Metal Ion Chaperone Function of the Soluble Cu(I) Receptor Atx1

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ATX1 was cloned into pET11d (Novagen) and expressed in E. coli strain BL21(DE3) after induction with IPTG. The protein was isolated by a freeze-thaw extraction and purified to homogeneity by DEAE-Sephacel batch treatment and subsequent chromatography on CM Sepharose FF (Pharmacia). Approximately 6 to 10 mg of pure protein was obtained per liter of bacterial culture. Protein concentration was determined from absorbance at 280 nm (with an extinction coefficient of 4400 M −1 cm −1 calculated on the basis of total amino acid composition) or by Bradford assay [M. Bradford Anal. Biochem. 72 248 (1976)] with immunoglobulin G as a standard. Electrospray mass spectrometry (ES-MS) of apo-Atx1 revealed a single peak of 8088.1 daltons which corresponds to full-length Atx1 lacking its NH 2 -terminal methionine; in addition ES-MS of Hg-Atx1 yielded one peak at 8287.6 daltons. Gel filtration on an HPLC QC-PAK TSK 200GL (TosoHaas) column yielded molecular sizes of 10 200 9840 and 9960 daltons for apo- Hg(II)- and Cu(I)-Atx1 respectively.

Cu(I)-Atx1 was typically prepared by incubation of apoprotein with a Cu(II) salt in the presence of a thiol reductant. All manipulations were performed in an inert atmosphere at 4°C. CuSO 4 (2.5 equivalents) was added with stirring to a solution of apo-Atx1 and DTT (20-fold molar excess relative to protein) in 50 mM tris (pH 8.0 adjusted with solid MES to avoid Cl − ). Excess metal was removed by ultrafiltration and the protein was exchanged into a solution containing 20 mM MES (pH 6.0) and 20% (v/v) glycerol. The final protein concentration was 7.84 mM with a Cu/protein ratio of 0.75 ± 0.02. Solutions (>2 mM) of the Cu(I)-protein can be handled in air at 4°C for 30 min or stored long term (>4 months) at −70°C with no observable formation of Cu(II).

XAS data were obtained at Stanford Synchrotron Radiation Laboratory (SSRL) beam line VII-3 as fluorescence excitation spectra with a Ge solid-state detector array. Samples (150 μl) were maintained at 10 K throughout measurements. Data reduction and analysis were performed according to standard procedures [

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]. Data were fitted with amplitude and phase functions calculated with FEFF 6.01 [

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] calibrated by fitting model compounds of known structure.

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199 Hg-Atx1 was prepared by the addition of 199 Hg(II) to apo-Atx1 in 20 mM MES (pH 6.0). DTT was removed from apo-Atx1 by ultrafiltration before the addition of mercury. 199 Hg(II) (1.2 equivalents) was added to apo-Atx1 on ice with stirring. Free metal was removed with several successive washes with buffer by ultrafiltration followed by exchange into D 2 O buffer. The final protein concentration was 2.40 mM with a 199 Hg/protein ratio of 0.83 ± 0.05 in a solution containing 25 mM sodium phosphate (pH* 6.6) 50 mM NaCl and 98% D 2 O. A similar protocol was used to prepare the EXAFS sample except that Cl − was not present in any of the buffers. The final protein concentration was 3.36 mM with a Hg/protein ratio of 0.75 ± 0.06 in 20 mM MES (pH 6.0).

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The 1 H{ 199 Hg} HMQC spectra of 199 Hg-Atx1 were obtained on a Bruker 600-MHz spectrometer (14.09 T 107.4 MHz for 199 Hg) equipped with a 5-mm tunable probe. The pulse sequence and optimal delays for Met Cys and His ligands have been described (27). Spectral widths of 7246 Hz for 1 H ( F2 ) and 32 221 Hz for 199 Hg ( F1 ) were used. A total of 64 t 1 blocks was accumulated with 1024 transients collected in F2 per block. The 1 H 90° pulse was 8.0 μs and the 199 Hg(90°) −1 value was 20.4 μs as calibrated with Hg(CH 3 ) 2 . Safety note: The latter compound is permeable to latex gloves and is exceedingly toxic. Discussion of alternative compounds can be found at

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The two-hybrid analysis [

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] was performed with the MATCHMAKER System (Clontech). Nucleotides +1 to +222 of ATX1 were amplified by PCR and inserted at the Bam HI and Pst I sites of the pGBT9 plasmid encoding the DNA-binding domain of Gal4 to yield pPS002. For preparation of vector pPS001 CCC2 nucleotides (nt) +1 to +745 were amplified and inserted at the Eco RI and Sal I sites of pGAD424 encoding the Gal4 activation domain. The remaining three Ccc2-Gal4 fusion constructs were obtained by amplifying the corresponding CCC2 sequences and inserting them into the Bam HI and Pst I sites of pGAD424. Plasmid pCF12 contains CCC2 nt +1163 to +1582 pCF34 contains nt +1731 to +2711 and pCF56 contains nt +2831 to +3294. Yeast strain SFY526 was cotransformed with the vectors based on pGBT9 and pGAD424 and grown in 5 ml of synthetic dextrose (SD) medium to an optical density at 600 nm of 4.0. Cells were harvested washed in water and resuspended in 1 ml of 50 mM sodium phosphate buffer (pH 7.0) containing 10 mM KCl 1 mM MgSO 4 and 40 mM β-mercaptoethanol. They were lysed by homogenization with glass beads after adding 45 μl of 0.1% SDS. The resulting extract (800 μl) was mixed with 200 μl of o -nitrophenyl-β- d -galactopyranoside (4 mg/ml) and incubated at 30°C overnight. After centrifugation β-Gal activity was measured in the supernatant at 420 nm. In some instances cells were grown in the presence of the Cu chelator BCS (3 mM). Results are representative of two to four independent samples with ranges of ≤15%. β-Gal activity units for the p53/SV40 positive controls (and vectors) were 9.75 (0.21) in the absence of BCS and 9.95 (0.27) in the presence of BCS.

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J. Gitschier and W. Fairbrother personal communication.

Cizweski Culotta V., et al., J. Biol. Chem. 272, 23469 (1997).

Supported by NIH: GM-54111 (T.V.O.) GM-38047 (J.E.P.-H.) GM-50016 (V.C.C.) Biophysics Training Grant T32GM08382 (C.P.S.) Training Grant ES 07141 (P.J.S.) and F32 DK-09305 (R.A.P.). SSRL is funded by the Department of Energy (Office of Basic Energy Sciences and Office of Health and Environmental Research) and by the NIH Biomedical Research Technology Program. The Northwestern University 600-MHz NMR Facility is funded by the W. M. Keck Foundation NSF NIH and the R. H. Lurie Cancer Center. We thank R. Scott for the Cu(SC 6 H 5 ) 3 2− XANES data D. Huffman for [Cu(II)(imidazole) 4 ](NO 3 ) 2 and A. Duncan for the color illustration.