Báo cáo khoa học: Coordination of three and four Cu(I) to the a- and b-domain of vertebrate Zn-metallothionein-1, respectively, induces significant structural changes doc

14 437 0
  • Loading ...
    Loading ...
    Loading ...

Tài liệu hạn chế xem trước, để xem đầy đủ mời bạn chọn Tải xuống

Tài liệu liên quan

Thông tin tài liệu

Ngày đăng: 07/03/2014, 09:20

Coordination of three and four Cu(I) to the a- and b-domainof vertebrate Zn-metallothionein-1, respectively, inducessignificant structural changesBenedikt Dolderer1, Hartmut Echner1, Alexander Beck1, Hans-Ju¨rgen Hartmann1, Ulrich Weser1,Claudio Luchinat2and Cristina Del Bianco21 Interfakulta¨res Institut fu¨r Biochemie, University of Tu¨bingen, Germany2 Magnetic Resonance Center, University of Florence, Sesto Fiorentino, ItalyThe first member of the metallothionein (MT) familywas isolated in 1957 [1]. Since then, a large numberof proteins have been described featuring commoncharacteristics. They include ubiquitous small cys-teine-rich proteins (50–70 amino acids) that are ableto bind many d10metal ions [2]. A wealth of differentbiological functions has been proposed and continuesto be discovered. Obviously, MTs play importantroles in minimizing the uncontrolled reactions ofheavy metal ions like cadmium and the homeostasisof essential metal ions including copper(I) and zinc(II)ions [2,3]. They are known to successfully cope withoxidative stress and ionizing radiation [4,5]. Otherfunctions may be associated with the occurrence oftissue-specific isoforms, such as the brain-specific iso-form MT-3, which acts as neuronal growth inhibitoryfactor [6,7].Both mammalian MT-1 and MT-2 are composed ofthe N-terminal b- and the C-terminal a-domain. Theyare predominantly isolated containing zinc or cadmiumexclusively bound to cysteine thiolates. The nine cys-teine residues of the b-domain accommodate a metal(M)(II)3S9cluster, while 11 cysteine residues contributeto the formation of a M(II)4S11cluster in the a-domain[8]. However, under certain physiological conditions,e.g. when isolated from fetal liver, mammalian MT-1Keywordscopper; domain; metallothionein; proteinstructure; 2D NMRCorrespondenceU. Weser, Anorganische Biochemie,Interfakulta¨res Institut fu¨r Biochemie,University of Tu¨bingen, Hoppe-Seyler-Str. 4,D-72076 Tu¨bingen, GermanyFax ⁄ Tel: +49 7071295564E-mail: ulrich.weser@uni-tuebingen.de(Received 17 January 2007, revised 28February 2007, accepted 5 March 2007)doi:10.1111/j.1742-4658.2007.05770.xVertebrate metallothioneins are found to contain Zn(II) and variableamounts of Cu(I), in vivo, and are believed to be important for d10-metalcontrol. To date, structural information is available for the Zn(II) andCd(II) forms, but not for the Cu(I) or mixed metal forms. Cu(I) binding tometallothionein-1 has been investigated by circular dichroism, luminescenceand1H NMR using two synthetic fragments representing the a- and theb-domain. The1H NMR data and thus the structures of Zn4a metallo-thionein (MT)-1 and Zn3bMT-1 were essentially the same as those alreadypublished for the corresponding domains of native Cd7MT-1. Cu(I) titra-tion of the Zn(II)-reconstituted domains provided clear evidence of stablepolypeptide folds of the three Cu(I)-containing a- and the four Cu(I)-con-taining b-domains. The solution structures of these two species are grosslydifferent from the structures of the starting Zn(II) complexes. Further addi-tion of Cu(I) to the two single domains led to the loss of defined domainstructures. Upon mixing of the separately prepared aqueous three and fourCu(I) loaded a- and b-domains, no interaction was seen between the twospecies. 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.AbbreviationsM, metal; MT, metallothionein.FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS 2349and MT-2 are also found to be enriched with Cu(I)[9]. For other members of the MT family, differentmetal cluster architectures were reported. The previ-ously mentioned MT-3, which is also a two-domainprotein, for example, is composed of a Cu(I)4S9clusterin the N-terminal b-domain and a Zn(II)4S11cluster inthe C-terminal a-domain [10,11]. Examples for solelyCu(I) binding MTs are Cu(I)8thionein from Saccharo-myces cerevisiae and Cu(I)6thionein from Neurosporacrassa [12–14]. Differently from other described MTs,these two fungal proteins consist only of a singledomain harbouring homometallic Cu(I) thiolate clus-ters [13,14].The three-dimensional structure of Cd5Zn2MT-2,isolated from rat liver after cadmium supplementa-tion, was determined using both NMR and X-raycrystallography [15]. The entire protein is dumbbell-shaped and contains two independent domains. Thepolypeptide backbone wraps around the metal thio-late core forming the scaffold of the two domains.All metal ions are tetrahedrally surrounded by fourthiolate sulphur atoms. In the N-terminal b-domain,the three metal ions and the three bridging thiolatesulphurs are ordered to form a distorted chair. TheC-terminal a-domain is characterized by an adaman-tane-like four-metal cluster. Solution structures of113Cd-substituted Cd7MT-2 from rabbit, rat andhuman are available and revealed structural identitywith the structure of Cd5Zn2MT-2 [8]. ComparativeNMR studies provided evidence that Zn(II) can iso-morphically replace Cd(II) in MT-2 [16]. This resultwas corroborated by NMR studies on cobalt substi-tuted MTs, as cobalt was often used as a zinc ana-logue in structural investigations [17–19]. The solutionstructure of murine113Cd7MT-1 showed high similar-ity with rat liver MT-2. Its b -domain, however,turned out to be more flexible than in the latterprotein, exhibiting enhanced cadmium–cadmiumexchange rates [20].The structural and spectroscopic data available onCd(II)-substituted human MT-3 indicated the forma-tion of two metal thiolate clusters, similar to thosefound in MT-1 and MT-2. Further investigation ofthat protein pointed towards a highly dynamic struc-ture [8]. Recently, a high-resolution solution structureof the C-terminal a-domain has become available. Thedata revealed a tertiary fold very similar to that ofMT-1 and MT-2, except for a loop that contains anacidic insertion that is highly conserved in these iso-forms. The structure of the b-domain has escaped itsexperimental solution, as no characteristic signalsattributable to its residues were observed using NMR.On the basis of homology modelling, a backbonearrangement virtually identical to the correspondingdomains in MT-1 and MT-2 was suggested [21].Despite the large number of structural data availablefor the MT family, only the structures of two MTscontaining Cu(I) were known until now. One of themis the aforementioned yeast MT whose structure wassuccessfully determined using both 2D NMR andX-ray diffraction [22–24]. This protein forms one singledomain that harbours eight Cu(I) ions. Six of them arecoordinated by three thiolate sulphur atoms, whereas alinear binding mode was observed for the remainingtwo. The solution structure of N. crassa MT backbone,in which, like yeast, the MT solely binds Cu(I), repre-sents the second known structure of a copper thionein[25]. Its polypeptide chain wraps around the coppersulphur cluster in a left-handed form in the N-terminalhalf of the molecule and in a right-handed form in theC-terminal half. Due to the lack of copper isotopeswith NMR-active spin ½, no metal–cysteine restraintswere available to solve the positions of Cu(I) withinthe N. crassa MT polypeptide fold.At present, the structural information on Cu(I)-loa-ded forms of mammalian MTs is rather limited.In vitro, Cu(I) titrations of isolated MT-2 and its sep-arate domains demonstrated that up to six Cu(I) ionscan bind to each domain [26]. In another extensive ti-tration study, it was postulated that zinc was requiredfor the in vivo and in vitro folding of the two domainsof copper MTs [27]. Replacement of Zn(II) by Cu(I)led to the proposal of the formation of Cu,Zn-MTintermediates and that, during the last steps of coppertitration, the two domains merge into one. However,earlier Cu(I) titration studies of rat liver MT clearlyshowed 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 ofCu(I) for binding to the b-domain is only partial andnot absolute, as widely accepted until now [27].It was of interest to shed some light on the changesof the molecular architecture of the two domains ofvertebrate MT when Cu(I) is added to them. For thistask, the synthetic murine aMT-1 and bMT-1 domainswere prepared for subsequent Cu(I) titrations. Employ-ing NMR, we obtained an interesting and unexpectedpicture of the Cu(I) binding to the two single domains.Results and DiscussionCu(I) titration of Zn4aMT-1 and Zn3bMT-1As both the structure of native Zn7MT-1 was known,and several Cu(I) binding stoichiometries for its twoMurine Cu(I)a- and Cu(I)b-MT-1 domains B. Dolderer et al.2350 FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBSdomains had been proposed, it was of interest to shedsome more light upon their reactivity towards the pres-ence of Cu(I). To this end, a Cu(I) titration study ofthe two separated domains was performed employingthe combined detection of luminescence, circulardichroism and1H NMR. Solid-phase peptide synthesiswas successfully employed to prepare the independenta- (residues 31–61) and b-domains (residues 1–30) ofmurine MT-1. Either domain was fully reconstitutedunder anaerobic conditions with Zn(II) to yieldZn4aMT-1 and Zn3bMT-1. For each Cu(I) titrationstep, a new sample was prepared in order to minimizethe risk of oxidation during sample manipulationand measurement. The Zn4aMT-1 and Zn3bMT-1derivatives were separately titrated with Cu(I) undera nitrogen atmosphere to yield Cu(I)–polypeptidestoichiometries from zero to six. The sample solutioncontained 20% (v ⁄ v) acetonitrile, as the presence ofsoft ligands prevents Cu(I) from disproportionation toCu(II) and Cu(0).CD and luminescence emission was measured inorder to assess the sample preparation quality and tocompare the obtained results with those previouslypublished [26,27]. These physicochemical properties areexclusively attributable to the metal-thiolate chro-mophores that have been proven to be essential for theproper polypeptide folding in other MTs [2]. The over-all shape of the CD spectra was essentially the same asreported before (Fig. 1). During the titration of the a-domain, two positive dichroic bands developed at 248and 300 nm, respectively, and one negative band at275 nm. The addition of Cu(I) to Zn3bMT-1 shiftedthe positive dichroic band at 248 to 260 nm. A secondpositive band at 300 nm, that was not present in thespectrum of Zn3bMT-1, appeared on addition ofCu(I). As in the case of the CD spectra, the results ofluminescence emission were comparable to earlier stud-ies (Fig. 2). An almost linear increase of intensity wasobserved until the addition of the third and fourthCu(I) ions to the a- and b-domain, respectively. Fur-ther Cu(I) addition led to a much more pronouncedincrease of intensity in both species.Two-dimensional1H-1H NOESY spectra of eachsample were acquired at 700 MHz (Figs 3 and 4). Thespectrum of Zn4aMT-1, corresponding to the startingpoint of the aMT-1 titration, was consistent with awell-folded polypeptide. Spin systems of the amideprotons spread from 6.8 to 9.2 p.p.m. Upon additionof the first equivalent Cu(I), the spin systems of thestarting point remained preserved, but additional newspin systems started to appear. In the spectra of thesamples containing two, three and four equivalents ofCu(I), these new spin systems were prevalent with themost and strongest signals observed for the threeCu(I)-containing sample. On further additions ofCu(I), the signals faded away such that the spectraof the six and seven Cu(I) titration steps were devoidof cross-peaks (not shown).For the b-domain similar results were obtained withthe difference that the first addition of Cu(I) led onlyto the reduction of signals in the NOESY spectrumand that new spin systems appeared only after the sec-ond equivalent Cu(I) was added. The spectra of thesamples containing three, four and five equivalents250 300 350 400-30-20-1001020250 300 350 400 / nm Zn4- -MT + 1 eq. Cu(I) + 2 eq. Cu(I) + 3 eq. Cu(I) + 4 eq. Cu(I) + 5 eq. Cu(I) + 6 eq. Cu(I)A / nm Zn3 MT + 1 eq. Cu(I) + 2 eq. Cu(I) + 3 eq. Cu(I) + 4 eq. Cu(I) + 5 eq. Cu(I) + 6 eq. Cu(I)BFig. 1. CD spectra of Zn4aMT (A) andZn3bMT (B) along the titration with Cu(I).Samples containing 35 lM of the respectivedomains dissolved in 15% (v ⁄ v) acetonitrile,20 mM sodium phosphate buffer pH 7.6were prepared under nitrogen containing<1 p.p.m. O2.B. Dolderer et al. Murine Cu(I)a- and Cu(I)b-MT-1 domainsFEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS 2351Cu(I) contained the same new spin systems. The mostand strongest NOEs were observed in the spectrumof the four Cu(I)-containing sample. As with thea-domain, progressive disappearance of NOEs withoutreappearance of any new signals was the result ofCu(I) to polypeptide stoichiometries higher than five.Taken together, the initial additions of Cu(I) to eachdomain caused the disappearance of a large set ofNOESY cross-peaks and the parallel appearance ofanother set of cross-peaks, until a clean 2D spectrumbelonging to a single species was obtained. Judgingfrom the highest number of NOEs and the strongestsignals in the respective NOESY spectra, the recoveryof a single species was completed after the addition ofthree Cu(I) equivalents to Zn4aMT-1 and of fourCu(I) equivalents to Zn3bMT-1. This result was surpri-sing insofar as structurally defined Cu(I)-containingspecies were identified with such unexpectedly low stoi-chiometries of Cu(I) to polypeptide. Several differentCu(I) binding stoichiometries had been proposed forthe two domains, among which Cu3aMT-1 andCu4bMT-1 had mostly been considered to be transientintermediates in the pathways describing the formationof the fully loaded domains [27–30]. Cu6aMT-1 andCu6bMT-1 had been the most prominent among thecandidates for the fully Cu(I) loaded domains [26]. Inthe present titration study, however, the distinct struc-tures disappear upon addition of more than three andfour Cu(I) equivalents without any sign of newly form-ing defined structures. We can only speculate whathappens at this stage of titration. One possible explan-ation for the disappearance of NOESY signals at highCu(I) concentration might be that two or more Cu(I)binding modes coexist in an intermediate exchangeregime, such that signals are exchange broadened andbecome invisible. Of course, there is still the possibilitythat the separated domains are simply incapable ofbinding more than three and four Cu(I) without aggre-gating and denaturing, whereas in the native MT-1,the presence of the other domain would help toaccommodate additional ions. We do not believe thatthis is very likely, however, because of the similarity ofthe Zn4aMT-1 and Zn3bMT-1 structures with thestructures of the domains of the intact protein (seebelow). [Correction added after publication 30 March2007: in the preceding sentence the first term,Zn3aMT-1 was corrected to Zn4aMT-1]. The biophysi-cal similarities of intact protein and separated domainswould also argue against this proposal [26].Luminescence titration series also provide notewor-thy information. Luminescence intensities increasedalmost linearly until Cu(I) stoichiometries of three andfour for the a- and b-domain, respectively. At thispoint, the curves were bent and further Cu(I) equiva-lents caused a much stronger, but also linear increaseof intensity. As luminescence intensity is a measure ofhow the Cu-thiolate luminophore is shielded fromsolvent quenching, the titrations indicate that theshielding of the metal-thiolate cluster in the newlyidentified structures is not optimal compared with thesituation with higher Cu(I):polypeptide stoichiometries.An explanation for this and the loss of structuralinformation might be the formation of polymolecularstructurally undefined aggregates of native MT or itssingle domains when they are overloaded with Cu(I) inthe presence of unphysiologically high Cu(I) concentra-500 550 600 650 7000102030405060500 550 600 650 700relative intensity / nmAB0 equivalents Cu(I)6 equivalents Cu(I)/ nm0 equivalents Cu(I)6 equivalents Cu(I)mole equiv. of Cu(I)0123456012345601000200030004000500001000200030004000500060007000relative intensityrelative intensitymole equiv. of Cu(I)Fig. 2. Luminescence emission spectra ofZn4aMT (A) and Zn3bMT (B) along the titra-tion with Cu(I). Samples containing 14 lM ofthe respective domains dissolved in 15%(v ⁄ v) acetonitrile, 20 mM sodium phosphatebuffer, pH 7.6, were prepared under nitro-gen containing <1 p.p.m. O2. Spectra wererecorded at 25 °C using slits of 15 and20 mm for excitation and emission mono-chromators, respectively. Excitation was atk ¼ 300 nm. The insets show the emissionintensities plotted against the respectivepolypeptide stoichiometries.Murine Cu(I)a- and Cu(I)b-MT-1 domains B. Dolderer et al.2352 FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBStions. Unlike the observed distinct stoichiometries ofthree and four Cu(I) leading to a sharp rise of theluminescence, only a very small dependency was seenin the circular dichroic measurements. This wasalso shown earlier by Bofill et al. [27], although CDspectrometry is obviously not sensitive enough todetect comparable significant changes as deduced fromluminescence data.1H NMR and solution structures of Zn4aMT-1 andZn3bMT-1From previous different studies on vertebrate Zn(II)-and Cd(II)-containing MTs, it was already known thatthe two domains form independently from each otherand do not interact with each other. Therefore, it wassuggested that the two single domains possess veryFig. 3. Upper-left parts of the1H-1H NOESYspectra of Zn4aMT (A), Zn4aMT +1 Cu(I) (B),Zn4aMT +2 Cu(I) (C), Zn4aMT +3 Cu(I) (D),Zn4aMT +4 Cu(I) (E) and Zn4aMT +5 Cu(I)(F). All samples contained 1 mM polypeptidedissolved in 15 mM acetate-d3, 15% aceto-nitrile-d3, 10% D2O, 50 mM potassium phos-phate, pH 6.5 and were prepared under anitrogen atmosphere containing less than1 p.p.m. O2. Measurements were per-formed at 283 K on a Bruker AVANCE 700spectrometer operating at 700.13 MHzusing 600 ms mixing time.B. Dolderer et al. Murine Cu(I)a- and Cu(I)b-MT-1 domainsFEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS 2353similar structures, if not identical, to their structure inthe intact protein. Analysis of the NOESY and TOC-SY (not shown) spectra of the two domains permittedthe full sequence-specific assignments, the identificationof the spin systems and the assignment of 398 and 252of the NOESY cross-peaks of the a- and b-domain,respectively. The comparison of the chemical shiftswith those reported for the cadmium derivativerevealed very close similarities (supplementary TablesS1 and S2). In the spectrum of the a-domain, the reso-nances were shifted marginally by some hundredths ofa p.p.m. The differences observed for the b-domainwere more pronounced, with some deviations of up to0.2 p.p.m., which is probably due to an increased flexi-bility in this domain. The spin system patterns repor-ted for the published cadmium protein, however, werepreserved in both domains. Most importantly, 22 ofthe 28 long-range NOEs that were reported forCd7MT-1 were also found in the spectra of the zinc-containing a- and b-domain (Table 1). It should benoted that three of the six missing long-range NOEswere assigned to residues of the linker region betweenFig. 4. Upper-left parts of the1H-1H-NOESYspectra of Zn3bMT (A), Zn3bMT +1 Cu(I) (B),Zn3bMT +2 Cu(I) (C), Zn3bMT +3 Cu(I) (D),Zn3bMT +4 Cu(I) (E), and Zn3bMT +5 Cu(I)(F). Sample and measurement conditionswere the same as described in Fig. 3.Murine Cu(I)a- and Cu(I)b-MT-1 domains B. Dolderer et al.2354 FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBSthe two domains. Therefore, the lack of the seconddomain seems to lead to increased flexibility at eitherend that would build up the linker region in nativeMT-1. The fact that the majority of the long-rangeNOEs observed in Cd7MT-1 is still present in the sin-gle zinc-containing domains suggests that the globalstructures of the two domains are preserved, regardlessof whether zinc or cadmium is bound to them and alsoregardless of the existence of the second domain.The assigned peaks of the a-domain were integratedand their consistency with the published solution struc-ture was checked using the program dyana and thepublished solution structure of the respective cad-mium-containing domain of the intact protein as astarting point. The resulting structure family had a tar-get function of 0.15 ± 0.11 A˚2and rmsd values of1.51 ± 0.27 A˚and 2.22 ± 0.30 A˚with respect to themean structure for the backbone and all heavy atoms,respectively. In the previous study on the Cd7deriv-ative, metal–sulphur connectivities were obtained usingthe NMR-active cadmium isotope113Cd and added tothe structure calculation process. Using these connec-tivities together with the data for Zn4aMT-1 resultedin a target function of 0.52 ± 0.13 A˚2and rmsd valuesof 1.04 ± 0.12 A˚and 1.51 ± 0.18 A˚for a new struc-ture family. Both mean structures were essentiallythe same, with rmsd values of 0.79 A˚and 1.19 A˚forthe backbone and heavy atoms, respectively. Thus, theaddition of metal–sulphur connectivities to the struc-ture calculation of Zn4aMT-1 resulted in a better-resolved structural family but did not change theoverall protein fold. Figure 5 shows the superpositionof the structural family obtained with these additionalconnectivities and the mean structure of the previouslypublished Cd4aMT-1, showing that they possess thesame structure, within the experimental uncertainty.A separate structure determination of Zn3bMT-1 onthe basis of the present NMR data was not attempted,as only a small number of NOEs and only four long-range NOEs were found in its NOESY spectrum. Onlywith the help of the metal–sulphur connectivities dis-covered using the cadmium-containing derivative coulda structure determination have been possible. How-ever, preserved chemical shifts and spin system pat-terns indicate an identical structure for Zn3bMT-1 asfor Cd3bMT-1. As anticipated, the structures of theseparated Zn(II)-containing domains are indistinguish-able from those of Cd7MT-1 and, knowing the appear-ance of the starting points, it was of interest to knowto what extent they would change in the presence ofcopper(I).1H NMR and solution structures of ZnxCu3aMT-1and ZnyCu4bMT-1The NOESY spectra of the ZnxCu3aMT-1 andZnyCu4bMT-1 domains are markedly different fromthe starting Zn4aMT-1 and Zn3bMT-1 derivatives,pointing to a different arrangement of the polypeptidechains, which is probably needed to accommodate theresulting Cu(I)- or mixed metal–sulphur clusters(Fig. 6). From a cursory inspection of the superim-posed spectra, it has already become clear that theaddition of Cu(I) not only leads to a completely differ-ent pattern of spin systems, but also to a significantlyhigher number of NOEs. Therefore, we expected thestructures of the newly identified Cu(I)-containingdomains to be more rigid and distinct from theirZn(II)-containing forms. The spectra of both the onlyZn(II)-containing b-domain and its Cu(I)4derivativeseem to be of lower quality with large areas of broadoverlapping peaks. This behaviour might be due tohigher flexibility within the b-domain which has beenreported already before [17–20]. TOCSY spectra (notTable 1. Comparison of the long-range NOEs (dijj > I +4) of Zn4-aMT and Zn3bMT with those observed for Cd7MT [20]. Presence(+) or absence (–) of NOEs is indicated.b-DomainProton 1 Proton 2Asn 4 (a) Lys 22 (b)+Asn 4 (a) Asn 23 (NH) +Asn 4 (b) Asn 23 (NH) +Cys 5 (a) Cys 21 (b)+Cys 5 (b) Cys 21 (b)+Asn 23 (b) Cys 29 (a)–a-DomainLys 1 (NH) Val 9 (c)–Lys 1 (a) Val 9 (c)+Ser 2 (b) Val 9 (b)+Ser 2 (HN) Val 9 (c)–Cys 3 (HN) Val 9 (c)+Cys 3 (a) Cys 18 (b)+Cys 3 (b) Cys 18 (HN) +Ser 5 (b) Asp 25 (HN) –Cys 6 (a) Asp 25 (HN) +Cys 6 (a) Asp 25 (a)–Cys 6 (a) Lys 26 (HN) +Cys 6 (b) Lys 26 (HN) +Cys 6 (b) Lys 26 (a)+Cys 6 (a) Cys 27 (HN) +Cys 6 (b) Cys 27 (HN) +Cys 6 (b) Cys 27 (a)–Cys 6 (b) Cys 27 (b)+Lys 13 (b) Val 19 (c)+Lys 13 (c) Val 19 (c)+Lys 13 (c) Cys 30 (a)+Cys 14 (NH) Val 19 (c)+Val 19 (c) Cys 19 (b)+B. Dolderer et al. Murine Cu(I)a- and Cu(I)b-MT-1 domainsFEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS 2355shown) were recorded for the ZnxCu3aMT-1 andZnyCu4bMT-1 derivatives and used, together with theNOESY spectra, to obtain their sequence specificassignments (supplementary Tables S1 and S2). Theresonances of the new Cu(I)-containing species differedmostly by several tenths of a p.p.m. from those oftheir starting points, thereby confirming the observa-tions mentioned above.In the NOESY spectrum of ZnxCu3aMT-1 502,NOEs were assigned, integrated and converted intodistance constraints. In the last dyana run, a set of200 structures was generated out of which the 20 bestwere combined to a structure family (Fig. 7). The tar-get function was 0.43 ± 0.10 A˚2, and the rmsd valueswere 0.70 ± 0.12 A˚and 1.03 ± 0.14 A˚for the poly-peptide backbone and heavy atoms, respectively. Like-wise, 500 NOEs of the ZnyCu4bMT-1 spectrum wereused to derive a structure family for this species(Fig. 8). In this case, a target function of 0.19 ±0.02 A˚2and rmsd values of 0.49 ± 0.21 A˚and0.72 ± 0.21 A˚for the backbone and heavy atoms wereobtained.As with other known structures of MTs, those pre-sented here also lack typical secondary structure ele-ments. The structure of ZnxCu3aMT-1 is roughly atwo-level structure (Fig. 7) where the segment 5–10 ofthe a-domain polypeptide backbone forms the firstlevel. The stretch 10–16 links the first with the secondFig. 5. Superposition of the present family of 30 structures of Zn4aMT (blue) with the published average structure of Cd4aMT (red). Thecoordinates of the Cd4aMT structure were extracted from the Brookhaven protein data bank (1DFS). In the last run of structure calculationsfor Zn4aMT 398 upper distance limits (upl) obtained from the assignment of its NOESY spectrum and the metal-sulphur connectivities repor-ted by Zangger et al. [20] for Cd4aMT were used as input for the program DYANA. Twenty out of 200 structures were combined into a struc-ture family with a target function of 0.52 ± 0.13 A˚2and RMSD values of 1.04 ± 0.12 A˚and 1.51 ± 0.18 A˚for the backbone and all heavyatoms, respectively.Fig. 6.1H-1H-NOESY spectra of Zn4aMT(red) superimposed with that of ZnxCu3aMT(green) (A) and of Zn3bMT (red) superim-posed with that of ZnyCu4bMT (green) (B).Sample and measurement conditions werethe same as described in Fig. 3.Murine Cu(I)a- and Cu(I)b-MT-1 domains B. Dolderer et al.2356 FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBSlevel, which is constituted by the second half of thepolypeptide chain. The region between residue 20 and26 includes a loop that is more disordered than therest of this domain, shielding the rear of its core. Thepositions of several cysteine sulphur atoms are notvery well defined (Fig. 7). Nevertheless, the 11 cyste-ines seem to form a somewhat broader cavity than inits Cd4aMT-1 counterpart, with the subgroup of Cys3,11 and 18 being rather isolated from the other eightcysteines.The polypeptide backbone of the b-domain wrapsaround its core in a right-handed fashion (Fig. 8). Thecentral part of its polypeptide chain, residues 8–24,limits an almost elliptical planar structure. At the pointwhere the two ends of the polypeptide chain wouldmeet, they leave the central plane and continue inopposite directions. Again, outside the uncertainty inthe positions of some of the sulphur atoms, the cavityencased by the cysteines is somewhat broader than itsCd3bMT-1 counterpart, although in this case the ninecysteines still point to a unique core.The superposition of the newly identified Cu(I)-con-taining domains onto the mean structures of theirCd(II)-coordinating forms highlights the striking struc-tural differences that are caused only by the bindingof different metal ions to the two domains of MT(Fig. 9). When the structures are superimposedthroughout the full length of their polypeptide chainsonly very faint similarities of some parts of the poly-peptide backbone folds could be observed. Separatesuperpositions of stretches 3–12, 11–20 and 18–28 werealso attempted. For both domains, the first and thirdstretches gave very poor overlap, while the central partshowed a more pronounced similarity. This could indi-cate that each domain adapts to host the additionalcopper(I) ions by opening up and rearranging itsN- and C-terminal parts, minimizing the structuralperturbation of its central part.The arrangement of the cysteine sulphur donoratoms within the two Cu(I)-containing domains is alsoshown in Figs 7 and 8. Although, from the presentdata, it is neither possible to guess the number ofFig. 7. Stereoview of the 20-structure familyof ZnxCu3aMT. Polypeptide backbone bondsare shown in grey, cysteinyl side chainbonds in blue and sulphur atoms as yellowspheres. 502 NOEs were converted into upland were used as input for the structurecalculation programDYANA. Twenty out of200 structures were combined into a struc-ture family with a target function of0.43 ± 0.10 A˚2and RMSD values of0.70 ± 0.12 A˚and 1.03 ± 0.14 A˚for thebackbone and all heavy atoms, respectively.Fig. 8. Stereoview on the family of 20 beststructures of ZnyCu4bMT. Polypeptide back-bone bonds are shown in grey, cysteinylside chain bonds in blue and sulphur atomsas yellow spheres. 500 NOEs were conver-ted into upl and were used as input for thestructure calculation programDYANA. Twentyout of 200 structures were combined into astructure family with a target function of0.19 ± 0.02 A˚2and rmsd values of0.49 ± 0.21 A˚and 0.72 ± 0.21 A˚for thebackbone and all heavy atoms, respectively.B. Dolderer et al. Murine Cu(I)a- and Cu(I)b-MT-1 domainsFEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS 2357residual Zn(II) ions in each structure nor the overalltopology of the clusters, it appears that all cysteine res-idues point towards the inside of the respectivedomain, as expected if all of them are to be involvedin metal coordination. In turn, the somewhat broadercavities encased by the cysteines are consistent with theincreased number of metals in each domain.At this point it was still an open question as towhether or not the single species ZnxCu3aMT-1 andZnyCu4bMT-1 would be stable when the other Cu(I)-containing domain was also present in solution. To thisend, both Cu(I)-containing domains were prepared asfor the titration experiment mentioned above, com-bined in equal amounts at a final concentration of1mm each and incubated for >48 h before the meas-urement of their1H NMR spectra. The observed NO-ESY and TOCSY spectra (not shown) consisted of thesum of the respective spectra of the single species. Thespectral resonances were assigned to all the protonspresent in the two domains and are listed in supple-mentary Tables S1 and S2. This result indicates thatthe two domains are stable and independent from eachother. Cu(I) is not transferred between the two domainsto form new species with higher and lower Cu(I):poly-peptide stoichiometries. As no additional NOEs and ⁄ orchanges of the spectral resonances were observed in theNOESY spectrum of the mixture, an interaction of thetwo single domains and the formation of one singleCu(I)-containing domain with the involvement ofboth domains could be excluded for the presentZn(x+y)Cu7MT-1 stoichiometry.ConclusionThe Cu(I) titration of the independent Zn(II)-loadeddomains of mouse MT-1 revealed Cu(I) stoichiometriesof three and four for the a- and b-domain, respectively.The presence of Cu(I) led to dramatic conformationalchanges of both polypeptide folds. Cu(I) stoichio-metries of up to six Cu(I) ions each led to the progressivedisappearance of the altered structures. [Correctionadded after publication 30 March 2007: in the preced-ing sentence, disappearing of the affered structurer, wascorrected to disappearance of the altered structures].Unfortunately, due to the lack of metal Æ sulphurconstraints, the Cu(I) positions within the resolvedpolypeptide folds remained unclear. Therefore, crystal-lization of the newly identified Cu(I)-containing speciesFig. 9. (A) Stereoview of the superpositionof the Cd4aMT-1 mean structure (red) to thestructure family of ZnxCu3aMT-1 (blue). (B)Stereoview of the superposition of theCd3bMT-1 mean structure (red) to the struc-ture family of ZnyCu4bMT-1 (blue).Murine Cu(I)a- and Cu(I)b-MT-1 domains B. Dolderer et al.2358 FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS[...]... Coordination of Cu(I) to acetonitrile prevented undesired Cu(I) disproportionation into Cu(II) and elemental copper Four and three molar equivalents of Zn(ClO4)2 were added to the a- and b-domains, respectively The pH was adjusted to pH 6.5 by adding potassium phosphate to a final concentration of 50 mm Seven different samples per domain were titrated using zero to six molar equivalents of Cu(I) The Cu(I) titration... 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... 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... consisted of 1024 data points in the F2 dimension and 2048 data points in the F1 dimension and were acquired using 16 scans per F1 increment Structure determination The program sparky was used for analysis of the 2D spectra [34] The sequential assignment of the spin systems in the TOCSY and NOESY spectra at 283 K was achieved employing standard procedures In the cases when only zinc was bound to the protein,... tubes, flushed with argon and sealed FEBS Journal 274 (2007) 2349–2362 ª 2007 The Authors Journal compilation ª 2007 FEBS 2359 Murine Cu(I )a- and Cu(I)b-MT-1 domains B Dolderer et al The preparation of samples for the CD measurements was carried out essentially identical to the protocol used to prepare the NMR samples Final polypeptide concentrations were adjusted to 35 lm and only undeuterated chemicals... 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 . Coordination of three and four Cu(I) to the a- and b-domain of vertebrate Zn-metallothionein-1, respectively, induces significant structural changes Benedikt. the identification of the spin systems and the assignment of 398 and 252 of the NOESY cross-peaks of the a- and b-domain, respectively. The comparison of
- Xem thêm -

Xem thêm: Báo cáo khoa học: Coordination of three and four Cu(I) to the a- and b-domain of vertebrate Zn-metallothionein-1, respectively, induces significant structural changes doc, Báo cáo khoa học: Coordination of three and four Cu(I) to the a- and b-domain of vertebrate Zn-metallothionein-1, respectively, induces significant structural changes doc, Báo cáo khoa học: Coordination of three and four Cu(I) to the a- and b-domain of vertebrate Zn-metallothionein-1, respectively, induces significant structural changes doc