Flexibility of the Metal-Binding Region in Apo-Cupredoxins

Flexibility of the metal-binding region in apo-cupredoxins

María-Eugenia Zaballaa, Luciano A. Abriataa, Antonio Donaireb,1, and Alejandro J. Vilaa,1

aInstituto de Biología Molecular y Celular de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina; and bDepartamento de Química Inorgánica, Facultad de Química, Universidad de Murcia, Campus Universitario de Espinardo, Apdo. 4021, 30100 Murcia, Spain

Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved April 18, 2012 (received for review November 30, 2011)

Protein-mediated electron transfer is an essential event in many the unusual functional and spectroscopic features observed in biochemical processes. Efficient electron transfer requires the reor- type 1 copper and CuA centers (15). This scenario, known as the ganization energy of the redox event to be minimized, which is entatic/rack-induced state hypothesis, was originally formulated ensured by the presence of rigid donor and acceptor sites. Electron for protein-bound cofactors by Lumry and Eyring in 1954 (16) transfer copper sites are present in the ubiquitous cupredoxin fold, and then extended to electron transfer metalloproteins by Mal- able to bind one or two copper ions. The low reorganization en- mström and Williams in the late 1960s (17, 18). This concept has ergy in these metal centers has been accounted for by assuming been matter of debate along many years (7, 15, 19–26). In parti- that the protein scaffold creates an entatic/rack-induced state, cular, Ryde et al. have reported in vacuum quantum calculations which gives rise to a rigid environment by means of a preformed suggesting the absence of any strain in the geometry adopted by metal chelating site. However, this notion is incompatible with the the copper in the protein framework (21, 22). need for an exposed metal-binding site and protein–protein inter- The entatic/rack-induced state hypothesis applied to ET cop- actions enabling metallochaperone-mediated assembly of the copper proteins was strongly supported by several X-ray structures of per site. Here we report an NMR study that reveals a high degree of type 1 copper proteins revealing that the conformation of the structural heterogeneity in the metal-binding region of the nonme- copper ligands in the metal-depleted (apo) form was identical

tallated CuA-binding cupredoxin domain, arising from microsecond to that found in the holoproteins (27–31). These results prompted CHEMISTRY to second dynamics that are quenched upon metal binding. We the idea that the protein fold determines the coordination also report similar dynamic features in apo-azurin, a paradigmatic geometry of the metal center by creating a preformed chelating blue copper protein, suggesting a general behavior. These findings site with very little flexibility, thus minimizing conformational reveal that the entatic/rack-induced state, governing the features changes that would normally be exhibited during Cu(II)/Cu(I) re- of the metal center in the copper-loaded protein, does not require a dox cycling (19). This idea is reinforced by the fact that all ET preformed metal-binding site. Instead, metal binding is a major copper centers are bound to a rigid Greek-key β-barrel domain, contributor to the rigidity of electron transfer copper centers. known as the cupredoxin fold (31–34). Instead, iron-sulfur and

These results reconcile the seemingly contradictory requirements heme centers are found in different folding motifs, with diverse BIOCHEMISTRY of a rigid, occluded center for electron transfer, and an accessible, intrinsic mobilities (35–37). dynamic site required for in vivo copper uptake. The notion of a preformed rigid binding site is conflicting with the finding that copper centers are loaded by specific metallocha- metalloproteins ∣ nuclear magnetic resonance ∣ protein dynamics perones (38, 39). The concept of metallochaperones is relatively new and it has replaced the idea that metal uptake depends ong-range electron transfer (ET) in proteins is a key chemical entirely on the chelating properties of the apoprotein. Several Levent in many essential biological processes such as cellular copper chaperones have been identified and characterized so far, respiration and photosynthesis (1–3). According to Marcus’ semi- requiring specific protein–protein interactions that can be metal- classical theory, the outstanding efficiency of these processes is mediated in some cases (40). In the latter situation, the metal ion based on the maximization of the superexchange coupling be- is simultaneously bound to ligands from both the metallochaper- tween donor and acceptor and the minimization of the reorgani- one and the target protein during the transfer step (40). In any zation energy, given the low driving forces found in most case, the assumption of a highly rigid metal site, which should also biological systems (4–6). Thus, with driving forces as low as 0.1 eV be hidden from the bulk solvent to prevent unwanted side reac- and distances larger than 10 Å, efficient long-range electron tions, does not favor metal delivery by another protein. Thus, the transfer is only possible if the nuclear reorganization energy of demands for efficient electron transfer and in vivo metallation are the reactants is below 1 eV (4, 7). hard to reconcile given the current knowledge in the field. Transition metal ions such as copper and iron are ubiquitous in We have recently elucidated the chaperone-mediated mechan- ETchains because their redox potentials and electronic structures ism of copper uptake by the CuA-binding cupredoxin domain in can be tuned by the protein environment to match the require- Thermus thermophilus ba3 cytochrome c oxidase (41). In that ments of different biological redox events. Cycling between the work, we observed that the NMR spectra of the apo and metal- coordination geometries preferred by these metals in each redox state would in principle lead to high reorganization energies (7). Electron transfer iron centers avoid this by using rigid cofactors Author contributions: M.-E.Z., L.A.A., A.D., and A.J.V. designed research; M.-E.Z., L.A.A., – and A.D. performed research; M.-E.Z., L.A.A., A.D., and A.J.V. analyzed data; and M.-E.Z., such as iron-sulfur clusters and heme groups (8 10). Instead, L.A.A., A.D., and A.J.V. wrote the paper. copper ions in ET centers are only bound to protein residues, as The authors declare no conflict of interest. observed in type 1 (blue) copper and Cu centers; and thus the A This article is a PNAS Direct Submission. protein fold around the metal ion is expected to impart rigidity to – Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, these otherwise flexible metal centers (10 12). Particularly, out- www.pdb.org (PDB ID code 2LLN). The NMR chemical shifts have been deposited in the er-sphere coordination involving hydrogen bonding networks has BioMagResBank, www.bmrb.wisc.edu (accession nos. 18081 and 18254). been proposed as responsible for the low reorganization energies 1To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. observed in copper centers in proteins (13, 14). The strain intro- This article contains supporting information online at www.pnas.org/lookup/suppl/ duced by the protein fold is then predicted to be responsible for doi:10.1073/pnas.1119460109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1119460109 PNAS Early Edition ∣ 1of6 Downloaded by guest on October 2, 2021 lated forms of this protein show significant differences, suggesting ing residues with duplicated correlations are located around the that there might be nonnegligible structural perturbations upon metal-binding site (Ala85, Ala87, Tyr90, Val112, Ile113, His117, metal binding, consistent with the finding that copper uptake is Gly120, Asn124, Gly154, Gly156, Met160-Thr163). Comparison mediated by a metallochaperone (Fig. S1). Here we report a de- of the assignments obtained for apoCuA with those available tailed NMR study showing the solution structure and dynamic for the reduced holoprotein revealed that most chemical shift features of this cupredoxin domain in the nonmetallated form. perturbations also map close to the metal-binding site (Fig. 1 We have found that not only is the metal-binding site disordered and Fig. S1). in the apoprotein but it also exhibits significant dynamics in the The calculated three-dimensional structure of apoCuA is well microsecond to second timescale. Moreover, to prove that our defined by 2,147 (including 879 long-range) NOE-based distance results can be extended to other cupredoxins, we report an NMR- restraints and 215 experimental torsion angle restraints (Fig. S2 based study of Pseudomonas aeruginosa apo-azurin, which has and Table S2). The atomic rmsd values for the 20 models in been considered as the paradigm of blue copper proteins and the refined structure are 1.0 0.2 and 1.4 0.1 Å for the back- long-range ET (42–44). Also in this case, we show there is signif- bone and all heavy atoms, respectively. Considering only residues icant dynamics in the metal-binding region only in the absence of from the rigid β-barrel domain (residues 53–148 and 162–168), the copper ion. Thus, our findings indicate that the rigid copper the rmsd values for backbone and all heavy atoms are 0.49 0.07 center needed for efficient long-range ET is not preformed in the and 1.01 0.08 Å, respectively. nonmetallated cupredoxin. Instead, metal binding does signifi- The apoprotein in solution adopts a β-barrel fold where most cantly contribute to the rigidity of these centers and the metal- regions are well-defined except the N terminus and loops 86–90 binding region in the apoprotein is flexible enough to allow in and 149–161 (ligand loop) (Fig. 1 and Fig. S3). The disorder vivo metallation. observed at the ligand loop might be due to the lack of experimental constraints (Fig. S2) and/or true mobility of this region in Results the absence of the metal ions. Indeed, the low number of con- The CuA-Binding Cupredoxin Domain. Subunit II of T. thermophilus straints in this region parallels the missing correlations in the ba3 oxidase consists of a soluble CuA-binding domain anchored to 1H, 15N-HSQC, suggestive of some degree of flexibility (see the membrane through an N-terminal transmembrane helix (32, below). It is then clear that the polypeptide presents a similar 45). This domain adopts a cupredoxin fold similar to the one Greek-key β-barrel fold both in the apo- and copper-loaded found in type 1 copper proteins, with a longer loop that allows forms, and that the structural perturbations between both forms binding of an additional metal ion (12). The two copper ions of of the protein are confined to the metal-binding region, with the the Cu site are bridged by two cysteine ligands (Cys149 and A ligand loop showing the highest degree of disorder. Cys153), giving rise to a rigid Cu2S2 core. Each copper ion is also coordinated to a terminal histidine residue (His114 and His157) Dynamics of apoCu and Reduced holoCu . Subnanosecond timescale. and a weak axial ligand: a methionine sulfur (Met160) and a back- A A The dynamics of apo- and reduced holoCuA in the pico-to-nano- bone carbonyl (Gln151), respectively. The soluble fragment here- 15 second timescale was studied by measuring NR1 and R2 and in studied comprises only the soluble cupredoxin domain of 1H-15N heteronuclear NOE. Average correlation times of 8.3 subunit II, where the transmembrane helix is not part of the pur- 0 1 8 2 0 1 ified protein. This fragment retains the structure observed in the . ns (apoCuA) and . . ns (holoCuA) were estimated and used for relaxation data analysis with the model-free approach whole oxidase (32, 45) and it is competent for ET with its biolo- – S2 gical redox partner (46, 47). Given that residues 1–42 are absent (49 51). Order parameter values ( ) for apo- and holoCuA are remarkably high along most of the sequence, averaging 0.9 in our protein construct, residue numbering starts at 43 in the 0 1 results presented here, following that employed in the X-ray . in both proteins and confirming the high level of rigidity of the β-barrel (Fig. S4). As expected, the order parameter values for structure of oxidized holoCuA [Protein Data Bank (PDB) ID 2CUA] (32).* both protein forms drop below 0.6 at the N terminus, due to the high mobility usually observed in this region. S2 values lower 1 13 than 0.6 are also observed for residues 154–156 in apoCu . How- Resonance Assignment and Solution Structure of apoCuA. H, C, A 15 S2 and N resonances were assigned for most residues in the non- ever, a slight decrease in values is also found in this region for metallated form of the soluble domain of T. thermophilus ba3 holoCuA. Overall, these data do not reveal significantly different oxidase subunit II (apoCuA, hereafter) (Table S1). Resonances corresponding to eight non-proline residues were missing in the 1H, 15N-heteronuclear single quantum coherence (HSQC) spectrum of apoCuA, whereas 19 residues presented duplicated NH cross-peaks (see below). Five of the residues with missing resonances (Cys149-Gln151, His157, and Gln158) are located at the loop bearing five out of the six copper ligands (ligand loop, hereafter) whereas the remaining correspond to His114 (the N-terminal histidine coordinated to one of the copper ions), its adjacent residue Gly115, and the N-terminal Met43. These eight residues are thus involved in an exchange process whose frequency lies right on an intermediate timescale that broadens signals beyond detection. Among residues with duplicated resonances in the 1H, 15N-HSQC spectrum, five are located in the N-terminal region (Val44, Ile45, Ala47, Lys49, and Leu50) and duplication here arises from the cis-trans isomerization of Pro46 Fig. 1. NMR-based solution structure of T. thermophilus apoCuA.(A) Car- in a slow regime, as reported for the reduced holoprotein [Bio- toon representation of the X-ray structure of oxidized holoCuA (from PDB MagResBank (BMRB) entry 5819; ref. 48]. Most of the remain- ID 2CUA; ref. 32). (B) Ribbon representation of the best 20 out of 80 structures calculated by CYANA for apoCuA in solution (this work, PDB ID 2LLN). Regions with significant chemical shift perturbations (Eq. S1) between apo- and *The PDB coordinates (PDB ID 2LLN) and resonance assignments (BioMagResBank entry holoCuA and structural disorder in the calculated structure of the apoprotein – – 18081) for apoCuA obtained during this work are deposited using the linear 1 126 num- are highlighted in blue (ligand loop, residues 149 161), and orange (residues bering. 86–90).

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1119460109 Zaballa et al. Downloaded by guest on October 2, 2021 dynamic features in the pico-to-nanosecond timescale between correspond to copper ligands and/or map to the surroundings the apo and holo forms of the CuA-binding domain. of the metal-binding site. We note that only one correlation is observed for all these residues in the 1H, 15N-HSQC spectrum Microsecond to millisecond timescale. Protein motions in the micro- of reduced holoCuA (BMRB entry 5819). Thus, slow dynamic to-millisecond timescale were probed by constant-time Carr– features on the copper ligands and surrounding residues are only Purcell–Meiboom–Gill (CPMG) relaxation dispersion experi- observed in the absence of the copper ions (Fig. 3B). ments (52, 53). Fig. 2A shows the ΔR2 profiles obtained for Δ apoCuA and reduced holoCuA,where R2 is the difference Dynamics of apo-azurin. In order to extend these results to other between R2 values measured at CPMG frequencies of 33 and cupredoxins, we have studied the solution dynamics of P. aerugi- 966 Hz, validated by fitting of the dispersion curves to Eq. S2 nosa apo-azurin. The blue copper protein azurin has been exten- Δ (Fig. 2, Inset and Fig. S5). The R2 profile for apoCuA shows sively studied as a paradigmatic protein for electron transfer two regions experiencing significant exchange in comparison to (42–44), and Cu(I)-azurin has been the subject of several NMR the holoprotein. Both regions map close to the metal-binding site, studies (55–58). Instead, apo-azurin has not been characterized as shown in Fig. 3A. Thus, residues in the metal-binding region by NMR. exhibit significant micro-to-millisecond dynamics in the apopro- Backbone 1H and 15N resonances were assigned for most tein, and this flexibility is lost upon binding of the metal ions. residues of P. aeruginosa apo-azurin. Resonances corresponding to 17 non-proline residues were missing in the 1H, 15N-HSQC 1 Protein compactness. Protein compactness was studied by a H, spectrum, indicating they are involved in an intermediate ex- 15N heterogeneity-band-selective optimized-flip-angle short- change process on the chemical shift timescale. These residues transient-heteronuclear multiple quantum coherence (HET- are Ala1-Cys3, Gly9-Asp11, Asn18, His35-Leu39, Gly45, His46, SOFAST-HMQC) experiment with irradiation of aliphatic pro- Gly88-Glu91, most of them mapping to the surroundings of the tons. The λ parameter calculated from this experiment metal-binding site (Fig. 4B). This behavior contrasts with that of NOE 1 15 reports on the proton density around the probed amide, with Cu(I)-azurin, whose H, N-HSQC spectrum shows one cross- λ 0 3 low NOE values (< . ) indicating well-structured compact seg- peak for every non-proline residue in the protein (except the ments and higher values indicating a less compact environment N-terminal Ala1) (56), indicating that this slow dynamic process (which might be due in turn to enhanced dynamics) (54). Fig. 2B takes place only in the nonmetallated form, in agreement with the λ shows the NOE profiles obtained for apo- and reduced holoCuA. findings on apoCuA. Moreover, most chemical shift perturbations CHEMISTRY Both protein forms present a similar global compactness along resulting from metal removal in azurin cluster close to the copper most of the sequence, except for a significant difference observed center (Fig. 4A and Fig. S6), again resembling the situation met in λ at the ligand loop, where the higher NOE values obtained for the the CuA-binding domain. apoprotein suggest a less compact structure in the absence of the Protein dynamics in the micro-to-millisecond timescale were copper ions. evaluated by relaxation dispersion experiments in apo-azurin. As observed in Fig. 4C, the ΔR2 profile, validated by fitting of Millisecond to second timescale. The absence (for eight non-proline the dispersion curves to Eq. S2 (Fig. S7), shows three defined regions in the micro-to-millisecond timescale. Residues in these

residues) and duplication (for 19 residues) of NH cross-peaks in BIOCHEMISTRY 1 15 1 the H, N-HSQC spectrum of apoCuA reveals chemical ex- regions are next to residues with missing correlations in the H, change in the intermediate and slow regimes on the chemical shift 15N-HSQC of apo-azurin, thus evidencing a common exchange timescale, respectively. As detailed above, most of these residues process. Comparison of these data with those reported by

Δ Fig. 2. Protein dynamics and compactness of apoCuA (black squares) and reduced holoCuA (gray triangles). (A) R2 profiles showing dynamics in the micro-to-millisecond timescale. Fits of experimental data to Eq. S2 for some apoCuA residues are shown as examples in the inset of the figure (Gln91, circles; λ Met160, triangles; and Gly162, squares, showing significant relaxation dispersion; and Ala101, diamonds, located in a region showing no exchange).(B) NOE profiles showing proton density around the backbone amides.

Zaballa et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on October 2, 2021 Discussion In this work, we present an NMR study on apoCuA, revealing that the metal-binding region (mainly the ligand loop) shows a disordered and less compact structure in the apoprotein compared to the holo form (Figs. 1 and 2B) and that this disorder, in turn, is due to a dynamic behavior of this region in the microsecond to second timescale (Figs. 2A and 3). The blue copper protein azurin displays similar dynamic features in its apo form which also map to the metal-binding region (Fig. 4). The absence of such dynamic features both in holoCuA and Cu(I)-azurin allows us to conclude that this region is significantly flexible in the apoproteins and that only metal binding quenches the dynamics of these loops in the cupredoxin fold. Given the different loop length and structure in CuA and azurin, these results strongly suggest that this behavior is a general feature of cupredoxins.

Fig. 3. Mapping of the dynamic features in apoCuA. Residues experiencing dynamics in the absence of the copper ions (i.e., in apoCu ) are highlighted in Dynamics of Apo-Cupredoxins and the Entatic/Rack-Induced State. A The entatic/rack-induced state in ET copper proteins refers to the structure of holoCuA (PDB ID 2CUA, ref. 32, used to evidence the proxi- mity of these residues to the metal ions). (A) Dynamics in the micro-to-milli- the organized protein structure around the metal-binding site −1 −1 second timescale. Residues with ΔR2 < 1.3 s , ΔR2 > 1.3 s , and missing that ensures a low reorganization energy for efficient electron residues are shown in blue, orange, and gray, respectively. (B) Slow dynamics. transfer (7). There have been numerous reports and reviews sup- Residues with zero (missing), one or two (duplicated) correlations in the 1H, 15 porting the entatic/rack-induced state hypothesis (7, 13, 19, 20, N-HSQC spectrum of apoCuA are shown in red, gray, and yellow, respec- 26, 59). On the other hand, theoretical and experimental studies tively. have questioned the role of the protein matrix in defining the geometry of the copper center (21, 22, 24, 25). X-ray studies on Orekhov and coworkers for Cu(I)-azurin (55, 57) reveals an in- the apo form of several cupredoxins, showing no changes in the creased mobility at the metal-binding region in the absence of the ligands’ conformations upon removal of the copper ion, were copper ion. considered as experimental evidence of the entatic/rack-induced

Fig. 4. NMR results on apo-azurin. Residues showing significant chemical shift perturbations or enhanced dynamic features are mapped on the tridimensional structure of Cu(II)-azurin (PDB ID 4AZU; ref. 33). (A) Chemical shift perturbations between apo-azurin and Cu(I)-azurin backbone amide resonances. Residues with chemical shift perturbations ðCSPÞ < 0.2, 0.2 < CSP < 0.5,CSP> 0.5 are shown in blue, yellow, and red, respectively. Residues with missing NH correlations −1 −1 are shown in gray. (B) Microsecond to second dynamics of apo-azurin. Residues with ΔR2 < 2 s , ΔR2 > 2 s are shown in blue and orange, respectively. 1 15 Residues with missing correlations in the H, N-HSQC spectrum are shown in red. In both representations, prolines are shown in gray. (C) ΔR2 profiles showing dynamics in the micro-to-millisecond timescale. Fits of experimental data to Eq. S2 for some apo-azurin residues are shown as examples in the inset of the figure (Ile87, circles; Met44, squares, showing significant relaxation dispersion; and Thr61, triangles, located in a region showing no exchange).

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1119460109 Zaballa et al. Downloaded by guest on October 2, 2021 hypothesis for ET copper proteins (27–31). In addition, those re- posed, dynamic metal-binding site allowing for efficient metallo- sults put forward the idea that the protein matrix creates a pre- chaperone-mediated copper binding in vivo. In turn, the fully formed rigid chelating site in the absence of the metal ions, thus conserved Greek-key β-barrel scaffold would be able to render entirely determining the geometry of the copper center (19). a rigid and efficient ET site once the copper ions are bound. The present NMR-based results provide compelling evidence Our results thus allow us to reconcile the seemingly contradictory of structural disorder, flexibility, and conformational exchange in requirements of a rigid, occluded center for electron transfer, and the metal-binding region of the apo form of the CuA-binding an accessible, dynamic site for in vivo copper uptake. Nature has cupredoxin domain. Flexibility and slow chemical exchange were solved this problem by exploiting the cupredoxin fold, which is also observed here for apo-azurin, supporting the extension of endowed at the same time with rigid and dynamic features that these findings to other cupredoxins and contrasting the men- are finely tuned by the binding of the metal ions. tioned X-ray studies. This discrepancy may be attributed to the fact that most of the reported structures of apo-cupredoxins were Materials and Methods

obtained by crystallization of the holoprotein and subsequent Protein Preparation. Samples of azurin and apo- and holoCuA proteins were metal removal from the crystal by soaking with a chelating agent uniformly labeled with 15Nor13C and 15N (Cambridge Isotope Laboratories, (28–30). Instead, direct crystallization of apo-azurin gave rise to Inc.) and produced as described elsewhere (40, 47, 64). The apoCuA protein some (minor) structural heterogeneity selectively on the copper was expressed and purified in the absence of copper, whereas azurin was ligands (27). However, analysis of the structure of apo-azurin purified as Cu(II)-azurin and the apoprotein was obtained by dialysis against · does not reveal a significant increase in the B factors in the metal- 50 mM KCN, 200 mM Tris HCl pH 8.5, followed by several dialysis steps with- binding region nor the lack of electron density, suggesting that the out the complexing agent. Protein samples for NMR experiments were pre- pared in 100 mM phosphate buffer pH 7.0 for azurin and pH 6.0 for the Cu absence of dynamics in these cases is due to crystal packing. A domain, adding 100 mM KCl in the case of the holoprotein or 2 mM DTT for We note that our results do not necessarily support nor refute the apoproteins in order to avoid oxidation of the cysteine ligands. Protein the entatic/rack-induced state hypothesis because they apply to concentration was about 1 mM. the nonmetallated protein and do not assess the strain the protein environment exerts on the Cu(I) and Cu(II) centers. Instead, our Nuclear Magnetic Resonance Spectroscopy. NMR experiments were carried out findings show that the entatic/rack-induced state, when operative on a 600 MHz Bruker Avance II Spectrometer equipped with a triple reso- on the metallated protein, does not require a preformed rigid nance inverse (TXI) probe head and on a 900 MHz Bruker Avance II Spectro- chelating site. This conception is in line with the results reported meter equipped with a triple resonance inverse (TCI) cryoprobe. All

by Wittung-Stafshede and coworkers, showing that a rigid, ET- experiments were carried out at 298 K using standard techniques, as de- CHEMISTRY functional blue copper center can be obtained from Cu(II) bind- scribed in SI Text. The obtained family of structures for apoCuA is deposited at the Protein Data Bank under PDB ID 2LLN. Chemical shifts for all 1H, 13C, ing to unfolded azurin, suggesting that the constraints imposed by 15 the protein to the geometry of the metal center do not require a and N nuclei assigned in this work are deposited at the Biological Magnetic preorganized binding site defined in the polypeptide (60–62). Resonance Data Bank under accession numbers 18081 and 18254 for apoCuA and apo-azurin, respectively. Resonance assignments for the reduced metal-

lated proteins, holoCuA and Cu(I)-azurin, were taken from the literature (48, Dynamics of Apo-Cupredoxins and in Vivo Copper Uptake. The copper 56) and transferred by us to the sample conditions used in this work. cargo in the CuA-binding cupredoxin domain of T. thermophilus

oxidase is due to the action of a specific Cu(I)-metallochaperone BIOCHEMISTRY ACKNOWLEDGMENTS. The authors thank J. H. Richards (Caltech) for providing (PCuAC) (41). Similar copper-loading mechanisms have been the plasmid for azurin expression. The Centro di Risonanze Magnetiche of proposed for the type 1 protein plastocyanin (63). Copper trans- Florence, Italy, and the European Program Bio-NMR (BIO-NMR-00012) are fer mediated by chaperones generally takes milliseconds to sec- acknowledged for the access to high magnetic field spectrometers. A.D. also onds, overlapping with the fluxional timescales measured in this thanks the Spanish Ministerio de Educación for a grant supporting a four months’ stay in Rosario, Argentina (PR2009-0479). M.E.Z. and L.A.A. are doc- work for the metal-binding region of apoCuA and apo-azurin toral and postdoctoral fellows from Consejo Nacional de Investigaciones (although no copper chaperones have been reported for azurin Científicas y Técnicas (CONICET), respectively. A.J.V. is an Howard Hughes to date). This observation suggests that the observed flexibility Medical Institute International Research Scholar and a staff member from of this region in the apo form of the protein allows the cupredoxin CONICET. The Bruker Avance II 600 MHz was purchased with funds from fold to sample different conformations, enabling copper transfer. Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) and CON- ICET. This research was funded by ANPCyT, Argentina (Proyecto de Investiga- Our unique picture of a folded apo-cupredoxin consisting in a ción Científica y Tecnológica 2007-314), the Spanish Ministerio de Economía y β-barrel with disordered flexible loops is fully consistent with the Competitividad, and Fundación Séneca de la Región de Murcia, Spain (project need of specific protein–protein interactions and defines an ex- numbers SAF2011-26611 and 15354/PI/10, respectively).

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