Coordination of three and four Cu(I) to the a- and b-domain of vertebrate Zn-metallothionein-1, respectively, induces significant structural changes Benedikt Dolderer1, Hartmut Echner1, Alexander Beck1, Hans-Jurgen Hartmann1, Ulrich Weser1, ă Claudio Luchinat2 and Cristina Del Bianco2 Interfakultares Institut fur Biochemie, University of Tubingen, Germany ă ă ă Magnetic Resonance Center, University of Florence, Sesto Fiorentino, Italy Keywords copper; domain; metallothionein; protein structure; 2D NMR Correspondence U Weser, Anorganische Biochemie, Interfakultares Institut fur Biochemie, ă ă University of Tubingen, Hoppe-Seyler-Str 4, ă D-72076 Tubingen, Germany ă Fax Tel: +49 7071295564 E-mail: ulrich.weser@uni-tuebingen.de (Received 17 January 2007, revised 28 February 2007, accepted March 2007) doi:10.1111/j.1742-4658.2007.05770.x Vertebrate metallothioneins are found to contain Zn(II) and variable amounts of Cu(I), in vivo, and are believed to be important for d10-metal control To date, structural information is available for the Zn(II) and Cd(II) forms, but not for the Cu(I) or mixed metal forms Cu(I) binding to metallothionein-1 has been investigated by circular dichroism, luminescence and 1H NMR using two synthetic fragments representing the a- and the b-domain The 1H NMR data and thus the structures of Zn4a metallothionein (MT)-1 and Zn3bMT-1 were essentially the same as those already published for the corresponding domains of native Cd7MT-1 Cu(I) titration of the Zn(II)-reconstituted domains provided clear evidence of stable polypeptide folds of the three Cu(I)-containing a- and the four Cu(I)-containing b-domains The solution structures of these two species are grossly different from the structures of the starting Zn(II) complexes Further addition of Cu(I) to the two single domains led to the loss of defined domain structures Upon mixing of the separately prepared aqueous three and four Cu(I) loaded a- and b-domains, no interaction was seen between the two species There was neither any indication for a net transfer of Cu(I) between the two domains nor for the formation of one large single Cu(I) cluster involving both domains The first member of the metallothionein (MT) family was isolated in 1957 [1] Since then, a large number of proteins have been described featuring common characteristics They include ubiquitous small cysteine-rich proteins (50–70 amino acids) that are able to bind many d10 metal ions [2] A wealth of different biological functions has been proposed and continues to be discovered Obviously, MTs play important roles in minimizing the uncontrolled reactions of heavy metal ions like cadmium and the homeostasis of essential metal ions including copper(I) and zinc(II) ions [2,3] They are known to successfully cope with oxidative stress and ionizing radiation [4,5] Other functions may be associated with the occurrence of tissue-specific isoforms, such as the brain-specific isoform MT-3, which acts as neuronal growth inhibitory factor [6,7] Both mammalian MT-1 and MT-2 are composed of the N-terminal b- and the C-terminal a-domain They are predominantly isolated containing zinc or cadmium exclusively bound to cysteine thiolates The nine cysteine residues of the b-domain accommodate a metal (M)(II)3S9 cluster, while 11 cysteine residues contribute to the formation of a M(II)4S11 cluster in the a-domain [8] However, under certain physiological conditions, e.g when isolated from fetal liver, mammalian MT-1 Abbreviations M, metal; MT, metallothionein FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS 2349 Murine Cu(I)a- and Cu(I)b-MT-1 domains B Dolderer et al and MT-2 are also found to be enriched with Cu(I) [9] For other members of the MT family, different metal cluster architectures were reported The previously mentioned MT-3, which is also a two-domain protein, for example, is composed of a Cu(I)4S9 cluster in the N-terminal b-domain and a Zn(II)4S11 cluster in the C-terminal a-domain [10,11] Examples for solely Cu(I) binding MTs are Cu(I)8 thionein from Saccharomyces cerevisiae and Cu(I)6 thionein from Neurospora crassa [12–14] Differently from other described MTs, these two fungal proteins consist only of a single domain harbouring homometallic Cu(I) thiolate clusters [13,14] The three-dimensional structure of Cd5Zn2MT-2, isolated from rat liver after cadmium supplementation, was determined using both NMR and X-ray crystallography [15] The entire protein is dumbbellshaped and contains two independent domains The polypeptide backbone wraps around the metal thiolate core forming the scaffold of the two domains All metal ions are tetrahedrally surrounded by four thiolate sulphur atoms In the N-terminal b-domain, the three metal ions and the three bridging thiolate sulphurs are ordered to form a distorted chair The C-terminal a-domain is characterized by an adamantane-like four-metal cluster Solution structures of 113 Cd-substituted Cd7MT-2 from rabbit, rat and human are available and revealed structural identity with the structure of Cd5Zn2MT-2 [8] Comparative NMR studies provided evidence that Zn(II) can isomorphically replace Cd(II) in MT-2 [16] This result was corroborated by NMR studies on cobalt substituted MTs, as cobalt was often used as a zinc analogue in structural investigations [17–19] The solution structure of murine 113Cd7MT-1 showed high similarity with rat liver MT-2 Its b-domain, however, turned out to be more flexible than in the latter protein, exhibiting enhanced cadmium–cadmium exchange rates [20] The structural and spectroscopic data available on Cd(II)-substituted human MT-3 indicated the formation of two metal thiolate clusters, similar to those found in MT-1 and MT-2 Further investigation of that protein pointed towards a highly dynamic structure [8] Recently, a high-resolution solution structure of the C-terminal a-domain has become available The data revealed a tertiary fold very similar to that of MT-1 and MT-2, except for a loop that contains an acidic insertion that is highly conserved in these isoforms The structure of the b-domain has escaped its experimental solution, as no characteristic signals attributable to its residues were observed using NMR On the basis of homology modelling, a backbone 2350 arrangement virtually identical to the corresponding domains in MT-1 and MT-2 was suggested [21] Despite the large number of structural data available for the MT family, only the structures of two MTs containing Cu(I) were known until now One of them is the aforementioned yeast MT whose structure was successfully determined using both 2D NMR and X-ray diffraction [22–24] This protein forms one single domain that harbours eight Cu(I) ions Six of them are coordinated by three thiolate sulphur atoms, whereas a linear binding mode was observed for the remaining two The solution structure of N crassa MT backbone, in which, like yeast, the MT solely binds Cu(I), represents the second known structure of a copper thionein [25] Its polypeptide chain wraps around the copper sulphur cluster in a left-handed form in the N-terminal half of the molecule and in a right-handed form in the C-terminal half Due to the lack of copper isotopes with NMR-active spin ½, no metal–cysteine restraints were available to solve the positions of Cu(I) within the N crassa MT polypeptide fold At present, the structural information on Cu(I)-loaded forms of mammalian MTs is rather limited In vitro, Cu(I) titrations of isolated MT-2 and its separate domains demonstrated that up to six Cu(I) ions can bind to each domain [26] In another extensive titration study, it was postulated that zinc was required for the in vivo and in vitro folding of the two domains of copper MTs [27] Replacement of Zn(II) by Cu(I) led to the proposal of the formation of Cu,Zn-MT intermediates and that, during the last steps of copper titration, the two domains merge into one However, earlier Cu(I) titration studies of rat liver MT clearly showed that the two domains remained separated [26] Additionally, the cooperative formation of (Cu3Zn2)a(Cu4Zn1)bMT)1 upon addition of Cu(I) to (Zn4)a(Cu4Zn1)bMT)1 indicated that the preference of Cu(I) for binding to the b-domain is only partial and not absolute, as widely accepted until now [27] It was of interest to shed some light on the changes of the molecular architecture of the two domains of vertebrate MT when Cu(I) is added to them For this task, the synthetic murine aMT-1 and bMT-1 domains were prepared for subsequent Cu(I) titrations Employing NMR, we obtained an interesting and unexpected picture of the Cu(I) binding to the two single domains Results and Discussion Cu(I) titration of Zn4aMT-1 and Zn3bMT-1 As both the structure of native Zn7MT-1 was known, and several Cu(I) binding stoichiometries for its two FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS B Dolderer et al Murine Cu(I)a- and Cu(I)b-MT-1 domains domains had been proposed, it was of interest to shed some more light upon their reactivity towards the presence of Cu(I) To this end, a Cu(I) titration study of the two separated domains was performed employing the combined detection of luminescence, circular dichroism and 1H NMR Solid-phase peptide synthesis was successfully employed to prepare the independent a- (residues 31–61) and b-domains (residues 1–30) of murine MT-1 Either domain was fully reconstituted under anaerobic conditions with Zn(II) to yield Zn4aMT-1 and Zn3bMT-1 For each Cu(I) titration step, a new sample was prepared in order to minimize the risk of oxidation during sample manipulation and measurement The Zn4aMT-1 and Zn3bMT-1 derivatives were separately titrated with Cu(I) under a nitrogen atmosphere to yield Cu(I)–polypeptide stoichiometries from zero to six The sample solution contained 20% (v ⁄ v) acetonitrile, as the presence of soft ligands prevents Cu(I) from disproportionation to Cu(II) and Cu(0) CD and luminescence emission was measured in order to assess the sample preparation quality and to compare the obtained results with those previously published [26,27] These physicochemical properties are exclusively attributable to the metal-thiolate chromophores that have been proven to be essential for the proper polypeptide folding in other MTs [2] The overall shape of the CD spectra was essentially the same as reported before (Fig 1) During the titration of the adomain, two positive dichroic bands developed at 248 and 300 nm, respectively, and one negative band at 275 nm The addition of Cu(I) to Zn3bMT-1 shifted the positive dichroic band at 248 to 260 nm A second positive band at 300 nm, that was not present in the spectrum of Zn3bMT-1, appeared on addition of Cu(I) As in the case of the CD spectra, the results of luminescence emission were comparable to earlier studies (Fig 2) An almost linear increase of intensity was observed until the addition of the third and fourth Cu(I) ions to the a- and b-domain, respectively Further Cu(I) addition led to a much more pronounced increase of intensity in both species Two-dimensional 1H-1H NOESY spectra of each sample were acquired at 700 MHz (Figs and 4) The spectrum of Zn4aMT-1, corresponding to the starting point of the aMT-1 titration, was consistent with a well-folded polypeptide Spin systems of the amide protons spread from 6.8 to 9.2 p.p.m Upon addition of the first equivalent Cu(I), the spin systems of the starting point remained preserved, but additional new spin systems started to appear In the spectra of the samples containing two, three and four equivalents of Cu(I), these new spin systems were prevalent with the most and strongest signals observed for the three Cu(I)-containing sample On further additions of Cu(I), the signals faded away such that the spectra of the six and seven Cu(I) titration steps were devoid of cross-peaks (not shown) For the b-domain similar results were obtained with the difference that the first addition of Cu(I) led only to the reduction of signals in the NOESY spectrum and that new spin systems appeared only after the second equivalent Cu(I) was added The spectra of the samples containing three, four and five equivalents A B 20 10 -10 Zn4- -MT Fig CD spectra of Zn4aMT (A) and Zn3bMT (B) along the titration with Cu(I) Samples containing 35 lM of the respective domains dissolved in 15% (v ⁄ v) acetonitrile, 20 mM sodium phosphate buffer pH 7.6 were prepared under nitrogen containing 48 h before the measurement of their 1H NMR spectra The observed NOESY and TOCSY spectra (not shown) consisted of the sum of the respective spectra of the single species The spectral resonances were assigned to all the protons present in the two domains and are listed in supplementary Tables S1 and S2 This result indicates that the two domains are stable and independent from each other Cu(I) is not transferred between the two domains to form new species with higher and lower Cu(I):poly2358 peptide stoichiometries As no additional NOEs and ⁄ or changes of the spectral resonances were observed in the NOESY spectrum of the mixture, an interaction of the two single domains and the formation of one single Cu(I)-containing domain with the involvement of both domains could be excluded for the present Zn(x+y)Cu7MT-1 stoichiometry Conclusion The Cu(I) titration of the independent Zn(II)-loaded domains of mouse MT-1 revealed Cu(I) stoichiometries of three and four for the a- and b-domain, respectively The presence of Cu(I) led to dramatic conformational changes of both polypeptide folds Cu(I) stoichiometries of up to six Cu(I) ions each led to the progressive disappearance of the altered structures [Correction added after publication 30 March 2007: in the preceding sentence, disappearing of the affered structurer, was corrected to disappearance of the altered structures] Unfortunately, due to the lack of metal Ỉ sulphur constraints, the Cu(I) positions within the resolved polypeptide folds remained unclear Therefore, crystallization of the newly identified Cu(I)-containing species FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS B Dolderer et al ZnxCu3aMT-1 and ZnyCu4bMT-1 seems to be the only promising way to determine how Cu(I) is coordinated by the vertebrate MT When ZnxCu3aMT-1 and ZnyCu4bMT-1 were prepared individually and combined at equal concentrations, the two domains did not affect each other The net transfer of Cu(I) and the possible formation of one single Cu(I)-containing domain were clearly excluded at least under the conditions of separated and not covalently linked domains Experimental procedures All chemicals were of analytical grade quality or better The protected amino acids were purchased from Novabiochem (Laufelfingen, Switzerland), the resin for peptide synthesis was from Rapp Polymere (Tubingen, Germany), and all ă other chemicals were from Merck (Darmstadt, Germany) Synthesis of the individual a- and b- domains The individual a- and b-domains (KSCCSCCPVGCSKCA QGCVCKGAADKCTCCA and MDPNCSCSTGGSCTCT NaCl ⁄ CitACKNCKCTSCK) of murine MT-1 consisting of residues 31–60 and 1–30 of the wild-type protein, respectively, were obtained on an Eppendorf ECOSYN P solid phase peptide synthesizer (Hamburg, Germany) The synthesis was begun from the resins containing the C-terminalprotected residues coupled to them, namely Fmoc-Ala-PHB TentaGel R and Fmoc-Lys(Boc)-PHB TentaGel R All amino acids were incorporated with the a-amino functions protected by the Fmoc group Side-chain functions were protected as tert-butyl esters (aspartic acid), tert-butyl ethers (serine, threonine), Boc derivatives (lysine) and trityl derivatives (asparagine, glutamine, cysteine) Coupling was performed using a four-fold excess in protected amino acids and the coupling reagent 2-(1H-benzotriazole-1-yl)1,1,3,3tetramethyluronium tetrafluoroborate (TBTU) over the resin loading plus 1.2 mL diisopropylethylamine (DIEA; 12.5% solution in dimethyl formamide) The cysteine residues were incorporated as their pentafluorphenylesters Before coupling of the protected amino acids, the Fmoc groups were removed from the last amino acid of the growing fragment using 25% piperidine in dimethyl formamide After cleavage of the N-terminal Fmoc group, the peptide was removed from the resin under simultaneous cleavage of the amino acid side-chain protecting groups This was accomplished by incubating the resin in a mixture of trifluoroacetic acid (12 mL), ethanedithiol (0.6 mL), thioanisole (0.3 mL) anisole (0.3 mL), water (0.3 mL) and triisopropylsilane (0.1 mL) for h The mixture was filtered, washed with trifluoroacetic acid and the combined filtrates precipitated with anhydrous ether The crude product was further purified by HPLC on a Nucleosil 100 C18 (7 lm) 250 · 10 mm column (Macherey & Nagel, Duren, ă Murine Cu(I)a- and Cu(I)b-MT-1 domains Germany) using a gradient from 10 to 90% solution B (solution A: 0.07% TFA ⁄ H2O; B: 0.059% trifluoroacetic acid in 80% acetonitrile) over 32 at a flow rate of 3.5 mLỈmin)1 The elution was monitored at 214 nm The polypeptides were assayed for purity by analytical HPLC and mass spectroscopy (supplementary Figs S1 and S2) For the measurement of ESI-MS control spectra, the dried peptides were dissolved in 100 lL of 50% methanol and 1% formic acid in water (v ⁄ v), and were analysed using syringe pump infusion The ESI-MS experiments were carried out on an Esquire3000plus ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a standard ESI source Dried peptides were dissolved in 100 lL of 50% methanol, 1% formic acid in water (v ⁄ v) and directly infused (5 lLỈmin)1 flow rate) into the ESI source using a syringe pump Mass spectra were acquired in the positive-ion mode The spray voltage was 3750 V, dry gas (6 LỈmin)1) temperature 300 °C and the nebulizer 172.37 kPa For the a-domain, an average molecular mass of 3021.3 Da (3021.7 Da, theoretical) was observed, calculated from the detected [M + 5H]5+ and [M + 4H]4+ quasi molecular ions at m ⁄ z 605.2 and 756.3, respectively For the b-domain, an average molecular mass of 3014.1 Da (3014.5 Da, theoretical) was observed ([M + 3H]3+ and [M + 2H]2+ at m ⁄ z 1005.6 and 1507.8) Copper titration conditions The lyophilized polypeptides were weighed prior to the subsequent metal additions in order to determine the correct polypeptide amount employed Being aware of the high sensitivity of apo-MT towards oxygen, especially under neutral or basic solvent conditions, all further manipulations were performed in a nitrogen atmosphere containing