Metal exchange in metallothioneins – a novel structurally significant Cd 5 species in the alpha domain of human metallothionein 1a Kelly E. Rigby Duncan, Christopher W. Kirby and Martin J. Stillman Department of Chemistry, The University of Western Ontario, London, Canada Cadmium is a known carcinogen that interferes with cellular signaling and the regulation of gene expression [1]. Metallothionein, a cysteine-rich metal-binding pro- tein, has been shown to protect the cell from toxicity by sequestering the cadmium ions via the cysteinyl thiolate ligands [2–4]. Cellular response to cadmium is dependent on the level of exposure, such that high concentrations induce cytotoxicity whereas low to moderate concentrations result in gene dysregulation and uncontrollable growth. The mechanism of cad- mium-induced toxicity is complex; however, evidence is mounting that suggests a role for Cd 2+ in the inhibi- tion of DNA repair processes [5]. Specifically, Cd 2+ is thought to impair DNA damage recognition by inter- fering with the interaction of key nucleotide excision repair component proteins at the damage site by Keywords 113 Cd NMR spectroscopy; circular dichroism spectroscopy; ESI mass spectrometry; metal exchange; metallothionein Correspondence M. J. Stillman, Department of Chemistry, Chemistry Building, The University of Western Ontario, London, ON, Canada N6A 5B7 Fax: +1 519 661 3022 Tel: +1 519 661 3821 E-mail: martin.stillman@uwo.ca Website: http://www.uwo.ca/chem/ (Received 7 January 2008, revised 27 February 2008, accepted 4 March 2008) doi:10.1111/j.1742-4658.2008.06375.x Metallothioneins (MTs) are cysteine-rich, metal-binding proteins known to provide protection against cadmium toxicity in mammals. Metal exchange of Zn 2+ ions for Cd 2+ ions in metallothioneins is a critical process for which no mechanistic or structural information is currently available. The recombinant human a domain of metallothionein isoform 1a, which encompasses the metal-binding cysteines between Cys33 and Cys60 of the a domain of native human metallothionein 1a, was studied. Characteristi- cally this fragment coordinates four Cd 2+ ions to the 11 cysteinyl sulfurs, and is shown to bind an additional Cd 2+ ion to form a novel Cd 5 a-MT species. This species is proposed here to represent an intermediate in the metal-exchange mechanism. The ESI mass spectrum shows the appearance of charge state peaks corresponding to a Cd 5 a species following addition of 5.0 molar equivalents of Cd 2+ to a solution of Cd 4 a-MT. Significantly, the structurally sensitive CD spectrum shows a sharp monophasic peak at 254 nm for the Cd 5 a species in contrast to the derivative-shaped spectrum of the Cd 4 a-MT species, with peak maxima at 260 nm (+) and 240 nm ()), indicating Cd-induced disruption of the exciton coupling between the original four Cd 2+ ions in the Cd 4 a species. The 113 Cd chemical shift of the fifth Cd 2+ is significantly shielded (approximately 400 p.p.m.) when compared with the data for the Cd 2+ ions in Cd 4 a-MT by both direct and indirect 113 Cd NMR spectroscopy. Three of the four original NMR peaks move significantly upon binding the fifth cadmium. Evidence from indirect 1 H- 113 Cd HSQC NMR spectra suggests that the coordination environment of the additional Cd 2+ is not tetrahedral to four thiolates, as is the case with the four Cd 2+ ions in the Cd 4 a-MT, but has two thiolate ligands as part of its ligand environment, with additional coordination to either water or anions in solution. Abbreviations MT, metallothionein; a-rhMT 1a, recombinantly prepared a domain of human metallothionein isoform 1a. FEBS Journal 275 (2008) 2227–2239 ª 2008 The Authors Journal compilation ª 2008 FEBS 2227 substitution for a Zn 2+ ion in the four-cysteine Zn-finger protein xeroderma pigmentosum group A protein. This is one of many examples where Cd 2+ has been shown to replace Zn 2+ in Zn-finger proteins, resulting in structural alterations and ultimately func- tional inhibition. Indeed, substitution of Cd 2+ for the Zn 2+ ions in the two-finger Tramtrack (TTK) peptide reduces the affinity of this peptide for its DNA-binding sequence [6]. However, incubation of the Cd-substi- tuted peptide with Zn-substituted metallothionein reverses the effect and restores DNA-binding ability. Similarly, Cd 2+ coordination to the Zn-finger protein TFIIIA was shown to inhibit DNA association at the internal control region of the 5S ribosomal RNA gene; however, metal exchange between Cd-TFIIIA and Zn-MT resulted in reconstitution of the functional Zn-finger protein. It is evident from these examples that metallothionein is the primary defender against Cd-induced toxicity, and that its role extends beyond merely sequestering the ‘free’ ion upon cellular expo- sure. Extraction of the Cd 2+ ion from the affected protein results in liberation of the essential Zn 2+ ion from the metallothionein pool; thus the metallothion- ein exhibits a dual function with respect to metal replacement. The physiological effects described above indicate that metal exchange or metal replacement in metallo- thioneins is a critically important process that requires mechanistic consideration; however, details of this pro- cess are completely lacking. Based on examination of the structures, we and others have previously proposed that the domain crevice acts as the initiation site for the exchange reaction due to exposure of one edge of the metal-thiolate cluster to the surrounding environ- ment [7–9]. Upon incorporation of the incoming Cd 2+ ion into the metal-thiolate cluster at the crevice site, rearrangement of the cluster is proposed to take place, resulting in expulsion of a previously coordinated Zn 2+ ion from the domain to reduce the stoichiometry back to four metal ions. This mechanism would require an intermediate that includes the metals of the domain plus the incoming metal. However, to date, no experimental data have been published to support this hypothesis. In this paper, the first structural evidence to support the formation of a cluster-expanded a domain is described. CD, NMR spectroscopic and MS data show the formation of a novel and structurally modified Cd 5 a-MT 1a species upon titration of Cd 4 a-MT 1a with a moderate excess of Cd 2+ . The additional Cd 2+ ion is proposed to coordinate to two cysteinyl sulfurs positioned near the crevice site of the domain, with the remainder of the ligand sphere probably completed with either water or chloride ions based on indirect 1 H- 113 Cd HSQC NMR data. We propose that this cluster-expanded Cd 5 a species represents a model for the intermediate in the Cd ⁄ Zn metal-exchange reaction pathway for this particular metallothionein isoform. Results The sequence of the thrombin-cleaved isolated a do- main prepared recombinantly in Escherichia coli as an S-tag fusion protein (herein referred to as a-rhMT 1a), as used in this study, is shown in Fig. 1A. This sequence encompasses the metal-binding cysteines between Cys33 and Cys60 of the a domain of native human metallothionein 1a but also includes amino acids not found in the native protein. The four diva- lent metal ions are labeled as 1, 5, 6 and 7 in accor- dance with the original NMR numbering for two-domain mammalian metallothionein (2, 3 and 4 are assigned to the three divalent metal ions in the b domain) [10,11]. The 11 cysteinyl sulfurs are labeled according to the residue number in the natural two-domain human metallothionein 1a sequence [12]. Existence of the Cd 4 (S cys ) 11 species is well docu- mented for the a domain of mammalian metallothione- ins as the result of structural characterization using a variety of techniques including NMR spectroscopy [11,13] and X-ray crystallography [14]. The isolated Cd 4 (S cys ) 11 cluster is shown in Fig. 1B: each cadmium ion (green sphere) coordinates tetrahedrally to four cysteinyl sulfurs (yellow spheres) such that five of the 11 cysteinyl sulfurs act as bridging ligands between two metal centers and the remaining six act as terminal ligands by coordinating to a single metal center. To date, this is the maximum structurally characterized Cd-to-cysteine stoichiometry observed for the single a domain. These results are based largely on studies carried out on a variety of mammalian MT species including rabbit, rat and human [15–17]. The numbering of the cadmium ions and the cyste- inyl sulfurs in Fig. 1B correspond with those in the sequence shown in Fig. 1A. Figure 1C shows the space-filling and ribbon model representations of Cd 4 a-rhMT 1a, emphasizing the wrapping of the poly- peptide backbone in a left-handed coil around the metal-thiolate cluster, which is shown in the space- filling model as located in the center of the domain. Metal exchange of Zn 4 a-rhMT 1a with Cd 2+ The exchange reaction of the Zn-substituted a domain with Cd 2+ was investigated by ESI mass spectrometry. The Zn-substituted metallothionein was prepared by A novel Cd 5 a metallothionein species K. E. Rigby Duncan et al. 2228 FEBS Journal 275 (2008) 2227–2239 ª 2008 The Authors Journal compilation ª 2008 FEBS demetallation of recombinantly isolated Cd 4 a-rhMT 1a at low pH followed by removal of the Cd 2+ ions using size-exclusion chromatography. Reconstitution with Zn 2+ was achieved by raising the pH in the presence of stoichiometric amounts of Zn 2+ . The top spectrum of Fig. 2 shows that the Zn 4 a species is the sole species formed in the reconstitution process. In all the spectra, the measured charge states were +4 and +5, with the +4 state predominant. Addition of 1.5 molar equivalents of Cd 2+ to the Zn 4 a sample results in the formation of mixed-metal species, with the Zn 3 Cd 1 a and Zn 2 Cd 2 a species predominating. The rel- ative abundance shifted to primarily the Zn 1 Cd 3 a and Cd 4 a species upon titration with 3.4 molar equivalents of Cd 2+ . When 4.7 molar equivalents of Cd 2+ are added, the Cd 4 a species is the predominant species, indicating a near stoichiometric replacement of the Zn 2+ ions with the incoming Cd 2+ ions in a non- cooperative manner. This is consistent with other reports for the Zn ⁄ Cd metal-exchange reaction [18,19]. However, titration with a moderate excess of Cd 2+ results in the appearance of a Cd 5 a species, which is shown in Fig. 2 to be present as a minor contributor upon addition of 4.7 molar equivalents, and is the dominating species with the addition of 8.2 molar equivalents of Cd 2+ . This newly identified Cd 5 a spe- cies was further characterized by CD and UV absorp- tion spectroscopy and ESI mass spectrometry by titration of recombinantly prepared Cd 4 a-rhMT 1a isolated directly from the E. coli source with excess Cd 2+ . Titration of Cd 4 a-rhMT 1a with excess Cd 2+ :CD and ESI-MS results The CD spectrum obtained for the Cd-coordinated a domain as isolated from the recombinant prepara- tion in E. coli is shown in Fig. 3A. A significant fea- ture of the spectrum with no excess of Cd 2+ is the biphasic, derivative-shaped signal, with positive extrema at 260 and 220 nm and a negative extremum at 240 nm. Many previous studies have reported this CD spectrum as characteristic of the mammalian Cd 4 a species, and it has been described as being due to exci- ton splitting between the symmetric pairs of [Cd(S cys ) 4 ] 2 groups in the Cd 4 (S cys ) 11 binding site [18– 22]. This result confirms the correct folding and domain stoichiometry of the recombinantly synthesized a domain as being the Cd 4 a species. However, closer inspection of the CD spectrum reveals a poorly defined, weak and atypical shoulder at 254 nm, indi- cating a coexisting secondary species of lower abun- dance that lacks the exciton coupling property, as a pure Cd 4 a sample results in a point of inflection at this wavelength. Such a species was found during metal replacement and cadmium-loading experiments for A BC Fig. 1. (A) Recombinant sequence of the a domain of human MT 1a showing the connectivities of the four divalent metal cations to the 11 cysteinyl sulfurs. (B) Isolated Cd 4 (S cys ) 11 cluster present in the a domain of human MT 1a. (C) Space-filling and ribbon representations of the recombinant Cd 4 a-rhMT 1a. Numbering of metal ions is based on NMR numbering of mammalian MT [11] and cysteine numbering is based on the natural human MT 1a sequence [12]. Atom legend: gray = C, blue = N, red = O, yellow = S, green = Cd. K. E. Rigby Duncan et al. A novel Cd 5 a metallothionein species FEBS Journal 275 (2008) 2227–2239 ª 2008 The Authors Journal compilation ª 2008 FEBS 2229 Cd < 4 molar equivalents; however, as we show below, the present peak is due to a species with Cd > 4 molar equivalents. Based on the ESI mass spectrometric data for the Zn ⁄ Cd metal exchange, this species can be identified as a Cd 5 a species, and conse- quently should increase in abundance upon titration with excess Cd 2+ . We note similar broadening of the CD spectrum for the Cd-substituted two-domain ba-MT 1a as isolated from E. coli [23]. Addition of Cd 2+ to the solution of Cd 4 a, up to 5.0 molar equivalents, resulting in a total of 9.0 molar equivalents of Cd 2+ in solution, results in a significant shift in the CD spectrum, leading stepwise to a mono- phasic peak at 254 nm and a reduction in peak inten- sity of the band at 223 nm to negative DA values. Despite the significant change observed in the CD spectrum, the corresponding UV absorption spectrum shows very little change upon addition of excess Cd 2+ (Fig. 3B). The loss of exciton coupling in the CD spec- trum following titration of excess Cd 2+ into the protein sample must be due to an alteration of the metal-thiolate cluster arrangement, which we associate Fig. 2. ESI mass spectra recorded for the titration of Zn 4 a-rhMT 1a with Cd 2+ at pH 7.4. Spectral changes were recorded as aliquots of Cd 2+ (3.3 mM) were titrated into a solution of Zn 4 a-rhMT 1a (15 lM) at 22 °C. Spectra were recorded at Cd 2+ molar equivalent values of 0.0, 1.5, 3.4, 4.7 and 8.2. A B C Fig. 3. (A) CD and (B) UV absorption spectral changes observed upon titrating Cd 4 a-rhMT 1a with an additional 5.0 molar equiva- lents of Cd 2+ at pH 7.4 and 22 °C. (C) The ratio of CD peak inten- sity at 254 nm per 264 nm versus molar equivalents of Cd 2+ added to a sample of Cd 4 a-rhMT 1a as a measure of Cd 5 a-rhMT 1a species formation. A novel Cd 5 a metallothionein species K. E. Rigby Duncan et al. 2230 FEBS Journal 275 (2008) 2227–2239 ª 2008 The Authors Journal compilation ª 2008 FEBS with loss of symmetry of the Cd 4 a structure. This spec- trum is reminiscent of the CD spectrum recorded for the a domain with up to three Cd 2+ ions [18,24]. Retention of the overall CD envelope shape indicates that no significant changes in the wrapping of the polypeptide backbone are induced by the additional Cd 2+ . We propose that titration of excess Cd 2+ alters only the metal-thiolate cluster stoichiometry to give the lower-symmetry Cd 5 a species (see below). Cycling the pH from neutral to acidic and then back to neutral pH results in demetallation and subsequent metallation of the protein, which can be monitored by both CD spectroscopy and ESI mass spectrometry. This reaction sequence, when applied to the Cd 4 a sam- ple, results in restoration of the Cd 4 stoichiometry upon raising the pH from 2 to 7 (data not shown). The presence of an additional 5.0 molar equivalents of Cd 2+ (total ratio of Cd 2+ : MT of 9.0) results in for- mation of the Cd 5 a species, which also reforms repro- ducibly upon cycling the pH (data not shown). A plot of the ratio DA 254 ⁄ DA 264 from the CD spec- trum approximates the ratio of Cd 5 a ⁄ Cd 4 a. Figure 3C shows that up to 5.0 additional molar equivalents are required for nearly all the metallothionein species to be converted into the Cd 5 a form. The ESI mass spectrum obtained for the purified Cd-coordinated a domain as isolated from the recom- binant preparation in E. coli is shown in Fig. 4A. The measured charge state distribution ranges from +3 to +5, with the +4 charge state as the predominant peak. Reconstruction of the mass spectrum results in a single, principal species with a measured mass of 4526.4 Da, corresponding to the Cd 4 a-rhMT species (calculated mass 4524.6 Da). Closer inspection of the original mass spectrum shows the presence of a minor peak with a measured m ⁄ z of 927, corresponding to the +5 charge state of a Cd 5 a-rhMT species. This result confirms the existence of a Cd 5 a species as a minor contributor to the equilibrium of the Cd-coordi- nated a domain of human MT 1a. Addition of 5.0 molar equivalents of Cd 2+ to the Cd 4 a-rhMT 1a solution results in the ESI mass spec- trum shown in Fig. 4B. Peaks corresponding to the Cd 4 a-rhMT species are no longer detected, and instead a new set of peaks are observed that are consistent with the formation of 100% Cd 5 a-rhMT species with a reconstructed mass of 4633.2 Da (cal- culated mass 4635.0 Da). The measured charge state distribution remains +3 to +5, with the +4 charge state predominating; however, the relative abundance of the +5 charge state has increased significantly compared to the corresponding peak in the mass spectrum of the Cd 4 a-rhMT species. Previous ESI-MS studies of globular proteins have shown a correlation between the observed charge state distribution and the solution polypeptide conformation [25–27]. In the case of metallothionein, Palumaa et al. have reported ESI-MS data showing a higher charge state distribu- tion for the Cd 4 a domain of human MT 3 compared with the Zn 4 a MT 3 by one unit [28]. Similarly, the Cd 3 b domain of human MT 3 was shown to be sig- nificantly more open in conformation compared with the Zn 3 b domain of MT 3, indicating non-isostructur- al replacement of Cd 2+ for Zn 2+ in this particular MT isoform. This charge state distribution was inter- preted by the authors as being due to a slight open- ing of the polypeptide backbone to accommodate the slightly larger cadmium ions. These data strongly sup- port our interpretation that the increased relative abundance of the higher +5 charge state species observed in the ESI mass spectrum of the Cd 5 a pre- sented here compared with the Cd 4 a species is due to expansion of the metal binding domain to accommo- date the fifth Cd 2+ . This suggests that the additional Cd 2+ ion is inserted into the core of the domain, becoming part of an expanded metal-thiolate cluster. 113 Cd NMR spectroscopy was therefore used to probe the metal-thiolate cluster arrangement in the newly identified Cd 5 a species. A B Fig. 4. (A) ESI mass spectrum observed for the Cd-coordinated a-rhMT 1a following isolation and purification of the recombinant protein from E. coli. Reconstruction of the mass spectrum results in a measured mass of 4526.4 Da corresponding to the Cd 4 a-rhMT species (calculated mass 4524.6 Da). (B) ESI mass spectral changes observed upon titrating the Cd 4 a-rhMT 1a sample from (A) with an additional 5.0 molar equivalents of Cd 2+ at pH 7.4 and 22 °C. The reconstructed mass for Cd 5 a-rhMT 1a was 4633.2 Da (calculated mass 4635.0 Da). K. E. Rigby Duncan et al. A novel Cd 5 a metallothionein species FEBS Journal 275 (2008) 2227–2239 ª 2008 The Authors Journal compilation ª 2008 FEBS 2231 Titration of Cd 4 a-rhMT 1a with excess Cd 2+ : 113 Cd NMR results 113 Cd NMR spectroscopy was used in this study to further investigate the nature of the metal-thiolate binding site in the a domain of human MT 1a both before and after the addition of excess Cd 2+ . Direct 1D 113 Cd NMR ( 1 H-decoupled) spectroscopic techniques to probe for formation of a novel Cd 5 a-rhMT 1a species The 1D 113 Cd NMR ( 1 H-decoupled) spectrum of Cd 4 a-rhMT 1a as isolated directly from recombinant overexpression in E. coli and prepared in 10 mm Tris ⁄ HCl buffer (pH 7.4) is shown in Fig. 5A. The natural isotopic abundance of 113 Cd was used in this experiment, despite the low value of 12.26%, in order to observe the naturally occurring speciation. Six sig- nals were observed in the Cd 4 a-rhMT 1a spectrum at 670, 633, 630, 626, 611 and 599 p.p.m. The chemical shift values of the five most deshielded peaks observed in the NMR spectrum are in the range of 670 to 611 p.p.m., and are in agreement with those reported previously for the four cadmium ions in the a domain of the native two-domain human MT isoform 1 [10]. These five peaks are labeled in Fig. 5A as 1, 5, 5¢,6 and 7, respectively, in accordance with the original NMR numbering assignments. Splitting of the peak assigned to the metal in site 5 has been noted previ- ously and is attributed to heterogeneity in that particu- lar site in the metal-thiolate cluster [10]. The sixth peak observed at 599 p.p.m. is more shielded than the other peaks and has not been reported previously for human MT 1 isoforms. Based on the ESI-MS and CD spectroscopic results, this peak is predicted to be due to the Cd 5 a-rhMT species, which has been shown in this report to be a minor contribu- tor to the equilibrium together with the Cd 4 a-rhMT species. Fig. 5. (A) Direct 1D 113 Cd NMR ( 1 H-decoupled) spectrum (133 MHz) for a-rhMT 1a following isolation and purification of the recombinant protein from E. coli with the natural isotopic abundance of 113 Cd, showing primarily the 113 Cd 4 a-rhMT 1a species. (B) Direct 1D 113 Cd NMR ( 1 H-decoupled) spectrum (133 MHz) for isotopically labeled 113 Cd 4 a-rhMT 1a titrated with an additional 10.0 molar equivalents of 113 Cd 2+ to form 113 Cd 5 a-rhMT 1a. The spectrum of 113 Cd 5 a-rhMT 1a (B) is a combination of two separate spectra acquired in the regions 585–705 p.p.m. and 220–245 p.p.m. Samples were prepared in 10 m M Tris ⁄ HCl pH 7.4, and buffer-exchanged into 10% D 2 O for the Cd 4 a- rhMT 1a sample and > 70% D 2 O for the 113 Cd 5 a-rhMT 1a sample. All spectra were acquired at 25 °C. A novel Cd 5 a metallothionein species K. E. Rigby Duncan et al. 2232 FEBS Journal 275 (2008) 2227–2239 ª 2008 The Authors Journal compilation ª 2008 FEBS A1D 113 Cd NMR ( 1 H-decoupled) spectrum of iso- topically enriched 113 Cd 4 a-rhMT 1a titrated with an additional 10.0 molar equivalents of 113 Cd 2+ (total ratio of Cd 2+ : MT of 14.0) is shown in Fig. 5B. For easier viewing, the spectrum is divided into two parts, the region on the left covers the chemical shift range 585–705 p.p.m. and that on the right covers the range 215–245 p.p.m. There are five relatively sharp signals at 685, 647, 630, 599 and 224 p.p.m. The peak detected at 599 p.p.m. confirms the presence of a small popula- tion of the Cd 5 a species in the naturally isolated Cd 4 a sample, as this peak was observed in the 1D spectrum of this sample. The four most deshielded peaks detected between 600–700 p.p.m. in the Cd 5 a spectrum are assigned to the four 113 Cd 2+ ions that are known to bind to the a domain of mammalian metallothion- ein in a tetrahedral coordination to four thiolate ligands. The relative assignment of these peaks to the four cadmium sites is comparable to that of the 113 Cd 4 a-rhMT species in that the order is 1, 5, 6 and 7 for the peaks 685, 647, 630 and 599, respectively. How- ever, the observed chemical shifts have changed signifi- cantly with the addition of the fifth Cd 2+ ion. Peaks 1, 5 and 6 have shifted upfield by 15, 14 and 3 p.p.m., respectively, while peak 7 has shifted downfield by 12 p.p.m. In addition, the peaks labeled 5 and 5¢ in the spectrum of Cd 4 a-rhMT 1a (Fig. 5A) have collapsed into a single peak in the spectrum acquired with excess 113 Cd 2+ (Fig. 5B), indicating a loss of heterogeneity at that site in the metal-thiolate cluster. This is expected if a fifth Cd 2+ ion results in strain in the binding site, reducing fluxionality of the metal cluster. The signal detected at 224 p.p.m. is assigned to the additional fifth Cd 2+ ion, confirming the CD spectro- scopic and mass spectrometric data regarding the for- mation of a Cd 5 a species. This peak is significantly shielded compared to the other four peaks in the spec- trum (approximately 400 p.p.m.), indicating that the coordination environment around this additional Cd 2+ is not tetrahedral to four thiolate ligands. A previous study reporting the chemical shifts of inorganic cad- mium(II)-thiolate complexes correlated signals with chemical shifts in the region of 224 p.p.m. with octahe- dral complexes of the form Cd(RS) 2 (OH 2 ) 4 [29,30]. Although the current data do not provide enough information to verify this exact ligand field assignment, as chloride ions are equally as likely as water molecules to participate as ligands, it does provide support for two thiolate groups acting as ligands for the fifth Cd 2+ ion and an increase in coordination number from four to six. This proposed ligand field assignment suggests partial insertion of the fifth Cd 2+ ion into the metal- thiolate cluster in a manner that allows solvent access. Given this restriction, the most likely position for the fifth Cd 2+ ion is the crevice site of the domain in which a number of the cysteinyl sulfurs that make up the metal-thiolate cluster are solvent-exposed. To further explore this possibility, indirect 2D NMR methods were employed as a means of probing the coordination environment around the additional Cd 2+ ion, with par- ticular emphasis on identifying the two cysteinyl sulfurs that are proposed to ligate the fifth Cd 2+ ion. Indirect 2D 1 H– 113 Cd NMR spectroscopic techniques for identification of the binding site for the fifth Cd 2+ ion in the Cd 5 a-rhMT 1a cluster The indirect 2D 1 H– 113 Cd NMR approach exploits the 3 J scalar coupling between the cysteine b protons and the coordinated cadmium ions as a means of mapping out the metal-thiolate cluster connectivities and identi- fying the cysteine residues that are coupled to the fifth Cd 2+ ion. By focusing on the 1 H chemical shift range of 2.3–3.6 p.p.m., corresponding to the cysteine H b protons only, the tetrathiolate connectivities of the Cd(S cys ) 4 units can be identified. Furthermore, sequence assignment of the cysteine residues is possible through identification of bridging versus terminal cysteine ligands in the 2D spectrum. Two-dimensional 1 H– 113 Cd HSQC NMR spectra were acquired for the 113 Cd 5 a-rhMT 1a formed by titration of 113 Cd 4 a-rhMT 1a with an additional 10.0 molar equivalents of 113 Cd 2+ (total ratio of Cd 2+ : MT of 14.0). Figure 6 shows a combination of two separate spectra acquired in the 113 Cd chemical shift ranges of 585–705 p.p.m. and 215–245 p.p.m. to allow visualization of the correlations between all five Cd 2+ ions in the Cd 5 a cluster with the 11 cysteinyl sul- fur residues. The H b – 113 Cd 3 J scalar couplings were set to 66 and 40 Hz for acquisition between 585 and 705 p.p.m. and 215 and 245 p.p.m., respectively. Four strong peaks were observed in the 113 Cd dimension of the 2D spectrum of 113 Cd 5 a-rhMT 1a in the range 585–705 p.p.m. (Fig. 6), at chemical shift values that are in agreement with those observed in the 1D 113 Cd NMR ( 1 H-decoupled) spectrum for the Cd 5 a species (Fig. 5B, sites 1, 5, 6 and 7). The fifth Cd 2+ peak was observed in the 2D spectrum acquired in the 113 Cd chemical shift range of 215–245 p.p.m., with an exact chemical shift of 224 p.p.m., which is also in agree- ment with the observed chemical shift in the 1D 113 Cd NMR ( 1 H-decoupled) spectrum (Fig. 5B, site X). Identification of the specific bridging versus terminal cysteine residues is possible in this 113 Cd-decoupled spectrum, and a nearly complete assignment of the cysteine residues in the 2D spectrum has been K. E. Rigby Duncan et al. A novel Cd 5 a metallothionein species FEBS Journal 275 (2008) 2227–2239 ª 2008 The Authors Journal compilation ª 2008 FEBS 2233 accomplished. The bridging cysteines are identified by a solid line in Fig. 6, because a single H b chemical shift correlates to two different 113 Cd atoms. The numbers written beside each peak in Fig. 6 correspond to the sequence number of the cysteine residues as labeled in Fig. 1A. This assignment is the most probable solution that satisfies the known connectivities in the metal- thiolate cluster (Fig. 1A,B). Although the H b – 113 Cd correlations are known for the four tetrahedral thiolate-coordinated 113 Cd 2+ ions, the unknown correlations of interest are those of the fifth Cd 2+ . The two peaks correlating to the fifth Cd 2+ at 224 p.p.m. were observed at 1 H chemical shift values of 2.97 and 3.59 p.p.m., which are consistent with the H b chemical shifts of cysteine residues 34 and either 57 or 59, respectively, as shown by the dotted lines in Fig. 6. This result substantiates our interpreta- tion of cluster expansion to a Cd 5 (S cys ) 11 species upon titration with excess Cd 2+ , as opposed to the fifth Cd 2+ ion attaching as an adduct to the surface of the domain. The detection of only two correlations also confirms that the coordination sphere of the fifth Cd 2+ ion includes two cysteine residues as predicted by the highly shielded chemical shift [30]. Figure 7 shows a space-filling model of Cd 4 a-rhMT 1a with a view of the crevice site showing the exposed edge of the metal-thiolate cluster. Cys34 is one of the sulfur atoms exposed in this site (highlighted in purple), which supports the NMR results indicating that this sulfur atom is involved in coordination of the fifth Cd 2+ ion. Cys57 and Cys59 are not present in the cre- vice site, so it is not immediately obvious how the sec- ond sulfur is involved in the coordination. One could envision a potential cluster rearrangement upon coor- dination to Cys34 that brings Cys57 or Cys59 into position for coordination. Discussion The in vitro reactivity of metallothionein with Cd 2+ has been well documented by reports on the native 1 H (p.p.m.) 113 CD (p.p.m.) Fig. 6. A combination of two indirect 2D 1 H– 113 Cd HSQC NMR spectra for isotopically enriched 113 Cd 4 a-rhMT 1a titrated with an additional 10.0 molar equivalents of 113 Cd 2+ to form the 113 Cd 5 a-rhMT 1a species. The spectra were recorded in the 1 H chemical shift range 2.3– 3.7 p.p.m. and the 113 Cd ranges 590–690 p.p.m. ( 3 J = 66 Hz) and 220–245 p.p.m. ( 3 J = 40 Hz). Both spectra were acquired at 25 °C using an inverse single-axis z-gradient HCX probe with X tuned to 113 Cd. Fig. 7. Space-filling model of Cd 4 a-rhMT 1a in two orientations rotated by 90°, emphasizing the crevice site on the domain. One of the proposed ligands of the fifth Cd 2+ ion, Cys34, is highlighted by the arrow and the sulfur atom of this residue is shown in purple. Atom legend: gray = C, blue = N, red = O, yellow = S, green = Cd. A novel Cd 5 a metallothionein species K. E. Rigby Duncan et al. 2234 FEBS Journal 275 (2008) 2227–2239 ª 2008 The Authors Journal compilation ª 2008 FEBS and ⁄ or recombinant two-domain protein, in addition to the isolated fragments, from many mammalian spe- cies including human, rat, rabbit and mouse [18,19, 31–33]. These spectroscopic studies involve reaction of Zn-containing or metal-free forms of metallothionein with sub-stoichiometric, stoichiometric or excess amounts of Cd 2+ . Significantly, titration of Zn 7 -MT with sub-stoichiometric molar equivalents of Cd 2+ resulted in a gradual red shift of the charge-transfer band in the CD spectrum, indicating mixed-metal speciation before saturation with seven Cd 2+ ions. This result is consistent with the isolation of the mixed-metal Cd 5 Zn 2 -MT species from in vivo sources such as rabbit liver upon exposure of these animals to Cd 2+ , indicating a non-cooperative mechanism of metal replacement [19]. The available X-ray, NMR and CD data are consistent with the Cd 4 (S cys ) 11 cluster in the a domain being adamantane-like in structure. Thus, the Cd 4 a domain observed from many mamma- lian species is characterized by a derivative-shaped CD signal and a distinct NMR spectrum. Despite the highly conserved positioning of the cysteine residues in the mammalian sequence of metal- lothionein and the structural consistencies, the behav- ior of Cd 7 ba-MT towards excess Cd 2+ has been shown to differ depending not only on the species but also on the isoform or sub-isoform of the protein within a particular species. Mouse MT 1, the sequence of which is shown in Fig. 8, has been shown by CD spectroscopy to have the capability of expanding beyond the typical seven Cd 2+ ions per metallothion- ein molecule [31,33]. Significant changes in the CD spectrum of the isolated b domain of mouse MT 1 when binding additional Cd 2+ were interpreted by the authors as the result of a Cd-induced, rearranged peptide conformation. Unfortunately there were no mass spectral data to confirm the actual number of Cd 2+ ions bound. Reaction of the isolated a domain of mouse MT 1 with excess Cd 2+ did not induce a change in the CD spectroscopic profile, indicating that this cluster did not expand beyond the Cd 4 (S cys ) 11 stoi- chiometry. The Cd-substituted two-domain human MT 3 has been shown to coordinate additional Cd 2+ ions with stoichiometries of up to Cd 13 ba-MT; how- ever, species with more than nine equivalents of Cd 2+ are reported as probably being due to adducts on the surface of the protein. The additional two Cd 2+ ions leading to the Cd 8 ba-MT and Cd 9 ba-MT species of human MT 3 are proposed to bind into the cluster regions; however, the ESI-MS results reported did not provide the structural information necessary for locali- zation of these metal ions [28,32,34]. ESI mass spectral data have been reported for the single a domain of human MT 2, prepared as a synthetic peptide, in the presence of excess Cd 2+ , in which Cd 5 a was detected as a minor species [35]. However, structural data on this species were not provided, leaving open the possi- bility that the fifth Cd 2+ acts as an adduct, as is com- monly observed in mass spectroscopy and would lead to the increased mass found in the ESI mass spectrum. Interestingly, the structurally sensitive CD spectro- scopic data reported for rabbit liver MT 2a and rat liver MT 2, isolated from natural sources, showed no change in the CD spectrum upon addition of excess Cd 2+ , indicating that these particular metallothionein species do not have the capability to expand to larger metal clusters at reasonably low levels of excess Cd 2+ [18,36]. ESI mass spectral data for the two-domain rabbit MT 1a also indicated that this protein was satu- rated at seven Cd 2+ ions, similar to the data for the rabbit MT 2a isoform [34]. While ESI-MS data have been reported previously that do show Cd 2+ binding greater than seven for the two-domain ba full chain of human MT 1a [23], no structural data have been provided to indicate the sig- nificance of the ‘over-loaded’ species. The experimental data presented here show unambiguously that a Cd 5 a species of recombinantly prepared human MT 1a is Fig. 8. Sequence comparison of human MT 1a with other isoforms of human metallothionein as well as metallothionein from other mamma- lian species for which spectroscopic data have been reported. K. E. Rigby Duncan et al. A novel Cd 5 a metallothionein species FEBS Journal 275 (2008) 2227–2239 ª 2008 The Authors Journal compilation ª 2008 FEBS 2235 formed that involves a metal binding site that is differ- ent from the Cd 4 a site. All three techniques used pro- vide specific information. First, the ESI-MS data confirm the increased metal binding stoichiometry and indicate a slight unwinding of the peptide backbone. Second, the CD spectroscopic data show a disruption of the exciton coupling with molar equivalents of Cd 2+ > 4, confirming disruption of the Cd 4 binding site. Finally, the NMR data verify the coordination of the fifth Cd 2+ ion by thiolate ligands within the clus- ter, and, more specifically, within the crevice site of the protein. The biological significance of the Cd 5 a species is an interesting question. We propose that the Cd 5 a species described here may be a model of the intermediate in the Cd ⁄ Zn metal-exchange reaction, a critically impor- tant process for cadmium detoxification. However, the data presented here only apply to the human metallo- thionein isoform 1a, although a number of reports of ‘over-loaded’ metallothioneins for other mammalian species are noted above. The ESI mass spectral data obtained for the replacement reaction of the Zn-substi- tuted a domain with Cd 2+ (Fig. 2) show a range of mixed-metal species with a stoichiometry of no more than four metal ions in any given species. The total loading of four divalent metal ions that is observed until all of the Zn 2+ ions have been replaced can be explained in terms of the relative binding constants of the two metals. The incoming metal by necessity has a higher binding constant than the metals already pres- ent, so the five-metal intermediate, when both Cd 2+ and Zn 2+ are present, is short-lived as Zn is immedi- ately displaced. In the case of human MT 1a, when four Cd 2+ ions are bound, we propose that the fifth Cd 2+ ion is simply trapped at the exchange site as no exchange can take place. Experimental procedures Materials The chemicals used were cadmium sulfate (Fisher Scientific, Ottawa, Canada), cadmium(113) chloride (Trace Sciences International Inc., Richmond Hill, Canada), deuterium oxide (Cambridge Isotopes Laboratories Inc., Andover, MA, USA), ultrapure Tris buffer, tris(hydroxymethyl)ami- nomethane (ICN Biomolecules, Irvine, CA, USA), zinc sulfate (Caledon Laboratory Chemicals, Georgetown, Canada), ammonium formate buffer (Sigma-Aldrich, Oakville, Canada), ammonium hydroxide (BDH Chemi- cals ⁄ VWR, Mississauga, Canada), formic acid (J. T. Baker Chemical Co., Phillipsburg, NJ, USA) and hydrochloric acid (Caledon Laboratory Chemicals). All solutions were produced using > 16 MWÆcm deionized water (Barnstead Nanopure Infinity, Van Nuys, CA, USA). HiTrapÔ SP HP ion-exchange columns (Amersham Biosciences ⁄ GE Health- care, Piscataway, NJ, USA), superfine G-25 Sephadex (Amersham Biosciences), a stirred ultrafiltration cell (Am- icon Bioseparations ⁄ Millipore, Bedford, MA, USA) with YM-3 membrane (3000 molecular weight cut-off) and a Mi- crocon YM-3 centrifugal filtration device (Amicon Biosepa- rations ⁄ Millipore) were used in the protein purification steps. Protein preparation The recombinant a domain of human metallothionein 1a (sequence shown in Fig. 1A) was produced by overexpres- sion in E. coli BL21(DE3) cells as an S-tag fusion protein in the presence of Cd 2+ as described previously [24]. Fol- lowing isolation and purification, the S-tag was cleaved from the protein by incubation with thrombin. Metal exchange of Zn 4 a-rhMT 1a with Cd 2+ Metal-free apo-a-rhMT was prepared by eluting the throm- bin-cleaved Cd-bound protein from a G-25 column equili- brated with deionized water that had been pH-adjusted with HCOOH to pH 2.8. Elution of the protein using an eluant of low pH effectively removes the metal ions from the protein, which are then separated from the protein band as a result of the size-exclusion processes. Preparation of apo-MT by this method simultaneously desalts the solution by the same size-exclusion process. The metal-free protein was reconstituted by adding 4.0 molar equivalents of Zn 2+ (3.0 mm stock solution) and increasing the pH to 7.4. The a-rhMT solution was determined to have a concentration of 15 lm based on UV absorption at 220 nm (e 220 = 40 000 LÆmol )1 Æcm )1 ) and atomic absorption spec- troscopy following complete metallation with Zn 2+ . Cad- mium solutions were prepared in 25 mm ammonium formate pH 7.4 (for MS studies) to a final concentration of 3.3 mm as determined by atomic absorption spectroscopy. The final samples were thoroughly evacuated and argon- saturated to remove the bulk of the oxygen from the solu- tions in order to deter oxidation of the protein. Cd 2+ was added incrementally to the solution of Zn 4 a to 8.2 molar equivalents, with thorough mixing after each titration. Mass spectra were acquired at each addition after a 2–5 min delay to allow the reaction to reach equilibrium conditions. Mass spectra were acquired on a Micromass LCT ESI-TOF mass spectrometer (Waters Micromass, Mississauga, Canada) at room temperature (22 °C), and recorded using the mass lynx software package version 4.0. The ESI-TOF spectrometer was calibrated with a solu- tion of NaI. The scan conditions for the spectrometer were: capillary, 3000.0 V; sample cone, 39.0 V; RF lens, 450.0 V; A novel Cd 5 a metallothionein species K. E. Rigby Duncan et al. 2236 FEBS Journal 275 (2008) 2227–2239 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... K-S, Onosaka S & Tanaka K (1985) Synthesis of a nonacosapeptide (betafragment) corresponding to the N-terminal sequence 1–2 9 of human liver metallothionein II and its heavy metal- binding properties FEBS Lett 183, 37 5–3 78 18 Stillman MJ, Cai W & Zelazowski AJ (1987) Cadmium binding to metallothioneins Domain specificity in reactions of a and b fragments, apometallothionein, and zinc metallothionein with... properties of metallothionein Met Ions Biol Syst 15, 21 3–2 73 23 Chan J, Huang Z, Watt I, Kille P & Stillman MJ (2007) Characterization of the conformational changes in recombinant human metallothioneins using ESI-MS and molecular modeling Can J Chem 85, 89 8–9 12 24 Rigby DuncanKE & Stillman MJ (2007) Evidence for non-cooperative metal binding to the alpha domain of human metallothionein FEBS J 274, 225 3–2 261... 113Cd 4a- rhMT 1a NMR sample The 113CdCl2 was added to the NMR sample in aliquots of 0.5, 1.0, 5.0 and 10.0 molar equivalents, and the resultant solution monitored by CD spectroscopy Following addition of the final aliquot of 113 CdCl2, the NMR sample was argon-saturated and sealed for analysis All of the NMR spectra were acquired on a Varian Inova 600 NMR spectrometer using vnmrj 1.1D software (Varian Canada... Binding excess cadmium(II) to Cd7 -metallothionein from recombinant mouse Zn7 -metallothionein 1 UV-VIS absorption and circular dichroism studies and theoretical location approach by surface accessibility analysis J Inorg Biochem 68, 15 7–1 66 Palumaa P, Eriste E, Njunkova O, Pokras L, Jornvall H & Sillard R (2002) Brain-specific metallothionein- 3 has higher metal- binding capacity than ubiquitous metallothioneins. .. metallothioneins and binds metals noncooperatively Biochemistry 41, 615 8–6 163 Capdevila M, Cols N, Romero-Isart N, GonzalezDuarte R, Atrian S & Gonzalez-Duarte P (1997) Recombinant synthesis of mouse Zn3-beta and Zn4 -alpha metallothionein 1 domains and characterization of their cadmium(II) binding capacity Cell Mol Life Sci 53, 68 1–6 88 Palumaa P, Eriste E, Kruusel K, Kangur L, Jornvall H & Sillard R (2003) Metal. .. binding to brain-specific metallothionein- 3 studied by electrospray ionization mass spectrometry Cell Mol Biol 49, 76 3–7 68 Dabrio M, Vyncht GV, Bordin G & Rodriguez AR (2001) Study of complexing properties of the alpha and beta metallothionein domains with cadmium and ⁄ or zinc using electrospray ionization mass spectrometry Anal Chim Acta 435, 31 9–3 30 Zelazowski AJ, Szymanska JA, Law AYC & Stillman... originÒ version 7.0383 (OrginLab Corp., Northampton, MA, USA) The CD spectra are measured as DA, which required conversion of the measured ellipticity in degrees divided by a factor of 33 UV spectra were acquired using a Cary 5G UV-Vis-NIR spectrophotometer (Varian Canada, Mississauga, Canada) in a 1 cm quartz cuvette at room temperature (22 °C) and recorded using the Cary win uv scan software application... the metal clusters in rabbit liver metallothionein Proc Natl Acad Sci USA 77, 709 4–7 098 12 Richards RI, Heguy A & Karin M (1984) Structural and functional analysis of the human metallothionein 1A gene: differential induction by metal ions and glucocorticoids Cell 37, 26 3–2 72 FEBS Journal 275 (2008) 222 7–2 239 ª 2008 The Authors Journal compilation ª 2008 FEBS K E Rigby Duncan et al 13 Otvos JD & Armitage... on a Jasco J810 spectropolarimeter (Jasco, Easton, MD, USA) in a 1 cm quartz cuvette at room temperature (22 °C) and recorded using spectra manager version 1.52.01 software (Jasco) The wavelength range of 20 0–3 00 nm was scanned continuously at a rate of 50 nmÆmin)1 with a band width of 2 nm All spectra were baseline-corrected using 10 mm Tris ⁄ HCl The spectral data were organized and plotted using... was A novel Cd 5a metallothionein species added as the sulfate salt dissolved in 25 mm ammonium formate buffer (pH 7.4) to a final concentration of 3.3 mm Mass spectra were acquired on a Micromass LCT ESI-TOF mass spectrometer (Waters Micromass) at room temperature (22 °C) and recorded using the mass lynx software package version 4.0 The observed spectra were reconstructed using the max ent 1 program . Metal exchange in metallothioneins – a novel structurally significant Cd 5 species in the alpha domain of human metallothionein 1a Kelly E. Rigby Duncan,. in Fig. 1A. This sequence encompasses the metal- binding cysteines between Cys33 and Cys60 of the a domain of native human metallothionein 1a but also includes