Classically, the metal ions studied in detail [mainly ZnII, CuI, CdII, HgII and AgI] are Keywords bioinorganic chemistry; copper; growth inhibitory factor; metallothionein; metal– thiola
Trang 1Neuronal growth-inhibitory factor (metallothionein-3):
reactivity and structure of metal–thiolate clusters*
Peter Faller1,2
1 CNRS, LCC (Laboratoire de Chimie de Coordination), Toulouse, France
2 Universite´ de Toulouse, UPS, INPT; LCC; Toulouse, France
Introduction
Metallothionin-3 (MT3) was originally dubbed
neuro-nal growth-inhibitory factor (GIF) [1] because of the
discovery that it is a factor in brain extract with the
ability to inhibit neuronal outgrowth Moreover, MT3
or GIF was reported to be down-regulated in extract
from Alzheimer’s disease (AD) brain Later on it
became clear that GIF belongs to the metallothionein
(MT) family based on its high cysteine and metal
con-tents In mammals, the MT family consists of four
dif-ferent subfamilies designated MT1 to MT4 [2–4]
Mammalian MTs are composed of a single
polypep-tide chain of 61–68 residues They are characterized by
a conserved array of 20 cysteines and the absence of
His and aromatic amino acids MT3 contains 68
amino acids with 70% sequence identity to the MT1 and MT2 (MT1⁄ 2) isoforms The MT3 sequence contains two inserts: an acidic hexapeptide in the C-terminal region and a Thr in position 5 Moreover, a conserved Cys-Pro-Cys-Pro motif between positions 6 and 9 is unique to MT3 [1,4,5]
All mammalian MTs can bind a variety of different mono-, di- and trivalent metal ions via their cysteine residues Most relevant under normal conditions in biology is the binding of Zn2+ and Cu+ However, MTs can also bind other metals (Cd2+, Hg2+, Ag+,
Pt2+, Pb2+ and Bi3+) when they are administered to animals Classically, the metal ions studied in detail [mainly Zn(II), Cu(I), Cd(II), Hg(II) and Ag(I)] are
Keywords
bioinorganic chemistry; copper; growth
inhibitory factor; metallothionein; metal–
thiolate clusters; protein structure; zinc
Correspondence
P Faller, CNRS, LCC 205, route de
Narbonne, 31077 Toulouse, France
Fax: +33 5 61 55 30 03
Tel: +33 5 61 33 31 62
E-mail: peter.faller@lcc-toulouse.fr
*This article is dedicated to Prof M Vasak
on the occasion of his retirement
(Received 3 December 2009, revised 4 May
2010, accepted 17 May 2010)
doi:10.1111/j.1742-4658.2010.07717.x
Metallothionein-3, also called neuronal growth-inhibitory factor, is one of the four members of the mammalian metallothionein family, which in turn belongs to the metallothionein, a class of ubiquitously occurring low-molecular-weight cysteine- and metal-rich proteins containing metal– thiolate clusters Mammalian metallothioneins contain two metal–thiolate clusters of the type M(II)3-Cys9 and M(II)4-Cys11 [or Cu(I)4-CysS6-9] Although metallothionein-3 shares these metal clusters with the well-characterized metallothionein-1 and metallothionein-2, it shows distinct bio-logical, structural and chemical properties This short review focuses on the recent developments regarding the chemistry of the metal clusters in metal-lothionein-3, in comparison to those in metallothionein-1 and metallothion-ein-2, and discusses the possible biological and functional implications
Abbreviations
Ab, b-amyloid; AD, Alzheimer’s disease; apo-T, apo-thionein; DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid)); GIF, neuronal growth-inhibitory factor; M(II), divalent metal ion; MT, metallothionein.
Trang 2bound in mammalian MTs collectively in two metal–
thiolate clusters located in independent domains
(a b-domain for the N-terminal cluster and an
a-domain for the C-terminal cluster) In the absence of
metals, apo-thionein (apo-T) is predominantly
unstruc-tured and only upon metal coordination is a defined
3D structure formed The cluster structures for the
divalent metals have been resolved for MT1⁄ 2 and they
were found to be highly unusual compared with other
proteins (Fig 1) The N-terminal b-domain (amino
acids 1-30) binds three divalent metal ions by nine
deprotonated cysteines in a hexane-like cluster [M(II)3
-Cys9], including three bridging and six terminal
cyste-ines The C-terminal a-domain (amino acids 31-61)
binds four divalent metal ions by 11 deprotonated
cysteines in an adamantane-like cluster [M(II)4-Cys11],
including five bridging and six terminal cysteines All
seven divalent metals are bound in tetrahedral
coordi-nation (Fig 1) [4] The precise cluster structure of the
Cu(I)X-CysSY moieties in mammalian MTs has not yet
been determined, and the only structure available is
from the Cu(I)8-Cys10cluster in yeast MT (Fig 2) [6]
It is unlikely, however, that mammalian MTs contain
such a cluster Spectroscopic experiments on
mamma-lian MT1⁄ 2 indicate that they contain Cu(I)4-CysS6-9,
Cu(I)6-CysS9-11 or Cu(I)3⁄ 4-ZnxCysSx clusters and
hence have a distinctly different cluster organization In
all cases, Cu(I) shows a preference (at least partial) for
binding to the N-terminal b-domain [4,7]
These metal–thiolate cluster structures are unusual
for metal-binding proteins and are responsible for the
typical reactivity properties of MTs First, the metal
binding is thermodynamically relatively stable, with
the following order Cu(I)>Cd(II)>Zn(II) The cluster
structure raises the possibility of a cooperative binding
of the metal ions Cooperative binding has been reported but is not always found, being dependent on the type of metal, the type of cluster and the pH [2,5,8] In contrast to Zn(II)-MT and Cd(II)-MT,
apo-T is is oxidatively unstable under aerobic conditions as
a result of the formation of disulfide bridges [2] Sec-ond, in contrast to the high thermodynamic stability, the binding is kinetically labile, allowing rapid intra-molecular and interintra-molecular metal transfer This is a direct consequence of the relatively high structural dynamics and flexibility typical of all MTs [5] Third, the deprotonated cysteines bound to the metal ions (i.e thiolates) are good nucleophiles, conferring a high reactivity with radical species (HO·, O2·), NO·) as well
as with alkylating and oxidizing agents Such reactions result in oxidation or derivatization of the cysteines with subsequent metal release [4]
Although MT3 belongs to the MT family and hence shares the unusual properties of their metal–thiolate clusters, there are important differences between MT3 and the well-characterized MT1⁄ 2 [4] Such chemical and structural differences are probably important for the biological roles of MT3, such as its growth-inhibitory activity, the non-inducibility of its gene by diverse metal ions and other compounds known to elicit the formation
of MT1⁄ 2, its predominant localization in the central nervous system with an accumulation in zinc-enriched neurons, and its possibility of being excreted into the extracellular space [4,9,10] Note, that the latter may not
be restricted to MT3 as evidence is accumulating that MT1⁄ 2 also occurs extracellularly [4] This is summa-rized in Table 1 and discussed in more detail below
Metal content of MT3 Initially, MT3 was isolated from human brain with a metal content of four Cu(I) and three Zn(II) per MT3
Fig 1 Scheme of the two metal–thiolate clusters containing
Zn ⁄ Cd in mammalian MT1 ⁄ 2 Left: four-metal cluster [M(II) 4 -Cys 11 ]
localized in the C-terminal a-domain Right: three-metal cluster
[M(II)3-Cys9] localized in the N-terminal b-domain The scheme is
based on structures obtained by X-ray and NMR for MT1 ⁄ 2 MT3
contains the same type of four-metal cluster M, divalent metal, S,
cysteine thiolate Adapted from a previous publication [4].
Fig 2 Scheme of the Cu(I) 8 (Cys) 10 cluster from yeast CuMT Cu, copper; S, cysteine thiolate Adapted from a previous publication [6].
Trang 3Table 1 Comparison between MT3 and MT1 ⁄ 2 NOS, nitric oxide synthase; ROS, reactive oxygen species.
Feature
Putative
biological
function
Involved in Zn and Cu metabolism Synthesis not inducible Synthesis induced by Zn, Cu, Cd
Growth-inhibitory activity ROS and NOS scavengers
(antioxidant)
Protects cultured neurons against
Ab toxicity [42]
Does not protect [42]
Localization Can occur extracellularly [14] Predominantly in the brain,
neurons and ⁄ or astrocytes? [3]
Ubiquitous; in the brain mostly in astrocytes [3]
20 cysteines, same arrangements Acidic 6-amino-acid insert in the
C-terminal domain
Absence of 6-amino-acid and Thr insert
Thr insert at position 4 Cys(6)-Pro-Cys-Pro(9) Cys(5)-Ser-Cys-Ala(8) Metal content Binds without metal exposure Zn
and perhaps Cu No heterometallic Zn ⁄ Cu clusters
Isolated as a mixture of Cu and Zn [1,11], but perhaps in vivo predominantly Zn [13]
Isolated predominantly as Zn only
Zn binding 7 Zn(II) bound to 20 thiolates Additional specific (eighth)
Zn-binding site [30,31]
Similar overall apparent K d of Zn Cu 4 Zn 4 : 4.2 · 10)12M[32] Zn7:
1.6 · 10)11M [17]
Zn7: 3.2 · 10)12M [17]
Individual Kdat
pH 7.4
Zn ⁄ Cd binding stronger in 4-metal cluster than in 3-metal cluster [18] Zn 3 and Zn 4 cluster:
non-cooperative? [21,22,30]
Not known Additional eighth site:
1 · 10)4M
7th Zn: 2.0 · 10)8M 6th Zn: 1.1 ·
10)10M 5th Zn: 3.5 · 10)11M First 4 Zn: 1.6 · 10)12M
Cd binding 7 Cd(II) bound to 20 thiolates Additional specific eighth binding
site [30]
No specific additional sites [30]
Cd binding similar to Zn Cd7form less compact than Zn7
[26]
Similar compactness [26]
Similar overall apparent K d of Cd Cd 7 : 5.0 · 10)15M [17] Cd 7 : 1.4 · 10)15M [17]
More non-cooperative Cd binding [17]
More cooperative Cd binding (at pH 7.4) [17]
Zn ⁄ Cd–thiolate
clusters
Cd ⁄ Zn 3 -CysS 9 in b-domain and
Cd ⁄ Zn 4 -CysS11in a-domain Same connectivities in Cd form in Cd4 cluster [5,23,24]
Dynamics Cd 3 -CysS 9 more dynamic than
Cd4-CysS11[5]
Cd 3 -CysS 9 very dynamic (precluded structure determination by NMR) [24,27,39]
Cd 3 -CysS 9 less dynamic, NMR structure available [5]
Reaction with NO Cysteine oxidation and Zn release Zn release is faster [38] Zn release is slower [38]
Reaction with
ROS
Cysteine-oxidation and Zn-release rates relatively similar [38]
Reaction with Pt
compounds
Reaction with Cys, Pt bound to Cys
Cisplatin and transplatin react faster [36]
Cisplatin and transplatin react slower [36]
Cu(I) binding Cooperative formation of Cu4
-CysS x [33,43] Further forms:
Cu8MT (two Cu4-CysSxclusters in each domain) Cu12MT (Cu6-CysS9 and Cu 6 -CysS 11 )[33]
Cu4-CysS8-9[32,33] Cu4-CysS6-7[44]
Established that first Cu 4 -CysS 8-9 is
in the N-terminal domain [33]
Not established, but evidence provided
Cluster stable in air [12,32] Cluster not stable in air [44] Redox-labile site in Zn 4 cluster of
Cu4Zn4MT-3 [32] formation of
Cu 4 Zn 3 MT-3 with disulfide
Not studied
K d of Cu(I) K d estimated to be 1 · 10)19M Stronger Cu(I) affinity? [35] Weaker Cu(I) affinity? [35] Cu(II) binding to
Zn7-MT
MT binds Cu(II) after reduction to Cu(I) through cysteine oxidation
Formation of Cu(I)4Zn4MT-3 with two disulfide bridges [37]
Zn form not studied
Trang 4[1] A mixture of Zn(II) and Cu(I) has also been found
in MT3 isolated from bovine and equine brain [11,12]
as well as from mice with disrupted Mt1 and Mt2
genes [13] In all cases only homometallic clusters have
been reported and the predominant Zn⁄ Cu form might
thus be Cu(I)4Zn(II)4-MT3[4] The physiological
accu-mulation of Cu in MT3 has been questioned as it was
speculated that in vivo MT3 binds Zn almost
exclu-sively [13] This draws support from the demonstration
that during the purification of Zn(II)-MT3, metal
exchange reactions do occur It could allow picking up
the more firmly binding Cu+during purification, but,
of course, yields no proof that MT3 functions solely as
a Zn protein(II) [13] Clearly, further investigations are
needed to clarify how much, and under what
condi-tions, Cu(I) is bound to MT3 The binding of Cu to
MT3 has to be considered to occur not only
intracellu-larly because it is now known that MT3 also occurs
extracellularly [14] and evidence is accumulating that
Cu can be released into the synaptic cleft [15]
More-over, there is evidence that MT3 can bind Cu(I) when
Cu homeostasis breaks down, such as in AD [16]
Binding of Zn and Cd to MT3
MT3 binds predominantly seven Zn(II) or Cd(II) ions
with overall apparent dissociation constants, at pH
7.4, of 1.6· 10)11m and 5.0· 10)15m, respectively
[17] The three-metal cluster was less stable than the
four-metal cluster for Zn(II) and Cd(II) [18] Initially,
information about the presence of two separate metal–
thiolate clusters came from spectroscopic studies and
comparison with other MTs of Zn(II)- and
Cd(II)-MT3, as well as their individual domains [12,19–22]
Precise structural data were obtained by NMR
show-ing a Cd(II)4-CysS11 cluster in the C-terminal domain
with Cd(II)-Cys connectivities identical to those found
in the structure of human MT2 [23,24] Such
informa-tion is still lacking for the N-terminal cluster, but
molecular dynamics simulation proposed a Cd(II)3
-CysS9 cluster structure essentially identical to that of
MT2 [9,25] Thus, apart from determining the Cd(II)3
-CysS9 structure in MT3, the confirmation that Cd
replaces Zn isostructurally (as shown for MT2) is still
required This seems important because the
isostructur-al replacement has been chisostructur-allenged for MT3 (but
not MT2) based on the observed difference in signal
intensity of the charge states of Zn(II)-MT3 and
Cd(II)-MT3 in ESI-MS, suggesting that with Cd(II)
the N-terminal domain [the M(II)3-CysS9 cluster] has a
less compact structure with Cd than with Zn [26] No
comparable difference was seen in MT2, indicating
that the more open structure in MT3 with Cd(II) is
not just the result of the larger ionic radius of Cd(II) over Zn(II) and that the difference in amino acid sequence plays a role
One of the most important aspects is the greater dynamics of the Cd(II)3-CysS9 cluster of MT3 It was observed that the resonances of Cd(II)3-CysS9 in the
113Cd(II) NMR were much less intense than those of Cd(II)4-CysS11 Increasing the temperature did sharpen them but their intensity was not enhanced [27] Recently, Wang et al [24] confirmed the low intensity
of the resonances of Cd(II)3-CysS9, although their dif-ference from those of Cd(II)4-CysS11 was smaller Moreover, NMR measurements of mouse MT3 and human MT confirmed a high dynamical structure caused by rapid internal motion (mostly of the first 12 amino acids) [24] and this was viewed as conforma-tional exchange broadening This dynamic structure, only observed in the Cd(II)3-CysS9 of MT3, is intrigu-ing because it correlates with the growth-inhibitory activity [9,28] as well with the higher chemical reactiv-ity of the metal cluster (e.g with NO·, see later) Moreover, it is also the reason why the determination
of the spatial structure of the N-terminal domain con-taining the Cd(II)3-CysS9 cluster was thus precluded For a more detailed discussion of the dynamics of the protein structure and its implication for the biological function, see the minireview by Huang et al in this minireview series [29]
What is the reason for the higher dynamics of the MT3 structure? Initially it was proposed that slow exchange occurs between alternative configurations involving CysS-Cd(II) bond breaking⁄ formation in the conversion, which may include a cis–trans isomeriza-tion of the Cys-Pro amide bonds in the Cys6 -Pro-Cys-Pro(6–9) motif [17] Only one configuration is detected
by 113Cd(II)-NMR, whereas the other is broadened beyond detection by fast exchange processes However, another possibility has been proposed by Palumaa
et al [30] They found, by ESI-MS, that Zn(II) and Cd(II) do not bind cooperatively to MT3 at pH 7.3 Upon adding seven equivalents of Cd(II) or Zn(II) to MT3, distributions of metal loading were detected, ranging from five to nine for Cd(II) and from six to eight for Zn(II) (note that identical experiments with MT1A showed more homogeneous M(II)-binding of seven metals per MT [30]) This would mean that Cd(II)7-MT3, and, to a lesser extent, also Zn(II)7 -MT3, are heterogeneous in their metal content, and that metal-exchange reactions between different metal-loaded forms could be the reason for the higher dynamics detected in NMR However, this conclusion has its limits, first because the MS analysis is not quantitative and, second, because more recent MS
Trang 5measurements did not confirm binding of more than
eight equivalents of Zn(II) [31] Importantly, the
bind-ing of the eighth metal ion was confirmed by other
methods and studied in more detail [31] An additional
equivalent of Zn(II) can bind to Zn(II)7-MT3 [but not
to Zn(II)7-MT2] with an apparent Kdof 0.1 mm
Simi-larly, Cd binds to Cd(II)7-MT3 but with an even
stron-ger affinity (exact Kd not reported) Binding of either
additional ion disturbs the CD signatures of the
thio-late clusters, indicating interference with the cluster
structure Moreover, a decrease in the Stokes radius
was observed, suggesting a mutual approach of the
two domains Also, the additional binding of M(II)
induced slow, but appreciable, non-covalent
dimeriza-tion of MT3 [40% for Zn(II) and 80% for Cd(II)]
Subsequent analysis of 113Cd(II) NMR revealed
com-plete loss of the Cd3-CysS9 resonances The reported
data indicate that the b-domain provides the binding
site for the eighth equivalent of Zn(II) [and Cd(II)]
Moreover, the occurrence of a weaker eighth binding
site in Cd(II)7-MT could be involved in
metal-exchange reactions and hence contribute to the
flexibil-ity of the Cd(II)3-CysS9 cluster This is supported by
the almost complete loss of resonance intensity upon
binding of an additional Cd(II)
Disulfide bond formation might be another way to
produce heterogeneity and⁄ or increased structural
dynamics in the b-domain, leading to partial disulfide
bonds and⁄ or to disulfide exchange reactions,
respec-tively No evidence for disulfide bond formation in
Zn(II)7-MT3 has been reported Nevertheless, it might
be worthwhile to re-investigate this point because
oxidation of cysteine was noted in the Zn(II)4-CysS11
cluster of freshly prepared Cu(I)4Zn(II)4-MT3 [32]
Definitive studies will be important to explore the
isostuctural replacement of Zn(II) with Cd(II), by
monitoring the peptide structure and the flexibility of
Zn(II) and Cd(II) by NMR spectroscopy Another
interesting (and to my knowledge not yet reported)
experiment would be assessment of the
growth-inhibi-tory activity of the Cd-containing form of MT3 If
affirmed, this would support true isostructural
replace-ment, as this MT3-specific activity is believed to be a
structure-dependent feature A problem with such a
measurement could be the toxicity of Cd(II)
Neverthe-less it might work as Cd(II) is tightly bound to MT-3
and Cd(II)-MT1⁄ 2 could be used as a control, the
issue of Cd toxicity may be overcome So far the
struc-ture determination of the b-domain of Cd7-MT-3 by
NMR was hampered by the high structural dynamics
and exchange reactions The recently gained insights
discussed above (dimerization, disulfide formation,
additional Zn(II)⁄ Cd(II) binding and Mg ⁄ Ca effects)
might be used to slow down or increase the time regime of exchange reactions as such that they are more favorable for NMR studies [30–32]
It seems clear now that Zn7-MT3 and Cd7-MT3 can bind specifically an additional eighth equivalent of Zn(II) or Cd(II) Binding of more Zn(II)⁄ Cd(II) is very likely to be non-specific Whether the binding of seven equivalents of Zn(II)⁄ Cd(II) in MT3 is more heteroge-neous than the binding of seven equivalents of Zn(II)⁄ Cd(II) to MT1 ⁄ 2 is still not known There are indications that binding of Cd is less cooperative in MT3 compared with MT2 [17] For Zn this is less clear
Binding of Cu(I) to MT-3 The spectroscopic characterization of MT3 isolated from bovine and equine brain showed that Cu is bound in the oxidation state I in a four Cu(I)–thiolate cluster, Cu(I)4-CysSX [12,20] Furthermore, reconstitu-tion experiments with human apo-T3 and its separate domains reproduced closely the features of the isolated native MT3 forms (bovine, equine) [32,33] Moreover, titration experiments of Cu(I) to T3 (or to apo-a- and apo-b-domains) showed cooperative formation
of the Cu–thiolate cluster involving eight or nine cyste-ines [i.e Cu(I)4-CysS8-9] [21,22,33] In apo-T3 the first cluster formed, Cu(I)4-CysS8-9, was localized in the N-terminal domain [33] The structure of this Cu(I)4 -CysS8-9 cluster is not known, but extended X-ray absorption spectroscopy data yield a Cu-Cu distance
of 2.67 A˚ and a Cu-S distance of 2.26 A˚ The latter points to mainly trigonal coordination of Cu(I) [the correlation would predict one or two digonal bound Cu(I)] This clearly suggests that Cu(I) and Zn(II)⁄ Cd(II) bind preferentially to the b- and a-domains, respectively, and that no heterometallic clusters con-taining both Cu(I) and Zn(II)⁄ Cd(II) are formed Addition of Cu(I) beyond four equivalents results in the cooperative formation of a Cu(I)4-CysS8-9 cluster
in the C-terminal domain After completion of the two Cu(I)4-CysS8-9 clusters, further addition of Cu(I) results in the formation of Cu(I)6-CysS9 clusters and Cu(I)6-CysS11 in the N-terminal and C-terminal domains, respectively [33]
The dissociation constant of Cu(I) to MT3 has not been measured, but by analogy with other MTs it can
be estimated to be around 10)19m [34] Judged from the higher reactivity of Cu(I)-MT1 with 5,5¢-dithio-bis(2-nitrobenzoic acid) (DTNB) compared with Cu(I)-MT3, it has been proposed that the affinity of MT3 for Cu(I) is higher than that of MT1⁄ 2 [35] One of the remarkable features of Cu(I)4-CysS8-9 clusters in the isolated Cu(I)4-Zn3-4MT-3 is its stability in air
Trang 6The spectroscopic features of isolated Cu(I)4-Zn3-4
MT-3 did not change over time in air [12] The Cu(I)4
clus-ter in freshly reconstituted Cu(I)4-Zn4MT-3 also
seemed to be stable, although oxidation occurred in
the Zn cluster [32] Indeed, exposure of Cu(I)4
-Zn4MT3 to air resulted in the slow formation of a
disulfide linkage by cysteine oxidation of the a-domain
and a concominant release of one Zn ion, but the
spec-troscopic features of the Cu(I)4 cluster did not change
This might indicate that in the isolated Cu(I)4-Zn4MT3
this oxidation had already occurred (for further
discus-sion see below) The molecular basis of the air stability
of the Cu(I)4 cluster is not known Cu(I)–thiolates are
normally oxidized by molecular oxygen, resulting in
the formation of disulfide bonds (Eqn 1) Thiyl
radi-cals have been observed as an intermediate The
mech-anism might be oxidation of Cu(I) by oxygen (Eqn 2)
The formed superoxide might oxidize a further
equiva-lent of Cu(I) (Eqn 3) A two-electron reaction of Cu(I)
with oxygen yielding H2O2 is probably favored (Eqn
4) as the reduction of O2to O2 )is thermodynamically
unfavored, but the two-electron oxidation of O2 to
H2O2is favored
2CuðIÞ þ 2Sþ O2þ 2Hþ! 2CuðIÞ þ S S þ H2O2 ð1Þ
CuðIÞ þ O2! CuðIIÞ þ O2 ð2Þ
CuðIÞ þ O2 þ 2Hþ! CuðIIÞ þ H2O2 ð3Þ
ðEqn1 þ Eqn3Þ2CuðIÞ þ O2þ 2Hþ! 2CuðIIÞ þ H2O2 ð4Þ
CuðIIÞ S! CuðIÞ S ð5Þ
Then, Cu(II) can oxidize thiolate to thiyl (Eqn 5)
and two thiyls form a disulfide bridge (Eqn 6) [It is
also possible for S•to react with a neighboring thiolate
to yield a disulfide radical anion (S-S•)), which can be
further oxidized to disulfide] In the framework of this
mechanism several possibilities can be envisaged to
explain the air-stability of Cu(I)4-Zn4MT The first
possibility is no accessibility of oxygen to the cluster
This is unlikely because MT3 is more dynamic and
hence more exposed to the solvent The second
possi-bility is steric hindrance to form disulfide bridges, and
the third is that the redox potential of Cu(I) [or the
Cu(I)–thiolate moiety] is high enough that it is not
oxi-dized by molecular oxygen In this context it is
note-worthy that absorption data indicate that a different
number of cysteines is bound to Cu(I) (i.e eight or
nine in MT3 and six or seven in MT1⁄ 2) This
indi-cates that the cluster structure is different More
cyste-ines involved means either fewer bridging cystecyste-ines or
a higher coordination number of MT3 compared with MT1⁄ 2, features that might be responsible for the sta-bility in air
Reactivity of the metal–thiolate clusters in MT3
First, some general considerations about the reactivity
of metal–thiolate clusters, normally concerning all metallothioneins, are given In the case of Zn (and Cd) the metal is bound by thiolates (i.e deprotonated thiol groups of cysteine) Thiolate is a soft ligand and therefore shows a preference for soft metals More-over the structures of metallothioneins are not rigid and hence there is little selectivity concerning the size
of the ion Thus, the affinity of the different metal ions in MTs is governed primarily by the thermody-namic stability of the thiolate–metal bond Therefore, soft metals such as Cu(I), Cd(II), Hg(II), Pb(II) and Pt(II) bind more strongly than Zn(II), leading to Zn(II) release
The cysteine side chains are also very reactive At physiological pH, free cysteine is predominantly pro-tonated The availability of Zn(II) leads to their depro-tonation at physiological pH, yielding Zn–thiolate complexes This renders thiolates more nucleophilic than thiols However, their binding to Zn(II) generally also inhibits the formation of disulfides under aerobic conditions The latter occurs with uncoordinated thio-lates Thus, Zn(II) binding elicits the formation of thiolates, which are more reactive than thiols, but less reactive than uncoordinated thiolates This can be con-sidered as the sulfur reactivity of MT on a biological time scale is controled by the Zn-binding state Thiols would react too slowly, whereas free thiolates would react too fast and be difficult to control
Metal-centered reactions of MT3
In the framework of considering thiolates as simple metal ligands, metal-exchange reactions such as Cd(II)
or Hg(II) with Zn(II)-MT have been studied with MT1⁄ 2 However, as MT3 synthesis is not inducible
by exposure to metal ions, a role in detoxification is less likely, which is probably the reason why metal-exchange reactions with Cd(II), Hg(II), Pb(II), etc., have not been studied so far Moreover, the interac-tion of Zn(II)7MT3 with biologically relevant Cu(I) has not been reported By contrast, the reaction of MT3 with cis-amminedichloridoplatinum(II) (cisplatin) and trans-amminedichloridoplatinum(II) (transplatin) has been studied These reactions are of interest because MTs play an important role in the acquired
Trang 7resistance of platinum-based anticancer drugs Here,
MT3 is important because its gene is overexpressed in
a number of cancer tissues ([36] and references
therein) It was shown that cisplatin and transplatin
react with the cysteines in Zn(II)7MT3, causing the
stoichiometric release of Zn(II) The reactions were
much faster than with MT2 Transplatin reacted more
quickly, but retained the two ligands By contrast, the
slow-reacting cisplatin had all ligands replaced
with thiolates Cisplatin binds preferentially to the
b-domain (but binding with transplatin has not been
determined)
A special case of metal-exchange reaction is Cu(II),
because it involves, in addition to its exchange with
Zn, also the reduction to Cu(I) by the oxidation of
cysteine Meloni et al [37] showed that Zn(II)7MT3
scavenges free Cu2+ ions through reduction to Cu(I)
and binding to the protein In this reaction, thiolate
ligands are oxidized to disulfides concomitant with
Zn2+ release The binding of the first four Cu2+ is
cooperative, forming a Cu(I)4–thiolate cluster in the
N-terminal domain of Cu(I)4,Zn(II)4MT3 together
with two disulfide bonds Because four zinc ions
remain bound, it seems likely that the four-metal Zn4–
thiolate cluster in the a-domain stayed intact As a
consequence the two disulfides would be localized in
the b-domain with the Cu(I)4–thiolate cluster The
formed Cu(I)4–thiolate cluster has spectroscopic
prop-erties similar to the isolated and the
Cu(I)-reconsti-tuted Cu(I)4Zn(II)4MT3 described above, including
stability in air The reaction of Zn(II)7MT3 with
Cu(II) has been proposed to be the underlying
mecha-nism for the protective effect of MT3 against
b-amy-loid (Ab) neurotoxicity linked to AD It was shown
that a metal swap between Zn(II)7MT3 and soluble
and aggregated Cu(II)–Ab abolishes the production of
reactive oxygen species and the related cellular toxicity
[16] Thus, MT3 might have a role in protecting Ab
from aberrant Cu binding
Thiolate-centered reactions (NO•,
reactive oxygen species, DTNB)
Metals in MTs are relatively deeply buried in the
pro-tein The modification of the surface-accessible sulfur
of cysteine ligands is thought to be the key that
unlocks the metals from the protein [38] Thus, in
gen-eral, reactions of the cysteine thiolates result in a
con-comitant release of Zn(II) A variety of reagents
reacting with thiolate have been studied in MTs [5],
but such reactions are limited for MT3 The reaction
with NO• has attracted particular attention [38,39]
Most interest in the reaction with NO•comes from the
possible role of MTs as NO• scavengers and in the conversion of a NO• signal to a Zn(II) signal The reaction with NO• providing S-nitrosothiols is sug-gested to be a transnitrosation (i.e translocation of
NO+from the S-nitrosothiols to the cysteine of MT), which then releases NO)during the formation of disul-fides [38] This means that NO) is not stable in the MTs and a storage function of MTs for NO· is less likely By comparing the reaction of NO and S-nitros-othiols in MT3 and MT1⁄ 2, Chen et al [38] found that MT3 was much more reactive, whereas the activi-ties with reactive oxygen species (H2O2, OCl), O2 )) were comparable In line with this, MT3 was also more potent in protecting rat embryonic cortical neurons against S-nitrosothiols The b-domain showed greatest reactivity in Zn(II) release and cell protection The higher reactivity of the b-domain in MT3 versus NO has been confirmed by NMR studies on Cd(II)7MT3 [39] Moreover, the NMR data suggest a non-selective release of the metals from the b-domain first, followed
by a partial release of two Cd(II) ions from the a-domain, without a significant change in the poly-peptide structure Further addition of NO resulted in a complete loss of protein structure
DTNB has often been used to probe the nucleophilic reactivity of thiolates in MTs DTNB contains an intra-molecular disulfide bond and, upon nucleophilic attack from MT, disulfide exchange occurs resulting in an in-termolecular disulfide bond between MT and 5-thio-2-nitrobenzoic acid It has been shown that Cd(II)7MT3 reacts faster with DTNB than MT1⁄ 2 does [35,40] For Zn(II)7MT3, similar kinetics have been reported for human MT3 and rat MT1, but this was monitored in the presence of EDTA and hence is more likely to reflect the reactivity of the unstructured apo-T than that of the metal-loaded form [19]
In general, it can be suggested that MT3 is more reactive than MT1⁄ 2, which is probably related to its more flexible b-domain and hence to a better access of compounds to the metal–thiolate cluster This is also
in line with the general (but not exclusive) observation that the difference in reactivity is more pronounced for larger molecules (DTNB, providing S-nitrosothiols) than for small molecules (H2O2, OCl), O2))
Conclusions and Perspectives MT3 is clearly a member of the MT family as it shares with them several biological and chemical properties; however, there are also very distinct chemical features that might be directly relevant to the particular biolog-ical properties of MT3 Therefore, comparison of the properties of MT3 with those of the well-studied
Trang 8MT1⁄ 2 forms could yield information on the structural
and chemical features responsible for the biological
peculiarities of MT3, such as its growth-inhibitory
activity
The fact that synthesis of MT3, in contrast to that
of MT1⁄ 2, is not inducible by metal ions (Zn, Cu, Cd,
etc.) suggests that it has no essential role in
sequester-ing toxic or an overload of essential metals This is in
line with a lower binding affinity to Cd(II) for MT3
compared with MT1⁄ 2
One of the most striking features is the greater
struc-tural dynamics and flexibility of MT3 and in particular
of the N-terminal b-domain This seems to be connected
with the lower metal-binding affinity and with the
higher reactivity towards nucleophilic reagents (NO•, Pt
compounds, DTNB) and the ease of Zn release One
could speculate that higher structural dynamics should
also result in a faster metal transfer from and to MT3
and hence is in line with an involvement in the
traffick-ing of Zn(II) in particular in zinc-containtraffick-ing neurons
[41] However, metal-exchange rates have not yet been
measured experimentally
Importantly, MT3 shows increased Zn(II) release
compared with MT1⁄ 2 upon reacting with NO• and
other compounds, supporting a function in Zn
meta-bolism It was suggested that MT3 could be involved
in turning a NO•signal into a Zn signal [38]
Although the question of whether Cu(I) is
physio-logically bound to MT3 is not as yet resolved (see
ear-lier), it is clear that MT3 has the capacity of binding
Cu(I) under conditions of Cu-homeostasis breakdown
There are several unanswered questions concerning
Cu(I)MT3 First the exact cluster structures of the
dif-ferent forms are not known [i.e Cu(I)4,Zn(II)4MT3
(with or without disulfides), Cu(I)4,Cu(I)4MT3 and
Cu(I)6,Cu(I)6MT3] The determination of a 3D
struc-ture seems crucial for understanding the differences
between Cu–MT3 and Cu–MT1⁄ 2 (structure also not
known) and the presence and role of the disulfide
bonds This would also give insight into the stability
of the Cu(I)4-CysSX cluster in the b-domain of MT3
towards oxidation by molecular oxygen The data seem
to be contradictory (see above) as the Cu(I)4 cluster is
stable in air, but freshly reconstituted Cu(I)4,Zn(II)4
MT3 shows an oxidation reaction forming disulfide
bonds in the Zn-loaded a-cluster One could ask why
this reaction seems not to occur in the Zn(II)7-MT-3,
as in Cu(I)4-Zn(II)4-MT-3 the Cu(I)4-cluster seems not
to be involved? Moreover, in the reaction of Cu(II)
with Zn(II)7MT3, a Cu(I)4 cluster and two disulfide
bridges are formed in the b-domain, while the
remain-ing Zn cluster seems stable The spectroscopic features
of this Cu(I)4cluster are very similar to those of the
reconstituted forms One partial explanation would be that the spectroscopic features of the Cu(I)4 cluster are not affected by the presence of disulfide bridges and this cluster is only stable in air in the presence of one
or two disulfide bonds This could explain the sensitiv-ity to oxidation of freshly reconstituted Cu(I)4, Zn(II)4MT3 and the stability of Cu(I)4, Zn(II)4MT3 [isolated, incubated or generated upon Cu(II) binding] in air However, this does not explain why the disulfide bridge is formed in the a-domain (instead of the b-domain) upon oxidation of freshly reconstituted Cu(I)4,Zn(II)4MT3 To shed more light
on this issue it might be worthwhile investigating the number and localization of disulfide bridges in diverse preparations of MT3
With regard to the putative role of MT3 in Cu traf-ficking, it would be important to determine the binding constants Very little is known about Cu(I) affinity in the MTs, and the values in the literature are mostly estimates This is mainly because of the very low disso-ciation constants, with Kd estimated to be about
10)19m, and the lack of suitable competing ligands with well-known binding constants However, even rel-ative affinities could give important insights, such as comparison of MT3 with other MTs The comparison reported in the literature, based on reactivity with DTNB, is indirect (see above) [35] Relative Cu(I) affinities between MT3 and other MTs should be mea-sured by a competition assay using MS, as accom-plished previously for Zn(II) and Cd(II) [18], or by NMR analysis
The high affinity of Cu(I) to MT3 (or to MTs in general) means that Cu(I) release into solvent, as a result of thiolate bonding, is too slow to be biologi-cally relevant Therefore, transfer of Cu(I) is impossi-ble via ‘free’ Cu(I) One way to transfer Cu(I) to another protein on a biologically relevant time scale is through the formation of a ternary complex [MT– Cu(I)–protein] (i.e by a coordination bridge forming
an interaction between MT and the acceptor protein) Other possibilities are that the transfer is assisted by cysteine oxidation⁄ modification, by protonation or by protein breakdown If the Cu(I) transfer did not taking place, MT would just be a sink for Cu(I) This could
be sufficient for a redox-silencing role of MT3 for Cu
In this context it might be interesting to search for possible binding partners of Cu(I)4MT3
Since the discovery of MT3 [1] almost 20 years ago,
it has been discovered that this member of the family has unusual biological and chemical properties, clearly distinct from the widely expressed MT1⁄ 2 This holds also for structure and reactivity of the metal–thiolate clusters, in particular for the cluster in the b-domain
Trang 9Several intriguing facets have been observed, such as
high dynamics, formation of disulfides, high reactivity,
stability of the Cu(I)4-cluster, etc A better
understand-ing of these features will help to shed light on the
specific biological roles of MT3
Acknowledgement
Gabriele Meloni (Caltech, USA) and Milan Vasak
(Univ Zu¨rich) are acknowledged for very helpful
discussion
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