and Inter-Repeat Copper Binding Modes within the ... - ACS Publications

Oct 23, 2008 - ... ReceiVed: August 30, 2008. The unique biology of prion proteins (PrPs) allied with the public-health risks posed by prion zoonoses,...
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15140

J. Phys. Chem. B 2008, 112, 15140–15150

Structural Characterization of the Intra- and Inter-Repeat Copper Binding Modes within the N-Terminal Region of “Prion Related Protein” (PrP-rel-2) of Zebrafish Elena Gaggelli,† Elzbieta Jankowska,‡ Henryk Kozlowski,*,§ Alina Marcinkowska,‡ Caterina Migliorini,† Pawel Stanczak,§ Daniela Valensin,† and Gianni Valensin*,† Department of Chemistry, UniVersity of Siena, Via Aldo Moro, 53-100 Siena, Italy, Faculty of Chemistry, UniVersity of Gdan˜sk, Gdan˜sk, Poland, and Faculty of Chemistry, UniVersity of Wrocław, F. Joliot-Curie 14, 50-383 Wrocław, Poland ReceiVed: May 29, 2008; ReVised Manuscript ReceiVed: August 30, 2008

The unique biology of prion proteins (PrPs) allied with the public-health risks posed by prion zoonoses, such as various animal neurodegenerations, has focused much attention on the molecular basis of the controls cross-species and on the similarities between PrPs from different species. Given the common feature of PrPs as Cu2+ binding proteins, it appears relevant to compare the impact of Cu2+ on the stability constants and structures of “physiological” complexes. After having comprehensively delineated the interaction of Cu2+ with mammalian and avian PrPs, the stabilities and molecular structures of species generated by Cu2+ interacting with the irregular repeated domain derived from Danio rerio zebrafish PrP-rel-2 were investigated. Copper complexes with different zebrafish PrP-rel-2 fragments were analyzed by potentiometric and spectroscopic techniques. The data were interpreted as to provide evidence of all investigated repeat units selectively binding Cu2+ via the His imidazole(s). The structural models obtained from paramagnetic NMR showed an intra- or inter-copper binding according to the number of the His in the sequence. In comparison to the mammalian and avian cases, the enzymatic function referred to SOD-like activity was shown to be rather faint in the fish PrPs cases. Introduction Prion diseases are fatal neurodegenerations in humans and animals, which may be of infectious, sporadic or inherited origin.1-3 All the diseases share the common feature of an aberrant metabolism of the prion protein, PrP, resulting in the PrPC f PrPSc transformation that implies a change in secondary structure elements. Even though the role of PrP in the pathogenesis is generally agreed, its normal function remains elusive. Evidence suggests that one of the most likely functions of the PrPC in presynaptic space is super oxide dismutase (SOD)like activity.4-8 This function may be abolished by the removal of the octarepeat region involved in the copper ion binding or by addition of copper chelators.6 The SOD-like activity was reported for both the full-length mouse and chicken recombinant PrPs, which were refolded in the presence of copper.5 Recently it has also been shown that a single Cu2+ ion bound to the 4 His side-chain imidazoles within the repeat region of mammalian and avian PrP shows SOD activity.9 Another possibility is that PrPC endocytosis transports copper from the extracellular space to the cell interior.10-13 Other studies have revealed that PrPC is also implicated in copper buffering,14 copper sensing,15 copper reductase activity,16 signal transduction,17 the suppression of apoptosis,18 and neuronal activity.19 The homologues of mammalian disease genes are likely to contribute to a deeper understanding of the physiological and pathogenic processes. Prion protein gene (PRNP) homologues discovered so far include (i) the Doppel gene (PRND) which * To whom correspondence should be addressed. E-mail: (G.V.) valensin@ unisi.it; (H.K.) [email protected]. † University of Siena. ‡ University of Gdan ˜ sk. § University of Wrocław.

lies adjacent to PRNP in the genomic sequence and encodes for the protein doppel Dp1,20,21 (ii) the testis-specific gene PRNT adjacent to PRND,22 not found in mouse, rat, or cow and (iii) the shadow of prion protein gene, SPRN, encoding for the shadow protein, annotated in eutherians and fish.23 Mammalian PrPC, that is predominantly expressed in the central nervous system, is a variably glycosylated protein tethered to the outer surface of the plasma membrane by a glycosylphosphatydylinositol (GPI) anchor located on its Cterminus to the outer surface of the plasma membrane. Residues 60-91 of the protein consist of four tandem repeats with the consensus sequence PHGGGWGQ that have a strong impact on the biology of copper.2,6,11,15,16,24-26 This region, known as the octapeptide repeat fragment was first discovered to bind Cu2+ ion.27 Also the N-terminal region of avian PrP contains mostly regular hexapeptide repeats that bind Cu2+,25,28-31 but its physiological function is much less examined. The family of fish prion proteins is characterized by sequences encompassing potential Cu2+ binding sites. In the Fugu rubripes StPrP-2 protein, the region encompassing residues 94-128, also known as Gly-Pro rich domain, contains three consecutive repeats connected by PGYG linkers. Recently, it has been shown that StPrP-2 96-128 fragment binds the copper ion even more effectively than the human octapeptide repeat region:25,32 coordination of Cu2+ implies multi imidazole donors and yields an inter- repeat binding mode stronger than that of the mammalian PrPs.25,32 Studying prion proteins from different species is expected to provide valuable information on PrP molecular evolution as well as on structure/function relationships. It may therefore be rather important to investigate copper coordination properties of prion proteins from species phylogenetically dissimilar.

10.1021/jp804759q CCC: $40.75  2008 American Chemical Society Published on Web 10/23/2008

Cu2+ Complexes with Fragments of PrP-rel-2 of Zebrafish It has recently been reported that the duplicate of PrP-rel genes (zebrafish PrP-like) yields two proteins: PrP-rel-1 and PrPrel-2 also known as zebrafish PrP3 or zebrafish PrP-like.33,34 It is worth noting that PrP-rel’s share very common features with PRNP and code short (188 aa), GPI anchored proteins containing PrP features, such as a highly conserved hydrophobic region, a β1 stretch, and irregular repeats. The repeat domain in PrPrel-2 encompassing residues 60-87, contains three hexapeptide HXGHXG repeats. The presence of seven His in the primary sequence of zPrP63-87 (Ac-PV-HTGHMG-HIGHTG-HTGHTGSSG-HG-NH2) and the occurrence of two His in just one hexapeptide makes the N terminal region of StPrP-2 and PrPrel-2 very similar suggesting a common copper binding chemistry and consequently a common biological role of Fugu rubripes StPrP-2 and zebrafish Danio rerio PrP-rel-2 proteins. In the present work, the structures and copper binding modes of four peptides derived from irregular repeat domain of PrPrel-2 from zebrafish Danio rerio have been investigated by potentiometric titrations as well as spectroscopic data (NMR and restrained MD simulation, CD, UV-vis, and EPR). The considered peptides are (i) zPrP63-70 (Ac-PV-HTG-HMGNH2), (ii) zPrP63-74 (Ac-PVHTGHMGHIGH-NH2), (iii) zPrP6380 (Ac-PVHTGHMGHIGHTGHTGH-NH2) and (iv) zPrP6387 (Ac-PVHTGHMGHIGHTGHTGHTGSSGHG-NH2). Also the SOD-like activity of the all analyzed peptides were measured and compared to those of chicken and human PrP fragments in order to make inferences on the potential physiological role of fish PrP’s. Materials and Methods Peptide Synthesis, Purification, and Characterization. Syntheses of peptide amides Ac-PVHTGHMG-NH2 (zPrP6370), Ac-PVHTGHMGHIGH-NH2 (zPrP63-74), Ac-PVHTGHMGHIGHTGHTGH-NH2 (zPrP63-80), and Ac-PVHTGHMGHIGHTGHTGHTGSSGHG-NH2 (zPrP63-87) were performed on Cross-Linked Ethoxylate Acrylate Resin (CLEAR-Amide Resin; Peptides International) using Fmoc (Fmoc ) 9-fluorenylmethoxycarbonyl)strategywithcontinuous-flowmethodology.35,36 Attachment of the first amino acid to the resin and next coupling steps were realized using diisopropylcarbodiimide (DIPCI) as a coupling reagent in the presence of 1-hydroxybenzotriazole (HOBt) in dimethylformamide (DMF)/N-methylpyrrolidone (NMP)/methylene chloride/Triton X-100 (33:33:33:1, v/v) mixture. Removal of Fmoc protecting group during peptide synthesis was achieved by means of 20% piperidine solution in DMF/ NMP (1:1, v/v) + 1% Triton X-100.37 N-terminal amino group was acetylated using 1 M acetylimidazole in DMF. All peptides were cleaved from the resin and deprotected by treatment with a mixture containing 88% of trifluoroacetic acid, 5% of phenol, 2% of triisopropylsilane, and 5% of water. The cleavage reaction was carried out for 2 h at room temperature.37 The crude peptides were purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C8 semipreparative Kromasil column (25 × 250 mm, 7 µm). The purity of the peptides was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and analytical RP-HPLC using a C8 Kromasil column (4.6 × 250 mm, 5 µm) and 60 min linear gradient of 0-80% acetonitrile in 0.1% aqueous trifluoroacetic acid as a mobile phase. Analytical data were as follows: (1) Rt ) 11.3 min, MS ) 876.0 [M+], calc. 876.0; (2) Rt )11.8 min, MS ) 1320.6 [M+], calc. 1320.5; (3) Rt ) 12.0 min, MS ) 1911.0 [M+], calc. 1911.1; (4) Rt ) 12.6 min, MS ) 2495.5 [M + H]+, calc. 2494.7.

J. Phys. Chem. B, Vol. 112, No. 47, 2008 15141 Potentiometric Measurements. Stability constants for both proton and Cu2+ complexes were calculated from five titrations carried out over the pH range 3-11 at 298 K using a total volume of 3 cm3. The purities and the exact concentration of the ligands solution were determined by the method of Gran.38 NaOH was added from a 0.500 cm3 micrometer syringe, which was calibrated by both weight titration and the titration of standard materials. Ligand concentrations were 2 × 10-3 mol dm-3 (for zPrP63-70) and 1 × 10-3 mol dm-3 (for others). The metal to ligand molar ratio was 1:1. The pH-metric titrations were performed at 298 K in (30:70, v/v) DMSO/water in 0.1 mol dm-3 KNO3 on a MOLSPIN pH-meter system using a normal Russel CMAW 711 semicombined electrode calibrated in proton concentrations using HNO3 dissolved in (30:70 v/v) DMSO/water, respectively.39 The calculated ionic product for DMSO/water solutions was 14.511. The SUPERQUAD and HYPERQUAD 2000 programs were used for the stability constant calculations.40,41 Standard deviations were computed by SUPERQUAD and HYPERQUAD 2000 and refer to random errors only. They are however, good indications of the importance of a particular species in the equilibrium. EPR, UV-Vis, and CD Measurements. Solutions were of similar concentrations to those used in the potentiometric studies and 30% ethylene glycol was used as a cryoprotectant for EPR measurements. Electron paramagnetic resonance (EPR) spectra were recorded on a Bruker ESP 300E spectrometer at X-band frequency (9.3 GHz) in liquid nitrogen. The EPR parameters were calculated for the spectra obtained at the maximum concentration of the particular species for which well-resolved separations were observed. The absorption spectra were recorded on a Beckman DU 650 spectrophotometer. Circular dichroism (CD) spectra were recorded on Jasco J 715 spectropolarimeter in the 750-240 nm range. The values of ∆ε (i.e., εl - εr) and ε were calculated at the maximum concentration of the particular species obtained from the potentiometric data. SOD-like Activity. The in vitro SOD activities of the copper complexes were evaluated at 298 K, in samples containing Cu(NO3)2 and peptides in the ratio 1:1 in Tris-HCl buffer (25 mM, pH 7.4, and pH 5.3, respectively). The enzymatic activity was examined indirectly using nitroblue tetrazolium (5 × 10-5 M) assay.42 The superoxide anion was generated in situ by the xanthine/xanthine oxidase reaction and detected spectrophotometrically by monitoring the reduction of NBT at 550 nm. The concentration of xanthine was 1 × 10-4 M, and the reaction was evoked by adding appropriate amount of xanthin oxidase to form ∆A550 ) 0.025-0.026 min-1. The reduction rate of NBT was measured in the presence as well as in the absence of the studied system ([Cu2+]tot ) 0-1.5 × 10-6 M). The control experiment was also carried out in the presence of horseradish Cu, Zn-SOD (0-7 × 10-8 M). Separately, the experiment in that the xanthine oxidase activity was monitored following urate production, spectrophotometrically at 298 nm was carried out in order to exclude any inhibition induced by the copper(II)peptide complexes. The SOD-like activity was then represented as IC50 values indicating the concentration that causes 50% inhibition of NBT reduction. The lower value of IC50 means higher SOD activity. NMR Spectroscopy. NMR spectra were performed at 14.1 T with a Bruker Avance 600 MHz Spectrometer at controlled temperatures ((0.1 K). Solutions were prepared either in 100% D2O (99.95% from Merck) or in 90% H2O/10% D2O mixture and were carefully deoxygenated through a freezing/vacuumpumping/sealing/thawing procedure. The pH was adjusted at desired values with either DCl or NaOD. The desired concentra-

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Gaggelli et al.

tion of copper ions was achieved by using a stock solution of copper nitrate (Sigma Chemical Co.) in deuterium oxide. TSPd4, 3-(trimethylsilyl)-[2,2,3,3-d4] propansulphonate, sodium salt, was used as internal reference standard. Suppression of residual water signal was achieved by presaturation or excitation sculpting. A typical 1H NMR spectrum required 8 transients acquired with a 9.2 µs 90° pulse, 6600 Hz spectral width, and 2.0 s recycling delay. The assignment was accomplished with total correlated spectroscopy (TOCSY), correlated spectroscopy (COSY), nuclear Overhauser enhancement spectroscopy (NOESY), and rotating frame Overhauser enhancement spectroscopy (ROESY) 2D experiments. TOCSY spectra were recorded with a total spin-locking time of 75 ms using a MLEV-17 mixing sequence. ROESY was performed at a mixing time ranging from 150 to 300 ms and the radio frequency strength for the spinlock field was 1.9 KHz. The spectral width of homonuclear 2D experiments was typically 6000 Hz in both F1 and F2 dimensions. Spin lattice relaxation rates were measured with inversion recovery pulse sequences. All rates were calculated by regression analysis of the initial recovery curves of longitudinal magnetization components leading to errors not larger than ( 3%. While the simple inversion recovery experiment is suitable for the wellisolated peaks, the IR-TOCSY sequence was used to calculate the relaxation rates of the overlapping 1H NMR signal. This was obtained by introducing a 1H 180° pulse followed by a variable delay in front of the TOCSY sequence. The T1 values were determined by a three-parameter fit of peak intensities to the following equation:

I(τ) ) I0[1 - (1 + B)] exp(-τ/T1) where B is a variable parameter that considers nonideal magnetization and has a value smaller than one. The obtained results were compared with those obtained from normal IR sequence. The agreement was found in the errors limit of both experiments. Heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments were carried out with standard pulse sequences. Spectral processing was performed on a Silicon Graphics O2 workstation using the XWINNMR 3.1 software. Extraction of Structures from NMR Data. In solutions of zPrP peptides containing substoichiometric amounts of paramagnetic ions, the measured spin-lattice relaxation rate was averaged over the values in the free (f) and metal-bound (b) environments43

R1obs )

1 T1obs

)

pf T1f +

k-1 on

+

pb T1b + k-1 off

(1)

where pf and pb are fractional populations and kon and koff are kinetic rate constants for entrance into and exit from the metal coordination sphere. Since entrance into the metal coordination sphere is a fast diffusion controlled process, kon-1 , T1f usually holds, which yields the so-called paramagnetic contribution to the spin-lattice relaxation rate43,44

R1p ) R1obs - pfR1f )

pb T1b + k-1 off

(2)

The paramagnetic contribution is easily measured and has the potential of providing T1b ) 1/R1b, which is the structure-

sensitive term and is given by the Solomon equation,45 here reported for systems with S ) 1/2

( )

{

2 2 2 τc 1 1 µ0 2 p γ1γS ) R1b ) + 6 T1b 10 4π r 1 + (ωI - ωS)2τ2c 6τc 3τc + 2 2 1 + ωI τc 1 + (ωI - ωS)2τ2c

}

(3)

where µ is the permeability of vacuum, γΙ and γS are the nuclear and electron magnetogyric ratios respectively, ωΙ and ωS the nuclear and electron Larmor frequencies, r is the metal–nucleus distance, and τc is the effective correlation time. In all the investigated systems τc was determined from the Stokes equation and the following values were used: zPrP63-70-Cu2+ τc ) 0.40 ns at T ) 298 K, zPrP63-74-Cu2+ τc ) 0.80 ns at T ) 298 K, zPrP63-80-Cu2+ τc ) 0.90 ns at T ) 298 K, zPrP63-87-Cu2+ τc ) 1.00 ns at T ) 298 K. When metal–nucleus distances are sought, T1b must be calculated from eq 2 and, therefore, the koff-1 value, if not negligible, must be evaluated. In all the analyzed systems the exchange off-rate was estimated by using Cu-H distances from which the corresponding T1b can be calculated from eq 3 and then inserted into eq 2 for obtaining koff-1.29,32,46,47 The considered fixed distances were (i) the Cu-Hε distance (0.31 nm) in His-anchored Cu2+-peptide complexes and (ii) the Cu-HR distance of the residue “X” (0.25-0.40 nm), in cases where Cu2+, binds to deprotonated amide nitrogen belonging to a “X” generic residue. The obtained exchange rates were then used to calculate all copper-proton distances from the R1p values measured in the presence of Cu2+ by using first eq 2 and then eq 3.29,32,46-48 Molecular Dynamics. The distance constraints obtained from relaxation measurements were used to build a pseudo potential energy for a molecular dynamics calculation. In this procedure the potential energy is a function of the difference between the distance constraints provided by the user and corresponding distances found in a given conformer (target function). No other potential energy terms are present except the Van der Waals repulsion. At the beginning of the calculation an arbitrary number of different conformers is generated by randomly varying torsional dihedral angles. Then, the potential energy is minimized by a simulated annealing procedure in the torsion angle space in which the system is brought to a high temperature to allow all possible high energy starting conformations and subsequently cooled down in order to stabilize it in those potential energy minima that better satisfy the imposed constraints. In particular we performed the calculation with the program DYANA,49 using 10 000 steps and 300 random relative starting positions of peptides and Cu2+. Since only one molecule can be given as input in the program, peptides were linked to Cu2+ through a long chain of linkers without Van der Waals radii. These linkers can freely rotate around their bonds and be enabled to sample all possible relative positions of the ligand with respect to copper before the minimization step. The obtained structure was then optimized through an energy minimization followed by a 250 ps restrained molecular dynamics simulation (50 ps to bring the system from 0 to 298 K, followed by 200 ps at a constant temperature of 298 K), both in water solvent, using the program Hyperchem with the AMBER force field.50 This was done in order to validate and refine the structure, especially regarding the position of the metal, using a molecular mechanics force field and thus taking

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Figure 1. Species distribution profile for Cu2+ complexes of (A) zPrP63-70 at 298 K and I ) 0.1 M KNO3. [Cu2+] ) 2 × 10-3, metal-to-ligand ratio of 1:1; (B) zPrP63-74 at 298 K and I ) 0.1 M KNO3. [Cu2+] ) 1 × 10-3, metal-to-ligand ratio of 1:1; (C) zPrP63-80 at 298 K and I ) 0.1 M KNO3. [Cu2+] ) 1 × 10-3, metal-to-ligand ratio of 1:1; (D) zPrP63-87 at 298 K and I ) 0.1 M KNO3. [Cu2+] ) 1 × 10-3, metal-to-ligand ratio of 1:1.

into account electrostatic and Van der Waals interactions besides the experimental data. These latter were included by imposing bonds between copper and the atoms involved in its binding and distance restraints for the experimentally obtained copperproton distances. Results zPrP63-70. zPrP63-70 exhibits two protonation constants assigned to two imidazole nitrogens of His residues. The log K values of 6.73 and 5.85, respectively, agree with literature data concerning similar peptides (Table 1s, Supporting Information).28,29,32 Calculations based on the pH titrations of equimolar zPrP6370-Cu2+ solutions imply the formation of five mononuclear species: CuHL, CuL, CuH-1L, CuH-2L, and CuH-3L (Figure 1A). CuHL is a minor species corresponding to copper anchored at the imidazole nitrogen of one His residue. This species transforms into CuL where a two-imidazole coordination pattern occurs. The log β value of this complex (5.90) closely matches the log K* values of previously reported two-imidazole complexes.28,29,32 The coordination pattern is also supported by the d–d transition centered at 621 nm and by EPR parameters (A| ) 165 G; g| ) 2.307). CuH-1L dominates in the pH range 5.5-6.0 and involves the {2Nim, N-} donor set as indicated by the blue shift of the d–d band to 588 nm, the change in EPR parameters (A| ) 187 G; g| ) 2.220) and also by the appearance

of the CD band at 304 nm corresponding to deprotonated amide N- f Cu2+ charge-transfer transition. Deprotonation of CuH-1L yields CuH-2L which dominates within the physiological pH range. The shift of the d–d transition to 573 nm and the slight changes in EPR parameters (A| ) 190 G; g| ) 2.218) may be consistent with the participation of an additional amide nitrogen in coordination resulting into the {2Nim, 2N-} donor set (Supporting Information, Table 1 and 1s). CuH-3L is successively formed through binding of a third amide nitrogen, as confirmed by the distinct changes in the spectroscopic parameters (Supporting Information, Table 1s). NMR experiments of zPrP63-70 were performed at pH 7.3 where predominance of CuH-2L had been shown by potentiometry. 1H and 13C NMR spectra were assigned by homo- and heteronuclear 2D experiments and the obtained chemical shifts are summarized in Supporting Information, Tables 2s and 3s. All the experiments show the existence of two isomers related to the trans (77%) or cis (23%) conformation of the Pro residue. With the addition of copper, large and selective broadening of 1H and 13C resonances was detected (Figures 2 and 3). The aromatic protons of His-65 and His-68 underwent a pronounced broadening and were almost washed out in the presence of 0.5 metal equivalents. Aliphatic resonances of His-65, Thr-66, Gly67, His-68, and Met-69 were also consistently broadened (Figures 2 and 3). In particular, the comparison of 1H–13C

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Figure 2. Aromatic region of (left) 1H 1D spectrum of zPrP63-70 0.8 mM in D2O at pH 7.3 and 298 K free (upper trace) and in the presence of increased copper equivalents (from 0.1 to 0.5); (right) 1H–13C HSQC spectrum of zPrP63-70 2.5 mM in D2O at pH 7.3 and 278 K, free (top) and in the presence of 0.6 Cu2+ eqs. (bottom).

correlations in the alpha-region of the HSQC spectra recorded in the absence and in the presence of Cu2+ showed that (i) His68 is no longer detectable, (ii) His-65, though largely broadened, is still observable, and (iii) Thr-66 and Met-69 intensities are noticeably reduced. The paramagnetic relaxation enhancements, R1p () R1obs xfR1f; xf ) fraction of unbound peptide) of most protons were obtained by measuring the spin-lattice relaxation rates in the free state (R1f) and in the presence of 0.5 Cu2+ equivalents (R1obs). Figure 4 shows that, besides the aromatic and beta protons of both histidines, the largest R1p values were measured for Thr-66 and Gly-67 protons. zPrP63-74. zPrP63-74 behaves as a H4L acid with protonation sites at the four imidazole nitrogens of His residues. All protonation constants vary from 5.33 to 7.05 similarly to those calculated for the tetrameric human octa-repeats and for the tetrameric chicken hexa-repeats (Supporting Information, Table 4s).9,28,29 Calculations based on potentiometric data of equimolar zPrP63-80-Cu2+ system show the formation of seven mononuclear species in solution (Figure 1B). The first minor species are formed by Cu2+ bound to two and three His imidazoles respectively, while the first major species CuL corresponds to the four-imidazole complex. The log K of CuL (9.63) is much higher than the log K* values measured for the four-imidazole complexes from human and chicken PrP, but this may be due to the peptide steric effect. The d–d transition at 580 nm and the EPR parameters (A| ) 185 G; g| ) 2.251) strongly support the {4Nim} coordination mode. The consecutive deprotonations (CuL f CuH-3L) lead to the substitution of His imidazole by adjacent amide nitrogens with consequent strong variations of the spectroscopic data (Supporting Information, Table 4s). Inclusion of the HIGH tail leaves the NMR spectra of zPrP6374 quite similar to those of zPrP63-70, being His-71, His-74, and Gly-73 completely overlapped to His-68 and Gly-70, respectively. Only the appearance of NMR signals belonging to Ile-72 determines a difference in the two 1H NMR spectra.

Gaggelli et al. Addition of copper at pH 7.3 selectively broadens proton signals, affecting all histidine protons (Figure 5). In the same way as with zPrP63-70, relaxation measurements allowed to calculate the R1p values, reported in Figure 6. The largest values belong to His moieties and to the residues immediately preceding or following the four His such as Val-64, Thr-66, Gly-67, Met-69, and Ile-72. zPrP63-80. zPrP63-80 behaves as a H6L acid with protonation sites at the six imidazole nitrogens of His residues. All protonation constants that varying in the range 5.32-7.22, are in good agreement with previously described His-rich peptides (Supporting Information, Table 5s).9,28,29,32 Calculations based on the potentiometric data of the zPrP63-80-Cu2+ system indicate the formation of eight species (Figure 1C). The first minor complex CuH4L corresponds to two-imidazole-bound complex. The deprotonation of CuH4L leads to a three-imidazole minor CuH3L. CuH2L dominates in the pH range 4.5-5.5. The log K* ) log βCuH2L - log βH2L ) 8.82 is typical for four imidazole nitrogen coordination,9,28 as also supported by the EPR parameters (A| ) 185 G; g| ) 2.252) and by the d–d transition at 595 nm. The next two deprotonation reactions give the very stable “physiological” CuL species dominating in the pH range 6.5-8.0 (Table 1 and Supporting Information, Table 5s). The log β ) 10.34 strongly differs from that of appropriate log K* values of four-imidazole bound species (e.g., 8.11 for the chicken tetrameric PrP peptide), supporting the involvement of five or even six-imidazole coordination pattern. The next minor species, CuH-1L and CuH-2L, make use of amide nitrogens in the coordination process. At pH > 8.0, CuH-3L predominates. The large change in spectroscopic data strongly suggests the {Nim, 3N-} donor set. NMR experiments of the zPrP63-80-Cu2+ system were focused on characterization of the CuL species dominating at pH 7.3. The assignment of the additional His, Thr, and Gly residues in either 1D or 2D NMR spectra was not possible because of extensive superimposition with the pre-existent His, Gly, and Thr spin systems. Addition of up to 0.6 Cu2+ equivalents determines extensive line broadening of 1H and 13C NMR spectra (Figures 5 and 7). As in the previous cases, the strongest effects were detected on His moieties, which, as expected, provide the main copper anchoring sites. Paramagnetic effects were monitored on Met-69 and Ile-72 resonances as well; whereas the selective line broadening of Gly and Thr signals was more troublesome to be interpreted because of the overlapping of all the five Gly and of three Thr crosspeaks. zPrP63-87. zPrP63-87 was found to behave as an H7L acid with protonation constants corresponding to the imidazole nitrogens of the seven His residues (Supporting Information, Table 6s). The potentiometric titrations of equimolar zPrP6387-Cu2+ solutions demonstrated a striking similarity with the zPrP63-80-Cu2+ system (Figure 1D). In the same way, the first six species (from CuH5L to CuL) correspond to multi-imidazole complexes involving from two to five or six His side chains. The log β ) 10.31 of CuL complex is identical to that described above for zPrP63-80. The next deprotonations lead to final CuH-3L species having the {Nim, 3N-} donor set (Supporting Information, Table 6s). Also in the case of zPrP63-87, NMR investigations were performed at pH 7.3 where CuL predominates. The 1H 1D spectrum was very similar to the others, except for the Ser signals which were easily assigned. As with zPrP63-74 and zPrP63-80, addition of copper determined selective line broadening, being the His aromatic protons again the most affected ones (Figure 5). The paramagnetic effects monitored on 1H

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Figure 3. Aliphatic region of (left) 1H 1D spectrum of zPrP63-70 0.8 mM in D2O at pH 7.3 and 298 K free (upper trace) and in the presence of increased copper equivalents (from 0.1 to 0.5); (right) 1H–13C HSQC spectrum of zPrP63-70 2.5 mM in D2O at pH 7.3 and 278 K, free (top) and in the presence of 0.6 Cu2+ equivalents (bottom).

Discussion

Figure 4. Proton paramagnetic relaxation contributions (R1p) of zPrP6370 0.8 mM pH 7.3, T 298 K in D2O calculated in presence of 0.5 Cu2+ equivalents.

NMR spectra of zPrP63-74, zPrP63-80, and zPrP63-87 in the presence of 0.2 metal equivalents were very similar to each other, as shown in Figure 5. Moreover, the comparison of the R1p values for all the three systems (Figure 8) indicates an almost identical trend, thus supporting a similar multiple imidazole Cu2+ binding mode for the three peptides with the involvement of 4 His residues in the planar metal coordination sphere (vide infra). SOD-like Activity. The IC50 values of the Cu2+ peptide complexes were calculated (Supporting Information, Table 7s). zPrP63-70 and st2PrP96-104 showed very similar values in agreement with the occurrence of almost identical metal donor set. zPrP63-87, zPrP63-80, and StPrP96-128 had very high IC50 values indicating a poor enzymatic activity. The only peptide showing a SOD-like activity comparable to that of human and chicken prion fragments9 is zPrP63-74, revealing that the 4 His imidazoles donor set possess the highest enzymatic activity.

All the findings provide evidence of all investigated repeat units specifically binding Cu2+ via the His imidazole(s). Potentiometric titrations allowed to determine the stoichiometry and the stabilities of the formed complexes and spectroscopic measurements permitted to define the metal binding donors. The Cu2+-peptide interactions were modulated by subtle changes in pH, and the binding mode shifted from the inter- to the intrarepeat mode, depending on the investigated peptide and on the metal concentration, as already reported for other prion sequences.25,28,29,32,51-53 Complete characterization of the metal complexes formed was made possible by combining potentiometric and spectroscopic measurements such as UV-vis, CD, EPR, and NMR, which finally provide the 3D structures of the complexes. Table 1 lists all the results obtained for the investigated Cu2+ complexes at physiological pH. As NMR measurements are usually performed in substoichiometric conditions, a possible shortcoming of this approach is represented by the eventual occurrence of complexes other than the desired 1:1. However, since, in the present case, NMR spectra could be detected in the presence of up to 0.6 metal equivalents without any apparent change in the binding mode, the occurrence of the 1:1 complex only is very likely to be verified. Potentiometric, UV-vis and EPR of zPrP63-70-Cu2+ complex at pH around 7 were consistent with the occurrence of the {2Nim, 2N-} donor set (Table 1 and Supporting Information, Table 1s). NMR results indicated that the largest paramagnetic effects were localized in the neighborhood of the two His and, more precisely, identified the 65-68 region as the metal binding site. The four metal coordinated nitrogens were assigned to imidazole nitrogens of both His and to Gly-67 and His-68 deprotonated amides, based on the following: (i) His aromatic signals showed the largest broadening and the largest R1p (Figures 2–4); (ii) His-68 alpha resonances was more affected

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Figure 5. 1H 1D spectrum of zPrP63-74 (black trace), zPrP63-80 (gray trace) and zPrP63-87 (blue trace) 0.8 mM in D2O at pH 7.3 and 298 K free (lower traces) and in the presence of 0.2 copper equivalents (upper traces).

Figure 6. Proton paramagnetic relaxation contributions (R1p) of zPrP6374 0.8 mM pH 7.3, T 298 K in D2O calculated in presence of 0.2 Cu2+ equivalents.

than His-65 (Figure 3); (iii) Gly-67 HR exhibited a slightly larger R1p than Thr-66 HR (Figure 4); (iv) deprotonation and coordination of amide nitrogens toward the N-terminal direction is generally preferred because of the formation of a more favored 6-membered {Nim, N-} chelate ring.54 The paramagnetic relaxation enhancements (R1p) reported in Figure 4 allowed the calculation of the exchange off-rates (Table 1 and Supporting Information, Table 8s) and the copper-proton distances (Supporting Information, Table 9s) of zPrP63-70-Cu2+ complex (see Materials and methods). A Cu2+-Hδ distance within the 0.5-0.6 nm range was found for both His, consistently with copper coordination to Nδ rather than Nε. All the calculated distances, together with the bond separation between Cu2+ and its coordinating atoms, were used as constraints for structure determination of the zPrP63-70-Cu2+ complex. The first 10 structures are reported in Figure 9a, where the 4 coordinated nitrogens (His-65 Nδ Gly-67 N-, His-68 N- and His-68 Nδ) are shown as blue spheres. A good resolution is apparent in the region encompassing the two His residues as provided by the low values of backbone rmsd (0.016 ( 0.012 nm) calculated in the region 65-69. His-65 aromatic ring exhibits a greater flexibility than His-68 consistently with the formation of the 6-membered {Nim, N-} chelate ring which

makes the His-68 side chain more rigid. The obtained mean structure was then optimized through an energy minimization in order to validate and refine the structure, especially for the location of the metal, using a molecular mechanics force field and thus taking into account also electrostatic and Van der Waals interactions. Experimental data were included by binding copper to all implied atoms and by restraining the Cu-H distances. The comparison between the two structures (Figure 9b) indicates that a large conformation rearrangement occurs in the region containing Gly-67 and His-68. All the results collected on the zPrP63-74-Cu2+ system at physiological pH strongly indicate the involvement of four imidazoles in metal binding. The R1p values measured for His Hε protons (Figure 6) were used to estimate the exchange offrate (Table 1 and Supporting Information, Table 8s), which allowed the calculation of Cu-H distances (Supporting Information, Table 10s). The Cu2+–Hδ distance obtained from the R1p averaged over all the four His was in the range 0.46-0.50 nm indicating, again, Nδ as the metal donor atom for all the four imidazole rings. All the distances reported in Supporting Information Table 10s were used to obtain the structures of the complex through energy minimization and 250 ps molecular dynamics calculations (Figure 10). The mean global backbone and heavy rmsd calculated on all the reported structures are 0.032 ( 0.013 and 0.045 ( 0.018 nm, respectively. Addition of further 2 or 3 His in zPrP63-80 and zPrP63-87 yielded larger log K than zPrP63-74, suggesting the involvement of five or six His in copper binding. NMR experiments performed on zPrP63-74, zPrP63-80 and zPrP63-87 systems indicate that the three peptides show comparable paramagnetic metal effects as pointed out by the similar line broadenings (Figure 5) and by the calculated exchange off-rates (Table 1 and Supporting Information, Table 8s). The superimposition of all the Thr and Gly spin systems does not allow to obtain any deeper insight into the residues nearby each His. However the calculations of R1p of the Thr’s and Gly’s signals (containing information averaged over all the overlapping protons) do not locate any preferred region in metal binding. It follows that either the simultaneous binding of six imidazoles or the formation of a 4N complex, with the involvement of 4 interchangeable His can be hypothesized. In fact, no preference

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Figure 7. 1H–13C HSQC spectrum of zPrP63-80 0.8 mM in D2O at pH 7.3 and 278 K, free (left) and in the presence of 0.4 (middle) and 0.6 (right) Cu2+ equivalents.

Figure 8. Proton paramagnetic relaxation contributions (R1p) of zPrP63-74 (black), zPrP63-80 (gray) and zPrP63-87 (blue) 0.8 mM pH 7.3, T 298 K in D2O calculated in presence of 0.2 Cu2+ equivalents.

of Cu2+ toward any His residue can be assumed, since (i) all of them have similar chemical environments and (ii) apart from His-86 in zPrP63-87, each His is separated from the other by just two residues. In any case the very similar exchange offrates (Table 1 and Supporting Information, Table 8s) calculated for all the three peptides support a common kinetic process, suggesting that the four His planar copper coordination plays a pivotal role in the formation of the complex. All the obtained results indicate the occurrence of diverse Cu2+ binding modes depending on the peptide sequence and

pH. At physiological pH and at the copper to ligand ration 1:1, binding of deprotonated amide nitrogens occurs only in the smallest fragment while the exclusive binding of His imidazole(s) takes place for the other systems. These binding modes are reminiscent of what happens for human, chicken, and fugu StPrP prion protein where intra- or inter-repeat copper binding modes have been characterized.25,28,29,32 In fact, all these systems show different copper binding modes depending on pH and Cu2+ concentration at physiological pH, and in equimolar metal solutions the multi imidazole(s) binding occurs only in the long

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TABLE 1: Potentiometric and Spectroscopic Features, Coordination Environments, Stoichiometry Kinetic Constants, and SOD-like Activities of the Investigated Cu2+ Systems at Physiological pH zPrP63-70 log β log K UV-vis CD

λ (nm) ε (M-1 cm-1) λ (nm)

∆ε (M-1 cm-1)

EPR

a

coordination environments

A| (G) g| atom donors

stochiometry kinetic

koff (s-1)

SOD-like activity

IC50 (µM)

-6.04 6.64 573a 75 628a 531a 335b 305c -0.068 0.165 0.136 -0.117 190 2.218 {2Nim, 2N-} Nδ His-65 N- Gly-67 N- His-68 Nδ His-68 1:1 147 21-22 0.368

zPrP63-74

zPrP63-80

zPrP63-87

9.63 4.72 580a 88 650a 543a 304b

10.34 6.55 575a 58 653a 540a 304b

10.31 6.93 575a 81 644a 541a 304b

-0.063 0.476 -0.463

-0.038 0.620 0.535

-0.035 0.557 -0.446

185 2.251 {4Nim} Nδ His-65 Nδ His-68 Nδ His-71 Nδ His-74 1:1 118.5

185 2.307 {4/6Nim} 4/6 His Nδ

185 2.228 {4/6Nim} 4/6 His Nδ

1:1 113.6

1.1 129.9

0.191

0.625

0.618

d–d transition. b Charge transfer transition Nim f Cu2+. c Charge transfer transition N- f Cu2+.

Figure 9. (a) The first 10 structures of the Cu2+-zPrP63-70 complex obtained through restrained simulated annealing. Color codes are the following: gray ) backbone, blue ) side-chains, copper ion is shown as green sphere, and the coordinating nitrogens of His-65, Gly-67, and His-68 are shown as blue spheres. (b) Comparison between the binding domain of the mean structure obtained by restrained simulated annealing (gray) and that one obtained after energy minimization (red). Copper and nitrogen atoms are shown as spheres or tetrahedrons in the energy minimized and DYANA structure respectively.

repeat fragments; on the contrary, the involvement of amide deprotonated nitrogens is present only in the single repeat unit.

Moreover the two fish prion peptides revealed the largest similarities, the presence of two His residues in zPrP(63-70) and stPrP(96-104) peptides makes the two fish prion related protein intrarepeat copper donor set {2Nim, 2N-} almost identical. Concerning the longest fragments (zebra PrP-2(6387) and fugu StPrP(96-128)) the occurrence of six or seven His yielded copper multiple imidazoles coordination at physiological pH and in the presence of 1:1 metal:ligand ratio. The SOD activity is likely to be the most substantiated PrPs’ function related to the Cu2+ binding ability. However nothing is known about the SOD-like activity of lower vertebrate PrPs. The comparison of the SOD-like activity among fish analogues of PrP (and related to mammalian and avian PrPs) shows that the previously described St2PrP as well as the presently investigated zebrafish PrP possess rather faint enzymatic activity (Table 1 and Supporting Information, Table 7s) at least at the used experimental conditions. The low activity of zPrP63-70 is consistent with the presence of {2Nim, 2N-} donor set which strongly differs from that observed in cellular Cu, Zn-SOD copper site.55 The high IC50 values found for St2PrP96-128 and zPrP63-87 copper complexes might be explained considering the sterical hindrance due to the six or five imidazole rings which could prevent the superoxide anion accessibility to the metal ion. On the other hand the four-imidazole species of zPrP6374 exhibited an enzymatic activity comparable to those measured for the human (4 Octapeptide) and chicken (4 Hexapeptide) prion copper complexes.9 The obtained structures indicated a four-imidazole coordination pattern (Figure 10), closely resembling the active center of Cu, Zn-SOD enzyme.55 The intra- and inter-repeat copper binding modes found in fish prion-related protein resulted to be more effective than those of the mammalian octa-repeat region25 and suggest a biological relevance of copper binding also in fish related prion protein. The SOD activity of the Cu2+ complexes from fish protein was found much lower than those detected for human and chicken prion protein, excluding a possible prion protein-associated superoxide dismutase activity in fish prion-related proteins. The lack of SOD-like activity and the high affinity copper binding

Cu2+ Complexes with Fragments of PrP-rel-2 of Zebrafish

J. Phys. Chem. B, Vol. 112, No. 47, 2008 15149 ratio ) 1:1; [Cu2+] ) 0.001 M. Table 7s. IC50 (µM) values of the Cu2+-peptide complexes in pH 7.4 with respect to the native Cu, Zn-SOD enzyme, and Cu(NO3)2 · 6H2O. Table 8s. R1p (S–1) and koff (S–1) values for zPrP Cu (II) complexes. Table 9s. Cu2+–H distances (nm) of zPrP63-70 derived from R1p values calculated in the presence of 0.5 Cu2+ equivalents (τM ) 46-47 ms). zPrP63-70 0.8 mM in D2O at pH 7.3 and 298 K. Table 10s. Cu2+–H distances (nm) of zPrP63-74 derived from R1p values calculated in presence of 0.4 Cu2+ equivalents (τM ) 9 ms). zPrP63-74 0.8 mM in D2O at pH 7.3 and 298 K. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes

Figure 10. Structures of the Cu2+-zPrP63-74 complex obtained through restrained simulated annealing and molecular dynamics; two different orientation are shown. The four coordinating His Nδ atoms are represented as blue tetrahedrons; Cu2+ is represented as a green sphere.

suggest a fish prion protein function consistent with copper transport or copper buffering rather than with SOD activity; moreover, the lack of SOD-like activity in fish PrP could exclude SOD activity also in mammal and chicken prion protein, since a throught species conserved function is expected. Acknowledgment. This work was supported by Polish Ministry of Education and Science (MEiN 1 T09A 008 30). We acknowledge the CIRMMP (Consorzio Interuniversitario Risonanze Magnetiche di Metalloproteine Paramagnetiche) and the MIUR (FIRB RBNE03PX83_003) for financial support. Supporting Information Available: Table 1s. Potentiometric and spectroscopic data for proton and Cu2+ complexes of zPrP63-70. Metal to ligand ratio ) 1:1; [Cu2+] ) 0.002 M. Table 2s. 1H chemical shift of zPrP63-70 0.8 mM in D2O at pH 7.3 and 298 K. Table 3s. 13C chemical shift of zPrP63-70 0.8 mM in D2O at pH 7.3 and 298 K. Table 4s. Potentiometric and spectroscopic data for proton and Cu2+ complexes of zPrP6374. Metal to ligand ratio ) 1:1; [Cu2+] ) 0.001 M. Table 5s. Potentiometric and spectroscopic data for proton and Cu2+ complexes of zPrP63-80. Metal to ligand ratio ) 1:1; [Cu2+] ) 0.001 M. Table 6s. Potentiometric and spectroscopic data for proton and Cu2+ complexes of zPrP63-87. Metal to ligand

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