Ctr1 Intracellular Loop Is Involved in the Copper ... - ACS Publications

Article pubs.acs.org/JPCB

Ctr1 Intracellular Loop Is Involved in the Copper Transfer Mechanism to the Atox1 Metallochaperone Ariel R. Levy, Matan Nissim, Netanel Mendelman, Jordan Chill, and Sharon Ruthstein* The Department of Chemistry, Faculty of Exact Science, Bar Ilan University, Ramat-Gan 5290002, Israel S Supporting Information *

ABSTRACT: Understanding the human copper cycle is essential to understand the role of metals in promoting neurological diseases and disorders. One of the cycles controlling the cellular concentration and distribution of copper involves the copper transporter, Ctr1; the metallochaperone, Atox1; and the ATP7B transporter. It has been shown that the C-terminus of Ctr1, specifically the last three amino acids, HCH, is involved in both copper coordination and the transfer mechanism to Atox1. In contrast, the role of the intracellular loop of Ctr1, which is an additional intracellular segment of Ctr1, in facilitating the copper transfer mechanism has not been investigated yet. Here, we combine various biophysical methods to explore the interaction between this Ctr1 segment and metallochaperone Atox1 and clearly demonstrate that the Ctr1 intracellular loop (1) can coordinate Cu(I) via interactions with the side chains of one histidine and two methionine residues and (2) closely interacts with the Atox1 metallochaperone. Our findings are another important step in elucidating the mechanistic details of the eukaryotic copper cycle.

■

INTRODUCTION Copper is an essential cofactor for many enzymes and indispensable for their function. Nevertheless, excess cellular copper is toxic due to its tendency to generate free radicals and interferes with neuronal and metabolic functions, leading to various neurological diseases and disorders. Neurodegenerative diseases are the fourth leading cause of death in developed countries. Hence, it becomes apparent that to reveal the consequences of dysregulation in cellular copper concentration, it is required to map the copper transfer mechanism at the molecular level. Moreover, the structure of this copper cycle may suggest new therapeutic approaches to combat these diseases.1−5 Cu(I) enters into the cell through the high-affinity copper transporter Ctr1.6−8 Ctr1 cycles between the plasma membrane and cytosolic vesicles in many cell types and regulates changes in its abundance or localization, allowing for the rate of Cu(I) uptake to be rapidly adjusted in response to altered cellular Cu(I) levels.7,9 Metallochaperone Atox1 is responsible for transferring copper from the Ctr1 to the N-terminal domains of ATP7A and ATP7B in the Golgi through a direct interaction.8−14 ATP7A and ATP7B have a dual role in cells: a biosynthetic role, delivering copper to the secretory pathway for metalation of cuproenzymes, and a homeostatic role, exporting excess copper from the cell. Some of these cuproenzymes, such as dopamine-β-hydroxylase and peptidylglycine α-amidating monooxygenase, play an important role in the central nervous system.15−20 Others have important functions in the connective tissue and blood vessel development (lysyl oxidase, superoxide-dismutase), as well as in iron and copper transport (ceruloplasmin, hepaestin).1,3,15,16,21,22 Therefore, the lack of functional ATP7A/B leads to a number © XXXX American Chemical Society

of severe symptoms, manifested in Menkes and Wilson diseases, and is probably connected to many other yet-to-bedefined neurological disorders. As a result, the Ctr1−Atox1− ATP7A/B cycle has been suggested to be essential for cell growth and is also involved in the modulation of various neurodegenerative diseases.5,16,20,23 Over the last few years, significant progress has been made toward understanding the copper transfer mechanism from Ctr1 to the Golgi apparatus. Structures of some of the Atox1, ATP7B, and Ctr1 domains have been resolved using NMR, Xray crystallography, and electron microscopy techniques,6,13,24,25 and kinetic information on the interaction between Atox1 and ATP7B has also been reported.26,27 The 6 Å resolution electron crystallography structure of human Ctr1, reported by the group of Unger,6,28 indicates that Ctr1 is a homotrimer, in which each monomer is a 23 kDa, 190-residue subunit containing (1) a 60 amino acid extracellular N-terminal domain, (2) an intracellular loop of 46 amino acids, connecting the first and second putative membrane-spanning helices, (3) three transmembrane helices, and (4) a short intracellular Cterminal domain with 15 amino acids. It was previously suggested that Atox1 copper metallochaperone can dock to Ctr1 as significant structural similarity exists between two Cu(I) chaperones in the cell, CCS and Atox1, proposing that both proteins can form similar interactions with Ctr1.2,29,30 We recently showed using electron paramagnetic resonance (EPR) spectroscopy that a specific interaction exists between the short C-terminal domain of Ctr1 and Atox1, in which the last three Received: October 10, 2016 Revised: November 9, 2016 Published: November 10, 2016 A

DOI: 10.1021/acs.jpcb.6b10222 J. Phys. Chem. B XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry B residues of Ctr1, HCH, control this interaction.31 Later, Kahara et al. showed that these last three amino acids form a Cu(I) binding site and mutations in this motif decrease the binding of Cu(I) to the intracellular segment of Ctr1 but do not eliminate it.32 As most studies to date have focused on the C-terminal domain of Ctr1 (see Figure 1), here we focus upon another

In this study, we utilize CW and pulsed EPR together with SDSL, circular dichroism (CD), and NMR to explore the interaction between the Ctr1 intracellular loop and Atox1. NMR experiments show that residues M85, M96, and H99 of the intracellular loop participate in Cu(I) coordination and that a peptide derived from the loop sequence binds Cu(I) at intermediate affinity estimated as ∼1−10 μM. Kinetic CD measurements together with the cross-linking experiment demonstrate that the Atox1−Ctr1 intracellular loop forms a stable complex. Finally, EPR experiments and SDSL monitor the conformational state of Atox1 while interacting with the Ctr1 intracellular loop. This study is an additional step toward mapping the full copper cycle in eukaryotic systems.

■

EXPERIMENTAL SECTION Peptide Synthesis, Purification, and Labeling. The nonlabeled Ctr1 intracellular loop (MPVPGPNGTILMETHKTVGQQM) and the spin-labeled Ctr1 intracellular loop (MPVPGPNGTILMETHKTVGQQMC−MTSSL) were synthesized on a rink amide resin (Applied Biosystems). Couplings of standard 9-fluorenylmethoxy-carbonyl (Fmoc)protected amino acids were achieved with (O-benzotriazol-1yl)-N,N,N′,N′-tetramethyluronium (D-chem) in N,N-dimethylformamide (Bio lab) in combination with N,N-diisopropylethylamine (Bio lab) for a 1 h cycle. Fmoc deprotection was achieved with piperidine (Bio lab). Side-chain deprotection and peptide cleavage from the resin were achieved by treating the resin-bound peptides with 5 mL of 95% mixed TFA solution (90% trifluoroacetic acid (TFA, Bio lab), 5% ethane dithiol (Alfa Aesar), 2.5% triisopropylsilane (Alfa Aesar), and 2.5% thioanisole (Alfa Aesar)) and 5% of water for 3.5 h under N2. An additional 65 μL of bromotrimethylsilane (Alfa Aesar) was added during the final 30 min to minimize methionine oxidation. The peptides were washed four times with cold diethyl ether, vortexed, and then centrifuged for 5 min at 4000 rpm. After evaporation of TFA under N2, 10 mM dithiothreitol (DTT, Sigma) was added to the peptide, and it was dissolved in HPLC water. The peptide was then purified by preparative reversed-phase HPLC (Vydac, C18, 5 cm). The mass of the peptide was confirmed either by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-Autoflex III-TOF/TOF mass spectrometer (Bruker, Bermen, Germany), equipped with a 337 nm nitrogen laser, or by electron spray ionization (ESI) mass spectrometry on a quadruple time of flight (Q-TOF) low-resolution micromass spectrometer (Micromass-Waters, Corp.). Peptide samples were typically mixed with dihydrobenzoic acid matrix solution, deposited onto the stainless steel target surfaces, and allowed to dry at room temperature. For SDSL, 1 mg of lyophilized peptide was dissolved in 1.0 mL of phosphate buffer (25 mM KPi; pH = 7.4−7.6). S(2,2,5,5-Tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate (MTSSL; TRC; 1.0 mg) dissolved in 15 μL of dimethyl sulfoxide (DMSO, BioLab) was added to the solution (1:10 peptide/MTSSL molar ratio). The spin label and peptide solution were then vortexed overnight at 4 °C. The free spin label was removed by semipreparative HPLC (Vydac, C18 1 cm). The mass of the spin-labeled peptide was confirmed by mass spectrometry to verify the above 95% purified spin-labeled peptide. Atox1 Cloning, Expression, Purification, and Labeling. The human Atox1 construct pYTB12−Atox1 was kindly given by the lab of Prof. Svetlana Lutsenko (Johns Hopkins

Figure 1. Schematic view presenting the interaction between the copper chaperone, Atox1, and the copper transporter, Ctr1, intracellular loop, which is explored in this study. The red dots on Atox1 mark the spin-labeling sites.

aspect in the copper cycle by exploring the contribution of the intracellular Ctr1 loop domain to the interaction with metallochaperone Atox1 using EPR spectroscopy. EPR spectroscopy has emerged as an excellent tool for resolving such systems, as it does not require crystallization and its abilities are independent of protein size. EPR measurement can be performed in buffer solution, and even weak interactions between proteins can be detected.33,34 EPR is sensitive to both atomic-level changes and nanoscale fluctuations. Continuous wave (CW) EPR measurements are sensitive to molecular-level changes of the paramagnetic species’ environment, and pulsed EPR experiments can measure intra- and interprotein distances of up to 8.0 nm between paramagnetic probes.35−43 The most common experiment for obtaining nanoscale structure information is the double electron−electron resonance experiment (DEER).38,44,45 The combination of CW and pulsed EPR with site-directed spin labeling (SDSL) has become widely used in biophysical research.46−50 The SDSL method has been developed by the group of Hubbell,51−53 in which a nitroxide spin label is attached to the cysteine residue in the protein; this, in turn, provides information on the local environment and the mobility of the protein domain. When multiple spin labels are attached, distance distributions between them can be derived.48−51,54−57 B


Article

The Journal of Physical Chemistry B

were measured in 0.8 mm capillary quartz tubes (Vitrocom). CW−EPR simulations were carried out on MATLAB, using the easyspin toolbox.59 The concentration of the spin-labeled protein or peptide was between 0.05 and 0.1 mM. The constant-time four-pulse DEER experiment π/2(νobs) − τ1 − π(νobs) − t′ − π(νpump) − (τ1 + τ2 − t′) − π(νobs) − τ2(νobs) − τ2 − echo was carried out at 80 ± 1.0 K on Q-band Elexsys E580 (equipped with a 2 mm probe head, bandwidth = 220 MHz). A two-step phase cycle was employed on the first pulse. The echo was measured as a function of t′, whereas τ2 was kept constant to eliminate relaxation effects. The observer pulse was set at 60 MHz higher than the pump pulse. The observer π/2 and π pulses had a length of 40 ns, the π pump pulse had a length of 40 ns as well, and the dwell time was 20 ns. The observer frequency was 33.80 GHz. The power of 40 ns π pulse was 20.0 mW. τ1 was set to 200 ns and τ2 to 1200 ns. Each DEER data was collected for 24−48 h. The spin concentration was between 0.01 and 0.02 mM. The samples were measured in 1.6 mm capillary quartz tubes (Wilmand). The data was analyzed using the DeerAnalysis 2015 program, using Tikhonov regularization.60 The regularization parameter in the L curve was optimized by examining the fit of the timedomain data. NMR. For NMR measurements, the nonlabeled Ctr1 intracellular loop was dissolved in a phosphate buffer (20 mM, pH 6.6) containing 10 mM NaCl and 0.02% NaN3 in 7 or 99.9% D2O. Typical peptide concentrations were 2−5 mM. Cu(I) was then titrated into the sample to 2:1 Cu(I)/peptide ratio. NMR measurements were conducted on a Bruker DRX700 spectrometer equipped with a cryogenic tripleresonance TCI probe with z-axis pulsed field gradients. All experiments were conducted at 16.4 T and 283 K. Backbone chemical shift assignments of the Ctr1 intracellular loop were obtained from homonuclear two-dimensional (2D) total correlation spectroscopy (TOCSY) and 2D nuclear overhauser effect spectroscopy (NOESY) spectra, both acquired with 256− 300 complex points and an acquisition time of 30.5−35.7 ms in the indirect 1H dimension and with 2048 complex points and an acquisition time of 209 ms in the observed 1H dimension. Mixing times were 150 and 500 ms for the TOCSY and NOESY experiments, respectively, and water suppression was achieved using the 3−9−19 scheme. Chemical shift assignments of 13C and 15N amides were obtained using 2D 1H,13Cheteronuclear single-quantum correlation (HMQC) and 1 15 H, N-heteronuclear single quantum coherence (HSQC) experiments acquired at natural abundance. The HMQC spectrum was acquired with 400 complex points and with an acquisition time of 35.5 ms in the 13C dimension and with 2048 complex points and an acquisition time of 209 ms in the observed dimension. The HSQC spectrum was acquired in an echo−antiecho fashion with 100 complex points and an acquisition time of 50.1 ms in the 15N dimension and with 1024 complex points and acquisition time of 90 ms in the observed dimension. All spectra were processed and analyzed using the Bruker TopSpin 3.1 program suite.

University). This construct encodes for the fusion protein composed of Atox1, with an intein- and a chitin-binding domain. It was transformed to the Escherichia coli strain BL21 (DE3). The Atox1 construct was expressed in BL21 cells, grown to an optical density of 0.5−0.8 at 600 nm, and induced with 1.0 mM isopropyl-β-D-thiogalactopyranoside (Calbiochem) for 18 h at 18 °C. The cells were then harvested by centrifugation and suspended in lysis buffer (25 mM Na2HPO4, 150 mM NaCl, 20 μM PMSF pH 7.5). The cells were sonicated over five cycles of 1 min each with a 1 min cooling time between each cycle (65% amplitude). After sonication, cells were centrifuged and the soluble fraction of the lysate was run through a chitin bead column (New England Biolabs), allowing the Atox1 fusion to bind to the resin via its chitin-binding domain. The resin was then washed with 30-column volumes of lysis buffer (pH 8.9). To induce the intein-mediated cleavage, the beads were incubated in 50 mM DTT, 25 mM Na2HPO4 (pH 8.9), and 150 mM NaCl for 40 h at room temperature. Atox1 was then collected in elution fractions and analyzed by SDS-PAGE (tricine 19%) and MALDI-TOF mass spectrometer (Bruker, Bermen, Germany) or by electron spray ionization (ESI) mass spectrometry on a quadruple time of flight (QTOF) low-resolution micromass spectrometer (MicromassWaters, Corp.). Before labeling, 10 mM DTT was added to 0.3 mM protein solution and mixed for 10 h at 4 °C. DTT was dialyzed out using 3.5 kDa dialysis cassettes (Pierce) for 12 h. MTSSL (TRC; 1.0 mg) dissolved in 15 μL of DMSO (Bio lab) was added to 1 mL protein solution (1:20 protein/MTSSL molar ratio). The protein solution was then vortexed overnight at 4 °C. The free spin label was removed by several dialysis cycles (in 3 kDa cassettes) over 4 days. The mass of the spinlabeled protein was confirmed by mass spectrometry (was ensured to be above 95% purified spin-labeled protein),31 and the concentration was determined by a Lowry assay.58 Addition of the Metal Ion. Cu(I) (tetrakis(acetonitrile) copper (I) hexafluorophosphate (Aldrich)) was added to a protein solution under nitrogen gas to preserve anaerobic conditions. Cu(II) EPR signal was not observed at any time. The sequence of addition of the metal ion and Ctr1 intracellular loop domain to the Atox1 protein solution was tested, and it was observed that similar CW−EPR spectra are obtained at any sequence of addition. Glutaraldehyde Cross-Linking. Treatment with glutaraldehyde was conducted by mixing 20 μg (10 μL) of interacting protein in 20 mM (35 μL) of sodium phosphate and 0.15 M NaCl solution at pH = 8 (PBS 10×), which was then reacted with 5 μL of glutaraldehyde solution, incubated, and shaken for 10 min at 37 °C. The reaction was terminated by the addition of 5 μL of 1 M Tris−HCl at pH = 8 followed by SDS-PAGE (19% tricine) analysis. CD. CD measurements were carried out using a Chirascan spectrometer (Applied Photophysics, UK). Measurements were carried out over a range of 20−90 °C in a 1.0 cm optical path length cell. The data was recorded from 190 to 260 nm with a step size and a bandwidth of 0.5 nm. The CD signal was averaged for three scans for each sample. The temperature measurements were performed with a rate of 1 °C/min. EPR. CW−EPR spectra were recorded using an E500 Elexsys Bruker spectrometer operating at 9.0−9.5 GHz equipped with Elexsys super-high-sensitivity probe head. The spectra were recorded at room temperature (295 ± 2 K) at a microwave power of 20.0 mW, modulation amplitude of 1.0 G, time constant of 60 ms, and receiver gain of 60.0 dB. The samples

■

RESULTS To explore the interaction between the Ctr1 intracellular loop and Atox1, we synthesized a 22-residue segment corresponding to Ctr1 residues 85−106, with the sequence MPVPGPNGTILMETHKTVGQQM. This segment was chosen because it includes three methionine residues as well as a histidine residue, C


Article


Figure 2. SDS 19% tricine gel of Atox1 and Ctr1 intracellular loop in the absence and presence of glutaraldehyde cross-linker, as a function of Cu(I) (at two molar ratios of Atox1/loop, 1:1 and 1:2).

which might serve as potential residues to coordinate Cu(I) ions.61,62 To target the interaction between the Ctr1 intracellular loop and Atox1 metallochaperone, we exposed the two polypeptides to glutaraldehyde, which cross-links proximal proteins via their lysine residues. Figure 2 (and Figure S3 in Supporting Information (SI)) shows an SDS-PAGE analysis of Atox1 and the Ctr1 intracellular loop in the presence and absence of the cross-linker. In this experiment, the effective electrophoretic size of Atox1 increases with exposure to glutaraldehyde and the 22-residue Ctr1 loop is undetectable. Whereas the non-crosslinked dimeric Atox1 runs as a monomer in the denaturing gel conditions (slightly under 10 kDa in the absence of a crosslinker and slightly above 10 kDa in its presence), cross-linking converts a fraction of Atox1 into a covalent dimeric band running between 15 and 25 kDa. More interesting is the noticeable shift to higher molecular weight of the Atox1 band observed upon addition of the loop peptide in the absence and presence of Cu(I) (Figure 2, compare lanes 2−5 to lanes 1 and 6), a trend that is echoed in the dimeric bands as well. The intensity of this elevated band increases in excess of the Ctr1 loop (see bands 5 and 3 compared to 4 and 2, respectively). These findings support an interaction between Atox1 and the Ctr1 intracellular loop. Moreover, it seems that the presence of Cu(I) increases the intensity of a diffuse band just above the cross-linked Atox1−Ctr1 loop (10−15 kDa, compare lanes 3 and 5). This may indicate that Cu(I) induces cross-linking or could be a result of the difference in mobility of Atox1 observed in the presence of Cu(I) (Figure S3, comparing lanes 7 and 9). CD Characterization. Atox1 has an overall βαββαβ fold structure, and its CD spectrum is characterized by a negative peak at 220 nm (see Figure 3A), which we utilized to monitor the effect of the Ctr1 loop upon thermal stability of the protein in the presence of Cu(I). In the presence of the Ctr1 loop, the CD spectrum was characterized by contribution of random coil structure (at wavelengths of 190−210 nm), owing to the presence of the disordered Ctr1 segment; however, the

secondary structure of Atox1 was not affected by this interaction (Figure 3A). CD chromatograms were recorded in the 20−90 °C range and with increasing temperature exhibited a gradual reduction of the 220 nm peak and a concomitant increase of the signal at 200 nm, which is indicative of unfolding (Figure 3B). These changes in Atox1, Atox1 + Cu(I), and Atox1 + Ctr1 intracellular loop in the absence and presence of Cu(I) are presented in Figure 3C−F, demonstrating that the Ctr1 intracellular loop exerts a stabilizing effect upon Atox1 (all CD traces are presented in the SI). In the absence of the Ctr1 loop, Atox1 and Atox1 + Cu(I) were more structured at ambient temperature, remained stably folded up to 45−50 °C, and the unfolding of Atox1 occurred at Tm of 60−70 °C. In the presence of the Ctr1 loop, Atox1 remained stably folded up to 55−60 °C and the unfolding of Atox1 occurred at a bit higher temperature, at Tm of about 70−75 °C. Addition of Cu(I) to the Atox1−Ctr1 loop solution increased the Tm to about 75− 85 °C. This evident effect confirms that Atox1 and the Ctr1 loop form a complex in the solution, especially in the presence of Cu(I), which results in a higher energy of unfolding for Atox1. Cu(I) Coordination to the Ctr1 Intracellular Loop Domain. To assess whether the Ctr1 intracellular loop segment can bind Cu(I), we performed both CW−EPR and NMR measurements. We spin-labeled the Ctr1 intracellular loop domain on an added Cys residue at its C-terminal end (MPVPGPNGTILMETHKTVGQQMC−MTSSL) and followed the change in the CW−EPR spectra as a function of Cu(I) concentration (see Figure 4). The CW−EPR spectra are characterized by fast isotropic dynamics as expected for a peptide labeled at its terminus. However, a slight reduction in the hyperfine value (aN) was observed from 15.9 ± 0.05 G in the absence of Cu(I) to 15.6 ± 0.05 G at [Cu(I)]/[loop] = 2:1. This reduction in the hyperfine value suggests that the peptide does coordinate Cu(I) and that the spin label is pointing to a more-hydrophobic environment upon Cu(I) coordinaD


Article


Figure 3. (A) CD spectra for Atox1, Atox1 + Cu(I), Atox1 + loop, and Atox1 + loop + Cu(I) in a ratio of 1:1:1 at room temperature. (B) Atox1 + Cu(I) at various temperatures, 20−90 °C. The Ctr1 intracellular loop’s sequence is MPVPGPNGTILMETHKTVGQQM. Temperature effect at 220 nm (black square) and 200 nm (blue triangular) for (C) Atox1, (D) Atox1 + Cu(I) at a ratio of 1:1, (E) Atox1 + loop at a ratio of 1:1, and (F) Atox1 + loop + Cu(I) at a ratio of 1:1:1. The solid lines were only drawn to guide the eyes (they are not a fit).

tion,31,33,63 indicating conformational changes in the peptide upon metal binding. In an attempt to identify the Cu(I)-induced change in the Ctr1 loop conformation, the far-UV CD spectra of our peptide were recorded. As shown in Figure 5, the peptide exhibits a negative peak at 197 nm, which is typical of a random coil structure, and the addition of Cu(I) to the peptide failed to

change this behavior. Thus, it is apparent that the slight shielding of the spin label at the C-terminus of the peptide is not accompanied by a significant change in the secondary structure. To further investigate the molecular basis for Cu(I) binding and its effects upon the loop peptide, we used NMR to follow Cu(I)-induced changes upon chemical shifts of the peptide. E


Article


Figure 4. CW−EPR spectra of the Ctr1 intracellular loop domain labeled at the C-terminus with MTSSL (MPVPGPNGTILMETHKTVGQQMC−MTSSL). The change in the hyperfine value, aN, for the Ctr1 intracellular loop domain as a function of [Cu(I)] concentration. Figure 6. 1H,15N-HSQC-observed chemical changes induced by Cu(I) binding. Numbering is based on the peptide sequence, with residues 1−22 corresponding to Ctr1 residues 85−106. Shifting of peaks is indicative of an interaction with Cu(I). (A) Comparison of the spectrum before (black) and after (red) addition of 2 equiv of Cu(I), reflecting an overall change in the conformation adopted by the peptide. (B) Summary of the spectral changes quantified as ΔNH = sqrt((ΔH)2 + 0.04(ΔN)2), where ΔH and ΔN are the changes in 1H and 15 N resonances, respectively. Residues M1 (fast-exchanging free amine) and P2, P4, and P6 (lacking an amide proton) are undetectable in this spectrum. A 5-residue moving average is shown in gray, and Cu(I)coordinating residues M12 and H15 are highlighted.

nitrogens,61,62,64 it is reasonable to assume that the loop peptide coordinates the cuprous ion via such moieties and in doing so adopts a more compact conformation, which would result in chemical shifts throughout the peptide. This is also consistent with the aforementioned Cu(I)-induced decrease of the C-terminal spin-label hyperfine interaction, as it supports a global change in peptide conformation, which would reduce the solvent exposure of MTSSL. For a more structural description of the Cu(I) coordination center, it is helpful to follow changes in the 13C,1H-HMQC spectrum as these groups are more strongly affected by the Cu(I) ligand field. The HMQC exhibited both shifting and broadening of peaks, suggesting that equilibrium between the Cu(I)-bound and free conformations of the loop peptide occurs on a fast-to-intermediate time scale. Therefore, the spectrum was interpreted under the assumption that broadened peaks (in some cases beyond the detection limit) represent the nuclei most affected by Cu(I) coordination, followed by strongly shifted peaks. Examples include (1) the disappearance of H15 aromatic protons and a significant shift in the M12 Hβ/γ protons are good indications that the imidazole ring and the methionine side chain are in direct contact with the Cu(I) ion (Figure 7A), (2) in the Hα−Cα region, broadening was observed for M1, N7, M12, E13, and H15, whereas the analogous cross-peaks of residues P6, V18, M22, and others were unaffected (Figure 7B),

Figure 5. CD spectra of the Ctr1 intracellular loop in the presence and absence of Cu(I).

Briefly, homonuclear TOCSY and NOESY spectra were acquired, allowing full assignment of all Ctr1 loop protons (Figure S1), and subsequent heteronuclear 2D experiments carried out at natural abundance provided assignments for all backbone 15N and the majority of 13C nuclei of the peptide. Particularly informative were comparisons of the heteronuclear 15 1 N, H-HSQC and 13C,1H-HMQC spectra, which correlate protons with their neighboring N- and C-nuclei, respectively, before and after addition of 2 equiv of Cu(I). The H−N correlation spectrum (from which the protonated N-terminal methionine and all proline residues are absent by definition) exhibits Cu(I)-induced changes throughout the peptide, with the strongest effect observed for the central region spanning residues M12ETHKTV18 (correspond to Ctr1 residues M96− V102, Figure 6). As known electron−donor ligands for Cu(I) include Cys and Met sulfur atoms and His imidazole F


Article


Figure 7. 1H,13C-HMQC-observed chemical changes induced by Cu(I) binding delineate Ctr1 residues involved in ion coordination. Numbering is based on the peptide sequence, with residues 1−22 corresponding to Ctr1 residues 85−106. Shifting and/or broadening of peaks is indicative of an interaction with Cu(I); spectra before and after addition of 2 equiv of Cu(I) are in black and red, respectively. (A) The Hβ/γ−Cβ/γ region, exhibiting changes for residues in the 12−15 segment, the insets show the aromatic region of the spectrum containing peaks from the H15 imidazole ring carbons C2 and C4, (B) the Hα−Cα region contrasting various residues along the peptide sequence, (C) the Hδ−Cδ region comparing residue P2 (affected by Cu(I)) and residues P4 and P6 (unaffected), (D) effect of Cu(I) upon the N7 Hβ−Cβ correlation, and (E) summary of Cu(I)-induced effects upon peptide residues, with green, orange, and red designating weak, significant, and strong effects; glycines (weakest effect) are in gray.

(3) in the Hδ−Cδ region of proline residues, P4 and P6 are unaffected by Cu(I), whereas P2 exhibits Cu(I)-induced changes (Figure 7C), and (4) the double-peak observed for the N7 Hβ/Cβ correlation collapses to a single shifted peak (Figure 7D). All Cu(I) effects are summarized in Figure 7E, revealing three Cu(I) “hotspots”, centered around residues M1, N7, and M12/H15. Notably, changes are not observed for terminal residue M22, indicating that it is not involved in coordination of Cu(I). On the basis of these results, it is posited that three of the four Cu(I)-coordinating ligands are M1 and M12 thioether groups and the H15 imidazole ring (corresponding to Ctr1 residues M85, M96, and H99). On the basis of geometric considerations, a reasonable candidate for the fourth ligand is a backbone carbonyl, potentially of residue E13, a fact which would account for the chemical shift change for the T14 amide group. Finally, it appears that N7 is not directly involved in coordination of Cu(I) and the observed changes are indirect effects of the conformational change in the peptide. Probing the Interaction between the Ctr1 Intracellular Loop Domain and Metallochaperone Atox1. To investigate the conformational changes that Atox1 and the Ctr1 intracellular loop undergo upon interaction and Cu(I) coordination, we monitored the CW−EPR spectra of both spin-labeled Atox1 (at position Cys41)31 and spin-labeled Ctr1

intracellular loop domain. Figure 8A shows the effect of Cu(I) and the Ctr1 intracellular loop domain upon the CW−EPR spectra of spin-labeled Atox1. These were simulated with a constant g-tensor ([2.0087 2.006 2.0022] ± 0.0001) using a slow-motion theory derived by Freed and co-workers65 as implemented in the Easyspin toolbox59 and are presented as dotted lines in Figure 8. The CW−EPR spectrum of Atox1 could be simulated with a correlation time of 2 × 10−9 s, an isotropic electron−electron interaction (ωee) of 4 MHz, line width of 1.8 G, and hyperfine constant of aN = 16.5 G and was independent of Cu(I) up to a ratio of Atox1/Cu(I) of 1:3. Addition of the Ctr1 intracellular loop at 1:1 ratio kept the same hyperfine coupling and tumbling rate, though removed the electron−electron interaction and also reduced the line width to 1.0 G. However, the spectrum of the Cu(I)−Atox1− Ctr1 intracellular loop (3:1:1 ratio) reintroduced the isotropic electron−electron interaction (ωee = 2 MHz) and increased the line width to 1.2 G (aN = 16.5 G, correlation time of 2 × 10−9 ± 0.5 × 10−9 s). This suggests that the distance between the two spin labels in the Atox1 dimer is dependent on the presence of the Ctr1 intracellular loop and Cu(I). Therefore, it demonstrates an interaction between the Ctr1 intracellular loop and Atox1 and that this interaction is dependent on Cu(I). G


Article


Figure 8. Room temperature X-band CW−EPR spectra of (A) spin-labeled Atox1 in the presence and absence of Ctr1 intracellular loop and Cu(I) (solid lines); the dotted lines are simulated spectra; (B) spin-labeled Ctr1 intracellular loop in the presence and absence of nonlabeled Atox1 and Cu(I); and (C) spin-labeled Atox1 in the presence of spin-labeled Ctr1 intracellular loop in the absence and presence of Cu(I) at two different concentrations. The arrows mark the characteristic signals of exchange interaction.

G) by addition of the spin-labeled (as opposed to unlabeled) loop, suggesting some interaction between the two proteins. However, in the presence of Cu(I), addition of the spin-labeled loop results in a clear indication of a dipolar interaction, and at a higher Cu(I) concentration, characteristic signals of exchange interaction appeared (see the arrows in Figure 8C). Together these findings confirm that Atox1 and the Ctr1 intracellular loop closely interact with each other, and this interaction is dependent on Cu(I). We have previously demonstrated by DEER that Atox1 can assume two different conformational states, in which the distances between the two spin labels in the dimer are approximately 2.8 ± 0.7 and 4.5 ± 0.4 nm. These two conformations were preserved while interacting with the 15residue Ctr1 intracellular C-terminal domain independently of Cu(I), although the C-terminal peptide did restrict Atox1

Figure 8B shows the CW−EPR spectra of the spin-labeled Ctr1 intracellular loop, labeled at the C-terminal of this domain, with nonlabeled Atox1. In this case, the spectra reflected fast isotropic dynamics, as expected for a peptide labeled at its terminus. The hyperfine value (aN) slightly increased from 15.9 ± 0.05 to 16.1 ± 0.05 G upon addition of Atox1, whereas in the presence of Cu(I), these values changed to 15.6 ± 0.05 and 15.8 ± 0.05 G, respectively. Large changes in the EPR spectra were not expected as the Ctr1 intracellular loop is a peptide labeled at its terminus with no confirmed structure; nevertheless, small changes in the hyperfine value were observed, confirming the interaction between Atox1 and the Ctr1 intracellular loop. Finally, the intermolecular effects of spin labels on both proteins were examined (Figure 8C). In the absence of Cu(I), the CW−EPR spectra of Atox1 were marginally broadened (0.1 H


Article


Figure 9. Q-band DEER signals and corresponding distance distribution functions for spin-labeled Atox1 in the presence of Cu(I) and nonlabeled (MPVPGPNGTILMETHKTVGQQM) and spin-labeled (MPVPGPNGTILMETHKTVGQQMC−MTSSL) Ctr1 intracellular loops. The ratio of Cu(I)/Atox1/loop is 3:1:1.

motions.31 Single-molecule FRET studies similarly observed two conformations of Atox1 with intermonomer distances between the two C69 residues of 2.66 and 4.3 nm.10 Here, we utilized the DEER experiment to measure the effects of the Ctr1 loop upon spin-labeled Atox1. Figure 9 shows the DEER time-domain signals between the spin labels on both Atox1 domains. Although the distance distribution in the presence of Ctr1 intracellular loop reveals two typical distances, a major contribution of 2.3 ± 0.3 and a minor contribution of 3.1 ± 0.2 nm, a conservative assessment of the distribution (considering potential fitting errors) is of a single broad population with distances in the 2.0−3.2 nm range. A comparison with DEER results for the apo form of Atox1 suggests that the Ctr1 loop stabilizes the more compact conformation of Atox1 at the expense of the more open one.31 In addition, the distance distribution function for the apo-Atox1 is much broader, indicating higher flexibility of Atox1 in the absence of the Ctr1 intracellular loop than in the presence of the Ctr1 intracellular loop. This suggests that when the Ctr1 intracellular loop was added to the solution, there was less contribution of smaller distances ( Δυ, whereas for the latter, koff ≈ Δυ, where koff is the Cu(I) off-rate and Δυ is the frequency difference between the nucleus in question in both states. On the basis of an examination of several shifted peaks, this leads to an estimate of ∼103 s−1 for the off-rate. Theory predicts the Cu(I) diffusion-limited on-rate to be kon = 4πNADta , where NA is Avogadro’s number, Dt is the translational diffusion rate, and a is the effective size of the binding site on the target peptide.75 Making a reasonable assumption for Brownian diffusion of the hydrated Cu(I) ion, 10−9 m2/s (half the diffusion rate of water), and an effective size of a ∼ 2 Å for the coordination site and considering that the Ctr1 peptide has no net charge, kon can be estimated on the order of 109 M−1 s−1, resulting in a low micromolar affinity for its complex with Cu(I). This is a reasonable prediction considering that four coordination bonds are formed with limited loss of entropy due to the biomolecular reaction and is consistent with the results obtained for other Cu(I)-binding peptides with similar coordination sites.61,66,67 The dependence of Atox1−Ctr1 intracellular loop interaction on [Cu(I)] and the intermediate affinity of Cu(I) to the Ctr1 intracellular loop may indicate that Cu(I) transfer can occur between the Ctr1 intracellular loop and Atox1 only when the cellular Cu(I) concentration is high. In this case, Atox1 assumes a more flexible conformation state that allows it to interact with this segment and to transfer Cu(I). This proposes that Atox1 is highly flexible and adjusts itself to its surroundings and its partner biomolecule. The flexibility of Atox1 might be associated with an efficient control mechanism to ensure a proper Cu(I) transfer to the various integral proteins at different cellular Cu(I) concentrations.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b10222. Assignment tables of proton and heteronuclear shifts for the Ctr1 loop peptide before addition of Cu(I) and a comparison of TOCSY spectra in H2O before and after addition of Cu(I). Moreover, raw data of the DEER signals and CD data are presented (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 972-3-7384329. Fax: 972-3-7384053. ORCID

Sharon Ruthstein: 0000-0002-1741-6892 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was supported by the Israel Science Foundation, grant no. 280/12. The Elexsys E580 Bruker EPR spectrometer was partially supported by the Israel Science Foundation, grant no. 564/12. The 700 MHz spectrometer system was partially funded by a generous donation from Fundacion Adar. We are thankful to Ortal Marciano for her assistance with the SDSPAGE gel experiments.

■

REFERENCES

(1) Kaplan, J. H.; Lutsenko, S. Copper Transport in Mammalian Cells:Special Care for Metal with Special Needs. J. Biol. Chem. 2009, 284, 25461−25465. (2) Lutsenko, S. Human Copper Homeostasis: A Network of Interconnected Pathways. Curr. Opin. Chem. Biol. 2010, 14, 211−217. (3) Madsen, E.; Gitlin, J. D. Copper and Iron Disorders of the Brain. Annu. Rev. Neurosci. 2007, 30, 317−337. (4) Peñ a , M. M. O.; Lee, J.; Thiele, D. J. A Delicate Balance:Homeostatic Control of Copper Uptake and Distribution. J.Nutr. 1999, 129, 1251−1260. (5) Prohaska, J. R. Role of Copper Transporters in Copper Homeostasis. Am. J. Clin. Nutr. 2008, 88, 826S−829S. (6) De Feo, C. J.; Aller, S. G.; Siluvai, G. S.; Blackburn, N. J.; Unger, V. M. Three-Dimensional Structure of the Human Copper Transporter Hctr1. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 4237−4242. (7) Eisses, J. F.; Kaplan, J. H. Molecular Characterization of Hctr1, the Human Copper Uptake Protein. J. Biol. Chem. 2002, 277, 29162− 29171. (8) Wee, N. K. Y.; Weinstein, D. C.; Fraser, S. T.; Assinder, S. J. The Mammalian Copper Transporters Ctr1 and Ctr2 and Their Roles in Development and Disease. Int. J. Biochem. Cell Biol. 2013, 45, 960− 963. (9) Maryon, E. B.; Molloy, S. A.; Ivy, K.; Yu, H.; Kaplan, J. H. Rate and Regulation of Copper Transport by Human Copper Transporter 1 (Hctr1). J. Biol. Chem. 2013, 288, 18035−18046. (10) Keller, A. M.; Benitez, J. J.; Klarin, D.; Zhong, L.; Goldfogel, M.; Yang, F.; Chen, T.-Y.; Chen, P. Dynamic Multibody Protein Interactions Suggest Versatile Pathways for Copper Trafficking. J. Am. Chem. Soc. 2012, 134, 8934−8943.

■

CONCLUSIONS Using CD, NMR, and EPR, we successfully showed that the Ctr1 intracellular loop can coordinate Cu(I) via residues M85, M96, and H99 at intermediate affinity estimated as ∼1−10 μM. Moreover, we indicate that the Ctr1 intracellular loop closely interacts with Atox1 metallochaperone in the presence of Cu(I) and Atox1 is more thermodynamically stable while interacting with the Ctr1 intracellular loop. We also observed that the conformation assumed by Atox1 in the presence of Ctr1 intracellular domain is different from the conformation assumed in the presence of Ctr1 C-terminal domain. Therefore, we propose that Atox1 is highly flexible and can accommodate its conformational state based on its surroundings and partner biomolecule. This study shows that the Ctr1 intracellular loop J


Article

The Journal of Physical Chemistry B (11) Kidane, T. Z.; Farhad, R.; Lee, K. J.; Santos, A.; Russo, E.; Linder, M. C. Uptake of Copper from Plasma Proteins in Cells Where Expression of Ctr1 Has Been Modulated. Biometals 2012, 25, 697− 709. (12) Rosenzweig, A. C. Copper Delivery by Metallochaperone Proteins. Acc. Chem. Res. 2001, 34, 119−128. (13) Scushan, M.; Bhattacherjee, A.; Ben-Tal, N.; Lutsenko, S. A Structural Model for the Copper Atpase Atp7b to Facilitate Analysis of Wilson Disease Causing Mutations and Studies of Transport Mechanism. Metallomics 2012, 4, 669−678. (14) Wernimont, A. K.; Yatsunyk, L. A.; Rosenzweig, A. C. Binding of Copper(I) by the Wilson Disease Protein and Its Copper Chaperone. J. Biol. Chem. 2004, 279, 12269. (15) Boal, A. K.; Rosenzweig, A. C. Structural Biology of Copper Trafficking. Chem. Rev. 2009, 109, 4760−4779. (16) Burkhead, J. L.; Gogolin Reynolds, K. A.; Abdel-Ghany, S. E.; Cohu, C. M.; Pilon, M. Copper Homeostasis. New Phytol. 2009, 182, 799−816. (17) Cerpa, W.; Varela-Nallar, L.; Reyes, A. E.; Minniti, A. N.; Inestrosa, N. C. Is There a Role for Copper in Neurodegenerative Diseases? Mol. Aspects Med. 2005, 26, 405−420. (18) Donnelly, P. S.; Xiao, Z. G.; Wedd, A. G. Copper and Alzheimer’s Disease. Curr. Opin. Chem. Biol. 2007, 11, 128−133. (19) Festa, R. A.; Thiele, D. J. Copper an Essential Metal in Biology. Curr. Biol. 2011, 21, R877−R883. (20) Gaggelli, E.; Kozlowski, H.; Valensin, D.; Valensin, G. Copper Homeostasis and Neurodegenerative Disorder (Alzheimer’s, Prion, and Parkinson’s Diseases and Amyotropic Lateral Sclerosis). Chem. Rev. 2006, 106, 1995−2044. (21) Adlard, P. A.; Bush, A. I. Metals and Alzheimer’s Disease. J. Alzheimers Dis. 2006, 10, 145−163. (22) Linder, M. C.; et al. Copper Transport. Am. J. Clin. Nutr. 1998, 67, 965S−971S. (23) Robinson, N. J.; Winge, D. R. Copper Metallochaperones. Annu. Rev. Biochem. 2010, 79, 537−562. (24) Arnesano, F.; Banci, L.; Bertini, I.; Felli, I. C.; Losacco, M.; Natile, G. Probing the Interaction of Cisplatin with the Human Copper Chaperone Atox1 by Solution and in-Cell NMR Spectroscopy. J. Am. Chem. Soc. 2011, 133, 18361−18369. (25) Arnesano, F.; Banci, L.; Bertini, I.; Huffman, D. L.; O’Halloran, T. V. Solution Strcuture of the Cu(I) and Apo Forms of the Yeast Metallochaperone, Atx1. Biochemistry 2001, 40, 1528−1539. (26) Benítez, J. J.; Keller, A. M.; Huffman, D. L.; Yatsunyk, L. A.; Rosenzweig, A. C.; Chen, P. Relating Dynamic Protein Interactions of Metallochaperones with Metal Transfer at the Single-Molecule Level. Faraday Discuss. 2011, 148, 71−82. (27) Benítez, J. J.; Keller, A. M.; Ochieng, P.; Yatsunyk, L. A.; Huffman, D. L.; Rosenzweig, A. C.; Chen, P. Probing Transient Copper Chaperone- Wilson Disease Protein Interactions at the SingleMolecule Level with Nanovesicel Trapping. J. Am. Chem. Soc. 2008, 130, 2446−2447. (28) Aller, S. G.; Unger, V. M. Projection Structure of the Human Copper Transporter Ctr1 at 6 Å Resolution Structure Reveals a Compact Trimer with a Novel Channel-Like Architecture. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3627−3632. (29) De Feo, C. J.; Aller, S. G.; Unger, V. M. A Structural Perspective on Copper Uptake in Eukaryotes. Biometals 2007, 20, 705−716. (30) Portnoy, M. E.; Rosenzweig, A. C.; Rae, T.; Huffman, D. L.; O’Halloran, T. V.; Culotta, V. C. Strcuture-Function Analyses of the Atx1 Metallochaperone. J. Biol. Chem. 1999, 274, 15041−15045. (31) Levy, A. R.; Yarmiayev, V.; Moskovitz, Y.; Ruthstein, S. Probing the Structural Flexibility of the Human Copper Metallochaperone Atox1 Dimer and Its Interaction with the Ctr1 C-Terminal Domain. J. Phys. Chem. B 2014, 118, 5832−5842. (32) Kahra, D.; Kovermann, M.; Wittung-Stafshede, P. The CTerminus of Human Copper Importer Ctr1 Acts as a Binding Site and Transferes Copper to Atox1. Biophys. J. 2016, 110, 95−102. (33) Shenberger, Y.; Yarmiayev, V.; Ruthstein, S. Exploring the Interaction between the Human Copper Transporter, Ctr1, C-

Terminal Domain and a Methionine Motif in the Presence of Cu(I) and Ag(I) Ions, Using EPR Spectrosocopy. Mol. Phys. 2013, 111, 2980−2991. (34) Jeschke, G.; Abbott, R. J. M.; Lea, S. M.; Timmel, C. R.; Banham, J. E. The Characterization of Weak Protein-Protein Interactions: Evidence from Deer for the Trimerization of a Von Willebrand Factor a Domain in Solution. Angew. Chem., Int. Ed. 2006, 45, 1058−1061. (35) Banham, J. E.; Baker, C. M.; Ceola, S.; Day, I. J.; Grant, G. H.; Groenen, E. J. J.; Rodgers, C. T.; Jeschke, G.; Timmel, C. R. Distance Measurements in the Borderline Region of Applicability of Cw EPR and DEER: A Model Study on a Homologous Series of Spin-Labelled Peptides. J. Magn. Reson. 2008, 191, 202−218. (36) Godt, A.; Franzen, C.; Veit, S.; Enkelmann, V.; Pannier, M.; Jeschke, G. EPR Probes with Well-Defined, Long Distances between Two or Three Unpaired Electrons. J. Org. Chem. 2000, 65, 7575− 7582. (37) Jeschke, G.; Polyhach, Y. Distance Measurements on SpinLabelled Biomacromolecules by Pulsed Electron Paramagnetic Resonance. Phys. Chem. Chem. Phys. 2007, 9, 1895−1910. (38) Pannier, M.; Veit, S.; Godt, A.; Jeschke, G.; Spiess, H. W. DeadTime Free Measurement of Dipole-Dipole Interactions between Electron Spins. J. Magn. Reson. 2000, 142, 331−340. (39) Raitsimring, A. M.; Gunanathan, C.; Potapov, A.; Efremenko, I.; Martin, J. M. L.; Milstein, D.; Goldfarb, D. Gd3+ Complexes as Potential Spin Labels for High Field Pulsed EPR Distance Measurements. J. Am. Chem. Soc. 2007, 129, 14138. (40) Schiemann, O.; Prisner, T. F. Long-Range Distance Determinations in Biomacromolecules by EPR Spectroscopy. Q. Rev. Biophys. 2007, 40, 1−53. (41) Xu, Q.; Ellena, J. F.; Kim, M.; Cafiso, D. S. Substrate-Dependent Unfolding of the Energy Coupling Motif of a Membrane Transport Protein Determined by Double Electron-Electron Resonance. Biochemistry 2006, 45, 10847−10854. (42) Aitha, M.; Moritz, L.; Sahu, I. D.; Sanyurah, O.; Roche, Z.; McCarrick, R. M.; Lorigan, G. A.; Bennett, B.; Crowder, M. W. Conformational Dynamics of Metallo-B-Lactamase Ccra During Catalysis Investigated by Using DEER Spectroscopy. J. Biol. Inorg. Chem. 2015, 20, 585−594. (43) Martorana, A.; Bellapadrona, G.; Feintuch, A.; Gregorio, E. D.; Aime, S.; Goldfarb, D. Probing Protein Conformation in Cells by Epr Distance Measurements Using Gd3+ Spin Labeling. J. Am. Chem. Soc. 2014, 136, 13458−13465. (44) Milov, A. D.; Maryasov, A. G.; Tsvetkov, Y. D.; Raap, J. Pulsed ELDOR in Spin-Labeled Polypeptides. Chem. Phys. Lett. 1999, 303, 135−143. (45) Larsen, R. G.; Singel, D. J. Double Electron-Electron Resonance Spin-Echo Modulation: Spectroscopic Measurements of Electron Spin Pair Separations in Orientationally Disordered Solids. J. Chem. Phys. 1993, 98, 5134−5146. (46) Cai, Q.; Kusnetzow, A. K.; Hubbell, W. L.; Haworth, I. S.; Gacho, G. P. C.; Van Eps, N.; Hideg, K.; Chambers, E. J.; Qin, P. Z. Site-Directed Spin Labeling Measurements of Nanometer Distances in Nucleic Acids Using a Sequence-Independent Nitroxide Probe. Nucleic Acids Res. 2006, 34, 4722−4730. (47) Columbus, L.; Hubbell, W. L. A New Spin on Protein Dynamics. Trends Biochem. Sci. 2002, 27, 288−295. (48) Mchaourab, H. S.; Oh, K. J.; Fang, C. J.; Hubbell, W. L. Conformation of T4 Lysozyme in Studied by Site-Directed Spin Labeling. Biochemistry 1997, 36, 307−316. (49) Shin, Y.-K.; Levinthal, C.; Levinthal, F.; Hubbell, W. L. Colicin E1 Binding to Membranes: Time-Resolved Studies of Spin-Labeled Mutants. Science 1993, 259, 960−963. (50) Vamvouka, M.; Cieslak, J.; Van Eps, N.; Hubbell, W.; Gross, A. The Structure of the Lipid-Embedded Potassium Channel Voltage Sensor Determined by Double-Electron-Electron Resonance Spectroscopy. Protein Sci. 2008, 17, 506−517. (51) Altenbach, C.; Kusnetzow, A. K.; Ernst, O. P.; Hofmann, K. P.; Hubbell, W. L. High Resolution Distance Mapping in Rhodopsin K


Article

The Journal of Physical Chemistry B Reveals the Pattern of Helix Movement Due to Activation. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 7439−7444. (52) Hubbell, W. L.; Gross, A.; Langen, R.; Lietzow, M. A. Recent Advances in Site-Directed Spin Labeling of Proteins. Curr. Opin. Struct. Biol. 1998, 8, 649−656. (53) Hubbell, W. L.; Lopez, C. J.; Altenbach, C.; Yang, Z. Technological Advances in Site-Directed Spin Labeling of Proteins. Curr. Opin. Struct. Biol. 2013, 23, 725−733. (54) Cornish, V. W.; Benson, D. R.; Altenbach, C. A.; Hideg, K.; Hubbell, W. L.; Schultz, P. G. Site-Specific Incorportaion of Biophysical Probes into Proteins. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 2910−2914. (55) Stone, K. M.; Townsend, J. E.; Sarver, J.; Sapienza, P. J.; Saxena, S.; Jen-Jacobson, L. Electron Spin Resonance Shows Common Structural Features for Different Classes of Ecori-DNA Complexes. Angew Chem., Int. Ed. 2008, 47, 10192−10194. (56) Zou, P.; Bortolus, M.; Mchaourab, H. S. Conformational Cycle of the ABC Transporter Msba in Liposoms: Detailed Analysis Using Double Electron Electron Resonance Spectroscopy. J. Mol. Biol. 2009, 393, 586−597. (57) Pliotas, C.; Ward, R.; Branigan, E.; Rasmussen, A.; Hagelueken, G.; Huang, H.; Balck, S. S.; Booth, I. R.; Schiemann, O.; Naismith, J. H. Conformational State of the Mscs Mechanosensitive Channel in Solution Revealed by Pulsed Electron-Electron Double Resonance (PELDOR) Spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, E2675−E2682. (58) Peterson, G. L. A Simplification of the Protein Assay Method of Lowry Et Al. Which Is More Generally Applicable. Anal. Biochem. 1977, 83, 346−356. (59) Stoll, S.; Schweiger, A. Easyspin, a Comprehensive Software Package for Spectral Simulation and Analysis in EPR. J. Magn. Reson. 2006, 178, 42−55. (60) Jeschke, G. Deeranalysis 2006: Distance Measurements on Nanoscopic Length Scales by Pulse ESR. Biol. Magn. Reson. 2007, 27, 287−288. (61) Pushie, M. J.; Shaw, K.; Franz, K. J.; Shearer, J.; Haas, K. L. Model Peptide Studies Reveal a Mixed Histidine-Methionine Cu(I) Binding Site at the N-Terminus of Human Copper Transporter 1. Inorg. Chem. 2015, 54, 8544−8551. (62) Schwab, S.; Shearer, J.; Conklin, S. E.; Alies, B.; Haas, K. L. Sequence Proximity between Cu(I) and Cu(I) Binding Sites of Human Copper Transporter 1 Model Peptides Defines Reactivity with Ascorbate and O2. J. Inorg. Biochem. 2016, 158, 70−76. (63) Shenberger, Y.; Gottlieb, H. E.; Ruthstein, S. EPR and NMR Spectroscopies Provide Input on the Coordination of Cu(I) and Ag(I) to a Disordered Methionine Segment. J. Biol. Inorg. Chem. 2015, 20, 719−727. (64) Haas, K. L.; Putterman, A. B.; White, D. R.; Thiele, D. J.; Franz, K. J. Model Peptides Provide New Insights into the Role of Histidine Residues as Potential Ligands in Human Cellular Copper Acquisition Via Ctr1. J. Am. Chem. Soc. 2011, 133, 4427−4437. (65) Schnider, D. J.; Freed, J. H. Calculating Slow Motional Magnetic Resonance Spectra: a User’s Guide. In Biological Magnetic Resonance: Spin Labeling, Theory and Applications; Berliner, L. J., Reuben, J., Eds.; Plenum: New York, 1989; Vol. 8, Chapter 1. (66) Jiang, J.; Nadas, I. A.; Kim, J. M.; Franz, K. J. A Mets Motif Peptide Found in Copper Transport Proteins Selectively Binds Cu(I) with Methionine-Only Coordination. Inorg. Chem. 2005, 44, 9787− 9794. (67) Rubino, J. T.; Riggs-Gelasco, P.; Franz, K. J. Methionine Motifs of Copper Transport Proteins Provide General and Flexible Thioether-Only Binding Sites for Cu(I) and Ag(I). J. Biol. Inorg. Chem. 2010, 15, 1033−1049. (68) Chen, K.; Yuldasheva, J. E.; Penner-Hahn, J. E.; O’Halloran, T. V. An Atypical Linear Cu(I)−S2 Center Constitutes the High-Affinity Metal-Sensing Site in the Cuer Metalloregulatory Protein. J. Am. Chem. Soc. 2003, 125, 12088−12089.

(69) Hussain, F.; Olson, J. S.; Wittung-Stafshede, P. Conserbed Residues Modulate Copper Release in Human Copper Chapeone Atox1. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 11158−11163. (70) Boal, A. K.; Rosenzweig, A. C. Crystal Structure of Cisplatin Bound to Human Copper Chaperone. J. Am. Chem. Soc. 2009, 131, 14196−14197. (71) Changela, A.; Chen, K.; Xue, Y.; Holschen, J.; Outten, C. E.; O’Halloran, T. V.; Mondragon, A. Molecular Basis of Metal-Ion Selectivity and Zeptomolar Sensitivity by Cuer. Science 2003, 301, 1383−1387. (72) Banci, L.; Cantini, F.; Kozyreva, T.; Rubino, J. T. Mechanistic Aspects of HSOD1 Maturation from the Solution Structure of CuiLoaded Hccs Domain 1 and Analysis of Disulfide-Free Hsod1 Mutants. ChemBioChem 2013, 14, 1839−1844. (73) Badarau, A.; Dennison, C. Copper Trafficking Mechanism of CXXC-Containing Domains: Insight from the Ph-Dependence of Their Cu(I) Affinities. J. Am. Chem. Soc. 2011, 133, 2983−2988. (74) Rodriguez-Granillo, A.; Crespo, A.; Estrin, D. A.; WittungStafshede, P. Copper-Transfer Mechanism from the Human Chaperone Atox1 to a Metal-Binding Domain of Wilson Disease Protein. J. Phys. Chem. B 2010, 114, 3698−3706. (75) Berg, O. G.; von Hippel, P. H. Diffusion-Controlled Macromolecular Interactions. Annu. Rev. Biophys. Biophys. Chem. 1985, 14, 131−158.

L


Ctr1 Intracellular Loop Is Involved in the Copper ... - ACS Publications

Recommend Documents