PAPER
www.rsc.org/dalton | Dalton Transactions
An NMR study on nickel binding sites in Cap43 protein fragments
Maria Antonietta Zoroddu,*a Massimiliano Peana,a Serenella Medicia and Roberto Aneddab
Received 23rd February 2009, Accepted 5th May 2009
First published as an Advance Article on the web 3rd June 2009
DOI: 10.1039/b903305j
NMR spectroscopy was used to study the interaction of Ni(II) ions with C-terminal sequence of Cap43
protein where, from Thr341 to Gly360 residue, a T1 R2 S3 R4 S5 H6 T7 S8 E9 G10 ten-amino acid fragment is
consecutively repeated three times. The behaviour of ends-blocked Ac-RSRSHTSEG-Am (pept1),
Ac-TRSRSHTSEG-Am (pept2), and the three repeats Ac-TRSRSHTSEG-TRSRSHTSEGTRSRSHTSEG-Am (pept3) peptides towards Ni(II) ions was examined at different pH values and, for
pept3, at different ligand-to-metal molar ratios. 1 H-1 H TOCSY, 1 H-13 C HSQC, 1 H-1 H NOESY and
1
H-1 H ROESY multidimensional NMR techniques were performed to understand the details of metal
binding sites and the conformational behaviour of the peptides. The results confirmed that each
mono-histidinic sequence of pept3 is able to independently coordinate one, two or three Ni(II) ions for
1:1, 1:2 and 1:3 ligand-to-metal molar ratios, respectively. At higher pH values, the coordination of
Ni(II) involves imidazole Nd of His6 and three preceding deprotonated peptide nitrogens from the
backbone, giving a {Nd, 3N- } chromophore in a square planar geometry. In addition, at lower pH
values, the involvement of g-O of carboxyl group from Glu9 residue with the formation of a
macrochelate giving a {Nd, g-O- , 4OH2 O } chromophore in an octahedral geometry, was evidenced.
NMR results allowed us to build a model for the structure of the major complex. Structural changes in
the conformation of the peptide with organized Arg4 and Thr7 side chain orientation promoted by
nickel coordination, were detected.
Introduction
One of the research interests of our group has been centred during
the past years on a stress responsive protein, named Cap43, which
seems to be correlated to a number of events occurring inside the
cell, mainly linked to cancerous states (it is in fact overexpressed
in a variety of cancer tissues) and hypoxia response.1–4
Besides, Cap43 gene is induced by a rise in free intracellular Ca2+
following nickel exposure, indicating that the protein is expressed
with marked specificity to Ni(II) ions. This fact points to Cap43
protein as an attractive target for a research carried out to cast
a light on the role of this protein in the events connected with
nickel toxicity and carcinogenesis, through the investigation of the
relations and interactions between its structure and the nickel ions.
The 10-amino acid (TRSRSHTSEG) mono-histidinic sequence
repeated consecutively three times looked very promising for an
efficient interaction with metal ions. In fact, the observation that in
prion proteins mono-histidinic octapeptide repeats play a critical
role in metal metabolism, using sets of histidines as the binding
sites for metal ions,5 suggested an analogous behaviour for our
mono-histidinic decapeptide fragment.
Pursuing our interest in peptides nickel coordination,6,7 we
previously reported on Ni(II) binding to the 20-(TRSRSHTSEGTRSRSHTSEG) and 30-(TRSRSHTSEG-TRSRSHTSEGTRSRSHTSEG) amino acid sequences of Cap43 protein by a
combined pH-metric and spectroscopic study.8,9
a
Department of Chemistry, University of Sassari, Italy. E-mail: zoroddu@
uniss.it
b
Porto Conte Ricerche, Porto Conte, Tramariglio, Alghero, Italy
This journal is © The Royal Society of Chemistry 2009
Here we discuss about a series of 1D and 2D TOCSY, 1 H-13 C
HSQC, NOESY and ROESY NMR experiments carried out to
get a better insight into the Ni(II) coordination environment and
to explore the effect of metal complexation on the conformational
behaviour of the 30-amino acid C-terminal sequence of Cap43 protein. In addition, we studied the 10-Ac-T1 R2 S3 R4 S5 H6 T7 S8 E9 G10 Am amino acid and the shorter 9-Ac-R2 S3 R4 S5 H6 T7 S8 E9 G10 -Am
amino acid fragment, in which the T1 residue was removed in order
to solve any ambiguity in the chemical shift assignments.
Experimental
Peptide synthesis
Peptides were chemically synthesized using solid phase Fmoc
(fluoren-9-ylmethoxycarbonyl) chemistry in an Applied Biosystems Synthesizer.10 Peptides were N-terminally acetylated and
C-terminally amidated in order to mimic this region of Cap43
within the full-length protein. The peptides were removed from
the resin and deprotected before purification by reverse-phase
HPLC. Fractions were collected and analyzed by MALDI-TOF
MS. Fractions containing the peptide of the expected molecular
weight were then pooled and lyophilized.
NMR spectroscopy
NMR experiments were performed on Bruker Avance 600 and
700 MHz spectrometers equipped with inverse quadruple (QXI)
and triple (TXI) resonance probes, respectively. Samples used for
NMR experiments were 5 mM in concentration and dissolved in
90/10 (v/v) H2 O/D2 O solutions. All acquisitions were performed
Dalton Trans., 2009, 5523–5534 | 5523
at 298 K using 5 mm NMR tubes. A series of 1D spectra of the
free peptide was recorded at various pH values ranging between
2.6 and 10.0 with steps of 1.0 pH units. The titration experiments
of Ni(II)-containing samples, peptide-to-metal molar ratios of 1:1
for pept1 and pept2, 1:1, 1:2 and 1:3 for pept3, were performed in
the pH range 5–10. The pH of the sample was adjusted to reach
the final pH value by addition of 1 N NaOH or 1 N HCl.
2D 1 H/13 C heteronuclear correlation spectra were acquired using an HSQC sequence implemented in the Bruker library. Briefly,
a phase-sensitive sequence using Echo-Antiecho-TPPI gradient
selection with a heteronuclear coupling constant J XH = 145 Hz and
shaped pulses for all 180◦ pulses on f2 channel with decoupling
during acquisition, sensitivity improvement and gradients in backinept were used.11–13 Relaxation delays of 2 s, 90◦ pulses of about
9.5 ms (pept3) were used for all experiments. ROESY 2D spectra14
were acquired with spin-lock pulses duration in the range 200–
250 ms (optimized for each peptide). 32–64 scans and 512–
1024 increments were acquired. 1 H-1 H TOCSY with excitation
sculpting with gradients or with WATERGATE to suppress the
resonance from water protons were performed, using mixing times
of 60 ms. The combination of TOCSY, HSQC and ROESY
experiments was used to assign the spectra of both free and Ni(II)bound peptides at different pHs. Solvent suppression for 1D and
ROESY experiments was achieved using WATERGATE pulse
sequence15 or using excitation sculpting with gradients,16 the best
results being observed with excitation sculpting, especially when
solutions containing paramagnetic metal were analyzed. All NMR
data were processed using XWINNMR (Bruker Instruments)
software and analyzed using the Sparky 3.11 and MestRe Nova
programs.
Model calculations
Due to the identical coordination mode of Ni(II) ions to the single
independent 10-amino acid fragment TRSRSHTSEG of the 30amino acid peptide (TRSRSHTSEG)3 , structure calculations for
the 4 N {NIm , 3N- } peptide-metal complex were performed for
the single mono-histidinic fragment (T1 R2 S3 R4 S5 H6 T7 S8 E9 G10 ) on
the basis of the ROE cross-correlations observed in the 2D 1 H-1 H
ROESY spectra at pH = 9.
The high pH value needed for the formation of the major metal–
peptide complex does not allow the detection of the labile HN
amidic protons in the aromatic region.
Meaningful ROEs involving a through-space correlation of the
side-chain aliphatic protons of Arg4 and Thr7 with the aromatic or
aliphatic b-protons of His6 were used as input data for structure
calculations. Furthermore, the model proposed has been restricted
to the residues directly involved in the complex formation for
which significant ROEs were detected (S3 R4 S5 H6 T7 ). This most
likely would provide useful information on the conformation of
the binding site, neglecting structural features not well supported
by experimental results. The intensities of the 2D cross-peaks of
1
H-1 H ROESY spectra of the peptide-Ni(II) system were transformed into the upper limit distances using the following calibration method. Upper bounds u on the distance between two
correlated hydrogen atoms were derived from the corresponding
ROESY cross peak volumes V according to calibration curves V =
k/u6 , where k was determined by using the cross-peak intensity
of histidine imidazole aromatic protons Hd2 -He1 as the reference
5524 | Dalton Trans., 2009, 5523–5534
(u = 4.25 Å).17 The interresidual constraints allowing for structural
analysis of Ni(II)–S3 R4 S5 H6 T7 complex were: R4 /Hb1 - H6 /He1
u = 3.5 Å, R4 /Hb2 - H6 /He1 u = 3.8 Å, R4 /Qg- H6 /He1 u = 5.1 Å,
R4 /Qd- H6 /He1 u = 4.5 Å, T7 /Qg- H6 /Hd2 u = 4.8 Å and T7 /
Qg- H6 /Hb2 u = 5.1 Å.
Low-spin Ni(II) binding to R4 S5 H6 sequence was restrained
to the geometry of analogous square-planar Ni(II) complexes
known from the literature,18,19 and introduced by a linker made
of pseudoatoms starting from the last residue G10 of the 10-amino
acid. The metal ion was also forced to be positioned within 1.8
and 2.0 Å from the four nitrogen donors (Nd1 , NH 6 , NH 5 , NH 4 , respectively). Backbone dihedral angles j and y of R4 S5 H6 sequence
were fixed in a square-planar geometry by using the torsion angle
values derived from the X-ray structure of Ni(II)(Glycyl-GlycylAlpha-Hydroxy-D,L-Histamine)·3H2 O complex.19 A number of
200 structures were generated for the model of peptide–nickel
complex by imposing the experimental constraints with the
simulated annealing program DYANA20 running on a Silicon
Graphics workstation. The minimized average structure of the
20 best converged structures calculated with lowest overall energy
was subjected to the molecular mechanics geometry optimization,
using an AMBER force field implemented in HyperChemTM
7.01 molecular modeling software.21 The displayed model of the
complex was analyzed with the MOLMOL 2.5.1 program22 and
generated with the HyperChemTM software.23
Results and discussion
Our previous studies included the investigation on nickel binding
to 30-TRSRSHTSEG-TRSRSHTSEG-TRSRSHTSEG amino
acid sequence of C-terminal of Cap43.8,9 The combined pHmetric and spectroscopic (UV-Vis, CD, NMR) study revealed
that each 10-amino acid fragment was able to independently
coordinate a single metal ion. Metal coordination started at
pH 5 from imidazole nitrogen of histidine residue and, with
increasing the pH, Ni(II) ion deprotonated peptidic nitrogen
atoms, forming Ni(II)–N- bonds, until a square-planar 4 N {NIm ,
3N- } species was obtained. It involves imidazolic nitrogens of each
histidine and deprotonated amidic nitrogens from histidine and
preceding serine and arginine residues, respectively. Above pH 9,
a further deprotonation was identified; it was attributed to the
deprotonation of a pyrrolic nitrogen of histidine or of an arginine
side-chain, suggesting a possible involvement of the residue in an
apical interaction with the metal.9
Starting from the information previous collected, we decided
to get a deeper insight into the coordination environment of
Ni(II) ions by exploiting solution state NMR multidimensional
techniques.
NMR Studies of free peptides
All the peptides were studied in the pH range from 2.6 to 10 using
1D and 2D 1 H NMR homonuclear TOCSY, NOESY, ROESY
and 1 H-13 C HSQC NMR experiments. Pept1 and pept2 showed
almost equal proton and carbon chemical shift values for all the
residues except for those of Arg2 , due to their different chemical
environment in the two peptides. The resonances of the 30-amino
acid sequence (pept3) reflect those found for the shorter 10-amino
This journal is © The Royal Society of Chemistry 2009
Table 1
1
H proton resonance assignment for Ac-TRSRSHTSEG-Am pept2, at pH of 2.6, 5, 7, 9
pH 2.6
Proton
Residue
HN
Ha
T1
R2
S3
R4
S5
H6
T7
S8
E9
G10
8.149
8.396
8.447
8.483
8.281
8.573
8.167
8.358
8.360
8.258
4.190
4.354
4.363
4.317
4.333
4.724
4.317
4.409
4.354
pH 5
Proton
Residue
HN
Ha
T1
R2
S3
R4
S5
H6
T7
S8
E9
G10
8.149
8.397
8.293
8.464
8.277
8.527
8.157
8.328
8.414
8.351
4.192
4.346
4.361
4.310
4.338
4.698
4.321
4.395
4.257
Qa
Hb
Hb1
Hb2
1.773
1.723
Qb
Qg
4.103
Hd1
Hd2
Qd
He
1.131
1.584
3.129
7.155
1.570
3.129
7.144
He1
He2
8.534
6.981
He1
He2
8.454
6.991
He1
He2
7.699
6.996
He1
He2
3.782
1.808
1.687
3.748
3.253
3.130
4.162
7.386
7.236
Hd1
Hd2
1.112
3.809
2.084
1.915
Hb1
Hb2
1.800
1.695
2.415
3.893
Qa
Hb
Qb
Qg
4.095
Qd
He
1.125
1.569
3.129
7.151
1.565
3.129
7.140
3.782
1.808
1.689
3.754
3.244
3.126
4.192
7.368
7.211
Hd1
Hd2
1.116
3.818
2.017
1.875
Hb1
Hb2
1.804
1.695
2.216
3.819
pH 7
Proton
Residue
HN
Ha
T1
R2
S3
R4
S5
H6
T7
S8
E9
G10
8.146
—
8.301
—
8.259
—
—
8.328
8.412
8.343
4.195
4.339
4.370
4.321
4.364
4.601
4.308
4.394
4.236
pH 9
Proton
Residue
HN
Ha
T1
R2
S3
R4
S5
H6
T7
S8
E9
G10
—
—
—
—
—
—
—
—
—
—
4.201
4.332
4.374
4.320
4.370
4.589
4.314
4.405
4.238
Qa
Hb
Qb
Qg
4.091
Qd
1.132
1.562
3.123
1.560
3.123
He
3.787
1.806
1.692
3.755
3.085
3.026
4.166
7.358
6.934
Hd1
Hd2
1.059
3.815
2.011
1.870
Hb1
Hb2
1.803
1.692
2.197
3.821
Qa
Hb
Qb
Qg
4.091
Qd
1.135
1.567
3.119
1.558
3.123
He
3.777
1.803
1.690
3.747
3.046
3.030
6.892
4.159
7.607
1.055
3.803
2.025
1.860
2.196
3.824
acid sequence (pept2), showing an almost coincident overlap in
the chemical shifts of all the protons.
For that reason NMR details only for the 10-amino
acid peptide, the minimal motif, have been reported in the
tables.
This fact points out the independent relation between each of
the three mono-histidinic fragments and the non-existence of any
conformational preference for the whole peptide.
This journal is © The Royal Society of Chemistry 2009
In Table 1 and Table 2, 1 H and 13 C assignments at the selected
pH of 2.6, 5, 7 and 9 for pept2, are reported.
By raising the pH from 5, the most affected protons are those of
His6 and Glu9 residue, as a result of the deprotonation of He1 on
the imidazole ring and of Qg on the carboxyl group, respectively.
His6 He1 is the most affected proton on imidazole ring with
a Dd = d(pH 9) - d(pH 2.6) = -0.927 ppm. At pH = 9,
in the aromatic region, only histidine He1 and Hd2 signals
Dalton Trans., 2009, 5523–5534 | 5525
Table 2
13
C carbon resonance assignment for Ac-TRSRSHTSEG-Am pept2 at selected pH of 2.6, 5, 7, 9
pH 2.6
Carbon
Residue
Ca
Cb
Cg
Cd
T1
R2
S3
R4
S5
H6
T7
S8
E9
G10
59.69
53.68
55.65
53.46
55.69
—
59.18
55.75
53.06
41.50
67.06
28.15
61.19
28.13
61.17
26.29
67.23
61.10
26.17
18.87
24.36
40.66
24.36
40.66
pH 7
Carbon
Residue
Ca
Cb
Cg
Cd
T1
R2
S3
R4
S5
H6
T7
S8
E9
G10
59.60
53.46
55.68
53.48
55.68
54.11
59.14
55.88
54.23
42.33
67.13
28.12
61.12
28.12
61.14
28.39
67.18
61.08
27.49
18.90
24.35
40.66
24.35
40.66
Cd2
117.47
Ce1
133.77
18.83
30.00
Cd2
117.20
Ce1
136.00
18.69
33.63
(d = 7.607 ppm and 6.892 ppm, respectively) were present. All the
amidic protons resonances were lost indicating a fast exchange
with water molecules; in the aliphatic region, the histidinic
Ha proton appeared under the water signal; its assignment at
4.600 ppm was based on the TOCSY spectrum and the correlation
between Ha and Hb1 /Hb2 (d = 3.046 and 3.030 ppm, respectively).
The charge redistribution on imidazole ring strongly affects also
Cb with a large downfield shift of Dd 2.35 ppm. Imidazole Ce1
resonance shifted downfield, Dd = d(pH 9) - d(pH 2.6) = 2.50,
while Cd2 exhibits an upfield shift, Dd = -0.30 ppm, respectively.
By raising the pH, a general downfield shift with maximum Dd
at pH 6, for 13 C, Cg > Cb > Ca (Dd = 3.64, 1.69 and 1.18 ppm,
respectively) and an upfield shift for 1 H, more pronounced for Qg
protons (Ddg = -0.219) of Glu9 residue was evidenced and could
be in agreement with the deprotonation of the carboxylic group.
Analysis of Ha, HN , Ca and Cb chemical shifts via Chemical
Shift Index (CSI) and bioinformatic tools (secondary structure
prediction) supported the presence of a random coil structure
for all the peptides investigated. Two-dimensional NOESY and
ROESY spectra did not show any inter-residual interaction. The
observation of only sequential daN or dbN ROE connectivities
together with intraresidue dipolar contacts, are not indicative of
an organized secondary structure in all the range of pH and are
consistent with an unstructured and flexible peptide backbone.
NMR Studies of Ni(II)–peptide complexes
NMR spectra of 1:1 Ni(II)-to-ligand molar ratio for pept1 and
pept2 and of 1:1, 1:2 and 1:3 solutions for pept3, were studied over
the pH range 5–10. The assignments were made by combination
of COSY and TOCSY experiments.
The relevant data collected for pept2 are listed in Table 3.
5526 | Dalton Trans., 2009, 5523–5534
pH 5
Carbon
Residue
Ca
Cb
Cg
Cd
T1
R2
S3
R4
S5
H6
T7
S8
E9
G10
59.64
53.62
55.70
53.53
55.65
—
59.27
55.87
54.09
42.32
67.09
28.08
61.12
28.08
61.10
26.42
67.20
61.08
27.38
18.90
24.37
40.66
24.37
40.66
pH 9
Carbon
Residue
Ca
Cb
Cg
Cd
T1
R2
S3
R4
S5
H6
T7
S8
E9
G10
59.59
53.40
55.70
53.36
55.54
54.27
58.92
55.87
54.24
42.32
67.13
28.15
61.10
28.15
61.10
28.64
67.18
61.08
27.86
18.90
24.32
40.65
24.34
40.63
Cd2
Ce1
117.50
133.90
Cd2
Ce1
117.17
136.27
18.87
33.29
18.67
33.64
Our previous potentiometric study on Ni(II) binding with
mono-histidinic
Ac-T1 R2 S3 R4 S5 H6 T7 S8 E9 G10 T11 R12 S13 R14 -Am
14-amino acid fragment8 evidenced, from pH 5 to pH 10, four
nickel species NiL, NiH-2 L, NiH-3 L and NiH-4 L, respectively.
NiL was identified as 1 N species, starting from pH 5 to 8,
maximum formation (~30% of the total metal in solution) at
pH 7.5. Above pH 8 consecutive deprotonation and coordination
of N amides take place.
From pH 6 to 7.5, NMR spectra of pept2:Ni(II) 1:1 showed 1 H
and 13 C changes in chemical shifts of His6 aromatic and aliphatic
signals together with a differential broadening of resonances. As
shown in Fig. 1A, aromatic He and Ce are the most affected
signals. The almost total disappearance of Ce and He indicates
coordination by the adjacent Nd of the imidazole ring (Hd less
affected is the opposite side of the ring to Nd). A vanishing of His6
Hb1 and Hb2 is visible, suggesting an approach of these protons to
the metal.
At the same time, a dramatic loss in intensity of Glu9 signals with
an almost total disappearance of g protons and a clear broadening
of b and a signals, was evidenced, but again g, b and a-Glu9
protons appear and sharpen when the pH raises above 7.
No other residues in the sequence showed a comparable metalinfluence, though a slight general line broadening, due to the
presence of paramagnetic species in solution, was evidenced.
Fig. 1B shows a comparison of selected 2D 1 H-13 C HSQC and
1D 1 H NMR aliphatic region of pept2 and Ni(II)–pept2 species
at 1:1 molar ratio at pH 6.
The spectroscopic evidence support the involvement of Nd of
His6 and g-O of Glu9 carboxyl group in the coordination to the
metal. Nd and g-O binding requires that both Thr7 and Ser8
residues approach the nickel atom. The observed vanishing of
Qg-Thr7 protons with the appearance of a new broad downfield
This journal is © The Royal Society of Chemistry 2009
Table 3 Chemical shifts of 1 H and 13 C of Ac-TRSRSHTSEG-Am pept2 and Ni(II)–pept2, in a peptide-to-metal ratio of 1:1
Residue
Proton Free pept2 (ppm)
Pept2:Ni(II) 1:1 (ppm) Dd (ppm)
T1
T1
T1
R2
R2
R2
R2
R2
S3
S3
R4
R4
R4
R4
R4
S5
S5
H6
H6
H6
H6
H6
T7
T7
T7
S8
S8
E9
E9
E9
E9
G10
Ha
Hb
Qg
Ha
Hb1
Hb2
Qg
Qd
Ha
Qb
Ha
Hb1
Hb2
Qg
Qd
Ha
Qb
Ha
Hb1
Hb2
Hd2
He1
Ha
Hb
Qg
Ha
Qb
Ha
Hb1
Hb2
Qg
Qa
4.199
4.103
1.118
4.152
1.706
1.609
1.538
3.105
4.059
3.6097
4.014
2.214
2.043
1.901
3.278
4.053
3.600
3.503
2.875
2.865
6.876
7.428
4.351
4.322
1.183
4.403
3.812
4.229
2.015
1.878
2.192
3.820
4.201
4.091
1.135
4.332
1.803
1.692
1.567
3.119
4.374
3.777
4.320
1.803
1.690
1.558
3.123
4.370
3.747
4.589
3.046
3.030
6.892
7.607
4.314
4.159
1.055
4.405
3.803
4.238
2.025
1.860
2.196
3.824
-0.002
0.012
-0.017
-0.180
-0.097
-0.083
-0.029
-0.014
-0.315
-0.167
-0.306
0.411
0.353
0.343
0.155
-0.317
-0.147
-1.086
-0.171
-0.165
-0.016
-0.179
0.037
0.163
0.128
-0.002
0.009
-0.009
-0.010
0.018
-0.004
-0.004
peak at 1.200 ppm (Dd = +0.14), together with a broadening
of b and a vanishing of a protons, suggest that Thr7 side-chain
can approach one of the axial position around the coordinated
metal. The vicinity of a paramagnetic centre enhanced relaxation
so that the signals from Qg-Thr7 protons did not yield detectable
cross peaks in COSY and TOCSY experiments. Nevertheless, they
could be easily identified by comparison with the NMR spectra of
the shorter 9-amino acid peptide and with the results obtained
for the diamagnetic species at high pH. The large effect on
Qg-Thr7 protons can suggest a change on hydrophobic packing
of the Thr7 side chain, rather than a variation in electronic density
due to the electronic charge on deprotonated and coordinated
ligand.
A slight broadening of Ha of Ser8 can be seen though, because of
its proximity to the water signal, is not amenable to interpretation.
The structure of the complex emerging from the NMR results
is presented in Fig. 2.
NiL species appears to be a distorted octahedral macrochelate
complex involving Nd imidazolic nitrogen of His6 and g-O
carboxyl oxygen of Glu9 in Ni(II) coordination; the sphere of
coordination around the nickel ion can be completed by water
molecules.
At low pH, amide deprotonation, even in the presence of
nickel ions, is unfavourable and the carboxylate coordination
is favoured. The results obtained support the preference, at
physiological pH, for the formation of a macrochelate species
besides the monodentate binding of the imidazolic function. This
is in agreement with the log b value previously reported for the
This journal is © The Royal Society of Chemistry 2009
Residue
Carbon Free pept2 (ppm)
Pept2:Ni(II) 1:1 (ppm) Dd (ppm)
T1
T1
T1
R2
R2
Ca
Cb
Cg
Ca
Cb
59.59
67.13
18.90
53.40
28.15
59.43
67.12
19.02
53.50
28.08
-0.16
-0.01
0.12
0.10
-0.06
R2
R2
S3
S3
R4
R4
Cg
Cd
Ca
Cb
Ca
Cb
24.32
40.65
55.70
61.12
53.36
28.15
24.38
40.65
62.21
62.36
64.44
24.61
0.06
0.00
6.51
1.24
11.07
-3.55
R4
R4
S5
S5
H6
H6
Cg
Cd
Ca
Cb
Ca
Cb
24.34
40.63
55.54
61.10
54.27
28.64
31.68
41.41
62.21
62.21
55.53
30.00
7.34
0.77
6.67
1.11
1.26
1.36
H6
H6
T7
T7
T7
S8
S8
E9
E9
Cd2
Ce1
Ca
Cb
Cg
Ca
Cb
Ca
Cb
117.17
136.27
58.92
67.18
18.67
55.87
61.08
54.24
27.86
113.94
137.29
58.64
67.09
18.99
55.92
61.23
54.26
27.45
-3.23
1.02
-0.28
-0.09
0.32
0.06
0.15
0.02
-0.41
E9
G10
Cg
Ca
33.64
42.32
33.68
42.35
0.04
0.04
14-amino acid sequence and the three repeats sequence which are
higher than those usually found in similar ligands with the only
monodentate binding of the imidazolic function and quite similar
to other macrochelates involving carboxylic coordination.7,24–26
By raising the pH up to 7, the signals of Glu9 residue increased
gradually in intensity and finally they resulted, at pH 9, almost
unaffected by Ni(II) coordination (Fig. 6A, see later). It is thus
clear that, by raising the pH, the macrochelate character of the
complex with the metal coordinated to Nd of His6 and g-O
carboxyl group of Glu9 is lost and replaced by a diamagnetic
4 N square-planar tetracoordinated complex. In fact, from pH 8
to 10, the involvement in coordination of deprotonated amidic
nitrogens N- from the backbone is clearly seen from the changes
in chemical shifts of Ha of His6 , Ser5 and Arg4 which are, together
with the aromatic and aliphatic Hb His6 , among the most affected
signals.
Ni(II) ions, at pH 9, caused the loss in intensity of a number of
resonances with an appearance of a new set of peaks. Generally, it
is known that Ni(II) binding to an imidazole ring located on the
equatorial plane leads to an upfield shift of the imidazole protons
signals.27
Fig. 3 shows the changes in 1 H 1D NMR spectra upon nickel
addition at pH 9 for pept 2.
The two sets of aromatic protons on the histidine His6 showed an
upfield shift with Dd = -0.179 ppm for He1 and Dd = -0.016 ppm
for Hd2 , respectively; the most affected protons are the same as
at physiological pH, confirming the monodentate coordination
at Nd of His6 . Dd = 1.02 ppm for Ce1 is in the range observed
Dalton Trans., 2009, 5523–5534 | 5527
Fig. 2 Scheme of Ni(II)–pept2 in a NO {NIm, COO- } coordination
mode.
Fig. 1 Comparison of selected 2D 1 H-13 C HSQC and 1D 1 H NMR spectra for pept2 free (red) and Ni(II)-pept2 at 1:1 molar ratio ligand-to-metal
(green) at pH 6, aromatic (A) and aliphatic region (B).
for imidazol bound-nickel in histidyl peptide complexes with slow
exchange.28
The formation of five membered chelate rings by consecutive
deprotonation of the amidic function is the driving force in the
coordination process. In the basic environment the main chain
“wraps” in the opposite direction, in comparison to the complex
obtained at the physiological pH, towards the metal.
Besides for imidazole residue, upfield chemical shifts are observed also for Ha protons of His6 , Ser5 and Arg4 characteristic of
the adjacent main chain amides being involved directly in chelating
Ni(II) ions. Shifts to high field for the Ha of residues involved
5528 | Dalton Trans., 2009, 5523–5534
in main chain coordination are: Ha-His6 Dd = -1.086 ppm,
Ha-Ser5 = -0.317, Ha-Arg4 = -0.306, respectively.
Ha of His6 residue is extremely shielded after deprotonation
and coordination of its neighbouring amidic nitrogen. Usually,
residues involved in direct amide coordination all exhibit shifts to
high field typically between 0.3 and 0.9 ppm, upon Ni binding.29
In Fig. 4 a plot of the observed chemical shift changes for 1 H
and 13 C of pept2 after metal coordination is reported.
The change in coordination and the formation of fused rings
around the metal ion by raising the pH, is confirmed by the
strong upfield shift of His Hb1 and Hb2 signals Dd = -0. 171
and -0.165 ppm. The expected two sets of doublets of doublets
are almost coalesced, though it is clear that all J-coupling values
are <7 Hz typical for ab1 , ab2 3 J-coupling within the normal range
in complexes with fused chelate rings.30,31
The residues directly involved in the coordination sphere,
R4 S5 H6 , show also large variations in carbon chemical shift
(-3.55 ppm ≥ Dd ≤ 11.07 ppm) as reported in Fig. 4.
All the NMR data confirm the basic 4 N coordination, including
His6 Nd nitrogen, His6 amide and the two main chain amides
preceding the histidine imidazol, Ser5 and Arg4 , producing a
diamagnetic low-spin square planar complex.
Ser3 residue, though not directly involved in the sphere of
coordination, showed a large shift in its Ha, Hb proton and carbon
signals, probably due to the influence of the close proximity to the
folding of the backbone around the metal centre.
It is interesting to note that, also at pH 9, Thr7 side chain is
still close to the coordination centre. This is confirmed by the size
of g-protons shifts and perturbations of its proton and carbon
resonances which are clearly visible in both 1D and 2D COSY
and TOCSY spectra. Thr7 side chain can approach one of the
axial position as in the species obtained at physiological pH and
modelled in Fig. 2.
Arg2 and Arg4 side chain showed overlap of their resonances
in the free peptide resulting from an equivalent chemical environment. On the contrary, Ni(II) coordination to the peptide at
pH 9 caused a strong differentiation of its signals, as shown in
the TOCSY and ROESY spectra. While all the Arg2 protons set
underwent a poor upfield shift, Arg4 protons were much more
affected, Dd = 0.411 ppm for Hb1 , 0.353 ppm for Hb2, 0.343 ppm
for Qg, and 0.155 ppm for Qd respectively, with a Dd decreasing
according to the b > g > d order. The entity and the relative
ordering of the shifts point out to suggest a structural involvement
of Arg4 side chain in the 4 N complex. Similar shifts and ordering
This journal is © The Royal Society of Chemistry 2009
Fig. 3
Comparison of 1 H 1D NMR spectra of pept2 (A) and (B) Ni(II)–pept2 at 1:1 molar ratio metal-to-peptide at pH 9.
Fig. 4 Plot of the observed chemical shift changes for the 1 H proton and 13 C carbon atoms of pept2 after metal coordination at pH 9.0.
This journal is © The Royal Society of Chemistry 2009
Dalton Trans., 2009, 5523–5534 | 5529
Fig. 5 Overlaid of aliphatic region of 1 H-1 H NMR TOCSY spectra for the free Ac-TRSRSHTSEG-TRSRSHTSEG-TRSRSHTSEG-Am peptide (red)
and Ni(II) bound peptide (green) at 1:3 peptide-to-nickel molar ratio. New cross-peaks are visible in the Ni(II) bound peptide spectra compared to the
metal-free status. New resonances due to Ni-binding have been labelled.
were found for Lys4 residue in human serum albumin bound to
Ni(II) in a 4 N environment. The position of Lys4 side chain over
the coordination plane was crucial for its identification by nickelspecific antibody in nickel allergy.32
We have already reported of an arginine residue showing a
downfield shift of all its protons but the Ha in a similar complex
of a histidine-containing peptide.26 In that case it was possible to
determine by NMR spectra and structural calculations that the
deshielding effect experienced by the b, g and d protons of the
arginine residue was due to the influence of both the metal and the
histidine ring current.
In addition, an increase in the difference in chemical shift
between the two Arg4 Hb protons, Ddb1b2 = 0.113 ppm for the
free peptide and Ddb1b2 = 0.171 ppm for the Ni-complex, can be
explained through a lower mobility of Arg4 side chain after nickel
coordination. On the contrary, Dd for Arg2 Hb protons remains
almost unaffected after complexation (Dd ~ 0.1 ppm in both
cases), suggesting an absence of limitation on Arg2 conformational
freedom.
While all the carbons are shifted downfield after nickel coordination at pH 9, imidazolic Cd2 of His6 and aliphatic Cb of Arg4
are shifted upfield, Dd = -3.23 ppm and -3.55 ppm, respectively.
The strong shift observed for Cd2 , which is in an opposite position
to the site of coordination, could be explained with its approach
to Arg4 Cb in a shielding area.
A similar behaviour, although less pronounced, was reported
for His Cd2 and Asp Cb in L-aspartyl-L-alanyl-L-histidine-Nmethyl amide (DAH) sequence of human albumin bound to
nickel (DdCd2 = -2.45 ppm and DdCb = -1.73 ppm, respectively).
5530 | Dalton Trans., 2009, 5523–5534
NMR data suggested a restricted mobility about the Asp Ca
Cb bond, consistent with the involvement of a COO- group in
coordination.33 In our case the observed shielding effect could be
consistent with an Arg4 side chain restricted mobility caused by its
structural involvement in an axial position over the plane of the
coordinated metal. Though, the stronger effect in our case could
take into account also a possible deprotonation on the Arg4 side
chain.
The NMR study on shorter peptides (pept1 and pept2) was
useful for a better understanding of the coordination behaviour
of Ni(II) ions towards the longer 30-amino acid fragment (pept3)
and in particular to study the possible involvement of multiple
histidinic sites in coordination events.
From 1:1 to 1:3 ligand-to-metal molar ratio, the chemical shift
changes in each of the three mono-histidinic fragments of pept3
reflected, identically in terms of type and intensity, the values
found for the pept2–Ni(II) system in a 1:1 ligand-to-metal molar
ratio. Fig. 5 shows an overlaid of aliphatic region of 1 H-1 H NMR
TOCSY spectra for the free pept3 and Ni(II) bound pept3 at 1:3
peptide-to-nickel molar ratio at pH 9.
It is clear that the longer chain of pept3, representing the three
decapeptide repeats, displays a unique mode of Ni(II) binding.
Pept3 is able to coordinate up to three Ni2+ ions in its three
equivalent sites, each involving Nd of His6+n and deprotonated
amides of His6+n itself and of the two adjacent Ser5+n and Arg4+n
residues (where n = 0, 10 or 20) at pH 9 (Fig. 5 and Fig. 6).
From the NMR analysis each of the three repeats appears to
behave in the same way towards the metal ion and the metal
binding sites appear to be isolated from each other.
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Fig. 6 Comparison of (A) aliphatic and (B) aromatic region of 1 H NMR spectra of the pept3–Ni(II) system in 1:0, 1:1, 1:2 and 1:3 peptide-to-metal
molar ratio. New resonances due to Ni-binding have been labelled.
The main chain must be highly flexible before ion coordination,
in order to be able to fold around the metal and facilitate the
coordination of one to three Ni(II) ions.
Separate resonances indicative of strong binding were observed
for free ligand and nickel complex, due to slow exchange on
the NMR timescale. From the chemical shift differences between
bound and free ligand resonances, an estimate of the lifetime of
the diamagnetic complex was derived. 13 ms can be assumed as
the shortest lifetime of the Ni–pept3 complex. This was evaluated
by considering that Hd2 of His shows a clearly resolved doublet
This journal is © The Royal Society of Chemistry 2009
signal at about 6.9 ppm. Dd = 11.92 Hz is the separation between
the two signals; (Dd)t m > 1/2p, i.e. t >> 1/2pDd, where t m is the
lifetime of the complex and Dd is the separation between the two
NMR lines.34
The large variation in chemical shifts and the appearance of
new ROEs indicated that the conformation of pept3 dramatically
changed upon metal coordination.
Fig. 7 shows a selected aromatic region of 1 H-1 H NMR ROESY
spectra for Ni(II) bound pept3 at 1:1 ligand-to-metal molar
ratio.
Dalton Trans., 2009, 5523–5534 | 5531
Fig. 7 Selected aromatic region of 1 H-1 H NMR ROESY spectra for Ni(II) bound pept3 at 1:1 ligand-to-metal molar ratio. The spatial correlations
between the aliphatic Hb1 , Hb2 , Qg and Qd proton of Arg4 and Qg of Thr7 with the aromatic He1 proton of His6 are visible only in the bound state of
peptide with Ni(II).
From the spectra it is possible to compare directly the difference
in terms of conformational arrangement induced by nickel on the
peptide. From the unordered conformation of the free ligand,
confirmed by the absence of meaningful spatial correlations, we
assist to the appearance of new ROEs involving Hb1 , Hb2 , Qg, Qd
of Arg4+n and Qg of Thr7+n side chains with He1 of the imidazole
ring. These findings suggest a more restricted conformational
space induced by the metal coordination, in comparison to the
free peptide, that lack in spatial closeness concerning these two
residues. This, reinforces the evidence that Arg4+n side chain, after
metal binding, might find itself positioned over the metal centre,
pointing towards the imidazole ring of the histidine residue. In
addition, also Thr7+n Qg protons point towards aromatic ring. In
this way both side chains may shield the complex from the action
of water molecules which can destabilize it. This evidence is also
supported by the model for the structure of our complex derived
by the ROEs spatial connectivities detected by the bidimensional
spectra.
5532 | Dalton Trans., 2009, 5523–5534
Fig. 8 shows the minimized average for the best 20 selected
structures with lowest overall energy calculated for the 4 N
diamagnetic Ni(II) complex, after molecular mechanics geometry
optimization was made.
Conclusions
The NMR study performed on the fragments of C-terminal of
Cap43, provided useful information about the coordination mode
of our peptides with nickel ion.
By using the 10-amino acid fragment containing a single histidine residue, we have shown that the mode of nickel coordination
has not changed for the three repeats, the 30-amino acid fragment,
showing that the decapeptide represents the minimum motif for
nickel binding.
The highly flexible unstructured main chain is able to wrap
around the metal in order to be able to coordinate from one up
to three metal ions. The most striking feature of the structure
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Acknowledgements
This work was supported by the Regione Autonoma Sardegna
“Master and Back” program and the Fondazione Banco di
Sardegna, Sassari, Sardegna, Italy. Porto Conte Ricerche is
gratefully acknowledged for the use of NMR facilities.
References
Fig. 8 Molecular model for the peptide fragment (S3 R4 S5 H6 T7 )–Ni(II)
consistent with NMR data.
of metal complexes is the ordering of side chains of Arg4+n and
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Although the pH values necessary to determine the major
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