www.sciencemag.org/cgi/content/full/1179907/DC1

Supporting Online Material for

Tetrathiomolybdate Inhibits Copper Trafficking Proteins Through Metal Cluster Formation

Hamsell M. Alvarez, Yi Xue, Chandler D. Robinson, Mónica A. Canalizo-Hernández, Rebecca G. Marvin, Rebekah A. Kelly, Alfonso Mondragón, James E. Penner-Hahn,

Thomas V. O’Halloran*

*To whom correspondence should be addressed. E-mail: t-ohalloran@northwestern.edu

Published 26 November 2009 on Science Express

DOI: 10.1126/science.1179907 This PDF file includes

Materials and Methods Figs. S1 to S16 Tables S1 to S4 References

Other Supporting Online Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/1179907/DC1)

Movie S1

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Materials and Methods:

TM Characterization and Characterization. TM was synthesized as described byMellor (34) and Laurie (35) (yield ~ 87%), and characterized by 95Mo-NMR (400MHz, D2O): δ 2260 p.p.m., using a 2 M Na2[MoO4] solution (in D2O, pH 11) as anexternal reference and a 90° pulse of 50 µs; IR (KBr, cm-1): 3125 (sh), 2360 (w),2336, (w), 1384 (m), 835 (w); and UV/Vis [H2O, λmax (nm), ε x 10-3 (M-1 cm-1): 241(24.7), 317 (16.7), 468 (11.8). Analysis (% calcd, % found for H8N2S4Mo): H (3.10,2.99), N (10.76, 10.80), S (49.28, 48.91), Mo (36.86, 36.78%).

Protein Preparation and Purification. The WT-Atx1, Ccc2a, and WT-Atox1protein were overexpressed, purified and metallated as previously described(20,21,25).

[TM][(Cu)(Cu-Atx1)3] Synthesis. The complex was prepared by mixing ca. 670 µlof 3.0 mM Cu-Atx1 or Cu-Atx1(SeMet) plus TM (1:0.4) (trxn = 30 min, aerobicconditions, T = 4°C) (20 mM MES, 150 mM NaCl, pH 6.0), and isolated using aSuperdex 75 HR 10/30 column and an AKTA FPLC (0.3 mL/min, 280 nm) (GE,Pisctaway, NJ) (fig. S14). Fractions corresponding to three different peaks werecollected (2 mL each) and concentrated to approximately 100 µL using YM3microcon ultrafiltration (fig. S16, inset). Fractions that correspond to the first peakshowed an approximate molecular weight of 10 kDa ([Atx1] = 0.65 mM, [Cu] = 0.36mM and no [Mo]; [Cu]:[Atx1] = 0.55), the second peak, 29 kDa (color: purple, [Atx1]= 3.1 mM, [Cu] = 3.2 mM and [Mo] = 0.95 mM; [Cu]:[Mo] = 3.4, [Cu]:[Atx1] = 1.0,[Atx1]:[Mo] = 3.3), and the third peak, 52 kDa (color: brown, [Atx1] = 1.3 mM, [Cu]= 1.1 mM, [Mo] = 0.29 mM; [Cu]:[Mo] = 3.8, [Cu]:[Atx1] = 0.85, [Atx1]:[Mo] =4.5). Metal and protein concentrations were determined by ICP-OES and Bradfordassay (correction factor = 0.54), respectively. Reactions carried out with Cu-Atox1gave similar products. The concentrated deep purple solution (peak # 2) representedthe [TM][(Cu)(Cu-Atx1)3], which was used further for crystallizations.

Atx1(SeMet) Preparation and Purification. A starting 250 mL culture of E. colicontaining WT-Atx1 overexpressing vector was prepared as previously described(20). A dilution of this culture was used to grow 15 L of E. coli in SeMet MM(minimal medium) (36) using a BIOFLO 4500 fermentor (New Brunswick Scientific,Edison, NJ). Atx1(SeMet) was purified and metallated following a previous protocol(20). MALDI-TOF-MS revealed a mass of m/zobs = 8136.0 Da, which is consistentwith m/zcalc = 8136.3 Da.

Crystallization and X-ray Studies. Purple crystals of the [TM][(Cu)(Cu-Atx1)3]complex were grown at 14°C by vapor diffusion equilibrated over 0.15 M DL-Malicacid pH 7.0 plus 20% polyethylene glycol 3350. Crystals reached maximumdimensions of 0.15 x 0.06 x 0.04 mm after 1 week. Before data collection, crystalswere soaked in the crystallization solution supplemented with 25% glycerol for 3minutes and flash-cooled in N2(l). The native (resolution = 2.3 Å) and Cu-MAD(resolution = 2.7 Å) data were collected at the SBC-CAT and IMCA-CAT beamlinesat the APS (Argonne National Laboratory), and processed using Denzo and Scalepack(37). A summary of the data-collection statistics is given in table S3. The crystals

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belong to spacegroup P21212. An initial Molecular Replacement solution wasobtained with Phaser (38) using monomeric Atx1 as the search model (PDB: 1CC8)(24). Twelve monomers of Atx1 were found arranged in 4 trimeric groups with alarge density feature (Fo-Fc map) on the three-fold axis and linking three proteinmonomers (fig. S15a). The MR model was not refined, but instead used to locate theCu atoms for subsequent MAD phasing (39). An anomalous difference map usingphases from the Molecular Replacement model clearly revealed 16 peaks with apattern of four peaks clustered together and overlapping the density feature at thethree-fold axis (fig. S15b). The 16 Cu sites positions were imported intoSharp/autoSharp (40) for heavy atom refinement, MAD phasing, and densitymodification. The MR model was rebuilt into the experimental electron density mapfollowed by restrained positional and temperature factor refinement with REFMAC5(41). After model building, refinement and Cu sites assignment, a clear tetrahedrallyshaped density was perfectly modelled by a [MoS4] moiety (fig. S15c). In order tofind the best refinement target values for the Cu-S, and Cu-Mo distances, twoprocedures were followed: 1) the target restraint bond lengths for the intramolecularCuI–S, CuI–Mo, and CuI-protein CuI–Sγ bonds were varied systematically using targetvalues between 2.2 Å – 2.5 Å, 2.6 Å – 3.0 Å, and 2.2 Å – 2.5 Å for the CuI–S,CuI–Mo, and CuI–Sγ bonds respectively, with a large (0.2 Å) target standarddeviation. This procedure allowed the atoms to move without much restraint. Themean bond lengths after this procedure were 2.296 Å (σ = 0.060 Å, n = 3000), 2.781(σ = 0.030 Å, n = 1500) Å and 2.283 Å (σ = 0.094 Å, n = 4500). 2) The targetrestraint distances for the same bond lengths were systematically varied butemploying a small (0.02Å) target standard deviation. After refinement, the set of bondlengths that produced the lowest Rfree was selected. The mean distances after thisprocedure were 2.303 Å (σ = 0.037 Å, n = 24), 2.786 Å (σ = 0.021 Å, n = 12), and2.321 Å (σ = 0.052 Å, n = 36). The two sets of bond lengths agree well and values of2.28 Å, 2.78 Å, and 2.28 Å were then selected as target restraint values in therefinement procedure with a standard deviation of 0.02 Å. TLS refinement (with eachtrimer as one TLS group) resulted in an additional 3% decrease in both R and Rfree. Asummary of the geometry parameters with standard deviations is listed in table S4.The structure was validated with MOLPROBITY (42) and the Ramachandran plot(fig. S16) indicates that all residues are in allowed regions and 98.3 % in favouredregions. The final model has an Rwork and Rfree of 20.2%/25.6% respectively. Theestimated overall coordinate uncertainty based on the Rfree is 0.24 Å (41). Modelbuilding was performed with Coot (43). All protein diagrams were generated usingPymol (44).

Native Gel Electrophoresis. A 0.8% agarose gel (8 cm x 5.5 cm x 3 cm) wasimmersed in an electrophoresis box containing native gel buffer (25 mM Tris-Cl pH8.5, 19.2 mM glycine buffer), placing the comb at the middle of the gel, and runningit at 50 V for 2 h. Protein mixtures (5 µg of each protein) were mixed with loadingbuffer (1:1) (20% glycerol, 0.2 % bromophenol blue, 0.12 M Tris base) (Vt = 10 µl)before being loaded in the gel. Gel was stained in Coomassie blue for 20 minutes anddried by sandwiching it between two pieces of cellophane and filter paper for 48 hwith weight compression (45). For protein gel extraction, four identical samples ofeach protein or mixture (5 µg each protein) were loaded on contiguous lanes. One of

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the four lanes was stained to identify the protein bands, and the unstained ones werecut out with a sharp glass. The isolated bands were transferred into an Ultrafree-DAunit (Millipore, Bedford, MA), and centrifuged at 5000 g for 10 min at 4°C. Proteinsamples in the eluate were further concentrated to a Vf ~ 30 µl by centrifugation at14000 g using YM3 microcon ultrafiltration (Millipore, Bedford, MA).

ESI-MS, ICP-MS and LA-ICP-MS. Results shown for native gel electrophoresis,ESI-MS, ICP-MS, and LA-ICP-MS experiments were performed with Atx1, wherethe Met is replace by a SeMet. Similar results were obtained using a native Atx1 (datanot shown). Concentrated protein samples were analyzed by ESI-MS using a FiniganLTQ-FT LC/MS/MS (m/z ± 0.8) (Thermo Scientific, Minneapolis, MN). The ESI-MS was previously calibrated with a Thermo Scientific calibration mixture (caffeicacid, peptide MRFA and ultramak). The 30 µl samples were injected in a PoroshellC3 0.2 x 75 mm reverse phase HPLC column (Agilent Technologies, Santa Clara,CA) and eluted using a mixture of 0.1% formic acid and 95% Milli-Q H2O and agradient of 35-85% methanol. The metal concentrations were analyzed by a ThermoFischer X Series II ICP-MS (Thermo Scientific, Minneapolis, MN) and a UP 213Nd:YAG LA (New Wave Research, Fremont, CA). For ICP-MS, concentrated proteinsamples were diluted with Milli-Q-water (Vf = 3 ml, 2% nitric acid), including anICP-MS internal standard (CPI International, Santa Rosa, CA). For gel analysis bylaser ablation coupled with ICP-MS (46), the gel (fig. S6) was first dried for 48 hbetween two cellophane sheets to avoid cracks, cut in lanes, and mounted onmicroscope slides. The laser (0.43 mJ, 45 %) was set to initiate a single line scanstarting at the top of each of the lanes of the gel with a scanning speed of 50 µm s-1,fluence of 5.44 J/cm2, a pulse repetition frequency of 10 Hz and a 100 µm holediameter. Helium gas was used as a carrier gas. Pre-ablation of each lane wasperformed at a reduced energy (20%). Tuning of the LA-ICP-MS integrated systemwas performed with a SRM 612 glass standard (NIST, Gaithersburg, MD). Glassstandard calibration resulted in an instrumental mass bias correction factor of 5.8e-4for Cu and 4.9e-4 for Mo.

Preparation of XAS samples. A sample of [TM][(Cu)(Cu-Atx1)3] was prepared forXAS analysis following the same protocol described before. Glycerol was added to afinal concentration of 30%. Samples were transferred to Lucite cuvettes with 40 µmKapton windows and frozen in liquid N2 at – 20°C and stored at – 80°C until XASmeasurements were made. Preparation and X-ray absorption data collection (Cu andMo K-edge EXAFS) of the kidney sample used for the Fourier transforms phase shiftoverlay (Fig. 3B) was previously described (29).

X-ray absorption data collection and analysis. The Cu X-ray absorption K-nearedge structure (XANES) and extended X-ray absorption fine structure (EXAFS) datawere collected at the Stanford Synchrotron Radiation Laboratory (SSRL) on beamline9-3 (3 GeV, ~ 90 mA), using a a Si(220) double-crystal monochromator with a Rh-coated mirror upstream for harmonic rejection. Incident intensity was measured usingan N2-filled ion chamber. Fluorescence excitation spectra were collected using a 30-element Ge solid-state detector array. The sample temperature was held at 12 K in anOxford liquid helium flow cryostat during the measurements. The total integrated

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count rate was held below 90 kHz to avoid saturating the detector, and the averagewindowed fluorescence count rate was ~ 7-12 KHz per channel in the EXAFS region.Each scan had 10 eV increments prior to the edge (8780 - 8970 eV), 0.5 eVincrements for the pre-edge and edge region (8970 - 9020 eV), 0.05 Å-1 incrementsfor the EXAFS region (2.29 – 13.1 Å-1) with integration times of 1 s in the pre-edgeand edge regions and 1-10 s (k3 weighted) in the EXAFS region for a total scan timeof ~ 35 minutes. Energy calibration was done using a Cu(s) foil as an internalstandard, with the first inflection point of the foil defined as 8980.3 eV. Each detectorchannel was checked for glitches and all good channels [~ 20-24] were averagedusing 4 scans. XANES data were normalized by fitting the data to the McMasterabsorption coefficients below and above the edge using a single backgroundpolynomial and scale factor (47,48). The EXAFS background correction wasperformed by fitting a three-region quartic spline for the copper sample. Fouriertransforms were calculated using k3 weighted data over a range of 1.13 – 13 Å-1 forthe copper sample. The data were then converted to k-space using

2

0)(2h

EEmk e −= where E0 = 9000 eV.

EXAFS data can be described by following equation, where χ(k) is the fractionalmodulation in the absorption coefficient above the edge, Ns is the number of scatterersat

))(2sin(expexp)()()()2()2(

2

22

kkRkR

kAkSNk asas

Rask

sas

sssas φχ λσ +=

−−∑

a distance Ras, As(k) is the backscattering amplitude, σas2 is the root-mean-square

variation in Ras, φas(k) is the phase shift experienced by the photoelectron wave inpassing through the potentials of the absorbing and backscattering atoms, λ is themean free path of the photoelectron and backscattering atoms, and Ss(k) is a scalefactor specific to the absorber-scattering pair and the sum is taken over all scatteringinteractions (49). The program FEFF v7.02 (50) was used to calculate amplitude andphase functions, As(k)exp(-2Ras/λ) and φas(k) for a copper-nitrogen interaction at 2.10Å, a copper-sulfur interaction at 2.26 Å, a copper-molybdenum interaction at 2.70 Åand a copper-copper interaction at 5.40 Å. E0 was initially set at 9000 eV and Ss, wasfixed at 0.89. Mo K-edge spectra were measured as above with an average windowedfluorescence count rate of ~ 2-7 KHz per channel in the EXAFS region. Each scanhad 10 eV increments prior to the edge (19788 - 19988 eV), 0.3 eV increments in theedge region (19989 - 20030 eV), 0.05 Å-1 increments for the EXAFS region (1.62 –16.1 Å-1) with integration times of 1 s in the pre-edge and edge regions and 1-20 s (k3

weighted) in the EXAFS region for a total scan time of ~ 26 minutes. Energycalibration was done using a Mo(s) foil as an internal standard, with the firstinflection point of the foil defined as 20003 eV. Each detector channel was checkedfor glitches and all good channels [25] were averaged using 6 scans. XANES and

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EXAFS analysis was as described above, with the exception that Fourier transformswere calculated over a range of 1-15.5 Å-1. FEFF calculations were performed for amolybdenum-sulfur interaction at 2.21 Å, and a molybdenum-copper interaction at2.70 Å. E0 was initially set at 20020 eV and Ss, was fixed at 0.9.

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Figures:

Fig. S1. Crystals of [TM][(Cu)(Cu-Atx1)3]. The crystals have approximatedimensions of 0.15 mm x 0.06 mm x 0.04 mm.

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Fig. S2. Overall structure of [TM][(Cu)(Cu-Atx1)3]: In the crystal asymmetric unit,12 Atx1 monomers are clustered in two hexameric groups, each hexamer is composedof two parallel layers of [TM][(Cu)(Cu-Atx1)3] trimers. Each trimer is related by a 3-fold non-crystallographic symmetry axis (arrows) and all three monomers in a trimerare colored identically. Atx1 monomers are shown in cartoon ribbon diagram. TheTM groups are represented as ball-and-stick, with the Mo atoms shown as cyanspheres, the sulfurs atoms shown as yellow spheres, and the Cu atoms shown as bluespheres.

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Fig. S3. Cu and Mo x-ray absorption K near-edge structure (XANES) spectra of[TM][(Cu)(Cu-Atx1)3]. (a) The Cu XANES spectrum does not exhibit the weak 1s →3d transition at 8979 eV that is characteristic of CuII, and has relatively low edgeenergy. Both observations are consistent with most of the Cu being present as CuI

although it is difficult to exclude the presence of some CuII (51). The Cu XANESspectrum lacks the distinct “1s → 4p” feature at ~ 8984 eV that is typical of digonalCuI, and to a lesser extent trigonal CuI, suggesting that the copper sites are at leastpartially four coordinate. (b) The Mo XANES strongly resembles that oftetrathiomolybdate (52) suggesting that the Mo in the [TM][(Cu)(Cu-Atx1)3] complexis four coordinate, has an oxidation state of 6+, and is not significantly different fromfree TM. The shoulder at 20006 eV attributed to the formally forbidden 1s → 4ptransition characteristic of a Mo–S in a tetrahedral environment (52) is slightly weakerin the complex than in TM, which may indicate some distortion in geometry from anideal tetrahedron. The XANES studies of the [CuSMo] cluster from the[TM][(Cu)(Cu-Atx1)3] agrees with the predicted structure by protein X-raycrystallography.

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Fig. S4. Interactions between the protein and the [Cu4MoS4] cluster. The proteininteracts with the cluster through a hydrogen bond network formed by Lys65, Thr14and Gly17 and through the positive α helix 2 dipole. These interactions help in theneutralization of the negatively charged [Cu4MoS4]4- cluster via the sulfides of TM,and the thiolates of Cys 18 and Cys 15 (Cu atom = blue sphere, S atom = yellowsphere and Mo atom = cyan sphere, hydrogen bond = yellow dashed line, and Cu–Sbond = green dashed line). The hydrogen bond network presents some similarities anddifferences to the ones observed for the Hg-Atx1 (24) and Cu-Atox1 (25). Forexample, very similar hydrogen bonds were found between the thiolates of the metalbinding site (Atx1: Cys15 and Cys 18; Atox1: Cys12 and Cys 15) and the backboneamides (Atx1: Thr14 and Gly17; Atox1: Thr11 and Gly14); but in contrast theCys18–SH–Lys65 hydrogen bond in the Cu-Atx1 monomer is shorter than the ones inHg-Atx1 (3.3 Å vs. 3.9 Å) and Atox1 (3.3 Å vs. 3.8/ 3.6 Å). All distances shownrepresent the average distances found in the 4 clusters.

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Fig. S5. Small inorganic analogue of the cluster from the [TM][(Cu)(Cu-Atx1)3]. (a)Overall x-ray structure of the [Cu12Mo8S32]4- anion from the [Bun

4N]4[Cu12Mo8S32]complex (27). (b ) Structure of the extracted [S6Cu3MoS4] cluster from the[Bun

4N]4[Cu12Mo8S32] complex (Cu atom = blue sphere, S atom = yellow sphere, andMo atom = cyan sphere). The structure of [S6Cu3MoS4] cluster from the[Bun

4N]4[Cu12Mo8S32] complex is identical to the [S6Cu4MoS4] cluster from the[TM][(Cu)(Cu-Atx1)3], except for the absence of the trigonally coordinated Cu atom,however the [S6Cu3MoS4] cluster already posses the right geometry to fit the extra Cuatom.

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Fig. S6. Native agarose gel electrophoresis with analysis of protein-protein complexformation. A: apo-Atx1(SeMet) (band C1 and C2), B: Cu-Atx1(SeMet) (band C3), C:apo-Ccc2a (band C4), D: Cu-Ccc2a (band C5), I: [TM][(Cu)(Cu-Atx1(SeMet))3](band 1), II: Cu-Atx1(SeMet) + apo-Ccc2a (1:1) (band 2, 3 and 4), and III:[TM][(Cu)(Cu-Atx1(SeMet))3] + apo-Ccc2a (1:1) (band 5, 6, 7 and 8). Atx1 proteinand its complex with TM migrated to the cathode due to their basic character(positively charged, pI = 8.64), while Ccc2a protein migrated to the anode due to theiracidic character (negatively charged, pI = 4.42). Results shown for native gelelectrophoresis (also for ESI-MS, ICP-MS, and LA-ICP-MS) were performed withAtx1, where the Met is replace by a SeMet. Similar results were obtained using anative Atx1 (data not shown).

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Fig. S7. ESI-MS spectra of bands C1, C2, and C3. (a) ESI-MS spectrum of band C1(∗) [apo-Atx1(SeMet): m/zcalc = 8136.4 (red), m/zcalc = 8134.4 (oxid), m/zobs =8134.2], (∗) [apo-Atx1(SeMet) + H2O: m/zcalc = 8154.4, m/zobs = 8150.5], (∗) [apo-Atx1(SeMet) + HCOOH: m/zcalc = 8178.4, m/zobs = 8179.0]. (b) ESI-MS spectrum ofband C2 (∗) [apo-Atx1(SeMet): m/zcalc = 8136.4 (red), m/zcalc = 8134.4 (oxid), m/zobs

= 8133.2]. (c) ESI-MS spectrum of band C3 (∗) [apo-Atx1: m/zcalc = 8089.5 (red),m/zcalc = 8087.5 (oxid), m/zobs = 8086.3, apo-Atx1(SeMet): m/zcalc = 8136.4 (red),m/zcalc = 8134.4 (oxid), m/zobs = 8134.3, Cu-Atx1: m/zcalc = 8151.0, m/zobs = 8150.3,Cu-Atx1(SeMet): m/zcalc = 8197.9, m/zobs = 8196.2] (apo-Atx1(SeMet) also shows alow proportion of apo-Atx1 due that the % of incorporation of SeMet in the apo-Atx1is high (~90%) but not complete), (∗) [apo-Atx1(SeMet) + MeOH: m/zcalc = 8168.4,m/zobs = 8169.0], (∗) [Cu-Atx1(SeMet) + HCOOH: m/zcalc = 8243.9, m/zobs = 8248.9].

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Fig. S8. ESI-MS spectra of bands C4, C5 and 1. (a) ESI-MS spectrum of band C4 (∗)[apo-Cc2a: m/zcalc = 7882.9 (red), m/zcalc = 7880.9 (oxid), m/zobs = 7891.7] (Thedifference of approximately 9 units between m/zcalc and m/zobs for apo-Ccc2a can beattributed to protonation caused by the mixture of solvents used for our experiment,apo-Ccc2a + 9H+: m/zcalc = 7891.9, m/zobs = 7891.7), (∗) [apo-Ccc2a with oxidationof arginine to glutamic acid (53,54) : m/zcalc = 7864.8, m/zobs = 7863.7], (∗) [apo-Ccc2a + Na: m/zcalc = 7914.9, m/zobs = 7913.7]. (b) ESI-MS spectrum of band C5 (∗)[apo-Cc2a: m/zcalc = 7882.9 (red), m/zcalc = 7880.9 (oxid), m/zobs = 7891.7, Cu-Ccc2a:m/zcalc = 7944.5, m/zobs = 7948.7], (∗) [apo-Ccc2a with oxidation of arginine toglutamic acid: m/zcalc = 7864.8, m/zobs = 7863.7], (∗) [apo-Ccc2a + H2O: m/zcalc =7909.9, m/zobs = 7908.7], (∗) apo-Ccc2a + MeOH: m/zcalc = 7923.9, m/zobs = 7925.0].(c) ESI-MS spectrum of band 1 (∗) [apo-Atx1: m/zcalc = 8089.5 (red), m/zcalc = 8087.5(oxid), m/zobs = 8087.3, apo-Atx1(SeMet): m/zcalc = 8136.4 (red), m/zcalc = 8134.4(oxid), m/zobs = 8134.2, Cu-Atx1: m/zcalc = 8151.0, m/zobs = 8157.2, Cu-Atx1(SeMet):m/zcalc = 8197.9, m/zobs = 8197.2], (∗) [Cu-Atx1(SeMet) + HCOOH: m/zcalc = 8243.9,m/zobs = 8248.0].

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Fig. S9. Control native gel lanes with corresponding LA-ICP-MS scans for Cu. Gellanes (A, B, C, and D) were cut from gel (fig. S6). (A) apo-Atx1(SeMet) (band C1and C2), (B) Cu-Atx1(SeMet) (band C3), (C) apo-Ccc2a (band C4), and (D) Cu-Ccc2a (band C5) (LA-ICP-MS scans are represented by the intensity (CPS: counts persecond) of 65Cu (x-axis), and length of the gel (mm) (y-axis), ↔: protein band length,↔: protein loading well).

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Fig. S10. ESI-MS spectra of bands 2, 3 and 4. (a) ESI-MS spectrum of band 2 (∗)[apo-Atx1: m/zcalc = 8089.5 (red), m/zcalc = 8087.5 (oxid) m/zobs = 8086.3, apo-Atx1(SeMet): m/zcalc = 8136.4 (red), m/zcalc = 8134.4 (oxid), m/zobs = 8133.2]. (b)ESI-MS spectrum of band 3 (∗) (RT: 11.38 min) [apo-Cc2a: m/zcalc = 7882.9 (red),m/zcalc = 7880.9 (oxid), m/zobs = 7891.7]. (c) ESI-MS spectrum of band 3 (∗) (RT:12.48 min) [apo-Atx1: m/zcalc = 8089.5 (red), m/zcalc = 8087.5 (oxid) m/zobs = 8087.3,apo-Atx1(SeMet): m/zcalc = 8136.4 (red), m/zcalc = 8134.4 (oxid), m/zobs = 8134.2]. (d)ESI-MS spectrum of band 4 (∗) [apo-Cc2a: m/zcalc = 7890.0 (red), m/zcalc = 7888.0(oxid), m/zobs = 7891.7, Cu-Ccc2a: m/zcalc = 7944.5, m/zobs = 7948.7], (∗) [apo-Ccc2a+ H2O: m/zcalc = 7909.9, m/zobs = 7908.7].

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Fig. S11. ESI-MS spectra of bands 5 and 6. (a) ESI-MS spectrum of band 5 (∗) [apo-Atx1: m/zcalc = 8089.5 (red), m/zcalc = 8087.5 (oxid) m/zobs = 8086.3, apo -Atx1(SeMet): m/zcalc = 8136.4 (red), m/zcalc = 8134.4 (oxid), m/zobs = 8133.2, Cu-Atx1: m/zcalc = 8151.0, m/zobs = 8151.2]. (b) ESI-MS spectrum of band 6 (∗) (RT:10.08 min) [apo-Cc2a: m/zcalc = 7882.9 (red), m/zcalc = 7880.9 (oxid), m/zobs = 7892.7,Cu-Ccc2a: m/zcalc = 7944.5, m/zobs = 7947.7]. (c) ESI-MS spectrum of band 6 (∗)(RT: 11.98 min) [apo-Atx1: m/zcalc = 8089.5 (red), m/zcalc = 8087.5 (oxid) m/zobs =8087.3, apo-Atx1(SeMet): m/zcalc = 8136.4 (red), m/zcalc = 8134.4 (oxid), m/zobs =8133.2].

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Fig. S12. ESI-MS spectra of bands 7 and 8. (a) ESI-MS spectrum of band 7: Atx1 (∗)+ Ccc2a (∗) (RT: 12.01) [apo-Cc2a: m/zcalc = 7882.9 (red), m/zcalc = 7880.9 (oxid),m/zobs = 7892.7; apo-Atx1: m/zcalc = 8089.5 (red), m/zcalc = 8087.5 (oxid), m/zobs =8086.3, apo-Atx1(SeMet): m/zcalc = 8136.4 (red), m/zcalc = 8134.4 (oxid), m/zobs =8134.2]. (b) ESI-MS spectrum of band 8 (∗) [apo-Cc2a: m/zcalc = 7882.9 (red), m/zcalc

= 7880.9 (oxid), m/zobs = 7891.7, Cu-Ccc2a: m/zcalc = 7944.5, m/zobs = 7950.7], (∗)[apo-Ccc2a with oxidation of arginine to glutamic acid: m/zcalc = 7864.8, m/zobs =7863.7], (∗) [apo-Ccc2a + H2O: m/zcalc = 7909.9, m/zobs = 7908.0], (∗) [apo-Ccc2a +Na: m/zcalc = 7914.9, m/zobs = 7913.7], (∗) [apo-Ccc2a + MeOH: m/zcalc = 7923.9,m/zobs = 7923.7].

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Fig. S13. Cu and Mo K-edge extended x-ray absorption fine structure (EXAFS)spectra and Fourier transforms of [TM][(Cu)(Cu-Atx1)3]. (a ) Cu K-edge EXAFSFourier transform shows a single, intense peak at ~ 2.0 Å that is best modeled with ashell of 3 S ligands (3 Cu—S) at 2.30 Å with σ2 = 0.0042, and a weak feature athigher R value that can be modeled by a single CuMo interaction (1 CuMo) at2.76 Å with σ2 = 0.0063. (b) Mo K-edge EXAFS Fourier transform data has anintense peak at ~ 2.0 Å that is best modeled with a shell of 4 S ligands (4 Mo—S) at2.24 Å with σ2 = 0.0033, and a second peak at ~ 2.4 Å that is best modeled with asecond shell of 2 Cu scatterers (2 MoCu) at 2.76 Å with σ2 = 0.0027. Models withtwo or three MoCu interactions gave essentially the same quality of fit, reflectingthe difficulty in determining the coordination number from EXAFS alone (data for amodel with 3 MoCu is not shown). Thus, the EXAFS analysis indicated that bothmetals have primarily sulfur ligands, and that each molybdenum has approximatelytwo or three copper neighbors, while every copper has approximately onemolybdenum neighbor. The EXAFS studies of the [CuSMo] cluster from the[TM][(Cu)(Cu-Atx1)3] agrees with the predicted structure by protein X-raycrystallography, with exception of the detection of the fourth additional copper. Thesolid lines show experimental data, while the broken lines show the best fits. Inset: Cuand Mo K-edge EXAFS spectra for the [TM][(Cu)(Cu-Atx1)3] complex.

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Fig. S14. Analytical gel filtration chromatogram for purification of [TM][(Cu)(Cu-protein)3] [protein: Atx1 (black) & Atox1 (red)]. Analytical gel filtrationchromatograms of 3.0 mM Cu-Atx1/Cu-Atox1 + TM (1:0.4). Inset: Concentratedsolution of [TM][(Cu)(Cu-Atx1)3] after purification by analytical gel filtration andconcentration by ultrafiltration. The oligomeric complex shown above maycorrespond to the hexamer, given the crystal packing described before wherein twotrimers associate to form a hexamer.

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Fig. S15. (a) Fo-Fc map of [TM][(Cu)(Cu-Atx1)3] calculated using the modelobtained by Molecular Replacement . The map clearly exhibits a density feature at theinterface of each trimer, indicating the positions of the Mo-Cu cluster. The Atx1monomers are shown in ribbon diagram and each trimer is colored identically. TheFo-Fc map is contoured at the 3σ level. (b) Anomalous difference Fourier mapcalculated using phases from the Molecular Replacement solution and the anomalousdata collected at 1.3799 Å (Cu peak data set). The map peaks reveal the Cu sites ineach cluster. There are 4 clusters for a total of 16 Cu peaks. Only one group is shownin the figure. The map was contoured at the 4σ (yellow) density and 10σ (blue)density levels. (c) Fo-Fc map of the cluster after building in the Cu atoms. Thetetrahedrally shaped density clearly reveals the position of the TM group. The map iscontoured at the 4σ level.

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Fig. S16. Ramachandran Plot of [TM][(Cu)(Cu-Atx1)3]. The regions in the blue lineindicate the generally allowed areas (100% of residues), while the regions in the aquablue indicate the favoured regions (98.3% of residues).

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Tables:

Table S1. Stoichiometry of Cu and Mo from protein gel extracts (µmol)

*NCu & †NMo represent the number of µmols of Cu & Mo before and after protein gel extraction andICP-MS. ‡Percentage of metal recovery after mass balance.

Band Protein Initial Final % Rec.‡

# (ESI-MS) NCu* NMo

† Cu/Mo NCu* NMo

† Cu/Mo Cu Mo

C1 Atx1 --- --- --- --- --- --- --- ---

C2 Atx1 --- --- --- --- --- --- --- ---

C3 Atx1 2.04 x 10-3 --- --- 4.53 x 10-4 --- --- 22.2 ---

C4 Ccc2a --- --- --- --- --- --- --- ---

C5 Ccc2a 2.24 x 10-3 --- --- 3.78 x 10-4 --- --- 16.9 ---

1 Atx1 2.57 x 10-3 6.80 x 10-4 3.8 1.63 x 10-3 4.41 x 10-4 3.6 63.4 64.9

2 Atx1 --- --- ---

3 Atx1/Ccc2a 2.04 x 10-3 --- --- 1.19 x 10-4 --- --- 13.5 ---

4 Ccc2a 1.57 x 10-4 --- ---

5 Atx1 --- --- ---

6 Atx1/Ccc2a 2.57 x 10-3 6.80 x 10-4 3.8 2.13 x 10-5 --- --- 35.4 41.2

7 Atx1/Ccc2a 8.89 x 10-4 2.80 x 10-4 3.1

8 Ccc2a --- --- ---

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Table S2. Results of Cu and Mo EXAFS Curve Fitting for [TM][(Cu)(Cu-Atx1)3]

CN* R† σ2‡ CN* R† σ2‡ F§

Cu 4S 2.31 6.3 0.338

3S 2.30 4.3 0.366

3S 2.30 4.2 1Mo 2.76 6.3 0.268

Mo 4S 2.24 3.3 2.512

4S 2.24 3.4 1Cu 2.76 4.7 1.064

4S 2.24 3.3 2Cu 2.76 2.7 0.839

*Integer coordination number. †Bond length in Å. ‡Debye-Waller factor x 103 in Å2. §Mean-square-deviation between the K3-weighted data and fit divided by (Nidp – Nvar) , where Nvar is the number ofvariables in the fit. F values provide a partial correction for the fact that more parameters invariablygive better fits (55).

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Table S3. Data collection and refinement statistics

Data collection Cu-MAD

Data set Native Peak InflectionLow

remote Beamline* 19-BM 17-ID-B 17-ID-B 17-ID-B Wavelength (Å) 0.9787 1.3799 1.3805 1.3850

Resolution limits (Å) 50.0 - 2.30 50.0 - 2.30 50.0 -2.5050.0 -2.72

Completeness (%)† 97.0 (84.0) 94.3 (64.0)99.1

(92.4)99.7

(98.5) Data redundancy† 5.0 (2.9) 3.3 (1.4) 3.6 (2.0) 3.7 (3.0) Rsym (%)†,‡ 8.1 (47.0) 8.3 (34.2) 6.9 (51.6) 6.8 (54.0) I/<σ>† 16.7 (2.0) 11.9 (1.7) 15.5 (1.6) 17.1 (2.1)

Space group P21212

Unit-cell parameters (Å, °)109.9 182.2

52.790.0 90.0

90.0

Phasing Statistics Peak InflectionLow

remote Phasing Power (iso)§ 0.44 1.42 N/A Phasing Power (ano)§ 1.51 0.86 0.17 Cu sites found 14 FOM after SHARP 0.33 FOM after DM 0.82

Refinement Statistics

Rwork /Rfree (%)†,20.2 (27.0) /25.6 (33.0)

R.m.s. bond lengths (Å) 0.020 R.m.s. bond angles (°) 1.758 Protein atoms 6746 No. of Clusters 4 Mo 4 S 16 Cu 16 No. of water molecules 372 No. of other molecules (malic acid) 18

Residues in Ramachandran plot regions (%)¶

Allowed 100.0 Most favoured 98.3 Disallowed 0.0

* All data sets were collected at 100 K at the Advance Photon Source. † Values in parentheses are forthe highest resolution shell. ‡Rsym = _|Iobs - Iavg|/ _Iobs.

§ The phasing power is defined as the ratio of therms value of heavy atom structure factor amplitudes to the rms value of the lack-of-closureerror.Rvalues = _|Fobs - Fcalc| / _Fobs, 5% of the reflections were reserved for the calculation of Rfree¶The validation was performed with program MOLPROBITY42

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Table S4. Geometry of [S6Cu4MoS4] cluster

Tetrahedral Mo Values Mean No. of Obs. S.D.‡

Mo-S 2.18-2.26 Å 2.22 Å 16 0.02 Å

S-Mo-S 103~116º 109º 24 4º

Tetrahedral Cu

Cu-S 2.21-2.44 Å 2.30 Å 48 0.05 Å

S-Cu-S 97-124º 109º 72 7º

Trigonal Cu

Cu-S 2.22-2.30 Å 2.26 Å 12 0.02 Å

S-Cu-S 113-126º 119º 12 4º

Metal-metal distances

Mo-Cu/4* 2.74-2.82 Å 2.77 Å 12 0.02 Å

Mo-Cu/3† 3.84-3.96 Å 3.92 Å 4 0.05 Å

Cu/4-Cu/4 3.80-3.90 Å 3.85 Å 12 0.04 Å

Cu/4-Cu/3 3.06-3.24 Å 3.18 Å 12 0.05 Å

*Cu/4 represents tetrahedrally-coordinated copper. †Cu/3 represents trigonally-coordinated copper. ‡Byconvention, the S.D. values for bond lengths and angles correspond to deviations from target values inthe refinement process. Given the estimated error of ~ 0.24 Å in the coordinates (see page S-3), theuncertainty in bonds and angles is greater.

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41. “The CCP4 suite: programs for protein crystallography”, Acta Cryst. D Biol.Cryst. 50, 760-763 (1994).

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43. P. Emsley, K. Cowtan, Acta Cryst. D Biol Cryst. 60, 2126-2132 (2004).44. W. L. DeLano (DeLano Scientific Press, San Carlos, CA, 2002).

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47. W. H. McMaster, N. K. Del Grande, J. H. Mallett, J. H. Hubbell, J. H.Compilation of x-ray cross sections (Natl. Bur. Stand. US UCRL-50174, Sec II, Rev.1, National Technical Information Service, U. S. Department of Commerce,Springfield, VA 1969).

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49. B. K. Teo, in EXAFS: Basic Principles and Data Analysis (Springer-Verlag, NewYork, 1986).

50. S. I. Zabinsky, J. J. Rehr, A. Aukudinov, R. Albers, M. J. Eller, Phys. Rev. B 52,2995-3009 (1995).

51. L. Kau, D. J. Spira-Solomon, J. E. Penner-Hahn, K. O. Hodgson, E. I. Solomon, J.Am. Chem. Soc 109, 6433-6442 (1987).

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54. J. R. Requena, C. C. Chao, R. L. Levine, E. R. Stadtman, Proc. Natl. Acad. Sci.98, 69 – 74 (2001).

55. K. Clark-Baldwin et al., J. Am. Chem. Soc. 120, 8401 – 8409 (1998).

Supporting Online Material Video Clip Files

Movie S1. [TM][(Cu)(Cu-Atx1)3] complex and [S6Cu4MoS4] cluster molecularstructure (video version of Fig. 1A & 1B, and Fig. 2) (QuickTime Movie,1179907S1.mov, 34.8 MB).