Critical Review
Copper Metallothioneins
Department of Chemistry and Biochemistry, University of Texas at Dallas,
Richardson, TX, USA
Abstract
Metallothioneins (MTs) are a class of low molecular weight
and cysteine-rich metal binding proteins present in all the
branches of the tree of life. MTs efficiently bind with high
affinity several essential and toxic divalent and monovalent
transition metals by forming characteristic polynuclear metal-
thiolate clusters within their structure. MTs fulfil multiple bio-
logical functions related to their metal binding properties, with
essential roles in both Zn(II) and Cu(I) homeostasis as well as
metal detoxification. Depending on the organism considered,
the primary sequence, and the specific physiological and
Keywords: Metallothioneins; Copper; Zinc; metal-thiolate clusters;
metal homeostasis
Metallothioneins (MTs) constitute a superfamily of cysteine-
rich metal-binding proteins and peptides characterized by the
presence of single or multiple metal-thiolate clusters in their
three dimensional structure (1). Metal clusters in MTs are usu-
ally formed through the preferential coordination of d10 metal
ions (Zn(II), Cd(II), and Cu(I)), by an array of closely spaced
cysteine thiolate ligands present in their primary sequence.
The presence of nonsulfur coordination residues (e.g., histi-
dine) in some members of the superfamily, such as bacterial
and plant MTs, has broken the dogma of the exclusive metal
coordination by cysteine thiolate residues in MTs. According to
Abbreviations: Ab, amyloid-b; AD, Alzheimer’s disease; a-Syn, a-synu-
clein; CNS, central nervous system ; ESI-MS, electrospray ionization mass
spectrometry; MymT, mycobacterial metallothionein; MT, metallothionein;
NMR, nuclear magnetic resonance ; ORF, open reading frame; ROS, reac-
tive oxygen species; 3D, three-dimensional
C 2017 International Union of Biochemistry and Molecular Biology
V mammalian MTs for which additional specialized roles in
Volume 69, Number 4, April 2017, Pages 236–245
*Address correspondence to: Gabriele Meloni, Chemistry and Biochemis-
try, University of Texas at Dallas, Richardson, TX 75080, USA. Tel: 972-
883-4207. Fax: 972-883-2925.
E-mail: gabriele.meloni@utdallas.edu
J.C. and H.J. contributed equally to the article.
Received 16 January 2017; Accepted 16 February 2017
DOI 10.1002/iub.1618
Published online 13 March 2017 in Wiley Online Library
(wileyonlinelibrary.com)
236
Jenifer Calvo
Hunmin Jung
Gabriele Meloni*
metabolic status, Cu(I)-bound MT isoforms have been isolated,
and their chemistry and biology characterized. Besides the rec-
ognized role in the biochemistry of divalent metals, it is
becoming evident that unique biological functions in selective-
ly controlling copper levels, its reactivity as well as copper-
mediated biochemical processes have evolved in some mem-
bers of the MT superfamily. Selected examples are reviewed
to highlight the peculiar chemical properties and biological
functions of copper MTs. V C 2017 IUBMB Life, 69(4):236–245,
2017
Binz and Ka € gi (2), MTs are classified into 15 families based on
taxonomic parameters and the patterns of distribution of Cys
residues in their sequence.
The recognized fundamental functions of MTs arise from
their capabilities to bind transition metals with high affinity,
and their primary biological roles include homeostasis of
essential trace metals zinc and copper, and sequestration and
protection from environmental toxic metals such as cadmium,
mercury, and lead (1). In addition, in light of the reactivity of
the metal-coordinating thiolate ligands, MTs play fundamental
roles in protection against oxidative stress including reactive
oxygen and nitrogen species and other free radicals (1,3–5).
However, additional specific MT functions arise from specific
biological needs and complexity of the organisms in which
they are expressed. Since 60 years from their discovery, it
emerged that metallothoineins indeed possess complex pleio-
tropic functions. This is exemplified by the better-studied
adaptation to stress, protection against brain injury, regulation
of neuronal outgrowth, antiapoptotic effects, and reactivity
and inactivation of metal-based chemotherapeutics leading to
resistance have been demonstrated (4).
As a result of the high number of thiolate coordinating res-
idues in MTs and the lack of defined three-dimensional (3D)
structures in the metal depleted apo forms, MTs can bind a
number of different monovalent and divalent metals both in
vitro and in vivo. The affinity of the metal ions for the binding
IUBMB Life
sites in MTs follows the order found for inorganic thiolates:
HgII > AgI  CuI > CdII > ZnII (6). The replacement of one metal
by another based on the relative affinities leads to the forma-
tion of mixed metal-MT complexes. As a result, homogeneous
or mixed species can be generated in vitro as a function of
metal concentrations and relative ratios (7). In vivo, the spe-
cific metal composition and the functional roles of isolated MT
appear to depend on the source organism, on the previous
exposure to metals, as well as on the primary sequence of MT
chain which can impose partial selectivity toward specific met-
als. Thus, homometallic or heterometallic M(II)MTs (with M(II)
indicating divalent transition metals such as Zn(II) and Cd(II))
are predominantly isolated in physiological conditions or upon
cadmium exposure. The biochemistry and structural biology of
MTs bound to divalent metal ions has been extensively investi-
gated and reviewed (4,5,8). In specific organisms, or under
specific physiological or pathological situations selected MT
isoforms are instead isolated as homometallic Cu(I)-MTs or
heterometallic Cu(I),Zn(II) species. Thus, an important role for
MTs in copper physiology has been revealed. Base on the
structural/functional relationships, a classification of MTs as
zinc–thioneins and copper–thioneins has been proposed in the
efforts to identify evolutionary features defining copper-
specific roles for MTs (9). However, many challenges remain
in understanding the structural and functional diversity the
copper MTs within the entire MT superfamily. This review
focuses on aspects concerning structural and biochemical fea-
tures in four classes of copper-binding MTs to exemplify their
unique chemical and biological properties in copper
biochemistry.
Bacterial Copper MTS
Bacterial MTs (Family 15) have been first identified in the
marine cyanobacterium Synechococcus sp. (strain RRIMP N1)
(10). Until recently, the only bacterial MT family identified
and characterized was the BmtA family. The prototypical bio-
chemical and biophysical properties of BmtA family members
have emerged by detailed spectroscopic, biophysical, and
structural investigation of the SmtA protein from Synechococcus
PCC 7942. The expression of MTs in Synechococcus sp. is upre-
gulated by exposure to increased levels of Cd(II) and Zn(II) and
only to a lesser extent by Cu(II). SmtA has been demonstrated to
be involved in Zn(II) and Cd(II) sequestration and detoxification
in vivo. The three-dimensional structure of zinc- and cadmium-
loaded Zn4SmtA revealed a single domain protein in which four
M(II) ions are coordinated in distorted tehtrahedral geometry by
nine terminal and l2-bridging cysteine thiolates sulfurs and two
histidine imidazole nitrogen ligands (11). Another important
feature is the presence of an inert Zn(II)Cys4 site surrounded by
b-strands and a-helix that form a zinc finger (11). SmtA has
been demonstrated to be capable of binding Cu(I) in vitro via
zinc displacement from Zn4SmtA to generate monomeric homo-
metallic Cu(I)7-SmtA in which significant conformational
changes occurs. However, no evidence has been obtained that
Calvo et al.
SmtA is directly involved in Cu(I) binding in vivo and no native
Cu(I)7-SmtA have been isolated. Based on these studies, it has
been assumed that prokaryotic MTs are exclusively involved in
Zn(II) homeostasis (11).
Surprisingly, in 2008, the discovery of a copper-binding
MT in the pathogen Mycobacterium tuberculosis demonstrated
the existence of prokaryotic MTs specifically evolved to control
copper metabolism (12). This key copper-binding protein
involved in M. tuberculosis copper homeostasis has been
named mycobacterial MT (MymT) based on the presence of
characteristic Cys-X-Cys motifs in its primary sequence and its
capability in efficiently coordinating transition metal ions.
MymT is the first MT from gram-positive bacteria for which a
defined physiological function has been discovered, which is to
partially protect M. tuberculosis from copper toxicity. M. tuber-
culosis, the pathogen that causes tuberculosis, shows a
remarkable capability of evading the immune system response
by being capable of surviving in the phagosome compartment
of hosts’ macrophages. Its virulence is strictly correlated to the
capability of controlling metal homeostasis within this com-
partment. During the M. tuberculosis infection, the host mac-
rophage activates key immune responses including metal over-
loading of the phagosome compartment. Copper is mobilized
toward site of infection to aid in bacterial killing, imparting
toxicity by catalyzing the metal-dependent production of reac-
tive oxygen species (ROS) and disrupting iron–sulfur clusters in
the infecting pathogen. However, M. tuberculosis counters this
copper toxicity by activation of copper resistance mechanisms
such as upregulation of copper export, copper sequestration,
and oxidation of Cu (I) to less toxic Cu (II) (12–14) (Fig. 1).
MymT is a 4.9-KDa polypeptide of 53 amino acids possess-
ing metal binding properties (with the first 5 amino acids pos-
sibly absent in the mature form) containing 7 Cys and 2 His
residues and conserved in other Mycobacterium homologue
proteins (12). MymT show poor amino acid sequence homology
with other known eukaryotic or prokaryotic MTs. Although the
Cys-X-His-X-X-Cys-X-Cys motif of MymT is conserved in SmtA,
the second metal binding motif, Cys-His-Cys-(X)3-Gly-(X)2-Tyr-
Arg-Cys-Thr-Cys, has no known counterpart in other MT
sequences (12). Biophysical studies on purified MymT obtained
by recombinant expression in Escherichia coli revealed that
Cu(I)-binding results in the formation of stable copper-bound
species while, differently from SmtA, stable zinc species could
not be formed (12). MymT generates stable polynuclear Cu(I)-
thiolate cluster as demonstrated by characteristic emissive
luminescence spectra typical of proteins containing solvent-
shielded polynuclear Cu(I)-thiolate cores (12). Cu(I) titration
studies coupled to metal–protein complexes analysis by nano
Electrospray Ionization Mass Spectrometry (ESI-MS) revealed
that the predominant species formed contains 4–6 copper ions.
Exposure to nitric oxide resulted in cluster disruption and met-
al release in accordance to S-nitrosylation of the coordinating
residues with concomitant metal dissociation (12). MymT
expression is induced by Cu, Cd, Co, Ni, and Zn. Nevertheless,
the existence of the Cu(I) bound form in vivo has been
237
 IUBMB LIFE

Copper homeostasis and MymT in M. tuberculosis. Copper is pumped into the macrophage, phagosome and transported into
FIG 1 the cytoplasm of M. tuberculosis via an unknown import mechanism. Elevated copper levels are sensed by the copper-
responsive transcriptional repressor CsoR which, by losing affinity to its DNA cognate sequence upon copper binding, triggers
operon activation, and expression of CtpV, a P-type ATPase of the inner membrane (IM) likely involved in copper efflux. In the
outer membrane (OM), Mctb prevents the accumulation of copper within the mycobacterial cell possibly by copper export. In
the cytoplasm, the copper-binding MT, Mymt, binds and sequesters copper, enabling growth at elevated copper levels. Copper
release from MymT occurs in the presence of reactive nitrogen species (RNS), further triggering the copper operon activation
via formation of Cu-CsoR (12–14).

demonstrated by comparing the characteristic Cu(I)-MymT
luminescence in vivo for copper-exposed wild-type M. tubercu-
losis with the one in mutants in which MymT has been
knocked-out and therefore absent. A significant decrease in
luminescence was attributed to the absence of Cu(I)-MymT
species in those cells. In agreement to its specific role in cop-
per homeostasis, MymT knock-out mutants are more sensitive
to high copper concentrations but not to other metals or reac-
tive oxygen and nitrogen species, further supporting the Cu(I)
binding nature of MymT in vivo. Thus, by binding and seques-
tration of Cu(I), the role of MymT is crucial in virulence and
resistance in M. tuberculosis (12).
Although the identification of prokaryotic copper MTs has
so far been restricted to MymT homologues exclusively present
in Mycobacterium species, it cannot be excluded that copper
MTs present in other bacterial classes remain to be discov-
ered. MT sequences are short and often display a low level of
complexity; thus, they can be easily overlooked in sequenced
bacterial genomes (15).
Yeast and Fungal Copper MTS
In yeasts and fungi, a large number of MT proteins have been
identified and studied. Most of the these yeast and fungal MTs
(belonging to families 8–13) are isolated exclusively as Cu-MTs
and their expression is induced by Cu(I) (16). A number of
studies have established their functional importance as major
intracellular copper chelators responsible for controlling intra-
cellular copper pools. As a consequence of their Cu(I)-binding
nature, MTs play key roles in regulating available copper
concentrations thereby preventing copper toxicity when copper
levels increase (16). Despite their sequential diversity, the
mechanisms of their copper-induced regulation follow shared
biochemical principles. When intracellular copper concentra-
tions rise, specific copper-sensing transcription factors are
activated by Cu(I)-binding to their metal binding domains
through conserved cysteine thiolates (16,17). Cu(I)-binding
triggers structural changes within their three-dimensional
structure, thus enabling them to bind to specific DNA motifs
and to induce the transcription of the downstream MT genes
(16,17).
The best characterized MT isolated from yeast is the Sac-
charomyces Cerevisiae MT. S. cerevisiae MT is encoded by the
CUP1 gene and the mature protein is 53 amino acids long, with
the first 8 amino acids encoded by the Open Reading Frame
(ORF) cleaved off and absent in the mature protein (18,19). MT
expression in S. cerevisiae is exclusively induced by increased
intracellular Cu(I) and Ag(I) levels, while Zn(II) and Cd(II) fail to
induce CUP1 transcription (18,19). Increased Cu(I) concentra-
tions result in Cu(I) binding (forming a Cu(I)-thiolate cluster) and
activation of the ACE1 transcription factor, which in turn binds
to the promoter region upstream of the CUP1 gene thereby trig-
gering MT gene transcription (17). According to the activation of
expression profile under physiological conditions, yeast MT is
isolated exclusively as Cu(I)-bound form.
The nuclear magnetic resolution (NMR) solution structure
was initially determined for the native copper-containing form
and for the NMR-active 109Ag(I) derivative, both showing that
10 of the 12 available cysteines bind 7 Cu(I) or Ag(I) ions in a
single cluster (20). However, as it was uncertain that Cu/Ag

238 Copper Metallothioneins

 Yeast MT. (A) Crystal structure of S. cerevisiae Cu(I)8-MT (PDB ID: 1RJU) (22). Cu(I) ions are shown as orange spheres, and
FIG 2 CysS-Cu(I) connectivities are presented as dashed lines. (B) Structure of the Cu(I)8–thiolate cluster in S. cerevisiae Cu(I)8-MT.
Thiolate sulfur atoms from Cys10 and Cys22 coordinate 3 Cu(I) and connect the two distorted Cu(I)- tetrahedron structures (22).
The figure was generated with Chimera.
isomorphous replacement can be assumed, it was realized
that the structure of the Cu7-cluster derived from the 109Ag(I)7-
MT derivative might have not been necessarily correct as the
coordination number for Cu(I) tends to be larger than that for
Ag(I) (21).
Structural determination by X-ray crystallography of the
Cu(I)8-MT has provided definitive details on the structural
properties of the metal-thiolate cluster in yeast Cu(I)-MT (22)
(Fig. 2A). The yeast MT sequence contains 12 cysteine resi-
dues. However, only 10 of the 12 residues are involved in Cu(I)
coordination. The two Cys residues located closest in the
sequence the C-terminus are not involved in metal binding.
The structure revealed a single Cu(I)8-thiolate cluster core sur-
rounded by the polypeptide chain in which defined secondary
structure elements are lacking. Six of the eight bound Cu(I)
are trigonally coordinated each by three cysteine thiolate sul-
fur atoms, while the two remaining Cu(I) are coordinated digo-
nally by two cysteine thiolate sulfurs (Fig. 2B). The Cu(I) atoms
are arranged in two groups of four, each assuming two con-
nected distorted tetrahedron structures. In one of these tetra-
hedron, the Cu–Cu distances range between 2.59-2-91 Å, while
in the other longer distances (2.78–3.12 Å) are observed. The
overall structure and the short Cu–Cu distances indicate the
presence of a compact Cu(I)-thiolate core shielded from solvent
access. Accordingly, strong luminescence emissive features
are observed in agreement with the shielded nature of copper-
thiolate cluster. Of the 10 coordinating Cys thiolate sulfurs,
8 coordinate 2 Cu(I) in a l2-bridging fashion while the remain-
ing 2 Cys sulfurs bind 3 Cu(I) and connect the 2 distorted tetra-
hedron structures.
Despite the structure of the yeast MT is the only structure
available for a copper MT other Cu(I)-MTs from yeasts and fungi
have been identified and characterized. The expression of Neu-
rospora crassa MT is exclusively induced by Cu(II) and not by
Zn(II), Cd(II), Co(II), or Ni(II), supporting its role as an exclusive
Calvo et al.
copper sequestering molecule in vivo similarly to S. cerevisiae
MT, despite in vitro also divalent metal ions can be bound. The
sequence of N. crassa MT is 25 amino acids long and contains 7
Cys residues all involved in metal coordination. Circular dichro-
ism, electronic absorption and luminescence spectroscopic
characterization revealed the presence of a Cu(I)6S-thiolate
cluster in which all the Cys residues are involved in metal coor-
dination. The fold of the polypetide chain has been determined
by solution NMR but the direct Cys-Cu(I) connectivity could not
be determined due to the NMR-insensitive nature of the Cu(I)
quadrupolar nuclei (23). Interestingly, the native-like Cu(I)6-MT
could be obtained in vitro only by metal substitution from the
Zn(II)3-MT. Contrarily, only Cu(I)4-MT species could be obtained
from the direct Cu(I) incorporation into the apo-MT, suggesting
diverse pathway for the cluster assembly in the two cases. The
structure revealed the lack of classic secondary structural ele-
ments with the N-terminal and C-terminal halves wound around
the Cu(I)-thiolate core in a left- and right-handed fashion,
respectively. A similar Cu-MT containing a Cu(I)6-thiolate core
has been isolated from the mushroom Agaricus bisporus, and
spectroscopic analysis suggested a similar cluster organization
as in N. crassa MT (24).
Additonal insights about the physiological roles of Cu(I) MTs
in fungi has been obtained in Podospora anserina. P. anserina
possesses a Cu(I)-inducible MT whose expression progressively
increases during aging. It has been demonstrated that alteration
in the turnover of mitochondrial respiratory complex proteins
during aging is responsible for the release of copper ions bound
to cytochrome c oxidase. Increasing copper concentration due to
these processes in turn causes the induction of P. anserina MT
expression, further supporting its role in copper sequestration
and detoxification in yeast and fungal MTs (25).
Other Cu(I) MTs have been identified in yeasts or fungi,
but detailed characterization of their structure, reactivity, and
physiological roles remain more elusive.
239

Snail MTs. Sequence alignment of the three MT isoforms from the Roman snail Helix Pomatia. The conserved Cys residues are
FIG 3 highlighted in yellow and the metal selectivity for each isoform is indicated on the left.
Copper MTS in Molluscs
Studies of MTs from pulmonate gasteropods have proven
instrumental to investigate how metal specificity can be
achieved in MTs. In the snail Helix Pomatia, two different MTs
showing selective binding of cadmium and copper, respective-
ly, have been identified. Interestingly, these two MT isoforms
possess specific tissue expression (26). A third MT isoform,
recovered as a mixed Cd21- and Cu1-containing complex, also
exists, but considering its low abundance, it probably is less
important to the snail’s metal metabolism. The first isoform
isolated from Cd-exposed Roman snails showed binding of
6 Cd(II) ions (27), while the second isoform from the same spe-
cies was isolated with 12 Cu(I) equivalents, and they have
been named HpCdMT and HpCuMT, respectively (28). These
two isoform are 66 and 64 amino acids long and possess a
conserved array of 18 Cys residues in corresponding positions
(Fig. 3). Studies of metal binding to the two isoforms expressed
in E. coli in the presence of the cognate metals confirmed the
homothallic nature of the isolated products. In HpCd6MT,
Cd(II) ions are bound in tetrahedral coordination each by four
thiolate ligands in the form oligonuclear metal thiolate clus-
ters, for which the structure remains to be determined. In
Cu12-HpCuMT, spectroscopic analysis indicate that Cu(I) is
organized in the form of oligonuclear Cu(I) thiolate clusters
with features very similar to mammalian Cu12-MT in which
metals are bound predominantly in trigonal coordination
(29,30). Stability of the expressed Cd6-HpCdMT and Cu12-
HpCuMT complexes was demonstrated by their metal
exchange inertness. Contrarily, expression in the presence of
the noncognate metal partners resulted in multiple heteroge-
neous heterometallic species containing both Cu(I) and Zn(II)/
Cd(II) ions (29,30). In light of the fact that heterothallic species
can be generated when exposed to noncognate metals, abso-
lute selectivity cannot result exclusively from difference in the
noncysteine residues in their primary sequence (29,30).
HpCuMT is constitutively expressed in a specialized molluscan
cell type, the rhogocyte, which is the site of synthesis of the
copper protein hemocyanin. It is likely that specific cellular
localization exposes each synthesized isoforms to environ-
ments with unique and diverse metal buffering capabilities. It
is expected that HpCuMT has an affinity for copper high
enough to efficiently compete with other copper buffering mol-
ecules in rhogocytes cytosol (31). On the other hand, expres-
sion of HpCdMT appears to be heavily regulated by exposure
to increasing concentrations of cadmium in the snail midgut
gland and epithelial cells of foot, gut, and kidney, thus playing
a major role in metal detoxification (31).
240
IUBMB LIFE
Overall, it appears that cell-type specific expression com-
bined with evolution of metal selectivity preferences achieved
by modulation of noncysteine amino acid positions result in
the generation of specific homometallic species, at least in
snail. As a result, functional HpCuMT and HpCdMT species
show a metal composition tailored to satisfy their specialized
localization and cellular function (i.e., HpCuMT in rhogocytes).
Mammalian Copper MTs
In mammals, four distinct MT isoforms have been identified.
They are designated MT-1 through MT-4.
In certain species, including Homo sapiens, genetic poly-
morphism occurs with at least seven MT-1 isoforms expressed,
while in others, such as Mus musculus, single MT gene exists
(MT-1, 22, 23, 24). Mammalian MTs are single polypetide
chains of 61–68 amino acids characterized by a conserved
array of 20 Cys residues, with no aromatic or histidine resi-
dues present in the sequence. Cysteine residues are clustered
in Cys–X–Cys, Cys–Cys and Cys–X–X–Cys motifs characteristic
of the vertebrate MT family (Family 1, according to Binz and
Ka€ gi) (2).
MT-1 and MT-2 are inducible proteins and ubiquitously
expressed in several organs including liver, kidney, intestine,
and brain. MT-3 and MT-4 expression is instead restricted
mainly to the central nervous system (CNS) and stratified epi-
thelia, respectively, and their expression is not inducible by
the classic stimuli of MT-1/-2 (4,32,33). MT-1 and MT-2 are
major zinc-binding proteins in the cells and they are overex-
pressed in response to heavy metal exposure. A large number
of toxicological studies have revealed that cadmium binding to
MT-1 and MT-2 isoforms represent a fundamental protective
effect for heavy metal cellular detoxification. In agreement to
this role, increased MT content in liver, kidney, and intestines
occurs following the parenteral or dietary administration of
cadmium, zinc, or copper to experimental animals (1,4). Cad-
mium detoxification is a property of MTs rather than its evolu-
tionary function. As MTs constitute a major fraction of zinc-
binding proteins in the cell under philological conditions,
mammalian MTs can potentially modulate many important
biological processes that involve zinc-requiring enzymes. It has
been demonstrated that MT-1/-2 can directly activate zinc
enzymes by direct zinc transfer, inactivate zinc-finger tran-
scription factors via zinc removal, and indirectly modulate
zinc-dependent processes by controlling zinc availability in the
cell (1).
As a consequence of their fundamental properties in regu-
lating physiological Zn(II) concentrations and their role in
Copper Metallothioneins
 Mammalian MTs. (A) Overview of the crystal structure of Cd5,Zn2-MT-2 isolated from rat (PDB ID: 4MT2). Two Zn(II) and one
FIG 4 Cd(II) are located in the N-terminus b-domain, and the other four Cd(II) in the C-terminus a-domain. The figure was generated
with Chimera. (B) Spectroscopic model of Cu(I)4CysS6-7 clusters in Cu(I)8-MT (40). (C) Sequence alignment of human MT-1, 22,
23, and 24 along with rat MT-2. The conserved 20 Cys residues are highlighted in yellow.

detoxification of divalent toxic metals (e.g., Cd(II)), a wealth of
information is available regarding the structural properties of
M(II)-bound mammalian MTs. The 3D structures have been
obtained for rat Cd5, Zn2MT-2 (34), and rat (35), rabbit (36),
and human (37) Cd7MT-2, as well as the mouse (38) and
human (39) a-domain of Cd4-MT-3. These structures showed
identical metal–thiolate clusters organization and very similar
protein folds. Thus, mammalian MTs show a dumbbell-like
topology with two separated domains linked by flexible hinge
region each encompassing a Metal-thiolate cluster. A
M(II)3CysS9 is present in the N-terminal b-domain and a
M(II)4CysS11 in its C-terminal domain (with M(II) 5 Cd(II) or
Zn(II)) with all the 20 conserved cysteines involved in metal
coordination. All metals in the clusters show tetrahedral coor-
dination by l2-bridging and terminal cysteine thiolates (Fig.
4A).
When mammalian MTs are isolated from livers of various
species MT-1 and MT-2 usually contain seven Zn(II) ions. MT-
3 and MT-4 are instead isolated from brain and epithelia,
respectively, as mixed species containing Zn(II) and Cu(I) (4).
However, in situations of cellular copper overload, such as
Wilson’s disease, MT plays major role in Cu(I) binding and
detoxification, and MT-1/-2 species containing predominantly
Cu(I) ions can be isolated from the liver (1).
Presently, no 3D structure of the Cu(I)-containing mamma-
lian MT-1 and MT-2 isoforms is available. However, spectro-
scopic and biophysical investigations have revealed that Cu(I)-
loaded MT can bind up to 12 Cu(I) ions organized in two sepa-
rated Cu(I)6-thiolate clusters, with even higher stoichiometries
reported. These clusters are located in the b- and a-domains
with all the conserved 20 Cys residues involved in metal
coordination. Differently from M(II) which are each coordinat-
ed in tetrahedral geometry by four Cys ligands, Cu(I) ions are
coordinated digonally and trigonally by 2–3 sulfurs from bridg-
ing and terminal cysteine ligands. Evidence for the existence
of a distinct stable MT complexes with 8 Cu(I) equivalents
bound (Cu(I)8-MT) has been obtained, indicating that two sepa-
rated Cu(I)4-thiolate clusters are formed in the pathway of
cluster assembly. In these clusters, Cu(I)–Cu(I) distances are
shorter than 2.8 Å allowing d10–d10 orbital overlap resulting in
a certain degree of metal–metal bonding character in the cop-
per core (Fig. 4B) . In the presence of increasing Cu(I)-to-pro-
tein ratios, the Cu(I)4S(6-7) cores can be expanded to Cu(I)6-
Cys9 clusters, resulting in an increase in Cu–Cu distance to
>2.8Å (40). Thus, cluster structures and metal coordination
geometries are different in Cu(I)-MT and Zn(II)-MT forms. As a
consequence, a different polypeptide arrangement around the
metal core of Cu(I)-MT and Zn(II)-MT occurs (41).
In the human brain, besides MT-1/MT-2, the MT-3 isoform
is also present. When isolated from human, bovine, and equine
brains, MT-3 contains four Cu(I) ions and 3–4 Zn(II). MT-3 has
been discovered by Uchida et al. and named neuronal growth
inhibitory factor, as MT-3 possess distinct biological functions
that are not shared by other MT isoforms (32). MT-3 antago-
nizes the ability of Alzheimer’s disease brain extract to stimu-
late survival and neuritic sprouting of cultured neurons, pos-
sesses extracellular growth inhibitory activity in neuronal
primary cultures, and protects neuronal cells from the toxic
effect of amyloid-b (Ab) peptide Ab1–40, suggesting that MT-3
may be involved in protection from pathogenic processes lead-
ing to Alzheimer’s disease (AD) (4). Besides the CNS, lower
MT-3 expression has been subsequently reported also in renal

Calvo et al. 241


epithelial cells (42) and human prostate (43) as well as in sev-
eral cancers cells including urinary bladder (44), breast (45),
and prostate(46), suggesting that MT-3 could represent a bio-
marker for cell growth and/or proliferation.
The differences in amino acid sequences are responsible
for the observed bioactivity in the CNS. MT-3 sequence con-
tains two unique features when compared with MT-1/-2 iso-
forms (Fig. 4C): (1) a threonine in position 5 in the N-terminal
b-domain followed by a conserved C6PCP9 motif and (2) an
acidic hexapeptide in the C-terminal domain. Native monomer-
ic Cu(I)4Zn3-4MT-3 contains two homometallic clusters, a Cu(I)-
thiolate cluster and a Zn(II) cluster. By direct reconstitution of
Cu(I) and Zn(II) in the recombinantly expressed apoMT-3, the
Cu(I)4-thiolate cluster was demonstrated to be located in the
N-terminal b-domain, while the Zn(II)-cluster in the C-terminal
domain. While the Cu(I)4-thiolate cluster shows a remarkable
stability toward oxidation, the C-terminal Zn(II)4-thiolate clus-
ter is susceptible to oxidation with consequent release of 1
Zn(II) ion and concomitant formation of Cu(I)4Zn3MT-3.
Details on Cu(I) binding to isolated MT-3 domains have
been characterized. Binding of Cu(I) to the N-terminal b-
domain revealed the initial cooperative formation of a
Cu(I)4S(6-7) cluster which could be subsequently expanded to a
Cu(I)6S9 core by further addition of Cu(I) (47). Contrarily, in
the a-domain, a Cu(I)4S7-8 cluster is formed that can be further
expanded to generate a Cu(I)6S11(48). Characterization of the
low-temperature luminescence properties confirmed that, as
observed for MT-1/-2, the expansion of the Cu(I)4-thiolate
cores to Cu(I)6-thiolate cores is responsible for increased Cu–
Cu distances and disruption of d10–d10 orbital overlap which
might be pivotal in the air-stability of the Cu(I)4 cluster toward
oxidation (47,48).
Besides being a neuronal intracellular protein, MT-3 can
be efficiently secreted in the brain extracellular space. A pro-
tective role of extracellular Zn7MT-3 from Cu(II) toxicity has
been suggested. Zn7MT-3 thiolates can efficiently reduce Cu(II)
to Cu(I) and bind it to form Cu(I)4Zn4MT-3 via cooperative for-
mation of an air stable Cu(I)4-thiolate cluster in its b-domain
(49). In this process, the three Zn(II) ions are released and two
disulfide bonds, likely located in the a-domain, are generated.
The formed Cu(I)4Zn4MT-3 species are redox inert, and the
observed quenching of the ascorbate-driven and Cu(II)-cata-
lyzed production of ROS highlight the stability of the protein
and its capability to act as a protective factor from Cu(II) toxic-
ity. Overall, these results indicate that MT-3 b-domain possess
higher reactivity and affinity toward copper ions over Zn(II)
compared with the a-domain (49).
In light of the reactivity of MT-3 toward Cu(II), a major
role played by MT-3 in controlling aberrant copper–protein
interactions in neurodegenerative disorders has been pro-
posed. Cu(II) binding to Ab peptides in AD (the major compo-
nent of extracellular amyloid plaques in Alzheimer’s diseases),
to prion protein in disorders like Creutzfeldt–Jakob disease,
and to a-synuclein (a-Syn) in Parkinson’s disease has been
implicated as disease-potentiating factor contributing to
242
IUBMB LIFE
protein misfolding, aggregation into soluble oligomers and
insoluble amyloid aggregates, and copper-catalyzed ROS pro-
duction via Haber-Weiss and Fenton-type chemistry. In these
neurodegenerative diseases, the expression of MT-3 is found
to be altered (4).
In light of its reactivity toward copper, Zn7MT-3 can effi-
ciently scavenge Cu(II) from soluble and aggregated Ab-Cu(II)
complexes as well as from soluble oligomers via a metal-swap
reaction generating the described redox-inert Cu(I)4Zn4MT-3
species and Ab-Zn(II) (50). These reaction underlie the abolish-
ment of copper redox cycling and concomitant radical produc-
tion, providing a protective effect against the toxicity of Ab-
Cu(II). Accordingly, the toxic effect of Ab1–40-Cu(II) in cell cul-
ture was abolished thereby providing a possible molecular
mechanism explaining the protective effect by MT-3 against
Ab toxicity shown in previous studies. Similar metal-swapping
reactions between a number of different PrP-Cu(II) complexes
as well as a-synuclein-Cu(II) complexes resulting in copper
scavenging and formation of Cu(I)4Zn4MT-3 substantiate a
putative and more general role played by MT-3 in controlling
aberrant protein-Cu(II) interaction in the brain (51,52).
The expression of the last mammalian isoform discovered
by Quaife and Palmiter et al., MT-4, is restricted to cornified
and stratified epithelia, where a role in metal metabolism dur-
ing differentiation has been proposed (33). When isolated from
tongue epithelium, MT-4 is purified as a mixed copper and
zinc form, substantiating its role in the homeostasis of both
metals (33,53). MT-4 expression in E. coli with media supple-
mented with zinc, cadmium, or copper indicated that MT-4
has a stronger preference for Cu(I) over divalent metal ions
Zn(II) and Cd(II) when compared with MT-1, resulting in the
formation of heterometallic mixed species. By in vitro titration,
it could be demonstrated that full-length MT-4 could bind up
to 10 Cu(I) to generate Cu(I)10-MT-4, while Cu(I)5-aMT-4 and
Cu(I)7-bMT-4 species could be formed and isolated in the a-
and b- domains, respectively (54). Based on these results and
in silico protein sequence analysis, MT-4 was classified as a
novel copper–thionein, made up of two copper–thionein
domains (54). The discovery of the participation of sulfide
anions as additional ligands in the metal clusters of Zn/Cd-MT-
4 forms, generated in vivo via recombinant expression, has
pioneered the discovery of S2- as additional metal ligands in a
series of other MTs from different species (recombinant and
native) (55). Despite S2- incorporation does not commonly
occur in Cu-bound MT forms, the discovery that the higher sul-
fide content in the cadmium-loaded MTs is obtained in MT-
isoforms possessing the higher the copper–thionein character
has provided to the community further criterion for the classi-
fication of MTs (56).
Other Characterized MTS
A number of other MT isoforms from different species have
been characterized and, for some, their Cu(I) binding ability
demonstrated, including MTs belonging to protozoan (57),
Copper Metallothioneins
arthropoda (58), crustacea (59), molluscs (26,60), echinoderms
(60), nematodes (61), and plants (some of which show Cu(I)-
thionein character) (62). Complete 3D structures are available
from the cadmium-bound forms of some of these MTs includ-
ing crustacean blue crab Cd6-MT-1 (Callinectes sapidus) (63),
lobster Cd6MT-1 (Homarus americanus) (64), sea urchin
Cd7MTA (Strongylocentrotus purpuratus) (65), and fish
Cd7MT_nc (Notothenia coriiceps) (66) (all of which are mono-
meric proteins with two globular domains each encompassing
a metal thiolate cluster), as well as wheat MT Ec-1 (67,68).
Although some investigations have been undertaken to under-
stand Cu(I) binding to some of these MTs, no other 3D struc-
tures of Cu(I)-bound MT has been obtained so far, limiting our
understanding on conserved and divergent features in the
structure, reactivity, and functional properties of copper MTs.
Conclusions
Since their discovery, MTs emerged as a central and conserved
protein class involved in both essential metal homeostasis and
toxic metal detoxification in all the kingdoms of life. Character-
ization of MTs isolated from a wide variety of organisms and
tissues, together with in silico protein sequence analysis sug-
gest that specific MT isoforms evolved to play preferential
roles in controlling copper homeostasis over other transition
metals. In recent years, it has been demonstrated that the
metal-selectivity and metal-composition in MTs builds on com-
plex factors arising from a strict interplay between the prima-
ry sequence, the pattern of the conserved coordinating cys-
teines, the nature of the neighboring non-coordinating amino
acids, the metal composition of the media/cellular milieu
where the MTs are expressed, and the nature of metals induc-
ing MT expression in vivo. The functional classification of MTs
as zinc- or copper-thioneins taking into account several of
these aspects has significantly contributed to our understand-
ing the pleiotropic chemistry of MTs (56). However, the a pri-
ori prediction of the metal binding character and properties
still remains challenging when sequence and functional homol-
ogy among different MTs are divergent. Presently, the under-
standing of the chemistry and biology of Cu-MTs has not yet
reached the level obtained for Zn(II)-MTs. Several questions
such as how protein sequence and structure control preferen-
tial metal selectivity in Cu-MTs, how metal buffering and avail-
ability in distinct cellular and tissue localizations determines
the generation of Cu-MTs, as well as specific and unique physi-
ological function of Cu-MTs remain to be answered. More
work is yet to be carried out to understand the chemistry and
biology of Cu-MTs and intriguing new findings are likely to
emerge.
References
[1] Vasa
k, M. and Romero-Isart, N. (2005) Metallothioneins. In Encyclopedia of
Inorganic Chemistry, 2nd ed. (King, R. B. ed.). pp. 3208 – 3221, Wiley, New
York.
Calvo et al.
[2] Binz, P. A. and Ka €gi, J. H. R. (1999) Metallothionein: molecular evolution and
classification. In Metallothioenein IV (Klaassen, C., ed.). pp. 7 – 13, Birkha €user
Verlag, Basel.
[3] Thornalley, P. J. and Va sak, M. (1985) Possible role for metallothionein in
protection against radiation-induced oxidative stress. Kinetics and mecha-
nism of its reaction with superoxide and hydroxyl radicals. Biochim. Biophys.
Acta 827, 36 – 44.
[4] Va k, M. and Meloni, G. (2011) Chemistry and biology of mammalian metal-
sa
lothioneins. J. Biol. Inorg. Chem. 16, 1067 – 1078.
[5] Stillman, M. J. (1995) Metallothioneins. Coord. Chem. Rev. 144, 461 – 511.
[6] Romero-Isart, N. and Va k, M. (2002) Advances in the structure and chemis-
sa
try of metallothioneins. J. Inorg. Biochem. 88, 388 – 396.
[7] Good, M., Hollenstein, R., and Va sak, M. (1991) Metal selectivity of clusters in
rabbit liver metallothionein. Eur. J. Biochem. 197, 655 – 659.
[8] Stillman, M. J. (1992) Metallothionein. In Metallothioneins (Stillman, M. J.,
Shaw, C. F. I., and Suzuki, K. T., eds.). pp. 55 – 127, VCH, New York.
[9] Valls, M., Bofill, R., Gonzalez-Duarte, R., Gonzalez-Duarte, P., Capdevila, M.,
et al. (2001) A new insight into metallothionein (MT) classification and evolu-
tion—the in vivo and in vitro metal binding features of Homarus americanus
recombinant MT. J. Biol. Chem. 276, 32835 – 32843.
[10] Olafson, R. W., Abel, K., and Sim, R. G. (1979) Prokaryotic metallothionein:
preliminary characterization of a blue-green alga heavy metal-binding pro-
tein. Biochem. Biophys. Res. Commun. 89, 36 – 43.
[11] Blindauer, C. A., Harrison, M. D., Parkinson, J. A., Robinson, A. K., Cavet, J.
S., et al. (2001) A metallothionein containing a zinc finger within a four-
metal cluster protects a bacterium from zinc toxicity, Proc. Natl. Acad. Sci.
USA 98, 9593 – 9598.
[12] Gold, B., Deng, H., Bryk, R., Vargas, D., Eliezer, D., et al. (2008) Identification
of a copper-binding metallothionein in pathogenic mycobacteria. Nat.
Chem. Biol. 4, 609 – 616.
[13] Robinson, N. J. (2008) A bacterial copper metallothionein. Nat. Chem. Biol.
4, 582 – 583.
[14] Rowland, J. L. and Niederweis, M. (2012) Resistance mechanisms of Myco-
bacterium tuberculosis against phagosomal copper overload. Tuberculosis
(Edinb) 92, 202 – 210.
[15] Blindauer, C. A. (2011) Bacterial metallothioneins: past, present, and ques-
tions for the future. J. Biol. Inorg. Chem. 16, 1011 – 1024.
[16] Dolderer, B., Hartmann, H. J., and Weser, U. (2009) Metallothioneins in
yeast and fungi. Met. Ions Life Sci. 5, 83 – 105.
[17] Furst, P., Hu, S., Hackett, R., and Hamer, D. (1988) Copper activates metallo-
thionein gene transcription by altering the conformation of a specific DNA
binding protein. Cell 55, 705 – 717.
[18] Butt, T. R., Sternberg, E. J., Gorman, J. A., Clark, P., Hamer, D., et al. (1984)
Copper metallothionein of yeast, structure of the gene, and regulation of
expression. Proc. Natl. Acad. Sci. USA 81, 3332 – 3336.
[19] Karin, M., Najarian, R., Haslinger, A., Valenzuela, P., Welch, J., et al. (1984)
Primary structure and transcription of an amplified genetic locus: the CUP1
locus of yeast. Proc. Natl. Acad. Sci. USA 81, 337 – 341.
[20] Peterson, C. W., Narula, S. S., and Armitage, I. M. (1996) 3D solution struc-
ture of copper and silver-substituted yeast metallothioneins. FEBS Lett. 379,
85 – 93.
[21] Bertini, I., Hartmann, H. J., Klein, T., Liu, G., Luchinat, C., et al. (2000) High
resolution solution structure of the protein part of Cu7 metallothionein. Eur.
J. Biochem. 267, 1008 – 1018.
[22] Calderone, V., Dolderer, B., Hartmann, H. J., Echner, H., Luchinat, C., et al.
(2005) The crystal structure of yeast copper thionein: the solution of a long-
lasting enigma. Proc. Natl. Acad. Sci. USA 102, 51 – 56.
[23] Cobine, P. A., McKay, R. T., Zangger, K., Dameron, C. T., and Armitage, I.
M. (2004) Solution structure of Cu6 metallothionein from the fungus Neu-
rospora crassa. Eur. J. Biochem. 271, 4213 – 4221.
[24] Munger, K. and Lerch, K. (1985) Copper Metallothionein from the fungus
Agaricus-Bisporus—chemical and Spectroscopic Properties. Biochemistry
24, 6751 – 6756.
[25] Averbeck, N. B., Borghouts, C., Hamann, A., Specke, V., and Osiewacz, H. D.
(2001) Molecular control of copper homeostasis in filamentous fungi:
243

increased expression of a metallothionein gene during aging of Podospora
anserina. Mol. Gen. Genet. 264, 604 – 612.
[26] Dallinger, R., Berger, B., Hunziker, P., and Ka €gi, J. H. (1997) Metallothionein
in snail Cd and Cu metabolism Nature 388, 237 – 238.
[27] Dallinger, R., Wang, Y. J., Berger, B., Mackay, E. A., and Ka €gi, J. H. R. (2001)
Spectroscopic characterization of metallothionein from the terrestrial snail,
Helix pomatia. Eur. J. Biochem. 268, 4126 – 4133.
[28] Gehrig, P. M., You, C., Dallinger, R., Gruber, C., Brouwer, M., et al. (2000)
Electrospray ionization mass spectrometry of zinc, cadmium, and copper
metallothioneins: evidence for metal-binding cooperativity. Protein Sci. 9,
395 – 402.
[29] Palacios, O., Pagani, A., Perez-Rafael, S., Egg, M., Hockner, M., et al. (2011)
Shaping mechanisms of metal specificity in a family of metazoan metallo-
thioneins: evolutionary differentiation of mollusc metallothioneins. BMC
Biol. 9, 4.
[30] Palacios, O., Perez-Rafael, S., Pagani, A., Dallinger, R., Atrian, S., et al.
(2014) Cognate and noncognate metal ion coordination in metal-specific
metallothioneins: the Helix pomatia system as a model. J. Biol. Inorg.
Chem. 19, 923 – 935.
[31] Foster, A. W. and Robinson, N. J. (2011) Promiscuity and preferences of
metallothioneins: the cell rules. BMC Biol. 9, 25.
[32] Uchida, Y., Takio, K., Titani, K., Ihara, Y., and Tomonaga, M. (1991) The
growth inhibitory factor that is deficient in the Alzheimer’s disease brain is
a 68 amino acid metallothionein-like protein. Neuron 7, 337 – 347.
[33] Quaife, C. J., Findley, S. D., Erickson, J. C., Froelick, G. J., Kelly, E. J., et al.
(1994) Induction of a new metallothionein isoform (MT-IV) occurs during
differentiation of stratified squamous epithelia. Biochemistry 33, 7250 –
7259.
[34] Braun, W., Vasak, M., Robbins, A. H., Stout, C. D., Wagner, G., et al. (1992)
Comparison of the NMR solution structure and the x-ray crystal structure of
rat metallothionein-2. Proc. Natl. Acad. Sci. USA 89, 10124 – 10128.
[35] Schultze, P., Wo € rgo
€ tter, E., Braun, W., Wagner, G., Va k, M., et al. (1988)
sa
Conformation of [Cd7]-metallothionein-2 from rat liver in aqueous solution
determined by nuclear magnetic resonance spectroscopy. J. Mol. Biol. 203,
251 – 268.
[36] Arseniev, A., Schultze, P., Wo € rgo
€ tter, E., Braun, W., Wagner, G., et al. (1988)
Three-dimensional structure of rabbit liver Cd7metallothionein-2a in aqueous
solution determined by nuclear magnetic resonance. J. Mol. Biol. 201, 637 –
657.
[37] Messerle, B. A., Scha €ffer, A., Va
sak, M., Ka €gi, J. H., and W€ uthrich, K. (1990)
Three-dimensional structure of human [113Cd7]metallothionein-2 in solution
determined by nuclear magnetic resonance spectroscopy. J. Mol. Biol. 214,
765 – 779.
[38] € G., Zangger, K., and Armitage, I. M. (2001) Three-dimensional structure
Oz,
and dynamics of a brain specific growth inhibitory factor: metallothionein-3.
Biochemistry 40, 11433 – 11441.
[39] Wang, H., Zhang, Q., Cai, B., Li, H., Sze, K. H., et al. (2006) Solution structure
and dynamics of human metallothionein-3 (MT-3). FEBS Lett. 580, 795 –
800.
[40] Pountney, D. L., Schauwecker, I., Zarn, J., and Va k, M. (1994) Formation
sa
of mammalian Cu8-metallothionein in vitro: evidence for the existence of
two Cu(I)4-thiolate clusters. Biochemistry 33, 9699 – 9705.
[41] Dolderer, B., Echner, H., Beck, A., Hartmann, H. J., Weser, U., et al. (2007)
Coordination of three and four Cu(I) to the a- and b-domain of vertebrate
Zn-metallothionein-1, respectively, induces significant structural changes.
FEBS J. 274, 2349
[42] Hoey, J. G., Garrett, S. H., Sens, M. A., Todd, J. H., and Sens, D. A. (1997)
Expression of MT-3 mRNA in human kidney, proximal tubule cell cultures,
and renal cell carcinoma. Toxicol. Lett. 92, 149 – 160.
[43] Garrett, S. H., Sens, M. A., Shukla, D., Nestor, S., Somji, S., et al. (1999)
Metallothionein isoform 3 expression in the human prostate and cancer-
derived cell lines. Prostate 41, 196 – 202.
[44] Sens, M. A., Somji, S., Lamm, D. L., Garrett, S. H., Slovinsky, F., et al. (2000)
Metallothionein isoform 3 as a potential biomarker for human bladder can-
cer. Environ. Health Perspect. 108, 413 – 418.
244
IUBMB LIFE
[45] Sens, M. A., Somji, S., Garrett, S. H., Beall, C. L., and Sens, D. A. (2001)
Metallothionein isoform 3 overexpression is associated with breast cancers
having a poor prognosis. Am. J. Pathol. 159, 21 – 26.
[46] Juang, H. H., Chung, L. C., Sung, H. C., Feng, T. H., Lee, Y. H., et al. (2013)
Metallothionein 3: an androgen-upregulated gene enhances cell invasion
and tumorigenesis of prostate carcinoma cells. Prostate 73, 1495 – 1506.
[47] Faller, P., and Va k, M. (1997) Distinct metal-thiolate clusters in the N-
sa
terminal domain of neuronal growth inhibitory factor. Biochemistry 36,
13341 – 13348.
[48] Hasler, D. W., Faller, P., and Va k, M. (1998) Metal-thiolate clusters in the
sa
C-terminal domain of human neuronal growth inhibitory factor (GIF). Bio-
chemistry 37, 14966 – 14973.
[49] Meloni, G., Faller, P., and Va k, M. (2007) Redox silencing of copper in
sa
metal-linked neurodegenerative disorders: reaction of Zn7metallothionein-3
with Cu21 ions. J. Biol. Chem. 282, 16068 – 16078.
[50] Meloni, G., Sonois, V., Delaine, T., Guilloreau, L., Gillet, A., et al. (2008) Met-
al swap between Zn7-metallothionein-3 and amyloid-b-Cu protects against
amyloid-b toxicity. Nat. Chem. Biol. 4, 366 – 372. [PMC][10.1038/nchem-
bio.89] [18454142]-
[51] Meloni, G., Crameri, A., Fritz, G., Davies, P., Brown, D. R., et al. (2012) The
catalytic redox activity of prion protein-Cu(II) is controlled by metal
exchange with the Zn(II)-thiolate clusters of Zn(7) metallothionein-3. Chem.
Bio. Chem. 13, 1261 – 1265.
[52] Meloni, G., and Va k, M. (2011) Redox activity of alpha-synuclein-Cu is
sa
silenced by Zn(7)-metallothionein-3. Free Radic. Biol. Med. 50, 1471 – 1479.
[53] Meloni, G., Zovo, K., Kazantseva, J., Palumaa, P., and Vasak, M. (2006)
Organization and assembly of metal-thiolate clusters in epithelium-specific
metallothionein-4. J. Biol. Chem. 281, 14588 – 14595.
[54] Tio, L., Villarreal, L., Atrian, S., and Capdevila, M. (2004) Functional differen-
tiation in the mammalian metallothionein gene family: metal binding fea-
tures of mouse MT4 and comparison with its paralog MT1. J. Biol. Chem.
279, 24403 – 24413.
[55] Capdevila, M., Domenech, J., Pagani, A., Tio, L., Villarreal, L., et al. (2005)
Zn- and Cd-metallothionein recombinant species from the most diverse
phyla may contain sulfide (S2-) ligands. Angew. Chem. Int. Ed. Engl. 44,
4618 – 4622.
[56] Palacios, O., Atrian, S., and Capdevila, M. (2011) Zn- and Cu-thioneins: a
functional classification for metallothioneins? J. Biol. Inorg. Chem. 16, 991 –
1009.
[57] Santovito, G., Irato, P., Palermo, S., Boldrin, F., Sack, R., et al. (2001) Identifi-
cation, cloning and characterisation of a novel copper-metallothionein in tet-
rahymena pigmentosa. Sequencing of cDNA and expression. Protist 152,
219 – 229.
[58] Valls, M., Bofill, R., Romero-Isart, N., Gonzalez-Duarte, R., Abian, J., et al.
(2000) Drosophila MTN: a metazoan copper-thionein related to fungal forms.
FEBS Lett. 467, 189 – 194.
[59] Brouwer, M., Syring, R., and Hoexum Brouwer, T. (2002) Role of a copper-
specific metallothionein of the blue crab, Callinectes sapidus, in copper
metabolism associated with degradation and synthesis of hemocyanin. J.
Inorg. Biochem. 88, 228 – 239.
[60] Vergani, L. (2009) Metallothioneins in aquatic organisms: fish, crustaceans,
molluscs, and echinoderms. Met Ions Life Sci 5, 199 – 237.
[61] Stuerzenbaum, S. (2009) Earthworm and nematode metallothioneins. Met.
Ions Life Sci 5, 183 – 197.
[62] Tarasava, K., Loebus, J., and Freisinger, E. (2016) Localization and spectro-
scopic analysis of the Cu(I) binding site in wheat metallothionein Ec-1. Int.
J. Mol. Sci. 17, 371.
[63] Narula, S. S., Brouwer, M., Hua, Y., and Armitage, I. M. (1995) Three-dimen-
sional solution structure of Callinectes sapidus metallothionein-1 deter-
mined by homonuclear and heteronuclear magnetic resonance
spectroscopy. Biochemistry 34, 620 – 631.
[64] Munoz, A., Forsterling, F. H., Shaw, C. F. III, and Petering, D. H. (2002) Struc-
ture of the (113)Cd(3)beta domains from Homarus americanus metallothio-
nein-1: hydrogen bonding and solvent accessibility of sulfur atoms. J. Biol.
Inorg. Chem. 7, 713 – 724.
Copper Metallothioneins
[65] Riek, R., Precheur, B., Wang, Y., Mackay, E. A., Wider, G., et al. (1999) NMR
structure of the sea urchin (Strongylocentrotus purpuratus) metallothionein
MTA. J. Mol. Biol. 291, 417 – 428.
[66] Capasso, C., Carginale, V., Crescenzi, O., Di Maro, D., Parisi, E., et al. (2003)
Solution structure of MT_nc, a novel metallothionein from the antarctic fish
Notothenia coriiceps. Structure 11, 435 – 443.
Calvo et al.
[67] Loebus, J., Peroza, E. A., Bluthgen, N., Fox, T., Meyer-Klaucke, W., et al. (2011)
Protein and metal cluster structure of the wheat metallothionein domain gam-
ma-E(c)21: the second part of the puzzle. J. Biol. Inorg. Chem. 16, 683 – 694.
[68] Peroza, E. A., Schmucki, R., Guntert, P., Freisinger, E., and Zerbe, O. (2009)
The beta(E)-domain of wheat E(c)-1 metallothionein: a metal-binding
domain with a distinctive structure. J. Mol. Biol. 387, 207 – 218.
245