Structure, Vol. 11, 435–443, April, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0969-2126(03)00044-3
Solution Structure of MT_nc, a Novel
Metallothionein from the Antarctic Fish
Notothenia coriiceps
but the high thermodynamic and low kinetic stability
suggest that they may bind the metal moiety with a
considerable stability, providing at the same time a facile
metal exchange with other proteins. Because of the un-
usual capability to sequester heavy metals, MTs may
Clemente Capasso,
1
Vincenzo Carginale,
1
Orlando Crescenzi,
2
Daniela Di Maro,
3
Elio Parisi,
1
Roberta Spadaccini,
2
and Piero Andrea Temussi
2,4,
*
1
CNR
Institute of Protein Biochemistry also act as a defense against the harmful effects of toxic
elements, primarily cadmium and mercury [2].via Marconi 10
I 80125 Naples A great deal of knowledge on the chemical features
of MTs has arisen from the determination of three-Italy
2
Department of Chemistry dimensional structures obtained mainly by NMR spec-
troscopy [3–9], but also by X-ray crystallography [10,University of Naples “Federico II”
via Cinthia 45 11]. The results reported insofar demonstrate that the 3D
solution structures of mammalian MTs are very similar,I 80126 Naples
Italy featuring identical cluster topology and polypeptide
folding. The three-dimensional structure of the sea ur-
3
BIOMODEM pscrl
via Fiorentina chin MT has a cluster topology similar to that of evolu-
tionary distant MTs but displays some differences inI 53100 Siena
Italy both backbone folding and connectivity pattern of the
sulfur-metal bonds [8]. A comparative study of the
4
National Institute for Medical Research
Medical Research Council chemical features of vertebrate MTs may contribute to
a better understanding of the biological functions ofThe Ridgeway
Mill Hill MTs. Such a study has never been performed because,
among vertebrates, the only MT structures available areLondon NW7 1AA
United Kingdom those of mammalian origin. At first glance, the striking
similarity of MT sequences from different classes of
vertebrates suggests the existence of an almost equal
likeness at the structural level. Indeed, all vertebrate
Summary
MTs display the same arrangement of the cysteine-con-
taining motifs, with the exception of fish MTs, in which
The structure of [
113
Cd
7
]-metallothionein (MT_nc) of the
the last CXCC motif in the
domain becomes CXXXCC
Antarctic fish Notothenia coriiceps, the first three-
[12]. Such a synapomorphism may have important struc-
dimensional structure of a fish metallothionein, was
tural and, possibly, functional consequences in fish MTs.
determined by homonuclear
1
H NMR experiments and
In previous papers, we have studied piscine MTs and,
heteronuclear [
1
H,
113
Cd]-correlation spectroscopy.
in particular, compared the physicochemical character-
MT_nc is composed of an N-terminal
domain with
istics of the MT of Notothenia coriiceps, an Antarctic
9 cysteines and 3 metal ions and a carboxy-terminal
fish, with those of the mouse MT [12–15]. The two pro-
-domain with 11 cysteines and 4 metal ions. The posi-
teins differ markedly in the amino acid residues placed
tion of the ninth Cys of the  domain of MT_nc is
between the cysteines and, consequently, in the value
different from the corresponding Cys of mammalian
of the hydrophobicity index. A preliminary study carried
MTs. As a result, the last CXCC motif in the mammalian
out on a fish MT with the use of NMR spectroscopy
MT sequence becomes CXXXCC in the fish MT. This
unveiled a selective broadening of the heteronuclear
difference leads to a structural change of the domain
spectra that may reflect a higher structural flexibility [15].
and, in turn, to a different charge distribution with
Two-dimensional [
1
H,
113
Cd]-correlation experiments of
respect to that observed in mammalian metallothio-
N. coriiceps MT_nc show a difference in intensity of the
neins.
[
1
H,
113
Cd] correlations between the
and domains that
is apparently higher than in the corresponding spectra of
Introduction
mammalian MTs.
The fact that the observed broadening does not affect
Among all known proteins, metallothioneins (MTs) have
the homonuclear spectra suggests an exchange phe-
the most striking peculiarities. They are small in size
nomenon involving the metal ions that is much more
(typically 6–7 kDa), rich in cysteines (20 residues per pro-
pronounced in fish MT than in mouse MT. NMR observa-
tein molecule), and lack well-defined secondary structure
tions are paralleled by both circular dichroism and dy-
elements. Indeed, their fold is dictated mostly by a clus-
namic fluorescence spectra of fish MT that are consider-
tered network of metal-thiolate bonds among the sulfur
ably influenced by temperature, whereas the mouse MT
atoms of the cysteine residues and the metal ions, usu-
is much less affected by heating. An additional distinct
ally represented by zinc, copper, and cadmium [1]. The
feature of fish MT is the more pronounced reactivity of
role played by MTs has been debated for a long time,
Key words: Antarctic fish; electrostatic potential; ion exchange;
NMR; poikilotherm organism; zinc homeostasis*Correspondence: pat@chemistry.unina.it

Structure
436
the metal-thiolate clusters in the presence of the redox were measured by E-COSY experiments [26]. In the
structure calculation with DYANA, spin coupling valuescouple formed by reduced and oxidized glutathione [15].
In the present report, we describe the first three- were input into HABAS to obtain the possible range of
torsion angles with simultaneous consideration of thedimensional structure of a fish MT. The sequential
1
H-NMR assignments and subsequent structure deter- NOE-derived distance restraints, to yield a total of 103
angular constraints. Additional constraints were fur-mination were performed on the recombinant Noto-
thenia coriiceps Cd
7
-MT_nc by homonuclear and heter- nished by Cd
2
-sulfur bonds [3].
Since we could not detect any long-range interdomainonuclear NMR spectroscopy. The position of the ninth
Cys of the
domain of MT_nc, as of other piscine MTs, NOEs, separate structure calculations were performed
for the two domains. For the  domain, the input for theis different from the corresponding Cys of mammalian
MTs. In the new structure, this difference leads to a final structure calculation with the program DYANA [22]
consisted of 253 NOE upper-limit distance constraints,structural change of the
domain and, in turn, to a
different charge distribution with respect to that ob- 12 Cd-S bond constraints, and 63 dihedral angle con-
straints; for the
domain there were 373 NOE distanceserved in mammalian metallothioneins.
constraints, 16 Cd-S bond constraints, and 50 dihedral
angle constraints, amounting to more than 14 constraints
Results
per residue. The good rmsd of the best 20 structures
(0.44 for the
domain) together with the small size and
Structure Determination
number of residual constraint violations in the DYANA
The NMR structure determination ofN. coriiceps [Cd
7
2
]-
runs shows that the input data represent a self-consis-
MT_nc was performed at pH 7.0 and 293 K with natural
tent set and that the constraints are well satisfied in
abundance or
113
Cd-labeled recombinant protein sam-
the calculated conformers. As a further refinement, we
ples [15]. All attempts to measure additional restraints
performed a restrained energy minimization on the 40
via
15
N and/or
13
C labeling were frustrated by the negligi-
DYANA structures with the lowest target function values
ble yields of properly folded protein expressed in mini-
with the SANDER module of the AMBER 5.0 package
mal media. This situation is not new in the field of MTs,
[27]. The best 20 structures were selected to represent
since all previous structural determinations have been
the solution structure. The quality of the structure deter-
performed only with NOEs collected in homonuclear
mination is reflected by global rmsd values relative to
experiments. This approach, although at variance with
the mean coordinates for the backbone atoms of both
state of the art structure determination of larger proteins
domains (Table 1). The core of the
domain is character-
by NMR, remains acceptable for small proteins, such
ized with greatest precision; the rmsd calculated for
as the domains of MTs, whose fold is dominated by the
the backbone heavy atoms of residues 31–59 is 0.44 A
˚
clustered network of metal-thiolate bonds among the
(Table 1). Figure 1 shows the bundle of the best 20
sulfur atoms of the cysteine residues and the metal ions.
structures for the two domains separately, since we
Resonance assignments were obtained by standard
did not detect any interdomain NOEs. The total lack of
procedures. COSY [16], TOCSY [17], and NOESY [18]
correlation between the two domains, although common
experiments were used for spin system identification.
to all other solution studies, is at variance with the corre-
The assignments for the backbone amide protons and
lation found in the crystal structure of rat liver MT2 [10],
the
-carbon hydrogen atoms are complete except for
where correlation may be induced by crystallization [28].
the two initial residues (Gly1 and Ser2) of the hexapep-
However, the observation that the two domains do
tide N-terminal segment added in the expression proto-
not interact in any significant way relies on the possibility
col. Most nonexchangeable side chain protons were
of detecting a number of weak NOEs between pairs of
assigned. Analysis of the
3
J
HN

and
3
J


coupling con-
protons belonging to different domains. Such effects, if
stants together with the corresponding intraresidual and
present, may be so weak as to be virtually undetectable
sequential NOEs with the program HABAS [19] yielded
among the stronger intraresidue NOEs in crowded 2D
stereo-specific assignments for 13 CH
2
groups out of
spectra. On the other hand, it can be predicted that
the 45 nondegenerate -methylene resonances.
even minor interactions between the two domains can
The distribution of the Cd
2
ions into two clusters of
affect the chemical shifts of protons of either domain,
four and three metals was based on the thiolate-metal
since the dependence of chemical shifts even on small
connectivities directly measured by means of 2D [
1
H,
local environmental changes is highly nonlinear. It is
113
Cd]-COSY spectra [20]. The two globular domains of
common experience in NMR spectroscopy of proteins
MT_nc were termed the  domain (N terminal, 9 cys-
that large differences in chemical shifts can occur as a
teines and 3 metal ions) and
domain (C terminal, 11
consequence of minor structural differences, whereas
cysteines and 4 metal ions), according to the convention
the opposite, i.e., small differences in chemical shifts
employed for mammalian MTs (e.g., see [5]).
between substantially different structures, is virtually
Interproton distances were derived from the cross-
impossible. Accordingly, we have compared the spectra
peak integrals on 2D NOESY spectra in H
2
Oor
2
H
2
O
of the isolated domains with the corresponding spectra
solution with a 150 ms mixing period. The peak integrals
of the same domain in the whole protein (data not
were evaluated with the program NMRView [21], trans- shown). The superposition of partial NOESY spectra of
ferred to the program package DYANA 1.5 [22], and
the isolated
domain and of the whole metallothionein
converted to a total of 1170 upper distance limits with showed conclusively that corresponding resonances
CALIBA [23]. The
3
J
NH-
CH
coupling constants were mea- occupy virtually the same position, a clear proof of com-
sured by the methods of Kim and Prestegard [24] and plete lack of correlation between the
and the  do-
mains of MT_nc.Titman and Keeler [25], and the
3
J


coupling constants