Báo cáo khoa học: Metal exchange in metallothioneins – a novel structurally significant Cd5 species in the alpha domain of human metallothionein 1a ppt

Significantly, the structurally sensitive CD spectrum shows a sharp monophasic peak at 254 nm for the Cd5a species in contrast to the derivative-shaped spectrum of the Cd4a-MT species, wi

significant Cd5 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 Cd2+ in the inhibi-tion of DNA repair processes [5] Specifically, Cd2+ 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 Zn2+ ions for Cd2+ 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 Cd2+ ions to the 11 cysteinyl sulfurs, and is shown to bind an additional Cd2+ ion to form a novel Cd5a-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 Cd5a species following addition

of 5.0 molar equivalents of Cd2+to a solution of Cd4a-MT Significantly, the structurally sensitive CD spectrum shows a sharp monophasic peak at

254 nm for the Cd5a species in contrast to the derivative-shaped spectrum

of the Cd4a-MT species, with peak maxima at 260 nm (+) and 240 nm ()), indicating Cd-induced disruption of the exciton coupling between the original four Cd2+ ions in the Cd4a species The 113Cd chemical shift of the fifth Cd2+ is significantly shielded (approximately 400 p.p.m.) when compared with the data for the Cd2+ions in Cd4a-MT by both direct and indirect 113Cd NMR spectroscopy Three of the four original NMR peaks move significantly upon binding the fifth cadmium Evidence from indirect

1H-113Cd HSQC NMR spectra suggests that the coordination environment

of the additional Cd2+ is not tetrahedral to four thiolates, as is the case with the four Cd2+ ions in the Cd4a-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.

substitution for a Zn2+ ion in the four-cysteine

Zn-finger protein xeroderma pigmentosum group A

protein This is one of many examples where Cd2+has

been shown to replace Zn2+ in Zn-finger proteins,

resulting in structural alterations and ultimately

func-tional inhibition Indeed, substitution of Cd2+ for the

Zn2+ 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, Cd2+ 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 Cd2+ ion from the affected

protein results in liberation of the essential Zn2+ 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 Cd2+

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

Zn2+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

Cd5a-MT 1a species upon titration of Cd4a-MT 1a

with a moderate excess of Cd2+ The additional Cd2+

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

1H-113Cd HSQC NMR data We propose that this cluster-expanded Cd5a 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 Cd4(Scys)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

Cd4(Scys)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

Cd4a-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 Zn4a-rhMT 1a with Cd2+ The exchange reaction of the Zn-substituted a domain with Cd2+was investigated by ESI mass spectrometry The Zn-substituted metallothionein was prepared by

demetallation of recombinantly isolated Cd4a-rhMT

1a at low pH followed by removal of the Cd2+ ions

using size-exclusion chromatography Reconstitution

with Zn2+ was achieved by raising the pH in the

presence of stoichiometric amounts of Zn2+ The top

spectrum of Fig 2 shows that the Zn4a 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 Cd2+ to the Zn4a sample results

in the formation of mixed-metal species, with the

Zn3Cd1a and Zn2Cd2a species predominating The

rel-ative abundance shifted to primarily the Zn1Cd3a and

Cd4a species upon titration with 3.4 molar equivalents

of Cd2+ When 4.7 molar equivalents of Cd2+ are

added, the Cd4a species is the predominant species,

indicating a near stoichiometric replacement of the

Zn2+ ions with the incoming Cd2+ 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 Cd2+

results in the appearance of a Cd5a 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 Cd2+ This newly identified Cd5a

spe-cies was further characterized by CD and UV

absorp-tion spectroscopy and ESI mass spectrometry by

titration of recombinantly prepared Cd4a-rhMT 1a isolated directly from the E coli source with excess

Cd2+

Titration of Cd4a-rhMT 1a with excess Cd2+: 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 Cd2+ 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 Cd4a species, and it has been described as being due to exci-ton splitting between the symmetric pairs of [Cd(Scys)4]2 groups in the Cd4(Scys)11 binding site [18– 22] This result confirms the correct folding and domain stoichiometry of the recombinantly synthesized

a domain as being the Cd4a 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 Cd4a sample results in a point of inflection at this wavelength Such a species was found during metal replacement and cadmium-loading experiments for

A

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 Cd4(Scys)11cluster 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.

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 Cd5a species, and

conse-quently should increase in abundance upon titration

with excess Cd2+ 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 Cd2+to the solution of Cd4a, up to 5.0

molar equivalents, resulting in a total of 9.0 molar

equivalents of Cd2+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 Cd2+ (Fig 3B) The loss of exciton coupling in the CD spec-trum following titration of excess Cd2+ 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 Zn4a-rhMT 1a

with Cd 2+ at pH 7.4 Spectral changes were recorded as aliquots of

Cd2+(3.3 m M ) were titrated into a solution of Zn 4 a-rhMT 1a (15 l M )

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 Cd4a-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 Cd2+added

to a sample of Cd4a-rhMT 1a as a measure of Cd5a-rhMT 1a species formation.

with loss of symmetry of the Cd4a structure This

spec-trum is reminiscent of the CD specspec-trum recorded for

the a domain with up to three Cd2+ 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

Cd2+ We propose that titration of excess Cd2+ alters

only the metal-thiolate cluster stoichiometry to give

the lower-symmetry Cd5a 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 Cd4a

sam-ple, results in restoration of the Cd4 stoichiometry

upon raising the pH from 2 to 7 (data not shown)

The presence of an additional 5.0 molar equivalents of

Cd2+ (total ratio of Cd2+: MT of 9.0) results in

for-mation of the Cd5a species, which also reforms

repro-ducibly upon cycling the pH (data not shown)

A plot of the ratio DA254⁄ DA264 from the CD

spec-trum approximates the ratio of Cd5a⁄ Cd4a Figure 3C

shows that up to 5.0 additional molar equivalents are

required for nearly all the metallothionein species to

be converted into the Cd5a 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 Cd4a-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 Cd5a-rhMT species This

result confirms the existence of a Cd5a 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 Cd2+ to the

Cd4a-rhMT 1a solution results in the ESI mass

spec-trum shown in Fig 4B Peaks corresponding to the

Cd4a-rhMT species are no longer detected, and

instead a new set of peaks are observed that are

consistent with the formation of 100% Cd5a-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 Cd4a-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 Cd4a domain of human MT 3 compared with the Zn4a MT 3 by one unit [28] Similarly, the

Cd3b domain of human MT 3 was shown to be sig-nificantly more open in conformation compared with the Zn3b domain of MT 3, indicating

non-isostructur-al replacement of Cd2+ for Zn2+ 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 Cd5a pre-sented here compared with the Cd4a species is due to expansion of the metal binding domain to accommo-date the fifth Cd2+ This suggests that the additional

Cd2+ ion is inserted into the core of the domain, becoming part of an expanded metal-thiolate cluster

113Cd NMR spectroscopy was therefore used to probe the metal-thiolate cluster arrangement in the newly identified Cd5a 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 Cd4a-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).

Titration of Cd4a-rhMT 1a with excess Cd2+:113Cd

NMR results

113Cd 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 Cd2+

Direct 1D 113Cd NMR (1H-decoupled) spectroscopic

techniques to probe for formation of a novel

Cd5a-rhMT 1a species

The 1D 113Cd NMR (1H-decoupled) spectrum of

Cd4a-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 113Cd 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 Cd4a-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 Cd5a-rhMT species, which has been shown in this report to be a minor contribu-tor to the equilibrium together with the Cd4a-rhMT species

Fig 5 (A) Direct 1D113Cd NMR (1H-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 Cd4a-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

Cd2+to form113Cd 5 a-rhMT 1a The spectrum of113Cd 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 Cd4 a-rhMT 1a sample and > 70% D2O for the 113 Cd5a-rhMT 1a sample All spectra were acquired at 25 C.

A 1D 113Cd NMR (1H-decoupled) spectrum of

iso-topically enriched 113Cd4a-rhMT 1a titrated with an

additional 10.0 molar equivalents of 113Cd2+ (total

ratio of Cd2+: 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 Cd5a species in the naturally isolated Cd4a

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 Cd5a spectrum

are assigned to the four 113Cd2+ 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

113Cd4a-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 Cd2+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 Cd4a-rhMT 1a (Fig 5A) have collapsed

into a single peak in the spectrum acquired with excess

113Cd2+(Fig 5B), indicating a loss of heterogeneity at

that site in the metal-thiolate cluster This is expected

if a fifth Cd2+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 Cd2+ion, confirming the CD

spectro-scopic and mass spectrometric data regarding the

for-mation of a Cd5a 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 Cd2+

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(OH2)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 Cd2+

ion and an increase in coordination number from four

to six This proposed ligand field assignment suggests

partial insertion of the fifth Cd2+ ion into the

metal-thiolate cluster in a manner that allows solvent access

Given this restriction, the most likely position for the fifth Cd2+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 Cd2+ion, with par-ticular emphasis on identifying the two cysteinyl sulfurs that are proposed to ligate the fifth Cd2+ion

Indirect 2D 1H–113Cd NMR spectroscopic techniques for identification of the binding site for the fifth Cd2+ ion in the Cd5a-rhMT 1a cluster

The indirect 2D1H–113Cd NMR approach exploits the

3J 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

Cd2+ ion By focusing on the 1H chemical shift range

of 2.3–3.6 p.p.m., corresponding to the cysteine Hb

protons only, the tetrathiolate connectivities of the Cd(Scys)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 1H–113Cd HSQC NMR spectra were acquired for the 113Cd5a-rhMT 1a formed by titration of 113Cd4a-rhMT 1a with an additional 10.0 molar equivalents of 113Cd2+ (total ratio of

Cd2+: MT of 14.0) Figure 6 shows a combination of two separate spectra acquired in the 113Cd chemical shift ranges of 585–705 p.p.m and 215–245 p.p.m to allow visualization of the correlations between all five

Cd2+ions in the Cd5a cluster with the 11 cysteinyl sul-fur residues The Hb–113Cd3Jscalar 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 113Cd dimension of the 2D spectrum of 113Cd5a-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 113Cd NMR (1H-decoupled) spectrum for the Cd5a species (Fig 5B, sites 1, 5, 6 and 7) The fifth Cd2+peak was observed in the 2D spectrum acquired in the 113Cd 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 113Cd NMR (1H-decoupled) spectrum (Fig 5B, site X) Identification of the specific bridging versus terminal cysteine residues is possible in this 113Cd-decoupled spectrum, and a nearly complete assignment of the cysteine residues in the 2D spectrum has been

accomplished The bridging cysteines are identified by

a solid line in Fig 6, because a single Hbchemical shift

correlates to two different 113Cd 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 Hb–113Cd correlations are known for

the four tetrahedral thiolate-coordinated113Cd2+ ions,

the unknown correlations of interest are those of the

fifth Cd2+ The two peaks correlating to the fifth

Cd2+at 224 p.p.m were observed at1H chemical shift

values of 2.97 and 3.59 p.p.m., which are consistent

with the Hb 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 Cd5(Scys)11species upon

titration with excess Cd2+, as opposed to the fifth

Cd2+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

Cd2+ ion includes two cysteine residues as predicted

by the highly shielded chemical shift [30] Figure 7

shows a space-filling model of Cd4a-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

Cd2+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 Cd2+ has been well documented by reports on the native

1H (p.p.m.)

Fig 6 A combination of two indirect 2D1H–113Cd HSQC NMR spectra for isotopically enriched113Cd 4 a-rhMT 1a titrated with an additional 10.0 molar equivalents of 113 Cd 2+ to form the 113 Cd5a-rhMT 1a species The spectra were recorded in the1H 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 Cd4a-rhMT 1a in two orientations rotated by 90, emphasizing the crevice site on the domain One of the proposed ligands of the fifth Cd2+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.

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 Cd2+ Significantly, titration of Zn7-MT

with sub-stoichiometric molar equivalents of Cd2+

resulted in a gradual red shift of the charge-transfer

band in the CD spectrum, indicating mixed-metal

speciation before saturation with seven Cd2+ ions

This result is consistent with the isolation of the

mixed-metal Cd5Zn2-MT species from in vivo sources

such as rabbit liver upon exposure of these animals to

Cd2+, indicating a non-cooperative mechanism of

metal replacement [19] The available X-ray, NMR

and CD data are consistent with the Cd4(Scys)11cluster

in the a domain being adamantane-like in structure

Thus, the Cd4a 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 Cd7ba-MT towards excess Cd2+ 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 Cd2+ 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 Cd2+ 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

Cd2+ ions bound Reaction of the isolated a domain

of mouse MT 1 with excess Cd2+ did not induce a

change in the CD spectroscopic profile, indicating that this cluster did not expand beyond the Cd4(Scys)11 stoi-chiometry The Cd-substituted two-domain human

MT 3 has been shown to coordinate additional Cd2+ ions with stoichiometries of up to Cd13ba-MT; how-ever, species with more than nine equivalents of Cd2+ are reported as probably being due to adducts on the surface of the protein The additional two Cd2+ ions leading to the Cd8ba-MT and Cd9ba-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 Cd2+, in which Cd5a was detected

as a minor species [35] However, structural data on this species were not provided, leaving open the possi-bility that the fifth Cd2+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

Cd2+, indicating that these particular metallothionein species do not have the capability to expand to larger metal clusters at reasonably low levels of excess Cd2+ [18,36] ESI mass spectral data for the two-domain rabbit MT 1a also indicated that this protein was satu-rated at seven Cd2+ ions, similar to the data for the rabbit MT 2a isoform [34]

While ESI-MS data have been reported previously that do show Cd2+ 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 Cd5a 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.

formed that involves a metal binding site that is

differ-ent from the Cd4a 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

Cd2+> 4, confirming disruption of the Cd4 binding

site Finally, the NMR data verify the coordination of

the fifth Cd2+ ion by thiolate ligands within the

clus-ter, and, more specifically, within the crevice site of the

protein

The biological significance of the Cd5a species is an

interesting question We propose that the Cd5a 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 Cd2+ (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 Zn2+ 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 Cd2+

and Zn2+ are present, is short-lived as Zn is

immedi-ately displaced In the case of human MT 1a, when

four Cd2+ ions are bound, we propose that the fifth

Cd2+ 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

Oakville, Canada), ammonium hydroxide (BDH

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) HiTrap SP HP

Health-care, Piscataway, NJ, USA), superfine G-25 Sephadex (Amersham Biosciences), a stirred ultrafiltration cell

YM-3 membrane (3000 molecular weight cut-off) and a Mi-crocon YM-3 centrifugal filtration device (Amicon

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 fuoverexpres-sion protein

Fol-lowing isolation and purification, the S-tag was cleaved from the protein by incubation with thrombin

Metal exchange of Zn4a-rhMT 1a with Cd2+ 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

(3.0 mm stock solution) and increasing the pH to 7.4 The a-rhMT solution was determined to have a concentration

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

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,

recorded using the mass lynx software package version 4.0 The ESI-TOF spectrometer was calibrated with a solu-tion of NaI The scan condisolu-tions for the spectrometer were: capillary, 3000.0 V; sample cone, 39.0 V; RF lens, 450.0 V;