Evidence for noncooperative metal binding to the
a domain of human metallothionein
Kelly E. Rigby Duncan and Martin J. Stillman
Department of Chemistry, The University of Western Ontario, London, ON, Canada
Over the past several decades, significant advances
have been made in the field of protein folding [1–4].
However, the direct and specific involvement of metal
ions in the folding process of metalloproteins has
received far less attention, despite the fact that one-
third of all known enzymes require metal ions for
structural or functional purposes [5]. Post-translational
metal-induced protein folding is a vital process that
still requires mechanistic elucidation. Metalloproteins
that bind multiple metals introduce an additional layer
of complexity, in that cooperative metal-binding mech-
anisms are possible in which the complete multiple
metal-binding site forms in preference to partially filled
binding sites.
Metallothionein (MT) is a metalloprotein found in
nearly all mammalian tissues coordinated to multiple
group 11 and 12 metal ions [6]. The high capacity of
MT to bind both essential and nonessential metal ions
in vivo and in vitro strongly suggests a role in metal
ion storage, metabolism and trafficking of Cu and Zn,
as well as sequestration of Cd and Hg; however, the
exact function of MT remains undefined. More
recently, MT has been implicated in brain tissue repair
through anti-inflammatory, antioxidant and antiapop-
totic roles [7–11], as well as in chemotherapy resistance
[12]. Domain-independent but metal ion-directed fold-
ing of MT results in the formation of discrete metal–

Correspondence
M. J. Stillman, Department of Chemistry,
Chemistry Building, The University of
Western Ontario, London, ON, Canada,
N6A 5B7
Fax: +1 519 661 3022
Tel: +1 519 661 3821
E-mail:
Website: />(Received 22 December 2006, revised 2
February 2007, accepted 1 March 2007)
doi:10.1111/j.1742-4658.2007.05762.x
In the present study, we investigated the metal-binding reactivity of the
isolated a domain of human metallothionein isoform 1a, with specific
emphasis on resolving the debate concerning the cooperative nature of
the metal-binding mechanism. The metallation reaction of the metal-free
a domain with Cd
2+
was unequivocally shown to proceed by a non-
cooperative mechanism at physiologic pH by CD and UV absorption
spectroscopy and ESI MS. The data clearly show the presence of interme-
diate partially metallated metallothionein species under limiting Cd
2+
con-
ditions. Titration with four molar equivalents of Cd
2+
was required for the
formation of the Cd
4
a species in 100% abundance. The implications of a
noncooperative metal-binding mechanism are that the partially metallated

[22,23]. Recent kinetic results for As
3+
binding to the
two isolated domains were also interpreted in terms of
a series of noncooperative bimolecular reactions [24].
The fact that Cd
2+
has been shown to coordinate to
the two-domain ba-MT in a domain-specific manner,
with a preference for the a domain, has been construed
as being an indicator of cooperative metal binding to
the a domain. This study focuses on the metallation of
the isolated a domain, with the purpose of clarifying
this point. Additionally, the reported concurrent metal-
lation of both domains in the two-domain protein by
Co
2+
[25] and Cd
2+
[23] provides an excellent example
of the complexity introduced by the presence of the b
domain in efforts to elucidate the potentially cooper-
ative nature of the metal-binding reaction within each
of the domains. Thus, the goal is to elucidate the meta-
llation mechanisms of the individual domains, in the
hope, initially, of simplifying the interpretation of
the metallation details of the two-domain protein. The
results presented here allow successful and complete
interpretation of the previous data in terms of non-
cooperative, domain-specific metal binding.

cluster, where each cadmium ion
(green spheres) coordinates tetrahedrally to four cystei-
nyl sulfurs (yellow spheres), such that five of the 11
cysteinyl sulfurs act as bridging ligands between two
metal centers, and the remaining six act as terminal
ligands by coordinating to a single metal center. The
numbering of the cadmium ions and the cysteinyl sul-
furs in Fig. 1C corresponds with that in the sequence
shown in Fig. 1A. Demetallation to produce the metal-
free apo-a-rhMT was carried out by eluting the
cadmium-containing domain through a size exclusion
column equilibrated with a low-pH eluant.
The term ‘positive cooperativity’ refers to an
increase in equilibrium constant (K) for each step of a
sequential reaction; in other words, coordination of
the first metal ion facilitates the binding of the second
metal ion, and so forth. Experimentally, this translates
into the detection of only the initial species, in this
case the metal-free protein, and the final species, which
is the fully metallated holoprotein, with no detectable
intermediate species. Thus, with substoichiometric
additions of Cd
2+
to apo-a-rhMT , the metal-free pro-
tein will be detected together with a corresponding
fraction of the metal-saturated Cd
4
a species if the met-
allation mechanism proceeds by a positively cooper-
ative pathway. Alternatively, the partially filled Cd

metal speciation after a few seconds of equilibration.
The spectroscopic data were acquired in this study
after a 2–5 min equilibration period at room tempera-
ture, to ensure that thermodynamic equilibrium was
achieved. The CD spectra measured during the metal-
binding reaction (Fig. 2A) at pH 7.3 show a concomit-
ant increase in CD signal intensity at 250 and 263 nm
with the addition of up to 2.4 molar equivalents of
Cd
2+
before a derivative-shaped signal, with band
maximum at 263 nm, begins to dominate at 3.2 equiv-
alents of Cd
2+
(Fig. 2A, inset). Finally, the full com-
plement of 4.0 molar equivalents of Cd
2+
is required
for the strong derivative signal to be observed with
DA
220
reaching positive values. The UV absorption
spectra (Fig. 2B) show an incremental increase in sig-
nal intensity at 250 nm with the addition of Cd
2+
to
the protein solution, reaching a maximum intensity at
4.0 molar equivalents of Cd
2+
, thus confirming the

(S
cys
)
11
binding site [27]. As noncoop-
erative metal binding is predicted to result in the for-
mation of intermediate, partially metallated, species,
alaalaalaala
lys
gly
met
sergly
A
M
4
(S
cys
)
11
Domain of Recombinant Human MT
1
2
7
6
5
4
11
8
3
10

shown in (A). Gray ¼ C; white ¼ H; blue ¼
N; red ¼ O; green ¼ Cd; yellow ¼ S.
Diagram adapted from Chan et al. [44].
K. E. Rigby Duncan and M. J. Stillman Noncooperative metallation of metallothionein
FEBS Journal 274 (2007) 2253–2261 ª 2007 The Authors Journal compilation ª 2007 FEBS 2255
these are qualitatively identifiable in the CD spectrum.
As is clearly observed in Fig. 2A, addition of less than
4.0 molar equivalents of Cd
2+
results in CD spectra
consistent with those observed for partially metallated
domain species, supporting the model of a noncooper-
ative metallation mechanism. Although a distinction
between partially metallated intermediates and the
fully metallated holoprotein can be made on the basis
of the acquired CD spectra, quantitative analysis of
the exact species being formed in the metallation reac-
tion requires supplementary MS analysis.
Figure 3 shows the corresponding MS data for the
titration of apo-a-rhMT-1a with Cd
2+
at pH 7.8 fol-
lowing a 2–5 min equilibration period at room tem-
perature following each metal addition. The spectra on
the left side of Fig. 3 are the original mass spectra,
with mass ⁄ charge (m ⁄ z) values on the x-axis illustra-
ting the charge state distributions of the protein spe-
cies. The spectra on the right side of Fig. 3 are the
deconvoluted spectra showing the mass and identity of
the species detected. The deconvoluted spectra on the

ting to the protein.
Discussion
In this report, we have unequivocally shown by CD
spectroscopy and ESI MS that metal binding to the a
domain of human MT-1a is a noncooperative process
at physiologic pH. This implies that the four equilib-
rium constants describing the sequential metallation
reaction are decreasing in magnitude (K
1
> K
2
>
K
3
> K
4
), albeit only marginally, as the reaction does
proceed to completion upon addition of 4.0 equiva-
lents of Cd
2+
. The previously described metallation of
the two-domain protein by Co
2+
indicated a simulta-
neous metallation of the a and b domains, with two
metal ions populating the a domain, and one in the b
domain [25]. All three of these metal ions were shown
to bind to independent tetrahedral tetrathiolate sites
within the two domains. This was followed by coordi-
nation of the fourth and fifth metal ions to the a

at pH 7.3. Spectral changes were recorded
as up to 4.0 equivalents of Cd
2+
(3.3 mM)
were titrated into a solution of apo-a-rhMT-
1a (15 l
M)at22°C. Spectra were recorded
at molar equivalent values of 0.0, 0.8, 1.6,
2.4, 3.2 and 4.0 of Cd
2+
at 22 °C. Inset: Plot
of changes in CD intensity monitored at
223, 240, 250 and 263 nm as a function of
molar equivalents of Cd
2+
up to a maximum
of 4.0 equivalents.
Noncooperative metallation of metallothionein K. E. Rigby Duncan and M. J. Stillman
2256 FEBS Journal 274 (2007) 2253–2261 ª 2007 The Authors Journal compilation ª 2007 FEBS
ligands, especially as the noncooperative metal binding
dictates that the K
eq
must be decreasing as the sequen-
tial reaction proceeds. Finally, K
3a
and K
4a
for the a
domain would have to be greater than K
2b

dues. The results showed the ability of the peptide to
coordinate a single metal ion, which induced a metal-
dependent fold of the peptide in the same configur-
ation as the holoprotein. Finally, results from a
computational molecular dynamics study carried out
A
B
C
D
E
F
Fig. 3. ESI mass spectra of the titration of
apo-a-rhMT-1a with Cd
2+
at pH 8.0. Spectral
changes were recorded as aliquots of Cd
2+
(3.3 mM) were titrated into a solution of
apo-a-rhMT-1a (21 l
M)at22°C. Spectra
were recorded at Cd
2+
molar equivalent val-
ues of (A) 0.0, (B) 0.8, (C) 1.6, (D) 2.4, (E)
3.2, and (F) 4.0. The left side of the figure
shows the measured mass spectra labeled
with the charge states of the molecular spe-
cies. The right side of the figure shows the
deconvoluted spectra with the reconstruc-
ted masses that correspond to the meas-

readily in vitro, the most well-studied being apo-car-
bonic anhydrase [30–33], and has been predicted to
occur in vivo on the basis of analysis of the Zn
2+
pools
in Erlich cells [34,35]; however, the fate of MT after
metal ion donation has not been determined. Degrada-
tion by cooperative demetallation of the remaining six
metal ions following the loss of the first Zn
2+
would
be, overall, an energetically expensive process, and
would therefore be expected to be highly unfavorable.
However, the demonstrated stability of the partially
metallated species in this study provides support for
the alternative scenario in which the partly demetall-
ated MT product persists in vivo following metal ion
donation. But if this is true, then what happens to the
remaining metal ions that are bound to the MT mole-
cule? Investigation into how the domain reacts in the
event of metal ion donation will be of significant value
for understanding the role of MT in the cellular meta-
bolism of Zn
2+
. Despite the relatively large thermo-
dynamic stability of the metal–thiolate clusters in MT,
the metals have been shown to be kinetically labile in
terms of both intramolecular and intermolecular metal
exchange reactions [36]. Thus, it is probable that a spe-
cific metal site is more labile than the others, and will

in which oxidative release of Zn
2+
from Zn
7
-MT
occurs by the formation of disulfide or S–O bonds
upon interaction with cellular oxidants [38–40]. This
proposal, however, is based on the assumption that
the metallation mechanism of apo-MT is cooperative,
and as such, only the metal-free and fully metallated
holoprotein are present in vivo [41]. Although strong
evidence exists for a critical balance between the
MT ⁄ thionein pair [42,43] the evidence presented in this
article demonstrates that alternative mechanisms for
Zn
2+
probably exist. Moreover, the highly reducing
environment of the cell, in which concentrations of
reduced glutathione as high as 3 mm have been detec-
ted, supports the theory generated by the data presen-
ted, in which partially metallated, yet reduced, forms
of MT can readily exist in the cell. In fact, the non-
cooperative metallation and the subsequently decrea-
sing equilibrium constants indicate that, from a
coordination chemistry point of view, it is not only
acceptable, but probable, that MT exists with a vacant
site in vivo in the presence of limiting concentrations of
free group 11 and 12 metal ions. Thus, it is proposed
that MT only resides in the fully metallated holopro-
tein upon influx of excess free metal ions into the cell.

metal ions. The vacant metal site(s) in the partially
metallated species offer free cysteinyl thiolate ligands
in the reducing environment of the cell for scavenging
Noncooperative metallation of metallothionein K. E. Rigby Duncan and M. J. Stillman
2258 FEBS Journal 274 (2007) 2253–2261 ª 2007 The Authors Journal compilation ª 2007 FEBS
of damaging reactive oxygen species, which supports
the proposal of MT as a potent antioxidant and anti-
apoptotic protein.
Experimental procedures
Materials
The chemicals used were: cadmium sulfate (Fisher Scientific,
Ottawa, ON, Canada); ultrapure Tris buffer (ICN Biomole-
cules, Irvine, CA, USA); ammonium formate buffer (Ald-
rich, Oakville, ON, Canada); isopropyl-b-d-thiogalactoside
(Sigma-Aldrich, Oakville, ON, Canada); ammonium hydrox-
ide (BDH Chemicals ⁄ VWR, Mississauga, ON, Canada); for-
mic acid (J. T. Baker Chemical Co., Phillipsburg, NJ, USA);
and hydrochloric acid (Caledon, Georgetown, ON, Canada).
All solutions were made with >16 MWÆcm
)1
deionized water
(Barnstead Nanopure Infinity, Dubuque, IA, USA).
HiTrap
TM
SP HP ion exchange columns (Amersham Bio-
sciences ⁄ GE Healthcare, Piscataway, NJ, USA), superfine
G-25 Sephadex (Pharmacia ⁄ Pfizer, Oakville, ON, Canada)
and a stirred ultrafiltration cell (Amicon Bioseparations ⁄
Millipore, Bedford, MA, USA) with a YM-3 membrane
(3000 MWCO) were used in the protein purification steps.

-substituted
MT was eluted with a gradient of 5–20% NaCl in 10 mm
Tris ⁄ HCl (pH 7.4). Protein fractions were collected on the
basis of strong UV absorption at 250 nm corresponding to
the ligand-to-metal charge transfer transitions of the SfiCd
of the metal–thiolate clusters. The pooled protein fractions
collected from the SP ion exchange column were concentra-
ted to a volume of 15 mL using the Amicon ultrafiltration
cell with a YM-3 cellulose membrane (3000 MWCO) under
N
2
pressure. The S-tag was cleaved from the concentrated
protein fraction using a Thrombin CleanCleave
TM
Kit
(Sigma) by stirring the protein with the thrombin-coated
beads under argon overnight at 4 °C. The cleaved protein
was separated from the thrombin beads, and eluted from a
superfine G-25 Sephadex column with Ar-saturated 10 mm
Tris buffer (pH 7.4) to desalt prior to loading onto the SP
ion exchange column for purification. The fractions collec-
ted from the SP were pooled and concentrated to 8 mL,
using the Amicon ultrafiltration cell.
Further protein preparation for metal-binding
studies
Metal-free apo-a-rhMT was prepared by eluting the throm-
bin-cleaved Cd-bound protein from a G-25 column equili-
brated with a low-pH eluant. Apo-MT prepared for the
CD studies was eluted with 10 mm Tris ⁄ HCl (pH 2.7),
whereas the apo-MT prepared for the MS studies was elut-

tion studies.
Metallation of apo-a-rhMT with Cd
2+
at pH 7
CD ⁄ UV absorption spectroscopy
The pH of apo-a-rhMT solution (13 lm) was raised from
2.7 to 7.3 by the addition of 10% NH
4
OH prior to the
addition of Cd
2+
(3.3 mm). Cd
2+
was added in 0.8 molar
equivalent increments up to 4.0 equivalents, with thorough
mixing after each titration. CD and UV absorption spectra
were recorded at each addition after a 2–5 min delay, in
which the reaction could reach equilibrium conditions.
K. E. Rigby Duncan and M. J. Stillman Noncooperative metallation of metallothionein
FEBS Journal 274 (2007) 2253–2261 ª 2007 The Authors Journal compilation ª 2007 FEBS 2259
MS
The pH of apo-a-rhMT solution (20 lm) was raised from
2.8 to 7.8 by the addition of 10% NH
4
OH prior to the
addition of Cd
2+
(3.3 mm). Cd
2+
was added in 0.8 molar

meter was calibrated with a solution of NaI. The scan con-
ditions for the spectrometer were: capillary, 3000.0 V;
sample cone, 39.0 V; RF lens, 450.0 V; extraction cone,
11.0 V; desolvation temperature, 20.0 °C; source tempera-
ture, 80.0 °C; cone gas flow, 51 LÆh
)1
; and desolvation gas
flow, 528 LÆh
)1
. The m ⁄ z range was 500.0–1600.0, the scan
mode was continuum, and the interscan delay was 0.10 s.
The observed spectra were reconstructed using the max
ent 1 program from the mass lynx v.4.0 software package.
Acknowledgements
We thank NSERC of Canada for financial support
(M. J. Stillman) and Postgraduate Scholarship (K. E.
Rigby Duncan). We also thank Professor R. J. Pudde-
phatt for use of the ESI mass spectrometer, funded by
the Canada Research Chair program, and Doug Hair-
sine for advice and discussion on the operation of the
ESI mass spectrometer.
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