Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Solution Structure of the Yeast Copper Transporter Domain Ccc2a in the Apo and Cu(I)-loaded States

Solution Structure of the Yeast Copper Transporter Domain Ccc2a in the Apo and Cu(I)-loaded States THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 11, Issue of March 16, pp. 8415–8426, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Solution Structure of the Yeast Copper Transporter Domain Ccc2a in the Apo and Cu(I)-loaded States* Received for publication, September 13, 2000, and in revised form, November 15, 2000 Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M008389200 Lucia Banci‡, Ivano Bertini‡§, Simone Ciofi-Baffoni‡, David L. Huffman¶, and Thomas V. O’Halloran¶i** From the ‡Magnetic Resonance Center and Department of Chemistry, University of Florence, Via Luigi Sacconi 6, Sesto Fiorentino, Florence, 50019, Italy and the Departments of ¶Chemistry and iBiochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208 Ccc2 is an intracellular copper transporter in Saccha- minus of the copper-transporting ATPases contains one or romyces cerevisiae and is a physiological target of the more metal-binding domains (2–7) characterized by a GMX- copper chaperone Atx1. Here we describe the solution CXXC motif. In humans, this intracellular copper pump is structure of the first N-terminal MTCXXC metal-binding encoded by the Wilson’s and Menkes’ disease genes (2– 6), and domain, Ccc2a, both in the presence and absence of a functional homologue (Ccc2) has been characterized in yeast Cu(I). For Cu(I)-Ccc2a, 1944 meaningful nuclear Over- (7). Copper is incorporated into trans-Golgi vesicles via the hauser effects were used to obtain a family of 35 struc- action of Ccc2 and ultimately into the multicopper oxidase tures with root mean square deviation to the average Fet3p, which translocates to the plasma membrane and works structure of 0.36 6 0.06 Å for the backbone and 0.79 6 in conjunction with an iron permease to mediate high affinity 0.05 Å for the heavy atoms. For apo-Ccc2a, 1970 mean- iron uptake (8, 9). Both the Wilson’s and Menkes’ disease ingful nuclear Overhauser effects have been used with proteins complement the function of Ccc2 in this pathway J to obtain a family of 35 structures with root HNHa (10 –12). The Menkes CPx-type ATPase contains six N-termi- mean square deviation to the average structure of 0.38 6 nal GMXCXXC motifs (2– 4), is located in the trans-Golgi net- 0.06 Å for the backbone and 0.82 6 0.07 Å for the heavy work, and translocates copper across intracellular membranes babbab, ferrodoxin-like atoms. The protein exhibits a into the secretory pathway (13). Metal binding studies on the fold similar to that of its target Atx1 and that of a human complete N-terminal cytoplasmic region have established that counterpart, the fourth metal-binding domain of the this region binds Cu(I) selectively (relative to cadmium, cobalt, Menkes protein. The overall fold remains unchanged or zinc) with a stoichiometry of one copper per metal-binding upon copper loading, but the copper-binding site itself domain (14). becomes less disordered. The helical context of the cop- The yeast metallochaperone Atx1 is a cytosolic Cu(I) receptor per-binding site, and the copper-induced conforma- that delivers its metal ion cargo to Ccc2 (15). The thermody- tional changes in Ccc2a differ from those in Atx1. Ccc2a presents a conserved acidic surface which complements namic gradient for metal transfer between Atx1 and the first the basic surface of Atx1 and a hydrophobic surface. metal-binding domain of Ccc2 (Ccc2a) is shallow, yet copper These results open new mechanistic aspects of copper transfer is facile, suggesting that Atx1 works like an enzyme to transporter domains with physiological copper donor catalyze the rate of copper transfer between partners (16). A and acceptor proteins. high resolution (1.02-Å) x-ray crystallographic structure of Hg(II)-Atx1 reveals that the mercury is coordinated in a biden- tate fashion from two cysteine sulfurs with a S–Hg–S bond Saccharomyces cerevisiae Ccc2 is a member of a class of angle of 167° (17). Mutation of several conserved lysines on the proteins that transport heavy metals across vesicular mem- surface of Atx1 greatly reduces the copper-dependent interac- branes (1). Members of this family, referred to as P-type or tion of Atx1 and Ccc2 in vivo (15, 18). The Atx1 metallochap- CPx-type ATPases, have been identified in a variety of bacte- erone (17), domain I of the copper chaperone for superoxide ria, yeast, nematodes, and mammals. The cytoplasmic N ter- dismutase (CCS) (19), and the fourth metal-binding domain of the Menkes protein (20) all adopt a babbab structural fold. This same structural fold is found in the mercury-binding pro- * This work was supported by European Community Contract HPRI- tein MerP (21, 22) and the putative copper chaperone CopZ CT-1999-00009, Italian Consiglio Nazionale delle Ricerche (Progetto Finalizzato Biotecnologie 99.00286.PF49), Ministero della Universita`e (23). In all of these domains, the cysteine ligands are located delle Ricera Scientifica e Tecnologica (MURST), National Institutes of within the first loop and the first helix. Health Grant GM 54111 (to T. V. O.), and Molecular Toxicology Train- The mechanism of copper transfer between Atx1 and Ccc2 is ing Program Postdoctoral Fellowship 5T32ES07284 (to D. L. H.). The proposed to involve a series of two- and three-coordinate inter- costs of publication of this article were defrayed in part by the payment mediates (15, 16). The factors responsible for facile and revers- of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate ible copper transfer between Atx1 and Ccc2a are probably this fact. mediated in part by metal-dependent conformational changes § To whom correspondence may be addressed: Prof. Ivano Bertini, in Atx1 and Ccc2. In Atx1, copper release involves a series of Magnetic Resonance Center and Department of Chemistry, University structural changes, in which both cysteines change conforma- of Florence, Via L. Sacconi 6, Sesto Fiorentino, Florence, 50019 Italy. Tel.: 39-055-4574272; Fax: 39-055-4574271; E-mail: bertini@ tion (46). In apo-Atx1, the loop is not well defined. We report cerm.unifi.it. here the solution structures of Cu(I)-Ccc2a from S. cerevisiae ** A fellow of the John Simon Guggenheim Foundation. To whom obtained for a nonlabeled sample and its reduced apo form, correspondence may be addressed: Dept. of Chemistry, Northwestern obtained for a N-labeled sample. Relative to Atx1, the first University, 2145 Sheridan Rd., Evanston, IL 60208. Tel.: 847-491-5060; Fax: 847-491-7713; E-mail: [email protected]. loop is well defined, and Cu(I) binding induces fewer changes in This paper is available on line at http://www.jbc.org 8415 This is an Open Access article under the CC BY license. 8416 Solution Structure of Cu(I) and Apo Forms of Ccc2a FIG.1. H 800-MHz NMR spectra of apo-Ccc2a (A) and Cu(I)-Ccc2a (B) proteins at 298 K and pH 7.0 in 100 mM phosphate buffer. The protein concentrations were 2.7 and 1.2 mM, respectively. respectively. Cu(I)-Ccc2a was prepared as previously described (16), the conformation of Ccc2a. Unlike Cu(I)-Atx1, the metal bind- and the buffer was exchanged by ultrafiltration into 100 mM sodium ing residues in Cu(I)-Ccc2a are on the surface of a helix, and phosphate, pH 7, 90% H O, 10% D O. The metal/protein ratio was 1.2 2 2 the copper cargo is more accessible to incoming ligands in the with a protein concentration of 1.2 mM. No exogenous thiols were added latter. Finally, a negative patch is observed on the surface of to the sample buffers. Approximately 0.6 ml of sample was loaded into Ccc2a in a site that corresponds to a positive patch on Atx1, 535-PP 5-mm quartz NMR tubes (Wilmad), which were capped with a suggesting that a complementary docking interface is em- latex serum cap in the VacAtmospheres chamber. ployed in the copper transfer mechanism. NMR Spectroscopy—The NMR spectra were acquired on Avance 800 and 600 Bruker spectrometers operating at a proton nominal frequency EXPERIMENTAL PROCEDURES of 800.13 and 600.13 MHz, respectively. A QXI probe has been used on Sample Isolation and Preparation—Ccc2a was uniformly labeled the Avance 800 spectrometer, and a triple resonance (TXI) 5-mm probe with N by expressing the protein in Escherichia coli strain BL21(DE3) has been used on the 600 spectrometer. All probes were equipped with (Novagen), transformed with pDLHV021, in minimal media supple- pulsed field gradients along the z axis. Total correlation spectroscopy mented with NH Cl. Unlabeled Ccc2a was isolated as described pre- (25, 26) spectra were recorded on the 600-MHz spectrometer with a viously (16). Ccc2a( N) was purified from the cell pellet by freeze-thaw spin-lock time of 100 ms, a recycle time of 1 s and a spectral window of extraction with 20 mM MES /Na, pH 6.0, followed by streptomycin 14 ppm. Two-dimensional NOESY maps (27, 28) were acquired on the sulfate precipitation (5%, w/v) of the extract. The supernatant was 800-MHz spectrometer with a mixing time of 100 ms, a recycle time of further purified by repetitive runs on Superdex 75 (Amersham Phar- 1 s, and a spectral window of 14 ppm. 15 15 1 macia Biotech). The yield of labeled protein was ;2 mg/liter of culture. On the N apo-Ccc2a sample, a two-dimensional N- H hetero- Protein samples were purified and stored in the presence of reducing nuclear single quantum coherence (29 –31) map was obtained at 800 agent. NMR samples were prepared in a Vac Atmospheres nitrogen MHz with an INEPT delay of 2.66 ms, a recycle time of 1 s, and spectral atmosphere chamber at 12 °C. Protein concentrations were determined 1 15 windows of 14 and 33 ppm for the H and N dimensions, respectively. by the Bradford assay and calibrated as described previously (16). 15 A three-dimensional NOESY- N heteronuclear multiple quantum co- Copper concentration was determined by ICP-AES. The NMR sample of 1 15 herence experiment (32) was recorded with 290 ( H) 3 96 ( N) 3 2048 apo-Ccc2a and apo-Ccc2a( N) was prepared by exchanging the reduced 1 ( H) data points on the 600-MHz spectrometer. The INEPT delay was form of the protein into 100 mM sodium phosphate, pH 7, 90% H O, 10% set to 5.4 ms, the mixing time was 100 ms, and the carrier frequency D O via ultrafiltration; the final concentrations were 2.7 and 3.3 mM, was set in the center of the amide proton region, at 7.35 ppm. Spectral windows of 14, 14, and 29 ppm were used for the direct H dimension 1 15 1 and the indirect H and N dimensions. A HNHA experiment (33) was The abbreviations used are: MES, 4-morpholineethanesulfonic acid; performed at 600 MHz to determine J coupling constants. The HNHa r.m.s., root mean square; NOE, nuclear Overhauser effect; NOESY, 1 15 1 spectrum was recorded as a 128 ( H) 3 80 ( N) 3 2048 ( H) data set nuclear Overhauser effect spectroscopy; INEPT, insensitive nuclei en- using pulsed field gradients along the z axis. The mixing time was 100 hanced by polarization transfer; WATERGATE, water suppression by gradient-tailored excitation; REM, restrained energy minimization; ms. Spectral windows of 14, 14, and 29 ppm were used, respectively, for 1 1 15 mbd4, metal-binding domain 4. direct H dimension and the indirect H and N dimensions. Solution Structure of Cu(I) and Apo Forms of Ccc2a 8417 FIG.2. Schematic representation of the sequential and medium range NOE connectivities involving NH, Ha, and Hb protons for apo-Ccc2a (A) and Cu(I)-Ccc2a (B). The thickness of the bar indicates the intensity of NOEs. For all the experiments, quadrature detection in the indirect dimen- Hydrogen bond constraints were introduced for backbone amide pro- sions was performed in the time-proportional phase incrementation tons that were found to be within hydrogen bond distance and to have mode (28), and water suppression was achieved through WATERGATE the correct orientation with respect to hydrogen bond acceptors in sequence (34). All two-dimensional data consisted of 4K data points in structural models obtained without inclusion of these constraints. The the acquisition dimension and of 1K experiments in the indirect dimen- distance between the NH proton and the hydrogen bond acceptor was sion. All three- and two-dimensional spectra were collected at 298 K, constrained to be in the 1.8 –2.4 Å interval by inclusion of the corre- processed using the standard Bruker software (XWINNMR), and ana- sponding upper and lower distance limits in structure calculations. In lyzed on IBM RISC 6000 computers through the XEASY program. addition, upper and lower distance limits of 3.0 and 2.7 Å between the Constraints Used in Structure Calculations—The peaks used for the N and the acceptor atoms were also included. structure calculations were integrated in the two-dimensional NOESY Structure Calculations—The structure calculations were performed map acquired at 298 K in H O. Intensities of dipolar connectivities were using DYANA (36). 200 random conformers were annealed in 10000 converted into upper distance limits, to be used as input for structure steps using NOE and J values (when available) constraints. The 35 calculations, by using the approach provided by the program CALIBA conformers with the lowest target function constitute the final family. (35). The calibration curves were adjusted iteratively as the structure The copper ion was included in the calculations by adding a new residue calculations proceeded. Stereospecific assignments of diastereotopic in the amino acid sequence, formed by a chain of dummy atoms that protons were obtained using the program GLOMSA (35). J cou- have their van der Waals radii set to 0 so that it can freely penetrate HNHa pling constants were correlated to the backbone torsion angle f by into the protein and one atom with a radius of 1.4 Å, which mimics the 13 16 means of the appropriate Karplus curve (33). These angles were used as copper ion. The sulfur atoms of Cys and Cys were linked to the metal constraints in the DYANA calculations and restrained energy minimi- ion through upper distance limits of 2.5 Å. This approach does not zation (REM) refinement. impose any fixed orientation of the ligands with respect to the copper. 8418 Solution Structure of Cu(I) and Apo Forms of Ccc2a FIG.4. A, number of meaningful NOEs per residue for apo-Ccc2a. White, gray, and black bars indicate intraresidue, sequential, and me- dium/long range connectivities, respectively; B, r.m.s. deviation per residue to the mean structure of apo-Ccc2a for the backbone (filled squares) and all heavy atoms (filled circles) of the REM structure family FIG.3. Schematic representation of long range connectivities of 35 conformers. for apo-Ccc2a (A) and Cu(I)-Ccc2a (B). Segments perpendicular to the diagonal of the plot represent pairs of anti-parallel b-strands. Assignments of the resonances of apo-Ccc2a started from the analysis of the N heteronuclear single quantum coherence REM was then applied within the molecular mechanics and dynamic 15 1 map, which allowed the identification of the N and HN module of SANDER (37). The force field parameters for the copper(I) ion were adapted from similar systems (38). In particular, no constraint on resonances. Then through the analysis of the three-dimen- 13 16 the S(Cys )–Cu-S(Cys ) angle was used. The values of NOE and sional NOESY-heteronuclear multiple quantum coherence and torsion angle potentials were calculated with force constants of 50 kcal of two-dimensional NOESY and total correlation spectroscopy, 21 22 mol Å . the sequence-specific assignment was performed. The assign- The program CORMA (39), which is based on relaxation matrix ment of the Cu(I)-Ccc2a derivative was performed through the calculations, was used to back calculate the NOESY cross-peaks from analysis of two-dimensional NOESY and total correlation spec- the calculated structure to check the consistency of the analysis. The quality of the structure was evaluated in terms of deviations from ideal troscopy maps only. Resonances for all 72 residues both for the bond lengths and bond angles and through Ramachandran plots, ob- apo and Cu(I) forms of Ccc2a have been assigned. In the apo tained using the programs PROCHECK (40) and PROCHECK-NMR and in the Cu(I) protein, about 97 and 98% of the proton (41). Structure calculations and analyses were performed on IBM RISC resonances, respectively, could be located in the maps, and all 6000 computers. of the N resonances have been assigned, with the exception of 14 15 14 RESULTS Ser and Ala in the apo form and with the exception of Ser 1 15 in the Cu(I) form. The H and N resonance assignments of the Sequence-specific Assignment of Apo-Ccc2a and Cu(I)- apo and Cu(I) forms are reported in Tables I and II of the Ccc2a—The H NMR spectra of apo-Ccc2a and Cu(I)-Ccc2a are supplementary materials, respectively. reported in Fig. 1, A and B, respectively. The most relevant Secondary Structure from NMR Data—The elements of sec- difference between apo and Cu(I) form is observed in the HN ondary structure were identified by analyzing the pattern of region. In particular, the HN resonance of Thr is broader in assigned NOEs. Backbone short, medium range NOEs were the apo form than in Cu(I)-Ccc2a and has a change in the shift used to generate Fig. 2, A and B. From their analysis, it is from 9.72 ppm, in the apo form, to 9.21 ppm, in the Cu(I) form. apparent that the secondary structure is not significantly af- In the H NMR spectrum of the Cu(I) form, it is also possible to identify the HN resonance of Ala at 9.50 ppm, a residue very close to the cysteine binding motif, that cannot be detected in Supplementary materials may be accessed at the CERM site, under the apo form. Structural Biology, on the World Wide Web. Solution Structure of Cu(I) and Apo Forms of Ccc2a 8419 TABLE I Statistical analysis of the final REM family and the mean structure of apo-Ccc2a from S. cerevisiae REM indicates the energy-minimized family of 35 structures; ,REM. is the energy-minimized average structure obtained from the coordinates of the individual REM structures. REM Parameters ,REM. (35 structures) r.s.m. violations per experimental distance constraint (Å) Intraresidue (257) 0.0092 6 0.0025 0.0074 Sequential (496) 0.0066 6 0.0010 0.0063 Medium range (511) 0.0082 6 0.0010 0.0073 Long range (706) 0.0058 6 0.0013 0.0047 Total (1970) 0.0073 6 0.0008 0.0062 Average number of violations per structure Intraresidue 3.6 6 1.3 4 Sequential 5.5 6 1.4 5 Medium range 6.5 6 1.2 5 Long range 6.2 6 2.2 4 Total 21.7 6 2.7 18 Average no. of NOE violations larger than 0.3 Å 0.00 6 0.00 0 Average no. of f violations larger than 5° 0.00 6 0.00 0 Largest residual NOE violation (Å) 0.23 0.09 Average NOE and torsion deviations (Å ) 0.14 6 0.02 0.11 Structural analysis Residues in most favourable regions (%) 80.0 82.1 Residues in allowed regions (%) 17.3 17.9 Residues in generously allowed regions (%) 2.0 0.0 Residues in disallowed regions (%) 0.8 0.0 The number of experimental constraints for each class is reported in parenthesis. Medium range distance constraints are those between residues (i, i 1 2), (i, i 1 3), (i, i 1 4), and (i, i 1 5). As it results from the Ramachandran plot analysis. fected by the presence or absence of the copper ion. In Ccc2a, two elements of helical secondary structure can be predicted, which are characterized by a high number of sequential and medium range connectivities such as d (i, i 1 1), d (i, i 1 2), NN NN d (i, i 1 3), d (i, i 1 4), and d (i, i 1 3). The two helices aN aN ab involve residues 14 –27 and 51– 63 in the apo form. The pres- ence of medium range connectivities such as d (i, i 1 2), d NN aN (i, i 1 2), and d (i, i 1 3) for residue Cys in the Cu(I) form NN with respect to the apo form gives evidence that Cys belongs a1 in the Cu(I) form. All of the to the the beginning of helix backbone NOEs were used to generate Fig. 3, A and B, from which it is possible to recognize the presence of four antipar- allel b-sheets in both apo and Cu(I) forms, involving residues 2– 8, 29 –35, 40 – 46, and 65–71. Fig. 3 also shows that the typical folding pattern of the copper chaperones, “open-faced b FIG.5. Backbone atoms for the solution structure of apo-Ccc2a -sandwich” fold (b1-a1-b2-b3-a2-b4) (17, 20, 21), is present also as a tube with variable radius, proportional to the backbone r.m.s. deviation value of each residue. The side chains of Cys and in this protein, both in the presence and absence of copper. Cys are also shown. The figure was generated with the program Solution Structure Calculations and Analysis of Apo- MOLMOL (45). Ccc2a—A total of 3785 NOESY cross-peaks were assigned, integrated, and transformed in upper distance limits with the program CALIBA (35). They corresponded to 2314 unique up- an average target function of 0.64 6 0.10 Å and average r.m.s. per distance limits, of which 1970 were found to be meaningful deviation values over all of the 72 residues with respect to the (nonmeaningful distance constraints are those that cannot be mean structure of 0.39 6 0.05 Å for the backbone and of 0.80 6 violated in any structure conformation and those involving 0.07 Å for the heavy atoms. The family of conformers was then proton pairs at fixed distance). The number of NOEs per resi- subjected to further refinement through energy minimization due is reported in Fig. 4A. The average number of NOEs per (37). The REM family has an average target function of 0.14 6 2 2 residue is 32 for apo-Ccc2a, of which 27 are meaningful. 35 0.02 Å , to which the NOE contribution is 0.13 Å , while the 3 2 torsional angle one is 0.01 Å . The average r.m.s. deviation J couplings were obtained from the HNHA three-dimen- HNHa sional spectrum, which were translated into dihedral angles values for the family with respect to the mean structure are through the standard equation. For 0.38 6 0.06 Å for the backbone and 0.82 6 0.07 Å for the heavy J values of .8 and HNHa ,4.5 Hz, the f angle was assumed to be between 2155 and atoms for all of the amino acids in the sequence. The r.m.s. 285° and between 280 and 230°, respectively (42, 43). Hydro- deviation values per residue of the final REM family to the gen bond constraints for 31 amide protons were used at later mean structure are shown in Fig. 4B. stages of structural calculations. A total of 38 proton pairs were The final family of conformers was analyzed with PRO- stereospecifically assigned through the program GLOMSA CHECK-NMR (41), and results are reported in Table I. Accord- (35). The constraints used for structure calculations and the ing to this program, the secondary structure elements in the stereospecific assignments are reported in the supplementary energy-minimized mean structure involve residues 2–9 (b1), materials. 13–26 (a1), 29 –36 (b2), 40 – 47 (b3), 51– 63 (a2), and 65–70 (b4). The 35 conformers constituting the final DYANA family had Analysis of the NOE patterns has led to somewhat similar 8420 Solution Structure of Cu(I) and Apo Forms of Ccc2a conclusion (see above). It is worth noting that in contrast to NOE secondary structure analysis above, Cys is assigned to helix a1 in the energy-minimized mean structure of the apo form. In this energy-minimized average structure, 82.1% of the residues are in the most favored regions of the Ramachandran plot, and 17.9% of the residues are in the allowed regions. No residues are in the disallowed regions (Table I). The structure of apo-Ccc2a, shown in Fig. 5, is well defined all over its sequence. All of the a-helices and antiparallel b-sheet are very well defined, with an average backbone r.m.s. deviation lower than 0.38 Å. The largest backbone r.m.s. devi- ation values are obtained for residues 12–14 and residue 48. The high r.m.s. deviation values of residues 12–14 (0.74 Å) are due to the paucity of NOEs in this region (Fig. 4A) that consti- tutes loop 1 and the beginning of helix a1. Indeed, this region 14 15 contains Ser and Ala , whose HN resonances have not been identified and are a break in the sequential connectivities. This region includes Cys , one of the copper ligands. The side chain of Cys is disordered in the apo state and spans different conformations due to the small number of NOEs. On the con- trary, the other copper ligand Cys , which belongs to the first a-helix, is very well defined (r.m.s. deviation BB, 0.22 Å; r.m.s. 13 16 deviation HA, 0.37 Å). The side chains of Cys and Cys are very close; the distance between the two sulfur atoms in the average structure is 5.6 Å. Solution Structure Calculations and Analysis of Cu(I)- Ccc2a—A total of 3866 NOESY cross-peaks were assigned, integrated, and transformed in upper distance limits with the program CALIBA (35). They corresponded to 2338 upper dis- tance limits, of which 1944 were found to be meaningful. The number of NOEs per residue is reported in Fig. 6A. The average number of NOEs per residue is 32, of which 27 are meaningful. Hydrogen bond constraints for 22 amide protons were used at later stages of structural calculations. A total of 47 proton pairs were stereospecifically assigned through the program FIG.6. A, number of meaningful NOEs per residue for Cu(I)-Ccc2a. GLOMSA (35). The constraints used for structure calculations White, gray, and black bars indicate intraresidue, sequential, and me- dium/long range connectivities, respectively. B, r.m.s. deviation per and the stereospecific assignments are reported in the supple- 2 residue to the mean structure of Cu(I)-Ccc2a for the backbone (filled mentary materials. squares) and all heavy atoms (filled circles) of the REM structure family The 35 conformers constituting the final DYANA family had of 35 conformers. an average target function of 0.48 6 0.11 Å and an average r.m.s. deviation value over all of the 72 residues with respect to form. Indeed, the backbone r.m.s. deviation values with respect the mean structure of 0.39 6 0.06 Å for the backbone and of to the mean structure for residues in the less well defined 0.79 6 0.04 Å for the heavy atom. The family of conformers was region of apo (10 –14) decrease from 0.64 Å in the apo form to then subjected to further refinement through energy minimi- 0.27 Å in the Cu(I) form, and the side chain of Cys that in the zation (37). The REM family has an average target function of apo form is disordered (r.m.s. deviation BB, 0.80 Å; r.m.s. 0.26 6 0.03 Å . The average r.m.s. deviation values for the deviation HA, 1.17 Å) is very well defined in the Cu(I) form family with respect to the mean structure are 0.36 6 0.06 Å for (r.m.s. deviation BB, 0.25 Å; r.m.s. deviation HA, 0.62 Å). the backbone and 0.79 6 0.05 Å for the heavy atoms for all of DISCUSSION the amino acids in the sequence. The r.m.s. deviation values per residue of the final REM family to the mean structure are Comparison between the Structures of Apo- and Cu-Ccc2a— shown in Fig. 6B. The solution structure of S. cerevisiae Ccc2a exhibits the The final family of conformers was analyzed with PRO- babbab folding pattern typical of copper chaperones (44) and CHECK-NMR (41), and results are reported in Table II. The the fourth metal-binding domain of the Menkes’ disease protein secondary structure elements in the energy-minimized mean (20). In the Cu-Ccc2a structure, the copper ion coordinates two 13 16 structure involve residues 2–9 (b1), 13–26 (a1), 29 –36 (b2), of the six cysteine residues, Cys and Cys . The global r.m.s. 40–47 (b3), 51– 62 (a2), and 65–70 (b4). Analysis of the NOE deviation values between the Cu(I)-bound and apo-averaged patterns has led to a similar conclusion (see above). In the minimized structures are 0.86 and 1.35 Å for backbone and energy-minimized average structure, 83.6% of the residues are heavy atoms, respectively. The r.m.s. deviation per residue is in the most favored regions of the Ramachandran plot, 13.4% of reported in Fig. 8 (dotted dashed line) and is compared with the the residues are in the allowed regions, and 3.0% are in the r.m.s. deviation values per residue for each family (apo-Ccc2a generously allowed regions. No residues are in the disallowed (solid line) or Cu(I)-Ccc2a (dotted line)) and the sum of the regions (Table II). r.m.s. deviation values of the two families (dashed line). The The structure of Cu(I)-Ccc2a, shown in Fig. 7, is also well structure definition within each family of structures is overall defined all over its sequence. All of the a-helices and antipar- very good and comparable between the two families; thus, a allel b-sheet are very well defined and also the loop that in- meaningful comparison can be undertaken. The highest back- cludes the copper binding site is more defined than in the apo bone r.m.s. deviation value between the two structures is 1.21 Solution Structure of Cu(I) and Apo Forms of Ccc2a 8421 ABLE II Statistical analysis of the final REM family and the mean structure of Cu(I)-Ccc2a from S. cerevisiae REM indicates the energy-minimized family of 35 structures; ,REM. is the energy-minimized average structure obtained from the coordinates of the individual REM structures. REM Parameters ,REM. (35 structures) r.s.m. violations per experimental distance constraint (Å) Intraresidue (272) 0.0153 6 0.0016 0.0160 Sequential (511) 0.0072 6 0.0013 0.0069 Medium range (490) 0.0103 6 0.0015 0.0102 Long range (671) 0.0102 6 0.0012 0.0106 Total (1944) 0.0105 6 0.0008 0.0106 Average number of violations per structure Intraresidue 10.4 6 2.4 11 Sequential 5.1 6 1.5 4 Medium range 9.3 6 1.9 11 Long range 12.2 6 2.0 13 Total 37.0 6 4.2 39 Average no. of NOE violations larger than 0.3 Å 0.00 6 0.00 0 Largest residual NOE violation (Å) 0.20 0.13 Average NOE deviations (Å ) 0.26 6 0.03 0.25 Structural analysis Residues in most favourable regions 79.0 83.6 Residues in allowed regions 18.6 13.4 Residues in generously allowed regions 2.0 3.0 Residues in disallowed regions 0.3 0.0 The number of experimental constraints for each class is reported in parenthesis. Medium range distance constraints are those between residues (i, i 1 2), (i, i 1 3), (i, i 1 4), and (i, i 1 5). As it results from the Ramachandran plot analysis. Å (found for Thr ). There are only few regions or amino acids in the protein where the r.m.s. deviation between the two average minimized structures is significantly larger than the r.m.s. deviation of each family when superimposing the whole structures (Fig. 8), thus indicating a meaningful differences between the two structures. Analysis of side chains reveals a significant r.m.s. deviation difference for Thr , which is in the copper binding pocket. Indeed, when copper is bound, the side 17 13 chain of Thr rotates closer to the backbone oxygen of Cys , and a hydrogen bond interaction can be readily formed, since it is found in many conformers of the family. This interaction can presumably be important to determine the optimal conforma- tion of Cys in the copper-bound state of the protein. The backbone r.m.s. deviation values for each secondary structure element are obtained either superimposing all resi- dues (global r.m.s. deviation) or three residues at a time (local r.m.s. deviation) and are reported in Table III. The highest FIG.7. Backbone atoms for the solution structure of Cu(I)- r.m.s. deviation values are found for strand b2, helix a2, and Ccc2a as a tube with variable radius, proportional to the back- loops 1, 3, and 4. The r.m.s. deviation values for all of these bone r.m.s. deviation value of each residue. The side chains of 13 16 regions but the last drop when the two structures are super- Cys , Cys , and the Cu(I) ion are also shown. The figure was generated with the program MOLMOL (45). imposed locally, indicating that the structural differences in these regions originate from some global translational displace- ments, since the local conformations are well maintained. On spect to the metal-bound state. Indeed, different NOE intensity 13 16 the contrary, loop 4 shows the highest difference in the local for proton pairs at fixed distances of Cys and Cys of Ccc2a r.m.s. deviation, and these local difference are due to variations has been observed in the two forms; Cys experiences a de- of dihedral angles. crease in intensity, while those of Cys show roughly the same Comparing the copper binding region in apo- and Cu(I)- intensity, suggesting that the binding of copper produces 13 16 Ccc2a, helix a1 (containing Cys and Cys ) is very well de- higher order in this region. Upon copper release, the most important change in the shal- fined in both forms, while loop 1, which is involved in the copper binding pocket, is less defined in the apo form (see Fig. low binding pocket is observed for the side chain of Cys ; the 8). The conformation of Cys sulfur flips away from the hydrophobic interior toward the is well defined in both structures, being the same in both the apo and metal-bound proteins, while surface (Fig. 9). Calculations of the solvent accessibility on the 13 13 Cys apo-Ccc2a structure show that exposure of Cys is remarkably is more disordered in the apo form than in the metal- bound protein. Indeed, in the apo-Ccc2a structure, loop 1 be- increased (from 23% in the copper-bound form to 36% accessi- comes more disordered (Fig. 5), and the amide NHs of residues ble surface in the apo form), while Cys is always more buried 14 and 15, belonging to helix a1, are not detected anymore, (with 7% in the copper-bound form and 11% accessible surface probably due to solvent exchange or to an increased mobility. in the apo form). The lower number of NMR constraints in the metal-binding The changes in HN and Ha chemical shifts observed between pocket (i.e. residues 12–17) of the apo form might be the result the apo and the Cu(I)-bound forms are plotted for each residue of increased conformational flexibility in this region with re- in Fig. 10, A and B, respectively, and confirm that only residues 8422 Solution Structure of Cu(I) and Apo Forms of Ccc2a FIG.8. r.m.s. deviation per residue (for backbone atoms) between the conformers of the family of apo- Ccc2a (solid line), between those of Cu(I)-Ccc2a (dotted line), and be- tween the mean structures of the two families (dashed dotted line). The sum of the r.m.s. deviation values per residue for the two families of conformers is also shown (dashed line). TABLE III Comparison of the solution structure of Cu(I)-Ccc2a with the reduced apo-Ccc2a solution structure Backbone r.m.s. deviation values for each secondary structure element are obtained either superimposing all residues (global r.m.s. deviation) or three residues at a time (local r.m.s. deviation). Secondary structure element Residue range Global backbone r.m.s. deviation to apo-Ccc2a Local backbone r.m.s. deviation to apo-Ccc2a ÅÅ b1 2–9 0.54 0.31 a1 13–26 0.54 0.12 b2 29–36 0.81 0.19 b3 40–46 0.55 0.19 a2 51–62 0.73 0.15 b4 65–70 0.58 0.22 Loop 1 10–12 0.78 0.24 Loop 2 27–28 0.66 0.21 Loop 3 37–39 0.95 0.28 Loop 4 47–50 0.75 0.43 Loop 5 63–64 0.60 0.20 FIG.9. A, backbone drawing of Cu(I)- Ccc2a (green) and apo-Ccc2a (blue). The copper ion is in yellow. Side chains of 21 64 11 38 Ile , Phe , Met , and Leu are re- ported. The secondary structure elements are indicated. The inset shows the copper binding region. very close to the metal binding pocket are affected by the loop, no significant conformational changes are detectable ei- presence of the copper ion. Indeed, several highly conserved ther as differences between their chemical shifts (Fig. 10, A and hydrophobic residues appear to play a role in maintaining an B) or as differences between the two average structures (Fig. 9). 64 11 optimal metal-ligand conformation. These include Phe and The methionine in the GMXCXXC metal binding loop (Met ) 20 16 Ile , which are in Van der Waals contact with Cys , and is highly conserved between other N-terminal, membrane-teth- 37 13 Leu , which contacts Cys and is highly conserved between ered domains of heavy metal ATPases and small metallochap- the Ccc2, Wilson, and Menkes domains as well as Atx1 and erones alike. Of all of the residues in the protein, Met shows CCS. While these side chains can stabilize the metal binding the greatest change in Ha resonances upon copper binding Solution Structure of Cu(I) and Apo Forms of Ccc2a 8423 (Fig. 10, A and B), but it is not directly involved in metal ion coordination. Instead, it points toward the hydrophobic core of the protein (Fig. 9). The contacts between this side chain and the residues surrounding it change little between the apo and Cu(I)-bound forms (Fig. 9) as judged from the observed medium and long range NOEs; however, copper occupancy does change the conformation of this residue. These results suggest that Met acts as a hydrophobic tether that anchors the metal- 37 64 binding loop via hydrophobic interactions with Leu , Phe , and Ile . The slight movements in the region are probably coupled to stabilization of the GMXCXXC domain in the pres- ence of bound metal ion. Comparison between the Solution Structures of the Fourth Metal Binding Cytosolic Domain from Menkes Copper-trans- porting ATPase and Cu(I)-Ccc2a—The structure of Cu(I)-Ccc2a is similar to the Ag(I)-bound solution structure of the fourth metal-binding domain (mbd4) of Menkes ATPase (20), the hu- man homologue of S. cerevisiae Ccc2. A sequence alignment of Ccc2a with this protein (Fig. 11A) reveals 29% identity. The structures were superimposed according to the sequence align- ment. Both proteins show the same ferrodoxin-like fold (Fig. 11B). The overall backbone r.m.s. deviation value is 1.15 Å between Cu(I)-Ccc2a and Ag(I)-mbd4. The major structural differences are represented by simple translations of the sec- ondary structure elements. All a-helices and b-strands are well superimposed or show slight displacements except for the short C-terminal b-strand. The conserved hydrophobic residues Ile , 37 64 Leu , and Phe , presumably important for maintaining opti- mal metal-binding loop conformation, have the same conforma- tion in the two structures. The biggest differences are observed in loop 1 and loop 4. Indeed, loop 1 differs in the vicinity of FIG. 10. Plot of the chemical shift differences (d(apo) 2 d(Cu(I)- 1 1 13 14 bound)) versus residue number for the HN (A) and Ha (B) Cys , which does not superimpose with Cys of mbd4. The 16 17 resonances. conformation of the other cysteine (Cys and Cys , respec- tively) is very similar between the two structures. In Cu(I)- 11C). The major structural differences are translations or Ccc2a, Cys is the first residue of helix a1, while in mbd4, changes in length of the secondary structure elements and of Cys belongs to the metal binding loop 1 (Fig. 11B). The the loops. The largest differences are found for helix a1, stabilization of the helix and the variation in the conformation b-strand 4, and loops 1, 2, and 4. The length of helix a1inthe of loop 1 in Cu(I)-Ccc2a with respect to Ag(I)-mbd4 may be Cu(I) form of both proteins is the same, but in Cu(I)-Atx1 this dictated by the different chemical properties of Cu(I) versus helix spans from residue 17 to 30, while in the Cu(I)-Ccc2a the Ag(I) and by the different kinds of residues in the vicinity of the same helix is slightly offset, starting from residue 13 and loop 1 region. ending at 26. Loop 1 exhibits conformational differences, in It is worth noting that in the refinement of the Ag(I)-mbd4 particular close to Cys . This cysteine in Cu(I)-Atx1 belongs to solution structure, a linear digonal coordination was imposed loop 1, whereas in Cu(I)-Ccc2a it belongs to helix a1. These by modifying the AMBER force field (20). In the data refine- differences in secondary structure lead to distinct positioning of ment for the Cu(I)-Ccc2a family of conformers, no S-Cu-S angle the metal-binding cysteines (see Fig. 11C) and allow the copper constraints were included. The resulting value of the S-Cu-S ion to be more exposed to solvent in Ccc2a with respect to Atx1 angle is 119 6 29°. When the S-Cu-S angle is constrained to (see below). In addition, the offset of helix a1, the different loop linearity, a digonal copper thiolate center can be refined. This size, and the presence of Pro in the Atx1 sequence (see Fig. suggests that coordination is not rigid but that Cu(I) in this 11A) determine a large conformational difference in loop 2, environment may represent a mixture of coordination numbers which is more extended toward the surface in the Cu(I)-Atx1 of two and higher or that a bent two-coordinate S-Cu-S geom- structure. etry is adopted. No other protein atoms appear close enough to The structures of apo-Ccc2a and apo-Atx1 show an overall be the third ligand in Ccc2a. A third coordinating atom, if there backbone r.m.s. deviation value of 2.93 Å (Fig. 12) and exhibit is one at all, can come only from an exogenous ligand, such as more differences than do the two copper-loaded forms (Fig. a buffer component. While no low molecular weight thiols (e.g. 11C). The b-strands 1, 2, and 3 and the loops 3 and 5 are well DTT, GSH) are present in the sample, other buffer components superimposed for the apoproteins; however, differences in could be coordinating to the Cu(I). The NMR data here do not other secondary structure elements are apparent. For example, allow us to distinguish between these possibilities. helix a1 is one full turn shorter in apo-Atx1 than in apo-Ccc2 Comparison between the Solution Structures of Ccc2a and (Fig. 12). Helix a2, which superimposes well in both forms of Atx1 for both Apo and Cu(I) States—The solution structures of Atx1 (46), is translated away from the copper-binding site in Cu(I)- and apo-Atx1 from S. cerevisiae have been recently apo-Atx1 relative to its position in apo-Ccc2a (Fig. 12). While solved (46). The global folding of Atx1 is very similar to that of the structure of Atx1 undergoes changes as a function of copper Ccc2a. When superimposing Cu(I)-Ccc2a and Cu(I)-Atx1 struc- capture and release, the Ccc2a structure remains relatively tures according to the sequence alignment (see Fig. 11A), the invariant, suggesting that the metal site in apo-Ccc2a is more overall backbone r.m.s. deviation value is 2.8 Å. Helix a2; preorganized than in apo-Atx1. This is one of the key structural b-strands 1, 2, 3; and loops 3 and 5 are well superimposed (Fig. differences between the Atx1 metallochaperone family and the 8424 Solution Structure of Cu(I) and Apo Forms of Ccc2a FIG. 11. A, sequence alignment of the Ccc2a amino acid sequence from S. cerevisiae with the sequences of the fourth metal-binding domain from Menkes-transporting ATPase (mbd4) (Protein Data Bank accession number 1aw0) (20) and of Atx1. The positions of the Ccc2a secondary structure elements (as found in the mean Cu(I) structure) are shown at the top. b-strands are in blue, a helices are shown in orange, and loop regions are in yellow. Each sequence is color-shaded according to secondary structure element, as found in their metal-bound structures. Residues that are highly similar or conserved are indicated, respectively, by the l and * below the sequences. B, comparison of the backbone of Cu(I)-Ccc2a (blue) and Ag(I)-mbd4 (green) structures (20). C, comparison of backbone of Cu(I)-Ccc2a (blue) and Cu(I)-Atx1 (green). The copper ion and the cysteine ligands are also shown. The secondary structure elements are indicated. homologous metal-binding domains of the copper-transporting P-type ATPases. Another important difference between these copper donor and acceptor proteins is apparent in Fig. 13, A and B, namely the accessibility of the copper to incoming nucleophiles. The solvent-accessible surface of the Cu(I) center in Cu-Atx1 is 9% but over 18% in Cu-Ccc2a. Access to the Cu(I) in Atx1 is partially obscured by Lys , which is highly conserved among copper chaperones. In Ccc2a, this residue corresponds to Phe , and the structure reveals that it extends into the hydrophobic core, away from the surface. In fact, a Phe (or a Tyr) is highly FIG. 12. Comparison of the backbone of apo-Ccc2a (blue) and conserved among all of the domains of the CPx ATPases, while apo-Atx1 (green). The cysteines involved in the copper binding are Lys is conserved among the diffusable Atx1 and CCS copper indicated in blue and green for apo-Ccc2a and apo-Atx1 structures, chaperone proteins (44). The Phe side chain packs adjacent to respectively. the conserved Met and is anticipated to contribute to the stability of the metal binding loop in Ccc2a. In contrast, the surface electrostatic potential distribution was also generated lysine at this position in Atx1 (Lys ) can access several con- for human mbd4 (Fig. 13B). A very similar negative patch, 67 62 63 formations and may play a role in partner recognition and formed by Asp , Glu , and Asp , which are conserved in the control of metal ion access. homologous yeast Ccc2a, and Asp is observed on one surface Electrostatic Surface and Structural Implications for Inter- of the protein. The electrostatic potential distribution of Cu(I)- action with Physiological Partners—Partnership between Atx1 Atx1 is almost complementary and shows the presence of 7 and Ccc2 in vivo requires several basic residues, which cluster lysines (46), in positions 24, 28, 59, 61, 62, 65, and 71, which in several sites on the surface of Atx1 (18). The structure of generate a positively charged face on the molecule, as shown in Ccc2a reveals a complementary set of acidic residues. The Fig. 13C. It is known that the ATPase Ccc2 is the target of surface electrostatic potential distribution was generated with copper delivery by Atx1 (15, 16, 24). The interaction between the program MOLMOL (45) using the refined coordinates of the Atx1 and Ccc2a could be therefore determined by the comple- Cu(I)-Ccc2a structure (Fig. 13A). This protein has a region mentary attractions between the positive cluster in Atx1 and comprising several glutamate and aspartate residues that gen- the negative region in Ccc2a. erate a negatively charged face on the protein surface in the Finally, a large “nonpolar” area is present for both Ccc2a and proximity of the copper binding region. These residues are mbd4 proteins adjacent to the metal binding site in a region 67 57 60 65 61 53 37 38 35 Glu , Glu , Glu , Asp , Asp , and Asp . In particular, composed of residues Leu , Val , and Ile . In fact the side 65 61 60 38 Asp , Asp , and Glu are conserved in the metal-binding chain of Val extends into the solvent, and in Atx1 this residue domain of a large number of metal-transporting ATPases. The is replaced with Glu (Fig. 11A). These solvent-exposed nonpolar Solution Structure of Cu(I) and Apo Forms of Ccc2a 8425 FIG. 13. Electrostatic potential sur- face of the Cu(I)-Ccc2a (A), Ag(I)- mdb4 (B), and Cu(I)-Atx1 (C). The pos- itively charged, negatively charged, and neutral amino acids are represented in blue, red, and white, respectively. Copper ion is represented in green, silver ion in teal, and cysteine sulfur in yellow.In A and C, the residues that might have a role in molecular recognition and copper transfer are indicated. In B, the negative residues that form a negative region close to metal binding loop are indicated. The figure was generated with the program MOLMOL (45). regions may be important after transfer of copper from Atx1 to and arginine residues on the Atx1 (17). A phenylalanine is Ccc2a. For instance, in the subsequent steps of the original found in Ccc2 in place of the pivotal Lys residue of Atx1. Side copper-trafficking mechanism, Cu(I) is transferred to a cytoso- chains at this site abut the metal binding loop and are antici- lic face of the membrane-spanning ATPase (15). The fate of the pated to not only play an important role in determining the Cu(I) bound by Ccc2a is not known, but it is speculated to be stability of this region of the protein but are likely to control transferred to a cation translocation site within the membrane key steps in the metal transfer mechanism. portions of Ccc2, such as the canonical CPC motif. In an exten- REFERENCES sion of this model, we propose that a hydrophobic patch on 1. Solioz, M. & Vulpe, C. (1996) Trends Biochem. Sci. 21, 237–241 mobile N-terminal domains of Ccc2a or mbd4 can serve as a site 2. Vulpe, C., Levinson, B., Whitney, S., Packman, S. & Gitschier, J. (1993) Nat. for interaction with other hydrophobic sites essential for copper Genet. 3, 7–13 movement across the membrane. After transfer and ATP-in- 3. Chelly, J., Tumer, Z., Tonnesen, T., Petterson, A., Ishikawa-Brush, Y., Tom- merup, N., Monaco, A. P. & Horn, N. (1993) Nat. Genet. 3, 14 –19 duced conformational changes, the Cu(I) would be subse- 4. Mercer, J. F., Livingston, J., Hall, B., Paynter, J. A., Begy, C., quently released to the interior of the ATPase-containing ves- Chandrasekharappa. S., Lockhart, P., Grimes, A., Bhave, M., Siemieniak, D. & Glover, T. W. (1993) Nat. Genet. 3, 20 –25 icle. Tests of this model are under way. 5. Bull, P. C., Thomas, G. R., Rommens, J. M., Forbes, J. R. & Cox, D. W. (1993) Nat. Genet. 5, 327–337 CONCLUSION 6. Tanzi, R. E., Petrukhin, K., Chernov, I., Pellequer, J. L., Wasco, W., Ross, B., This paper presents the first report of the solution structure Romano, D. M., Parano, E., Pavone, L., Brzustowicz, L. M., Devoto, M., Peppercorn, J., Bush, A. I., Sternlieb, I., Pirastu, M., Gusella, J. F., of a copper-transporting ATPase domain bound to the native Evgrafov, O., Penchaszadeh, G. K., Honig, B., Edelman, I. S., Soares, M. B., metal ion Cu(I). The fold is similar to its physiological partner, Scheinberg, I. H. & Gilliam, T. C. (1993) Nat. Genet. 5, 344 –350 7. Fu, D., Beeler, T. J. & Dunn, T. M. (1995) Yeast 11, 283–293 the cytoplasmic copper chaperone Atx1 (17) and is even more 8. Yuan, D. S., Stearman, R., Dancis, A., Dunn, T., Beeler, T. & Klausner, R. D. similar to the structure of a metal-binding domain of one of its (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2632–2636 human orthologs, the Menkes’ disease protein (20). Unlike 9. Stearman, R., Yuan, D., Yamaguchi-Iwan, Y., Klausner, R. D. & Dancis, A. (1996) Science 271, 1552–1557 other structurally characterized members of this family, both of 10. Hung, I. H., Suzuki, M., Yamaguchi, Y., Yuan, D. S., Klausner, R. D. & Gitlin, the metal-binding cysteines of Ccc2a are located in a helical J. D. (1997) J. Biol. Chem. 272, 21461–21466 11. Forbes, J. R., Hsi, G. & Cox, D. W. (1999) J. Biol. Chem. 274, 12408 –12413 region, and the metal is more accessible to solution. Compari- 12. Larin, D., Mekios, C., Das, K., Ross, B., Yang, A. S. & Gilliam, C. T. (1999) sons of the cytosolic domains of the transporters with the J. Biol. Chem. 274, 28497–28504 chaperones reveal complementary features that are important 13. Yamaguchi, Y., Heiny, M. E., Suzuki, M. & Gitlin, J. D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14030 –14035 in their respective copper-trafficking functions. First, unlike 14. Lutsenko, S., Petrukhin, K., Cooper, M. J., Gilliam, C. T. & Kaplan, J. H. Atx1, few conformational changes are observed in Ccc2a upon (1997) J. Biol. Chem. 272, 14030 –14035 copper release, and the metal binding region is well defined in 15. Pufahl, R., Singer, C. P., Peariso, K. L., Lin, S.-J., Schmidt, P. J., Fahrni, C. J., Cizewski Culotta, V., Penner-Hahn, J. E. & O’Halloran, T. V. (1997) Science both the apo and holo forms of this domain. In the case of 278, 853– 856 apo-Atx1, the amide NH for residues close to the copper bind- 16. Huffman, D. L. & O’Halloran, T. V. (2000) J. Biol. Chem. 275, 18611–18614 17. Rosenzweig, A. C., Huffman, D. L., Hou, M. Y., Wernimont, A. K., Pufahl, R. A. ing, in particular Cys , are not detected, and this disappear- & O’Halloran, T. V. (1999) Structure 7, 605– 617 ance could be ascribed to an increased disorder/mobility of the 18. Portnoy, M. E., Rosenzweig, A. C., Rae, T., Huffman, D. L., O’Halloran, T. V. loop region. In contrast, apo-Ccc2a exhibits a higher degree of & Cizewski Culotta, V. (1999) J. Biol. Chem. 274, 15041–15045 19. Lamb, A. L., Wernimont, A. K., Pufahl, R. A., Culotta, V. C., O’Halloran, T. V. order and both amide HN of the metal-binding cysteines are & Rosenzweig, A. C. (1999) Nat. Struct. Biol. 6, 724 –729 detected. Furthermore, NOE signal intensity is significant for 20. Gitschier, J., Moffat, B., Reilly, D., Wood, W. I. & Fairbrother, W. J. (1998) Nat. 13 16 Struct. Biol. 5, 47–54 both Cys and Cys in apo-Ccc2. Together, these results sug- 21. Steele, R. A. & Opella, S. J. (1997) Biochemistry 36, 6885– 6895 gest a significantly greater stability of the Ccc2a domain with 22. Eriksson, P.-O. & Sahlman, L. (1993) J. Biomol. NMR 3, 613– 626 respect to Atx1. 23. Wimmer, R., Herrmann, T., Solioz, M. & Wu ¨ thrich, K. (1999) J. Biol. Chem. 274, 22597–22603 Finally, there are several mechanistically important aspects 24. Lin, S. J., Pufahl, R., Dancis, A., O’Halloran, T. V. & Culotta, V. C. (1997) of the Ccc2a surface that differ from Atx1. One prominent J. Biol. Chem. 272, 9215–9220 25. Bax, A. & Davis, D. G. (1985) J. Magn. Reson. 63, 207–213 feature is a negative patch (Fig. 13) formed by glutamate and 26. Griesinger, C., Otting, G., Wu ¨ thrich, K. & Ernst, R. R. (1988) J. Am. Chem. aspartate residues in the proximity of the copper binding re- Soc. 110, 7870 –7872 gion that could interact with complementary array of lysine 27. Macura, S., Wu ¨ thrich, K. & Ernst, R. R. (1982) J. Magn. Reson. 47, 351–357 8426 Solution Structure of Cu(I) and Apo Forms of Ccc2a 28. Marion, D. & Wu ¨ thrich, K. (1983) Biochem. Biophys. Res. Commun. 113, 38. Banci, L., Benedetto, M., Bertini, I., Del Conte, R., Piccioli, M. & Viezzoli, M. S. 967–974 (1998) Biochemistry 37, 11780 –11791 29. Palmer, A. G., III, Cavanagh, J., Wright, P. E. & Rance, M. (1991) J. Magn. 39. Borgias, B., Thomas, P. D. & James, T. L. (1989) Complete Relaxation Matrix Reson. 93, 151–170 Analysis (CORMA), University of California, San Francisco 30. Kay, L. E., Keifer, P. & Saarinen, T. (1992) J. Am. Chem. Soc. 114, 40. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. (1993) 10663–10665 J. Appl. Crystallogr. 26, 283–291 31. Schleucher, J., Schwendinger, M., Sattler, M., Schmidt, P., Schedletzky, O., 41. Laskowski, R. A., Rullmann, J. A. C., MacArthur, M. W., Kaptein, R. & Glaser, S. J., Sorensen, O. W. & Griesinger, C. (1994) J. Biomol. NMR 4, Thornton, J. M. (1996) J. Biomol. NMR 8, 477– 486 301–306 42. Bax, A. & Wang, A. C. (1995) J. Am. Chem. Soc. 117, 1810 –1813 32. Kay, L. E., Marion, D. & Bax, A. (1989) J. Magn. Reson. 84, 72– 84 43. Weisemann, R., Ru ¨ terjans, H., Schwalbe, H., Schleucher, J., Bermel, W. & 33. Vuister, G. W. & Bax, A. (1993) J. Am. Chem. Soc. 115, 7772–7777 Griesinger, C. (1994) J. Biomol. NMR 4, 231–240 34. Piotto, M., Saudek, V. & Sklenar, V. (1992) J. Biomol. NMR 2, 661– 666 44. Rosenzweig, A. C. & O’Halloran, T. V. (2000) Curr. Opin. Chem. Biol. 4, 35. Gu ¨ ntert, P., Braun, W. & Wu ¨ thrich, K. (1991) J. Mol. Biol. 217, 517–530 140 –147 36. Gu ¨ ntert, P., Mumenthaler, C. & Wu ¨ thrich, K. (1997) J. Mol. Biol. 273, 283–298 45. Koradi, R., Billeter, M. & Wu ¨ thrich, K. (1996) J. Mol. Graphics 14, 51–55 37. Pearlman, D. A., Case, D. A., Caldwell, J. W., Ross, W. S., Cheatham, T. E., Ferguson, D. M., Seibel, G. L., Singh, U. C., Weiner, P. K. & Kollman, P. A. 46. Arnesano, F., Banci, L., Bertini, I., Huffman, D. L., & O’Halloran, T. V. (2001) (1997) AMBER, version 5.0, University of California, San Francisco Biochemistry 40, 1528 –1539 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Solution Structure of the Yeast Copper Transporter Domain Ccc2a in the Apo and Cu(I)-loaded States

Banci, Lucia; Bertini, Ivano; Ciofi-Baffoni, Simone; Huffman, David L.; O'Halloran, Thomas V.
Journal of Biological Chemistry – Mar 1, 2001
Download PDF
12 pages

Loading next page...
 
/lp/unpaywall/solution-structure-of-the-yeast-copper-transporter-domain-ccc2a-in-the-tc7P9Fgg0D

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Unpaywall
ISSN
0021-9258
DOI
10.1074/jbc.m008389200
Publisher site
See Article on Publisher Site

Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 11, Issue of March 16, pp. 8415–8426, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Solution Structure of the Yeast Copper Transporter Domain Ccc2a in the Apo and Cu(I)-loaded States* Received for publication, September 13, 2000, and in revised form, November 15, 2000 Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M008389200 Lucia Banci‡, Ivano Bertini‡§, Simone Ciofi-Baffoni‡, David L. Huffman¶, and Thomas V. O’Halloran¶i** From the ‡Magnetic Resonance Center and Department of Chemistry, University of Florence, Via Luigi Sacconi 6, Sesto Fiorentino, Florence, 50019, Italy and the Departments of ¶Chemistry and iBiochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208 Ccc2 is an intracellular copper transporter in Saccha- minus of the copper-transporting ATPases contains one or romyces cerevisiae and is a physiological target of the more metal-binding domains (2–7) characterized by a GMX- copper chaperone Atx1. Here we describe the solution CXXC motif. In humans, this intracellular copper pump is structure of the first N-terminal MTCXXC metal-binding encoded by the Wilson’s and Menkes’ disease genes (2– 6), and domain, Ccc2a, both in the presence and absence of a functional homologue (Ccc2) has been characterized in yeast Cu(I). For Cu(I)-Ccc2a, 1944 meaningful nuclear Over- (7). Copper is incorporated into trans-Golgi vesicles via the hauser effects were used to obtain a family of 35 struc- action of Ccc2 and ultimately into the multicopper oxidase tures with root mean square deviation to the average Fet3p, which translocates to the plasma membrane and works structure of 0.36 6 0.06 Å for the backbone and 0.79 6 in conjunction with an iron permease to mediate high affinity 0.05 Å for the heavy atoms. For apo-Ccc2a, 1970 mean- iron uptake (8, 9). Both the Wilson’s and Menkes’ disease ingful nuclear Overhauser effects have been used with proteins complement the function of Ccc2 in this pathway J to obtain a family of 35 structures with root HNHa (10 –12). The Menkes CPx-type ATPase contains six N-termi- mean square deviation to the average structure of 0.38 6 nal GMXCXXC motifs (2– 4), is located in the trans-Golgi net- 0.06 Å for the backbone and 0.82 6 0.07 Å for the heavy work, and translocates copper across intracellular membranes babbab, ferrodoxin-like atoms. The protein exhibits a into the secretory pathway (13). Metal binding studies on the fold similar to that of its target Atx1 and that of a human complete N-terminal cytoplasmic region have established that counterpart, the fourth metal-binding domain of the this region binds Cu(I) selectively (relative to cadmium, cobalt, Menkes protein. The overall fold remains unchanged or zinc) with a stoichiometry of one copper per metal-binding upon copper loading, but the copper-binding site itself domain (14). becomes less disordered. The helical context of the cop- The yeast metallochaperone Atx1 is a cytosolic Cu(I) receptor per-binding site, and the copper-induced conforma- that delivers its metal ion cargo to Ccc2 (15). The thermody- tional changes in Ccc2a differ from those in Atx1. Ccc2a presents a conserved acidic surface which complements namic gradient for metal transfer between Atx1 and the first the basic surface of Atx1 and a hydrophobic surface. metal-binding domain of Ccc2 (Ccc2a) is shallow, yet copper These results open new mechanistic aspects of copper transfer is facile, suggesting that Atx1 works like an enzyme to transporter domains with physiological copper donor catalyze the rate of copper transfer between partners (16). A and acceptor proteins. high resolution (1.02-Å) x-ray crystallographic structure of Hg(II)-Atx1 reveals that the mercury is coordinated in a biden- tate fashion from two cysteine sulfurs with a S–Hg–S bond Saccharomyces cerevisiae Ccc2 is a member of a class of angle of 167° (17). Mutation of several conserved lysines on the proteins that transport heavy metals across vesicular mem- surface of Atx1 greatly reduces the copper-dependent interac- branes (1). Members of this family, referred to as P-type or tion of Atx1 and Ccc2 in vivo (15, 18). The Atx1 metallochap- CPx-type ATPases, have been identified in a variety of bacte- erone (17), domain I of the copper chaperone for superoxide ria, yeast, nematodes, and mammals. The cytoplasmic N ter- dismutase (CCS) (19), and the fourth metal-binding domain of the Menkes protein (20) all adopt a babbab structural fold. This same structural fold is found in the mercury-binding pro- * This work was supported by European Community Contract HPRI- tein MerP (21, 22) and the putative copper chaperone CopZ CT-1999-00009, Italian Consiglio Nazionale delle Ricerche (Progetto Finalizzato Biotecnologie 99.00286.PF49), Ministero della Universita`e (23). In all of these domains, the cysteine ligands are located delle Ricera Scientifica e Tecnologica (MURST), National Institutes of within the first loop and the first helix. Health Grant GM 54111 (to T. V. O.), and Molecular Toxicology Train- The mechanism of copper transfer between Atx1 and Ccc2 is ing Program Postdoctoral Fellowship 5T32ES07284 (to D. L. H.). The proposed to involve a series of two- and three-coordinate inter- costs of publication of this article were defrayed in part by the payment mediates (15, 16). The factors responsible for facile and revers- of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate ible copper transfer between Atx1 and Ccc2a are probably this fact. mediated in part by metal-dependent conformational changes § To whom correspondence may be addressed: Prof. Ivano Bertini, in Atx1 and Ccc2. In Atx1, copper release involves a series of Magnetic Resonance Center and Department of Chemistry, University structural changes, in which both cysteines change conforma- of Florence, Via L. Sacconi 6, Sesto Fiorentino, Florence, 50019 Italy. Tel.: 39-055-4574272; Fax: 39-055-4574271; E-mail: bertini@ tion (46). In apo-Atx1, the loop is not well defined. We report cerm.unifi.it. here the solution structures of Cu(I)-Ccc2a from S. cerevisiae ** A fellow of the John Simon Guggenheim Foundation. To whom obtained for a nonlabeled sample and its reduced apo form, correspondence may be addressed: Dept. of Chemistry, Northwestern obtained for a N-labeled sample. Relative to Atx1, the first University, 2145 Sheridan Rd., Evanston, IL 60208. Tel.: 847-491-5060; Fax: 847-491-7713; E-mail: [email protected]. loop is well defined, and Cu(I) binding induces fewer changes in This paper is available on line at http://www.jbc.org 8415 This is an Open Access article under the CC BY license. 8416 Solution Structure of Cu(I) and Apo Forms of Ccc2a FIG.1. H 800-MHz NMR spectra of apo-Ccc2a (A) and Cu(I)-Ccc2a (B) proteins at 298 K and pH 7.0 in 100 mM phosphate buffer. The protein concentrations were 2.7 and 1.2 mM, respectively. respectively. Cu(I)-Ccc2a was prepared as previously described (16), the conformation of Ccc2a. Unlike Cu(I)-Atx1, the metal bind- and the buffer was exchanged by ultrafiltration into 100 mM sodium ing residues in Cu(I)-Ccc2a are on the surface of a helix, and phosphate, pH 7, 90% H O, 10% D O. The metal/protein ratio was 1.2 2 2 the copper cargo is more accessible to incoming ligands in the with a protein concentration of 1.2 mM. No exogenous thiols were added latter. Finally, a negative patch is observed on the surface of to the sample buffers. Approximately 0.6 ml of sample was loaded into Ccc2a in a site that corresponds to a positive patch on Atx1, 535-PP 5-mm quartz NMR tubes (Wilmad), which were capped with a suggesting that a complementary docking interface is em- latex serum cap in the VacAtmospheres chamber. ployed in the copper transfer mechanism. NMR Spectroscopy—The NMR spectra were acquired on Avance 800 and 600 Bruker spectrometers operating at a proton nominal frequency EXPERIMENTAL PROCEDURES of 800.13 and 600.13 MHz, respectively. A QXI probe has been used on Sample Isolation and Preparation—Ccc2a was uniformly labeled the Avance 800 spectrometer, and a triple resonance (TXI) 5-mm probe with N by expressing the protein in Escherichia coli strain BL21(DE3) has been used on the 600 spectrometer. All probes were equipped with (Novagen), transformed with pDLHV021, in minimal media supple- pulsed field gradients along the z axis. Total correlation spectroscopy mented with NH Cl. Unlabeled Ccc2a was isolated as described pre- (25, 26) spectra were recorded on the 600-MHz spectrometer with a viously (16). Ccc2a( N) was purified from the cell pellet by freeze-thaw spin-lock time of 100 ms, a recycle time of 1 s and a spectral window of extraction with 20 mM MES /Na, pH 6.0, followed by streptomycin 14 ppm. Two-dimensional NOESY maps (27, 28) were acquired on the sulfate precipitation (5%, w/v) of the extract. The supernatant was 800-MHz spectrometer with a mixing time of 100 ms, a recycle time of further purified by repetitive runs on Superdex 75 (Amersham Phar- 1 s, and a spectral window of 14 ppm. 15 15 1 macia Biotech). The yield of labeled protein was ;2 mg/liter of culture. On the N apo-Ccc2a sample, a two-dimensional N- H hetero- Protein samples were purified and stored in the presence of reducing nuclear single quantum coherence (29 –31) map was obtained at 800 agent. NMR samples were prepared in a Vac Atmospheres nitrogen MHz with an INEPT delay of 2.66 ms, a recycle time of 1 s, and spectral atmosphere chamber at 12 °C. Protein concentrations were determined 1 15 windows of 14 and 33 ppm for the H and N dimensions, respectively. by the Bradford assay and calibrated as described previously (16). 15 A three-dimensional NOESY- N heteronuclear multiple quantum co- Copper concentration was determined by ICP-AES. The NMR sample of 1 15 herence experiment (32) was recorded with 290 ( H) 3 96 ( N) 3 2048 apo-Ccc2a and apo-Ccc2a( N) was prepared by exchanging the reduced 1 ( H) data points on the 600-MHz spectrometer. The INEPT delay was form of the protein into 100 mM sodium phosphate, pH 7, 90% H O, 10% set to 5.4 ms, the mixing time was 100 ms, and the carrier frequency D O via ultrafiltration; the final concentrations were 2.7 and 3.3 mM, was set in the center of the amide proton region, at 7.35 ppm. Spectral windows of 14, 14, and 29 ppm were used for the direct H dimension 1 15 1 and the indirect H and N dimensions. A HNHA experiment (33) was The abbreviations used are: MES, 4-morpholineethanesulfonic acid; performed at 600 MHz to determine J coupling constants. The HNHa r.m.s., root mean square; NOE, nuclear Overhauser effect; NOESY, 1 15 1 spectrum was recorded as a 128 ( H) 3 80 ( N) 3 2048 ( H) data set nuclear Overhauser effect spectroscopy; INEPT, insensitive nuclei en- using pulsed field gradients along the z axis. The mixing time was 100 hanced by polarization transfer; WATERGATE, water suppression by gradient-tailored excitation; REM, restrained energy minimization; ms. Spectral windows of 14, 14, and 29 ppm were used, respectively, for 1 1 15 mbd4, metal-binding domain 4. direct H dimension and the indirect H and N dimensions. Solution Structure of Cu(I) and Apo Forms of Ccc2a 8417 FIG.2. Schematic representation of the sequential and medium range NOE connectivities involving NH, Ha, and Hb protons for apo-Ccc2a (A) and Cu(I)-Ccc2a (B). The thickness of the bar indicates the intensity of NOEs. For all the experiments, quadrature detection in the indirect dimen- Hydrogen bond constraints were introduced for backbone amide pro- sions was performed in the time-proportional phase incrementation tons that were found to be within hydrogen bond distance and to have mode (28), and water suppression was achieved through WATERGATE the correct orientation with respect to hydrogen bond acceptors in sequence (34). All two-dimensional data consisted of 4K data points in structural models obtained without inclusion of these constraints. The the acquisition dimension and of 1K experiments in the indirect dimen- distance between the NH proton and the hydrogen bond acceptor was sion. All three- and two-dimensional spectra were collected at 298 K, constrained to be in the 1.8 –2.4 Å interval by inclusion of the corre- processed using the standard Bruker software (XWINNMR), and ana- sponding upper and lower distance limits in structure calculations. In lyzed on IBM RISC 6000 computers through the XEASY program. addition, upper and lower distance limits of 3.0 and 2.7 Å between the Constraints Used in Structure Calculations—The peaks used for the N and the acceptor atoms were also included. structure calculations were integrated in the two-dimensional NOESY Structure Calculations—The structure calculations were performed map acquired at 298 K in H O. Intensities of dipolar connectivities were using DYANA (36). 200 random conformers were annealed in 10000 converted into upper distance limits, to be used as input for structure steps using NOE and J values (when available) constraints. The 35 calculations, by using the approach provided by the program CALIBA conformers with the lowest target function constitute the final family. (35). The calibration curves were adjusted iteratively as the structure The copper ion was included in the calculations by adding a new residue calculations proceeded. Stereospecific assignments of diastereotopic in the amino acid sequence, formed by a chain of dummy atoms that protons were obtained using the program GLOMSA (35). J cou- have their van der Waals radii set to 0 so that it can freely penetrate HNHa pling constants were correlated to the backbone torsion angle f by into the protein and one atom with a radius of 1.4 Å, which mimics the 13 16 means of the appropriate Karplus curve (33). These angles were used as copper ion. The sulfur atoms of Cys and Cys were linked to the metal constraints in the DYANA calculations and restrained energy minimi- ion through upper distance limits of 2.5 Å. This approach does not zation (REM) refinement. impose any fixed orientation of the ligands with respect to the copper. 8418 Solution Structure of Cu(I) and Apo Forms of Ccc2a FIG.4. A, number of meaningful NOEs per residue for apo-Ccc2a. White, gray, and black bars indicate intraresidue, sequential, and me- dium/long range connectivities, respectively; B, r.m.s. deviation per residue to the mean structure of apo-Ccc2a for the backbone (filled squares) and all heavy atoms (filled circles) of the REM structure family FIG.3. Schematic representation of long range connectivities of 35 conformers. for apo-Ccc2a (A) and Cu(I)-Ccc2a (B). Segments perpendicular to the diagonal of the plot represent pairs of anti-parallel b-strands. Assignments of the resonances of apo-Ccc2a started from the analysis of the N heteronuclear single quantum coherence REM was then applied within the molecular mechanics and dynamic 15 1 map, which allowed the identification of the N and HN module of SANDER (37). The force field parameters for the copper(I) ion were adapted from similar systems (38). In particular, no constraint on resonances. Then through the analysis of the three-dimen- 13 16 the S(Cys )–Cu-S(Cys ) angle was used. The values of NOE and sional NOESY-heteronuclear multiple quantum coherence and torsion angle potentials were calculated with force constants of 50 kcal of two-dimensional NOESY and total correlation spectroscopy, 21 22 mol Å . the sequence-specific assignment was performed. The assign- The program CORMA (39), which is based on relaxation matrix ment of the Cu(I)-Ccc2a derivative was performed through the calculations, was used to back calculate the NOESY cross-peaks from analysis of two-dimensional NOESY and total correlation spec- the calculated structure to check the consistency of the analysis. The quality of the structure was evaluated in terms of deviations from ideal troscopy maps only. Resonances for all 72 residues both for the bond lengths and bond angles and through Ramachandran plots, ob- apo and Cu(I) forms of Ccc2a have been assigned. In the apo tained using the programs PROCHECK (40) and PROCHECK-NMR and in the Cu(I) protein, about 97 and 98% of the proton (41). Structure calculations and analyses were performed on IBM RISC resonances, respectively, could be located in the maps, and all 6000 computers. of the N resonances have been assigned, with the exception of 14 15 14 RESULTS Ser and Ala in the apo form and with the exception of Ser 1 15 in the Cu(I) form. The H and N resonance assignments of the Sequence-specific Assignment of Apo-Ccc2a and Cu(I)- apo and Cu(I) forms are reported in Tables I and II of the Ccc2a—The H NMR spectra of apo-Ccc2a and Cu(I)-Ccc2a are supplementary materials, respectively. reported in Fig. 1, A and B, respectively. The most relevant Secondary Structure from NMR Data—The elements of sec- difference between apo and Cu(I) form is observed in the HN ondary structure were identified by analyzing the pattern of region. In particular, the HN resonance of Thr is broader in assigned NOEs. Backbone short, medium range NOEs were the apo form than in Cu(I)-Ccc2a and has a change in the shift used to generate Fig. 2, A and B. From their analysis, it is from 9.72 ppm, in the apo form, to 9.21 ppm, in the Cu(I) form. apparent that the secondary structure is not significantly af- In the H NMR spectrum of the Cu(I) form, it is also possible to identify the HN resonance of Ala at 9.50 ppm, a residue very close to the cysteine binding motif, that cannot be detected in Supplementary materials may be accessed at the CERM site, under the apo form. Structural Biology, on the World Wide Web. Solution Structure of Cu(I) and Apo Forms of Ccc2a 8419 TABLE I Statistical analysis of the final REM family and the mean structure of apo-Ccc2a from S. cerevisiae REM indicates the energy-minimized family of 35 structures; ,REM. is the energy-minimized average structure obtained from the coordinates of the individual REM structures. REM Parameters ,REM. (35 structures) r.s.m. violations per experimental distance constraint (Å) Intraresidue (257) 0.0092 6 0.0025 0.0074 Sequential (496) 0.0066 6 0.0010 0.0063 Medium range (511) 0.0082 6 0.0010 0.0073 Long range (706) 0.0058 6 0.0013 0.0047 Total (1970) 0.0073 6 0.0008 0.0062 Average number of violations per structure Intraresidue 3.6 6 1.3 4 Sequential 5.5 6 1.4 5 Medium range 6.5 6 1.2 5 Long range 6.2 6 2.2 4 Total 21.7 6 2.7 18 Average no. of NOE violations larger than 0.3 Å 0.00 6 0.00 0 Average no. of f violations larger than 5° 0.00 6 0.00 0 Largest residual NOE violation (Å) 0.23 0.09 Average NOE and torsion deviations (Å ) 0.14 6 0.02 0.11 Structural analysis Residues in most favourable regions (%) 80.0 82.1 Residues in allowed regions (%) 17.3 17.9 Residues in generously allowed regions (%) 2.0 0.0 Residues in disallowed regions (%) 0.8 0.0 The number of experimental constraints for each class is reported in parenthesis. Medium range distance constraints are those between residues (i, i 1 2), (i, i 1 3), (i, i 1 4), and (i, i 1 5). As it results from the Ramachandran plot analysis. fected by the presence or absence of the copper ion. In Ccc2a, two elements of helical secondary structure can be predicted, which are characterized by a high number of sequential and medium range connectivities such as d (i, i 1 1), d (i, i 1 2), NN NN d (i, i 1 3), d (i, i 1 4), and d (i, i 1 3). The two helices aN aN ab involve residues 14 –27 and 51– 63 in the apo form. The pres- ence of medium range connectivities such as d (i, i 1 2), d NN aN (i, i 1 2), and d (i, i 1 3) for residue Cys in the Cu(I) form NN with respect to the apo form gives evidence that Cys belongs a1 in the Cu(I) form. All of the to the the beginning of helix backbone NOEs were used to generate Fig. 3, A and B, from which it is possible to recognize the presence of four antipar- allel b-sheets in both apo and Cu(I) forms, involving residues 2– 8, 29 –35, 40 – 46, and 65–71. Fig. 3 also shows that the typical folding pattern of the copper chaperones, “open-faced b FIG.5. Backbone atoms for the solution structure of apo-Ccc2a -sandwich” fold (b1-a1-b2-b3-a2-b4) (17, 20, 21), is present also as a tube with variable radius, proportional to the backbone r.m.s. deviation value of each residue. The side chains of Cys and in this protein, both in the presence and absence of copper. Cys are also shown. The figure was generated with the program Solution Structure Calculations and Analysis of Apo- MOLMOL (45). Ccc2a—A total of 3785 NOESY cross-peaks were assigned, integrated, and transformed in upper distance limits with the program CALIBA (35). They corresponded to 2314 unique up- an average target function of 0.64 6 0.10 Å and average r.m.s. per distance limits, of which 1970 were found to be meaningful deviation values over all of the 72 residues with respect to the (nonmeaningful distance constraints are those that cannot be mean structure of 0.39 6 0.05 Å for the backbone and of 0.80 6 violated in any structure conformation and those involving 0.07 Å for the heavy atoms. The family of conformers was then proton pairs at fixed distance). The number of NOEs per resi- subjected to further refinement through energy minimization due is reported in Fig. 4A. The average number of NOEs per (37). The REM family has an average target function of 0.14 6 2 2 residue is 32 for apo-Ccc2a, of which 27 are meaningful. 35 0.02 Å , to which the NOE contribution is 0.13 Å , while the 3 2 torsional angle one is 0.01 Å . The average r.m.s. deviation J couplings were obtained from the HNHA three-dimen- HNHa sional spectrum, which were translated into dihedral angles values for the family with respect to the mean structure are through the standard equation. For 0.38 6 0.06 Å for the backbone and 0.82 6 0.07 Å for the heavy J values of .8 and HNHa ,4.5 Hz, the f angle was assumed to be between 2155 and atoms for all of the amino acids in the sequence. The r.m.s. 285° and between 280 and 230°, respectively (42, 43). Hydro- deviation values per residue of the final REM family to the gen bond constraints for 31 amide protons were used at later mean structure are shown in Fig. 4B. stages of structural calculations. A total of 38 proton pairs were The final family of conformers was analyzed with PRO- stereospecifically assigned through the program GLOMSA CHECK-NMR (41), and results are reported in Table I. Accord- (35). The constraints used for structure calculations and the ing to this program, the secondary structure elements in the stereospecific assignments are reported in the supplementary energy-minimized mean structure involve residues 2–9 (b1), materials. 13–26 (a1), 29 –36 (b2), 40 – 47 (b3), 51– 63 (a2), and 65–70 (b4). The 35 conformers constituting the final DYANA family had Analysis of the NOE patterns has led to somewhat similar 8420 Solution Structure of Cu(I) and Apo Forms of Ccc2a conclusion (see above). It is worth noting that in contrast to NOE secondary structure analysis above, Cys is assigned to helix a1 in the energy-minimized mean structure of the apo form. In this energy-minimized average structure, 82.1% of the residues are in the most favored regions of the Ramachandran plot, and 17.9% of the residues are in the allowed regions. No residues are in the disallowed regions (Table I). The structure of apo-Ccc2a, shown in Fig. 5, is well defined all over its sequence. All of the a-helices and antiparallel b-sheet are very well defined, with an average backbone r.m.s. deviation lower than 0.38 Å. The largest backbone r.m.s. devi- ation values are obtained for residues 12–14 and residue 48. The high r.m.s. deviation values of residues 12–14 (0.74 Å) are due to the paucity of NOEs in this region (Fig. 4A) that consti- tutes loop 1 and the beginning of helix a1. Indeed, this region 14 15 contains Ser and Ala , whose HN resonances have not been identified and are a break in the sequential connectivities. This region includes Cys , one of the copper ligands. The side chain of Cys is disordered in the apo state and spans different conformations due to the small number of NOEs. On the con- trary, the other copper ligand Cys , which belongs to the first a-helix, is very well defined (r.m.s. deviation BB, 0.22 Å; r.m.s. 13 16 deviation HA, 0.37 Å). The side chains of Cys and Cys are very close; the distance between the two sulfur atoms in the average structure is 5.6 Å. Solution Structure Calculations and Analysis of Cu(I)- Ccc2a—A total of 3866 NOESY cross-peaks were assigned, integrated, and transformed in upper distance limits with the program CALIBA (35). They corresponded to 2338 upper dis- tance limits, of which 1944 were found to be meaningful. The number of NOEs per residue is reported in Fig. 6A. The average number of NOEs per residue is 32, of which 27 are meaningful. Hydrogen bond constraints for 22 amide protons were used at later stages of structural calculations. A total of 47 proton pairs were stereospecifically assigned through the program FIG.6. A, number of meaningful NOEs per residue for Cu(I)-Ccc2a. GLOMSA (35). The constraints used for structure calculations White, gray, and black bars indicate intraresidue, sequential, and me- dium/long range connectivities, respectively. B, r.m.s. deviation per and the stereospecific assignments are reported in the supple- 2 residue to the mean structure of Cu(I)-Ccc2a for the backbone (filled mentary materials. squares) and all heavy atoms (filled circles) of the REM structure family The 35 conformers constituting the final DYANA family had of 35 conformers. an average target function of 0.48 6 0.11 Å and an average r.m.s. deviation value over all of the 72 residues with respect to form. Indeed, the backbone r.m.s. deviation values with respect the mean structure of 0.39 6 0.06 Å for the backbone and of to the mean structure for residues in the less well defined 0.79 6 0.04 Å for the heavy atom. The family of conformers was region of apo (10 –14) decrease from 0.64 Å in the apo form to then subjected to further refinement through energy minimi- 0.27 Å in the Cu(I) form, and the side chain of Cys that in the zation (37). The REM family has an average target function of apo form is disordered (r.m.s. deviation BB, 0.80 Å; r.m.s. 0.26 6 0.03 Å . The average r.m.s. deviation values for the deviation HA, 1.17 Å) is very well defined in the Cu(I) form family with respect to the mean structure are 0.36 6 0.06 Å for (r.m.s. deviation BB, 0.25 Å; r.m.s. deviation HA, 0.62 Å). the backbone and 0.79 6 0.05 Å for the heavy atoms for all of DISCUSSION the amino acids in the sequence. The r.m.s. deviation values per residue of the final REM family to the mean structure are Comparison between the Structures of Apo- and Cu-Ccc2a— shown in Fig. 6B. The solution structure of S. cerevisiae Ccc2a exhibits the The final family of conformers was analyzed with PRO- babbab folding pattern typical of copper chaperones (44) and CHECK-NMR (41), and results are reported in Table II. The the fourth metal-binding domain of the Menkes’ disease protein secondary structure elements in the energy-minimized mean (20). In the Cu-Ccc2a structure, the copper ion coordinates two 13 16 structure involve residues 2–9 (b1), 13–26 (a1), 29 –36 (b2), of the six cysteine residues, Cys and Cys . The global r.m.s. 40–47 (b3), 51– 62 (a2), and 65–70 (b4). Analysis of the NOE deviation values between the Cu(I)-bound and apo-averaged patterns has led to a similar conclusion (see above). In the minimized structures are 0.86 and 1.35 Å for backbone and energy-minimized average structure, 83.6% of the residues are heavy atoms, respectively. The r.m.s. deviation per residue is in the most favored regions of the Ramachandran plot, 13.4% of reported in Fig. 8 (dotted dashed line) and is compared with the the residues are in the allowed regions, and 3.0% are in the r.m.s. deviation values per residue for each family (apo-Ccc2a generously allowed regions. No residues are in the disallowed (solid line) or Cu(I)-Ccc2a (dotted line)) and the sum of the regions (Table II). r.m.s. deviation values of the two families (dashed line). The The structure of Cu(I)-Ccc2a, shown in Fig. 7, is also well structure definition within each family of structures is overall defined all over its sequence. All of the a-helices and antipar- very good and comparable between the two families; thus, a allel b-sheet are very well defined and also the loop that in- meaningful comparison can be undertaken. The highest back- cludes the copper binding site is more defined than in the apo bone r.m.s. deviation value between the two structures is 1.21 Solution Structure of Cu(I) and Apo Forms of Ccc2a 8421 ABLE II Statistical analysis of the final REM family and the mean structure of Cu(I)-Ccc2a from S. cerevisiae REM indicates the energy-minimized family of 35 structures; ,REM. is the energy-minimized average structure obtained from the coordinates of the individual REM structures. REM Parameters ,REM. (35 structures) r.s.m. violations per experimental distance constraint (Å) Intraresidue (272) 0.0153 6 0.0016 0.0160 Sequential (511) 0.0072 6 0.0013 0.0069 Medium range (490) 0.0103 6 0.0015 0.0102 Long range (671) 0.0102 6 0.0012 0.0106 Total (1944) 0.0105 6 0.0008 0.0106 Average number of violations per structure Intraresidue 10.4 6 2.4 11 Sequential 5.1 6 1.5 4 Medium range 9.3 6 1.9 11 Long range 12.2 6 2.0 13 Total 37.0 6 4.2 39 Average no. of NOE violations larger than 0.3 Å 0.00 6 0.00 0 Largest residual NOE violation (Å) 0.20 0.13 Average NOE deviations (Å ) 0.26 6 0.03 0.25 Structural analysis Residues in most favourable regions 79.0 83.6 Residues in allowed regions 18.6 13.4 Residues in generously allowed regions 2.0 3.0 Residues in disallowed regions 0.3 0.0 The number of experimental constraints for each class is reported in parenthesis. Medium range distance constraints are those between residues (i, i 1 2), (i, i 1 3), (i, i 1 4), and (i, i 1 5). As it results from the Ramachandran plot analysis. Å (found for Thr ). There are only few regions or amino acids in the protein where the r.m.s. deviation between the two average minimized structures is significantly larger than the r.m.s. deviation of each family when superimposing the whole structures (Fig. 8), thus indicating a meaningful differences between the two structures. Analysis of side chains reveals a significant r.m.s. deviation difference for Thr , which is in the copper binding pocket. Indeed, when copper is bound, the side 17 13 chain of Thr rotates closer to the backbone oxygen of Cys , and a hydrogen bond interaction can be readily formed, since it is found in many conformers of the family. This interaction can presumably be important to determine the optimal conforma- tion of Cys in the copper-bound state of the protein. The backbone r.m.s. deviation values for each secondary structure element are obtained either superimposing all resi- dues (global r.m.s. deviation) or three residues at a time (local r.m.s. deviation) and are reported in Table III. The highest FIG.7. Backbone atoms for the solution structure of Cu(I)- r.m.s. deviation values are found for strand b2, helix a2, and Ccc2a as a tube with variable radius, proportional to the back- loops 1, 3, and 4. The r.m.s. deviation values for all of these bone r.m.s. deviation value of each residue. The side chains of 13 16 regions but the last drop when the two structures are super- Cys , Cys , and the Cu(I) ion are also shown. The figure was generated with the program MOLMOL (45). imposed locally, indicating that the structural differences in these regions originate from some global translational displace- ments, since the local conformations are well maintained. On spect to the metal-bound state. Indeed, different NOE intensity 13 16 the contrary, loop 4 shows the highest difference in the local for proton pairs at fixed distances of Cys and Cys of Ccc2a r.m.s. deviation, and these local difference are due to variations has been observed in the two forms; Cys experiences a de- of dihedral angles. crease in intensity, while those of Cys show roughly the same Comparing the copper binding region in apo- and Cu(I)- intensity, suggesting that the binding of copper produces 13 16 Ccc2a, helix a1 (containing Cys and Cys ) is very well de- higher order in this region. Upon copper release, the most important change in the shal- fined in both forms, while loop 1, which is involved in the copper binding pocket, is less defined in the apo form (see Fig. low binding pocket is observed for the side chain of Cys ; the 8). The conformation of Cys sulfur flips away from the hydrophobic interior toward the is well defined in both structures, being the same in both the apo and metal-bound proteins, while surface (Fig. 9). Calculations of the solvent accessibility on the 13 13 Cys apo-Ccc2a structure show that exposure of Cys is remarkably is more disordered in the apo form than in the metal- bound protein. Indeed, in the apo-Ccc2a structure, loop 1 be- increased (from 23% in the copper-bound form to 36% accessi- comes more disordered (Fig. 5), and the amide NHs of residues ble surface in the apo form), while Cys is always more buried 14 and 15, belonging to helix a1, are not detected anymore, (with 7% in the copper-bound form and 11% accessible surface probably due to solvent exchange or to an increased mobility. in the apo form). The lower number of NMR constraints in the metal-binding The changes in HN and Ha chemical shifts observed between pocket (i.e. residues 12–17) of the apo form might be the result the apo and the Cu(I)-bound forms are plotted for each residue of increased conformational flexibility in this region with re- in Fig. 10, A and B, respectively, and confirm that only residues 8422 Solution Structure of Cu(I) and Apo Forms of Ccc2a FIG.8. r.m.s. deviation per residue (for backbone atoms) between the conformers of the family of apo- Ccc2a (solid line), between those of Cu(I)-Ccc2a (dotted line), and be- tween the mean structures of the two families (dashed dotted line). The sum of the r.m.s. deviation values per residue for the two families of conformers is also shown (dashed line). TABLE III Comparison of the solution structure of Cu(I)-Ccc2a with the reduced apo-Ccc2a solution structure Backbone r.m.s. deviation values for each secondary structure element are obtained either superimposing all residues (global r.m.s. deviation) or three residues at a time (local r.m.s. deviation). Secondary structure element Residue range Global backbone r.m.s. deviation to apo-Ccc2a Local backbone r.m.s. deviation to apo-Ccc2a ÅÅ b1 2–9 0.54 0.31 a1 13–26 0.54 0.12 b2 29–36 0.81 0.19 b3 40–46 0.55 0.19 a2 51–62 0.73 0.15 b4 65–70 0.58 0.22 Loop 1 10–12 0.78 0.24 Loop 2 27–28 0.66 0.21 Loop 3 37–39 0.95 0.28 Loop 4 47–50 0.75 0.43 Loop 5 63–64 0.60 0.20 FIG.9. A, backbone drawing of Cu(I)- Ccc2a (green) and apo-Ccc2a (blue). The copper ion is in yellow. Side chains of 21 64 11 38 Ile , Phe , Met , and Leu are re- ported. The secondary structure elements are indicated. The inset shows the copper binding region. very close to the metal binding pocket are affected by the loop, no significant conformational changes are detectable ei- presence of the copper ion. Indeed, several highly conserved ther as differences between their chemical shifts (Fig. 10, A and hydrophobic residues appear to play a role in maintaining an B) or as differences between the two average structures (Fig. 9). 64 11 optimal metal-ligand conformation. These include Phe and The methionine in the GMXCXXC metal binding loop (Met ) 20 16 Ile , which are in Van der Waals contact with Cys , and is highly conserved between other N-terminal, membrane-teth- 37 13 Leu , which contacts Cys and is highly conserved between ered domains of heavy metal ATPases and small metallochap- the Ccc2, Wilson, and Menkes domains as well as Atx1 and erones alike. Of all of the residues in the protein, Met shows CCS. While these side chains can stabilize the metal binding the greatest change in Ha resonances upon copper binding Solution Structure of Cu(I) and Apo Forms of Ccc2a 8423 (Fig. 10, A and B), but it is not directly involved in metal ion coordination. Instead, it points toward the hydrophobic core of the protein (Fig. 9). The contacts between this side chain and the residues surrounding it change little between the apo and Cu(I)-bound forms (Fig. 9) as judged from the observed medium and long range NOEs; however, copper occupancy does change the conformation of this residue. These results suggest that Met acts as a hydrophobic tether that anchors the metal- 37 64 binding loop via hydrophobic interactions with Leu , Phe , and Ile . The slight movements in the region are probably coupled to stabilization of the GMXCXXC domain in the pres- ence of bound metal ion. Comparison between the Solution Structures of the Fourth Metal Binding Cytosolic Domain from Menkes Copper-trans- porting ATPase and Cu(I)-Ccc2a—The structure of Cu(I)-Ccc2a is similar to the Ag(I)-bound solution structure of the fourth metal-binding domain (mbd4) of Menkes ATPase (20), the hu- man homologue of S. cerevisiae Ccc2. A sequence alignment of Ccc2a with this protein (Fig. 11A) reveals 29% identity. The structures were superimposed according to the sequence align- ment. Both proteins show the same ferrodoxin-like fold (Fig. 11B). The overall backbone r.m.s. deviation value is 1.15 Å between Cu(I)-Ccc2a and Ag(I)-mbd4. The major structural differences are represented by simple translations of the sec- ondary structure elements. All a-helices and b-strands are well superimposed or show slight displacements except for the short C-terminal b-strand. The conserved hydrophobic residues Ile , 37 64 Leu , and Phe , presumably important for maintaining opti- mal metal-binding loop conformation, have the same conforma- tion in the two structures. The biggest differences are observed in loop 1 and loop 4. Indeed, loop 1 differs in the vicinity of FIG. 10. Plot of the chemical shift differences (d(apo) 2 d(Cu(I)- 1 1 13 14 bound)) versus residue number for the HN (A) and Ha (B) Cys , which does not superimpose with Cys of mbd4. The 16 17 resonances. conformation of the other cysteine (Cys and Cys , respec- tively) is very similar between the two structures. In Cu(I)- 11C). The major structural differences are translations or Ccc2a, Cys is the first residue of helix a1, while in mbd4, changes in length of the secondary structure elements and of Cys belongs to the metal binding loop 1 (Fig. 11B). The the loops. The largest differences are found for helix a1, stabilization of the helix and the variation in the conformation b-strand 4, and loops 1, 2, and 4. The length of helix a1inthe of loop 1 in Cu(I)-Ccc2a with respect to Ag(I)-mbd4 may be Cu(I) form of both proteins is the same, but in Cu(I)-Atx1 this dictated by the different chemical properties of Cu(I) versus helix spans from residue 17 to 30, while in the Cu(I)-Ccc2a the Ag(I) and by the different kinds of residues in the vicinity of the same helix is slightly offset, starting from residue 13 and loop 1 region. ending at 26. Loop 1 exhibits conformational differences, in It is worth noting that in the refinement of the Ag(I)-mbd4 particular close to Cys . This cysteine in Cu(I)-Atx1 belongs to solution structure, a linear digonal coordination was imposed loop 1, whereas in Cu(I)-Ccc2a it belongs to helix a1. These by modifying the AMBER force field (20). In the data refine- differences in secondary structure lead to distinct positioning of ment for the Cu(I)-Ccc2a family of conformers, no S-Cu-S angle the metal-binding cysteines (see Fig. 11C) and allow the copper constraints were included. The resulting value of the S-Cu-S ion to be more exposed to solvent in Ccc2a with respect to Atx1 angle is 119 6 29°. When the S-Cu-S angle is constrained to (see below). In addition, the offset of helix a1, the different loop linearity, a digonal copper thiolate center can be refined. This size, and the presence of Pro in the Atx1 sequence (see Fig. suggests that coordination is not rigid but that Cu(I) in this 11A) determine a large conformational difference in loop 2, environment may represent a mixture of coordination numbers which is more extended toward the surface in the Cu(I)-Atx1 of two and higher or that a bent two-coordinate S-Cu-S geom- structure. etry is adopted. No other protein atoms appear close enough to The structures of apo-Ccc2a and apo-Atx1 show an overall be the third ligand in Ccc2a. A third coordinating atom, if there backbone r.m.s. deviation value of 2.93 Å (Fig. 12) and exhibit is one at all, can come only from an exogenous ligand, such as more differences than do the two copper-loaded forms (Fig. a buffer component. While no low molecular weight thiols (e.g. 11C). The b-strands 1, 2, and 3 and the loops 3 and 5 are well DTT, GSH) are present in the sample, other buffer components superimposed for the apoproteins; however, differences in could be coordinating to the Cu(I). The NMR data here do not other secondary structure elements are apparent. For example, allow us to distinguish between these possibilities. helix a1 is one full turn shorter in apo-Atx1 than in apo-Ccc2 Comparison between the Solution Structures of Ccc2a and (Fig. 12). Helix a2, which superimposes well in both forms of Atx1 for both Apo and Cu(I) States—The solution structures of Atx1 (46), is translated away from the copper-binding site in Cu(I)- and apo-Atx1 from S. cerevisiae have been recently apo-Atx1 relative to its position in apo-Ccc2a (Fig. 12). While solved (46). The global folding of Atx1 is very similar to that of the structure of Atx1 undergoes changes as a function of copper Ccc2a. When superimposing Cu(I)-Ccc2a and Cu(I)-Atx1 struc- capture and release, the Ccc2a structure remains relatively tures according to the sequence alignment (see Fig. 11A), the invariant, suggesting that the metal site in apo-Ccc2a is more overall backbone r.m.s. deviation value is 2.8 Å. Helix a2; preorganized than in apo-Atx1. This is one of the key structural b-strands 1, 2, 3; and loops 3 and 5 are well superimposed (Fig. differences between the Atx1 metallochaperone family and the 8424 Solution Structure of Cu(I) and Apo Forms of Ccc2a FIG. 11. A, sequence alignment of the Ccc2a amino acid sequence from S. cerevisiae with the sequences of the fourth metal-binding domain from Menkes-transporting ATPase (mbd4) (Protein Data Bank accession number 1aw0) (20) and of Atx1. The positions of the Ccc2a secondary structure elements (as found in the mean Cu(I) structure) are shown at the top. b-strands are in blue, a helices are shown in orange, and loop regions are in yellow. Each sequence is color-shaded according to secondary structure element, as found in their metal-bound structures. Residues that are highly similar or conserved are indicated, respectively, by the l and * below the sequences. B, comparison of the backbone of Cu(I)-Ccc2a (blue) and Ag(I)-mbd4 (green) structures (20). C, comparison of backbone of Cu(I)-Ccc2a (blue) and Cu(I)-Atx1 (green). The copper ion and the cysteine ligands are also shown. The secondary structure elements are indicated. homologous metal-binding domains of the copper-transporting P-type ATPases. Another important difference between these copper donor and acceptor proteins is apparent in Fig. 13, A and B, namely the accessibility of the copper to incoming nucleophiles. The solvent-accessible surface of the Cu(I) center in Cu-Atx1 is 9% but over 18% in Cu-Ccc2a. Access to the Cu(I) in Atx1 is partially obscured by Lys , which is highly conserved among copper chaperones. In Ccc2a, this residue corresponds to Phe , and the structure reveals that it extends into the hydrophobic core, away from the surface. In fact, a Phe (or a Tyr) is highly FIG. 12. Comparison of the backbone of apo-Ccc2a (blue) and conserved among all of the domains of the CPx ATPases, while apo-Atx1 (green). The cysteines involved in the copper binding are Lys is conserved among the diffusable Atx1 and CCS copper indicated in blue and green for apo-Ccc2a and apo-Atx1 structures, chaperone proteins (44). The Phe side chain packs adjacent to respectively. the conserved Met and is anticipated to contribute to the stability of the metal binding loop in Ccc2a. In contrast, the surface electrostatic potential distribution was also generated lysine at this position in Atx1 (Lys ) can access several con- for human mbd4 (Fig. 13B). A very similar negative patch, 67 62 63 formations and may play a role in partner recognition and formed by Asp , Glu , and Asp , which are conserved in the control of metal ion access. homologous yeast Ccc2a, and Asp is observed on one surface Electrostatic Surface and Structural Implications for Inter- of the protein. The electrostatic potential distribution of Cu(I)- action with Physiological Partners—Partnership between Atx1 Atx1 is almost complementary and shows the presence of 7 and Ccc2 in vivo requires several basic residues, which cluster lysines (46), in positions 24, 28, 59, 61, 62, 65, and 71, which in several sites on the surface of Atx1 (18). The structure of generate a positively charged face on the molecule, as shown in Ccc2a reveals a complementary set of acidic residues. The Fig. 13C. It is known that the ATPase Ccc2 is the target of surface electrostatic potential distribution was generated with copper delivery by Atx1 (15, 16, 24). The interaction between the program MOLMOL (45) using the refined coordinates of the Atx1 and Ccc2a could be therefore determined by the comple- Cu(I)-Ccc2a structure (Fig. 13A). This protein has a region mentary attractions between the positive cluster in Atx1 and comprising several glutamate and aspartate residues that gen- the negative region in Ccc2a. erate a negatively charged face on the protein surface in the Finally, a large “nonpolar” area is present for both Ccc2a and proximity of the copper binding region. These residues are mbd4 proteins adjacent to the metal binding site in a region 67 57 60 65 61 53 37 38 35 Glu , Glu , Glu , Asp , Asp , and Asp . In particular, composed of residues Leu , Val , and Ile . In fact the side 65 61 60 38 Asp , Asp , and Glu are conserved in the metal-binding chain of Val extends into the solvent, and in Atx1 this residue domain of a large number of metal-transporting ATPases. The is replaced with Glu (Fig. 11A). These solvent-exposed nonpolar Solution Structure of Cu(I) and Apo Forms of Ccc2a 8425 FIG. 13. Electrostatic potential sur- face of the Cu(I)-Ccc2a (A), Ag(I)- mdb4 (B), and Cu(I)-Atx1 (C). The pos- itively charged, negatively charged, and neutral amino acids are represented in blue, red, and white, respectively. Copper ion is represented in green, silver ion in teal, and cysteine sulfur in yellow.In A and C, the residues that might have a role in molecular recognition and copper transfer are indicated. In B, the negative residues that form a negative region close to metal binding loop are indicated. The figure was generated with the program MOLMOL (45). regions may be important after transfer of copper from Atx1 to and arginine residues on the Atx1 (17). A phenylalanine is Ccc2a. For instance, in the subsequent steps of the original found in Ccc2 in place of the pivotal Lys residue of Atx1. Side copper-trafficking mechanism, Cu(I) is transferred to a cytoso- chains at this site abut the metal binding loop and are antici- lic face of the membrane-spanning ATPase (15). The fate of the pated to not only play an important role in determining the Cu(I) bound by Ccc2a is not known, but it is speculated to be stability of this region of the protein but are likely to control transferred to a cation translocation site within the membrane key steps in the metal transfer mechanism. portions of Ccc2, such as the canonical CPC motif. In an exten- REFERENCES sion of this model, we propose that a hydrophobic patch on 1. Solioz, M. & Vulpe, C. (1996) Trends Biochem. Sci. 21, 237–241 mobile N-terminal domains of Ccc2a or mbd4 can serve as a site 2. Vulpe, C., Levinson, B., Whitney, S., Packman, S. & Gitschier, J. (1993) Nat. for interaction with other hydrophobic sites essential for copper Genet. 3, 7–13 movement across the membrane. After transfer and ATP-in- 3. Chelly, J., Tumer, Z., Tonnesen, T., Petterson, A., Ishikawa-Brush, Y., Tom- merup, N., Monaco, A. P. & Horn, N. (1993) Nat. Genet. 3, 14 –19 duced conformational changes, the Cu(I) would be subse- 4. Mercer, J. F., Livingston, J., Hall, B., Paynter, J. A., Begy, C., quently released to the interior of the ATPase-containing ves- Chandrasekharappa. S., Lockhart, P., Grimes, A., Bhave, M., Siemieniak, D. & Glover, T. W. (1993) Nat. Genet. 3, 20 –25 icle. Tests of this model are under way. 5. Bull, P. C., Thomas, G. R., Rommens, J. M., Forbes, J. R. & Cox, D. W. (1993) Nat. Genet. 5, 327–337 CONCLUSION 6. Tanzi, R. E., Petrukhin, K., Chernov, I., Pellequer, J. L., Wasco, W., Ross, B., This paper presents the first report of the solution structure Romano, D. M., Parano, E., Pavone, L., Brzustowicz, L. M., Devoto, M., Peppercorn, J., Bush, A. I., Sternlieb, I., Pirastu, M., Gusella, J. F., of a copper-transporting ATPase domain bound to the native Evgrafov, O., Penchaszadeh, G. K., Honig, B., Edelman, I. S., Soares, M. B., metal ion Cu(I). The fold is similar to its physiological partner, Scheinberg, I. H. & Gilliam, T. C. (1993) Nat. Genet. 5, 344 –350 7. Fu, D., Beeler, T. J. & Dunn, T. M. (1995) Yeast 11, 283–293 the cytoplasmic copper chaperone Atx1 (17) and is even more 8. Yuan, D. S., Stearman, R., Dancis, A., Dunn, T., Beeler, T. & Klausner, R. D. similar to the structure of a metal-binding domain of one of its (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2632–2636 human orthologs, the Menkes’ disease protein (20). Unlike 9. Stearman, R., Yuan, D., Yamaguchi-Iwan, Y., Klausner, R. D. & Dancis, A. (1996) Science 271, 1552–1557 other structurally characterized members of this family, both of 10. Hung, I. H., Suzuki, M., Yamaguchi, Y., Yuan, D. S., Klausner, R. D. & Gitlin, the metal-binding cysteines of Ccc2a are located in a helical J. D. (1997) J. Biol. Chem. 272, 21461–21466 11. Forbes, J. R., Hsi, G. & Cox, D. W. (1999) J. Biol. Chem. 274, 12408 –12413 region, and the metal is more accessible to solution. Compari- 12. Larin, D., Mekios, C., Das, K., Ross, B., Yang, A. S. & Gilliam, C. T. (1999) sons of the cytosolic domains of the transporters with the J. Biol. Chem. 274, 28497–28504 chaperones reveal complementary features that are important 13. Yamaguchi, Y., Heiny, M. E., Suzuki, M. & Gitlin, J. D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14030 –14035 in their respective copper-trafficking functions. First, unlike 14. Lutsenko, S., Petrukhin, K., Cooper, M. J., Gilliam, C. T. & Kaplan, J. H. Atx1, few conformational changes are observed in Ccc2a upon (1997) J. Biol. Chem. 272, 14030 –14035 copper release, and the metal binding region is well defined in 15. Pufahl, R., Singer, C. P., Peariso, K. L., Lin, S.-J., Schmidt, P. J., Fahrni, C. J., Cizewski Culotta, V., Penner-Hahn, J. E. & O’Halloran, T. V. (1997) Science both the apo and holo forms of this domain. In the case of 278, 853– 856 apo-Atx1, the amide NH for residues close to the copper bind- 16. Huffman, D. L. & O’Halloran, T. V. (2000) J. Biol. Chem. 275, 18611–18614 17. Rosenzweig, A. C., Huffman, D. L., Hou, M. Y., Wernimont, A. K., Pufahl, R. A. ing, in particular Cys , are not detected, and this disappear- & O’Halloran, T. V. (1999) Structure 7, 605– 617 ance could be ascribed to an increased disorder/mobility of the 18. Portnoy, M. E., Rosenzweig, A. C., Rae, T., Huffman, D. L., O’Halloran, T. V. loop region. In contrast, apo-Ccc2a exhibits a higher degree of & Cizewski Culotta, V. (1999) J. Biol. Chem. 274, 15041–15045 19. Lamb, A. L., Wernimont, A. K., Pufahl, R. A., Culotta, V. C., O’Halloran, T. V. order and both amide HN of the metal-binding cysteines are & Rosenzweig, A. C. (1999) Nat. Struct. Biol. 6, 724 –729 detected. Furthermore, NOE signal intensity is significant for 20. Gitschier, J., Moffat, B., Reilly, D., Wood, W. I. & Fairbrother, W. J. (1998) Nat. 13 16 Struct. Biol. 5, 47–54 both Cys and Cys in apo-Ccc2. Together, these results sug- 21. Steele, R. A. & Opella, S. J. (1997) Biochemistry 36, 6885– 6895 gest a significantly greater stability of the Ccc2a domain with 22. Eriksson, P.-O. & Sahlman, L. (1993) J. Biomol. NMR 3, 613– 626 respect to Atx1. 23. Wimmer, R., Herrmann, T., Solioz, M. & Wu ¨ thrich, K. (1999) J. Biol. Chem. 274, 22597–22603 Finally, there are several mechanistically important aspects 24. Lin, S. J., Pufahl, R., Dancis, A., O’Halloran, T. V. & Culotta, V. C. (1997) of the Ccc2a surface that differ from Atx1. One prominent J. Biol. Chem. 272, 9215–9220 25. Bax, A. & Davis, D. G. (1985) J. Magn. Reson. 63, 207–213 feature is a negative patch (Fig. 13) formed by glutamate and 26. Griesinger, C., Otting, G., Wu ¨ thrich, K. & Ernst, R. R. (1988) J. Am. Chem. aspartate residues in the proximity of the copper binding re- Soc. 110, 7870 –7872 gion that could interact with complementary array of lysine 27. Macura, S., Wu ¨ thrich, K. & Ernst, R. R. (1982) J. Magn. Reson. 47, 351–357 8426 Solution Structure of Cu(I) and Apo Forms of Ccc2a 28. Marion, D. & Wu ¨ thrich, K. (1983) Biochem. Biophys. Res. Commun. 113, 38. Banci, L., Benedetto, M., Bertini, I., Del Conte, R., Piccioli, M. & Viezzoli, M. S. 967–974 (1998) Biochemistry 37, 11780 –11791 29. Palmer, A. G., III, Cavanagh, J., Wright, P. E. & Rance, M. (1991) J. Magn. 39. Borgias, B., Thomas, P. D. & James, T. L. (1989) Complete Relaxation Matrix Reson. 93, 151–170 Analysis (CORMA), University of California, San Francisco 30. Kay, L. E., Keifer, P. & Saarinen, T. (1992) J. Am. Chem. Soc. 114, 40. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. (1993) 10663–10665 J. Appl. Crystallogr. 26, 283–291 31. Schleucher, J., Schwendinger, M., Sattler, M., Schmidt, P., Schedletzky, O., 41. Laskowski, R. A., Rullmann, J. A. C., MacArthur, M. W., Kaptein, R. & Glaser, S. J., Sorensen, O. W. & Griesinger, C. (1994) J. Biomol. NMR 4, Thornton, J. M. (1996) J. Biomol. NMR 8, 477– 486 301–306 42. Bax, A. & Wang, A. C. (1995) J. Am. Chem. Soc. 117, 1810 –1813 32. Kay, L. E., Marion, D. & Bax, A. (1989) J. Magn. Reson. 84, 72– 84 43. Weisemann, R., Ru ¨ terjans, H., Schwalbe, H., Schleucher, J., Bermel, W. & 33. Vuister, G. W. & Bax, A. (1993) J. Am. Chem. Soc. 115, 7772–7777 Griesinger, C. (1994) J. Biomol. NMR 4, 231–240 34. Piotto, M., Saudek, V. & Sklenar, V. (1992) J. Biomol. NMR 2, 661– 666 44. Rosenzweig, A. C. & O’Halloran, T. V. (2000) Curr. Opin. Chem. Biol. 4, 35. Gu ¨ ntert, P., Braun, W. & Wu ¨ thrich, K. (1991) J. Mol. Biol. 217, 517–530 140 –147 36. Gu ¨ ntert, P., Mumenthaler, C. & Wu ¨ thrich, K. (1997) J. Mol. Biol. 273, 283–298 45. Koradi, R., Billeter, M. & Wu ¨ thrich, K. (1996) J. Mol. Graphics 14, 51–55 37. Pearlman, D. A., Case, D. A., Caldwell, J. W., Ross, W. S., Cheatham, T. E., Ferguson, D. M., Seibel, G. L., Singh, U. C., Weiner, P. K. & Kollman, P. A. 46. Arnesano, F., Banci, L., Bertini, I., Huffman, D. L., & O’Halloran, T. V. (2001) (1997) AMBER, version 5.0, University of California, San Francisco Biochemistry 40, 1528 –1539

Journal

Journal of Biological Chemistry – Unpaywall

Published: Mar 1, 2001

There are no references for this article.