EPR Spectroscopy Shows that the Blood Carrier Protein, Human

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EPR Spectroscopy Shows that the Blood Carrier Protein, Human Serum Albumin, Closely Interacts with the Nterminal Domain of the Copper Transporter, CTR1 Yulia Shenberger, Amit Shimshi, and Sharon Ruthstein J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b00091 • Publication Date (Web): 20 Mar 2015 Downloaded from http://pubs.acs.org on March 22, 2015

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The Journal of Physical Chemistry

EPR Spectroscopy Shows that the Blood Carrier Protein, Human Serum Albumin, Closely Interacts with the N-Terminal Domain of the Copper Transporter, Ctr1. Yulia Shenberger, Amit Shimshi, Sharon Ruthstein*

Department of Chemistry, Faculty of Exact Sciences, Bar Ilan University, Ramat-Gan, Israel 5290002 *corresponding author: [email protected] Tel. 972-3-7384329, Fax. 972-3-7384053

1 ACS Paragon Plus Environment


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Abstract Copper is an essential metal whose localization within the cells must be carefully controlled to avoid copper dependent redox cycling. Although most of the key proteins involved in cellular copper transfer have been identified, fundamental questions regarding the copper transfer mechanism have yet to be resolved. One of the blood carrier proteins believed to be involved in copper transfer to the cell is human serum albumin (HSA). However, direct evidence for close interaction between HSA and the extracellular domain of the copper transporter Ctr1 has not yet been found. By utilizing EPR spectroscopy, we show here that HSA closely interacts with the first 14 amino acids of the Ctr1, even without the presence of copper ions.

Keywords: Copper transfer • Ctr1 • human serum albumin • EPR spectroscopy • DEER


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Introduction Copper is required for various cellular processes and functions as a cofactor in specific proteins, catalyzing electron transfer reactions required for cellular metabolism.1-7 This metal is crucial to the development of the central nervous system, and disruption of copper homeostasis during fetal life leads to potential mortality, severe growth retardation, and neurodegeneration. An excess of copper can result in the unregulated oxidation of proteins, lipids, and other cellular components causing subsequent tissue injury.8-11 Copper coordination has also been linked to the promotion of peptide aggregation to form the amyloid plaques characteristic of Alzheimer's disease and the amyloid fibrils implicated in Parkinson's disease and the prion diseases. Thus, pathways of copper metabolism have evolved to ensure adequate quantities of copper for cellular survival, while protecting the organism from the consequences of metal excess. Today, it is known that Cu(II) is accumulated in the body through diet, and is then reduced to Cu(I) and delivered to the cell by the copper transporter (Ctr1).12-14 When Cu(I) is transferred into the cell, specific Cu chaperones are responsible for delivering it to specific cellular pathways.2, 7, 15, 16 However, the way copper is delivered into mammalian cells from the blood system is still unknown. Two of the three known protein carriers in the blood plasma are human serum albumin (HSA) and transcuprein,17-23 the main components of the exchangeable plasma copper pool, which bind Cu(II) directly and with high affinity. The third is ceruloplasmin, the protein responsible for up to 70% of copper in humans.19, 24 HSA and transcuperin have accessible binding sites for Cu(II) and can rapidly exchange copper between themselves; in ceruloplasmin, however, copper is buried in the structure, not dialyzable, and only extractable with disruptive procedures. Hence, it is believed that HSA and transcuperin deliver copper to the Ctr1.20 HSA coordinates one Cu(II) atom with 1 pM affinity17 through its first three amino residues Asp-Ala-His.17, 25 A sequence with a histidine in the third position is known as an ATCUN motif, and was found to bind Cu(II) and Ni(II) with high affinity.26, 27 Du et al.28 suggested that 20% of Cu(II) was transferred from HSA to the extracellular domain of Ctr1 upon incubation of the extracellular domain of Ctr1 and 1 equiv. of Cu(II)-HSA for 1 hr. This work suggests a close interaction between HSA and Ctr1. Haas et al.29 recently showed that the first 14 amino acids of the Ctr1 can coordinate one Cu(II) ion with an affinity of 10 pM at pH 7.4. This high affinity for Cu(II) should allow the Ctr1 protein to compete with biologically relevant extracellular Cu(II) carriers, such as HSA, and should form the first Cu(II) coordination site. How exactly are Cu(II) ions transferred 3 ACS Paragon Plus Environment


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from the blood carrier protein to the N-terminal segment of Ctr1? Is it by direct interaction between the two proteins? Such questions have not yet been resolved, and their answer is crucial to the understanding of the copper transfer mechanism. Electron paramagnetic resonance (EPR) spectroscopy has emerged as an excellent tool for resolving complex systems such as protein-protein interaction.30-32 EPR measurement can be performed in buffer solution, and even weak interactions between proteins can be detected. EPR can characterize properties such as redox state and ligand geometry for different functional states of the protein.33-37 In addition, EPR can measure distances between paramagnetic probes within the protein, and between proteins, up to 80 Å.31, 38-40 The most common experiment for obtaining nanoscale structure information is the pulsed electron double resonance (PELDOR) also commonly referred to as the double electron-electron resonance experiment (DEER). Pulsed EPR experiments can measure nanometer distances between paramagnetic probes, and continuous wave (CW) EPR can derive the dynamics of protein chains. The combination of CW and pulsed EPR has become widely used in biophysical research,32, 41-44 since an electron spin introduced into diamagnetic proteins can provide information on their local environment and on the mobility of the protein domain. In this study, EPR spectroscopy is applied to provide new insights on the copper uptake mechanism from the blood system to the N-terminal of Ctr1. We concentrate on the first 14 residues of Ctr1, which has been suggested to play a major role in Cu(II) coordination.29 The Ctr1 N-terminal domain segment has been synthesized and labeled, and conformational and structural changes upon interaction with HSA are monitored. This study presents one step towards a complete understanding of the cellular copper uptake mechanism in the human body.

Experimental Section Peptide synthesis, purification, and labeling- Table 1 lists the sequences of the peptides used in this study. All peptides were synthesized on a rink amide resin (Applied Biosystems). Couplings of standard Fmoc (9-fluorenylmethoxy-carbonyl)-protected amino acids were achieved with (O- Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HBTU, Dchem)

in

N,N-dimethylformamide

(DMF,

Bio

lab)

in

combination

with

N,N-

Diisopropylethylamine (DIPEA, Bio lab) for a one hour 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 a 5 mL cocktail of 95% (90% 4 ACS Paragon Plus Environment

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trifluoroacetic acid (TFA, Bio lab), 5% ethane dithiol (EDT, Alfa Aesar), and 2.5% triisopropylsilane (TIS, Alfa Aesar), 2.5% thioanisole (Alfa Aesar), for 2.5 h under N2. An additional 65 µL of bromotrimethylsilane (TMSBr. Alfa Aesar) were 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 3500 rpm. After evaporation of TFA under N2, 10mM DTT (Dithiothreitol, Sigma) were 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 MALDI-TOF MS-Autoflex IIITOF/TOF mass spectrometer (Bruker, Bermen, Germany) equipped with a 337 nm nitrogen laser, or with ESI (electron spray ionization) mass spectrometry on a Q-TOF (quadruple time of flight) low-resolution micromass spectrometer (Micromass-Waters, Corp.)

Peptide

samples were typically mixed with two volumes of permade dihydrobenzoic acid (DHB) matrix solution, deposited onto stainless steel target surfaces, and allowed to dry at room temperature. For SDSL: 1 mg lyophilized peptide was dissolved in 0.8 ml phosphate buffer (25mM KPi) (pH=7.4-7.6). 0.25 mg of S-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate (MTSSL, TRC) dissolved in 15µL Dimethyl sulfoxide (DMSO, Bio lab) was added to the solution (50-fold molar excess of MTSSL). The spin label and peptide solution was then vortexed overnight at 4°C. The free spin label was removed by HPLC semi preparative (Vydac, C18 1cm). The mass of the spin-labeled peptide was confirmed by mass spectrometer. Human serum albumin (HSA) was purchased from sigma-aldrich and was spinlabeled as described above. The free spin label was removed by several dialysis cycles over 6 days. HSA was added to a peptide solution dissolved in 25mM Kpi solution in a ratio of 1:1. EPR CW-EPR (continuous wave EPR) spectra were recorded using an E500 Elexsys Bruker spectrometer operating at 9.0-9.5 GHz equipped with a super high-sensitive CW resonator. The spectra were recorded at room-temperature (295±2K) at microwave power of 20.0 mW, modulation amplitude of 1.0 G, a time constant of 80 ms, and receiver gain of 60.0 dB. The samples were measured in 1.0 mm capillary quartz tubes (vitrocom). Spin-labeled peptide concentration for all samples was 0.3 mM. In the presence of non-labeled HSA, the spin-labeled peptide concentration was 0.15 mM. In the presence of spin-labeled HSA, the



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total concentration of the nitroxide radicals was 0.3 mM. For low temperature CW EPR measurements, 30% glycerol was added to the protein solution. The constant time four-pulse DEER experiment π/2(νobs)-τ1-π(νobs)-t’-π(νpump)-(τ1+τ2t’)-π(νobs)-τ2(νobs)-τ2-echo was carried out at (80 ± 1.0K) 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’, while τ2 was kept constant to eliminate relaxation effects. The pump pulse was set at the maximum of the nitroxide spectrum, and the observer pulse was set 60 MHz higher than the pump pulse (see Figure S1). The observer π/2 and π pulses had lengths of 40 ns, and the π pump pulse also had a length of 40 ns; the dwell time was 20 ns. The observer frequency was 33.65 GHz. The power of 40 ns π pulse was 20.0 mW. τ1 was set to 200 ns, repetition time was 5 ms, and shot per points was set to 30. The samples' concentration was similar to the concentration of the samples measured at the CW-EPR at low temperature and were measured in 1.6 mm capillary quartz tubes (Wilmand). The data was analyzed using the DeerAnalysis 2013 program, using Tikhonov regularization.45 The regularization parameter in the L curve was optimized by examining the compatibility of the time domain data and was found to be L=100 for all samples. The data presented in this manuscript is after 3D homogeneous background subtraction. CD: Circular Dichroism (CD) measurements were performed using a Chirascan spectrometer (Applied Photophysics, UK) at room temperature. Measurements were carried out in a 1 mm optical path length cell. The peptide concentration was 0.2 mM. The data was recorded from 190-260 nm with a step size and a bandwidth of 0.5 nm. The CD signal was averaged for 3 scans for each sample and the background was subtracted. Table 1. Sequences of Ctr1 peptides. Peptide designation

Sequence

Pep1

MDHSHHMGMSYMDS

Pep2

MDHSHHMGMSYMDSC-MTSSL

Pep3

MTSSL-CMDHSHHMGMSYMDS

Pep4

MTSSL-CMDHSHHMGMSYMDSC-MTSSL


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Results and Discussion HSA is a blood carrier protein, which carries Cu(II) ions in its N-terminal domain. Previous studies suggest that Ctr1 coordinates Cu(II) ion in its N-terminal domain involving the first 14 amino residues (see Figure 1), and before transferring copper into the cell reduces it to Cu(I) in the presence of ascorbate, in a mechanism that has yet to be resolved. 12, 13 In order to explore whether Ctr1 closely interacts with the Albumin (HSA), we synthesized four different peptides, as listed in Table 1. This study uses the site-directed spin labeling (SDSL) method.46-50 Thiol-reactive methanethiolsulfonate (MTSSL), a common nitroxide spin label, is chemically attached to the cysteine as illustrated in inset in Figure 1. HSA has one free cysteine residue, Cys34, which is reduced and accessible for labeling. Hence, HSA has been labeled with an MTSSL at this position. Figure 2 shows the CD spectra of the various synthesized peptides. All spectra are identical, and remain disordered upon spin-labeling. The CD spectra of the various peptides are characterized by a broad negative peak centered at 195 nm. Spectral analysis by CDNN software51 suggested that at least 50% of the peptide structure is disordered.

Figure 1 : A schematic view presenting the interaction between the HSA and the copper transporter, Ctr1, N-terminal domain which is explored in this study. Cys34 of HSA will be labeled with a MTSSL. The inset shows the sitedirected spin labeling (SDSL) method using methanethiolsulfonate (MTSSL) spin-label.

EPR measurements were performed to target the conformational changes that the peptides undergo while interacting with HSA. Figure 3 shows the CW-EPR spectra of the various spinlabeled peptides, with and without the presence of HSA and spin-labeled HSA. The CW-EPR spectra are characterized by fast motion of the MTSSL which is attached to a flexible peptide. Pep4 is spin-labeled at both ends of the peptide, hence the CW spectrum of pep4 is broader than the spectrum of pep2 or pep3, due to dipolar interaction between the two spin labels. Addition of non-labeled HSA to the peptide solution slightly increases the hyperfine coupling 7 ACS Paragon Plus Environment


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for pep2 by 0.2 G, and for pep3 by 0.3 G. The increase in the hyperfine coupling suggests that upon interaction with HSA, the spin-labels at the terminus of the peptides are exposed to a more hydrophilic environment. Moreover, the addition of HSA slightly restricts the motion of the spin-label attached to the peptide, causing a change in the intensity of the mI=-1 resonance line (high field absorption). Therefore, we calculated the reduction (R) in the intensity (I) of the resonance line at mI=-1 over the intensity of the resonance line at mI=0, R =

I ( mI =−1) I ( mI =0 )

, for

the various peptides with and without the presence of HSA. A higher reduction suggests that HSA further restricts the dynamic of the spin-label, suggesting that the close environment of the spin-label is less dynamic. For pep2, a reduction from R to 0.85R was observed in the presence of HSA, when compared to its absence. For pep3, we observed a smaller reduction from R to 0.93R in the presence of HSA, whereas, for pep4, a reduction of R to 0.75R was observed in the presence of HSA. This indicates that the motion of the spin-label attached to C15 is more restricted than that of the spin-label attached to C1 in the presence of HSA. The changes in the CW-EPR spectra upon addition of HSA to the peptide solution confirm that HSA and the N-terminal domain of Ctr1 closely interact with each other, and that the coordination site of HSA-Ctr1 is closer to S14 than to the first methionine residue. Incorporation of labeled HSA to the peptide solutions clearly shows characteristic features of broadening due to exchange interactions for all peptides. For pep3, the exchange features seemed to be a bit more dominant when compared to pep2, which might be associated with its higher dynamics at room temperature, allowing a spin-label at C1 position to assume conformations that are closer to the HSA spin-label site than spin-label at C15 position, which is more restricted in space. The broadening in the CW-EPR spectra suggests that paramagnetic centers are in close proximity to each other (< 2.0 nm), indicating close interaction between the spin-label attached to Cys34 of HSA to the spin-label attached to the N-terminal domain of Ctr1.

Figure 2: CD spectra of the various peptides used in this study at 298 K.


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Figure 3: X-band CW-EPR spectra at room temperature, ν=9.82 GHz, for pep2, pep3, and pep4 solutions (black solid lines), in the presence of HSA (red solid lines), and in the presence of spinlabeled HSA (green solid lines). The black arrows mark the characteristic signals corresponding to exchange interaction.

The CW-EPR at room temperature was able to target the close interaction between Ctr1 N-terminal domain and HSA. Moreover, the CW-EPR spectra suggest that the region of S14 is more restricted in the presence of HSA than is M1. To further explore the Ctr1 N-terminal and HSA interaction, we carried out CW-EPR measurements at 120K. Figure 4A shows the CW-EPR low temperature, 120 K, spectra of the various peptides with and without the presence of spin-labeled HSA. A clear change in the center line-width (∆H0) is observed for pep2 and pep4, whereas for pep3 no changes in the spectrum line shape were observed in the presence of labeled HSA. Spectral broadening at low temperature suggests that the distance between two spin-labels is smaller than 1.8 nm. The CW-EPR data at 120K indicates that the spin-label attached to C15 may assume conformations where the interdistance between it and the spin-label attached to Cys34 of HSA is smaller than 1.8 nm. Figure 4B shows the changes in ∆H0 for the various peptides in the presence of unlabeled and labeled HSA. For comparison, ∆H0 for a solution of solely spin-labeled HSA was found to be 12.3 ± 0.05G. The error in ∆H0 was determined based on the number of points (2048 points) and the magnetic 9 ACS Paragon Plus Environment


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field spectral width (200 G). Addition of unlabeled HSA to the peptide solution did not greatly affect the CW-EPR spectra at low temperature, as expected.

Figure 4: A. CW EPR spectra at 120K, ν=9.31 GHz, for pep2, pep3, and pep4 solutions, and in the presence of spin-labeled HSA. B. The change in ∆H0 for the various peptide solutions, and in the presence of HSA and labeled HSA.

To further investigate this structural information, double electron-electron resonance (DEER) experiments were carried out (Figure 5). The DEER time domain signal of pep2 in the presence of spin-labeled HSA resolves a clear time domain modulation of 800 ns, corresponding to 1.25 MHz dipolar interaction, as is observed in the FT spectra. A distance distribution of about 3.5 ± 0.3 nm was obtained by Tikhonov regularization. However, since the Tikhonov regularization does not adequately fit at the first 200 ns of the DEER signal, it might be that smaller distances, shorter than 2.0 nm, which could not be excited by the 40 ns mw pulses, also contribute to the DEER signal, as was further detected in the low temperature CW-EPR data. The Tikhonov validation (performed with the DeerAnalysis2013 program) of this distribution (noise, background start and dimensionality (between 2.0 to 3.0)) is shown by gray shadow in Figure 5. The DEER data of pep3:HSA initiated a broader distance distribution function between 2.0-5.0 nm, which was confirmed by Tikhonov validation and the less resolved FT spectrum compared to the FT spectrum of pep2:HSA. The DEER data of pep2 and pep3 in the presence of labeled HSA indicates that the N-terminal domain of Ctr1 can assume various conformations. Nevertheless, the comparable broad distance distribution function obtained for pep3_labeled HSA compared to pep2_labeled HSA suggests that the first residues of the N-terminal domain of Ctr1 are more flexible, while C15 residue is more fixed in space after interacting with HSA. This is further supported by the CW-EPR spectra at room temperature, which shows a reduction in the dynamic of the spin-label attached to C15 upon interacting with unlabeled HSA for pep2. For pep4 alone, the distance distribution 10 ACS Paragon Plus Environment

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between the two spin-labels attached to the two ends of the segment was found to be 2.8 ± 0.7 nm. The DEER data of pep4 in the presence of spin-labeled HSA reveals a broad distance distribution between 2.0–5.0 nm, corresponding to the intramolecular interaction between the two spin-labels attached to pep4, and additional distributions correspond to the intermolecular interaction between pep4 and spin-labeled HSA. Since it is a three-spin system, ghost distributions may exist. Hence, we also used the ghost suppression method for multi-spin system (implemented in the DeerAnalysis 2013 program), developed primarily for rigid symmetric systems.52 However, the change in the distance distributions was found to be negligible, as is presented by the dashed line in Figure 5D. The DEER data for all peptides indicates that the interdistance distribution of a spin-label attached to the termini of Ctr1 Nterminal domain and a spin-label attached to Cys34 of HSA is within the broad distribution of 3.5 ± 1.5 nm. Taking a closer look at the DEER data, it can be observed that the DEER excitation probability factor of pep2, pep3 in the presence of HSA, is similar to the excitation probability between the two spin-labels attached to pep4 (~ 0.04) (see Figure 5). This similar excitation profile suggests that almost all of the HSA proteins directly coordinated to the Nterminal domain of Ctr1, show high affinity between this segment and the HSA protein. This is supported, moreover, by the CW-EPR data, which shows larger dipolar interaction and a reduction in dynamic in the presence of labeled HSA for the various peptide segments. We recently studied the interaction between the Ctr1 cytoplasmic domain and a methionine segment, a general copper binding site,53 as well as the interaction between the Ctr1 cytoplasmic domain and the copper metallochaperone, Atox1 by EPR spectroscopy and DEER.32 There, however, the excitation probability for the interdipolar interaction between the Ctr1 cytoplasmic domain and the target protein was 60-75% lower, compared to the intradipolar interaction in the targeted protein. This suggests that the affinity between the first fifteen amino residues of Ctr1 and the blood carrier protein, HSA, is much higher than the affinity between the Ctr1 intracellular domain and a cytoplasmic target protein, which was reported to be in the µM range.54 This might further suggest that the time scale of the copper uptake is shorter than the time scale of the cellular copper import mechanism by the Ctr1.



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Conclusions In this study, EPR spectroscopy succeeded in showing a close and specific interaction between HSA and the first fifteen amino acids of the extracellular domain of Ctr1, even without the presence of copper ions. It has shown that the Ctr1 N-terminal segment can assume various conformations upon interacting with HSA, however, a spin-label attached to C15 is more fixed in space upon interaction with HSA than a spin-label attached to C1. This indicates that the binding site of HSA is closer to S14 than to the first methionine residue. Moreover, comparing the data obtained in this study with previous studies suggests that the affinity between the Ctr1 extracellular domain and HSA is larger than the affinity between the Ctr1 cytoplasmic domain and a cellular target protein.

Acknowledgements This work was supported by the Israel Science Foundation (ISF), grant no, 280/12. The Elexsys E580 Bruker EPR spectrometer was partially supported by the Israel Science Foundation, grant no. 564/12.

Supporting Information The supporting information includes EPR raw data. This material is available free of charge via the Internet at http://pubs.acs.org.


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Figure 5: Q-band DEER measurements for spin-labeled pep2 and pep3 in the presence of spin-labeled HSA, pep4, and pep4 in the presence of spin-labeled HSA, corresponding FT spectra, and distance distribution obtained from Tikhonov regularization. Tikhonov validation was applied to evaluate the error in the distance distribution functions, taking into account: white noise, background start and dimensionality (550 trials). The dashed line in the distance distribution for pep4+HSA (D) corresponds to the distribution obtained after ghost suppression method for a multiple-spin system (number of spins > 2).



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EPR Spectroscopy Shows that the Blood Carrier Protein, Human

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