J. Mol. Biol. (1991) 221, 1269-1293

Refined Crystal StructuFe of Cd, Zn Metallothionein at 2-O A Resolution A. H. Robbins’, D. E. McRee’, M. Williamson3 S. A. Collett3, N. H. Xuong3, W. F. Furey4T5 B. C. Wang’ and C. D. Stout’ l Miles Research Center West Haven, CT 06516, U.S.A. Research Institute

‘Department of Scripps

‘Departments University

VA Medical

of Molecular Biology Clinic, La Jolla, CA 92037, U.S.A.

of Chemistry,

“Biocrystallography Center, University Drive

University

5Department of Pittsburgh,

(Received 12 July

Physics

and Biology U.S.A.

San Diego, CA 92093,

of California,

Laboratory C, Pittsburgh,

PA 15240, U.S.,4.

of Crystallography Pittsburgh PA 15260, U.S.A.

1990; accepted 30 April

1991)

The crystal structure of Cd,,Zn,-metallothionein from rat liver has been refined at 2.0 x resolution of a R-value of 0.176 for all observed data. The five Cd positions in the asymmetric unit of the crystal create a pseudo-centrosymmetric constellation about’ a crystallographic 2-fold axis. Consequently, the distribution of anomalous differences is almost ideally centrosymmetrie. Therefore, the previously reported metal positions and the protein model derived therefrom are incorrect. Direct methods were applied to the protein amplitudes to locate the Cd positions. The new positions were used to calculate a new electron density map based on the Cd anomalous scattering and partial structure to model the metal clusters and the protein. Phases calculated from this model predict the positions of three sites in a (NHJ2WS, derivative. Single isomorphous replacement phases calculated with these tungsten sites confirm the positions of the Cd sites from the new direct methods calculations. The refined metallothionein structure has a root-mean-square deviation of 0.016 d from ideality of bonds and normal stereochemistry of 4, cp and x torsion angles. The metallothionein crystal structure is in agreement with the structures for the a and /I domains in solution derived by nuclear magnetic resonance methods. The overall chain folds and all metal to cysteine bonds are the same in the t,wo structure determinations. The handedness of a short helix in the a-domain (residues 41 to 45) is the same in bot.h structures. The crystal structure provides information concerning the metal cluster geometry and cysteine solvent accessibility and side-chain stereochemistry. Short cysteine peptide sequences repeated in the struct’ure adopt restri&ed conformations which favor the formation of amide to sulfur hydrogen bonds. The crystal packing reveals intimate association of molecules about t’he diagonal 2-fold axes and trapped ions of crystallization (modeled as phosphate and sodium). Variation in the chemical and structural environments of the metal sites is in accord with data for metal exchange reactions in metallothioneins. Keywords: direct methods; metal-sulfur

clusters; cysteine thiolate

ligands

amino acid residues, 20 of which are cysteine, and bind seven to 12 metal ions per polypeptide. The Metallothioneins are unusual metalloproteins chemistry of metal binding, physiological roles, consisting of onethird cysteine residues. The mamtoxicology, molecular biology and spectroscopy of malian proteins are comprised of approximately 60 metallothioneins have been studied in detail (Kagi 1269 1. Introduction

0022-1836/91

po I%9-1.5

$03.W/O

0

1991 Academi(~

Press Limited

1270

A. H. Robbins et al.

& Nordberg, 1979; Kagi & Kojima, 1985; Kagi & Schiiffer, 1988; Dalgarno & Armitage, 1984; Otvos et al., 1989; Vasik & Kagi, 1983; Hamer, 1986; Karin, 1985; Nielson et al., 1985). Although the physiological function of mammalian metallothionein (MT?) has not been firmly established, it is believed to be essential in maintenance of metal ion homeostasis, especially for Zn and Cu. The protein is found in a variety of tissues, is abundant in liver and kidney, and expression of MT genes is regulated by metal ion concentrations. MT also functions to sequester toxic metals, such as Cd and Hg. Basic features of the structure of MT have been established by chemical and spectroscopic methods. The formation of two distinct metal clusters in rabbit liver MT was first’ shown by rr3Cd nuclear magnetic resonance (n.m.r.) (Otvos & Armitage, 1980). The seven Cd ions are bound in clusters of four and three metals formed by bridging and terminal cysteine thiolate ligands (Dalgarno & Armitage, 1984). All 20 cysteines participate in metal binding, and each of the seven metal ions is bound tetrahedrally (Vasak & Kagi, 1981, 1983). Rat liver MT can be cleaved into two domains. which bind metals individually in the same manner as in the intact protein (Winge & Miklossy, 1982). The N-t,erminal domain of residues 1 to 30, termed nine cysteine residues and forms a B. contains cluster of three Cd or Zn ions liganded by three bridging and six terminal cysteine ligands; the C-terminal domain of residues 31 to 61, termed ~1, contains 11 cysteine residues and forms a cluster of four*Cd or Zn ions liganded by five bridging and six terminal cysteine ligands (Boulanger et al., 1982). The c( domain binds Cd co-operatively; the j domain binds both Cd and Zn (Nielson & Winge, 1983; Byrd & Winge, 1986). Each domain binds six Cu ions if the protein is reconstituted with t’his metal (Nielson $ Winge: 1984). The structure of MT in solution has been studied in detail bv two-dimensional [113Cd-1H] n.m.r. methods (Withrich, 1989). Three dimensional st,ruc:tures have been determined for MT from three species: rabbit liver Cd,-MT (isoform 2a; Arseniev et al.. 1988); rat liver Cd,-MT (isoform 2; Schultze rt al., 1988) and human liver Cd,-MT (Messerle et al.. 1990). These investigations have established the cysteine ligand t,o metal co-ordination pattern for the t,hree-metal and four-metal clusters, described hydrogen bonding within each domain and shown that all three proteins adopt a similar conformation. A st)ructure for Cd,Zn,-MT (isoform 2) from rat liver was report,ed on t’he basis of an electron density map at 2.3 A (1 A = 0.1 nm) resolution (Furey et al.. 1986). This structure analysis relied on the anomalous scattering of Cd in the protein for experiment’al phase informat’ion. as an extensive survey for heavy-at’om derivatives was unsuc t Abbreviations used: MT, metallothionein; n.m.r.. nuclear magnetic resonance; ISAS, iterative singlewavelength anomalous scatterer; ISTR, iterative single isomorphous replacement,.

cessful. Here, we shvw that the interpretation of the Cd positions is complicated by pesudo-symmetry. The structure model previously reported is not in agreement with the MT structures derived by n.m.r. methods (Arseniev et al., 1988; Schultze et al., 1988): in particular, the metal to cyst~eine co-ordination patterns in both clusters differ markedly. Here. we report, a refined structure for Cd&,-MT that’ is in overall agreement with the n.m.r. structure. The crystal structure was redetermined with new data collected from the same crystal fortn as previously studied. The correctness of the new crystal structure solution is supported by a heavy-&on1 derivative interpreted with knowledge of t,he correct’ (Id positions. A comparison of the solution and crystal structures will be presented elsewhere.

2. Structure Determination (a) ( ‘r?/dals Single crystals of Cd,Znz-MT (isoform 2) were grown as described (Melis et al.. 1983). The protein. as isolated from rat liver, has a metal composition of 5 mol Cd and :! mol Zn per mol of MT. Samples from dissolved crystals have t,he same metal composition (Melis et al.. 1983). The amino acid sequence consists of 61 residues, including 20 cyst,eine residues. and contains ,V-acetyl-methionine at the X terminus (Winge et al.. 1984). The crystals are tetragonal with unit cell constants a = h = 399 A, c = 120.4 .4 and 1 tnolerule/asymmetric unit (b) Lspac” g?mp Diffraction patterns indicate the space group of the MT P4,2,2 (Melis el crystals to be P4,2,2 or its enantiomer. al.. 1983). The space group derived in the previous analysis was P4,2,2 (Furey et al.. 1986). This analysis shows that the correct space group is P4,2,2. The error in spacr group assignment was caused by error in the positions of the Cd sites used to calculate phases. The location of the Cd sites is complicated by a pseudo-centrosymmetric constellation of the heavy atoms due to packing of pairs of MT molecules about a Z-fold axis. The pseudo symmetry diminishes the expected magnitude of the anomalous differences and introduces ambiguity int#o space group assignment based on t,he (‘d positions alone. Therefore. the error in space group assignment in the MT structure originally report-ed was due to incorrect location of the Cd sites. an error masked by pseudo-symmetry.

A new data set was collected for the structure rrdet)ermination using the multiwire area detector diffract,ometei at the Ilniversity of (California at San Diego (Hamlin. 1985; Howard el al.. 1985). The data were collect,rd using a (ix-6 rotating anode with Vu target and graphite monochromator. The crystals were maintained at 4°C’ and the diffracted beam path was purged w-it)h He. Data were collect,ed on 2 detectors set at low and high 28 and Kijvort pairs were collected in alternate runs at x, 4 and -x. ++ 180’ as described (?(uong rl al.. l!l%). ,I total of 21.272 observat,ions wit,h IFI >Oa(F) to 29 A were collected from 1 crystal in 36 h. The average l/a([) for this dat’a set was 13.7. I/@ I) for the data in t,he she11 2.1 to

Crystal Structure of Cd, Zn Metallothionein 2.0 A was 42. For 6704 reflections in point group 422, the R,,,,(I) was 4.6%. Subsequently, 7 additional crystals were used to collect Bijvoet pairs with the detectors set symmetrically at + 20 and -20 for 2.0 A resolution data. For each run frames were acquired for 120 s/frame versus 40 s/frame in the 1st data set. A total of * 30,000 observations with IFJ > Oa(F) were measured from these 7 crystals. After each data set was individually processed (Howard et al.. 1985), the number of scaled reflect,ions from the 7 long count data sets was 15,521. Because each of the long count data sets was incomplete, they were scaled individually to the reference data set (6704 reflections). The IF’\ and (F-1 values were scaled sepa,rately, and the data were scaled in shells of sinf?/L. After applying scale factors. the (&‘+I and (F-1 values were merged into a master file conta.ining 22,225 (6704+ 15,521) scaled reflections from the 8 crystals. The redundant values of IP+ ) and IF-1 from the 8 crystals were then averaged to yield a single value of JF+( and IF-1 for each of 4415 unique reflections in 4/mmm t,o 2.0 A resolution. The number of possible reflections to 2.0 A is 4457.

suggested that the previously determined Cd positions were incorrect (Furey et aE., 1986). The application of direct methods was suggested by our previous experiments (Furey et al., 1986), and by success with heavyatom derivative anomalous and isomorphous differences (Mukherjee et al., 1989; Wilson, 1978). The application of Multan to the protein IFI data was to our knowledge unprecedented, but was attempted after failure to locate the Cd positions from the new IAFl data. The 5 Cd positions represent 38% of the total scattering by the protein. Both (F( and IAFl values were normalized by assuming the contents of the asymmetric unit to be 5 carbon atoms (Table 1). The E distribution based on IA,F( is strongly centrosymmetric. While E distributions based on differences tend to be centrosymmetric (Wilson. 1978), the data in Table 1 are also consistent with a centrosymmetric arrangement of the Cd sites. The E distribut,ion based on (FI is intermediate between centrosymmetric and noncentrosymmetric, indicating the large influence of the Cd to the total scattering. The phase sets generated by Multan for the 300 largest E values were evaluated by examining the E maps. All of the maps based on the JAFI data had a single very large peak on the diagonal 2-fold axis. The E map based on the IFI data for the solution with the highest absolute figure-of-merit ((r395) had 5 high peaks indicating the Cd positions and 2 lower peaks for the Zn positions (Fig. 1). That this new solution is correct was suggested by 2 facts: the number of peaks and t,heir arrangement. which is consistent, with the known cluster structure. When t.he calculations were repeat,ed in 1’4,2,2 using the IFI data. the E map for t,he solution with 2nd highest

(d) Direct methods The new data were used for direct methods calculations with the program Multan (Karle, 1986). Calculations were done in 1’4,2,2 with the data to 2.5 A using both the averaged IFI values for the protein (2337 centric and acentric reflections) and the IAFI = I(P+I-IF-11 values (1522 acentric reflections) in an effort to locate the Cd positions. A variety of indicators and test, calculations, as well as the n.m.r. st.ructure (Schultze ef al.. 1988).

,Vormalized

1271

Table 1 2.5 A data from multiwire

structure factors for metallothionein

Theoretical

Experimental

No. of reflections (EZ) (I@-



From (FI

From /AFIt

Crntrosymmetric

2237 1400 08178 08643

I %2 1~000 09798 07906

09680 019x0

Percentages of total E

la00

values

greater than E limit

E limit 72.2 645 553 47.5 406 339 27.9 22.2

60.8 54.8 48.7 41.4 36.1 31.0 21.9

61.7 54.9 48.4 424 36% 31-7 27. I 23.0

1.4 IFJ

17.6 13.8 11.4

18.1 154

19.4 16.1

I.6

87

13.0 IO.6

13.4 11.0

7.0

8.9

8.9

56 46 3.1 2.2 I.7 1.3 0.9 0.6

7.6 6.2 50 4.1 3.4 2.x 20 1.8

7.2 5.7 46 3.6 2.8 2.1 1.A 1.2

(b.5

0.6 0.5 OX 0.9 I.0 I.1 1.2 1,J

I.7 1%

1.9 2.0 2.1 2.2 2.3 24 25

area diffractometer

261

~011.c~ntr~s\‘rnrnetrir

A. H. Robbins et al.

1272

Figure 1. The regions of the E map in P4,2,2 showing the peaks for all 7 metal sites in Cd,,Zn,-MT. Coordinat’es for the metals from the refined structure are shown connected by virtual bonds. Left side, Cd, cluster: rights side. CdZn, cluster. Contoured at 0.30 t,imes the maximum density.

combined figure-of-merit also contained 5 high peaks for t’he Cd sites (at inverted positions from the P4,2,2 solution). However. t,he P4,2,2 E map had less contrast on the Cd peaks and no rlear peaks for t,he Zn sites. (e) Phase calculations The 5 (‘d positions from the direvt methods solutions were refined against, the 25”)+, largest) IAF[ values

(Hendrickson & Teeter. 1981). The 2.5 A acrntric, data were used, occupancy and qz were refined with an overall R-factor of 15.0 A2 and the E-value at ronvrrgenne was 0.31. These positions were used to calculate phases in bot,h P4,2,2 and P4,2,2 hy interativr single-wavelength anomalous scatterer (ISAS) met’hods (Wang. 1985). The starting figure-of-merit, for 2518 acentric, reflections to 2.0 a resolution was 0.37. Phases calculated based on the (‘d partial structure for 2518 acentric and 1800 centric. reflections were combined with t,he ISAS ac+entric* phases using phase probahilitjg profiles derived from t)hr Sitn wrights (program Pmodrl. 13. (‘. Wang, personal (‘onmuniration). After phase combination the figure-of-merit for 4318 reflections to 2.0 A resolution was 0.55. The R-factor for t,hr Cd partial structure was 0~W4. The caomhined 20 A TSAS/partial structure phases wert’ improved 1)~ solvent flattening methods (Wang, 19851 assuming a solvent, content of 4O’j;, (ohsf~rveti c*onteni 54”/;,). Fire cycles of refinement, were done for acentric, rrflec%ions. followed by ext-ension to centric reflect’ions for A cycles: the process was repeated for 2 solvrnt~ masks. The resulting values of tigure-of-merit and map inversion E-value were 0.77 and 0207 respectively. in 1’4,2,:! 111 P4,2,2 1hese values were slightly better: W7!) ant1 0.18X The assignment of space group as 1’4$,P was rnadf, following rxamina.tion of the electron density maps. Thii assignment is consistent wit)h t)he dir& methods and ISAS ralcula,tions. In P4,2,2. thr map showed den+ R)r segments of the polypeptide chain. carbony bulges, all 7 metal sites and peaks for t*he sulfurs of all 20 (*ystr*int residues (Figs r’. 3). In c.ontrast. the P4,2,2 map had if number of breaks in the density for thtl protein. IOU.~I

0.5000

.?

0~0000 0.0000

x

2.0000

Figure 2. A 2.0 a resolution solvent Hattened electron density map calculat,etl with experimental phases tirrivc~tl from the Cd anomalous scattering and partial structure. The sections shown enclose 2 asymmetric units (7.725 x in the ,q dirertion). Contours start at @04 of the maximum density.

Crystal

Xtructure

of Cd, Zn Metallothionein

(b)

Fig. 3.

1273

A. H. Robbins et al.

1274

(dl

Figure 3. A 2.0 a resolution electron density map calculated with native data and rxperimental phast>s dc~rivc~l I’ronr (‘d positions. Co-ordinates of the refined model are shown. (a) Density of the Cd, (Sy),, (*luster. (‘ontoured at O+E a.tr(l O-28 of the maximum density. (b) Den&y for the CdZn,(P), cluster. The Cd4 site is at top vrntrr. (‘ontourcd at PO7 anal P28 of the maximum density. (c) Density for residues 41 to 48 t.hat includes 2 turns of an a/3,, helix (bot)torn to tofl) and Cd6 and Cd5 sites. Contoured at tiO6 of the maximum density. (d) Density for residues 6 to 13 (t,op c&enter to left renter) and Cd4 and Zn2 sites. Contoured at, 0.06 of the maximum densitr. solvent contrast. a peak for only 1 of the Zn sites and missing density for some of the cysteine sulfur atoms. especially at the Cd site in the B-domain cluster (Cd4 site). Co-ordinates for the structure of rat liver MT-Z derived by n.m.r. (K. Wiithrich. personal communication) were compared to the 2.0 A ma,ps. Superposition of the coordinates for t’he 4-metal cluster on the map supported the space group assignment. il’o superposition of the 4 Cd sites in P4’4,2,2 placed all the sulfur atoms in density. due to t,he chirality of the pucker of the 2 6-membered rings in this cluster. However. in P4,2,% the C!d4(SY),, model could be fit to the density such that all Sy atoms were in den&y. Similarly. the n.m.r. model for the 3-metal cluster was superposed on the map. until all 9 Sy atoms fit the density.

(g) l-kfinemrtct

The starting model of 291 atoms was retint~l with Splo~(Kriinger st al., 1989). The R-factor. for all 4318 reflection:: with IFI > Oo(F) in t.he range +(I to 2.0 :\ resolut,ion dropped from 0.45 to 035 in 1 myrtle (40 timesteps of 00005 ps) using an overall B-fact,or of I5 A’. Structurr factors and phases calculated from thts part,ially retinr,rl structure were combined with the ohst~rvrd dat>a tc, generate Sim-weight.ed coefficients (Stuart Ki Artyrniuk. 1985). rl Fourier map was calcula.ted with these c.orl-ticients using the c.ombinrd figure-of~rnrrit~s and phase+ (Robbins & Stout. 1989). The map wah used to build side>chains into the tnndel according t.o the scAquen”e ( WingtL P/ al.. 1984). Refinement and rebuilding of the model using a

(f) Model Ouildiny The 2.0 w electron density map in P4,2,2 was interprrtable due to the presence of all the metal and sulfur peaks and carbonyl bulges (Fig. 3). Using the 2 oriented cluster models as a reference? the n.m.r. co-ordinates of the separate a and fl domains were superposed in the density. The resulting model for the polypeptide was found to ht the density well for CXC (where X is a non-cysteine amino acid residue) sequences, but that concerted rot,ations and translations were required to fit the intervening larger loops. The program Frodo (Jones. 1978) was used to adjust the model for the individual tl and fi domains, and appllcatlon of symmetry allowed docking of the domains at residues 30 and 31 (Ca atoms 3-8 A apart) to complete the model for one asymmetric unit. Because the n.m.r. model we used had side-chain atoms only for the cysteine residues. the starting model for refinement consisted of the clusters. the 20 cvsteine residues and the 41 remaining residues in the protein as glyeine. The 3-metal cluster was modeled with 1 Cd and 2 Zn based on the composition of the crystals (Melis rt al., 1983). the suggested position ot Cd in this cluster (Fig. 1). and the anomalous scattering electron densit,y (Fig. 4).

*

Figure 4. Two regions of the 2.0 4 rt~solution Bijvoet diff‘erence Fourier map calculated with native data and phases from thr refined model showing peaks for thr 5 Pti sites only of thr Od4(SY),, and (‘dZn,(S’), cslustrrs;. (‘ontoured at 0.36 of the maxirnurn densit,y: Ko significant peaks are observed at, the Zn or S’ posItIons abovr the noise level (018 of the maximurn densit,v).

Crystal

I

151 0

IO

Structure

I 20

of Cd, Zn Metallothionein

I 30 Residue

/ 40

50

1275

I 60

1

70

number

Figure 5. Plot of average B-factor for all atoms of each residue ver.suS residue number for the refined structure,

2nd combined coefficients map reduced the R value to @31 for 414 atoms of the protein and the 2.0 data. Isotropic B-factors were refined for the complete prot’ein model (414 atoms). Water molecules and ions of crystallization were included in the model, refined, edited and refit,. The model contains 33 water molecules, all of which have R Oo(F) in the range 5.0 to 2.0 A (Table 2). For this model the root-mean-square deviations from ideality of bonds and angles are 0016 A During refinement Cd-V and and 3.1’. respectively. Zn-SY bonds were restrained to have bond lengths of restraint 2.4 A, respectively (bond 2.5 A and 300 kcaljmol). and tetrahedral bond angles (angle

Table 2 R-factor for the metallothionein Resolution range (A) 354-500 P99- 3.54 %68--2.99 2.47F2.68 .‘$31--2.47 219-.2,31 2+)8--2.19 Lwo- 2m

Number

of reflections (Fol > 0.0 1048 1013 982 959 959 939 702 560

structure R-factor 01305 0.1487 01716 01943 0.1999 02448 0.2449 0.2694

restraint 50 kcal/mol; 1 cal = 4184 J). Cysteine SY atoms were constrained to be bonded to the appropriate metal site and the CB-SY-Cd/Zn bond angles were restrained to be tetrahedral (angle restraint 50 kcal/mol). The refined phases were used to calculate a Bijvoet difference Fourier map (Strahs & Kraut, 1968) using the native data. This map (Fig. 4) also indicates the positions of the 5 Cd sites and that there is no significant occupation of Cd in the 2 Zn sites of the 3-metal cluster (for CuKcc f”(Cd)-5.0, f”(Zn) -06, f”(S) -@5). Co-ordinates have been deposited with the Protein Data Bank (Bernst’ein et al.. 1977). arression number 3MT2. (h) Tungsten

derivative

We reported that the compound (NH,),WS, when soaked into MT crystals stains them yellow and introduces significant isomorphous intensity differences (Furey et al., 1986) (samples of the compound were generously provided by E. Steifel, Exxon Research Laboratories). In addition, assay of soaked crystals showed that >07 mol of tungsten was bound per mol of protein. A 2.3 A oscillation camera data set had been collected but we were unable to solve the difference Patterson map (Furey et al., 1986). The WS:- derivative film data were scaled to the native data set (Emerge= 012). An isomorphous difference Fourier map calculated with the refined native phases revealed 3 WSi- sites, all near z = l/2, with 1 site on the diagonal X-fold axis (Fig. 6). The sites were refined against the origin-removed Patterson map (Terwilliger et al.. 1983) and used to calculate iterative single isomorphous replacement (ISIR) phases (Wang, 1985). For 530 cent,ric reflections Ris, was 0.675. and for 2120 reflect.iona to 2.3 A (data set 72% complete) the starting figure-of-merit was 026. Following ISTR refinement by solvent flatt,ening the figure-of-merit was @69. The electron densit,y map based on the 2.3 A WS:- TSIR phases also indicates the positions of the metal sites in both clusters as the highest peaks in the map (Fig. 7). The WS:- derivative supports the results of the new direct methods analysis on the protein JF( data (Fig. I). One could argue that the WS:sites carry no information about the original direct m&hods solution (Cd sites) and that these 2 “derivatives”

A. H. Robbins et al.

1276

Figure 6. Isomorphous difference Fourier map for WSi- derivative data to 2.3 A resolution using phases calculated from the refined native structure. The peaks shown are the only significant peaks in the asymmetric unit. Map shown encompasses l/2 of a unit cell along II‘ and y (down and across, respectively) and l/2 of a cell on z (normal to Figure). The origin (upper left-hand corner) is at l/2. l/2. 1jZ. One WS:- site lies on the diagonal 2-fold axis at z = l/2.

(W’S;- and Cd) are independent as defined by Dickerson et al. (1967). However, the presence of pseudo-symmetry (see below) and the novel manner in which the Cd sites were obtained does not, allow an unequivocal answer to this question. The native structure was refined against t,he WS:- data set (WSZ- sites not included in the model) resulting in an &factor of 0168 for all data between 20 and 2.3 B. Thr Fourier map for the WS:- data and refined phases is shown in Fig. 8. This map agreed with the protein model and was useful in modeling a chain trace for residues 51 to 56. as the density in this region was somewhat stronger than in the native map. Ordering of the 51 to 56 loop may be due to binding of the WS:- sites adjacent to K51 and 453. rl network of interactions is formed in the solvent channel at z = l/2. The 1st WS:- site (at 0.717. 0.717, @500) lies symmetrically between the XC atoms of 2-fold related K30 residues, 3.95 L4 from each. This distance suggests ionic interaction of the lysine side-chains and the W’S, ion. The second WS:- site (at 0.517.0.786.0494) lies in a hydrohobic pocket formed by P38. G40 and A53 on svmmetry related molecules. This sit~e is also 43 A from 6 of K51 (side-chain density weak) and 6.1 A from I\;’ of K30. The 3rd site (at 0694, 0585, 0.461) also lies in a hydrophobic pocket formed by methyl groups from symmetry-related side-chains of EacMl, A8. T9 and V39. and is 49 L% from Xi of K30. The carbon to WS:-contacts for the 2nd and 3rd sit,es are in the range 3.9 to 5.1 A. (i)

Pseudo-symmetry

qf (‘d positions

Fig. 9 shows the disposition of 10 (:d sites in 2 asynmetric units about the diagonal a-fold axis at z = l/4. This arrangement results from crystal packing of the I-domain MT molecules. A consequence of this packing is that a

pseudo-center of symmetry is csreatrtl where vrc%ors connecting the pairs of clusters nearly intersect,. That the Cd constellation is pseudo-centrosymmetric is suggested by the following observations. First, the distribution of E values derived from (AFI is almost ideally caentrospnmetric (Table 1). While this is generally true for differem? data (Wilson, 1978). the effect may be enhancaed in MT due to the special arrangement of the (Id at,oms. Sccontl. the Cd constellation may diminish the magnitude of tht* anomalous differences as well. The ratio of (jAB’j)/(lE’I) in the scaled data is 004 whereas t’he expetsted value for 5 (‘ti atoms is 021 (Hendrickson $ Teeter. 1981). Third. rrtiutt ment of t,hr protein structure with Xplor with anomalous scattering factors for (Id included in thr strut%ure fatatot calculation affects the overall M-factor by less t,han I (I(, Tn this refinement thr calculated values of’ IF’ 1 and IF / correctly predict t,hr sign of 49 of 50 Rijvort tlitYerenc,rs for the 50 st#rongcst acent,ric rrflections in thr dat’a set III other words. it is our opinion that the data arch a(:t‘urat,t but the observed Bijvort differences art’ small dw to pseudo-symmetry. The presence of pseudo-symmetry it1 I he (‘tl strut*turth may be the reason why the previous direct methods analysis based on film AF data yielded an inc~orrrot solu. tion (Furey of rtl.. 1986). In t’hat case. thtl (‘(1 praks wct‘rtl unequally weighted. and the model had t,o br translated to find an H m,n,mum.Moreover, previous tlilrct mr~t~hods solutions also yielded E maps with a very large peak ON the P-fold axis. A similar phenomenon was observed with the new JAFI data (see a,bovr). The pseudo-symmetry may havr hindrrr~d Ioc*aticm tbt the (‘d sites bv other m&hods and at the same tinit, introduced ambiguity in discriminating a correcat solution. An tj-fac%or search using all the IArI data to (PO A \vas performrd with 1 sitar as a probe for th(a t&enter of thtb gravit,y of the 4-metal cluster using the program HprcuIt+ (D. E. McR,er. unpublished results). The 1)rogram had been successfully t,ested using anomalous differrntar anti isomorphous replacement dat,a from known protc,ili structures. Thr IZrnl”lrn”rnoccurred at a positian translatf~tl - 6 4 on z from the originally reported solut,ion (Furry t,f /I/.. t 986). (An ambiguity of t (so-ordinat,ca was also observed previouslyv.) Repetition of thtA K-factor xrarcih procedure lrtl t,o a &site model for which the (.orrc:lation c~oeftic~irnt for observed LY~SILSc~alculated Bijvoet ditfrr tln~e l’att,erson maps was 0.76. The previously reportt%tl value at 6.0 -4 w’as 0.75 (Furey et al.. 19H6) and yet both solutions are wrong. At, 3.0 A resolution the c.orrelatiotl coeficient,s were 0.47 (Furey et (11.. 1986) and 0.59 (using program Hercaules). For thr present solution thr c.orr& tion c*oefficGents are 0.60 at 6.0 A and 061 al 3.0 :\. 11 appears that there arr mult,iple incorrect solutions fi)r the (Id sit,es in the structure. especially at low rrsolutiolr Rigid body staarcsht~alt~ulations using Nplor. thca li.tl~r’ model for the l-mt%al c*lust’er and thtx IP] data also failed to -irId t hr c.orrec1 solution. Again. t-hr. H“,~llIlO~ l’lat~etl thr c.lusttAr on the 2-fold axis under a variety of c.ornputittional caonditions. While the n.m.r. a,ntl t*rystal t.o-or(li nat,es for the clustrrs are very similar. t0iis search ula>. have suffered from omission of the 5th (‘(1 site. ~!olr~~ular replacement c.alculations using Merlot (l’itzgerald. 19X8) and the n.m.r. rnodrl for t)htx entire a tlonlain failt~tl t,o yield a consistent solution. This approach suffered from omission of the ,Cdomain. as thr rralat’ivr orientation ot the 2 domains is not known from the n.m.r. tlata (Schultz+ rf nl.. 1988). In addition. the K-factor oi’thr n.m.r. J ar~tl [i domain models. after titting both t,he elrt%ron tlensit~? map (see above) and rigid body rrfinemrnt, is only (1.47 At the same time, ;i survey of 30 addit#ional hravy-atr>nl

Crystal Structure

qf Cd,

Zn Metallothionein

1277

(bl

Figure 7. A 2.3 A resolution electron density map calculated with native data and phases derived from 3 WS:- sites. Portions of the refined model shown are in the same orientations as in Fig. 3. The shape of the electron density peaks is distorted due to incompleteness of the oscillation data set for WS:- derivative. (a) Density for the Cd,(S”),, cluster. Contoured as in Fig. 3(a). (b) Density for the CdZn,(SY)9 cluster. The Cd4 site is at top center. Contoured as in Fig. 3(b).

Figure 8. A 2.3 A resolution electron density map calculated with data from the WS:- derivative crystal and phases from the model refined against the WS:- data (R = @168). The main-chain atoms and some side-rhain atoms for residues 30 (upper left) to 40 (upper right) are shown. Contoured at, PO6 of the maximum density.

A. Ii. Robbins et al.

1278

CD4b

CD4b

1 \\

< a-fold

axis

CD7a

CD7a

Figure 9. Illustration of the pseudo-symmetric constellation of Cd sites in the crystal structure. 0111~(‘d-(Id virtual bonds are shown for the Cd, clusters. Lines connect pairs of Cd4 and Cd7 sites in 2-fold related molectules at (.r. !I, z) (molecule a) and (1 -y, l---2, 112-z) (molecule b). The 2 vectors intersect the diagonal 2-fold axis within 0.15 A of each other creating a center of pseudo-symmetry. However. in this Figure the Z-fold axis is not in the plane of the paper.

compounds and/or soaking conditions failed to yield any useful derivative other than (NH,)2WS, (.J. Soman, unpublished results). The originally reported 2.3 Lh ISAS map (Furey et al.. 1986) was of rather poor quality (figure-of-merit 0.67). Nevertheless, it was interpreted on the basis of the indications that’ the Cd sites were correct, and given that the structure of the metal clusters (Otvos & Armitage, 1980) and the primary sequence (Winge et al., 1984) were known. The choice of space group rested on the difference in ISAS refinement results whereas these could not have been meaningful given the incorrect Cd positions. Subsequent refinement of the incorrect model did not reduce R below @37 for all observed 2.0 A data with (8’1> Oa(F) without the addition of water molecules and individual B-factors. The inability of the model to refine. the disagreement with the n.m.r. structure (Schultze et al.. 1988) and the ambiguous results obtained in different attempts to subsequently locate the Cd sites all indicated that the originally reported st’ructure was incorrect.

3. Results (a) Domain

structure

The MT structure is illustrated in Figures 10 and II. Figure 10 depicts two views of the molecule highlighting the backbone of the polypeptide chain. Figure 1 l(a) and (b) shows all atoms of the protein in orthogonal views, emphasizing the disk shape of each domain. The directions through the minimum dimension of each domain are roughly orthogonal. The j? domain is defined as residues 1 to 29, CI is residues 33 to 61 and the linker is residues 30 to 32. There are no contacts between the a and fl domains; K31 in the linker region interacts with the carbonyl groups of residues 19 and 21. Figure 11 (c) and (d) show the domains individually and the cysteine co-

ordination of each metal site. The overall chain fold of both domains and the metal co-ordination of the seven metal sites by the 20 cysteine residues is the same as in the solution structure of rat liver (!d,-MT determined using n.m.r. methods (Schultze it nl.. 1988). A comparison of the solution and cry&al structures will be reported elsewhere. The Cp, rc/ angles for the MT st’ructure are within the normal ranges except, for two residues in very weak density, S54 and D55 (Fig. 12). The Cp. $ angles of ~416 (39”. 62”) are close t,o the values commonly observed for non-b-branched. non glycine residues in the left-handed a-helical conformation (Nicholson et al., 1989). Presumably, t,his conformation is recluired for the folding of the fl domain. The three glycine residues in MT (Gl 1. G I7 and G47) have 4, $ angles commonly observed for glycine residues outside the normal ranges in other structures (Nicholson et al., 1989). (b) J4etal clusters In the following discussion S’ refers to an> cysteine sulfur, Qb to sulfur t’hat bridges between two metals, and 8, to sulfur that is a t)erminal ligand to a metal. The structures of the isolated met’al clusters, Cd,(EY),, in the cr-domain and CdZn,(V), in the P-domain, are shown in Figure 13. The metal sites are labeled to correspond to the n.m.r. spectral assignments and numbering (Schultze et al., 1988). The n.m.r. sites II and III correspond to Znl and Zn2 (Fig. 13(b)). The six-membered rings (Fig. 14) of’ t!htx metal-sulfur clusters show distortions from ideal chair conformations. The Cd,Zn,(S,), ring and ring B of the 4-metal cluster have a left-handed twist,-

Crystal Structure of Cd, 2% Metallothionein

1279

(a)

lb) Figure 10. (a) Schematic representation of the MT structure showing the main-chain, cysteine side-chains and metal sites. The N terminus of the /I domain is at the left, C terminus of the a domain is at the right. (b) View of the MT structure showing all atoms of the protein and clusters and highlighting the main-chain. The p domain is on the left, a domain on the right.

1280

A. H. Robbins et al.

(al

(b)

Fig. 11.

Crystal Structure of Cd, 2% Metallothionein

1281

(d)

Figure 11. Stereo views of the MT structure showing all atoms of the protein and clusters. CP-Sy and SYm-metalbonds are highlighted. (a) View normal to the a-metal cluster of the /I domain (top). The linker region, residues 30 to 32, is in the center of the Figure, and the CIdomain is at. the bottom. (b) View orthogonal to that in (a) emphasizing that the short dimensions of the a and B domains are roughly normal to each other. (c) The fi domain showing the arrangement of the 9 cysteine ligands around the 3 metal sites. Side-chain atoms of the non-cysteine residues have been omitted for clarity. (d) The a domain showing the arrangement of the 11 cysteine ligands around the 4 Cd sites. Side-chain atoms of the noncysteine residues have been omitted for clarity.

boat conformation. Ring A of the 4-metal cluster has an envelope conformation with only the Cd7 site out of plane. Pairwise superposition to obtain maximum alignment of the cluster rings reveals the greatest similarity between the Cd,Zn,(S,J3 and B rings (Fig. 14). If the Cd,Zn2(S& and B rings are superposed an interesting alignment of the protein domains results (Fig. 15). Essentially the same alignment also occurs if the backbones are optimally superposed without consideration of the cluster structures. In either case, the polypeptide chains are roughly in-register up to a crossover point at residues 11 to 14142 to 45 where the chain folding around the clusters is reversed. The o! domain, following one turn of an u-helix at residues 41 to 45, continues in a left-handed manner, whereas the p domain, following Gil, continues in a right-handed manner.

C5, C7- and Cl3 lie at the base of a cleft,. In the tl domain, a similar cleft contains three exposed, contiguous S’ atoms from C37, C41 and C57 (Fig. 16). Because some sulfur atoms are exposed to the solvent they make hydrogen bonds to ordered water molecules. The sulfur atoms also make a number of hydrogen bonds with peptide amide groups and other protein side-chains (see below). The polar contacts to the sulfur atoms are grouped into categories in Table 4. The contacts are also divided into type I and type II, as defined by Parthasarathy in a

(c) Cysteine sulfur atoms The cysteine sulfur atoms in MT vary greatly in their solvent accessibility (Table 3). All seven metal sites, by virtue of being liganded tetrahedrally by sulfur atoms, are sequested from the solvent. The extent of solvent accessibility for the sulfur atoms surrounding a given metal varies considerably in the /3 domain. The total solvent accessibility of the four sulfur at,oms bonded to Znl is 5.05 A2, whereas the value is 35.83 A2 at Zn2 and 2968 A2 at Cd4 (Table 3). The a domain sites have more uniform values of 1990 to 27.19 A2. Each domain of MT has a solvent exposed cleft containing three cysteine residues. Figure 16 shows two space-filling views of the molecule. In the jl domain. a triad of solvent exDosed Sy atoms from I

D55 .

-180

I

-120

-60

0

60

120

I

I80

@ (deg.1

Figure 12. Ramachandran

plot for the refined metallothionein structure. Residues 554 and D55 are in very weak density where the model is tentative.

A. H. Robbins et al.

1282

-

--.

(b)

Figure 13. Stereo views of the metal clusters in MT. Sy atoms are labeled by residue number. (a) I domain (‘d,(S’), , cluster. (b) /I domain CdZnz(SY), cluster crystal structures of smaI1 molecule (Rosenfield et al., 1977). Type I contacts are made by electrophiles that approach the sulfur from above or below the plane defined by CB-SY-metal.

survey

i.e. toward the lone pair. Type II contacts are made by nucleophiles that approach the sulfur along the extension of a CJ bond. The predominance of contacts to sulfur atoms of termina,l ligands is

Table 3 Solvent

accessibility of cysteinr sulfur (I. 14 d probe sphere

Cd,&,),

Ring A

Ring B

Sy atomst

tmsed m

r domain

/I domain Cd#%,

atoms

Surface area A2

H,O Kmtact

tf97

0124

Surfaw S’ atoms? area .A*

H,(f Nmtac~t,

(a)

5

%(a&,

A

B

Cd, Z;l]

(b)

Figure 14. Comparison of 3 6-atom rings in the metal clusters. Cd sites are labeled with Roman numerals. Sy atoms with integers by residue number. (a) Cluster ring torsion angles (deg.). (b) Cluster ring superpositions.

7* 13 1*-i*

'3% 1247 0

19 21

I) #X6

14* 26 29

0 IF51 545

33 34* 36 37*

.41

0124 Oil

44* 48 50* :iT 59

60*

17.09 0 I .5$2 61-i 13.73 0

(M6,096 096f OlM

0101

1010 0 80X I 30.5

oioti

08‘2

0

t By residue number; *, bridging cynteine SY. $096 425 i9 from F(34) and 335 A from Sy(33): H -241

AZ

Crystal Structure of Cd, Zn Metallothionein

1283

Figure 15. Superposition of LXdomain (thin line) and fl domain (heavy line) that results when the J-metal cluster is superposed on ring B on the 4-metal cluster. Only N, C” and C atoms of the backbone are shown.

(Table 4). All but one of the amide contacts are type T; H,O contacts are both type I and II. The behavior is consistent with the chemical natures of the contacting atoms.

apparent

(d) Cysteine side-chains Chakrabarti (1989) has summarized the geometry of interaction of metal ions with cysteine residues in known crystal structures by plotting both the x1

and xZ tosion angles. A x,, x2 plot for the 20 cysteine residues in MT is given in Figure 17. Because there are eight bridging and 12 terminal cysteine ligands, 28 torsion angles are plotted altogether. The MT cysteine residues occupy all but one of the nine possible regions of the plot; only the region at 180”, 270” is unpopulated. The cysteine residues with x1 of 180”, and those at xi, x2 of 90”, 270” are typical of other proteins. The remaining cysteine residues with xi of 90” and 270” are unique

Figure 16. Stereo space filling representations of the MT structure calculated with 100% van der Waals’ radii on all non-hydrogen atoms. (Top) View showing 3 solvent exposed Sy atoms on the surface of the a domain (left of center: P(57), SY(37) and P(41) from top to bottom). (Bottom) View showing 3 solvent exposed Sy atoms in a cleft of the j? domain (top center: Sy(5), S’(7) and S’(13) from left to right).

1284

A. H. Robbins et al.

--

Table 4 Non-bonded

contacts to sulfur

~4.0 A involving

electrophiles (‘ontacting

and nucleophiles atom Type

Category of cgsteine sulfur atom

II

H,O

W to Zn site Sy to Cd@,)2 site Sy to Cd(S,)3 site All S, 811 8, S, with 2 1 NH S, with 2 1 H,O Sy in /3 domain Sy in G(domain

I 5 :i I 7 :1 7 3 3

Other --___“(of:. 3%) 0 (1 l(t)(;) IjSZ) I(S%) 0 P(o(:. NZ) 0

Total

X

2

t No contacts to Sy of cysteine residues 7, 50 and 60 $ Main-chain amide group.

to MT, except for three examples at x1 of 270” in other proteins (Chakrabarti, 1989). The MT data show that bridging cysteine residues prefer x1 of 90” or 270” while xZ is unconstrained. In MT x1 values for consecutive cysteine residues in CXC or CXXC sequences almost always differ, as observed for other proteins (Chakrabarti, 1989), presumably due to steric constraints of packing the cysteine side-chains about a common metal. Exceptions to this rule occur in the sequences C33CSC36C and C57SCC60 where C33, C34 and C36, and also C57 and C59 have the same x1 value. Apparently, co-operative binding to Cd can override the steric constraint. Terminal cysteine ligands show flexibility by having x1, x2 values in six regions of the plot. There is no obvious preference of x1, xZ angles for cysteine Sy atoms that are more

solvent exposed. buried or have ordered contacts (Tables 3 and 4). (e) C‘ysteine peptides

The MT structure can be broken down int,o short cysteine-containing peptides. It is of interest to analyze the structure this way because these sequences occur commonly in other metalloproteins. Figure 18 tabulates the 18 total consecutive

,-/MomahResidueSequenceCysteine S’

--a.Domein 1 C(XLJ ( ResidueSequence Cysteine S’ n Number

# - cysteble residue

O-bridging

I

I 55

Sy

I 09

I 124

I I 158 193 x2 (deg.)

I

I

227

262

C”-CD.-SY-M

Figure 17. Plot of the torsion angles (Chakrabarti, angles are defined along respectively.

cysteine side-chain to metal 1989). The x1 and xz torsion the ordinate and abscissa.

I

Figure 18. Summary of the sequence of all possible cysteine peptides in metallothionein of t,he type C(X)/,C where R ranges from 0 to 6. The amino acid sequence (Winge et al., 1984) is shown vertically for each domain in l-letter code. S,. terminal ligand; H,. bridging ligand: X. any amino acid residue except cysteine.

Crystal Structure of Cd, Z?z Metaltothionein

1285

(b)

Figure 19. Superposition of cysteine peptides within the MT structure CXC sequences aligned with respect to the 1st peptide bond. (b) Five central peptide bond (side-chains of K22 and K43 truncated for clarity). Cd5 and Cd7 (thin lines and dots) and C57SC59C60 co-ordinated to Cdl

showing their similar conformations. (a) Seven CXXC sequences aligned with respect to the (c) Best fit of C33C34SC36C37 coordinated to (heavy lines and dots).

1286

A. H. Robbins et al.

Table 5 Intramolecular

hydrogen bonds excluding sulfur atoms

Donor Residue N4 S12 s12 Cl6 518 618 K22 K22 Q23 K25 K25 c29 K31 K31 S32 532 c34 c41 K43 c44 s45 s45 s45 G47 A53

cysteine

Acceptor Atom

Residue

Atom

Distance (A)

N OG OG N N OG N NZ N NZ NZ x NZ NZ OG OG s N NZ N N N OG N N

D2 DIO DlO S28 Cl?5 c 15 N4 P3 N4

ODl ODl OD2 OG 0 0 0 0 0 OD2 0 0 0 0 0 0 oc: 0 0 0 0 0 0 0 0

3.03 3.08 2.74 3.46 L?93 2.85 284 309 2.85 3.28 2.83 311 314 2.87 2.94 2.74 3.0.5 3.12 2.82 3.37 3.35 308 263 3.16 3.25

Donor-H-acceptor

& C26 Cl9 C21 (~34 C37 S32 P38 (‘59 C41 C41

A42 A42 c44 K51

distance < 3.5 A. angle 180” + 4OO”.

cysteine peptides in the sequence (Winge et al., 1984) and the number of each type of peptide as defined by the number of residues intervening between the cysteine residues (central box, Fig. 18). The /I domain contains four CXC repeats whereas the u domain contains CXCC sequences at the N and C termini. Perhaps fortuitously, there is a nearly

-

of terminal and bridging alternation regular cysteine residues in each domain. There are seven peptides with the sequence (‘Xc’ in MT. All seven have similar 4, $ angles at the first C and at the X residue. This is illust,rat~ed in Figure 19(a) where the peptides are superposed with respect) to the first peptide bond. The average 4. $ angle at the first (’ are - 97”. 145’ and at X are -98”. 1”. The same conformation is ohaerved in Fe-S proteins where the sequence is (‘XYC’ (Adman et al.. 1975). In MT, the seven CX(: peptides t,hus mimic a type I reverse turn. This conformation favors a NH-S hydrogen bond from the second cysteine amide to the firsts cysteine Sy (Adman it ul. 1975). (lonsequently. the Sy positions of t,he first cysteine a,re more restricted. The preferred CS(‘ peptide conformation also places the Sy atoms in van der Waals’ contact, whether or not they cahelatc to the same metal site. When five CX,X,C peptidrs (including both CSCC sequences) are aligned (Fig. 19(b)) conformational restriction is also observed, especially for the side-chains of the X residues. even if X is cyst,eirre. The 4. II/ angles of the intervening Xi. X, residues are typicA of type I reverse turns: the average values at X Ir X, are -72”, -33” and --87”. -2”. respectively. Within the CX,X,C pept)ides there are three NH-S and two NH-O hydrogen bonds (see below). There is striking similarity in the co-ordination of Cd by the sequence CSCC at residues 34 to 37 and 57 to 60. This is illustrated in Figure 19(c) where these peptide fragments and the metal they bind are superposed. The two peptide fragments are isostruc:tural. Viewed along a common SY(50)-Cd bond. the chirality of ligation of Cdl by Sy(57). SY(59) and Sy(60) is the same as for Cd7 by Sy(34), Sy(36) and Sy(37). This feature offers a possible structural basis for the observed co-operative binding of Cd in the

Table 6 Reverse turns in metallothionein H-bond(s) Residues

Sequence

2-5 9-13

DPNC TDGSC

15-18 21~24 26-29 34-37 38-41 44-47 51-54 53-57 57-60

CAGS CKQC CTSC cscc PVGC CSQG KEAS ASDKC cscc

Donor

Acceptor

Distance (A)

N4 N S12 OG 512 OG S18 N

D2 ODl DlO ODI DlO OD2 Cl5 0

303 308 >74 293

c29 N

C26 0

3.1 1

C41 N G47 N A53 N K56 N C6ON

P38 0 c44 0 K51 0 S540 C57 SG

3, I 2 3.16 3.25 2.85 335

t Turn type as defined by Richardson (1981). $ Peptide plane of S12/C13 inverted nersu~ standard y-turn. 5 cp, JI of Al6 (+39”, +62”). (( cp, + of K22 (-78”, -39”); cp, $ of Q23 (- 144”, +78”). 1 Peptide plane of A531554 inverted versus standard y-turn.

Typet

l-4(5) (1” (‘” distance (A) 6.16 i.89

I rl

5.41 5 10 .559 590 566 5.24 6.30 8.74 6.46

Crystal Structure of Cd, Zn Metallothionein

Table 7

a domain (Nielson & Winge, 1983; Byrd & Winge, 1986). Analysis of the co-ordination of the other metal sites with respect to their liganding cysteine peptides reveals no other obvious patterns. For co-ordination of the two Zn sites is instance, dissimilar. (f ) Intramolecular

Intramolecular

Residue

hydrogen bonds

S6 CT Cl5 G17 c21 K25 C26 K31 c37 C44 149 c50 c60

residues. Lysine side-chains are involved in interactions or hydrogen bonds to cysteine

N-H-S

NH-S hydrogen, bonds in metallothionein Acceptor

Donor

The MT structure contains 25 potential hydrogen bonds between N/O donor/acceptor atoms (Table 5). All but two are intradomain; K31 NC to carbonyl groups of Cl9 and C21 is unique as a linker to p domain contact. Residues 41 to 47 form a twoturn a/3,, helix (Fig. 11(d)). There are no other occurrences of repetitive secondary structure interactions. Serine side-chains are involved in hydrogen bonds within loops (e.g. S12-DlO, Fig. 3(d)) and to cysteine tertiary

1287

Atom N N N N N N N NZ N N N N N

Residue c21 c5 Cl3 c29 Cl9 C5 C24 c19 (~34 C41 WI C48 c57

Atom SG SG SG SG SG SC sd SG SC SC; SC SC: SC,

Distance (A) 3.18 352 331 3+M 381 3.39 3.61 335 368 3.66 391 3.52 3.35

distance hree in c( (K43, K51, K56. see Fig. 1 I). Both features of the st,ructure could st,abilize the unliganded thiolate state of the cystleine residues, promoting greater metal exchange in /l. The ligands of the Znl site alone have t$hree NH-S and one NC--S interaction, consistent with t,he n.m.r. observed lability of this site. The /? domain may also be able to exchange metal more readily because its cluster is less crosslinked (Kggi & Schtiffer. 1988), even though the sulfur atoms are more buried from solvent on average (Table 3). Moreover. the net charge of the fi cluster is -2 Uersus --3 for the OLdomain if the K31 contact to SY(19) is counted.

Crystal

Structure

qf Cd,

Further, the unfolded p domain contains four CXC sequences which could act as local metal binding sites in the absence of longer range order, stabilizing partially unfolded conformations.

Zn Metallothionein

1291

E. Steifel for samples of (NH,),WS, and M. Pique and G. Gippert for computer graphics. We thank D. R. Winge, J. D. Otvos, I. M. Armitage and A. Briinger for discussions. This research supported by NIH grant GM-36535

(e) MT dimers Metal exchange occurs between pairs of MT molecules such that mixing of Cd,-MT and %,-MT leads to an equilibrium state of native Cd,,Zn,-MT (Nett,esheim et al., 1985). Moreover, dimers are formed when Zn,-MT is treated with free Cd’+ (Otvos et al., 1985). The presence of both monomers and dimers, and rapid exchange of Cd and Zn among sites (Otvos et al., 1985; Nettesheim et al., 1985) is apparent as heterogeneity in n.m.r. spectra of reconstituted MTs (Vasak et aE., 1985). Metal exchange also occurs with Cd and Hg between pairs of MT molecules (Johnson & Armitage, 1987). The implication is that MT molecules may exchange metals via direct intermolecular contacts. Two aspects of the crystal structure of Cd,,Zn,-MT support the idea that MT could exist as a dimer in solution. First, the crystal packing contains intimately associated pairs of molecules related by the S-fold axes, and second, cations are trapped between these Z-fold related MT molecules (2 per “dimer”). Together these interactions could stabilize a MT dimer in solution. (f ) Sequence comparisons The highly conserved sequences of MTs (Kagi & SchLffer, 1988) suggest that other MTs are isostructural with rat liver MT, as indeed is shown by the solution structures of rabbit, rat and human MT (Arseniev et al., 1988; Schultze et al., 1988; Messerle et al., 1990). The sequence of crab MT is uniquely different, however, having nine cysteine residues in each domain (Lerch et aE., 1982). The ‘13Cd n.m.r. structure of crab MT shows that each domain contains a three-metal cluster (Otvos et al., 1982). By comparison to the rat liver structure, crab MT has a CXXC peptide at the N terminus of its c1domain rather than CCXCC and also lacks C48, suggesting that the Cd5 site is lost (Fig. 13(a)) to make a thee-metal cluster. Sequence analysis with respect to sea urchin MT (Nemer et al., 1985) has shown that a “central segment” of CXCXXXCXC is conserved in all classes of MTs (with the exception of crab and yeast MTs). This sequence is C13SC15AGSClSKC21 in the structure, suggesting that the unique 4, cp angles of residue 16 are required for proper disposition of CXC moieties in the folding of B domains. All MT sequences possess either a CXC or CC sequence, or both. Based on three occurrences, CC sequences are predicted to bind the same metal (Fig.

13(a)). CXC sequences are

predicted to bind the same metal or the same cluster, based on the restricted stereochemistry observed in the crystal structure. We are indebted

protein, K. Wiithrich

to D. R. Winge for samples of the for co-ordinates of the n.m.r. model!

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