Báo cáo khoa học: Solution structure of Cu6 metallothionein from the fungus Neurospora crassa pptx

Solution structure of Cu
6
metallothionein from the fungus
Neurospora crassa
Paul A. Cobine
1
, Ryan T. McKay
2,
*, Klaus Zangger
2,
†, Charles T. Dameron
3
and Ian M. Armitage
2
1
Health Science Center, University of Utah, Salt Lake City, UT, USA;
2
Department of Biochemistry, Molecular Biology and
Biophysics, University of Minnesota, Minneapolis, MN, USA;
3
Department of Chemistry and Biochemistry, Duquesne University,
Pittsburgh, PA, USA
The 3 D-solution structu re o f Neu rospora crassa Cu
6
-metal-
lothionein (NcMT) polypeptide backbone was determined
using homonuclear, multidimensional
1
H-NMR spectros-
copy. It r epresents a new metallothionein (MT) fold with a
protein chain where the N-terminal half is left-handed and

the C-terminal half right-handedly folded around a cop-
per(I)-sulfur c luster. As seen with other MTs, the protein
lacks definable s econdary structural elements; however, the
polypeptide fold is unique. The metal coordination and
the cysteine spacing defines t his unique fold. NcMT is only
the second MT in the copper-bound form to be structurally
characterized and the first containing the -CxCxxxxxCxC-
motif. This motif i s found in a variety of mammalian MTs
and metalloregulatory proteins. The in vitro formation of the
Cu
6
NcMT identical to the native Cu
6
NcMT was dependent
upon the prior formation of the Zn
3
NcMT and its titration
with Cu(I). The enhanced sensitivity and resolution of the
800 MHz
1
H-NMR spectral data p ermitted the 3 D structure
determination of the polypeptide backbone without the
substitution and utilization of the NMR active spin 1/2
metals such as
113
Cd and
109
Ag. These restraints have been
necessary to estab lish s pecific metal to cysteine restraints in
3D structural studies o n this family of proteins when using

lower field, less sensitive
1
H-NMR spectral data. The accu-
racy of the structure calculated without these constraints is,
however, supported by the similarities of the 800 MHz
structures of the a-domain of mouse MT1 compared to the
one recalculated without metal–cysteine connectivities.
Keywords: copper; metallothionein; Neurospora crassa;
NMR; solution s tructure.
Metallothioneins (MTs) are a ubiquitous class of proteins
occurring in both prokaryotes and eukaryotes [1]. MTs a re
known for their small size (< 7 kDa), the ability to
coordinate a diverse range of metals, a lack of definable
secondary structure, high cysteine content ( 30%), and
degeneracy in the remaining residues (e.g. predominance of
cysteine, serine, lysine and no aromatic residues). The high
cysteine content and their spacing give the MTs a high
affinity for m etals (e.g. K
a
of Zn–MT  1 · 10
12
M
)[2,3].
While an essential physiological role has yet to be ascribed
to MT, there is no question that MTs are involved in the
protection of cells against metal intoxication through t he
sequestration of the excess essential metal ions like copper
and zinc, as well as nonessential metal ions, like cadmium,
mercury and silver [4]. Although the essential metals have
critical structural, catalytic and regulatory r oles in proteins,

their cellular concentration must be carefully maintained
through the use of pumps and s equestering peptides a nd
proteins to avoid toxic effects. Despite the high affinity
for metals, MTs are also postulated to participate in an
undetermined mechanism o f facile metal e xchange (i.e.
kinetically labile yet thermodynamically stable) with other
proteins possessing substantially lower metal affinities [5–8].
Since the MTs have little or no repetitive secondary
structure, their tertiary structure is dependent on the
number and type of metal ions they coordinate.
The MT from the fungus Neurospora crassa,isasingle
domain MT m ade u p o f 2 5 r esidues, seven of which are
cysteine residues [9]. This small peptide coordinates six
Cu(I) a toms via the seven c ysteinyl sulfurs [ 10]. N. crassa
metallothionein (NcMT) binds copper in a solvent-shiel-
ded environment, which p roduces a luminescent core [11].
In vivo , t he N cMT is induced only b y c opper; other
transition metals (zinc, cadmium, cobalt, and nickel) do
not induce transcription of NcMT mRNA although the
protein will bind these metal ions in vitro [12,13]. The
in vitro metal ion binding characteristics of N cMT mimic
that of the mammalian b-domain, i.e. NcMT b inds
zinc(II), c admium(II) and cobalt(II) with a 3 : 1 metal to
Correspondence to I. M. Armitage, Department of Biochemistry,
Molecular Biology and Biophysics, University of Minnesota, 6-155
Jackson Hall, 321 Church Street S.E., Minneapolis, MN 55455, USA.
Fax: +1 612 625 2163, Tel.: +1 612 624 5977,
E-mail:
Abbreviations: MT, metallothionein; NcMT, Neurospora crassa met-
allothionein; aMT-1, a-domain of mouse MT-1; s

c
, correlation time.
Note: The PDB file has been assigned the Brookhaven Protein Data
Bank Accession no. 1T2Y and the chemical shifts have been deposited
in the BMRB data bank under accession number 62 90.
*Present address: 101 NANUC, University of Alberta, Ed monton,
AB Canada T6G 2E1.
Present address: Institute of Chemistry/Organic and Bioorganic
Chemistry, University of Gra z, Heinrichstrasse 28, A-8010 Graz,
Austria.
(Received 3 0 June 2004, revised 2 September 2004,
accepted 7 September 2004)
Eur. J. Biochem. 271, 4213–4221 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04361.x
protein stoichiometry and Cu(I) with a 6 : 1 stoichiometry
[12]. The NcMT cysteine arrangement is identical to the
first seven amino-terminal cysteines of the b-domain of
human MT (Fig. 1), thou gh the human MT has t wo
additional cysteines in this domain. Similar cysteine
spacing is seen in the metal binding motifs of the
metalloregulatory proteins from Enterococcus hirae
(CopY), Saccharomyces cerevisiae (Ace1 and Mac1) and
Candida albicans (AMT1) though these proteins do not
share a ny other significant homology, Fig. 1. Along with
the metal coordination number, the positioning of the
cysteines in t he primary s tructure has been determined to
be the critical f actor in determining the global fold of t he
MTs [14]. The structures of several MT isoforms have
been determined including that from yeast [15,16], crab
[17], sea urchin [14], an antarctic fish [18] and various
mammals [19–23]. With the exception of the yeast Cu

7
MT
[16], the previou s NMR solution structures o f MTs,
including yeast MT [15], all relied on the substitution of
NMR active s pin 1/2 metals such as
113
Cd and
109
Ag for
the determination of specific metal–cysteine restraints,
which were included in the subsequent structure calcula-
tions [24]. While the mammalian copper(I) MTs have
evaded 3D structural elucidation by NMR and X-ray
crystallography [1], recent CD spectroscopic studies on the
Cu(I) and Ag(I) substituted b-fragmentofmouseMT1
have revealed structural differences that may reflect a
different conformational f old for the Ag(I) substituted
b-do main compared to the native Cu(I) containing
fragment [25]. The only copper(I) MT structure deter-
mined has been that of the yeast MT which has a
distinctly different cysteine arrangement, Fig. 1 [15,16].
However, disagreement exists in the comparison of this
NMR derived structure of the yeast MT without the 13
C-terminal residues and the small angle X-ray scattering
pattern of the full length protein which has been attributed
to the core of t he NMR structure being too compact
[15,16,26]. The similarity in the metal binding properties
and cysteine spacing in NcMT with mammalian MT
makes it a candidate for the modelling of the Cu(I)–sulfur
cluster o f the b-domainofmammalianCu

6
MT and
potentially useful for other copper(I)-regulated proteins
that contain t he - CxCxxxxCxC- and - CxCxxxxxCxC-
motifs, F ig. 1.
The
1
H NMR resonance assignments f or the C u
6
NcMT
purified from N. crassa have been reported [ 27] and these
were used as a template to compare and confirm a similar
overall fold for the in vitro reco nstituted, synthesized protein
used in this study. W e report here the 3D NMR solution
structure o f the Cu
6
NcMT, obtained without establishing
any metal–cysteine restraints, and discuss the formation
of the Z n
3
NcMT precursor m olecule t hat was needed to
obtain the Cu
6
NcMT structure.
Materials and methods
Proteins and peptides
The NcMT w as synthesized chemically to avoid its prob-
lematic induction and purification. As noted previously by
another group [28], N. crassa can use several or a mixture of
copper resistance mechanisms to detoxify excess copper. In

initial attempts, we found that exposure to copper did not
consistently result in the induction of MT. Therefore, a
synthetic p eptide corresponding to the N cMT w as s ynthes-
ized using B oc-Pam resin and Boc -chemistry protocols [29].
The peptide was deprotected and cleaved from the resin
using anhydrous liquid hydrogen fluoride, para-cre sol and
para-thiocresol (269–271 K for 70 min). Purification inclu-
ded a diethyl ether wash under a nitrogen atmosphere and
preparative trifluoroacetic acid (TFA)/acetonitrile [buffer
A: 0.1% (v/v) TFA/water; buffer B 90% acetonitrile/10%
water and 0.1% TFA, v/v/v) linear gradient H PLC (0–50%
buffer B over 40 min) on a reverse phase HPLC column
(Waters Delta-Pak PrepPak C18; 40 mm · 100 mm). Elu-
tion was monitored at 214 nm, and the eluted p eptide was
freeze-dried for storage. Subsequent analysis showed that
the p eptide was oxidized by the cleavage/purification
treatment, ther efore t he peptide was reduced by suspension
in 6
M
guanidine/HCl, 100 m
M
Tris pH 8.5, and 150 m
M
dithiothreitol with incubation at 42 °C for 2 h. The reduced
protein s ample w as then loaded onto a G25 (Pharmacia)
size exclusion column (200 mm · 15 mm) previously equili-
brated with 0.2% (v/v) TFA. The protein fraction was
collected, pooled and sealed anaerobically. P rotein concen-
tration was determined by amino acid analysis and the
reduction state of the sulfhydryls confirmed by dithiodi-

pyridine assay [30]. All subsequent manipulations of the
apo- a nd metallated N cMT were p erformed under anaer-
obic conditions, 5% H
2
and 95% N
2
(v/v), in a glove box
(Atomspure Protector Glove Box, Labconco, Kansas City,
MO, USA).
Metal titrations
Copper(I) titrations were performed as d escribed by Byrd
et al. [31]. Protein samples with reduction state > 95%
were used for all titrations. Sequential additions of
copper(I), as [Cu(CH
3
CN)
4
]ClO
4
in 200 m
M
ammonium
acetatepH7.9,weremadetotheapo-NcMT. Identical
titrations were performed with Zn(II)
3
NcMT instead of the
apo-NcMT. The Zn
3
NcMT was prepared from ap o-NcMT
by adding 3.5 molar equivalents of Zn(II) (added as Z n(II)-

acetate) in a 10-fold excess of free cysteine to stabilize the
Fig. 1. Amino acid sequence alignment. Sequences shown are tho se of
N. crassa MT (residues 2–26, NCBI accession No. CAA26793), the
b-domain o f Homo sapiens MT-2 A (residues 1–30, NCBI accession
No. P 02795), the b-domain o f Mus m usculus MT1 ( residues 1–3 0,
NCBI Accession No. AAH36990), AMT1 from Candida glabrata
(residues 70–113, NCBI accession No. P41772), ACE1 from Sac-
charomyces cerevisiae (residues 70–110, NCBI accession No.
NP_011349), CopY from Enterococcus hirae (residues 123–145, NCBI
accession No. Q47839), MAC1 from S. ce revisiae (residues 150–187,
NCBI accession No. NP_013734) and MT from S. cerevisiae (Cup1–1)
(residues 9–49, NCBI accession No. AAS56843). The cysteine residues
are highlighted in bold and the consensus sequence repeated.
4214 P. A. Cobine et al. ( Eur. J. Biochem. 271) Ó FEBS 2004
excess z inc. Prior to the copper(I) titration o f t he NcMT
sample, it was treated with 50 lL of a 1 : 1 slurry of
Chelex-100 (Bio-Rad) in degassed MilliQ water to r emove
any excess unbound/displaced metals. The Chelex-100 was
removed by centrifugation. The Cu(I)-thiolate emission
was monitored at 580 nm (excitation at 295 nm) with a
Perkin-Elmer LS 50B luminescence spec trometer. The
spectra were collected at 23 °C in sealed screw-topped
fluorescence cuvettes (Spectrocell). The spectrometer was
equipped w ith a 350 nm band-pass filter to avoid second
order effects and used settings of 5 and 20 nm, respectively,
for the excitation and emission slits. Cu(I)–NcMT for
NMR analysis was desalted on PD-10 ( Pharmacia, G-25)
columns equilibrated with MilliQ water, pooled and
lyophilized. Copper stoichiometry was quantified by flame
atomic absorption spectroscopy and amino acid analys is.

The final Cu
6
NcMT co ntained 1 .8 lmol N cMT and
10.9 lmol Cu(I).
NMR sample preparation
Lyophilized Cu
6
NcMT (handled under an argon atmo-
sphere) was dissolved in 500 lL 90% H
2
O, 10% D
2
O,
pH 6.5, 0.1 m
M
2,2-dimethyl-2-silapentane-5-sulfonate (as
an internal reference) [32], and 0.02% NaN
3
that was
degassed prior to protein addition under high vacuum and
re-pressurized to 1 atm under argon to avoid oxidation/
disproportionation of the C u(I)–protein. T he sample was
loaded into a 5 mm NMR tube, c apped a nd sealed with
parafilm.
NMR spectroscopy and assignments of NcMT
A2D
1
H,
1
H-TOCSY [33,34] and 2D

1
H,
1
H-NOESY were
collected at 10 °C on a Varian Inova 800 MHz NMR
spectrometer equipped with a triple-axis gradient, triple-
resonance p robe. T he sweep widths f or both experiments
were 9000 Hz with 1024 and 512 complex points collected
in the directly and indirectly detected dimensions, respect-
ively. The TOCSY was collected with 64 transients per
increment (40 ms mixing time with 300 ms delay between
acquisitions), and the NOESY was collected with 32
transients per increment (300 ms mixing period with
200 ms delay between acquisitions). Spectra were zero-
filled to twice the number of collected points a nd apodized
using a p/3 shifted sine bell before Fourier transformation.
New
1
H-chemical shift assignments at 10 °Cfrom2D-
NOESY and TOCSY s pectra were performed as described
previously [35]. All spectra were processed u sing
NMRPIPE
[36], and analysed using t he program
PIPP
[37]. Coupling
constants for backbone amide to a hydrogen atoms (i.e.
3
J H
N
H

a
) were obtained from 1D-proton and 2D-COSY
spectra using d econvolution. Due to t he different temper-
atures used during the NMR experiments (10 °Cinthe
present s tudy and 25 °C i n [ 27]) t here are expected t o b e
small differences in chemical shifts, typically larger for NH
protons. However, the overall very good agreement in
these shifts can be confidently attributed to the protein
forming the same structure. Gradient NOESY spectra
were acquired w ith WaterGate solvent s uppression taken
from the ÔgnoesywgÕ pulse sequence in ÔProtein PackÕ as
supplied b y Varian I nc.
Structure calculations for NcMT
Distance restraints (50 kcalÆmol
)1
ÆA
˚
)2
) for NcMT structure
calculations were classified as short (1.8–2.7 A
˚
), medium
(1.8–3.3 A
˚
), and long (1.8–5.0 A
˚
) based on their N OE
intensities. The upper bound of an NOE restraint was
extended by 0.5 A
˚

for each p seudoatom methylene, o r
methyl group. Residues e xhibiting
3
JH
N
H
a
coupling
constants of < 5 Hz, 5–8 Hz, 8–9 Hz, a nd > 9 Hz were
assigned dihedral angle restraints of )60° ± 30, )105° ±
55, ) 120° ± 40, and )120° ± 30, respectively, with a
force constant of 500 kcalÆmol
)1
Ærad
)1
.Nometalatoms
were included in the structure calculation. Structures were
calculated using
X
-
PLOR
3.851 [38] on an Octane Power
Desktop (SGI R12000 with
IRIX
6.5) using the hybrid
distance geometry-dynamical simulated annealing protocol
[39] as described for mouse MT1 [23]. Out of the 30
calculated structures, 12 were selected based on the complete
absence of NOE violations greater than 0.5 A
˚

and r.m.s.d.
for bond and angle deviations from ideality of less than
0.01 A
˚
and 5°, respec tively.
Structure calculations for mouse aMT
All structure calculations involving the a-domain of the
mouse MT, aMT-1, were performed as previously reported
[23] with the exception that the metal–cysteine restraints
were not used and therefore all metals w ere absent in the
calculations. To determine the precision [how well the
individual structures calculated with a limited set of l ong-
range N OEs (d
ij
, j > i + 4) compare to each other ] and
accuracy (the simila rity of the calculated structures with a
reduced set o f long-range NOEs to the structure calculated
with all NOEs) of the calculated structures, all 22 long-range
NOEs were first removed and then, by a random selection
process reintroduced one by one for the structure calcula-
tions. T hereby, for each number of long-range NOEs, 1 0
random selections of reintroduced long-range NOE sets
were made and 10 structures calculated for each set, giving a
total of 100 structures calculated for each point in Fig. 4.
Results
Metal binding stoichiometry of NcMT
N. crassa can express MT in response to excessive concen-
trations of copper in the growth media. NcMT purified
from the mycelium of t he fungus contains 6 Cu(I) ions per
mole of protein [9]. The copper(I) ions are bound to the

cysteinyl sulfurs in a solvent-shielded Cu–S core [40]. Direct
titration of copper(I) into a synthesized apo-NcMT resulted
in a 4-Cu(I)NcMT. This complex was luminescent, excita-
tion at 295 nm led to an emission maximum a t 5 80 nm,
which is evidence for solvent protected Cu(I)-thiolates but
the luminescence increased up to a plateau at 4 mole
equivalents of Cu(I) per protein (Fig. 2A,B). The 4 : 1
stoichiometry conflicts with the known native species.
Titrations with a variety of copper(I) sources, different
types a nd concentrations of reductant, and with or without
thermal treatments as used b y Stillman and coworkers [41]
to select stable forms of the rabbit CuMT all resulted in the
formation of Cu
4
NcMT. T he 1 D
1
H NMR spectrum of the
Ó FEBS 2004 NMR structure of N. crassa Cu6MT (Eur. J. Biochem. 271) 4215
Cu
4
NcMT, prepared under any of these conditions, was
very broad and in contrast to the well defined native
Cu
6
NcMT
1
H NMR spectrum [27]. The unresolved
1
H
NMR spectrum would be co nsistent with the sample

containing a mixture of inter converting NcMT structures
and/or structural instabilities. In an attempt to restrict the
family of conformers formed during the copper(I) titrations,
we first titrated zinc into the apo-protein to form a
Zn
3
NcMT precursor. The excess zinc was removed by
treatment of the sample with Chelex-100. The zinc stoi-
chiometry of the resultant protein, 3-Zn(II):1 NcMT, was as
expected from previous studies of NcMT and by analogy to
the well s tudied b-domains of the mammalian MTs [12].
Sequential titration of the Zn
3
NcMT with Cu(I) salts as
before yielded a luminescent core but in this case, the
stoichiometry w as 6 Cu(I):1 NcMT, Fig. 2C,D. Most
importantly, t he 1D
1
H-NMR p attern for the Cu
6
NcMT
is equivalent to the native Cu(I) proton spectrum [27].
Attempts to prepare the Ag(I) derivative of N cMT d id not
result in a stoichiometry of 6 metals p er mole of protein.
Attempts to displace all of the z inc from the Zn
3
NcMT by
silver were unsuccessful and always resulted in the forma-
tion of a mixed metal s pecies inappropriate for s tructural
studies. The structure determination of Zn

3
NcMT was not
pursued as to our knowledge there are n o reports of this
metal form from natural sources.
Metal-cysteine restraints
To explore the feasibility of determining an MT structure
without metal–cysteine restraints, t he structure o f the
a-do main of mouse MT-1 without the experimentally
determined
113
Cd–Cys restraints was re-calculated. Fig. 3
shows the backbone fold of the average energy minimized
structures for the a-domain of mouse MT-1 with (dark
strand) and without (light strand) the inclusion of experi-
mentally determined
113
Cd–Cys restraints. T he super-
imposed backbone structures are r emarkably similar with
an r.m.s.d. of 1.94 A
˚
over all backbone atoms. This value is
very similar to t he r.m.s.d. comparison between the
a-d omain of mouse MT-1 and the a-domain of rat MT-2,
2.14 A
˚
[23], suggesting that accurate and precise MT
structure d eterminations are possible without using metal
to cysteine restraints providing one has a sufficient number
of NOE constraints. The lack of regular secondary structure
in MTs places an increased importance upon the long-range

contacts for precision in structure calculations. This can be
quantitatively evaluated by recalculating the mouse MT1
a-d omain structure in the absence of all long-range NOE
restraints and
113
Cd–Cys connectivities and then syste-
matically and randomly reintroducing long-range NMR
Fig. 3. Ribbon backbone diagrams of the a-domain of mouse MT-1
calculated with (dark) and without (grey)
113
Cd–Cys restraints. The Cd
atoms (rendered to van der Waals radii), cysteine sulfur, and Cd-S
bonds are indicated by the large spheres, sma ll sphere s and black lines,
respectively. The N and C termini are labelled for orientation. Dia-
grams were generat ed using t he program
INSIGHTII
(Molecular Simu-
lations, Inc.).
Fig. 2. In vitro copper reconstitutions o f
N. crassa MT monitoring Cu(I)-S luminescence
at 580 nm after excitation at 295 nm. (A) The
reconstitutions at 10 nmolÆmL
)1
of apo-
NcMT with copper(I) the increasing additions
of copper the luminescence makes uniform
steps (B). (C) Titratio ns of 50 nm olÆmL
)1
Zn
3

NcMT with copper(I) t o a stoichiometry
of 6 Cu(I) per mole of prote in. The sequential
increase plotted in (D) r eveals a plateau at six
equivalents and that t he quantum yield of
relative luminescence per mole of protein is
equal for the 4 and 6 Cu(I)-N cMT .
4216 P. A. Cobine et al. ( Eur. J. Biochem. 271) Ó FEBS 2004
restraints to determine the effect on the precision and
accuracy of the generated structure. Figure 4A shows the
r.m.s.d. of 10 sets of s tructures, generated as described in
Materials and methods, to the average minimized structure,
for each set of reintroduced long-range NOEs. Determining
the deviation of the generated structure with a reduced
number of NOEs from the published MT1 structure
provides a measure of accuracy shown in Fig. 4B. As can
be seen, the structure of MT1 without metal–cysteine
restraints is sufficiently well defined as long as 10–15 long-
range NOEs are used for the structure calculation and the
backbone r.m.s.d. is kept below 2 A
˚
.
NcMT structure
Two-dimensional
1
H,
1
H-TOCSY and NOESY NMR
spectroscopy was used to assign the
1
H resonances and

provide interproton distance restraints, r espectively, on
NcMT. The chemical shifts of NcMT determined here at
10 °C a re similar t o t hose of a previous study perfo rmed at
25 °C [27]. A lmost a ll the
1
H chemical shifts were v isible in
the T OCSY with the exception of the side c hain of Cys5
(assigned from the N OESY), and a ll resonances of Gly1
(not identified i n either experiment). Assignment of Gly1
was not possible; this was most likely due to rapid exchange
of the amide proton of Gly1 with the s olvent, coupled with
chemical shift overlap reported previously [27]. The chem-
ical shifts have been deposited in the BMRB data bank
under accession number 6290. Examination of the chemical
shifts showed no indication of secondary structure [42,43].
Line widths of 1D-
1
H NMR resonances have been used as
an efficient method for determining the aggregation state of
a protein [44,45]. The average line width of NcMT amide
resonances from the 1D-
1
H spectrum was determined to be
5.6 Hz. When six Cu(I) atoms are considered bound to
NcMT, the correlation time (s
c
) and line width for the
monomer are expected to be 2.6–3.7 n s and  4.5–6 Hz,
respectively [34]. The observed line widths for the reconsti-
tuted Cu

6
NcMT are consistent with the sample being
monomeric. A summary of the observed NOEs is presented
in Fig. 5A. Inspection of the NOE patterns shows no
evidence of regular s econdary st ructure elements, which is in
agreement with the information from the chemical shifts
Fig. 4. Effect of the inclusion of long-range NMR restraints f or aMT-
1 structure determination in the absence of metal–Cys restraints.
Long-range NMR restraints were removed and t hen systematically
and randomly re-introduced for mouse aMT-1 structure calcula-
tions. (A) Precision indicated via the r .m.s.d. o f the family of 10
structures generated (compared to the minimized average) for each
number of long-range NOEs inclu ded in calculations. (B) Relative
accuracy of the calculations indicated by comparing the aMT-1
minimized average structure for each number of long-range NOEs
included (in the absence of metal–Cys restraints), to the average
minimized stru cture of aMT-1 calculated with all NOEs and inclu-
ding the m etal–Cys restraints. Error b ars in both panels indicate the
standard deviation.
d
αN
(i,i+3)
d
NN
(i,i+3)
d
NN
(i,i+2)
d
NN

(i,i+1)
G
20
10 20
10
Residue Number
Number of NOEs
20
15
10
5
0
DCGC SGASS CNCGSGCSCSNCGS K
d
βN
(i,i+1)
d
αN
(i,i+1)
d
αN
(i,i+2)
A
B
Fig. 5. NOE map for NcMT. (A) Summary of inter-residue N OEs
determined for NcMT that are typically used to ind icate secondary
structure. Strong, medium a nd weak intensity NOE cross-peaks are
indicated by tall dark, medium grey, and small white boxes, respect-
ively. Th e primary sequence is shown at the b ottom in the o ne letter
code. (B) T he total number of NO Es assigned for NcMT displayed on

a per residue basis. Intra-residue, sequential (d
ij
,j ¼ i+1),medium
(d
ij
,i+1<j<i+4),long(d
ij
, j > i + 4) range NOEs are des-
cribed by solid, dotted crosshatched, whit e, and thick crosshatched
columns, respectively.
Ó FEBS 2004 NMR structure of N. crassa Cu6MT (Eur. J. Biochem. 271) 4217
and is typical for this family of proteins. The total number
of intraresidue, s equential, medium, and long-range NOEs
for each residue in the NcMT sequence is shown in Fig. 5B.
The majority of critical long-range restraints involve the side
chains of Ala8, Asn12, and Cys21. Despite the almost
complete assignment of
1
H resonances, the number of long-
range contacts was low. Utilizing the 152 NOEs and 13
dihedral angle restraints, a total of 30 structures was
generated using the program
XPLOR
3.851 [38]. From these
generated structures, 12 resulted in no NOE or dihedral
angle violations and all showed the same backbone fold.
Subsequently, the 10 accepted structures (no NOE violation
>0.5 A
˚
, a n r.m.s.d. for bond deviations from ideality of

less than 0.01 A
˚
and an r.m.s.d. for angle deviations from
ideality of less than 5 °) with the lowest energy were selected
which yielded a backbone r.m.s.d. to the average minimized
structure of 0.79 A
˚
for the well-defined region (residues
5–20) and 1.59 A
˚
for the entire length of the protein. The
final family of 10 NcMT structures is shown in Fig. 6 with
the N - and C-termini labelled and the c ysteine sulfur atoms
in the Ôclosest to meanÕ structure drawn as spheres. The
structure of NcMT has been deposited in the Protein Data
Bank under accession number 1T2Y. The structural statis-
tics for NcMT are presented in Table 1. The program
PROCHECK
-
NMR
[46] showed that > 90% of the ø, w angles
for the 10 structures fell into the core or a llowed regions.
Despite the complete absence of elements of r egular
secondary structure, which is a quite common situation
for MTs, the backbone global fold of the 25-residue peptide
is well defined if one excludes the N and C termini. It shows
a new polypeptide structure with t he backbone being
wrapped around an empty space containing the copper–
sulfur cluster, on going from the N to C terminus, in a left
handed form for the first half of the molecule then in a right

handed f orm for the second half. Such a mixed h andedness
of the peptide part around the metal–cysteine cluster has so
far been found only in the C-terminal domain of blue c rab
MT [17], whose overall fold is quite different from that of
NcMT. While NcMT shares considerable sequence similar-
ities and a conservative positioning of the seven cysteine
residues with o ther members of t he MT family of proteins,
none of the oth er structurally characterized MTs show the
same fold. This is reflected in the r.m.s.d. differences in
cysteine residue positions (backbone heavy a toms and sulfur
atoms) between NcMT and lobster [47], yeast [15,16], mouse
[23], blue crab [ 17], fish [18] and se a urchin [14] M Ts as
shown in Table 2. Such r.m.s.d. differences in cysteine
positions are c haracteristic of unrelated MT structures. A
good example to illustrate this point is the r.m.s.d.
differences between the a-andb-domains of sea urchin
MT of 4.98 A
˚
for the cysteine backbone heavy atoms and
2.92 A
˚
for the sulfur atoms. For c omparison the cys teine
Fig. 6. Stereo drawing of a line representation
of the b ackbone of 10 N cMT structures which
possessed the lowest overall energy, out of 12
generated t hat contained n o NOE or dihedral
angle violations. Theensembleisleast-squares
fitted to the first structure and the N and C
termini are labelled f or orientation. The cys-
teine sulfur atoms of th e Ôclo sest to meanÕ

structure are shown as yellow spheres.
Table 1. Structural statistics for NcMT, for the 10 lowest overall energy
structures out of 12 without a ny NOE or d ihedral violations.
NOE restraints
Total 152
Intraresidue 63
Sequential (|i-j| ¼ 1) 53
Medium (2 6 |i-j| 6 4) 25
Long range (|i-j| P 5) 11
Dihedral restraints (ø) 13
r.m.s.d. to average structure (A
˚
)
Well-defined regions (N,C
a
,C)
a
0.79
All regions (N,C
a
,C) 1.59
All heavy atoms 1.97
Energies (kcalÆmol
)1
)
E
overall
82.40 ± 7.95
E
bonds

6.07 ± 0.74
E
angles
29.67 ± 2.35
E
vdw
7.92 ± 1.53
E
NOE
29.13 ± 2.62
E
dihedral
1.75 ± 0.58
E
improper
7.86 ± 0.46
r.m.s.d. from idealized covalent geometry used within
X
-
PLOR
Bonds (A
˚
) 0.0047 ± 0.0003
Angles (deg) 0.64 ± 0.03
Impropers (deg) 0.63 ± 0.02
NOEs (A
˚
) 0.062 ± 0.003
Dihedral (deg) 1.46 ± 0.3
Procheck-NMR

b
[46]
In most favoured regions 44.4% (80)
In additional allowed 44.4% (80)
In generously allowed 5.6% (10)
In disallowed regions 5.6% (10)
a
Residues 5–20.
b
Number of residues out of all 10 structures.
Total non-glycine and non-proline is 180. Number of glycines is 60,
with 10 end-residues, for a total of 250 residues.
4218 P. A. Cobine et al. ( Eur. J. Biochem. 271) Ó FEBS 2004
residue position r.m.s.d. between the structurally related
b-domain of mouse MT1 and human MT2 a re 0.57 and
2.10 A
˚
for the backbone heavy atoms and the sulfur atoms,
respectively.
Discussion
The solution structure of the Cu
6
NcMT, which was solved
without the acquisition and inclusion of specific metal–
cysteine NMR r estraints, shows a novel polypeptide fold
and represents only the second copper MT s tructure to be
elucidated [15,16]. Although other MTs show a strong
sequence similarity to NcMT (e.g. 32% with the b-domain
of human MT2) the 3D structure of the polypep tide
backbone i s completely different. The unique fold of this

CuMT is a clear demonstration that MT protein folds are
largely d etermined by t he constraints of metal–sulfur
connections and not the amino acid sequence. N. crassa
MT has a -CxCxxxxxCxC- motif that is found in a variety
of Cu(I) binding proteins (Fig. 1). The lack of repetitive
secondary structures in the NcMT peptide backbone along
with the unsuccessful efforts to prepare the isomorphic and
homogeneous NMR active spin 1/2 Ag(I) derivative o f the
native Cu(I) NcMT were factors which had inhibited its
structure elucidation. A hallmark of the MT structures is the
dependence on the sequential position of the cysteine in the
primary sequence, the identity of the coordinated metal and
its c oordination number [14,48]. The paper by Bertini et al.
[16] was the first to show that the acquisition of NMR data
at 800 MHz allows for the determ ination o f MT structures
without metal–cysteine constraints. The comparative study
on mouse MT1 at 800 MHz in this paper helps to validate
these studies. The successful in vitro reconstitution of
Zn
3
NcMT with six equivalents of Cu(I) indicated that the
metal binding sto ichiometry w as the s ame as the b-domain
of mammalian M T [12] even though NcMT lacks the two
C-terminal cysteines found in the mammalian MTs. These
two missing cysteines and therefore less metal coordination
mightbethereasonwhyNcMTneedstobeintheZn
3
-form
before it successfully binds six a toms of copper(I). Thus,
Zn

3
–NcMT might constitute a scaffold where the zinc can
be replaced by Cu(I) without the need for large structural
rearrangements. In other words the less favourable enthalpy
changes by binding 6 Cu (I) atoms to only seven cysteines in
NcMT are compensated by the smaller differences in
entropy when copper substitutes zinc i n Zn
3
–NcMT, rather
than binding to apo-NcMT.
The variable coordination number of c opper, which can
adopt a linear two-coordinate (digonal) or a distorted
planar trigonal three-coordinate geometry with sulfur
ligands [49] adds an addition al level of complexity in solving
the copper MT structures. These coordination possibilities
of the copp er(I) ions are perhaps responsible for the titration
of the apo-NcMT to a non-native (4 : 1) stoichiometry. In
thecaseoftheNcMT,itseemsthatthecoordinationofzinc
in Zn
3
NcMT was necessary to conform/stabilize a structure
such that the Cu(I) titration produced the native Cu
6
Cys
7
metal–thiolate cluster rather than the Cu
4
Cys
7
made from

apo-NcMT which gave rise to a f amily of rapidly i ntercon-
verting s tructures. The resultant Cu
6
Cys
7
core appears to
constrain the peptide sufficiently to enable its 3D solution
structure to be determined. The requirement that the
synthetic Cu(I)MT be made from a Zn-protein in vitro to
obtain an NMR spectrum identical t o t hat of t he native
Cu(I)MT raises questions about how the organism controls
the formation of the Cu(I)NcMT in vivo. If the Zn(II)NcMT
were to form in vivo when NcMT was induced by copper
exposure one might expect that it would be a very short
Table 2. C ysteine r.m.s.d. comparison ( in A
˚
)tootherMTs.
PDB number Cys-N,Ca,C¢ Cys-Sc
1J5M
a
4.88 5.47
1FMY
b
3.53 4.10
1DFT
c
4.95 4.60
1DMC
d
3.71 4.37

1DME
e
5.22 5.31
1M0G
f
3.49 4.45
1M0J
g
3.90 4.98
1QJK
h
4.47 5.29
1QJL
i
3.85 4.77
a
b-domain of lobster Homarus americanus MT.
b
Cu(I)-yeast
Saccharomyces cerevisiae MT.
c
b-domain of mouse Mus musculus
MT1.
d
a-domain of blue crab Callinectes sapidus MT.
e
b-domain
of blue crab Callinectes sapidus MT.
f
a-domain of the fish Noto-

thenia coriiceps MT.
g
b-domain of the fish Notothenia coriiceps
MT.
h
a-domain of sea urchin Strongylocentrotus purpuratus MT.
i
b-domain of sea urchin Strongylocentrotus purpuratus MT.
Fig. 7. Ribbon diagram of the NMR solution structure o f NcMT with
six Cu(I ) atoms modelled into possible positions within th e structure and
the cysteine side chains turned to point towards the protein’s centre. Cu(I)
atoms are rendered according to their van d er Waals radii. The Cys
side-chains are rendered as grey sticks with th e Cys sulfu r atom shown
in light grey. The modelling o f Cu(I) binding as depicted was acc om-
plished by a ttempting to best satisfy k nown b ond lengths and altering
the Cys chi side- chain a ngle to po int the sulfur atoms towards the
centre. The figure was prepared using
INSIGHTII
(Molecular Simula-
tions, Inc.). The backbone fold o f the NcMT protein is shown in black
with the carbons and su lfur of the cysteine side chains shown in dark
grey and light grey, respectively. The Cu(I) atoms are rendered a s
medium grey spheres with a van der Waals radiu s of 0.95 A
˚
.Taking
into consideration t he Cu(I)–S l uminesc ence which indicate a pro-
tected copper core, we have manually rotated the Cys s ide-chain 1
cys
and 2
cys

angles to direct the sul fur atoms into the core o f the protein.
Ó FEBS 2004 NMR structure of N. crassa Cu6MT (Eur. J. Biochem. 271) 4219
lived and hard to i solate species a s copper i ons would b e
expected to readily displace the Zn(II). However, there are
no in vivo data t o indicate that the o rgan ism has to form a
zinc precursor. The requirement of Zn(II) in the f ormation
of Cu(I)MT clusters, in particular the b-domain, has been
observed for mouse MT [50]. Additionally, the same spacing
of cys teine residues i s also f ound in the repressor protein
CopY from Enterococcus hirae and it is apparent that the
metal-binding properties of this protein may require
the binding of Zn(II) in this site [51,52]. The stability of
the Cu(I)-S core and the DNA-binding activity are
dependent o n zinc binding. In addition, the copper chap-
erone CopZ is a specific source of copper for the Zn-form
of CopY [51]. However, apo-CopY does not demonstrate
this specificity suggesting a potential loss of structure that
confers this property. A combination of the data suggests
that Zn(II)-binding to these cysteine-rich, copper-binding
sites orders a structure that is required for activity.
A model structure of Cu
6
NcMT demonstrating the
shielding of the copper core, using one model with copper
atoms satisfying the Cu–S bond distances and minimizing
any interactions within the NMR structure, is shown in
Fig. 7. While there is probably a multitude of models that
would fit the NMR data, there is no way to co nfirm any
from NMR s tudies using t he NMR inactive Cu(I) metal
form of the protein. The NcMT backbone structure

presented here uses no specific metal–Cys restraints and a
low number of long-range NOE constraints. However, the
mouse a-MT-1 s tructures calculated with and without
specific metal–Cys restraints support the assertion that
metal–Cys restraints are not required for precise and
accurate structure determination of the protein backbone
with NMR data collected at 800 MHz. The a-domain of
MT-1 was a lso u sed t o a ssess the requirements for long-
range NMR restraints in structure c alculatio ns. Although
both the length of the a-domain of mouse MT1 (31 vs. 25
amino acids in NcMT) and the number of NOEs (256 vs.
152 for NcMT) is larger for mouse MT1, t he back-
bone r.m.s.d. between structures calculated without
metal–cysteine restraints is comparable for NcMT (1.94 A
˚
for mouse MT1 and 1.59 A
˚
for NcMT).
Evaluation of the relationship between the number of
long-range NOEs and the resultin g r.m.s.d. is relevant i n
MTs because of their lack of defined s econdary structure. In
more typical proteins, the translational and rotational
positions of the residues contained in a secondary structural
element (a-helix, b-sheet) might be spatially defined by as
little as a single long-range contact to the rest of the molecule.
However, in MT, which is composed mostly of coils and
loops, the spatial orientation of relatively large regions
cannot be positioned b y such a single critical restraint.
In conclusion we have described a new MT fold for the
N. crassa Cu

6
MT which consists of a half left- and half
right-handedly polypeptide backbone wrapped around the
copper(I)-cysteine cluster. No direct information about the
metal–sulfur connectivities could be obtained b y using an
isomorphic, NMR active metal substitute for Cu
+
,suchas
Ag
+
, because a stable homogenous Ag substituted NcMT
could not be prepared. Nevertheless, the use of data at
800 MHz on th e reconstituted Cu
6
NcMT was sufficient to
allow for an accurate backbone fold to be determined.
Acknowledgements
This work was supported by NIH grant DK18778 to I.M.A. NMR
instrumentation was provided with funds from the NS F (BIR-961477)
and t he University of Minnesota Medic al School. K.Z. thanks the
Austrian Science Foundation FWF for financial support under project
number P15289. We would like to thank Dr David Live and Dr Be verly
G. Ostrowski for maintenance of the spectrometer facility and
computers, Matt Vetting for help with software in modeling of the
Cu(I)-NcMT an d expertise in use of the A rgon Chamber for NMR
sample preparation, and Gu
¨
lin O
¨
z for critical reading of the

manuscript. We also gratefully acknowledge the University of Minne-
sota Supe rcomputing Institute for Digital Simulation and Advanced
Computation for use of their facilities for processing/analysis of NMR
data and subsequent structure calculations.
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