J. Mol. Biol. (1990) 214, 165-779

Three-dimensional Structure of Human [113Cd,]Metallothionein-2 in Solution Determined by Nuclear Magnetic Resonance Spectroscopy Barbara

A. Messerlel,

Andreas

Schtiffer2t, Milan Kurt Wiithrichl

Va89k2, Jeremias H. R. Kfgi2

and

fiir Molekularbiologie und Biophysik Eidgeniissische Technische Hochschule-Hbnggerberg CH-8093 Ziirich, Switzerland ‘Institut

2Biochemisches Winterthurerstrasse

Institut der Universittit 190, CH-8057 Ziirich,

(Received 7 November

Ziirich

Switzerland

1989; accepted 4 April

1990)

was determined by The three-dimensional structure of human [ ‘13Cd,]metallothionein-2 nuclear magnetic resonance spectroscopy in solution. Sequence-specific ‘H resonance assignmenk were obtained using the sequential assignment method. The input for the structure calculations consisted of the metal-cysteine co-ordinative bonds identified with ‘H-‘H distance constraints from nuclear heteronuclear correlation spectroscopy, Overhauser enhancement spectroscopy, and spin-spin coupling constants 3JHNa and 3Jap. The molecule consists of two domains, the o-domain including amino acid residues 1 to 30 and three metal ions, and the a-domain including residues 31 to 61 and four metal ions. The nuclear magnetic resonance data present no evidence for a preferred relative orientation of the two domains. The polypeptide-to-metal co-ordinative bonds in human metallothionein-2 are identical to those in the previously determined solution structures of rat metallothionein-2 and rabbit metallothionein-2a, and the polypeptide conformations in the three proteins are also closely similar.

1. Introduction

Schk;ffer, 1988). In the naturally occurring form; the cysteinyl residues bind seven divalent metal ions, in particular Zn2+, and also Cd2+ or Hg2+ (Va%k & KB;gi, 1983). The three-dimensional structures of rat [Cd,]MT-2 and rabbit [Cd,]MT-2a in solution have been determined with n.m.r. spectroscopy (Schultze et al., 1988; Arseniev et al., 1988). Rat MT-2 differs from rabbit MT-2a in ten amino acid substitutions and one deletion, but the three-dimensional solution structures were found to be very similar. The a,mino acid sequence of human MT-2 differs from rat MT-2 by seven substitutions, and from rabbit MT-2 by four substitutions and one deletion. This paper describes the determination of the three-dimensional structure of human MT-2, and presents a comparison of this solution structure with those of MT-2a from rabbit and MT-2 from rat. In addition to the solution structures of rat MT-2 and rabbit MT-2a, an X-ray crystal structure (of rat [Cd,, Zn,]MT-2 has been reported (Furey et al., 1986, 1987), which was found to be different from the corresponding solution structure. The crystal

Human metallothionein-2 (MT-2$) belongs to a family of homologous metallothioneins (K&gi & Schk;ffer, 1988). These proteins consist of a polypeptide chain with 61 or 62 amino acid residues, including 20 cysteinyl residues, which are strictly conserved in all mammals studied so far (Kk;gi & 7 Present address: Ciba-Geigy Ltd., Agricultural Division, Basel, Switzerland. $ Abbreviations used: MT? metallothionein; n.m.r.; nuclear magnetic resonance; KOE, nuclear Overhauser enhancement; 2D. 2-dimensional; 311, 3-dimensional; NOESY, P-dimensional nuclear Overhauser enhancement spectroscopy; COW, Z-dimensiona,l correlated spectroscopy; SQF-COSY. S-dimensional 2quantum filtered correlated spectroscopy; RELAYEDCOSY, %dimensional relayed coherence transfer spectroscopy; E. COSY, S-dimensional exclusive correlation spectroscopy; TOCSY j 2-dimensional total correlation spectroscopy; TPPI. time proportional phase incrementation; r.m.s.d., root-mean-square difference; p.p.m., parts per million.

765 Oozz-2836/90/150765-lj

$03.00/O

0 1990 Academic Press Limited

B. A. Messerle

766

structure is under reinvestigation (Stout et al., 1989). A detailed comparison of the rat MT-2 structure in solution and the structure reported for rat MT-2 in single crystals has been given (Schultze et a~.; 1988) and no further comparisons between solut,ion structures and crystal st)ructure will be made here.

2. Materials

and Methods

Metallothionein was isolated from human livers as described (Biihler & KSgi, 1974). The ;MT-2 isoprotein used for t,he present n.m.r. studies was subjected to an additional ion-exchange chromatography step (Biogel; BIORAD) and eluted with a linear gradient of 10 mM to 30 mM-Tris.HCl (pH 8.6). Each preparation was characterized by amino acid analysis (Durrum 500) and by metal analysis using atomic absorption spectroscopy (Instrumentation Laboratory: model IL 157). All MT-2 preparations cont,ained 7 mol of zinc/m01 protein. The protein concentration was determined spectrophotometritally by measuring the absorbance of t’he apoprotein at 220 nm in 0.01 M-HCl (&,,,=48190 Mm* cm-‘: Biihler. 1974). Homogeneous [Cd,]MT-2 samples containing either Harwell) were ‘13Cd or l12Cd ( > 95 “/b enrichment, prepared as described (VaBBk et al., 1987). Before the n.m.r. measurements, the MT-2 samples were passed over a Sephadex G-50 column to ensure size homogeneity. In a subsequent step, the concentrated NIT-2 solution was transferred to a medium containing 20 mx-[2H11]Tris. HCl (pH 7.0), 20 mrw-KC1 using an Amicon ultrafiltration apparatus with a YM-2 membrane. For the n.m.r. measurements in ‘H,O. the sample was twice lyophilized, first from H,O and then from 2H,0; and then redissolved in 100% 2H,0. A small portion of each sample was used to verify the homogeneity of the final protein solution by reverse phase high-pressure liquid chromatography. In the n.m.r. samples, the protein concentration was 8 mM, and the aqueous solution further contained 20 mM-[2H, ,]Tris . HCI (pH 7.0). 20 rnfil KCl. Proton n.m.r. spectra were recorded on Bruker AM600 and AM500 spectrometers. Heteronuclear ‘13Cd-‘H n.m.r. spectra were recorded on Bruker AM360 and AM600 spectrometers. One-dimensional ‘13Cd n.m.r. spectra were recorded with broad-band ‘H decoupling during data acquisition. Throughout, the pure phase absorption mode was used with time proportional phase incrementation (TPPI: Redfield & Kunz, 1975; Marion & Wiithrich, 1983). Standard procedures were used for the following experiments, which were needed for the determination of the sequence-specific assignments and the preparation of the input for the structure determination: SQF-COSY (Ra,nee et al.; 1983; Neuhaus et al., 1985), 2quantum spectroscopy with a mixing time of ~,=30 ms (Wagner & Zuiderweg, 1983; Otting & Wiithrich, 1986), TOCSY with ~,=80 ms (Braunschweiler & Ernst, 1983; Bax 8r. Davis, 1985), RELAYED-COSY with z,=25 ms (Wagner; 1983). R’OESY with ~,=40 rns? 80 ms and 150 ms (Anil-Kumar et al., 1980; Jeener et al., 1979), and E. COSY (Griesinger et al., 1985). The typical data size was 2048 x 512 points in the time domain. with zero filling to 4096 x 2048 points. In order to obtain a complete set of cysteine proton-to- ’ 13Cd scalar connectivities, [’ 13Cd, ‘HI-COSY experiments were acquired at both 25°C and 36°C with mixing times of z,= 15 ms, 30 ms and 50 ms. The same experimental conditions as described by Frey et al. (1985) were employed, whereby only the pulse

et ai. sequence with a (n/2),( ‘H) pulse before detection was used (see Fig. 3 of Frey et al. (1985)). For the acquisition of NOESY spectra, zero-quantum coherence was suppressed using a modification of the technique described by Rance et al. (1985). For the T;OESY dat,a, a cosine window was applied as the weighting function prior to Fourier t,ransformation. Baseline distortions were eliminated using a polynomial fit of order 3. Sequence-specific ‘H n.m.r. assignments were obtained using the standard sequential assignment method (Billeter et al.; 1982; Wagner & Wiithrich, 1982; Wider et al., 1982; Wiit’hrich, 1986). The calibration of XOE intensities versus ‘H-‘H distances needed for the preparation of the input, for the structure calculations was established in 2 steps. First, for the initial structure calculations relative peak intensities were determined by counting the number of exponentially spaced contour levels at’ the peak maxima, and relat,ionships between peak intensity and upper distance limit, were determined using similar arguments to those described in detail by Arseniev et al. (1988; see also Wiithrich, 1986). .Four structures were computed from this input, and the average was computed for all ‘H-‘H distances in these structures. For the proton pairs where the standard deviation for this average distance was less t’han 0.2 a (1 8=0.1 nm): the distance between the protons was t’aken as “well defined”. Well defined average dist’ances between backbone protons that were shorter than 5.0 A were then related to the observed volume integrals for the corresponding PU’OESY cross-peaks. Peak integration was achieved using the EASY program (C. Eccles, P. Giintert. M. Billeter & K. Wiithrich, unpublished results). The resulting calibration curve corresponded closely to a l/r6 dependence of the NOE intensities on the proton-proton dist,ance r. St,ereospecific assignments for diastereotopic groups of protons were obtained from 3 different sources. The first was the program HABAS (Giintert et al., 1989), which uses exclusively local constraints, i.e. the spin-spin coupling constants 3JHNn and 3Jols. and intraresidual and sequential NOES. Second. additional stereospecific assignments were obtained using medium-range KOE distance constraints in addition to the int,raresidual and sequential constraints (Widmer et al., 1989). To this end. t~he program DISMAN (Braun & Go, 1985) was run 200 times up to level 4, i.e. considering interactions between residue pairs (i.j) with Ii-j\ 14. In these 200 siructures. all tripeptide segments were analyzed for residual constraint violations and for variations of the dihedral angles. From all the struct’ures that contained no residual constraint, violations in any of the residues (i-l), i and (ii- 1) that, exceeded 0.25 ,!% for KOE constraints and sterir constraints, or 5” for dihedral angle constraints, the dihedral angle values for the residue i were included int,o a set, of data of which the ext,reme values were then used as -,he upper and lower bounds on these angles in subsequent structure calculations. In some instances: where these new dihedral angle constraint’s were significantly tighter than those obtained from HABXS, additional stereospecific assignments for fi-methylene groups were obtained. Third, some additional stereospecific a,ssignments were obtained from the analysis of long-range SOEs in the structures calculated (see below) with the program DIS&liZX (Senn et al.: 1984; Zuiderweg et al.. 1985; Kline et al., 1988). The structural interpretation of t’he n.m.r. data was done using the program DISMAl\u (Braun B Go, 1985). 4s no iSOE constraint could be found between the 2 domains of the protein, the structure calculations were done with 2 completely independent domains: i.e. t’he P-domain with

Human

Metallothionein

Conformation

in Xolution

767

for rabbit MT-2a (Arseniev et al., 1988) arrd rat MT-2 (Schultze et al., 1988), except for the use of more sophisticated approaches in the preparation of the input for the structure calculations (see Materials and Methods). Therefore, the following description of the structure determination is rather brief and makes frequent reference to the earlier work with the homologous metallothioneins. (a) Resonance assignments

5.0

4.2

4.6 w2 (p.p.m.) (al

1.8

, 2.0

7 E 2 3

2.2

2.4 ,“,

,,a,

‘i>J I

1

4.6

1

I

44 w2 (p.p.m.)

I

,

4.2'

(b)

Figure 1. Homonuclear proton 2&F-COSY spectrum of (protein concn 8 mM in ‘H,O, human [ “‘Cd,]MT-2 20 mix-[2H,,]Tris.HC1 (p2H 7.0), 20 mivr-KCl, t=25”C, ‘H frequency 500 MHz). (a) Spectral region containing the cysteinyl HE-H0 cross-peaks. (b) Spectral region containing the Ha-HP cross-peaks of the amino acid residues with long side-chains. The peak assignments are indicated by the l-letter symbol for the amino acid residues and the sequence number. For cysteinyl residues 7; 15, 19 and 48, which are in a crowded spectral region, the w1 positions are identified at the top of the spectrum in (a).

residues 1 to 30, and the a-domain with residues 31 to 61. This treatment is analogous to that used previously for rabbit MT-2a (Arseniev et al., 1988) and rat MT-2 (Schultze et al.. 1988).

3. Results and Discussion In the n.m.r. spectral analysis and the structural interpretation of the n.m.r. dat’a for human MT-2, we followed closely the procedures used previously

Human MT-2 was reconstituted with both n.m.r.llZCdZf and n.m.r.-observable 1131Cd2f. inactive Comparison of the two spectra in the regions of the Ha-H” cross-peaks (Fig. 1) and the H”-H” crosspeaks enabled the identification of the ‘H n.m.r. of the metal-bound eysteinyl residues lines (Neuhaus et al., 1984). The identification of the Cys spin systems was then further confirmed with the use of an X-filt,ered [‘H, lH]-COSY experime:nt for the singly bound cysteinyl residues, and 2X-half[ X - ’ 13Cd) for the bridging filter [lH, rH]-COSY cysteinyl residues (Wiirgijtter et al., 1988). On comparison of the 2QF-COSY spectrum in ‘H,O of human MT-2 with those of rabbit MT-2a and rat MT-2, it was observed that the resonance positions for the cross-peaks due to non-labile protons of corresponding residues varied in virtually all instances by less than 0.1 p.p.m, among the three species. This greatly facilitated the further tracing of the spectra, both for the spin system identifications using BQF-COSY, RELAYED-COSY and and the sequential assignments using TOCSY, NOESY with a mixing time of 150 milliseconds (Wiithrich, 1986). In order to resolve ambiguities in some assignments of overlapping amide proton resonances, NOESY spectra were acquired at temperatures of 7”C, 10°C and 14”C, as had also been found necessary for rat MT-2 (Worgijtter et al., 1987). The sequential NOE connectivities obt,ained are presented in Figure 2, and the sequence-specific ‘H n.m.r. assignments are listed in Table 1. For the non-labile protons, the assignments are complete for the entire polypeptide chain, except that for fSerl2, Serl8, Ser28, Lys43 and Cys59 only a range of chemical shifts for the /I-methylene protons was obtained because of strong spin-spin coupling, and for the peripheral methylene groups of the eight lysyl side-chains some assignments could not be established because of chemical shift degen.eracy and spectral overlap. Furthermore, all the backbone amide protons and all the side-chain amide protons of Asn and Gln were assigned. ‘13Cd n.m.r. spectrum of The one-dimensional human MT-2 is similar to those of rabbit MT-2a and rat MT-2. The resonance III is broadened relative to the remaining resonances in the spectrum (Fig. 3), as was also observed for rabbit MT-2a (Frey et al., 1985). For human MT-2, only the correlation between Cd111 and Cys26 and Cysl3 could therefore spectrum at be observed in the [ ‘13Cd, ‘HI-COSY 25°C. On increasing the temperature to 36”C, the Cd111 resonance became sharper, and the additional

B. A. Xesserle et al.

768

n-d --hth-

-iLI/

daN

-+h-+

MDPNCSCAAGDSCTCAGSCKCKECKCTS 1 10

20

-5-t-e --hhKSCCSCCPVGCAK6iQGCI

CKG

-3

-

30

--h--

40

-

310-

CSCCA 60

50

Figure 2. Survey of the sequential and medium-range backbone NOES for human [ ” 2Cd,:J1T-2 (for the notation used. see Wiithrich; 1986). In a semi-quantitative analysis of the NOESY spectra, the intensities of t’he sequent,ial XOEs were classified as strong or weak. represented by thick or thin bars, respectively. Similarly, the thickness of the lines indicat,es the observed intensity of the medium-range P;OEs. The locations of 3,, helices (3,,) and half turns (h) are indicated below t#he sequence. t indicates the presence of a turn that was not further characterized (see the text).

correlations to Cysl5 and Cys’i could be established (Fig. 4). Figure 5 affords a survey of the metal-tocysteinyl co-ordinative bonds in human MT-2, which are identical to those found previously in rabbit MT-2a (Wagner et al.. 1987) and rat MT-2 (Va%k et al.: 1987).

P

660

640 6 (p.p.m.1

Figure 3. One-dimensional ‘H-decoupled l13Cd n.m.r. spectrum of human [ lL3Cd,]MT-2 (protein concn 8 m&f in ‘H,O, 20 m~r-[ZH,,]Tris~ HCI (pH 7.0). 20 mlw-KCl. t= 25”C, frequency 80 MHz). The ‘13Cd signals are identified with roman numerals according t’o decreasing chemical shift.

(b) fdenti$cation

of secondary

structure

eletned~

The secondary structure elements were documented using approximate ‘H-lH distance constraints obtained from a qualitative analysis of t,he NOESY spectra (Fig. 2: Wiithrich et al., 1984). The secondary structure elements thus identified are very similar to those observed for rabbit MT-2a and rat MT-2 (Wagner et al., 19866; Schultze et al.. 8988). Two 3,, helices are present’ in the same parts of all three molecules, from residues 42 to 47, and from residues 58 to 61. The posit!ions of six turns in human MT-2 were identified from the expected patt,ern of short distances d&2,3), d&3,4) and d,,(i,i+2) (Wiithrich et al.: 1984). The posit,ions of these turns are Pro3-Cys5, Cys5&Cys’i, ThrP& Alal6, Ser32-Cys34, Cys34-Cys36, Val39-Cys4i and Cys48-Cys50. With the exception of Val39--Cys41, where 3JHNa could not be measured (residue 40 is Gly. and stereospecific assignments for C’H, were not obt-ained), the coupling constant 3JHNZ for the third residue of each turn was greaker than 8 Hz; which identified the turns as half-turns (Wagner et al.. 19866). The number of turns iden& tied with this pattern recognition approach differs somewhat’ among the three species of Figure 5. For exampl.e, the turns Thr 14-Ala 16 and Ser32-Cys34

Human Metallothionein

Co%formation

in Solution

Table 1 ‘H chemical shifts for human [Cd ,]MT-2 Amino acid residue

at pH 7.0

Chemical shift (p.p.m.) NH

H”

Met1

8.39

443

2.00, 206

Asp2 Pro3 Asn4 cys5 Sex-6 cys7 Ala8 Ala9 GlylO Asp1 1 Serl2 cys13 Thr14 cys15 Ala16 Gly17 Serl8 Cysl9 Lys20 cys21 Lys22 Glu23 Cys24 Lys25 Cys26 Thr27 SW28 cys29 Lys30 Lys31 Ser32 cys33 cys34 Ser35 Cys36 cys37 Pro38 Va139 Gly40 cys41 Ala42 Lys43 cys44 Ala45 Gln46 Gly47 Cys48 Ile49 cys50 Lys5 1 Gly52 Ala53 Ser54

8.62

492 441 475 531 477 4.33 438 427 392, 4.23 4.66 4.56 445 469 431 4.03 383, 416 453 4.31 4.66 407 411 437 4.27 441 4.23 4.00 4.27 4.38 4.34 4.50 4.43 4.47 5.06 4.42 4.50 5.15 471 3.83 3.65, 4.10 408 414 4.24 4.66 414 456 3.61, 438 435 471 443 428 387. 4.07 4.47 4.63 436 470 518 468 459 474 412

2.58, 3.17 1.92, 231 2.79, 2.93 291, 337 388, 418 2.91, 2.98 1.43 1.43

Lyi56 cys57 Ser58 cys59 Cys60 Ala61

769

8.73 7.47 8.86 8.45 8.76 8.41 8.39 8.48 8.26 802 852 8.70 8.07 8.51 7.95 8.31 914 8.67 8.73 8.94 %94 941 859 900 8.40 7.39 7.55 8.48 8.85 8.14 836 8.97 8.54 7.22 8.58 8.96 7.03 941 8.32 7.57 7.09 8.18 7.39 %86 7.23 914 %55 862 8.18 8.19 8.58 7.90 852 9.13 8.43 7.74 7.15

Others?

@-I

2.70, 2.78 3.8%3.931 3.10, 3.15 4.63 3.06, 3.25 1.36 3.81-3.904 2.92, 3.05 1.74, 2.11 3.00, 3.61 1.82, 1.90 1.74, 1.89 2.88, 3.11 2.05, 2.05 3.00, 3.18 429 3.90-3.980 2.81: 3.15 1.76, 1.76 1.76, 1.86 3.91, 4.01 3.12, 328 351, 3.61 3.86, 3.99 2.76, 3.21 3.03, 3.10 2.05, 2.30 1.94

CYH,: 2.54, 2.60; C”H,: 2.11; N-AC-CH,: 2.04 CYH,: 1.94, 2.04; C’H,: NSH,: 7.00, 7.93

3.87, 3.88

CYH3: 1.24

CYH,: 1.47,.

; C6H,: 1.65,

CYH,: 1.45, 1.56; CSH,: 1.72,. CYH,: 2.09, 2.09 CYH,: 1.58,.

.;C”H,: 2.98, 2.98 ; C’H,: 301, 3.01

; C6H,: 1.70,.

CYH,: 1.31 UH,: 1.40, 1.47; C&H,: 1.63,. CYH,: 1.48, 1.68; @H,: 1.71,.

; C”H,: 2.95, 295 ; C&H,: 3-02, 3.02

CYH,: 1.85, 2.07; C6H,: 377, 3.80 CYH,: 0.90, o-96

3.16, 3.19 1.55 2.00-2.08s 2.62, 3.74 1.51 1.98, 2.41

CYH,: 1.56,.

2.91, 3.00 2.25 2.63, 312 1.83, 1.83

CYH,: 1.04; CYH2: 1.00, 1.42; C6H,: 0.95

1.45 3.86, 3.92 2.69, 2.78 1.69, 1.78 3.58, 3.63 3.89, 3.98 3,21X%28$ 2.66, 3.13 1.40

; C?H,: 1.71, 1.80; C&H,: 3.02; 3.02

C’H,: 2.36, 2.36; N&H,: 6.89, 7.60

CYH,: 1.39, 1.47: C’H,:

1.66,.

; C”H,: 2.99, 2.99

CEH,: 2.99, 2.99

The amide proton chemical shifts were measured at lO”C, and all others at 25°C. t For methylene groups, 2 chemical shifts are given wherever 2 resolved signals were observed, or where the presence of 2 degenerate signals had been established unambiguously. f A range of chemical shifts is given for the methylene groups that showed strong coupling fine structure patterns.

B. A. Messerle

et al.

570

I 5.0

I 3.0

I 4.0

w

CO, ('H,

p.p.m.)

(protein concn 10 mM in ‘H,O, Figure 4. Heteronuclear [rr3Cd, ‘HI-COSY spectrum of human [‘13Cd,]MT-2 20 m~-[2H,,]Tris~HCl (p2H 7.0), 20 mM-KCl, t=36”C, ‘H frequency 600 MHz). The tuning delay was set to 30 ms. and the pulse sequence in Fig. 3 of Frey et al. (1985) with a (7[/2)?(‘H) pulse before detection was used. The spect8rum was acquired over a period of 12 h with 122 points in t, and 4096 points in t,. Zero filling to 512 points was used in t,, and to 8096 points in t,. The r13Cd resonance assignments (see Fig. 3) are indicated on the left, and the ‘H resonance assignments of the cysteine c” protons, and two Cys Cp protons, which were essential for the identification of the co-ordinative bonds, are indicated at the top by their sequence positions.

were not observed in the secondary structure of rabbit MT-2a, and three turns observed in the rabbit MT-2a secondary structure could not be identified positively in human MT-2 because the NOE cross-peaks corresponding to d,,(i,i + 2) could not be identified. Independent, additional experimental evidence for t,he presence of Dhe first 310 helix indicated in Figure 2 was obtained from studies of amide proton exchange rates in human MT-2 (Messerle et al., 1990).

(c) Determination human

of the solution structure metallothionein-2

of

The input for the structure calculations consisted bonds of the [ 1l3 C d ] cysteine sulfur co-ordinative (Fig. 5), upper distance constraints derived from NOESY, and dihedral angle constraints. For the metal-sulfur connectivities we assumed tetrahedral symmetry for the Cd ions, and the distance constraints used to represent the co-ordinative bonds were exactly the same as in Table 1 of Arseniev et al. (1988).

were Upper constraints on ‘H-” H distances obtained from NOESY spect,ra in ‘H,O and II,0 acquired at 10°C with mixing times of 40 milliseconds and 80 milliseconds. The majority of constraints were obtained from the NOESY spectra acquired with a mixing time of 40 milliseconds, only five additional constra,ints were obtained from the NOESY spectrum in H,O acquired with a mixing time of 80 milliseconds. Ambiguities in assignment of NOESY cross-peaks were resolved by going through two cycles of resonance assignments and structure calculations, as described in Ma.terials and Methods. This procedure also ena.bled a refined calibration of the relations between NOE intensities and the corresponding ‘H-‘H distances. As part of the preparat,ion of the input, data, stereospecific assignments were obtained for some diast’erotopic groups of hydrogen atoms and methyl groups. In addition to the NOE distance constraints, spin-spin coupling data were collect’ed for this purpose. The spin-spin coupling constants 3JHi%were obtained from a 2QF-COSY spectrum measured in A,0 solution, where the final digital resolution along w2 was 0.28 Hz. For the measure-

Human

HumanMT-2 Rabbit MT-2a Rat MT-2

CoEformation in Solution

Metallothionein

5

7

I

I

13

II

15

19

771

21

24

II

IIII

3637

41

44

48

I I

HumanMT-2 KSCCSCCPVGCAKCAQGCI P Rabbit MT-2a Rat MT-2

29

II

MDPNCSCAAGDSCTCAGSCKCKECKCTSCK N T A’ TDG S

3334

26

I

A Q

50

57 5960

II CKGASDKCSCCAI II

S

E

Figure 5. Comparison of the amino acid sequences of human [ “3Cd,]MT-2, rabbit MT-2a of the human protein is given in full, and for the other 2 species only the residues differing

and rat MT-2. The sequence from the human protein are

indicated below the sequence. Rabbit MT-2a contains 62 amino acid residues (Wagner et al., 1986a) but, since the insertion is labeled as A%, the numeration of the residues remains the same for all 3 proteins. The metal-to-cysteine coordinative bonds are indicated above the sequence. They were found to be the same for all 3 proteins.

ment of spin-spin coupling constants 3J,a, a phasesensitive E. COSY spectrum was used (Griesinger et al., 1985). Some stereospecific assignments were then obtained with the program HABAS. The data obtained from the program HABAS further provided supplementary conformational constraints in the form of allowed ranges of dihedral angles for the input for the calculation of the protein structure (Giintert et al., 1989). For the determination of these

allowed dihedral angle ranges, a precision of the measurements of 3JHNa and 3Jas of 2 2.0 Hz was used. Additional stereospecific assignments were obtained in the course of the structure calculations, using long-range and medium-range NOE distance constraints. A survey of all the stereospecific assignments found is given in Table 2. A survey of the input data collected for human MT-2 is given in Table 3. Complete listings of the

Table 3

Table 2 Stereospeci$c

‘H

Residue

Method?

cys19 CysPl Cys24 Ser32 Va139 cys50 cys57 Cys60

n.m.r. assignments [Cd ,]MT-2

305 361 311 401 W’H, 990 312 363 313

in

human

292 3.00 2.88 3.91 CYZH, 0.96 2.63 3.58 266

t Method of stereospecific assignment: H, using HABAS; A; using long-range and medium-range NOE information in the results of the initial structure calculation with the program DISMAN at level 4 and at the final level of the target function (see the text).

Experimental input data used for the structure calculations of human [Cd ,]MT-2

Upper distance limits (A) Intraresidual Sequential backbone Medium-range and long-range backbone Interresidual with side chain protons Dihedral 4 x1

angle constraints

B-Domain

cc-Domain

38 20

27 28

9 49

21 68

13 12

21 12

(deg.)?

t Only those constraints are considered that were derived directly from experimental measurements of spin-spin coupling constants, assuming that these were measured with a precision of +2.0 Hz.

B. A. Messede et al.

772

Table 4 Residual

constraint

violations

in the 10 best DISMA,V

stw&.wex

oj human

MT-%

Number of violationst

Sum of violationsf A. P-Domain 8.7

NOE constraints 0.3-05 A >05 B

Cd-S co-ordination§ 0.3-05 A >05 A

Non-bonded contacts >@I A

Dihedral angleslj >5”

2

0

4

l(O.6)

4(0.20)

0

8.0

0

0

2

l(O.6)

2(026)

1(6)

8.0

2

0

5

0

5(926)

0

8.0

0

l(O.6)

1

0

4(@25)

0

7.1

%

0

5

l(0.6)

t5(@%4)

0

10.1 10.9 9.5 11.6 11.9

0 2 % % 2

l(O.6) 0 l(0.6) 0 l(O.7)

6 3 5 5 5

%(0.9) 3(0.5) iqO.9) 3(W) 2(0.7)

2(026) 2(0.26) 7(025) 8(026) lO(O23)

0 2(9) 2(B) 3(12) 2(7)

iu. U-Domain~ 142 15.4 17.3 168 161 19.9 21.8 196 18.7 23.2

4 3 4 3 % I 2 1

l(0.6) l(O.8) 3(0.7) 2(0.6) 3(OS) l(O.6) 2(0.6) 1(o-7) 2(0.9) l(O.7)

4 4 8 5 6 11 9 9 12 14

3(W8) 4(1.0) 3(@8) 8(@8) %(l.O) S(1.0) ll(O.9) 9( 1.0) 6(1.1) ll(1.0)

5(026) Z(Ol2) 2(026) Z(O.25) lO(o-32) 9(@17) i(O.14) 7(0.25) 8(@25) 6(0.27)

463)

i

i(7) 5(12) f(8) 2(14) 2(16) W) 3(7) l(l6)

4W

t In each column, the number of const,raint violations in the ranges indicated are listed. The numbers in parentheses are the maximal violations, $ The violations included in this sum are those resulting from SOE constraints and Cd-S co-ordination. The structures have been ordered according to increasing target function, which also accounts for residual violations of steric constraints and dihedral angle constraints (Braun & Go, 1985). 6 Number of upper and lower distance violations on the constraints representing Cd-S connectivities, with t,he assumption of tetrahedral Cd-S co-ordination and a Cd-S distance of 2.6 A. The metal co-ordination is identical to t,hat of both rabbit MT-2a and rat MT-2 (Arseniev et al.: 1988; Schultze et al., 1988). 11Violations of the angular constraints derived directly from the measured spin-spin coupling constants.

NOE distance constraint’s and the supplementary constraints obtained in the form of allowed ranges of dihedral angles from the HARAS treatment and the statistical analysis of the DTSMAN data at level 4 will be submitted to the protein data bank, together with t’he atomic co-ordinates of human MT-2. The program DTSMAN (Braun & Go, 1985) was used for the structure calculations. For the P-domain, 300 starting struct’ures were calculated up to target level 4, with 250 cycles per level. Of these, the 120 structures with the lowest target function values were used for calculations up to a final level encompassing the complete polypeptide chain. For the a-domain, 200 starting structures were generated and 80 were calculated up to the final level. The structures were checked for distance constraint violations in excess of 0.3 A at the target function levels 18 and at the final level. Finally, ten structures with target functions of less than 7 for the P-domain, and less than 12 for t,he a-domain were subjected to a further minimization of 500

iterations at the final level only, where the dihedral angle constraints obtained from HABAS but not, derived direct,ly from coupling constants were eliminated from the input. The residual constraint violations for the ten best structures of each domain are listed in Table 4. The sum of violations varies between 8.7 ‘4 and I 1.9 A for the structures of the &domain. The major contributions to these numbers are from violations of the ideal tetrahedral Cd-S co-ordination used in the input. The same occurred for the a-domain. where the sum of violat,ions was significantly higher than for the ,&domain, with values from 142 x t,o 232 A. The larger residual vioIat,ions must be rationa.lized by the fact that a significantly larger number of constraints was available for the calculations of the E-domain structures (Table 3). The r.m.s.d. values obtained from a comparison of the ten best DlSMA?lr’ structures obtained for each domain of human MT-2 are given in Table 5. The P-doma,in has a relatively high r.m.s.d. value when the whole backbone is considered. but when

Human

Metallothionein

Conformation

in Solution

K30

773

K30

(b)

Figure 6. Stereo view of a superposition of the 10 DISMAP; structures of human MT-2 with the lowest residual error functions (Table 4). The superposition is for minimum pairwise r.m.s.d. of the best structure with each of the others. Only the backbone is shown. (a) D-Domain (residues 1 to 30). Only residues 13 to 30 were considered in the calculation of the superposition. (b) n-Domain from residues 31 to 61. The orientation of the two domains relative to each other could not be determined from the n.m.r. data. the poorly constrained region between residues 1 and 12 is left out of the calculation, an r.m.s.d. value comparable to that obtained for the a-domain is obtained. The higher r.m.s.d. in the region l-12 can be attributed to the fact that there are only few

Table 5 Average r.m.s.d. values for pairwise comparison of the 10 best DISMAN structures of human MT-2 Atoms

Domain

consideredt

r.m.s.d.

(A)$

/I (residues

l-30)

C”, c’, N c”, C’, N, CB, W, Cd W. Cd

2.6kO.5 2.3+05 o-5+0.1

/I (residues

13-30)

C”, c’, N C”, C’, X, C?, Sy. Cd

15+w4 1.4+04

a (residues

31-61)

c”; c’, N C”, C’, N; C?, Sy, Cd Sy, Cd

1.5kO.3 1.6iO.3 0%+0.2

t Where Cfl atoms are indicated, only those of the cysteinyl residues were considered. $ The r.m.s.d. given is the average over 45 pairwise minimal r.m.s.d. values +/the standard deviation.

constraints for residues 7 to 12, as is also clearly seen in Figure 2. Overall stereo views of the structures obtained are shown in Figures 6 and 7. Tn Figure 6 it can be seen that the individual structures of the b-domain are quite variable bet’ween residues 7 and 12. The group of ten structures of the a-domain shows a good fit over the full length of the chain, with somewhat increased variability at the amino-terminal residues 31-32. As no NOE was observed

between

protons

located

in

the

two

different domains, no n.m.r. information was obtained on the relative spatial orientations of the two domains. The indirect implication is that the two domains are flexibly linked by the peptide segment 30-32. A superposition of the Cd ions and the sulfur atoms of the metal-sulfur clusters in the ten best DISMAN structures (Table 4) of both domains of human MT-2 is shown in Figure 8. The sixmembered ring formed by the three Cd ions and the three bridging sulfur atoms of the three-lmetal cluster of the b-domain has a boat conformation. The two fused six-membered rings of the four-lmetal cluster in the a-domain form somewhat distorted boat conformations. The bridging sulfur atoms have

B. A. Messerie -

(a)

K30

et al.

K30

Figure 7. Stereo view of the polypeptide backbone (thick line) and side-chain heavy-atom posit,ions of the cysteinyl residues in the best DISMAPj structure of human MT-2. (a) B-Domain, with cysteinyl residues in positions 5%7: 13, 15. 19, 21, 24; 26 and 29. (b) E-Domain, with cysteinyl residues in positions 33; 34, 36, 37, 41, 44, 48, 50, 57, 69 and 60. The Cd ions are represented as spheres of radius @9 8. The same viewing angle is used as for Fig. 6.

a tetrahedral environment of two cadmium atoms, one Cp atom and a lone electron pair. For some of the bridging sulfur atoms, for example Cys37 and Cys44 in the a-domain, both possible chiral conDISMAN figurations occur in the individual structures. (d) A comparison of the solution structures of human MT-2 rabbit MT-2a and rat MT-2 The amino acid sequences of the three metallothioneins are shown in Figure 5. The sequences of rabbit MT-2a and human MT-2 have the closest homology; with only four amino acid substitutions, and one insertion in the rabbit sequence between positions 8 and 9 (Wagner et al., 1986a; K%gi & Kojima, 1987; KSgi & SchB;ffer, 1988). Rat MT-2 has seven substitutions relative to human MT-2, and ten substitutions and the deletion 8’ relative to rabbit MT-2a, and human MT-Z has four substitutions and the deletion 8’ relative to rabbit MT-2a. The sequence homology between rabbit, MT-2a and rat MT-2 is thus 82%, between rabbit MT-2a and human MT-2 92 o/O, and between rat MT-2 and human MT-2 89%. Nearly all subst,itutions occur in the P-domain. The cysteinyl residues are strictly

conserved between the three species, and the metalto-cysteine co-ordination was also found to be the same for the proteins of all three species in solution (Fig. 5; Frey et al., 1985; Va%k et al., 1987). The quality of the st’ructure determinations; ad characterized by the residual constraint violations (Table 4) and the global r.s.m.d. values (Table 5) is nearly the same for the three proteins. This includes also the fact that for all three species the chiralit,y at individual bridging cyst,eine sulfur atoms was not uniquely defined (Fig. 8; Arseniev et al., 1988; Schultze et al.: 1988). In the following. the similarity of the global structures of the proteins from all three species is substantiat,ed with several more precise comparative analyses. A visual comparison of the global backbone structures in the three species is afforded by a superposition of the mean of the ten best DISMAX structures for each protein (Fig. 9). The r.m.s.d. values between the mean structures for the polypeptide backbone from residues 13 to 30 are 1.0 A between human MT-2 and rat MT-2, 0.9 L%between human MT-2 and rabbit MT-2a, and @9 L%between rabbit MT-2a and rat MT-2. For the a-domain. the r.m.s.d. values calculated for residues 31 to 61 are 1.1 d between human MT-2 and rat MT-2, 1.3 &

Human

Metallothionein

Conformation

in Solution

775

(b)

Figure 8. Cd&S clusters in human MT-2 in solution. (a) The Cd-S eonnectivities and the chirality at the Cd ions in the 2 domains. Same orientation as for the structures in (b) and (c). The Cd atoms are identified with arbitrary reman numerals corresponding to increasing ‘13Cd chemical shift (see Fig. 3). (b) and (c) Superposition of the 10 best DI8MAN structures of the S-metal cluster and the 4-metal cluster, which are located in the /? and cc-domains, respectivel,y. The superposition is for minimum pairwise r.m.s.d. of the cadmium and the cysteinyl sulfur positions of the best structure (Table 4) with each of the other structures.

between human MT-2 and rabbit MT-2a, and 0.9 A between rabbit MT-2a and rat MT-2. Clearly, the global fold of the polypeptide chain is virtually the same for all three structures. As is shown in Figure 7 for human MT-2, in all three species the polypeptide chain winds in a right-handed sense around the three-metal cluster in the P-domain, and in a left-handed sense around the four-metal cluster in the a-domain. I?or a more quantitative comparison, the r.m.s.d. values were calculated for all possible pairs among the ten best DISMAN structures each of human MT-2, rat MT-2 and rabbit MT-2a (Table 6). For the P-domain, two calculations were performed, one with the entire backbone including residues 1 to 30 (where residue Ala8’ of rabbit MT-2a had been omitted), and the other considering only the well-defined segment containing residues 13

to 30. For the well-defined residues 13 to 30, the average r.m.s.d. values between the DISMAN structures of the three proteins are not significantly different from the average r.m.s.d. values between the individual DISMAN structures of any single species. For the less well-defined region including residues 1 to 13, there are significantly greater differences between the structures of human MT-2 and rabbit MT-Sa, human MT-2 and rat MT-2, and between rat MT-2 and rabbit MT-2a, respectively. Here, one must also consider the influence of the insertion of Ala8’ in the rabbit protein. Flor the a-domain, the r.m.s.d. values obtained between the structures of the different species are nearly the same as those between the different structures of the same species. Close similarity of the three proteins is also found

B. A. Messerle

776

et al.

K30

la)

K31

Figure 9. Stereo view of a superposition

of the mean of the 10 best DISMAN st,ructures of rat MT-2 (thinnest. line; (medium thickness line; Arseniev et al., 1988) and human MT-2 (thickest line). The fit was achieved by obtaining minimum pairwise r.m.s.d. values for the backbone heavy-atoms between rabbit, MT-2a and human MT-2, and between rat MT-2 and human MT-2. (a) P-D omain. Only residues 13 to 30 were used for the r.m.s.d. Schultze

et al.; 1988), rabbit

computations.

MT-2a

(b) cc-Domain.

in local structural features. The secondary structure elements are very similar for all three proteins; in addition, the chirality at the Cd ions with respect’ to the bound sulfur at’oms is the same for human MT-2 (Fig. 8) as for rabbit MT-2a ands rat MT-Z. To compare local fea,tures of the polypeptide conformations of the three proteins, all pentapeplocally superimposed for tide segments were minimum r.m.s.d., which was calculated for the backbone atoms N, C” and C’. In Figure 10, these r.m.s.d. values are plotted versus the sequence, where each pentapeptide is represented by its central residue. In Figure 10(a): the average of the pairwise r.m.s.d. values between the ten best DISMAK structures of the same species is plotted. Overall, the same general trends prevail in these plots for human MT-Z, rabbit MT-2a and rat MT-Z.

Minor differences are that the rat protein has lower r.m.s.d. values from residues 7 to 12, and that from residues 44 to 48 the r.m.s,d. values for rat MT-2 are higher than for rabbit’ MT-2a and huma,n MT-2. The indicat’ion of increased static or dynamic disorder in the region from residues 7 to 15 thus indicated for both human MT-2 and rabbit MT-2a is discussed in more detail in the accompanying paper (Messerle ot al., 1990). Anot’her observation is that rabbit MT-2a is locally bet’ter defined between residues 36 and 43 than either huma,n MT-2 or rat MT-2. This could be due t’o the presence of two consecutive Pro residues in positions 38 and 39 in rabbit, MT-2a (Fig. 5). The local pairwise r.m.s.d. values between the mean structures of the three species (Fig. 9) are plotted in Figure 10(b). For residues 15 to 61, the local structures are very similar in all three species, and od)

Human

I

I

I

I,,,

8

CoFformation in Xolution

Metallothionein

I

,,,I,,

16

ll

24

I

I

‘32

Amino

acid

I

I,

40

I,

I

I

I

I

48

I

I

L

56

sequence

Figure 10. Local r.m.s.d. values calculated for pentapeptide segments and plotted for their central residues! for a comparison of the backbone structures of human MT-Z, rabbit MT-2a and rat MT-2. (a) Average of the pairwise local r.m.s.d. values among the 10 best DISMAPj structures of human MT-2 (O), rabbit MT-2a (+) (Arseniev et al., 1988), and rat MT-2 (0) (Schultze et al., 1988). (b) Pairwise local r.m.s.d. values calculated between the mean DISMAN structures (Fig. 9) of human MT-2 and rabbit MT-2a (O), human MT-2 and rat MT-2 (+), and rabbit MT-2a and rat MT-2 (0).

Average

Table 6 comparisons between the 10 best DISMAN r.m.s.d. values from pairwise structures of human MT-2, rabbit MT-2a and rat MT-2

Polypeptide segments compared

RMSD at Species

Human MT-2

Rabbit MT-2a

Rat MT-2

Residues l-30$

Human MT-2 Rabbit MT-2a Rat MT-2

2.3f0.5

32+@6 2.6 f 0.7

2.3 f 0.4 2.8 + 0.5 lG3fO.3

Residues 13-30

Human MT-2 Rabbit MT-2a Rat MT-2

1.4kO.4

1.7iO.4 1.5*05

1.7 kO.3 1.6kO.4 1.4f0.3

Residues 31-61

Human MT-2 Rabbit MT-2a Rat MT-2

1.5 f0.3

1.820.3 1.4kO.2

1.8kO.3 1.7 kO.2 1.6*0-2

The atoms used for the calculation of the r.m.s.d. values are N, c”; C’, CB and Sy of the cysteinyl residues, and Cd2 + 7 The r.m.s.d. values indicated for the DISMAN structures of the individual species are the averages of the 45 pairwise minimal r.m.s.d. values, those for the comparisons of different species are the average of the 100 pairwise minimal r.m.s.d. values. $ For rabbit MT-2a, Ala8’ was not used in the r.m.s.d. calculation.

B. A. Messerle

778

the r.m.s.d. values between the mean structures of human MT-2 and rat MT-2 are slightly increased from residues 49 to 56. Significantly higher r.m.s.d. values were found for residues 7 to 13. However, comparison with Figure 10(a) shows that throughout the sequence the local pairwise r.m.s.d. values between the mean structures of any two different species is lower than the average local r.m.s.d. values between the ten best structures of a single species. In conclusion, we know that the three metallothionein structures determined so far by n.m.r. in solution are very similar in both their global and local features. This includes identical metal-to-polypeptide co-ordinative bonds in all three proteins, nearly identical polypeptide secondary structures, and closely similar global polypeptide folds. This result contrasts with the important differences observed when the solution structure of rat MT-2 is compared with t’he structure reported for the same protein in single crystals (Furey et al.: 1986, 1987; for a detailed comparison, see Schultze et al., 1988). It appears that further discussion of the apparent discrepancies between metallothionein structures in solution and in single cryst’als should be deferred until the moment when the reinvestigation of the crystal structure (Stout et al., 1989) is completed.

Financial support by the Schweizerischer Nationalfonds (projects 31.2517488 and 3.160.88). a Kundesstipendium der Eidgenossischen Stipendienkommission and an Eleanor Sophia Wood Travelling Fellowship from the University of Sydney, Australia (to B.A.M.) is gratefully acknowledged. We thank Dr W. Braun for the use of the DISMAN program, and Mrs E. Huber and Mr R. Marani for careful processing of the manuscript.

References Bnil-Kumar, Ernst, R. R. & Wiithrich, K. (1980). Biothem. Biophys. Res. Commun. 95, 1-6. Arseniev, A., Schultze, P., Wijrgotter, E., Braun, W., Wagner, G.; Va%k, M., KSigi, J. H. R. & Wiithrich, K. (1988). J. Mol. Biol. 201, 637-657. Bax. A. & Davis; D. G. (1985). J. Magn. Reson. 65, 335360. Billeter, M., Braun, W. & Wiithrich, K. (1982). J. Mol. Biol. 155, 321-346. Braun, W. & Go, N. (1985). J. Mol. Biol. 186, 611-626. Braunsehweiler, L. & Ernst, R. R. (1983). J. Magn. Reson. 53, 52-528. Biihler, R. H. 0. (1974). Dissertation, University of Ziirich. Biihler, R. H. 0. & KB;gi, J. H. R. (1974). FEBS Letters, 39, 229-234. Frey, M. H., Wagner, G., VaEiak, M. Sorensen, 0. W., Neuhaus, D., WargStter, E., KLgi, J. H. R., Ernst, R. R. & Wiithrich, K. (1985). J. Amer. Chem. Sot.

107, 6847-6851. Furey, W. F., Robbins, A. H., Clancy, L. L., Winge, D. R., Wang, B. C. & Stout, C. D. (1986). Xcience,

231, 704-710. Furey, W. F., Robbins, A. H., D. R., Wang, B. C. & Stout,

Clancy, L. L., Winge, C. D. (1987). In Metal-

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Iothionein 11 (Kagi, J. H. R. & Kojima, Y., eds), pp. 139-148, Birkhfiuser Verlag, Basel. Griesinger, C., Sorensen, 0. W. & Ernst, R. R. (1985). J. Amer. Chem. Sot. 107, 6394-6396. Giintert, P.; Braun, W., Billeter, MCI. & Wiithrich, K. (1989). J. Amer. Chem. Sot. 111: 3997-4004. Jeener; J.; Meier, B. H., Bachmann, P. & Ernst, R. R. (1979). J. Chem. Phys. 7. 4546-4553. Kagi, J. H. R. & Kojima, Y. (1987). Metallothionein Ii (Klgi, J. H. R. & Kojima, Y., eds), pp. 25-61, Birkhauser Verlag, Basel. Kagi, J. H. R. & Schaffer, A. (1988). Biochemistry, 27, 8509-8515. Kline, A. D., Braun. W. $ Wiithrich, K. (1988). J. Moi. Biol. 204: 67&724. Marion, D. & Wiithrich, K. (1983). Biochem. Biophys. Res. Commun. 113, 967-974. Messerle, B. A., Boa, M.; Schaffer, A., VaBak, M., Kagi, J. H. R. & Wiithrieh, K. (1990). J. MoZ. BioE. 214. 781-786. Neuhaus, D., Wagner, G., VaBBk, M.: Kagi, J. H. R. $ Wiithrich, K. (1984). Eur. J. Biochem. 143, 659-667. Neuhaus, D., Wagner, G.: Va%k, M., Kiigi, J. H. R. & Wiithrich, K. (1985). Eur. J. Biochem. 151, 257-273. Otting, G. & Wiithrich, K., (1986). J. Magn. Reson. 46, 359-363. Rance, M., Sorensen, 0. W.. Bodenhausen, G., Wagner. G., Ernst, R. R. & Wiithrich, K. (1983). Biochem. Biophys. Res. Commun. 117, 479-485. Rance, M., Bodenhausen, G., Wagner, G., Wiithrich, K. & Ernst, R. R. (1985). J. ivagn. Reson. 62, 497QjlO. Redfield, A. G. & Kunz, S. D. (1955). J. Magn. Reson. 19. 250-254. Schultze, P., Wijrgijtter, E., Braun, W.; Wagner, G. VaBbk, M., KLgi, J. H. R. & Wiithrich, K. (1988). k. Mol. Biol. 203, 251-268. Senn, H., Billeter, M. & Wiithrich. K. (1984). Eur. Biophys. J. 11, 3-15. Stout, C. D., McRee, D. E., Robbins, A. H., Collett. S. A., Williamson, M. & Xuong, X. H. (1989). Abstr. Int. Chem. Congr. of Paci$c Basin Sot. Honolulu, Hawaii, U.S.A. vol. 1; pp. 4-57. Vasak, M.; & Kagi, J. H. R. (1983). In Metal Ions in Biological Systems (Sigel, N., vol. 15, ed.), pp. 213-273, Dekker, New York. Va%k; M., Wiirgijtter, E., Wagner, G., Kagi, J. H. R. Ss Wiithrich, K. (1987). J. Mol. Biol. 196: 711-719. Wagner. G. (1983). J. Magn. Reson. 55, 151-156. Wagner; G. & Wiithrich? K. (1982). J. MOE. Biol. 155; 347-366. Wagner, G. & Zuiderweg, F,. R. P. (1983). Bioehem. Biophys. Res. Commun. 113, 854-860. Wagner, G.? Neuhaus, D., WBrgGtter, E., Vasik. M.. KGgi, J. H. R. & Wiithrich, K. (1986a). Eur. J. Biochem. 157, 275-289. Wagner, G., Neuhaus, D.; WiirgGtter, E., VaSdk; M.. Klgi, J. H. R. & Wiithrich, K. (1986b). J. Mol. Biol. 187. 131-135. Wagner. G., Frey, M. H., Neuhaus, D., Worgijtter, E., Braun, W., Va%k, M.. Kagi, J. H. R. & Wiithrich, K. (1987). In Metallothionein II (KLgi, J. H. R. & Kojima, Y., eds); pp. 149-157. Birkhauser Verlag, Basel. Wider, G., Lee, K. H. & Wiithrich, K. (1982). J. MOE. Biol. 155, 367-388. Widmer; H.; Billeter, M. & Wiithrich, K. (1989). Proteins, 6, 357