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METALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY © World Scientific Publishing Co. Pte. Ltd. Chapter 1 METALLOTHIONEIN STRUCTURE AND REACTIVITY Milan Vašák and Gabriele Meloni The structure and chemistry of mammalian metallothioneins (MTs) with divalent (Zn II , Cd II ) and monovalent (Cu I ) metal ions pertinent to their role in biological systems are discussed. In human, four MT isoforms designated MT-1 through MT-4 are found. The characteristic feature of these cysteine- and metal-rich pro- teins is the presence of two metal-thiolate clusters located in independent protein domains. The structure of these clusters is highly dynamic, allowing a fast metal exchange and metal transfer to modulate the activity and function of zinc-binding proteins. Despite the fact that the protein thiolates are involved in metal binding, they show a high reactivity toward electrophiles and free radicals, leading to cysteine oxidation and/or modification and metal release. The unusual structural properties of MT-3 are responsible for its neuronal growth inhibitory activity, involvement in trafficking of zinc vesicles in the central nervous system (CNS), and protection against copper-mediated toxicity inAlzheimer’s disease. MT-1/MT-2 also play a role in cellular resistance against a number of metal-based drugs. Keywords: Metallothionein; three-dimensional structure; structure dynamics; metal-thiolate cluster; metal-cluster reactivity; zinc; copper. 1. Introduction Metallothioneins (MTs) are a superfamily of low-molecular-mass cysteine- and metal-rich proteins or polypeptides conserved through evolution and present in all eukaryotes and certain prokaryotes. They were discovered initially in the search for the tissue constituent responsible for the natural accumulation of cadmium in equine and human kidney in 1957 by Margoshes and Vallee (Margoshes and 3

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METALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch01Chapter 1METALLOTHIONEINSTRUCTURE AND REACTIVITYMilan Vak and Gabriele MeloniThe structure and chemistry of mammalian metallothioneins (MTs) with divalent(ZnII, CdII) and monovalent (CuI) metal ions pertinent to their role in biologicalsystemsarediscussed.Inhuman,fourMTisoformsdesignatedMT-1throughMT-4 are found. The characteristic feature of these cysteine- and metal-rich pro-teins is the presence of two metal-thiolate clusters located in independent proteindomains. The structure of these clusters is highly dynamic, allowing a fast metalexchange and metal transfer to modulate the activity and function of zinc-bindingproteins. Despite the fact that the protein thiolates are involved in metal binding,theyshowahighreactivitytowardelectrophilesandfreeradicals, leadingtocysteine oxidation and/or modication and metal release. The unusual structuralpropertiesofMT-3areresponsibleforitsneuronalgrowthinhibitoryactivity,involvement intrafckingof zinc vesicles inthe central nervous system(CNS), andprotection against copper-mediated toxicity in Alzheimers disease. MT-1/MT-2also play a role in cellular resistance against a number of metal-based drugs.Keywords: Metallothionein; three-dimensional structure; structure dynamics;metal-thiolate cluster; metal-cluster reactivity; zinc; copper.1. IntroductionMetallothioneins(MTs)areasuperfamilyoflow-molecular-masscysteine- and metal-rich proteins or polypeptides conserved throughevolution and present in all eukaryotes and certain prokaryotes. Theywerediscoveredinitiallyinthesearchfor thetissueconstituentresponsible for the natural accumulation of cadmium in equine andhumankidneyin1957byMargoshesand Vallee(Margoshesand3METALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch014 M. Vak and G. MeloniVallee, 1957). This proteinwas subsequentlynamedmetalloth-ionein to reect the extremely high thiolate sulfur and metal content,both of the order of 10% (w/w) (Kgi and Vallee, 1960). AlthoughMTs are still the only macromolecules in which cadmium accumu-latesnaturally, thismetalisonlyoneofseveraloptionalmetalliccomponents of this protein, the others being usually zinc and copper.In fact, in subsequent studies, zinc was found to be the most abundantandoftensole metallic component inmammaliantissues undernormal physiological conditions. Besides ZnII, CuI, and CdII, MTsbindinvitroandincertaincasesalsoinvivo(e.g. PtIIincancerchemotherapy) a variety of other metal ions such as CoII, NiII, FeII,HgII, PtII, AuI, AgI, InIII, SbIII, BiIII, AsIII, and TcOIII(Vak andRomero-Isart,2005). Atpresent,itisbecomingincreasinglyclearthatMTsfullldifferentfunctionsinanumberofbiologicalpro-cesses. In mammalian cells, these include homeostasis and transportof physiologicallyessential metals (Zn, Cu), metal detoxication(Cd,Hg), protection against oxidative stress, maintenance of intracellularredoxbalance, regulationofcellproliferationandapoptosis, pro-tection against neuronal injury and degeneration, and regulation ofneuronal outgrowth (reviewed in Palmiter, 1998; Maret, 2000; Mileset al., 2000; Hidalgo et al., 2001). The binding of copper to mam-malian MTs plays mainly a role in copper sequestration in copper-related diseases such as Menkes andWilsons diseases (Tapiero et al.,2003). MTs also play a critical role in the chemotherapy of certaincancers, both in the development of tolerance to chemotherapeuticsand as an adjunct to reduce toxic side-effects (Cherian et al., 2003;Theocharis et al., 2004).Differential expressionof mammalianMTisoformsistightlyregulated during development and in pathological situations (Mileset al., 2000; Theochariset al., 2004). Inmammals, four distinctMT isoforms exist designated MT-1 through MT-4. They are acidic67-kDa proteins possessing a novel type of metal-thiolate cluster.MT-1andMT-2areexpressedinalmostalltissues. Theirbiosyn-thesis is inducible by a variety of stress conditions and compoundsMETALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch01Metallothionein Structure and Reactivity 5includingglucocorticoids,cytokines,reactiveoxygenspecies,andmetalions(Milesetal., 2000);whereasMT-3andMT-4arerel-ativelyunresponsivetotheseinducers. AlthoughMT-1/MT-2arecytosolic proteins, during development they have also been detectedin the nucleus (Nartey et al., 1987). MT-3, also known as the growthinhibitoryfactor(GIF), hasbeendiscoveredasafactordecientinthebrainofAlzheimers diseasepatients. Theproteinoccursintracellularly and extracellularly, and exhibits neuronal growthinhibitoryactivityinneuronal cell cultures(Uchidaet al., 1991).MT-3 expression is primarily conned to the central nervous system(CNS), where it represents a major component of the intracellularZnIIpoolinzinc-enrichedneurons(ZENs)(Mastersetal.,1994).LowerexpressionlevelsofMT-3havealsobeenreportedinpan-creas, kidney, stomach, heart, salivary glands, organs of the repro-ductivesystem,andmaternaldeciduum(Sogawaetal.,2001;Irieet al., 2004). The expression of the lastly identied mammalian met-allothionein isoform, MT-4, has been found restricted to cornied andstratied squamous epithelium (Quaife et al., 1994). The develop-mentally regulated MT-4 expression in maternal deciduum togetherwith the expression of the entire MT gene locus have been reportedin mouse (Liang et al., 1996).2. Gene Structure and RegulationBased on the classication by Binz and Kgi (1999), the mammalianMT gene family belongs to the vertebrate family (family 1), whichis characterized by the consensus sequence K-Xaa(1,2)-C-C-Xaa-C-C-P-Xaa(2)-C, where Xaa stands for other amino acids. Within thisfamily, mammalianmetallothioneinsconstitutefourdifferentsub-families designated m1 (MT-1) through m4 (MT-4). In Homo sapiens,a signicant genetic polymorphism exists. The human MT genes arelocatedonchromosome 16q13andare encodedbya multigene clusteroftightlylinkedgenes. Thereareat least sevenfunctional MT-1genes (MT-1A, MT-B, MT-E, MT-F, MT-G, MT-H, and MT-X) and aMETALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch016 M. Vak and G. Melonisingle gene encoding the other MT isoforms MT-2 (MT-2A), MT-3,and MT-4 (Cherian et al., 2003). A number of other MT or MT-likegenes and pseudogenes with a signicant homology to functional MTgenes exist within the human genome, but their functionality is so farunknown. The MT genes encode for two-domain proteins. The threeexons composing the human MT genes sequentially encode for theN-terminal region of the -domain (exon 1), the rest of the -domain(fromresidue 11 or 12; exon 2), and all of the -domain (fromresidue31 or 32; exon 3). Exons 2 and 3 are spliced at the junction of codonsfor the Lys/Lys or Arg/Lys residues in the interdomain region.The promoter regions of the MT-1 and MT-2 genes contain severalmetal-responsiveelements(MREs) andglucocorticoid-responsiveelements(GREs)aswellaselementsinvolvedinbasalleveltran-scription (BLEs). MT expression is also regulated by oxidative stressby antioxidant responsive elements (AREs) or by MREs which arealso responsive to oxidants. The metal-regulatory transcription factor(MTF-1), which is essential for basal expression and induction byzinc, bindstoproximal promoter MREs. MTF-1bindstoMREsthrough its six C2H2 zinc ngers (Heuchel et al., 1994). The DNAsequences responsible for cell-specic regulation of MT-3 and MT-4genes are currently unknown. The regulation of tissue-specic MT-3gene expression does not appear to involve a repressor; thus, othermechanisms such as chromatin organization and epigenetic modica-tions may account for the presence or absence of MT-3 transcription.3. Structure of Mammalian Metallothioneins3.1. Metallothionein-1 and Metallothionein-2The metal-free protein, also named apo-metallothionein or thionein,possessesapredominantlydisorderedstructure, whichrendersitvulnerable to proteolysis (Winge and Miklossy, 1982). However, awell-dened structure develops upon metal binding. The structuralfeaturesofmammalianmetallothioneinswereforthcomingalmostMETALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch01Metallothionein Structure and Reactivity 7exclusivelyfromspectroscopicstudies. Therst direct evidencefortheexistenceofmetal-thiolateclustersinMTscamefromthe113Cdnuclearmagneticresonance(NMR)studiesofthereconsti-tuted 113Cd7MT-2 (Otvos and Armitage, 1980). The studies revealedthat20cysteineresiduesand7divalentmetalionsarepartitionedbetween two metal-thiolate clusters, a cyclohexane-like three-metalcluster (MII3(Cys)9) in the N-terminal -domain (residues 130) andanadamantane-relatedfour-metal cluster (MII4(Cys)11) intheC-terminal -domain (residues 3161). In these clusters, the metal ionsarecoordinatedbybothterminaland2-bridgingthiolateligands(Otvos and Armitage, 1980) in a tetrahedral-type symmetry (Vak,1980).Agreat deal ofknowledgeonthechemical featuresofMT-1/MT-2 pertinent to their function has arisen from the determination oftheir three-dimensional (3D) structures. The 3D structures of MT-1/MT-2fromvariousspecieswereobtainedmainlybyNMRspec-troscopy (Arseniev et al., 1988; Schultze et al., 1988; Messerle et al.,1990b; Zangger et al., 1999), but alsobyX-raycrystallography(Robbins et al., 1991; Braun et al., 1992) (Figs. 1 and 2). The reported3D structures reveal a similar monomeric two-domain protein of aFig. 1 Schematic drawing of the two metal-thiolate clusters in mammalian Zn2Cd5MT-2(Robbins et al., 1991). The metal atoms (MII) are shown as shaded spheres con-nectedtosulfur atoms. Themodels weregeneratedwiththeprogramPyMOLv0.99( using the Protein Data Bank (PDB) coordinate 4mt2.METALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch018 M. Vak and G. MeloniFig. 2 The three-dimensional crystal structure of rat Zn2Cd5MT-2 (top) (Robbins et al.,1991) and the NMR solution structure of the-domain of human113Cd7MT-3 (bottom)(Wangetal., 2006).TheCd2+andZn2+ionsareshownasshadedspheresconnectedto the protein backbone by cysteine thiolate ligands. The models were generated with theprogramPyMOLv0.99 ( using the PDBcoordinates 4mt2and 2f5h.dumbbell-like shape featuring the cluster topology shown in Fig. 1andanidenticalpolypeptidefolding.ThetwoproteindomainsinMT-1/MT-2 are connected by a exible hinge region of a conservedLys-Lys sequence in the middle of the polypeptide chain.Theexibilityoftheproteinbackbonestructureenfoldingthemetal core in MT-1/MT-2 is well documented. Both the calculatedroot meansquaredeviation(RMSD)valuesfromNMRdataandthe crystallographic B-factors indicate that a considerable degree ofdynamic structural disorder exists (Schultze et al., 1988). Direct evi-dence for the nonrigid MT structure came from the 1H NMR studiesof 1H2H amide exchange in Cd7-MT-1/Cd7-MT-2 (Messerle et al.,1990a; Zangger et al., 1999). In these studies, the enhanced exibilityMETALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch01Metallothionein Structure and Reactivity 9ofthelessstructurallyconstrained-domaincomparedtothe-domain in both isoforms has been demonstrated. Molecular dynamicssimulations of the-domain of rat liver MT-2 in aqueous solutionalso showthat the polypeptide loops between cysteine ligands exhibitan extraordinary exibility without disrupting the geometry of thethree-metal cluster (Berweger et al., 2000).Because of the exibility of the polypeptide loops between cys-teine ligands, it was generally assumed that the protein could accom-modate a wide range of divalent metal ions of different sizes withoutany selectivity. However, in inorganic polynuclear adamantane-likecages, both the metal size and the varying length of the metal-thiolatebondsgiverisetowidelydifferingcluster sizesandthusclustervolumes (Hagen et al., 1982). As a consequence of this effect, thebinding of different metal ions to MT should lead to an expansion ofthe cluster core, thus inuencing the energetics of the folding process.The metal selectivity of MT-1/MT-2 structures has been studied byoffering seven equivalents of two divalent metal ions i.e. CoII/CdII,ZnII/CdII, CoII/ZnII, and FeII/CdII in a 3:4 ratio to the apoprotein,followed by the determination of the respective metal distributionswithinandbetweenthe clusters byelectronic absorption, mag-netic circular dichroism (MCD), 113Cd NMR, 57Fe Mssbauer spec-troscopy, and electron paramagnetic resonance (EPR) spectroscopy,asrequired(Goodet al., 1991;Pountneyand Vak, 1992). Asaresult, offering CoII/CdIIions resulted in CoII3Cd4MT-2 in which twohomometallic clusters, i.e. the CoII3-thiolate cluster in the -domainandtheCd4-thiolateclusterinthe-domain, werepresent. Con-versely, in the case of FeII/CdII, the homometallic species FeII7MT-1andCd7MT-1intheratioof addedmetal ions wereformed.Although CoII/ZnIIand ZnII/CdIIgave rise to heterometallic clustersin CoII3Zn4MT-1 and Zn3Cd4MT-2, they showed a distinct metal dis-tribution. Thus, anappreciable selectivityinmetal partitioningamongandwithinthe clusters is indeedobservedinthis protein. These resultsare opposite to those obtained in the studies of inorganic adamantane-like cages with monodentate thiolate ligands of the general formulaMETALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch0110 M. Vak and G. Meloni[M4(SPh)10]2 (M=CdII, ZnII, CoII, FeII) where, despite the differ-ences in the metal-thiolate afnities and in the homometallic clustervolumes, a simple mixing of two homometallic cages always pro-duced heterometallic cage complexes with an almost statistical dis-tributionofbothmetalions(Hagenetal., 1982). Sincethesamemetal ions were employed in the studies of MT-1/MT-2, propertiesof the MT structure are responsible for this effect. It has been con-cluded that interplay between both the chemistry of metal ions and thesteric requirements of the protein structure determines the diversityof metal-thiolate cluster structures in MT-1/MT-2. In this context, itshould be noted that small structural differences between Zn7-MT-2and Cd7-MT-2, due to differences in the cluster volumes (approxi-mately 20%), have been observed (Braun et al., 1992).The coordination of the metal ions is the major determinant in thefolding of the polypeptide chain around the two clusters of MT-1/MT-2.ThepathwayofclusterformationhasbeendeterminedforCd7MT-2andCo7MT-2by113CdNMRandEPR/paramagnetic1HNMR, respectively. Becauseofthesimilarsizeandchemistryof CoIIand ZnII, the isostructural substitution of ZnIIby CoIIis awidely used method to probe the spectroscopically silent zinc sites inproteins. The studies revealed that the formation of both clusters inCd7MT-2 is cooperative and sequential, with the four-metal clusterin the -domain being formed rst (Good et al., 1988). However, theformation of CoIIclusters in Co7MT-2, and by inference also ZnIIclusters, proceeds by a different pathway; in this case, prior to thecluster formation in the-domain, the rst four CoIIare bound inindependent CoIIsites in tetrathiolate coordination (Vak and Kgi,1981; Bertini et al., 1989).At neutral pH, closelysimilar averageapparent stabilitycon-stants have been determined for MT-1/MT-2 by different methods,withvaluesoftheorderof1011M1and1014M1forZnIIandCdII, respectively (reviewed in Romero-Isart and Vak, 2002). Therecent development of uorescent metal chelators withdifferentmetal afnities allowed the dissection of average apparent stabilityMETALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch01Metallothionein Structure and Reactivity 11constants. Althougheachof thesevenZnIIions inZn7MT-2isbound in a tetrathiolate coordination environment, analysis of ZnIIbinding to thionein revealed at least three classes of binding sites withafnities that differ by four orders of magnitude (Krezel and Maret,2007). One ZnIIion in Zn7MT-2 is relatively weakly bound (log K=7.7), making MT a zinc donor. Moreover, it has been suggested thatphysiological ligands, including thionein, would further modulate themetal binding and the redox reactivity of thiols in the cellular envi-ronment. These chemical characteristics suggest how the molecularstructures and redox chemistries of fully and partially metallated MTand thionein may contribute to the variety of different functions thatMT may serve in the cell.Copper binds readily to MTin vivo and in vitro (Kgi and Schffer,1988). Both Wilsons and Menkes diseases in human are inborn dis-ordersofcoppermetabolisminwhichexcesscopperaccumulatesintracellularlyinMT.Inallinstancesexaminedtodate,copperisbound to MT as CuI. The only available 3D crystal structure of CuI-thiolate clusters in MT is that of yeast Cu8MT. The structure showsthe largest known oligonuclear CuI8-thiolate cluster in biomolecules,consisting of six trigonally and two diagonally coordinated CuIions(Calderone et al., 2005). However, the 3Dstructure of CuIcontainingmammalian MT-1/MT-2 is so far unknown. The structural features ofCuI-thiolate clusters inmammalianMTs have beenstudiedbyvariousspectroscopictechniques(Stillman,1995). Thecurrentknowledgeis limited to the fact that fully CuI-loaded MT-1/MT-2 bind 12 CuIions into two metal-thiolate clusters, where, in contrast to divalentmetal ions, the monovalent copper ions are coordinated by two orthree cysteine ligands forming two independent CuI6-thiolate clusters(Nielson et al., 1985; Stillman, 1995). In these studies, the Zn7MT-1 form was titrated with CuIions. However, the binding studies ofCuIto thionein revealed that, at a lower CuI/protein stoichiometry,two distinct CuI4-thiolate clusters are formed in both protein domains(Pountney et al., 1994). There is evidence to suggest that CuIbindspreferably to the less structurally constrained -domain (Nielson andMETALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch0112 M. Vak and G. MeloniWinge, 1984), and CdIIand ZnIIto the -domain (Nielson andWinge,1983). Arecent NMRinvestigation of both synthetic domains of MT-1 lled with CuIor ZnIIions revealed signicant differences in theirrespective polypeptide folds. This observation signies the differentcoordination geometries required for the binding of monovalent anddivalent metal ions (Dolderer et al., 2007).3.2. Metallothionein-3Although MT-1/MT-2 and MT-3 share a conserved array of 20 cys-teine residues, evolutionarily conserved changes in the primarystructure of MT-3 exist. These are a T5 insert followed by a uniqueC6-P-C-P9 sequence within the -domain, and an acidic hexapeptideinsert within the -domain (Fig. 3).Thestructural studiesonrecombinant Zn7MT-3andCd7MT-3established the presence of two mutually interacting protein domains,resemblingthosereportedforMT-1andMT-2,witheachdomainencompassing a metal-thiolate cluster (Faller andVak, 1997;Bogumil et al., 1998; Hasler et al., 1998; Faller et al., 1999).Thepresenceofathree-metalclusterintheN-terminal -domain(residues 131) and a four-metal cluster in the C-terminal -domain(residues 32-68) of ZnIIand CdIIcontaining MII7MT-3 was inferredfrom the studies of separate- and-domains. The metal-bindingafnity of MT-3 is weaker than that of MT-1/MT-2, and the metalbindingtoMT-3isnoncooperative(Hasleret al., 2000; Palumaaetal., 2002).WhencomparedtoCd7MT-1/Cd7MT-2, amarkedlyincreased structure exibility and cluster dynamics exist in Cd7MT-3.This informationwas forthcomingfrom113CdNMRstudies ofFig. 3 KALIGN amino acid sequence alignment of four human metallothionein isoforms,with the conserved residues highlighted. The gure was generated with the programESPriptversion 2.2.METALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch01Metallothionein Structure and Reactivity 13Cd7MT-3, in which a signicant broadening of all NMR resonancesand a very lowand temperature-independent intensity of the Cd3Cys9cluster resonances have been interpreted in terms of dynamic pro-cesses acting on two different NMR time scales: (1) fast exchangebetweenconformational clustersubstatesand(2)additional, veryslow exchange processes between congurational cluster substatesin the -domain (Faller et al., 1999). The changes in conformationalsubstatesmaybevisualizedasminordynamicuctuationsofthemetal coordinationenvironment, andthoseofthecongurationalsubstates as major structural alterations brought about by temporarybreaking and reforming of the metal-thiolate bonds. Therefore, onlythe 3D structure of the -domain of mouse and human 113Cd7MT-3could be determined by NMR (Fig. 2, bottom). The structure revealsa peptide fold and a cluster organization very similar to those found inCd7MT-1/Cd7MT-2, with the exception of an extended exible loopencompassing the acidic hexapeptide insert (z et al., 2001; Wanget al., 2006).Since the mutation of conserved proline residues in the T5CPCP9motif of MT-3 to Ala and Ser the amino acids present in MT-2 abolished the neuroinhibitory activity and cluster dynamics, it hasbeensuggestedthat dynamic events centeredat the three-metalcluster of MT-3 would include a partial unfolding of the-domainand that the cis/trans interconversion of Cys-Pro amide bonds wouldgovern the kinetics of this process (Hasler et al., 2000). Additionalinsightsintothisprocesswereprovidedbyamoleculardynamics(MD)simulationofthe-domainofCd7MT-3(Ni et al., 2007).The studies revealed that, due to the structural constraints introducedby the T5CPCP9 motif, an unusual conformation of the N-terminalfragment (aa 113) is formed when compared with Cd7MT-2, andthattheformationofthetrans/transisomerisenergeticallymorefavorable. Further simulation of the partial unfolding supported theproposed role for cis/trans interconversion of Cys-Pro amide bondsin the folding/unfolding process of the -domain. Other studies usingthe chimeric MT forms, generated by swapping the MT domains ofMETALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch0114 M. Vak and G. MeloniMT-3 and MT-1, showed that the-domain via domaindomaininteractions modulates the bioactivity of the-domain of MT-3and that, besides T5, P7, and P9 mutations (Hasler et al., 2000), theE23K mutation also abolishes the growth inhibitory activity of MT-3 (Ding et al., 2006; Ding et al., 2007). Although the mechanismsunderlining these effects remain to be elucidated, the results obtainedsofarsuggestthatthestructureofthe-domainofMT-3issub-jected to a ne tuning. Taken together, the changes in the primarystructure of MT-3 in comparison with MT-1/MT-2 result in extraor-dinary and unprecedented structure dynamics and thus in the alter-ationof surfacetopologyimportant for itsbioactivity, playingaputative role in proteinprotein or proteinreceptor interactions.The specic binding of intracellularly occurring neuronalZn7MT-3tothe small GTPase Rab3Ainits GDP-boundstate has beendemonstrated, and the nature of the complex characterized. Rab3Aiscritically involved in the exo-endocytotic cycle of synaptic vesicles,including neuronal zinc vesicles. This nding indicates that Zn7MT-3actively participates in the synaptic vesicle trafcking upstream ofvesicle fusion, playing a chaperone, sensor, and/or effector role inthisprocess(Knippetal., 2005).TheidenticationofMT-3asacomponent of a brain multiprotein complex with heat shock protein84 (HSP84) and creatine kinase (CK) has been linked to the peculiarstructural properties of the protein described above (El Ghazi et al.,2006).As isolated from the brain, MT-3 contains both CuIand ZnIIionsinCu4,Zn34MT-3. TheextendedX-rayabsorptionnestructure(EXAFS) studies of this species revealed the presence of twohomometallicclusters:aZn34-thiolateclusterandaCuI4-thiolateclusterwithZnIIandCuIionstetrahedrallyandtrigonallycoordi-nated, respectively (Bogumil et al., 1998). A striking feature of theCuI4-thiolate cluster, which by immunochemical methods was foundto be localized in the -domain (Roschitzki and Vak, 2002), is itsremarkablestabilityagainstairoxidation.Bycontrast,thestudieson a well-dened Cu4Zn4MT-3 form prepared by a selective metalMETALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch01Metallothionein Structure and Reactivity 15reconstitutionofbothdomainsinvitrorevealedthepresenceofaredox-labilezincsiteintheZn4-thiolateclusterlocatedinthe-domain. Whileunder anaerobicor reducingconditions theZn4-thiolate cluster is stable, a partial oxidation of specic thiolate ligandsin air results in ZnIIrelease, giving rise to a Zn3-thiolate cluster inthe -domain (Roschitzki and Vak, 2003). It may be noted that theneuroinhibitory activity of MT-3 in cell cultures was found for bothCu4Zn34MT-3andZn7MT-3metalloforms(Uchidaet al., 1991;Erickson et al., 1994). However, comparative biological studies onwell-dened metalloforms are currently lacking.In metal-linked neurodegenerative disorders likeAlzheimersdisease, a dysregulated copper homeostasis and related neurotoxicityarelinkedtotheproductionofreactiveoxygenspecies(ROS). Aprotective role of extracellularly occurring Zn7MT-3 against copper-mediated toxicity has been suggested based on its reactivity with freeCuIIions in vitro. The studies showed that Zn7MT-3, through CuIIreductiontoCuIbythiolate ligands andbindingtothe proteinforminganair-stableCu(I)4Zn4MT-3species,canefcientlyscavengeandredox-silence the toxic free CuIIions (Meloni et al., 2007).3.3. Metallothionein-4The last identied member of the mammalian MT family is MT-4.This isoformconsists of 62aminoacids, showinganinsert ofGluinposition5relativetoMT-1/MT-2proteins(Fig. 3). MT-4appears to be present exclusively in cornied and stratied squamousepithelium. Much of the information on MT-4 deals with gene reg-ulationmolecular biologyandexpressionprolesinmammalianmaternal deciduum(Liang et al., 1996), and during epitheliumdevel-opmentandphysiology(Quaifeetal.,1994;SchlakeandBoehm,2001). All of these studies revealed that the MT-4 gene is subject toa strict developmental regulation. However, the question of whetherMT-4 is involved in copper or zinc metabolism in epithelia is stilldebated.METALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch0116 M. Vak and G. MeloniInsights into the function of a protein have been inferred from itsstructural properties. From the studies of metal-binding abilities ofMT-4 using heterologously expressed MT-4 with zinc, cadmium, andcopper in combination with the in silico protein sequence analyses,thecopper-bindingnaturehasbeensuggested(Tioet al., 2004).However, from biological studies, a special role of MT-4 in the reg-ulation of zinc-dependent processes in keratinocytes has also beenproposed(Quaifeet al., 1994). Thisfunctionissupportedbythestructural studiesonCd7MT-4inwhichsimilaraverageapparentmetal-bindingafnitiesoftwometal-thiolateclustersinCd7MT-4and Cd7MT-1/Cd7MT-2, overall similarities in cluster topologies tothatdeterminedforCd7MT-1/Cd7MT-2(Fig. 2), andthepathwayofclusterassemblyhavebeenfound(Melonietal., 2006).Thus,MT-4maybe involvedinbothzinc andcopper metabolisminkeratinocytes.4. Reactivity of Metal-Thiolate ClustersAlthoughthethiol groupsinMTaremaskedthroughtheirinter-action with metal ions, they retain a substantial degree of the nucleo-philicity seen with the metal-free protein. This property is reectedby the extremely high reactivity of the coordinated cysteine ligandswith alkylating and oxidizing agents such as iodoacetamide or 5,5

-dithiobis-(2-nitrobenzoicacid)(DTNB),respectively(Shawetal.,1991). Different sulfur reactivities in both domains have also beenreported. Kinetic, mass spectrometric, and NMR studies have shownthat the kinetically preferred reaction of mammalian MT with elec-trophiles may be localized in either the - or the -domain, dependingonthespecicattackingreagent. For instance, whereas iodoac-etamide andp-(hydroxymercuri)benzoate have been found to reactpreferentially with the -domain, DTNB, aurothiomalate, melphalan,and chlorambucil have been shown to react preferentially with the -domain (Yu et al., 1995; Zaia et al., 1996; Muoz et al., 1999). Cys-teine residues of the zinc-thiolate clusters in Zn7MT-1/Zn7MT-2 canMETALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch01Metallothionein Structure and Reactivity 17also be oxidized by mild cellular oxidants like oxidized glutathione(GSSG), releasingboundmetalionsinthisprocess(Jacobetal.,1998).ExperimentsinthepresenceoftheGSH/GSSGredoxpairprovided evidence for an oxidoreductive mechanism modulating thezinc afnity of the cysteine thiolate ligands in Zn7MT in vitro (Jianget al., 1998; Maret andVallee, 1998). The importance of redox-activecysteine ligands in zinc proteins, especially Zn7MTs, in convertingredox signals to zinc signals in a cellular environment has also beendiscussed (Krezel et al., 2007).Another interesting aspect of MT reactivity is its ability to reactwith radical species. Thus, it has been shown that mammalian MII7MT(M =ZnIIand/orCdII)areefcientscavengersoftheROSsuchashydroxyl (OH)andsuperoxide(O2 )radicalsorthereactivenitrogen species (RNS) such as nitric oxide (NO) (Thornalley andVak, 1985; Krncke et al., 1994). In all instances, the free radicalattackoccursat themetal-boundthiolates, leadingtotheproteinoxidation and/or modication and subsequent metal release. Inter-estingly, inmanyinstances, theseeffectscouldbereversedunderreductive conditions and in the presence of the appropriate metal ion.The protective role of MTs against ROSdamage in biological systemsis well documented (reviewed in Fabisiak et al., 2002). Although thereactivity of MT-1/MT-2/MT-3 with ROS is comparable, MT-3 canscavenge free NO and NO fromS-nitrosothiols more efciently thanMT-1 and MT-2. In this process, the zinc-thiolate bonds are targetsfor both a direct attack by free NO andS-nitrosothiols, leading tothe CysS-NO formation and metal release. Further reaction of theseCysS-NO groups results in the formation of intramolecular disuldebonds in the MT structure. 1H and 113Cd NMR studies of Cd7MT-1revealed that, whereas NO selectively releases the metals from theN-terminal-domain, the C-terminal-domain is less sensitive tothe NO attack (Zangger et al., 2001); in contrast, S-nitrosothiols ef-ciently release zinc fromboth the - and -domains of Zn7MT-3. TheS-nitrosylation of MT-3 byS-nitrosothiols occurs via a transnitro-sation reaction. The increased reactivity of MT-3 with free NO andMETALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch0118 M. Vak and G. MeloniS-nitrosothiolsledtotheproposalthatZn7MT-3mayspecicallyconvert NO signals into zinc signals (Chen et al., 2002).Studies on ligand substitution reactivity have revealed thatsmall multidentate ligands (e.g. polyamines, polyaminocarboxylates,bis(thiosemicarbazones)) are effective competitors for zinc bound toMT in reactions which are much faster than the dissociation rate con-stant for zinc in Zn7MT-1/Zn7MT-2, implying a direct competitionmechanism (Petering et al., 1992). Biphasic kinetics and differentialreactivityof thetwometal clusters, - and-domains, inmam-malian MII7MT have been measured with ethylenediaminetetraaceticacid(EDTA)showing>(Ganetal., 1995), andinoppositeorder> with nitrilotriacetic acid (NTA) (Li and Otvos, 1998);on the other hand, bidentate ligands such as ethylenediaminediaceticacid and triethylenetetramine were found to be ineffective, even atthermodynamically competent concentrations. From these studies, ithas been suggested that a tripod conguration of chelating ligandsisrequiredinthiolatesubstitutionandthat onlyspecicregionsoftheproteindomainsmayprovideaneasyaccesstothemetalclusters. In this context, it may be noted that, in the crystal structureof Zn2Cd5MT-2, each domain contains a solvent-exposed cleft con-taining three accessible cysteine sulfurs. In summary, the reactivityof the MT structure, which is pertinent to its function, is dominatedby the chemistry of the nucleophilic thiolate groups.5. Structure DynamicsDespite the high thermodynamic stability of the metal-thiolateclustersinMT, andasaconsequenceofthestructuredynamics,theyarekineticallyverylabile, i.e. thethiolateligands allowarapid metallation and demetallation. The rate of metal exchange inMT-1/MT-2followstheorderHgII>CdII>ZnII. Acomparisonwith similar studies on inorganic complexes affords the most plau-sible explanation for the kinetics of metal exchange (Martell et al.,1994). In the latter studies, a correlation between the level of ligandMETALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch01Metallothionein Structure and Reactivity 19preorganizationandcomplexlabilityrevealedthat a decreasingrigidity of the ligand structure results in an increasing lability of themetal complexes. Based on the already-discussed properties of metal-free and metal-containing MTs, the apoprotein with multidentate cys-teine thiolate ligands resembles chelating inorganic ligands with longbridges, for which a low level of ligand preorganization and hence ahigh kinetic lability has been shown. Evidence for the kinetic labilityof metals in Cd7MT was provided by 113Cd NMR saturation transferexperiments, which established the presence of intermolecular and/orintramolecular metal exchange within the three-metal cluster of the-domain with a half-life of the order of 0.5 seconds. The occurrenceof similar processes taking place within the four-metal cluster, butwith a half-life of about 16 minutes, was afforded by metal exchangestudies using the radioactive 109Cd isotope (Otvos et al., 1993). Theimportanceofproteindynamicsformetalexchangehasbeenrec-ognizedintheNMRstudiesofCd7MT-1inwhichtheenhancedbackbone exibility, when compared to Cd7MT-2, resulted in a muchfaster intersite cadmiumexchange in the three-metal cluster (Zanggeret al., 1999). In this context, it may be noted that in zinc enzymessuch as alkaline phosphatase and carboxypeptidase, where a well-dened catalytic site is present in a rather rigid protein structure, theexchange half-life of zinc is in the order of hours and days. Thus, theactual exchange rates of metal ions in proteins are not an intrinsicattribute of their binding properties, but rather are determined by theenergetics and kinetics of protein folding.IntermolecularzinctransferbetweenzincproteinsandZn7MTin vitro has also been studied, leading to the apoenzyme activation andthe modulation of zinc-dependent transcription factors (Jacob et al.,1998; Roesijadi et al., 1998). Moreover, Zn7MT has been shown tobe able to transfer one zinc ion to apoenzymes possessing a lowerafnity compared to the apparent average binding constant for zincin Zn7MT. However, the presence of one weakly bound metal ion inthe structure of Zn7MT has recently been demonstrated (Krezel andMaret, 2007).METALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch0120 M. Vak and G. Meloni6. Metallothioneins in Inorganic PharmacologyMetallothionein, owing to its afnity to a wide range of metal ions,can through a direct interaction with inorganic drugs inuence theirefcacy. Amongothers,theMT-1/MT-2reactivitywithanticancerplatinum-baseddrugs, bismuthdrugs, andgold-containingdrugsused in rheumatoid arthritis has attracted particular interest. Platinumdrugs are effective chemotherapeutic agents for the treatment of tes-ticular cancer and are used in combination therapy for a variety ofother tumors. However, the occurrence of intrinsic resistance in sometumors, and that acquired after initial treatment, are the major draw-backs of these chemotherapeutics. Among other responses leadingto the resistance, the high reactivity of platinum compounds with themajorintracellularthiolsglutathione(GSH)andMTconferresis-tance to these drugs. However, the cysteine ligands in Zn7MT reactwith cisplatin much faster than the thiol group in GSH. As a conse-quence, in an anticancer treatment with platinum-based electrophylicantineoplastic drugs including cisplatin, an overexpression of MTsin cancer cells is observed. A direct interaction of these drugs withMTsisbelievedtobetheprimarycauseoftumorcell resistance(Kelley et al., 1988). A detailed study on the interaction of Zn7MT-2with a number of cis- and trans-PtIIcompounds, including a new-generation PtIIdrug with potential use in the clinic, showed that allligands in cis-PtIIcompounds, including cisplatin, are replaced bycysteine thiolates of the protein, thereby fully deactivating the drug(Knipp et al., 2007). In contrast, the protein-bound trans-PtIIcom-pounds retained their N-donor ligands, thus remaining in a potentiallyactive form; however, no trans-PtIItransfer from MT-2 to plasmidDNA was observed. During the binding process of PtII, the corre-spondingamount ofZnIIisreleasedfromMT-2. Thebindingofreleased zinc to the metal-responsive transcription factor (MTF-1),which activates MT-2 expression, represents an unrecognized aspectcontributingtothecellularresistanceagainstplatinum-basedanti-cancer drugs.METALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch01Metallothionein Structure and Reactivity 21The preinduction of MT-1/MT-2 by bismuth compounds, whichinduce these proteins almost exclusively in the kidney, can reducetherenaltoxicityofcisplatin.Inaddition,bismuthcomplexesarealsousedasantiulcerdrugs.BiIIIbindsstronglytoMTs,formingBiIII7MT. EXAFS studies of BiIII7MT revealed that the metal coordi-nation sphere is composed of three to four sulfur atoms with someadditional oxygen atoms. This suggests that a widely different MTstructure with BiIIIions exists when compared with those reportedfor ZnII- and CdII-containing MTs.It has been suggested that MT, besides playing a possible role inthe detoxication of gold drugs in kidney, may also contribute to theretention and localization of gold in the tissues during rheumatoidarthritistreatmentwith AuIcompounds.Moreover,ahighcontentof MT in cells is apparently responsible for their resistance to thegrowthinhibitoryeffectsofAuI-auranon(arheumatoidarthritisdrug). The acquiredresistance tothe golddrugaurothiomalate maybedue in part to MT induction by a mechanism described above for PtIIcompounds. The structural studies of gold-containing MT, upon thereactions of MII7MT with gold thiomalate (AuSTm), revealed that anexcess of Zn7MT results in the formation of an AuI(SCys)2 complexinwhichthethiomalateligandisreplacedbythethiolateligand.However, an excess of AuSTm gave rise to a monodentate AuISCyscoordination and the retention of the thiomalate ligand, i.e.CysS-Au-STm (Laib et al., 1985). Furthermore, mass spectrometry mea-surements revealed that a gold-binding mechanism to MT is similarto that reported for the formation of gold nanoclusters (Mercoglianoand DeRosier, 2006).Structural informationonothermetal derivativesofMT, suchas NiII, FeII, HgII, AgI, InIII, SbIII, AsIII, and TcOIII, is alsoavailable (Vak and Romero-Isart, 2005). In view of an increasingnumber of newmetal-baseddrugs for cancer chemotherapyandtreatment of other pathologies, withsomedrugs alreadyinpre-clinical or clinical testing, further studies of MT reactivity with themetal-baseddrugsarerequiredtoshedlightonthechemicalandMETALLOTHIONEINS IN BIOCHEMISTRY AND PATHOLOGY World Scientific Puli!"in# Co$ Pte$ Ltd$"tt%&''((($(orld!cioo)!$co*'*ed!ci'+++,$"t*lAugust 18, 2008 11:32 9in x 6in B-653 ch0122 M. Vak and G. Melonibiological rationale behind the mode of action and efcacy of thesecompounds.ReferencesArsenievA, SchultzeP, WrgtterE, et al. Three-dimensional structureofrabbit liverCd7metallothionein-2a in aqueous solution determined by nuclear magnetic resonance.J Mol Biol 1988; 201:637657.Bertini I, Luchinat C, Messori L, Vak M. Proton NMRstudies of the cobalt(II)-metallothionein system. J Am Chem Soc 1989; 111:72967300.BerwegerCD,Thiel W, vanGunsteren WF. 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