Báo cáo khoa học: Coordination of three and four Cu(I) to the a- and b-domain of vertebrate Zn-metallothionein-1, respectively, induces significant structural changes doc

Coordination of three and four Cu(I) to the a- and b-domain
of vertebrate Zn-metallothionein-1, respectively, induces
significant structural changes
Benedikt Dolderer
1
, Hartmut Echner
1
, Alexander Beck
1
, Hans-Ju
¨
rgen Hartmann
1
, Ulrich Weser
1
,
Claudio Luchinat
2
and Cristina Del Bianco
2
1 Interfakulta
¨
res Institut fu
¨
r Biochemie, University of Tu
¨
bingen, Germany
2 Magnetic Resonance Center, University of Florence, Sesto Fiorentino, Italy
The first member of the metallothionein (MT) family
was isolated in 1957 [1]. Since then, a large number
of proteins have been described featuring common

characteristics. They include ubiquitous small cys-
teine-rich proteins (50–70 amino acids) that are able
to bind many d
10
metal ions [2]. A wealth of different
biological functions has been proposed and continues
to be discovered. Obviously, MTs play important
roles in minimizing the uncontrolled reactions of
heavy metal ions like cadmium and the homeostasis
of essential metal ions including copper(I) and zinc(II)
ions [2,3]. They are known to successfully cope with
oxidative stress and ionizing radiation [4,5]. Other
functions may be associated with the occurrence of
tissue-specific isoforms, such as the brain-specific iso-
form MT-3, which acts as neuronal growth inhibitory
factor [6,7].
Both mammalian MT-1 and MT-2 are composed of
the N-terminal b- and the C-terminal a-domain. They
are predominantly isolated containing zinc or cadmium
exclusively bound to cysteine thiolates. The nine cys-
teine residues of the b-domain accommodate a metal
(M)(II)
3
S
9
cluster, while 11 cysteine residues contribute
to the formation of a M(II)
4
S
11

cluster in the a-domain
[8]. However, under certain physiological conditions,
e.g. when isolated from fetal liver, mammalian MT-1
Keywords
copper; domain; metallothionein; protein
structure; 2D NMR
Correspondence
U. Weser, Anorganische Biochemie,
Interfakulta
¨
res Institut fu
¨
r Biochemie,
University of Tu
¨
bingen, Hoppe-Seyler-Str. 4,
D-72076 Tu
¨
bingen, Germany
Fax ⁄ Tel: +49 7071295564
E-mail:
(Received 17 January 2007, revised 28
February 2007, accepted 5 March 2007)
doi:10.1111/j.1742-4658.2007.05770.x
Vertebrate metallothioneins are found to contain Zn(II) and variable
amounts of Cu(I), in vivo, and are believed to be important for d
10
-metal
control. To date, structural information is available for the Zn(II) and
Cd(II) forms, but not for the Cu(I) or mixed metal forms. Cu(I) binding to

metallothionein-1 has been investigated by circular dichroism, luminescence
and
1
H NMR using two synthetic fragments representing the a- and the
b-domain. The
1
H NMR data and thus the structures of Zn
4
a metallo-
thionein (MT)-1 and Zn
3
bMT-1 were essentially the same as those already
published for the corresponding domains of native Cd
7
MT-1. Cu(I) titra-
tion of the Zn(II)-reconstituted domains provided clear evidence of stable
polypeptide folds of the three Cu(I)-containing a- and the four Cu(I)-con-
taining b-domains. The solution structures of these two species are grossly
different from the structures of the starting Zn(II) complexes. Further addi-
tion of Cu(I) to the two single domains led to the loss of defined domain
structures. Upon mixing of the separately prepared aqueous three and four
Cu(I) loaded a- and b-domains, no interaction was seen between the two
species. There was neither any indication for a net transfer of Cu(I)
between the two domains nor for the formation of one large single Cu(I)
cluster involving both domains.
Abbreviations
M, metal; MT, metallothionein.
FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS 2349
and MT-2 are also found to be enriched with Cu(I)
[9]. For other members of the MT family, different

metal cluster architectures were reported. The previ-
ously mentioned MT-3, which is also a two-domain
protein, for example, is composed of a Cu(I)
4
S
9
cluster
in the N-terminal b-domain and a Zn(II)
4
S
11
cluster in
the C-terminal a-domain [10,11]. Examples for solely
Cu(I) binding MTs are Cu(I)
8
thionein from Saccharo-
myces cerevisiae and Cu(I)
6
thionein from Neurospora
crassa [12–14]. Differently from other described MTs,
these two fungal proteins consist only of a single
domain harbouring homometallic Cu(I) thiolate clus-
ters [13,14].
The three-dimensional structure of Cd
5
Zn
2
MT-2,
isolated from rat liver after cadmium supplementa-
tion, was determined using both NMR and X-ray

crystallography [15]. The entire protein is dumbbell-
shaped and contains two independent domains. The
polypeptide backbone wraps around the metal thio-
late core forming the scaffold of the two domains.
All metal ions are tetrahedrally surrounded by four
thiolate sulphur atoms. In the N-terminal b-domain,
the three metal ions and the three bridging thiolate
sulphurs are ordered to form a distorted chair. The
C-terminal a-domain is characterized by an adaman-
tane-like four-metal cluster. Solution structures of
113
Cd-substituted Cd
7
MT-2 from rabbit, rat and
human are available and revealed structural identity
with the structure of Cd
5
Zn
2
MT-2 [8]. Comparative
NMR studies provided evidence that Zn(II) can iso-
morphically replace Cd(II) in MT-2 [16]. This result
was corroborated by NMR studies on cobalt substi-
tuted MTs, as cobalt was often used as a zinc ana-
logue in structural investigations [17–19]. The solution
structure of murine
113
Cd
7
MT-1 showed high similar-

ity with rat liver MT-2. Its b -domain, however,
turned out to be more flexible than in the latter
protein, exhibiting enhanced cadmium–cadmium
exchange rates [20].
The structural and spectroscopic data available on
Cd(II)-substituted human MT-3 indicated the forma-
tion of two metal thiolate clusters, similar to those
found in MT-1 and MT-2. Further investigation of
that protein pointed towards a highly dynamic struc-
ture [8]. Recently, a high-resolution solution structure
of the C-terminal a-domain has become available. The
data revealed a tertiary fold very similar to that of
MT-1 and MT-2, except for a loop that contains an
acidic insertion that is highly conserved in these iso-
forms. The structure of the b-domain has escaped its
experimental solution, as no characteristic signals
attributable to its residues were observed using NMR.
On the basis of homology modelling, a backbone
arrangement virtually identical to the corresponding
domains in MT-1 and MT-2 was suggested [21].
Despite the large number of structural data available
for the MT family, only the structures of two MTs
containing Cu(I) were known until now. One of them
is the aforementioned yeast MT whose structure was
successfully determined using both 2D NMR and
X-ray diffraction [22–24]. This protein forms one single
domain that harbours eight Cu(I) ions. Six of them are
coordinated by three thiolate sulphur atoms, whereas a
linear binding mode was observed for the remaining
two. The solution structure of N. crassa MT backbone,

in which, like yeast, the MT solely binds Cu(I), repre-
sents the second known structure of a copper thionein
[25]. Its polypeptide chain wraps around the copper
sulphur cluster in a left-handed form in the N-terminal
half of the molecule and in a right-handed form in the
C-terminal half. Due to the lack of copper isotopes
with NMR-active spin ½, no metal–cysteine restraints
were available to solve the positions of Cu(I) within
the N. crassa MT polypeptide fold.
At present, the structural information on Cu(I)-loa-
ded forms of mammalian MTs is rather limited.
In vitro, Cu(I) titrations of isolated MT-2 and its sep-
arate domains demonstrated that up to six Cu(I) ions
can bind to each domain [26]. In another extensive ti-
tration study, it was postulated that zinc was required
for the in vivo and in vitro folding of the two domains
of copper MTs [27]. Replacement of Zn(II) by Cu(I)
led to the proposal of the formation of Cu,Zn-MT
intermediates and that, during the last steps of copper
titration, the two domains merge into one. However,
earlier Cu(I) titration studies of rat liver MT clearly
showed that the two domains remained separated
[26]. Additionally, the cooperative formation of
(Cu
3
Zn
2
)a(Cu
4
Zn

1
)bMT)1 upon addition of Cu(I) to
(Zn
4
)a(Cu
4
Zn
1
)bMT)1 indicated that the preference of
Cu(I) for binding to the b-domain is only partial and
not absolute, as widely accepted until now [27].
It was of interest to shed some light on the changes
of the molecular architecture of the two domains of
vertebrate MT when Cu(I) is added to them. For this
task, the synthetic murine aMT-1 and bMT-1 domains
were prepared for subsequent Cu(I) titrations. Employ-
ing NMR, we obtained an interesting and unexpected
picture of the Cu(I) binding to the two single domains.
Results and Discussion
Cu(I) titration of Zn
4
aMT-1 and Zn
3
bMT-1
As both the structure of native Zn
7
MT-1 was known,
and several Cu(I) binding stoichiometries for its two
Murine Cu(I)a- and Cu(I)b-MT-1 domains B. Dolderer et al.
2350 FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS

domains had been proposed, it was of interest to shed
some more light upon their reactivity towards the pres-
ence of Cu(I). To this end, a Cu(I) titration study of
the two separated domains was performed employing
the combined detection of luminescence, circular
dichroism and
1
H NMR. Solid-phase peptide synthesis
was successfully employed to prepare the independent
a- (residues 31–61) and b-domains (residues 1–30) of
murine MT-1. Either domain was fully reconstituted
under anaerobic conditions with Zn(II) to yield
Zn
4
aMT-1 and Zn
3
bMT-1. For each Cu(I) titration
step, a new sample was prepared in order to minimize
the risk of oxidation during sample manipulation
and measurement. The Zn
4
aMT-1 and Zn
3
bMT-1
derivatives were separately titrated with Cu(I) under
a nitrogen atmosphere to yield Cu(I)–polypeptide
stoichiometries from zero to six. The sample solution
contained 20% (v ⁄ v) acetonitrile, as the presence of
soft ligands prevents Cu(I) from disproportionation to
Cu(II) and Cu(0).

CD and luminescence emission was measured in
order to assess the sample preparation quality and to
compare the obtained results with those previously
published [26,27]. These physicochemical properties are
exclusively attributable to the metal-thiolate chro-
mophores that have been proven to be essential for the
proper polypeptide folding in other MTs [2]. The over-
all shape of the CD spectra was essentially the same as
reported before (Fig. 1). During the titration of the a-
domain, two positive dichroic bands developed at 248
and 300 nm, respectively, and one negative band at
275 nm. The addition of Cu(I) to Zn
3
bMT-1 shifted
the positive dichroic band at 248 to 260 nm. A second
positive band at 300 nm, that was not present in the
spectrum of Zn
3
bMT-1, appeared on addition of
Cu(I). As in the case of the CD spectra, the results of
luminescence emission were comparable to earlier stud-
ies (Fig. 2). An almost linear increase of intensity was
observed until the addition of the third and fourth
Cu(I) ions to the a- and b-domain, respectively. Fur-
ther Cu(I) addition led to a much more pronounced
increase of intensity in both species.
Two-dimensional
1
H-
1

H NOESY spectra of each
sample were acquired at 700 MHz (Figs 3 and 4). The
spectrum of Zn
4
aMT-1, corresponding to the starting
point of the aMT-1 titration, was consistent with a
well-folded polypeptide. Spin systems of the amide
protons spread from 6.8 to 9.2 p.p.m. Upon addition
of the first equivalent Cu(I), the spin systems of the
starting point remained preserved, but additional new
spin systems started to appear. In the spectra of the
samples containing two, three and four equivalents of
Cu(I), these new spin systems were prevalent with the
most and strongest signals observed for the three
Cu(I)-containing sample. On further additions of
Cu(I), the signals faded away such that the spectra
of the six and seven Cu(I) titration steps were devoid
of cross-peaks (not shown).
For the b-domain similar results were obtained with
the difference that the first addition of Cu(I) led only
to the reduction of signals in the NOESY spectrum
and that new spin systems appeared only after the sec-
ond equivalent Cu(I) was added. The spectra of the
samples containing three, four and five equivalents
250 300 350 400
-30
-20
-10
0
10

20
250 300 350 400
/ nm
Zn
4
- -MT
+ 1 eq. Cu(I)
+ 2 eq. Cu(I)
+ 3 eq. Cu(I)
+ 4 eq. Cu(I)
+ 5 eq. Cu(I)
+ 6 eq. Cu(I)
A
/ nm
Zn
3
MT
+ 1 eq. Cu(I)
+ 2 eq. Cu(I)
+ 3 eq. Cu(I)
+ 4 eq. Cu(I)
+ 5 eq. Cu(I)
+ 6 eq. Cu(I)
B
Fig. 1. CD spectra of Zn
4
aMT (A) and
Zn
3
bMT (B) along the titration with Cu(I).

Samples containing 35 l
M of the respective
domains dissolved in 15% (v ⁄ v) acetonitrile,
20 m
M sodium phosphate buffer pH 7.6
were prepared under nitrogen containing
<1 p.p.m. O
2
.
B. Dolderer et al. Murine Cu(I)a- and Cu(I)b-MT-1 domains
FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS 2351
Cu(I) contained the same new spin systems. The most
and strongest NOEs were observed in the spectrum
of the four Cu(I)-containing sample. As with the
a-domain, progressive disappearance of NOEs without
reappearance of any new signals was the result of
Cu(I) to polypeptide stoichiometries higher than five.
Taken together, the initial additions of Cu(I) to each
domain caused the disappearance of a large set of
NOESY cross-peaks and the parallel appearance of
another set of cross-peaks, until a clean 2D spectrum
belonging to a single species was obtained. Judging
from the highest number of NOEs and the strongest
signals in the respective NOESY spectra, the recovery
of a single species was completed after the addition of
three Cu(I) equivalents to Zn
4
aMT-1 and of four
Cu(I) equivalents to Zn
3

bMT-1. This result was surpri-
sing insofar as structurally defined Cu(I)-containing
species were identified with such unexpectedly low stoi-
chiometries of Cu(I) to polypeptide. Several different
Cu(I) binding stoichiometries had been proposed for
the two domains, among which Cu
3
aMT-1 and
Cu
4
bMT-1 had mostly been considered to be transient
intermediates in the pathways describing the formation
of the fully loaded domains [27–30]. Cu
6
aMT-1 and
Cu
6
bMT-1 had been the most prominent among the
candidates for the fully Cu(I) loaded domains [26]. In
the present titration study, however, the distinct struc-
tures disappear upon addition of more than three and
four Cu(I) equivalents without any sign of newly form-
ing defined structures. We can only speculate what
happens at this stage of titration. One possible explan-
ation for the disappearance of NOESY signals at high
Cu(I) concentration might be that two or more Cu(I)
binding modes coexist in an intermediate exchange
regime, such that signals are exchange broadened and
become invisible. Of course, there is still the possibility
that the separated domains are simply incapable of

binding more than three and four Cu(I) without aggre-
gating and denaturing, whereas in the native MT-1,
the presence of the other domain would help to
accommodate additional ions. We do not believe that
this is very likely, however, because of the similarity of
the Zn
4
aMT-1 and Zn
3
bMT-1 structures with the
structures of the domains of the intact protein (see
below). [Correction added after publication 30 March
2007: in the preceding sentence the first term,
Zn
3
aMT-1 was corrected to Zn
4
aMT-1]. The biophysi-
cal similarities of intact protein and separated domains
would also argue against this proposal [26].
Luminescence titration series also provide notewor-
thy information. Luminescence intensities increased
almost linearly until Cu(I) stoichiometries of three and
four for the a- and b-domain, respectively. At this
point, the curves were bent and further Cu(I) equiva-
lents caused a much stronger, but also linear increase
of intensity. As luminescence intensity is a measure of
how the Cu-thiolate luminophore is shielded from
solvent quenching, the titrations indicate that the
shielding of the metal-thiolate cluster in the newly

identified structures is not optimal compared with the
situation with higher Cu(I):polypeptide stoichiometries.
An explanation for this and the loss of structural
information might be the formation of polymolecular
structurally undefined aggregates of native MT or its
single domains when they are overloaded with Cu(I) in
the presence of unphysiologically high Cu(I) concentra-
500 550 600 650 700
0
10
20
30
40
50
60
500 550 600 650 700
relative intensity
/ nm
AB
0 equivalents Cu(I)
6 equivalents Cu(I)
/ nm
0 equivalents Cu(I)
6 equivalents Cu(I)
mole equiv. of Cu(I)
0123456
0123456
0
1000
2000

3000
4000
5000
0
1000
2000
3000
4000
5000
6000
7000
relative intensity
relative intensity
mole equiv. of Cu(I)
Fig. 2. Luminescence emission spectra of
Zn
4
aMT (A) and Zn
3
bMT (B) along the titra-
tion with Cu(I). Samples containing 14 l
M of
the respective domains dissolved in 15%
(v ⁄ v) acetonitrile, 20 m
M sodium phosphate
buffer, pH 7.6, were prepared under nitro-
gen containing <1 p.p.m. O
2
. Spectra were
recorded at 25 °C using slits of 15 and

20 mm for excitation and emission mono-
chromators, respectively. Excitation was at
k ¼ 300 nm. The insets show the emission
intensities plotted against the respective
polypeptide stoichiometries.
Murine Cu(I)a- and Cu(I)b-MT-1 domains B. Dolderer et al.
2352 FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS
tions. Unlike the observed distinct stoichiometries of
three and four Cu(I) leading to a sharp rise of the
luminescence, only a very small dependency was seen
in the circular dichroic measurements. This was
also shown earlier by Bofill et al. [27], although CD
spectrometry is obviously not sensitive enough to
detect comparable significant changes as deduced from
luminescence data.
1
H NMR and solution structures of Zn
4
aMT-1 and
Zn
3
bMT-1
From previous different studies on vertebrate Zn(II)-
and Cd(II)-containing MTs, it was already known that
the two domains form independently from each other
and do not interact with each other. Therefore, it was
suggested that the two single domains possess very
Fig. 3. Upper-left parts of the
1
H-

1
H NOESY
spectra of Zn
4
aMT (A), Zn
4
aMT +1 Cu(I) (B),
Zn
4
aMT +2 Cu(I) (C), Zn
4
aMT +3 Cu(I) (D),
Zn
4
aMT +4 Cu(I) (E) and Zn
4
aMT +5 Cu(I)
(F). All samples contained 1 m
M polypeptide
dissolved in 15 m
M acetate-d
3
, 15% aceto-
nitrile-d
3
, 10% D
2
O, 50 mM potassium phos-
phate, pH 6.5 and were prepared under a
nitrogen atmosphere containing less than

1 p.p.m. O
2
. Measurements were per-
formed at 283 K on a Bruker AVANCE 700
spectrometer operating at 700.13 MHz
using 600 ms mixing time.
B. Dolderer et al. Murine Cu(I)a- and Cu(I)b-MT-1 domains
FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS 2353
similar structures, if not identical, to their structure in
the intact protein. Analysis of the NOESY and TOC-
SY (not shown) spectra of the two domains permitted
the full sequence-specific assignments, the identification
of the spin systems and the assignment of 398 and 252
of the NOESY cross-peaks of the a- and b-domain,
respectively. The comparison of the chemical shifts
with those reported for the cadmium derivative
revealed very close similarities (supplementary Tables
S1 and S2). In the spectrum of the a-domain, the reso-
nances were shifted marginally by some hundredths of
a p.p.m. The differences observed for the b-domain
were more pronounced, with some deviations of up to
0.2 p.p.m., which is probably due to an increased flexi-
bility in this domain. The spin system patterns repor-
ted for the published cadmium protein, however, were
preserved in both domains. Most importantly, 22 of
the 28 long-range NOEs that were reported for
Cd
7
MT-1 were also found in the spectra of the zinc-
containing a- and b-domain (Table 1). It should be

noted that three of the six missing long-range NOEs
were assigned to residues of the linker region between
Fig. 4. Upper-left parts of the
1
H-
1
H-NOESY
spectra of Zn
3
bMT (A), Zn
3
bMT +1 Cu(I) (B),
Zn
3
bMT +2 Cu(I) (C), Zn
3
bMT +3 Cu(I) (D),
Zn
3
bMT +4 Cu(I) (E), and Zn
3
bMT +5 Cu(I)
(F). Sample and measurement conditions
were the same as described in Fig. 3.
Murine Cu(I)a- and Cu(I)b-MT-1 domains B. Dolderer et al.
2354 FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS
the two domains. Therefore, the lack of the second
domain seems to lead to increased flexibility at either
end that would build up the linker region in native
MT-1. The fact that the majority of the long-range

NOEs observed in Cd
7
MT-1 is still present in the sin-
gle zinc-containing domains suggests that the global
structures of the two domains are preserved, regardless
of whether zinc or cadmium is bound to them and also
regardless of the existence of the second domain.
The assigned peaks of the a-domain were integrated
and their consistency with the published solution struc-
ture was checked using the program dyana and the
published solution structure of the respective cad-
mium-containing domain of the intact protein as a
starting point. The resulting structure family had a tar-
get function of 0.15 ± 0.11 A
˚
2
and rmsd values of
1.51 ± 0.27 A
˚
and 2.22 ± 0.30 A
˚
with respect to the
mean structure for the backbone and all heavy atoms,
respectively. In the previous study on the Cd
7
deriv-
ative, metal–sulphur connectivities were obtained using
the NMR-active cadmium isotope
113
Cd and added to

the structure calculation process. Using these connec-
tivities together with the data for Zn
4
aMT-1 resulted
in a target function of 0.52 ± 0.13 A
˚
2
and rmsd values
of 1.04 ± 0.12 A
˚
and 1.51 ± 0.18 A
˚
for a new struc-
ture family. Both mean structures were essentially
the same, with rmsd values of 0.79 A
˚
and 1.19 A
˚
for
the backbone and heavy atoms, respectively. Thus, the
addition of metal–sulphur connectivities to the struc-
ture calculation of Zn
4
aMT-1 resulted in a better-
resolved structural family but did not change the
overall protein fold. Figure 5 shows the superposition
of the structural family obtained with these additional
connectivities and the mean structure of the previously
published Cd
4

aMT-1, showing that they possess the
same structure, within the experimental uncertainty.
A separate structure determination of Zn
3
bMT-1 on
the basis of the present NMR data was not attempted,
as only a small number of NOEs and only four long-
range NOEs were found in its NOESY spectrum. Only
with the help of the metal–sulphur connectivities dis-
covered using the cadmium-containing derivative could
a structure determination have been possible. How-
ever, preserved chemical shifts and spin system pat-
terns indicate an identical structure for Zn
3
bMT-1 as
for Cd
3
bMT-1. As anticipated, the structures of the
separated Zn(II)-containing domains are indistinguish-
able from those of Cd
7
MT-1 and, knowing the appear-
ance of the starting points, it was of interest to know
to what extent they would change in the presence of
copper(I).
1
H NMR and solution structures of Zn
x
Cu
3

aMT-1
and Zn
y
Cu
4
bMT-1
The NOESY spectra of the Zn
x
Cu
3
aMT-1 and
Zn
y
Cu
4
bMT-1 domains are markedly different from
the starting Zn
4
aMT-1 and Zn
3
bMT-1 derivatives,
pointing to a different arrangement of the polypeptide
chains, which is probably needed to accommodate the
resulting Cu(I)- or mixed metal–sulphur clusters
(Fig. 6). From a cursory inspection of the superim-
posed spectra, it has already become clear that the
addition of Cu(I) not only leads to a completely differ-
ent pattern of spin systems, but also to a significantly
higher number of NOEs. Therefore, we expected the
structures of the newly identified Cu(I)-containing

domains to be more rigid and distinct from their
Zn(II)-containing forms. The spectra of both the only
Zn(II)-containing b-domain and its Cu(I)
4
derivative
seem to be of lower quality with large areas of broad
overlapping peaks. This behaviour might be due to
higher flexibility within the b-domain which has been
reported already before [17–20]. TOCSY spectra (not
Table 1. Comparison of the long-range NOEs (d
ij
j > I +4) of Zn
4
-
aMT and Zn
3
bMT with those observed for Cd
7
MT [20]. Presence
(+) or absence (–) of NOEs is indicated.
b-Domain
Proton 1 Proton 2
Asn 4 (a) Lys 22 (b)+
Asn 4 (a) Asn 23 (NH) +
Asn 4 (b) Asn 23 (NH) +
Cys 5 (a) Cys 21 (b)+
Cys 5 (b) Cys 21 (b)+
Asn 23 (b) Cys 29 (a)–
a-Domain
Lys 1 (NH) Val 9 (c)–

Lys 1 (a) Val 9 (c)+
Ser 2 (b) Val 9 (b)+
Ser 2 (HN) Val 9 (c)–
Cys 3 (HN) Val 9 (c)+
Cys 3 (a) Cys 18 (b)+
Cys 3 (b) Cys 18 (HN) +
Ser 5 (b) Asp 25 (HN) –
Cys 6 (a) Asp 25 (HN) +
Cys 6 (a) Asp 25 (a)–
Cys 6 (a) Lys 26 (HN) +
Cys 6 (b) Lys 26 (HN) +
Cys 6 (b) Lys 26 (a)+
Cys 6 (a) Cys 27 (HN) +
Cys 6 (b) Cys 27 (HN) +
Cys 6 (b) Cys 27 (a)–
Cys 6 (b) Cys 27 (b)+
Lys 13 (b) Val 19 (
c)+
Lys 13 (c) Val 19 (c)+
Lys 13 (c) Cys 30 (a)+
Cys 14 (NH) Val 19 (c)+
Val 19 (c) Cys 19 (b)+
B. Dolderer et al. Murine Cu(I)a- and Cu(I)b-MT-1 domains
FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS 2355
shown) were recorded for the Zn
x
Cu
3
aMT-1 and
Zn

y
Cu
4
bMT-1 derivatives and used, together with the
NOESY spectra, to obtain their sequence specific
assignments (supplementary Tables S1 and S2). The
resonances of the new Cu(I)-containing species differed
mostly by several tenths of a p.p.m. from those of
their starting points, thereby confirming the observa-
tions mentioned above.
In the NOESY spectrum of Zn
x
Cu
3
aMT-1 502,
NOEs were assigned, integrated and converted into
distance constraints. In the last dyana run, a set of
200 structures was generated out of which the 20 best
were combined to a structure family (Fig. 7). The tar-
get function was 0.43 ± 0.10 A
˚
2
, and the rmsd values
were 0.70 ± 0.12 A
˚
and 1.03 ± 0.14 A
˚
for the poly-
peptide backbone and heavy atoms, respectively. Like-
wise, 500 NOEs of the Zn

y
Cu
4
bMT-1 spectrum were
used to derive a structure family for this species
(Fig. 8). In this case, a target function of 0.19 ±
0.02 A
˚
2
and rmsd values of 0.49 ± 0.21 A
˚
and
0.72 ± 0.21 A
˚
for the backbone and heavy atoms were
obtained.
As with other known structures of MTs, those pre-
sented here also lack typical secondary structure ele-
ments. The structure of Zn
x
Cu
3
aMT-1 is roughly a
two-level structure (Fig. 7) where the segment 5–10 of
the a-domain polypeptide backbone forms the first
level. The stretch 10–16 links the first with the second
Fig. 5. Superposition of the present family of 30 structures of Zn
4
aMT (blue) with the published average structure of Cd
4

aMT (red). The
coordinates of the Cd
4
aMT structure were extracted from the Brookhaven protein data bank (1DFS). In the last run of structure calculations
for Zn
4
aMT 398 upper distance limits (upl) obtained from the assignment of its NOESY spectrum and the metal-sulphur connectivities repor-
ted by Zangger et al. [20] for Cd
4
aMT were used as input for the program DYANA. Twenty out of 200 structures were combined into a struc-
ture family with a target function of 0.52 ± 0.13 A
˚
2
and RMSD values of 1.04 ± 0.12 A
˚
and 1.51 ± 0.18 A
˚
for the backbone and all heavy
atoms, respectively.
Fig. 6.
1
H-
1
H-NOESY spectra of Zn
4
aMT
(red) superimposed with that of Zn
x
Cu
3

aMT
(green) (A) and of Zn
3
bMT (red) superim-
posed with that of Zn
y
Cu
4
bMT (green) (B).
Sample and measurement conditions were
the same as described in Fig. 3.
Murine Cu(I)a- and Cu(I)b-MT-1 domains B. Dolderer et al.
2356 FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS
level, which is constituted by the second half of the
polypeptide chain. The region between residue 20 and
26 includes a loop that is more disordered than the
rest of this domain, shielding the rear of its core. The
positions of several cysteine sulphur atoms are not
very well defined (Fig. 7). Nevertheless, the 11 cyste-
ines seem to form a somewhat broader cavity than in
its Cd
4
aMT-1 counterpart, with the subgroup of Cys3,
11 and 18 being rather isolated from the other eight
cysteines.
The polypeptide backbone of the b-domain wraps
around its core in a right-handed fashion (Fig. 8). The
central part of its polypeptide chain, residues 8–24,
limits an almost elliptical planar structure. At the point
where the two ends of the polypeptide chain would

meet, they leave the central plane and continue in
opposite directions. Again, outside the uncertainty in
the positions of some of the sulphur atoms, the cavity
encased by the cysteines is somewhat broader than its
Cd
3
bMT-1 counterpart, although in this case the nine
cysteines still point to a unique core.
The superposition of the newly identified Cu(I)-con-
taining domains onto the mean structures of their
Cd(II)-coordinating forms highlights the striking struc-
tural differences that are caused only by the binding
of different metal ions to the two domains of MT
(Fig. 9). When the structures are superimposed
throughout the full length of their polypeptide chains
only very faint similarities of some parts of the poly-
peptide backbone folds could be observed. Separate
superpositions of stretches 3–12, 11–20 and 18–28 were
also attempted. For both domains, the first and third
stretches gave very poor overlap, while the central part
showed a more pronounced similarity. This could indi-
cate that each domain adapts to host the additional
copper(I) ions by opening up and rearranging its
N- and C-terminal parts, minimizing the structural
perturbation of its central part.
The arrangement of the cysteine sulphur donor
atoms within the two Cu(I)-containing domains is also
shown in Figs 7 and 8. Although, from the present
data, it is neither possible to guess the number of
Fig. 7. Stereoview of the 20-structure family

of Zn
x
Cu
3
aMT. Polypeptide backbone bonds
are shown in grey, cysteinyl side chain
bonds in blue and sulphur atoms as yellow
spheres. 502 NOEs were converted into upl
and were used as input for the structure
calculation program
DYANA. Twenty out of
200 structures were combined into a struc-
ture family with a target function of
0.43 ± 0.10 A
˚
2
and RMSD values of
0.70 ± 0.12 A
˚
and 1.03 ± 0.14 A
˚
for the
backbone and all heavy atoms, respectively.
Fig. 8. Stereoview on the family of 20 best
structures of Zn
y
Cu
4
bMT. Polypeptide back-
bone bonds are shown in grey, cysteinyl

side chain bonds in blue and sulphur atoms
as yellow spheres. 500 NOEs were conver-
ted into upl and were used as input for the
structure calculation program
DYANA. Twenty
out of 200 structures were combined into a
structure family with a target function of
0.19 ± 0.02 A
˚
2
and rmsd values of
0.49 ± 0.21 A
˚
and 0.72 ± 0.21 A
˚
for the
backbone and all heavy atoms, respectively.
B. Dolderer et al. Murine Cu(I)a- and Cu(I)b-MT-1 domains
FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS 2357
residual Zn(II) ions in each structure nor the overall
topology of the clusters, it appears that all cysteine res-
idues point towards the inside of the respective
domain, as expected if all of them are to be involved
in metal coordination. In turn, the somewhat broader
cavities encased by the cysteines are consistent with the
increased number of metals in each domain.
At this point it was still an open question as to
whether or not the single species Zn
x
Cu

3
aMT-1 and
Zn
y
Cu
4
bMT-1 would be stable when the other Cu(I)-
containing domain was also present in solution. To this
end, both Cu(I)-containing domains were prepared as
for the titration experiment mentioned above, com-
bined in equal amounts at a final concentration of
1mm each and incubated for >48 h before the meas-
urement of their
1
H NMR spectra. The observed NO-
ESY and TOCSY spectra (not shown) consisted of the
sum of the respective spectra of the single species. The
spectral resonances were assigned to all the protons
present in the two domains and are listed in supple-
mentary Tables S1 and S2. This result indicates that
the two domains are stable and independent from each
other. Cu(I) is not transferred between the two domains
to form new species with higher and lower Cu(I):poly-
peptide stoichiometries. As no additional NOEs and ⁄ or
changes of the spectral resonances were observed in the
NOESY spectrum of the mixture, an interaction of the
two single domains and the formation of one single
Cu(I)-containing domain with the involvement of
both domains could be excluded for the present
Zn

(x+y)
Cu
7
MT-1 stoichiometry.
Conclusion
The Cu(I) titration of the independent Zn(II)-loaded
domains of mouse MT-1 revealed Cu(I) stoichiometries
of three and four for the a- and b-domain, respectively.
The presence of Cu(I) led to dramatic conformational
changes of both polypeptide folds. Cu(I) stoichio-
metries of up to six Cu(I) ions each led to the progressive
disappearance of the altered structures. [Correction
added after publication 30 March 2007: in the preced-
ing sentence, disappearing of the affered structurer, was
corrected to disappearance of the altered structures].
Unfortunately, due to the lack of metal Æ sulphur
constraints, the Cu(I) positions within the resolved
polypeptide folds remained unclear. Therefore, crystal-
lization of the newly identified Cu(I)-containing species
Fig. 9. (A) Stereoview of the superposition
of the Cd
4
aMT-1 mean structure (red) to the
structure family of Zn
x
Cu
3
aMT-1 (blue). (B)
Stereoview of the superposition of the
Cd

3
bMT-1 mean structure (red) to the struc-
ture family of Zn
y
Cu
4
bMT-1 (blue).
Murine Cu(I)a- and Cu(I)b-MT-1 domains B. Dolderer et al.
2358 FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS
Zn
x
Cu
3
aMT-1 and Zn
y
Cu
4
bMT-1 seems to be the only
promising way to determine how Cu(I) is coordinated
by the vertebrate MT. When Zn
x
Cu
3
aMT-1 and
Zn
y
Cu
4
bMT-1 were prepared individually and com-
bined at equal concentrations, the two domains did not

affect each other. The net transfer of Cu(I) and the
possible formation of one single Cu(I)-containing
domain were clearly excluded at least under the condi-
tions of separated and not covalently linked domains.
Experimental procedures
All chemicals were of analytical grade quality or better. The
protected amino acids were purchased from Novabiochem
(Laufelfingen, Switzerland), the resin for peptide synthesis
was from Rapp Polymere (Tu
¨
bingen, Germany), and all
other chemicals were from Merck (Darmstadt, Germany).
Synthesis of the individual a- and b- domains
The individual a- and b-domains (KSCCSCCPVGCSKCA
QGCVCKGAADKCTCCA and MDPNCSCSTGGSCTCT
NaCl ⁄ CitACKNCKCTSCK) of murine MT-1 consisting of
residues 31–60 and 1–30 of the wild-type protein, respect-
ively, were obtained on an Eppendorf ECOSYN P solid
phase peptide synthesizer (Hamburg, Germany). The syn-
thesis was begun from the resins containing the C-terminal-
protected residues coupled to them, namely Fmoc-Ala-PHB
TentaGel R and Fmoc-Lys(Boc)-PHB TentaGel R. All
amino acids were incorporated with the a-amino functions
protected by the Fmoc group. Side-chain functions were
protected as tert-butyl esters (aspartic acid), tert-butyl
ethers (serine, threonine), Boc derivatives (lysine) and trityl
derivatives (asparagine, glutamine, cysteine). Coupling was
performed using a four-fold excess in protected amino acids
and the coupling reagent 2-(1H-benzotriazole-1-yl)1,1,3,3-
tetramethyluronium tetrafluoroborate (TBTU) over the

resin loading plus 1.2 mL diisopropylethylamine (DIEA;
12.5% solution in dimethyl formamide). The cysteine resi-
dues were incorporated as their pentafluorphenylesters.
Before coupling of the protected amino acids, the Fmoc
groups were removed from the last amino acid of the grow-
ing fragment using 25% piperidine in dimethyl formamide.
After cleavage of the N-terminal Fmoc group, the peptide
was removed from the resin under simultaneous cleavage of
the amino acid side-chain protecting groups. This was
accomplished by incubating the resin in a mixture of tri-
fluoroacetic acid (12 mL), ethanedithiol (0.6 mL), thioani-
sole (0.3 mL) anisole (0.3 mL), water (0.3 mL) and
triisopropylsilane (0.1 mL) for 3 h. The mixture was fil-
tered, washed with trifluoroacetic acid and the combined fil-
trates precipitated with anhydrous ether. The crude product
was further purified by HPLC on a Nucleosil 100 C18
(7 lm) 250 · 10 mm column (Macherey & Nagel, Du
¨
ren,
Germany) using a gradient from 10 to 90% solution B
(solution A: 0.07% TFA ⁄ H
2
O; B: 0.059% trifluoroacetic
acid in 80% acetonitrile) over 32 min at a flow rate of
3.5 mLÆmin
)1
. The elution was monitored at 214 nm. The
polypeptides were assayed for purity by analytical HPLC
and mass spectroscopy (supplementary Figs S1 and S2).
For the measurement of ESI-MS control spectra, the

dried peptides were dissolved in 100 lL of 50% methanol
and 1% formic acid in water (v ⁄ v), and were analysed
using syringe pump infusion. The ESI-MS experiments were
carried out on an Esquire3000plus ion trap mass spectro-
meter (Bruker Daltonics, Bremen, Germany) equipped with
a standard ESI source. Dried peptides were dissolved in
100 lL of 50% methanol, 1% formic acid in water (v ⁄ v)
and directly infused (5 lLÆmin
)1
flow rate) into the ESI
source using a syringe pump. Mass spectra were acquired
in the positive-ion mode. The spray voltage was 3750 V,
dry gas (6 LÆmin
)1
) temperature 300 °C and the nebulizer
172.37 kPa. For the a-domain, an average molecular mass
of 3021.3 Da (3021.7 Da, theoretical) was observed, calcu-
lated from the detected [M + 5H]
5+
and [M + 4H]
4+
quasi molecular ions at m ⁄ z 605.2 and 756.3, respectively.
For the b-domain, an average molecular mass of 3014.1 Da
(3014.5 Da, theoretical) was observed ([M + 3H]
3+
and
[M + 2H]
2+
at m ⁄ z 1005.6 and 1507.8).
Copper titration conditions

The lyophilized polypeptides were weighed prior to the sub-
sequent metal additions in order to determine the correct
polypeptide amount employed. Being aware of the high
sensitivity of apo-MT towards oxygen, especially under
neutral or basic solvent conditions, all further manipula-
tions were performed in a nitrogen atmosphere containing
<1 p.p.m. of oxygen. Furthermore, the peptides were first
dissolved in a buffer of 15 mm deuterated acetate, 20%
(v ⁄ v) deuterated acetonitrile, 15% D
2
O (pH 5.0) to yield a
final concentration of 1 mm. Coordination of Cu(I) to
acetonitrile prevented undesired Cu(I) disproportionation
into Cu(II) and elemental copper. Four and three molar
equivalents of Zn(ClO
4
)
2
were added to the a- and
b-domains, respectively. The pH was adjusted to pH 6.5 by
adding potassium phosphate to a final concentration of
50 mm. Seven different samples per domain were titra-
ted using zero to six molar equivalents of Cu(I). The
Cu(I) titration solution consisted of freshly produced crys-
tals of [Cu(CD
3
CN)
4
]ClO
4

dissolved in 50% (v ⁄ v) CD
3
CN ⁄
H
2
O. Copper concentrations were quantified by atomic
absorption spectrometry. Alternatively, the reactivity of
bathocuproinsulphonate with Cu(I) was monitored spectro-
photometrically. The copper concentrations in the titration
solutions were in the range of 10
-2
)10
-1
M. All samples
were transferred into high precision 5-mm NMR tubes,
flushed with argon and sealed.
B. Dolderer et al. Murine Cu(I)a- and Cu(I)b-MT-1 domains
FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS 2359
The preparation of samples for the CD measurements
was carried out essentially identical to the protocol used to
prepare the NMR samples. Final polypeptide concentra-
tions were adjusted to 35 lm and only undeuterated chemi-
cals were used. For luminescence measurements the CD
samples were further diluted 1 : 2.5 with 20% acetonitrile
(v ⁄ v)–sodium phosphate buffer, pH 7.0.
Spectrometry
Copper concentrations of the copper titration reagent
were determined using a 400 S atomic absorption spectro-
meter furnished with a HGA 76 B temperature control
unit (Perkin-Elmer, U

¨
berlingen, Germany). CD spectra
were recorded on a J-720 spectropolarimeter at 22 °C
(Jasco, Gross-Umstadt, Germany). The sample chamber
was kept under N
2
, thereby minimizing the formation of
ozone induced by the xenon lamp. Luminescence emission
spectra, excited at 300 nm, were recorded on a LS 50
fluorimeter at 22 °C (Perkin-Elmer). The second-order
emission of the excitation source was suppressed using an
edge filter at 390 nm.
Time-proportional-phase incrementation
1
H-
1
H NOESY
spectra [31,32] were acquired on a AVANCE 700 spectro-
meter operating at 700.13 MHz (Bruker, Karlsruhe, Ger-
many). The water signal was suppressed using water
presaturation during both relaxation delay and mixing time.
The measurements were performed at 283 and 298 K at a
spectral width of 14 p.p.m. using recycle and mixing times
of 2.0 s and 600 ms, respectively. Time-proportional-phase
incrementation TOCSY spectra [33] were recorded at 283 K
employing a spin-lock time of 60 ms. All two-dimensional
spectra consisted of 1024 data points in the F2 dimension
and 2048 data points in the F1 dimension and were
acquired using 16 scans per F1 increment.
Structure determination

The program sparky was used for analysis of the 2D spec-
tra [34]. The sequential assignment of the spin systems in
the TOCSY and NOESY spectra at 283 K was achieved
employing standard procedures. In the cases when only zinc
was bound to the protein, previously published data for the
cadmium-containing MT could be used to help identifying
proton resonances. Intensities of assigned cross-peaks were
obtained using the integration routine embedded into the
program Sparky. After creating peak lists and proton lists
in dyana format, the volumes were converted into upper
distance limits by the program caliba [35]. Those distance
constraints were used to generate protein conformers
employing the torsion angle dynamic program dyana
(ETH Zurich, Switzerland) with a simulated annealing algo-
rithm [36]. Stereospecific assignments of diastereotopic
groups were added with the help of the program glomsa
[35]. The resulting structures were subjected to a few
iterations of calibration and stereospecific assignments, until
self-consistency. The program molmol was used for struc-
ture display [37].
Acknowledgements
Support of the European Community Access to
Research Infrastructure action of the Improving
Human Potential (LSF user project) is gratefully
acknowledged. BD was recipient of a predoctoral fel-
lowship of the Konrad-Adenauer-Stiftung. This work
was partly supported by EC programs SPINE II
(LSHG-CT-2006–031220) and UPMAN (LSHG-CT-
2004–512052), and by Ente CR Firenze. Stimulating
discussions with Professor Pilar Gonza

´
lez-Duarte, Pro-
fessor Merce
´
Capdevila, and Professor Sı
´
lvia Atrian
(Barcelona) are warmly acknowledged.
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Supplementary material
The following supplementary material is available
online:
Table S1. Proton chemical shifts (p.p.m.) of all exam-
ined a-domain species of murine MT-1.
B. Dolderer et al. Murine Cu(I)a- and Cu(I)b-MT-1 domains
FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS 2361
Table S2. Proton chemical shifts (p.p.m.) of all exam-
ined b-domain species of murine MT-1.
Fig. S1. Averaged positive-ion mode ESI mass spectra
acquired from the synthetic a-domain of murine
MT-1.
Fig. S2. Averaged positive-ion mode ESI mass spectra
acquired from the synthetic b-domain of murine
MT-1.
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Murine Cu(I)a- and Cu(I)b-MT-1 domains B. Dolderer et al.
2362 FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS