Solution structure of Cu 6 metallothionein from the fungus Neurospora crassa Paul A. Cobine 1 , Ryan T. McKay 2, *, Klaus Zangger 2, †, Charles T. Dameron 3 and Ian M. Armitage 2 1 Health Science Center, University of Utah, Salt Lake City, UT, USA; 2 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA; 3 Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA, USA The 3 D-solution structu re o f Neu rospora crassa Cu 6 -metal- lothionein (NcMT) polypeptide backbone was determined using homonuclear, multidimensional 1 H-NMR spectros- copy. It r epresents a new metallothionein (MT) fold with a protein chain where the N-terminal half is left-handed and the C-terminal half right-handedly folded around a cop- per(I)-sulfur c luster. As seen with other MTs, the protein lacks definable s econdary structural elements; however, the polypeptide fold is unique. The metal coordination and the cysteine spacing defines t his unique fold. NcMT is only the second MT in the copper-bound form to be structurally characterized and the first containing the -CxCxxxxxCxC- motif. This motif i s found in a variety of mammalian MTs and metalloregulatory proteins. The in vitro formation of the Cu 6 NcMT identical to the native Cu 6 NcMT was dependent upon the prior formation of the Zn 3 NcMT and its titration with Cu(I). The enhanced sensitivity and resolution of the 800 MHz 1 H-NMR spectral data p ermitted the 3 D structure determination of the polypeptide backbone without the substitution and utilization of the NMR active spin 1/2 metals such as 113 Cd and 109 Ag. These restraints have been necessary to estab lish s pecific metal to cysteine restraints in 3D structural studies o n this family of proteins when using lower field, less sensitive 1 H-NMR spectral data. The accu- racy of the structure calculated without these constraints is, however, supported by the similarities of the 800 MHz structures of the a-domain of mouse MT1 compared to the one recalculated without metal–cysteine connectivities. Keywords: copper; metallothionein; Neurospora crassa; NMR; solution s tructure. Metallothioneins (MTs) are a ubiquitous class of proteins occurring in both prokaryotes and eukaryotes [1]. MTs a re known for their small size (< 7 kDa), the ability to coordinate a diverse range of metals, a lack of definable secondary structure, high cysteine content ( 30%), and degeneracy in the remaining residues (e.g. predominance of cysteine, serine, lysine and no aromatic residues). The high cysteine content and their spacing give the MTs a high affinity for m etals (e.g. K a of Zn–MT  1 · 10 12 M )[2,3]. While an essential physiological role has yet to be ascribed to MT, there is no question that MTs are involved in the protection of cells against metal intoxication through t he sequestration of the excess essential metal ions like copper and zinc, as well as nonessential metal ions, like cadmium, mercury and silver [4]. Although the essential metals have critical structural, catalytic and regulatory r oles in proteins, their cellular concentration must be carefully maintained through the use of pumps and s equestering peptides a nd proteins to avoid toxic effects. Despite the high affinity for metals, MTs are also postulated to participate in an undetermined mechanism o f facile metal e xchange (i.e. kinetically labile yet thermodynamically stable) with other proteins possessing substantially lower metal affinities [5–8]. Since the MTs have little or no repetitive secondary structure, their tertiary structure is dependent on the number and type of metal ions they coordinate. The MT from the fungus Neurospora crassa,isasingle domain MT m ade u p o f 2 5 r esidues, seven of which are cysteine residues [9]. This small peptide coordinates six Cu(I) a toms via the seven c ysteinyl sulfurs [ 10]. N. crassa metallothionein (NcMT) binds copper in a solvent-shiel- ded environment, which p roduces a luminescent core [11]. In vivo , t he N cMT is induced only b y c opper; other transition metals (zinc, cadmium, cobalt, and nickel) do not induce transcription of NcMT mRNA although the protein will bind these metal ions in vitro [12,13]. The in vitro metal ion binding characteristics of N cMT mimic that of the mammalian b-domain, i.e. NcMT b inds zinc(II), c admium(II) and cobalt(II) with a 3 : 1 metal to Correspondence to I. M. Armitage, Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church Street S.E., Minneapolis, MN 55455, USA. Fax: +1 612 625 2163, Tel.: +1 612 624 5977, E-mail: armitage@msi.umn.edu Abbreviations: MT, metallothionein; NcMT, Neurospora crassa met- allothionein; aMT-1, a-domain of mouse MT-1; s c , correlation time. Note: The PDB file has been assigned the Brookhaven Protein Data Bank Accession no. 1T2Y and the chemical shifts have been deposited in the BMRB data bank under accession number 62 90. *Present address: 101 NANUC, University of Alberta, Ed monton, AB Canada T6G 2E1. Present address: Institute of Chemistry/Organic and Bioorganic Chemistry, University of Gra z, Heinrichstrasse 28, A-8010 Graz, Austria. (Received 3 0 June 2004, revised 2 September 2004, accepted 7 September 2004) Eur. J. Biochem. 271, 4213–4221 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04361.x protein stoichiometry and Cu(I) with a 6 : 1 stoichiometry [12]. The NcMT cysteine arrangement is identical to the first seven amino-terminal cysteines of the b-domain of human MT (Fig. 1), thou gh the human MT has t wo additional cysteines in this domain. Similar cysteine spacing is seen in the metal binding motifs of the metalloregulatory proteins from Enterococcus hirae (CopY), Saccharomyces cerevisiae (Ace1 and Mac1) and Candida albicans (AMT1) though these proteins do not share a ny other significant homology, Fig. 1. Along with the metal coordination number, the positioning of the cysteines in t he primary s tructure has been determined to be the critical f actor in determining the global fold of t he MTs [14]. The structures of several MT isoforms have been determined including that from yeast [15,16], crab [17], sea urchin [14], an antarctic fish [18] and various mammals [19–23]. With the exception of the yeast Cu 7 MT [16], the previou s NMR solution structures o f MTs, including yeast MT [15], all relied on the substitution of NMR active s pin 1/2 metals such as 113 Cd and 109 Ag for the determination of specific metal–cysteine restraints, which were included in the subsequent structure calcula- tions [24]. While the mammalian copper(I) MTs have evaded 3D structural elucidation by NMR and X-ray crystallography [1], recent CD spectroscopic studies on the Cu(I) and Ag(I) substituted b-fragmentofmouseMT1 have revealed structural differences that may reflect a different conformational f old for the Ag(I) substituted b-do main compared to the native Cu(I) containing fragment [25]. The only copper(I) MT structure deter- mined has been that of the yeast MT which has a distinctly different cysteine arrangement, Fig. 1 [15,16]. However, disagreement exists in the comparison of this NMR derived structure of the yeast MT without the 13 C-terminal residues and the small angle X-ray scattering pattern of the full length protein which has been attributed to the core of t he NMR structure being too compact [15,16,26]. The similarity in the metal binding properties and cysteine spacing in NcMT with mammalian MT makes it a candidate for the modelling of the Cu(I)–sulfur cluster o f the b-domainofmammalianCu 6 MT and potentially useful for other copper(I)-regulated proteins that contain t he - CxCxxxxCxC- and - CxCxxxxxCxC- motifs, F ig. 1. The 1 H NMR resonance assignments f or the C u 6 NcMT purified from N. crassa have been reported [ 27] and these were used as a template to compare and confirm a similar overall fold for the in vitro reco nstituted, synthesized protein used in this study. W e report here the 3D NMR solution structure o f the Cu 6 NcMT, obtained without establishing any metal–cysteine restraints, and discuss the formation of the Z n 3 NcMT precursor m olecule t hat was needed to obtain the Cu 6 NcMT structure. Materials and methods Proteins and peptides The NcMT w as synthesized chemically to avoid its prob- lematic induction and purification. As noted previously by another group [28], N. crassa can use several or a mixture of copper resistance mechanisms to detoxify excess copper. In initial attempts, we found that exposure to copper did not consistently result in the induction of MT. Therefore, a synthetic p eptide corresponding to the N cMT w as s ynthes- ized using B oc-Pam resin and Boc -chemistry protocols [29]. The peptide was deprotected and cleaved from the resin using anhydrous liquid hydrogen fluoride, para-cre sol and para-thiocresol (269–271 K for 70 min). Purification inclu- ded a diethyl ether wash under a nitrogen atmosphere and preparative trifluoroacetic acid (TFA)/acetonitrile [buffer A: 0.1% (v/v) TFA/water; buffer B 90% acetonitrile/10% water and 0.1% TFA, v/v/v) linear gradient H PLC (0–50% buffer B over 40 min) on a reverse phase HPLC column (Waters Delta-Pak PrepPak C18; 40 mm · 100 mm). Elu- tion was monitored at 214 nm, and the eluted p eptide was freeze-dried for storage. Subsequent analysis showed that the p eptide was oxidized by the cleavage/purification treatment, ther efore t he peptide was reduced by suspension in 6 M guanidine/HCl, 100 m M Tris pH 8.5, and 150 m M dithiothreitol with incubation at 42 °C for 2 h. The reduced protein s ample w as then loaded onto a G25 (Pharmacia) size exclusion column (200 mm · 15 mm) previously equili- brated with 0.2% (v/v) TFA. The protein fraction was collected, pooled and sealed anaerobically. P rotein concen- tration was determined by amino acid analysis and the reduction state of the sulfhydryls confirmed by dithiodi- pyridine assay [30]. All subsequent manipulations of the apo- a nd metallated N cMT were p erformed under anaer- obic conditions, 5% H 2 and 95% N 2 (v/v), in a glove box (Atomspure Protector Glove Box, Labconco, Kansas City, MO, USA). Metal titrations Copper(I) titrations were performed as d escribed by Byrd et al. [31]. Protein samples with reduction state > 95% were used for all titrations. Sequential additions of copper(I), as [Cu(CH 3 CN) 4 ]ClO 4 in 200 m M ammonium acetatepH7.9,weremadetotheapo-NcMT. Identical titrations were performed with Zn(II) 3 NcMT instead of the apo-NcMT. The Zn 3 NcMT was prepared from ap o-NcMT by adding 3.5 molar equivalents of Zn(II) (added as Z n(II)- acetate) in a 10-fold excess of free cysteine to stabilize the Fig. 1. Amino acid sequence alignment. Sequences shown are tho se of N. crassa MT (residues 2–26, NCBI accession No. CAA26793), the b-domain o f Homo sapiens MT-2 A (residues 1–30, NCBI accession No. P 02795), the b-domain o f Mus m usculus MT1 ( residues 1–3 0, NCBI Accession No. AAH36990), AMT1 from Candida glabrata (residues 70–113, NCBI accession No. P41772), ACE1 from Sac- charomyces cerevisiae (residues 70–110, NCBI accession No. NP_011349), CopY from Enterococcus hirae (residues 123–145, NCBI accession No. Q47839), MAC1 from S. ce revisiae (residues 150–187, NCBI accession No. NP_013734) and MT from S. cerevisiae (Cup1–1) (residues 9–49, NCBI accession No. AAS56843). The cysteine residues are highlighted in bold and the consensus sequence repeated. 4214 P. A. Cobine et al. ( Eur. J. Biochem. 271) Ó FEBS 2004 excess z inc. Prior to the copper(I) titration o f t he NcMT sample, it was treated with 50 lL of a 1 : 1 slurry of Chelex-100 (Bio-Rad) in degassed MilliQ water to r emove any excess unbound/displaced metals. The Chelex-100 was removed by centrifugation. The Cu(I)-thiolate emission was monitored at 580 nm (excitation at 295 nm) with a Perkin-Elmer LS 50B luminescence spec trometer. The spectra were collected at 23 °C in sealed screw-topped fluorescence cuvettes (Spectrocell). The spectrometer was equipped w ith a 350 nm band-pass filter to avoid second order effects and used settings of 5 and 20 nm, respectively, for the excitation and emission slits. Cu(I)–NcMT for NMR analysis was desalted on PD-10 ( Pharmacia, G-25) columns equilibrated with MilliQ water, pooled and lyophilized. Copper stoichiometry was quantified by flame atomic absorption spectroscopy and amino acid analys is. The final Cu 6 NcMT co ntained 1 .8 lmol N cMT and 10.9 lmol Cu(I). NMR sample preparation Lyophilized Cu 6 NcMT (handled under an argon atmo- sphere) was dissolved in 500 lL 90% H 2 O, 10% D 2 O, pH 6.5, 0.1 m M 2,2-dimethyl-2-silapentane-5-sulfonate (as an internal reference) [32], and 0.02% NaN 3 that was degassed prior to protein addition under high vacuum and re-pressurized to 1 atm under argon to avoid oxidation/ disproportionation of the C u(I)–protein. T he sample was loaded into a 5 mm NMR tube, c apped a nd sealed with parafilm. NMR spectroscopy and assignments of NcMT A2D 1 H, 1 H-TOCSY [33,34] and 2D 1 H, 1 H-NOESY were collected at 10 °C on a Varian Inova 800 MHz NMR spectrometer equipped with a triple-axis gradient, triple- resonance p robe. T he sweep widths f or both experiments were 9000 Hz with 1024 and 512 complex points collected in the directly and indirectly detected dimensions, respect- ively. The TOCSY was collected with 64 transients per increment (40 ms mixing time with 300 ms delay between acquisitions), and the NOESY was collected with 32 transients per increment (300 ms mixing period with 200 ms delay between acquisitions). Spectra were zero- filled to twice the number of collected points a nd apodized using a p/3 shifted sine bell before Fourier transformation. New 1 H-chemical shift assignments at 10 °Cfrom2D- NOESY and TOCSY s pectra were performed as described previously [35]. All spectra were processed u sing NMRPIPE [36], and analysed using t he program PIPP [37]. Coupling constants for backbone amide to a hydrogen atoms (i.e. 3 J H N H a ) were obtained from 1D-proton and 2D-COSY spectra using d econvolution. Due to t he different temper- atures used during the NMR experiments (10 °Cinthe present s tudy and 25 °C i n [ 27]) t here are expected t o b e small differences in chemical shifts, typically larger for NH protons. However, the overall very good agreement in these shifts can be confidently attributed to the protein forming the same structure. Gradient NOESY spectra were acquired w ith WaterGate solvent s uppression taken from the ÔgnoesywgÕ pulse sequence in ÔProtein PackÕ as supplied b y Varian I nc. Structure calculations for NcMT Distance restraints (50 kcalÆmol )1 ÆA ˚ )2 ) for NcMT structure calculations were classified as short (1.8–2.7 A ˚ ), medium (1.8–3.3 A ˚ ), and long (1.8–5.0 A ˚ ) based on their N OE intensities. The upper bound of an NOE restraint was extended by 0.5 A ˚ for each p seudoatom methylene, o r methyl group. Residues e xhibiting 3 JH N H a coupling constants of < 5 Hz, 5–8 Hz, 8–9 Hz, a nd > 9 Hz were assigned dihedral angle restraints of )60° ± 30, )105° ± 55, ) 120° ± 40, and )120° ± 30, respectively, with a force constant of 500 kcalÆmol )1 Ærad )1 .Nometalatoms were included in the structure calculation. Structures were calculated using X - PLOR 3.851 [38] on an Octane Power Desktop (SGI R12000 with IRIX 6.5) using the hybrid distance geometry-dynamical simulated annealing protocol [39] as described for mouse MT1 [23]. Out of the 30 calculated structures, 12 were selected based on the complete absence of NOE violations greater than 0.5 A ˚ and r.m.s.d. for bond and angle deviations from ideality of less than 0.01 A ˚ and 5°, respec tively. Structure calculations for mouse aMT All structure calculations involving the a-domain of the mouse MT, aMT-1, were performed as previously reported [23] with the exception that the metal–cysteine restraints were not used and therefore all metals w ere absent in the calculations. To determine the precision [how well the individual structures calculated with a limited set of l ong- range N OEs (d ij , j > i + 4) compare to each other ] and accuracy (the simila rity of the calculated structures with a reduced set o f long-range NOEs to the structure calculated with all NOEs) of the calculated structures, all 22 long-range NOEs were first removed and then, by a random selection process reintroduced one by one for the structure calcula- tions. T hereby, for each number of long-range NOEs, 1 0 random selections of reintroduced long-range NOE sets were made and 10 structures calculated for each set, giving a total of 100 structures calculated for each point in Fig. 4. Results Metal binding stoichiometry of NcMT N. crassa can express MT in response to excessive concen- trations of copper in the growth media. NcMT purified from the mycelium of t he fungus contains 6 Cu(I) ions per mole of protein [9]. The copper(I) ions are bound to the cysteinyl sulfurs in a solvent-shielded Cu–S core [40]. Direct titration of copper(I) into a synthesized apo-NcMT resulted in a 4-Cu(I)NcMT. This complex was luminescent, excita- tion at 295 nm led to an emission maximum a t 5 80 nm, which is evidence for solvent protected Cu(I)-thiolates but the luminescence increased up to a plateau at 4 mole equivalents of Cu(I) per protein (Fig. 2A,B). The 4 : 1 stoichiometry conflicts with the known native species. Titrations with a variety of copper(I) sources, different types a nd concentrations of reductant, and with or without thermal treatments as used b y Stillman and coworkers [41] to select stable forms of the rabbit CuMT all resulted in the formation of Cu 4 NcMT. T he 1 D 1 H NMR spectrum of the Ó FEBS 2004 NMR structure of N. crassa Cu6MT (Eur. J. Biochem. 271) 4215 Cu 4 NcMT, prepared under any of these conditions, was very broad and in contrast to the well defined native Cu 6 NcMT 1 H NMR spectrum [27]. The unresolved 1 H NMR spectrum would be co nsistent with the sample containing a mixture of inter converting NcMT structures and/or structural instabilities. In an attempt to restrict the family of conformers formed during the copper(I) titrations, we first titrated zinc into the apo-protein to form a Zn 3 NcMT precursor. The excess zinc was removed by treatment of the sample with Chelex-100. The zinc stoi- chiometry of the resultant protein, 3-Zn(II):1 NcMT, was as expected from previous studies of NcMT and by analogy to the well s tudied b-domains of the mammalian MTs [12]. Sequential titration of the Zn 3 NcMT with Cu(I) salts as before yielded a luminescent core but in this case, the stoichiometry w as 6 Cu(I):1 NcMT, Fig. 2C,D. Most importantly, t he 1D 1 H-NMR p attern for the Cu 6 NcMT is equivalent to the native Cu(I) proton spectrum [27]. Attempts to prepare the Ag(I) derivative of N cMT d id not result in a stoichiometry of 6 metals p er mole of protein. Attempts to displace all of the z inc from the Zn 3 NcMT by silver were unsuccessful and always resulted in the forma- tion of a mixed metal s pecies inappropriate for s tructural studies. The structure determination of Zn 3 NcMT was not pursued as to our knowledge there are n o reports of this metal form from natural sources. Metal-cysteine restraints To explore the feasibility of determining an MT structure without metal–cysteine restraints, t he structure o f the a-do main of mouse MT-1 without the experimentally determined 113 Cd–Cys restraints was re-calculated. Fig. 3 shows the backbone fold of the average energy minimized structures for the a-domain of mouse MT-1 with (dark strand) and without (light strand) the inclusion of experi- mentally determined 113 Cd–Cys restraints. T he super- imposed backbone structures are r emarkably similar with an r.m.s.d. of 1.94 A ˚ over all backbone atoms. This value is very similar to t he r.m.s.d. comparison between the a-d omain of mouse MT-1 and the a-domain of rat MT-2, 2.14 A ˚ [23], suggesting that accurate and precise MT structure d eterminations are possible without using metal to cysteine restraints providing one has a sufficient number of NOE constraints. The lack of regular secondary structure in MTs places an increased importance upon the long-range contacts for precision in structure calculations. This can be quantitatively evaluated by recalculating the mouse MT1 a-d omain structure in the absence of all long-range NOE restraints and 113 Cd–Cys connectivities and then syste- matically and randomly reintroducing long-range NMR Fig. 3. Ribbon backbone diagrams of the a-domain of mouse MT-1 calculated with (dark) and without (grey) 113 Cd–Cys restraints. The Cd atoms (rendered to van der Waals radii), cysteine sulfur, and Cd-S bonds are indicated by the large spheres, sma ll sphere s and black lines, respectively. The N and C termini are labelled for orientation. Dia- grams were generat ed using t he program INSIGHTII (Molecular Simu- lations, Inc.). Fig. 2. In vitro copper reconstitutions o f N. crassa MT monitoring Cu(I)-S luminescence at 580 nm after excitation at 295 nm. (A) The reconstitutions at 10 nmolÆmL )1 of apo- NcMT with copper(I) the increasing additions of copper the luminescence makes uniform steps (B). (C) Titratio ns of 50 nm olÆmL )1 Zn 3 NcMT with copper(I) t o a stoichiometry of 6 Cu(I) per mole of prote in. The sequential increase plotted in (D) r eveals a plateau at six equivalents and that t he quantum yield of relative luminescence per mole of protein is equal for the 4 and 6 Cu(I)-N cMT . 4216 P. A. Cobine et al. ( Eur. J. Biochem. 271) Ó FEBS 2004 restraints to determine the effect on the precision and accuracy of the generated structure. Figure 4A shows the r.m.s.d. of 10 sets of s tructures, generated as described in Materials and methods, to the average minimized structure, for each set of reintroduced long-range NOEs. Determining the deviation of the generated structure with a reduced number of NOEs from the published MT1 structure provides a measure of accuracy shown in Fig. 4B. As can be seen, the structure of MT1 without metal–cysteine restraints is sufficiently well defined as long as 10–15 long- range NOEs are used for the structure calculation and the backbone r.m.s.d. is kept below 2 A ˚ . NcMT structure Two-dimensional 1 H, 1 H-TOCSY and NOESY NMR spectroscopy was used to assign the 1 H resonances and provide interproton distance restraints, r espectively, on NcMT. The chemical shifts of NcMT determined here at 10 °C a re similar t o t hose of a previous study perfo rmed at 25 °C [27]. A lmost a ll the 1 H chemical shifts were v isible in the T OCSY with the exception of the side c hain of Cys5 (assigned from the N OESY), and a ll resonances of Gly1 (not identified i n either experiment). Assignment of Gly1 was not possible; this was most likely due to rapid exchange of the amide proton of Gly1 with the s olvent, coupled with chemical shift overlap reported previously [27]. The chem- ical shifts have been deposited in the BMRB data bank under accession number 6290. Examination of the chemical shifts showed no indication of secondary structure [42,43]. Line widths of 1D- 1 H NMR resonances have been used as an efficient method for determining the aggregation state of a protein [44,45]. The average line width of NcMT amide resonances from the 1D- 1 H spectrum was determined to be 5.6 Hz. When six Cu(I) atoms are considered bound to NcMT, the correlation time (s c ) and line width for the monomer are expected to be 2.6–3.7 n s and  4.5–6 Hz, respectively [34]. The observed line widths for the reconsti- tuted Cu 6 NcMT are consistent with the sample being monomeric. A summary of the observed NOEs is presented in Fig. 5A. Inspection of the NOE patterns shows no evidence of regular s econdary st ructure elements, which is in agreement with the information from the chemical shifts Fig. 4. Effect of the inclusion of long-range NMR restraints f or aMT- 1 structure determination in the absence of metal–Cys restraints. Long-range NMR restraints were removed and t hen systematically and randomly re-introduced for mouse aMT-1 structure calcula- tions. (A) Precision indicated via the r .m.s.d. o f the family of 10 structures generated (compared to the minimized average) for each number of long-range NOEs inclu ded in calculations. (B) Relative accuracy of the calculations indicated by comparing the aMT-1 minimized average structure for each number of long-range NOEs included (in the absence of metal–Cys restraints), to the average minimized stru cture of aMT-1 calculated with all NOEs and inclu- ding the m etal–Cys restraints. Error b ars in both panels indicate the standard deviation. d αN (i,i+3) d NN (i,i+3) d NN (i,i+2) d NN (i,i+1) G 20 10 20 10 Residue Number Number of NOEs 20 15 10 5 0 DCGC SGASS CNCGSGCSCSNCGS K d βN (i,i+1) d αN (i,i+1) d αN (i,i+2) A B Fig. 5. NOE map for NcMT. (A) Summary of inter-residue N OEs determined for NcMT that are typically used to ind icate secondary structure. Strong, medium a nd weak intensity NOE cross-peaks are indicated by tall dark, medium grey, and small white boxes, respect- ively. Th e primary sequence is shown at the b ottom in the o ne letter code. (B) T he total number of NO Es assigned for NcMT displayed on a per residue basis. Intra-residue, sequential (d ij ,j ¼ i+1),medium (d ij ,i+1<j<i+4),long(d ij , j > i + 4) range NOEs are des- cribed by solid, dotted crosshatched, whit e, and thick crosshatched columns, respectively. Ó FEBS 2004 NMR structure of N. crassa Cu6MT (Eur. J. Biochem. 271) 4217 and is typical for this family of proteins. The total number of intraresidue, s equential, medium, and long-range NOEs for each residue in the NcMT sequence is shown in Fig. 5B. The majority of critical long-range restraints involve the side chains of Ala8, Asn12, and Cys21. Despite the almost complete assignment of 1 H resonances, the number of long- range contacts was low. Utilizing the 152 NOEs and 13 dihedral angle restraints, a total of 30 structures was generated using the program XPLOR 3.851 [38]. From these generated structures, 12 resulted in no NOE or dihedral angle violations and all showed the same backbone fold. Subsequently, the 10 accepted structures (no NOE violation >0.5 A ˚ , a n r.m.s.d. for bond deviations from ideality of less than 0.01 A ˚ and an r.m.s.d. for angle deviations from ideality of less than 5 °) with the lowest energy were selected which yielded a backbone r.m.s.d. to the average minimized structure of 0.79 A ˚ for the well-defined region (residues 5–20) and 1.59 A ˚ for the entire length of the protein. The final family of 10 NcMT structures is shown in Fig. 6 with the N - and C-termini labelled and the c ysteine sulfur atoms in the Ôclosest to meanÕ structure drawn as spheres. The structure of NcMT has been deposited in the Protein Data Bank under accession number 1T2Y. The structural statis- tics for NcMT are presented in Table 1. The program PROCHECK - NMR [46] showed that > 90% of the ø, w angles for the 10 structures fell into the core or a llowed regions. Despite the complete absence of elements of r egular secondary structure, which is a quite common situation for MTs, the backbone global fold of the 25-residue peptide is well defined if one excludes the N and C termini. It shows a new polypeptide structure with t he backbone being wrapped around an empty space containing the copper– sulfur cluster, on going from the N to C terminus, in a left handed form for the first half of the molecule then in a right handed f orm for the second half. Such a mixed h andedness of the peptide part around the metal–cysteine cluster has so far been found only in the C-terminal domain of blue c rab MT [17], whose overall fold is quite different from that of NcMT. While NcMT shares considerable sequence similar- ities and a conservative positioning of the seven cysteine residues with o ther members of t he MT family of proteins, none of the oth er structurally characterized MTs show the same fold. This is reflected in the r.m.s.d. differences in cysteine residue positions (backbone heavy a toms and sulfur atoms) between NcMT and lobster [47], yeast [15,16], mouse [23], blue crab [ 17], fish [18] and se a urchin [14] M Ts as shown in Table 2. Such r.m.s.d. differences in cysteine positions are c haracteristic of unrelated MT structures. A good example to illustrate this point is the r.m.s.d. differences between the a-andb-domains of sea urchin MT of 4.98 A ˚ for the cysteine backbone heavy atoms and 2.92 A ˚ for the sulfur atoms. For c omparison the cys teine Fig. 6. Stereo drawing of a line representation of the b ackbone of 10 N cMT structures which possessed the lowest overall energy, out of 12 generated t hat contained n o NOE or dihedral angle violations. Theensembleisleast-squares fitted to the first structure and the N and C termini are labelled f or orientation. The cys- teine sulfur atoms of th e Ôclo sest to meanÕ structure are shown as yellow spheres. Table 1. Structural statistics for NcMT, for the 10 lowest overall energy structures out of 12 without a ny NOE or d ihedral violations. NOE restraints Total 152 Intraresidue 63 Sequential (|i-j| ¼ 1) 53 Medium (2 6 |i-j| 6 4) 25 Long range (|i-j| P 5) 11 Dihedral restraints (ø) 13 r.m.s.d. to average structure (A ˚ ) Well-defined regions (N,C a ,C) a 0.79 All regions (N,C a ,C) 1.59 All heavy atoms 1.97 Energies (kcalÆmol )1 ) E overall 82.40 ± 7.95 E bonds 6.07 ± 0.74 E angles 29.67 ± 2.35 E vdw 7.92 ± 1.53 E NOE 29.13 ± 2.62 E dihedral 1.75 ± 0.58 E improper 7.86 ± 0.46 r.m.s.d. from idealized covalent geometry used within X - PLOR Bonds (A ˚ ) 0.0047 ± 0.0003 Angles (deg) 0.64 ± 0.03 Impropers (deg) 0.63 ± 0.02 NOEs (A ˚ ) 0.062 ± 0.003 Dihedral (deg) 1.46 ± 0.3 Procheck-NMR b [46] In most favoured regions 44.4% (80) In additional allowed 44.4% (80) In generously allowed 5.6% (10) In disallowed regions 5.6% (10) a Residues 5–20. b Number of residues out of all 10 structures. Total non-glycine and non-proline is 180. Number of glycines is 60, with 10 end-residues, for a total of 250 residues. 4218 P. A. Cobine et al. ( Eur. J. Biochem. 271) Ó FEBS 2004 residue position r.m.s.d. between the structurally related b-domain of mouse MT1 and human MT2 a re 0.57 and 2.10 A ˚ for the backbone heavy atoms and the sulfur atoms, respectively. Discussion The solution structure of the Cu 6 NcMT, which was solved without the acquisition and inclusion of specific metal– cysteine NMR r estraints, shows a novel polypeptide fold and represents only the second copper MT s tructure to be elucidated [15,16]. Although other MTs show a strong sequence similarity to NcMT (e.g. 32% with the b-domain of human MT2) the 3D structure of the polypep tide backbone i s completely different. The unique fold of this CuMT is a clear demonstration that MT protein folds are largely d etermined by t he constraints of metal–sulfur connections and not the amino acid sequence. N. crassa MT has a -CxCxxxxxCxC- motif that is found in a variety of Cu(I) binding proteins (Fig. 1). The lack of repetitive secondary structures in the NcMT peptide backbone along with the unsuccessful efforts to prepare the isomorphic and homogeneous NMR active spin 1/2 Ag(I) derivative o f the native Cu(I) NcMT were factors which had inhibited its structure elucidation. A hallmark of the MT structures is the dependence on the sequential position of the cysteine in the primary sequence, the identity of the coordinated metal and its c oordination number [14,48]. The paper by Bertini et al. [16] was the first to show that the acquisition of NMR data at 800 MHz allows for the determ ination o f MT structures without metal–cysteine constraints. The comparative study on mouse MT1 at 800 MHz in this paper helps to validate these studies. The successful in vitro reconstitution of Zn 3 NcMT with six equivalents of Cu(I) indicated that the metal binding sto ichiometry w as the s ame as the b-domain of mammalian M T [12] even though NcMT lacks the two C-terminal cysteines found in the mammalian MTs. These two missing cysteines and therefore less metal coordination mightbethereasonwhyNcMTneedstobeintheZn 3 -form before it successfully binds six a toms of copper(I). Thus, Zn 3 –NcMT might constitute a scaffold where the zinc can be replaced by Cu(I) without the need for large structural rearrangements. In other words the less favourable enthalpy changes by binding 6 Cu (I) atoms to only seven cysteines in NcMT are compensated by the smaller differences in entropy when copper substitutes zinc i n Zn 3 –NcMT, rather than binding to apo-NcMT. The variable coordination number of c opper, which can adopt a linear two-coordinate (digonal) or a distorted planar trigonal three-coordinate geometry with sulfur ligands [49] adds an addition al level of complexity in solving the copper MT structures. These coordination possibilities of the copp er(I) ions are perhaps responsible for the titration of the apo-NcMT to a non-native (4 : 1) stoichiometry. In thecaseoftheNcMT,itseemsthatthecoordinationofzinc in Zn 3 NcMT was necessary to conform/stabilize a structure such that the Cu(I) titration produced the native Cu 6 Cys 7 metal–thiolate cluster rather than the Cu 4 Cys 7 made from apo-NcMT which gave rise to a f amily of rapidly i ntercon- verting s tructures. The resultant Cu 6 Cys 7 core appears to constrain the peptide sufficiently to enable its 3D solution structure to be determined. The requirement that the synthetic Cu(I)MT be made from a Zn-protein in vitro to obtain an NMR spectrum identical t o t hat of t he native Cu(I)MT raises questions about how the organism controls the formation of the Cu(I)NcMT in vivo. If the Zn(II)NcMT were to form in vivo when NcMT was induced by copper exposure one might expect that it would be a very short Table 2. C ysteine r.m.s.d. comparison ( in A ˚ )tootherMTs. PDB number Cys-N,Ca,C¢ Cys-Sc 1J5M a 4.88 5.47 1FMY b 3.53 4.10 1DFT c 4.95 4.60 1DMC d 3.71 4.37 1DME e 5.22 5.31 1M0G f 3.49 4.45 1M0J g 3.90 4.98 1QJK h 4.47 5.29 1QJL i 3.85 4.77 a b-domain of lobster Homarus americanus MT. b Cu(I)-yeast Saccharomyces cerevisiae MT. c b-domain of mouse Mus musculus MT1. d a-domain of blue crab Callinectes sapidus MT. e b-domain of blue crab Callinectes sapidus MT. f a-domain of the fish Noto- thenia coriiceps MT. g b-domain of the fish Notothenia coriiceps MT. h a-domain of sea urchin Strongylocentrotus purpuratus MT. i b-domain of sea urchin Strongylocentrotus purpuratus MT. Fig. 7. Ribbon diagram of the NMR solution structure o f NcMT with six Cu(I ) atoms modelled into possible positions within th e structure and the cysteine side chains turned to point towards the protein’s centre. Cu(I) atoms are rendered according to their van d er Waals radii. The Cys side-chains are rendered as grey sticks with th e Cys sulfu r atom shown in light grey. The modelling o f Cu(I) binding as depicted was acc om- plished by a ttempting to best satisfy k nown b ond lengths and altering the Cys chi side- chain a ngle to po int the sulfur atoms towards the centre. The figure was prepared using INSIGHTII (Molecular Simula- tions, Inc.). The backbone fold o f the NcMT protein is shown in black with the carbons and su lfur of the cysteine side chains shown in dark grey and light grey, respectively. The Cu(I) atoms are rendered a s medium grey spheres with a van der Waals radiu s of 0.95 A ˚ .Taking into consideration t he Cu(I)–S l uminesc ence which indicate a pro- tected copper core, we have manually rotated the Cys s ide-chain 1 cys and 2 cys angles to direct the sul fur atoms into the core o f the protein. Ó FEBS 2004 NMR structure of N. crassa Cu6MT (Eur. J. Biochem. 271) 4219 lived and hard to i solate species a s copper i ons would b e expected to readily displace the Zn(II). However, there are no in vivo data t o indicate that the o rgan ism has to form a zinc precursor. The requirement of Zn(II) in the f ormation of Cu(I)MT clusters, in particular the b-domain, has been observed for mouse MT [50]. Additionally, the same spacing of cys teine residues i s also f ound in the repressor protein CopY from Enterococcus hirae and it is apparent that the metal-binding properties of this protein may require the binding of Zn(II) in this site [51,52]. The stability of the Cu(I)-S core and the DNA-binding activity are dependent o n zinc binding. In addition, the copper chap- erone CopZ is a specific source of copper for the Zn-form of CopY [51]. However, apo-CopY does not demonstrate this specificity suggesting a potential loss of structure that confers this property. A combination of the data suggests that Zn(II)-binding to these cysteine-rich, copper-binding sites orders a structure that is required for activity. A model structure of Cu 6 NcMT demonstrating the shielding of the copper core, using one model with copper atoms satisfying the Cu–S bond distances and minimizing any interactions within the NMR structure, is shown in Fig. 7. While there is probably a multitude of models that would fit the NMR data, there is no way to co nfirm any from NMR s tudies using t he NMR inactive Cu(I) metal form of the protein. The NcMT backbone structure presented here uses no specific metal–Cys restraints and a low number of long-range NOE constraints. However, the mouse a-MT-1 s tructures calculated with and without specific metal–Cys restraints support the assertion that metal–Cys restraints are not required for precise and accurate structure determination of the protein backbone with NMR data collected at 800 MHz. The a-domain of MT-1 was a lso u sed t o a ssess the requirements for long- range NMR restraints in structure c alculatio ns. Although both the length of the a-domain of mouse MT1 (31 vs. 25 amino acids in NcMT) and the number of NOEs (256 vs. 152 for NcMT) is larger for mouse MT1, t he back- bone r.m.s.d. between structures calculated without metal–cysteine restraints is comparable for NcMT (1.94 A ˚ for mouse MT1 and 1.59 A ˚ for NcMT). Evaluation of the relationship between the number of long-range NOEs and the resultin g r.m.s.d. is relevant i n MTs because of their lack of defined s econdary structure. In more typical proteins, the translational and rotational positions of the residues contained in a secondary structural element (a-helix, b-sheet) might be spatially defined by as little as a single long-range contact to the rest of the molecule. However, in MT, which is composed mostly of coils and loops, the spatial orientation of relatively large regions cannot be positioned b y such a single critical restraint. In conclusion we have described a new MT fold for the N. crassa Cu 6 MT which consists of a half left- and half right-handedly polypeptide backbone wrapped around the copper(I)-cysteine cluster. No direct information about the metal–sulfur connectivities could be obtained b y using an isomorphic, NMR active metal substitute for Cu + ,suchas Ag + , because a stable homogenous Ag substituted NcMT could not be prepared. Nevertheless, the use of data at 800 MHz on th e reconstituted Cu 6 NcMT was sufficient to allow for an accurate backbone fold to be determined. Acknowledgements This work was supported by NIH grant DK18778 to I.M.A. NMR instrumentation was provided with funds from the NS F (BIR-961477) and t he University of Minnesota Medic al School. K.Z. thanks the Austrian Science Foundation FWF for financial support under project number P15289. We would like to thank Dr David Live and Dr Be verly G. 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(1999) The Enterococcus hirae copp er chaperone CopZ delivers copper (I) to the CopY repressor. FEBS Lett. 445, 27–30. 52. Cobine, P .A., Jones, C.E. & Dameron, C.T. (2002) Role for zinc (II) i n the copper (I) regulated p rotein CopY. J. Inorg. Biochem. 88, 192–196. Ó FEBS 2004 NMR structure of N. crassa Cu6MT (Eur. J. Biochem. 271) 4221 . secondary structure, their tertiary structure is dependent on the number and type of metal ions they coordinate. The MT from the fungus Neurospora crassa, isasingle domain. stable forms of the rabbit CuMT all resulted in the formation of Cu 4 NcMT. T he 1 D 1 H NMR spectrum of the Ó FEBS 2004 NMR structure of N. crassa Cu6MT (Eur.