
results suggest a significantly greater stability of the Ccc2a domain
with respect to Atx1.
Finally, there are several mechanistically important aspects of the
Ccc2a surface that differ from Atx1. One prominent feature is a
negative patch (Fig. 13) formed by glutamate and aspartate residues in
the proximity of the copper binding region that could interact with
complementary array of lysine and arginine residues on the Atx1 (17). A
phenylalanine is found in Ccc2 in place of the pivotal
Lys65 residue of Atx1. Side chains at this site abut the
metal binding loop and are anticipated to not only play an important
role in determining the stability of this region of the protein but are likely to control key steps in the metal transfer mechanism.
to obtain a family of 35 structures
with root mean square deviation to the average structure of 0.38 ± 0.06 Å for the backbone and 0.82 ± 0.07 Å for the heavy
atoms. The protein exhibits a
, ferrodoxin-like fold
similar to that of its target Atx1 and that of a human counterpart, the
fourth metal-binding domain of the Menkes protein. The overall fold
remains unchanged upon copper loading, but the copper-binding site
itself becomes less disordered. The helical context of the
copper-binding site, and the copper-induced conformational changes in
Ccc2a differ from those in Atx1. Ccc2a presents a conserved acidic
surface which complements the basic surface of Atx1 and a hydrophobic
surface. These results open new mechanistic aspects of copper
transporter domains with physiological copper donor and acceptor proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
structural fold. This same structural fold is found
in the mercury-binding protein MerP (21, 22) and the putative copper
chaperone CopZ (23). In all of these domains, the cysteine ligands are
located within the first loop and the first helix.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
coupling constants. The spectrum was
recorded as a 128 (1H) × 80 (15N) × 2048 (1H) data set using pulsed field gradients along the
z axis. The mixing time was 100 ms. Spectral windows of 14, 14, and 29 ppm were used, respectively, for direct 1H
dimension and the indirect 1H and 15N dimensions.
coupling constants were correlated to
the backbone torsion angle
by means of the appropriate Karplus
curve (33). These angles were used as constraints in the DYANA
calculations and restrained energy minimization (REM) refinement.
1
Å
2.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
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Fig. 1.
1H 800-MHz NMR spectra of
apo-Ccc2a (A) and Cu(I)-Ccc2a (B)
proteins at 298 K and pH 7.0 in 100 mM phosphate
buffer. The protein concentrations were 2.7 and 1.2 mM, respectively.
N(i, i + 3), d
N(i, i + 4), and d
(i, i + 3). The two helices involve residues 14-27 and 51-63 in the apo form.
The presence of medium range connectivities such as
dNN(i, i + 2),
d
N (i, i + 2), and dNN(i, i + 3) for
residue Cys13 in the Cu(I) form with respect to the apo
form gives evidence that Cys13 belongs to the the beginning
of helix
1 in the Cu(I) form. All of the backbone NOEs were used to
generate Fig. 3, A and
B, from which it is possible to recognize the presence of
four antiparallel
-sheets in both apo and Cu(I) forms, involving
residues 2-8, 29-35, 40-46, and 65-71. Fig. 3 also shows that the
typical folding pattern of the copper chaperones, "open-faced
-sandwich" fold (
1-
1-
2-
3-
2-
4) (17, 20, 21), is
present also in this protein, both in the presence and absence of
copper.
View larger version (22K):
[in a new window]
Fig. 2.
Schematic representation of the sequential
and medium range NOE connectivities involving NH,
H , and H
protons for
apo-Ccc2a (A) and Cu(I)-Ccc2a
(B). The thickness of the
bar indicates the intensity of NOEs.
View larger version (39K):
[in a new window]
Fig. 3.
Schematic representation of long range
connectivities for apo-Ccc2a (A) and Cu(I)-Ccc2a
(B). Segments perpendicular to the
diagonal of the plot represent pairs of anti-parallel
-strands.
couplings were obtained from the
HNHA three-dimensional spectrum, which were translated into
dihedral angles through the standard equation. For
3JHNH
values of >8 and <4.5 Hz, the
angle was assumed to be between
155 and
85° and between
80 and
30°, respectively (42, 43). Hydrogen bond constraints for 31 amide
protons were used at later stages of structural calculations. A total
of 38 proton pairs were stereospecifically assigned through the program GLOMSA (35). The constraints used for structure calculations and the
stereospecific assignments are reported in the supplementary materials.2
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Fig. 4.
A, number of meaningful NOEs per residue
for apo-Ccc2a. White, gray, and black
bars indicate intraresidue, sequential, and medium/long
range connectivities, respectively; B, r.m.s. deviation per
residue to the mean structure of apo-Ccc2a for the backbone
(filled squares) and all heavy atoms
(filled circles) of the REM structure family of
35 conformers.
1), 13-26
(
1), 29-36 (
2), 40-47 (
3), 51-63 (
2), and 65-70 (
4).
Analysis of the NOE patterns has led to somewhat similar conclusion
(see above). It is worth noting that in contrast to NOE secondary
structure analysis above, Cys13 is assigned to helix
1
in the energy-minimized mean structure of the apo form. In this
energy-minimized average structure, 82.1% of the residues are in the
most favored regions of the Ramachandran plot, and 17.9% of the
residues are in the allowed regions. No residues are in the disallowed
regions (Table I).
Statistical analysis of the final REM family and the mean structure of
apo-Ccc2a from S. cerevisiae
-helices and antiparallel
-sheet are very well
defined, with an average backbone r.m.s. deviation lower than 0.38 Å.
The largest backbone r.m.s. deviation values are obtained for residues
12-14 and residue 48. The high r.m.s. deviation values of residues
12-14 (0.74 Å) are due to the paucity of NOEs in this region (Fig.
4A) that constitutes loop 1 and the beginning of helix
1.
Indeed, this region contains Ser14 and Ala15,
whose HN resonances have not been identified and are a break in the
sequential connectivities. This region includes Cys13, one
of the copper ligands. The side chain of Cys13 is
disordered in the apo state and spans different conformations due to
the small number of NOEs. On the contrary, the other copper ligand
Cys16, which belongs to the first
-helix, is very well
defined (r.m.s. deviation BB, 0.22 Å; r.m.s. deviation HA, 0.37 Å).
The side chains of Cys13 and Cys16 are very
close; the distance between the two sulfur atoms in the average
structure is 5.6 Å.
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Fig. 5.
Backbone atoms for the solution structure of
apo-Ccc2a as a tube with variable radius, proportional to the backbone
r.m.s. deviation value of each residue. The side chains of
Cys13 and Cys16 are also shown. The
figure was generated with the program MOLMOL (45).
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Fig. 6.
A, number of meaningful NOEs per residue
for Cu(I)-Ccc2a. White, gray, and
black bars indicate intraresidue, sequential, and
medium/long range connectivities, respectively. B, r.m.s.
deviation per residue to the mean structure of Cu(I)-Ccc2a for the
backbone (filled squares) and all heavy atoms
(filled circles) of the REM structure family of
35 conformers.
1), 13-26 (
1), 29-36 (
2), 40-47
(
3), 51-62 (
2), and 65-70 (
4). Analysis of the NOE patterns
has led to a similar conclusion (see above). In the energy-minimized
average structure, 83.6% of the residues are in the most favored
regions of the Ramachandran plot, 13.4% of the residues are in the
allowed regions, and 3.0% are in the generously allowed regions. No
residues are in the disallowed regions (Table II).
Statistical analysis of the final REM family and the mean structure of
Cu(I)-Ccc2a from S. cerevisiae
-helices and antiparallel
-sheet are very
well defined and also the loop that includes the copper binding site is
more defined than in the apo form. Indeed, the backbone r.m.s.
deviation values with respect to the mean structure for residues in the
less well defined region of apo (10-14) decrease from 0.64 Å in the
apo form to 0.27 Å in the Cu(I) form, and the side chain of
Cys13 that in the apo form is disordered (r.m.s. deviation
BB, 0.80 Å; r.m.s. deviation HA, 1.17 Å) is very well defined in the
Cu(I) form (r.m.s. deviation BB, 0.25 Å; r.m.s. deviation HA, 0.62 Å).
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Fig. 7.
Backbone atoms for the solution structure of
Cu(I)-Ccc2a as a tube with variable radius, proportional to the
backbone r.m.s. deviation value of each residue. The side chains
of Cys13, Cys16, and the Cu(I) ion are also
shown. The figure was generated with the program MOLMOL
(45).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
folding pattern typical of copper chaperones (44)
and the fourth metal-binding domain of the Menkes' disease protein (20). In the Cu-Ccc2a structure, the copper ion coordinates two of the
six cysteine residues, Cys13 and Cys16. The
global r.m.s. deviation values between the Cu(I)-bound and apo-averaged
minimized structures are 0.86 and 1.35 Å for backbone and heavy atoms,
respectively. The r.m.s. deviation per residue is reported in Fig.
8 (dotted dashed
line) and is compared with the r.m.s. deviation values per
residue for each family (apo-Ccc2a (solid line)
or Cu(I)-Ccc2a (dotted line)) and the sum of the r.m.s. deviation values of the two families (dashed
line). The structure definition within each family of
structures is overall very good and comparable between the two
families; thus, a meaningful comparison can be undertaken. The highest
backbone r.m.s. deviation value between the two structures is 1.21 Å (found for Thr51). There are only few regions or amino
acids in the protein where the r.m.s. deviation between the two average
minimized structures is significantly larger than the r.m.s. deviation
of each family when superimposing the whole structures (Fig. 8), thus
indicating a meaningful differences between the two structures.
Analysis of side chains reveals a significant r.m.s. deviation
difference for Thr17, which is in the copper binding
pocket. Indeed, when copper is bound, the side chain of
Thr17 rotates closer to the backbone oxygen of
Cys13, and a hydrogen bond interaction can be readily
formed, since it is found in many conformers of the family. This
interaction can presumably be important to determine the optimal
conformation of Cys13 in the copper-bound state of the
protein.
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Fig. 8.
r.m.s. deviation per residue (for backbone
atoms) between the conformers of the family of apo-Ccc2a
(solid line), between those of Cu(I)-Ccc2a
(dotted line), and between the mean
structures of the two families (dashed dotted
line). The sum of the r.m.s. deviation values per
residue for the two families of conformers is also shown
(dashed line).
2, helix
2, and loops 1, 3, and 4. The r.m.s. deviation values for all of these regions but the
last drop when the two structures are superimposed locally, indicating that the structural differences in these regions originate from some
global translational displacements, since the local conformations are
well maintained. On the contrary, loop 4 shows the highest difference
in the local r.m.s. deviation, and these local difference are due to
variations of dihedral angles.
Comparison of the solution structure of Cu(I)-Ccc2a with the reduced
apo-Ccc2a solution structure
1
(containing Cys13 and Cys16) is very well
defined in both forms, while loop 1, which is involved in the copper
binding pocket, is less defined in the apo form (see Fig. 8). The
conformation of Cys16 is well defined in both structures,
being the same in both the apo and metal-bound proteins, while
Cys13 is more disordered in the apo form than in the
metal-bound protein. Indeed, in the apo-Ccc2a structure, loop 1 becomes
more disordered (Fig. 5), and the amide NHs of residues 14 and 15, belonging to helix
1, are not detected anymore, probably due to
solvent exchange or to an increased mobility. The lower number of NMR
constraints in the metal-binding pocket (i.e. residues
12-17) of the apo form might be the result of increased conformational
flexibility in this region with respect to the metal-bound state.
Indeed, different NOE intensity for proton pairs at fixed distances of
Cys13 and Cys16 of Ccc2a has been observed in
the two forms; Cys13 experiences a decrease in intensity,
while those of Cys16 show roughly the same intensity,
suggesting that the binding of copper produces higher order in this region.
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Fig. 9.
A, backbone drawing of Cu(I)-Ccc2a
(green) and apo-Ccc2a (blue). The copper ion is
in yellow. Side chains of Ile21,
Phe64, Met11, and Leu38 are
reported. The secondary structure elements are indicated. The
inset shows the copper binding region.
chemical shifts observed between the apo and
the Cu(I)-bound forms are plotted for each residue in Fig.
10, A and B,
respectively, and confirm that only residues very close to the metal
binding pocket are affected by the presence of the copper ion. Indeed,
several highly conserved hydrophobic residues appear to play a role in
maintaining an optimal metal-ligand conformation. These include
Phe64 and Ile20, which are in Van der Waals
contact with Cys16, and Leu37, which contacts
Cys13 and is highly conserved between the Ccc2, Wilson, and
Menkes domains as well as Atx1 and CCS. While these side chains can
stabilize the metal binding loop, no significant conformational changes are detectable either as differences between their chemical shifts (Fig. 10, A and B) or as differences between the
two average structures (Fig. 9).
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Fig. 10.
Plot of the chemical shift differences
( (apo)
(Cu(I)-bound)) versus residue
number for the 1HN (A) and
1H
(B)
resonances.
resonances upon copper binding (Fig. 10, A and
B), but it is not directly involved in metal ion
coordination. Instead, it points toward the hydrophobic core of the
protein (Fig. 9). The contacts between this side chain and the residues
surrounding it change little between the apo and Cu(I)-bound forms
(Fig. 9) as judged from the observed medium and long range NOEs;
however, copper occupancy does change the conformation of this residue.
These results suggest that Met11 acts as a hydrophobic
tether that anchors the metal-binding loop via hydrophobic interactions
with Leu37, Phe64, and Ile20. The
slight movements in the region are probably coupled to stabilization of
the GMXCXXC domain in the presence of bound metal ion.
-helices and
-strands are well superimposed or show
slight displacements except for the short C-terminal
-strand. The
conserved hydrophobic residues Ile20, Leu37,
and Phe64, presumably important for maintaining optimal
metal-binding loop conformation, have the same conformation in the two
structures. The biggest differences are observed in loop 1 and loop 4. Indeed, loop 1 differs in the vicinity of Cys13, which does
not superimpose with Cys14 of mbd4. The conformation of the
other cysteine (Cys16 and Cys17, respectively)
is very similar between the two structures. In Cu(I)-Ccc2a,
Cys13 is the first residue of helix
1, while in mbd4,
Cys14 belongs to the metal binding loop 1 (Fig.
11B). The stabilization of the helix and the variation in
the conformation of loop 1 in Cu(I)-Ccc2a with respect to Ag(I)-mbd4
may be dictated by the different chemical properties of Cu(I)
versus Ag(I) and by the different kinds of residues in the
vicinity of the loop 1 region.
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Fig. 11.
A, sequence alignment of the Ccc2a
amino acid sequence from S. cerevisiae with the sequences of
the fourth metal-binding domain from Menkes-transporting ATPase (mbd4)
(Protein Data Bank accession number 1aw0) (20) and of Atx1. The
positions of the Ccc2a secondary structure elements (as found in the
mean Cu(I) structure) are shown at the top. -strands are
in blue,
helices are shown in orange, and
loop regions are in yellow. Each sequence is
color-shaded according to secondary structure element, as
found in their metal-bound structures. Residues that are highly similar
or conserved are indicated, respectively, by the
and *
below the sequences. B, comparison of
the backbone of Cu(I)-Ccc2a (blue) and Ag(I)-mbd4
(green) structures (20). C, comparison of
backbone of Cu(I)-Ccc2a (blue) and Cu(I)-Atx1
(green). The copper ion and the cysteine ligands are also
shown. The secondary structure elements are indicated.
2;
-strands 1, 2, 3; and
loops 3 and 5 are well superimposed (Fig. 11C). The major
structural differences are translations or changes in length of the
secondary structure elements and of the loops. The largest differences
are found for helix
1,
-strand 4, and loops 1, 2, and 4. The
length of helix
1 in the Cu(I) form of both proteins is the same,
but in Cu(I)-Atx1 this helix spans from residue 17 to 30, while in the
Cu(I)-Ccc2a the same helix is slightly offset, starting from residue 13 and ending at 26. Loop 1 exhibits conformational differences, in
particular close to Cys15. This cysteine in Cu(I)-Atx1
belongs to loop 1, whereas in Cu(I)-Ccc2a it belongs to helix
1.
These differences in secondary structure lead to distinct positioning
of the metal-binding cysteines (see Fig. 11C) and allow the
copper ion to be more exposed to solvent in Ccc2a with respect to Atx1
(see below). In addition, the offset of helix
1, the different loop
size, and the presence of Pro31 in the Atx1 sequence (see
Fig. 11A) determine a large conformational difference in
loop 2, which is more extended toward the surface in the Cu(I)-Atx1 structure.
-strands
1, 2, and 3 and the loops 3 and 5 are well superimposed for the
apoproteins; however, differences in other secondary structure elements
are apparent. For example, helix
1 is one full turn shorter in
apo-Atx1 than in apo-Ccc2 (Fig. 12). Helix
2, which superimposes
well in both forms of Atx1 (46), is translated away from the
copper-binding site in apo-Atx1 relative to its position in apo-Ccc2a
(Fig. 12). While the structure of Atx1 undergoes changes as a function
of copper capture and release, the Ccc2a structure remains relatively
invariant, suggesting that the metal site in apo-Ccc2a is more
preorganized than in apo-Atx1. This is one of the key structural
differences between the Atx1 metallochaperone family and the homologous
metal-binding domains of the copper-transporting P-type ATPases.
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Fig. 12.
Comparison of the backbone of apo-Ccc2a
(blue) and apo-Atx1 (green). The
cysteines involved in the copper binding are indicated in
blue and green for apo-Ccc2a and
apo-Atx11 structures, respectively.
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Fig. 13.
Electrostatic potential surface of the
Cu(I)-Ccc2a (A), Ag(I)-mdb4 (B), and
Cu(I)-Atx1 (C). The positively charged,
negatively charged, and neutral amino acids are represented in
blue, red, and white, respectively.
Copper ion is represented in green, silver ion in
teal, and cysteine sulfur in yellow. In
A and C, the residues that might have a role in
molecular recognition and copper transfer are indicated. In
B, the negative residues that form a negative region close
to metal binding loop are indicated. The figure was
generated with the program MOLMOL (45).
CONCLUSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
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FOOTNOTES |
---|
* This work was supported by European Community Contract HPRI-CT-1999-00009, Italian Consiglio Nazionale delle Ricerche (Progetto Finalizzato Biotecnologie 99.00286.PF49), Ministero della Università e delle Ricera Scientifica e Tecnologica (MURST), National Institutes of Health Grant GM 54111 (to T. V. O.), and Molecular Toxicology Training Program Postdoctoral Fellowship 5T32ES07284 (to D. L. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence may be addressed: Prof. Ivano Bertini, Magnetic Resonance Center and Department of Chemistry, University of Florence, Via L. Sacconi 6, Sesto Fiorentino, Florence, 50019 Italy. Tel.: 39-055-4574272; Fax: 39-055-4574271; E-mail: bertini@cerm.unifi.it.
** A fellow of the John Simon Guggenheim Foundation. To whom correspondence may be addressed: Dept. of Chemistry, Northwestern University, 2145 Sheridan Rd., Evanston, IL 60208. Tel.: 847-491-5060; Fax: 847-491-7713; E-mail: t-ohalloran@northwestern.edu.
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M008389200
2 F. Arnesano, L. Banci, I. Bertini, D. L. Huffman & T. V. O'Halloran, (2001) Biochemistry 40, 1528-39..
2 Supplementary materials may be accessed at the CERM site, under Structural Biology, on the World Wide Web.
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ABBREVIATIONS |
---|
The abbreviations used are: MES, 4-morpholineethanesulfonic acid; r.m.s., root mean square; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; INEPT, insensitive nuclei enhanced by polarization transfer; WATERGATE, water suppression by gradient-tailored excitation; REM, restrained energy minimization; mbd4, metal-binding domain 4.
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REFERENCES |
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