- Email: [email protected]
NMR mapping of copper binding sites in alpha-synuclein
Biochimica et Biophysica Acta 1764 (2006) 5 – 12 http://www.elsevier.com/locate/bba
Rapid report
NMR mapping of copper binding sites in alpha-synuclein Yoon-hui Sung, Carla Rospigliosi, David Eliezer * Department of Biochemistry and Program in Structural Biology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA Received 7 February 2005; received in revised form 18 October 2005; accepted 1 November 2005 Available online 28 November 2005
Abstract Copper binding to the Parkinson disease-linked protein alpha-synuclein (aS) has been shown to accelerate its oligomerization in vitro and may therefore play a role in aS-mediated pathology in vivo. We use NMR spectroscopy to identify a number of independent copper binding sites in both the lipid-binding N-terminal domain and the highly acidic C-terminal domain of aS. Most of the sites appear to involve negatively charged amino acid side chains, but binding is also observed to the sole histidine residue located at position 50 and to the N-terminal amino group. Both the N-terminal and the histidine sites, as well as the sites in the C-terminal tail, can also bind copper in the more highly structured conformation adopted by aS upon binding to detergent micelles or lipid vesicles. There is no evidence for the formation of any sites requiring long-range order in the protein. D 2005 Elsevier B.V. All rights reserved. Keywords: Synuclein; Parkinson’s disease; Copper binding; Protein aggregation
Alpha-synuclein (aS) is the first of a number of recently identified proteins genetically linked to Parkinson’s disease, with three different aS mutations now associated with familial PD [1,2] or a closely related disorder [3]. In addition, triplication of the wild type aS gene also results in early onset PD [4] and aS is a primary component of the Lewy body deposits that are a pathological hallmark of both familial and sporadic PD [5]. Within Lewy bodies, aS is found in an aggregated fibrillar state [6] and similar aS fibrils formed in vitro possess all of the characteristics of amyloid fibrils found in other amyloid disorders [7 –9]. Therefore, aggregation of aS is thought to play a role in the pathogenesis of PD. Various metals, including copper, have long been implicated in the pathogenesis of neurodegenerative syndromes such as Alzheimer’s disease and the Prion disorders, and a number of amyloidogenic proteins, including aS [10,11], the human prion protein [12,13] and the amyloid-h peptide of Alzheimer’s disease [14,15] have been shown to bind copper in vitro. In the case of aS, copper binding has been demonstrated to increase the rate at which the protein polymerizes in vitro [10,11], suggesting a possible role for copper binding in aS toxicity. Here we use NMR spectros* Corresponding author. Tel.: +1 212 746 6557; fax: +1 212 746 4843. E-mail address: [email protected] (D. Eliezer). 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.11.003
copy to identify copper binding sites in aS. The negatively charged C-terminal tail of the protein appears to contain a number of copper-binding sites associated with negatively charged side chains, while the N-terminal domain also features several binding sites, including two independent binding sites involving the N-terminus of the protein and the only histidine in the protein sequence, His 50. Based on prior reports of copper binding to aS and other amyloid proteins, we examined the effects of CuCl2 on the 600 MHz proton-nitrogen NMR spectrum of free aS at pH 7.4, 10 -C, for which we have previously obtained resonance assignments [16]. Because of the low solubility product constant of copper and phosphate, we worked in 20 mM MOPS, 20 mM NaCl instead of PBS. Broadening of NMR resonances by paramagnetic Copper II has been used to identify copper binding sites in the prion protein [17,18] and amyloid-h peptide [19]. We found that a number of specific backbone resonances of aS were significantly broadened in the presence of a stoichiometric excess of CuCl2, as illustrated in Fig. 1 for one particular region of the aS spectrum. Although copper can induce aS aggregation, under our conditions, no aS aggregation occurred over period of at several days as judged by a lack of light scattering at 300 nm (data now shown) and the absence of any changes in the location or intensity of resonances in our NMR spectra. This may be attributed to the lower copper
6
Y. Sung et al. / Biochimica et Biophysica Acta 1764 (2006) 5 – 12
Fig. 1. One region of a 2D proton – nitrogen correlation spectrum of aS in the absence (solid lines) and presence (dashed lines) of a stoichiometric excess of CuCl2, showing the disappearance of resonances corresponding to positions 48 and 52.
concentrations used in our studies, to the lower sample temperature, and to the lack of agitation. Because of the crowded nature of 2D NMR spectra of free aS, we collected 3D HNCO NMR spectra of matched aS samples with and without a CuCl2. HNCO resonance intensities in the presence of a 3:4 ratio of CuCl2:aS were normalized to the corresponding HNCO resonance intensities from a copper free sample and are shown as a function of sequence position in Fig. 2. A decrease in resonance intensity is visible at many locations in the acidic Cterminal tail of aS, as might be anticipated based on reports of electrostatic interactions between this region of the protein and calcium ions [20] as well as various polycations
[21]. Minima in the intensity data in the C-terminal tail correspond closely to the presence of negatively charged side chains in the protein sequence, as seen for E104E105, E110, E114D115, D119P120D121N122E123, E130E131, and D135, suggesting that these locations constitute copper binding sites (the sequence of aS is shown in supplementary material figure S1). Although a number of copper-binding sites are apparent as intensity minima in the C-terminal tail, the most severe broadening of NMR resonances is observed in the N-terminal region of the protein. There are deep and broad minima at the very N-terminus, of the protein and at position 50, and a deep minimum at position 20. In addition, there is a slightly less deep minimum at position 42, and distinct but lesser minima at positions 14, 27, 35 and 60. With the exception of the attenuated signal at position 42, each of these minima corresponds to an expected favorable interaction between a nearby sidechain and a copper ion. Specifically, E13, E20, E28, E35 and E61 carry a negative charge, while the primary alpha amino and imidazole groups present at the N-terminus and at residue H50, respectively, are known to readily coordinate copper [22]. There is also a negative charge at residue D2 that could contribute to binding at the N-terminus. To determine the concentration dependence of copper binding to the different sites aS, we used 2D NMR spectra to monitor resonance intensities in a copper titration (Fig. 3). Copper was added as aliquots of a 5-mM stock solution to achieve a final aS concentration of 35 AM. The lower resolution of the 2D spectra resulted in a number of overlapped resonances, the intensity of which could not be reliably determined. Nevertheless, essentially all of the features present in the HNCO-based data of Fig. 2 are visible in Fig. 3 as well. In addition, from the copper concentration series, intensity minima were also evident at
Fig. 2. Normalized NMR resonance intensities as a function of aS sequence position from matched HNCO spectra collected in the absence and presence of a stoichiometric amount of CuCl2. aS was prepared at 35 AM concentration in 20 mM MOPS, 20 mM NaCl, pH 7.4 and CuCl2 was added to a final Cu:aS ratio of 3:4. Spectra were collected on a Varian 600 MHz NMR spectrometer as previously described [16]. Resonances from positions 1 and 41 were not observed reliably. Resonances from positions 107, 116, 119, 127, and 137 (marked with filled diamonds) are absent because they are proline-preceding residues.
Y. Sung et al. / Biochimica et Biophysica Acta 1764 (2006) 5 – 12
7
Fig. 3. NMR resonance intensities as a function of aS sequence position from 2D proton – nitrogen correlation spectra collected at Cu:aS ratios of 1:2 (triangles), 3:4 (open circles), 1:1 (filled circles), and 2:1 (crosses) normalized by the intensities observed in the absence of copper, for the full-length protein (top panel), the truncated protein (middle panel) and the full-length H50A mutant (bottom panel).
sites corresponding to residues E83 and D98, which were not clearly seen in the HNCO data. Fig. 4 shows plots of resonance intensity versus copper concentration for a number of individual residues in aS. These plots show that at the protein concentration used, the intensity decrease for sites in the N-terminal region of the protein is essentially linear until saturation. This implies that the effective dissociation constants associated with copper binding to these sites is considerably lower than the protein concentration used (i.e., all copper is bound as it is added). In contrast, sites in the Cterminal tail of the protein show a less linear response and
require higher concentrations of copper to achieve complete broadening, suggesting a higher dissociation constant. Each curve was fit to equation 1, which provides an analytical expression for the occupancy of a single binding site in a simple two-component interaction [23]. F ¼ 1=ð1 þ ½ L=Kd Þ
ð1Þ
F is the fraction of bound protein, K d is the dissociation constant, [L]={-([P]0-[L]0 + K d)+sqrt(([ P]0 [L]0 + K d)2 + 4[L]0K d)}/2 is the concentration of free ligand (copper in this case) and [P]0 and [L]0 are the total concentrations of protein and ligand (copper)
Fig. 4. Normalized intensities of individual resonances from 2D HSQC spectra collected as a function of increasing copper concentration for full-length wild type (open circles) and H50A mutant (filled squares) aS. Fits to an independent binding sites model, as described in the text, are shown in solid lines for the wild type and hashed lines for the H50A mutant data.
8
Y. Sung et al. / Biochimica et Biophysica Acta 1764 (2006) 5 – 12
respectively. Although the presence of multiple binding sites in synuclein means that applying this equation is an oversimplification [23], reasonable fits were obtained, as shown in the figure, and allowed for the extraction of apparent binding constants. However, in addition to the inaccuracy introduced by ignoring the presence of multiple binding sites, it is important to note that the intensity of each NMR resonance is a function not only of the copper-bound and copper-free populations, but also of the rate of exchange between the two populations. Thus, in the absence of information regarding the dynamics of copper binding and release, it is not possible to extract quantitatively accurate binding constant information. Therefore, it must be emphasized that the fits obtained cannot be directly related to the true binding constants, and we consider them only in qualitative terms. To establish that copper binding is indeed reversible, 2D NMR spectra were collected from copper-containing samples to which EDTA had been added. No resonance broadening was observed in these spectra. A number of recent reports have suggested that long-range interactions may exist between the C- and N-terminal regions of aS [24,25]. To determine if any of the N-terminal copperbinding sites depended on such an interaction, we repeated the 2D NMR monitored copper titration using a truncated form of aS consisting of residues 1– 102. Despite the fact that Cterminally truncated aS has been reported to aggregate more rapidly than the full-length protein [26], both light scattering and NMR data again showed no indications of aggregation in our samples over a period of days. Fig. 3 shows that removal of the C-terminal tail did not eliminate any of the major binding sites in the N-terminal region, although broadening at positions 83 and 98 was diminished. Instead, the primary sites in the truncated protein appear to become more sensitive, with a greater degree of broadening apparent at the lowest copper concentration. This may be a result of reduced competition for copper ions in the absence of the C-terminal sites. The broad copper binding-induced intensity minima around position 50 and at the N-terminus of the protein suggested that these sites might feature a somewhat compact structure that would bring several backbone amide sites within the range of the broadening influence of the bound copper ions. In light of the similar binding curves obtained for positions 5 and 50 (Fig. 4), we also considered that these two sites could potentially comprise a single binding site involving residue H50 and Nterminal amino group. To test this idea, we produced an aS mutant in which residue H50 was replaced by an alanine. Fig. 3 shows the 2D NMR monitored copper titration for the H50A mutant. Broadening at the N-terminus of the protein, as well as at positions 20 and 28, is not abolished for the mutant protein and instead appears to be slightly enhanced. Clearly, the removal of the imidazole group at position 50 does not preclude copper binding to the N-terminus of the protein, nor to the sites at positions 21 and 28. Therefore, it is highly unlikely that H50 and the N-terminal amino group form a single longrange copper binding site in aS. Binding to the C-terminal sites is also essentially unaltered or perhaps slightly enhanced. Surprisingly, while this paper was under review a similar study appeared concluding, in contradiction to our own results,
that a long-range copper-binding site involving residue H50 and the N-terminal amino group is in fact present in aS [27]. In drawing this conclusion, this report relied heavily on the observation that DEPC treatment of aS resulted in elimination of copper binding both at position 50 and at the N-terminus of the protein. DEPC is known to modify histidine sidechains and prevent them from being able to coordinate copper. Therefore, it was reasoned that DEPC treatment would selectively inhibit copper binding to H50, and should not affect binding to the Nterminus unless such binding required the participation of H50. However, besides histidines, DEPC is known to modify several other sidechain groups, as well at the N-terminal primary amino group of proteins [28]. Therefore, we deemed it likely that the elimination of copper binding at both position 50 and at the N-terminus of aS by DEPC treatment was caused by modification of the protein at both of these sites. To test this, we incubated our aS constructs with DEPC and analyzed the samples using mass spectrometry. The results clearly demonstrated that DEPC modifies both full length and C-terminally truncated aS at multiple sites (data not shown), and is therefore not specific to H50. To further show that the N-terminal copper-binding site is directly targeted by DEPC, we incubated wild type full length, wild type C-terminally truncated, and H50A full-length aS with DEPC prior to the addition of copper and monitored copper binding by 2D NMR. Fig. 5 shows that DEPC does indeed eliminate copper binding to both the site around position 50 and to the N-terminal site in both full-length and truncated wild type aS, but that it also eliminates copperbinding to the N-terminal site in the H50A mutant, where modification of H50 is not possible. In addition, we note that DEPC treatment eliminated copper binding at other sites, both in the N-terminal and C-terminal region of the protein, consistent with the mass spectrometry data and with the ability of this reagent to modify a number of different groups. Interestingly, copper binding to the site around position 121 is greatly enhanced by DEPC modification in both the wild type and H50A proteins. This site is therefore clearly not blocked by DEPC, and as in the case of C-terminal truncation or the H50A substitution we attribute the enhanced copper sensitivity to the elimination of competition from other sites, in this case from those blocked by DEPC. After showing that copper binding to the N-terminal site in aS does not require the participation of H50, we also investigated the idea that H50 and the N-terminus could be involved in a long range contact even in the absence of copper. This was suggested to be the case based on the observation that DEPC modification leads to chemical shift changes both around position 50 and at the N-terminus of aS [27]. Our results show that the N-terminus of aS is also modified by DEPC, providing a more direct explanation for the chemical shift changes at this site. Nevertheless, we constructed an H50C mutant of truncated aS and produced samples in which the Cys residue at position 50 was modified with the paramagnetic spin label MTSL (methanethiosulfonate, Toronto Research Chemicals). The presence of such a spin label leads to severe broadening of NMR resonances from sites that are ˚ of the labeled site. Fig. 6 shows the intensity within 15– 20 A
Y. Sung et al. / Biochimica et Biophysica Acta 1764 (2006) 5 – 12
9
Fig. 5. Normalized NMR resonance intensities as a function of aS sequence position from 2D proton – nitrogen correlation spectra collected at a Cu:aS ratio of 3:4 for unmodified (filled circles) and DEPC-modified (open squares) full-length (top panel), truncated (middle panel) and full-length H50A mutant (bottom panel) aS.
of resonances in spin-labeled H50C truncated aS normalized to the intensity observed for concentration matched samples in which spin labeling was prevented by an excess of the reducing agent DTT. Although there is dramatic broadening of resonances that are close in sequence to position 50, resonances at the N-terminus are not dramatically broadened, confirming that there is no persistent long-range contact between position 50 and the N-terminus of aS. Instead, a weak broadening is observed throughout the regions both N- and C-terminal to those that are dramatically broadened by labeling at position 50. This likely results from an overall preference for compact
conformations within the ensemble sampled by the N-terminal lipid-binding domain of aS [16,29], and is consistent with early reports of residual structure within this region [29], as well as more recent constrained simulations of the behavior of the fulllength protein [24]. The N-terminal 100 residues of aS adopt a more ordered highly helical structure upon binding to lipid surfaces or detergent micelles [30 – 32], which is likely to mediate the normal function(s) of the protein. To explore whether copper bound to this folded conformation of the protein as well, we compared 2D NMR spectra of the micelle-bound protein in the
Fig. 6. NMR resonance intensities as a function of aS sequence position from proton – nitrogen correlation spectra collected from truncated MTSL spin-labeled H50C aS normalized by the intensities observed for an equivalent reduced sample.
10
Y. Sung et al. / Biochimica et Biophysica Acta 1764 (2006) 5 – 12
absence and presence of copper. Although the negatively charged surfaces of detergent micelles are likely to attract copper ions, increasing the effective copper concentration in the vicinity of micelle-bound aS [33], this is unlikely to result in any residue-specific broadening effects in the spectrum of micelle-bound aS. Fig. 7 shows the normalized resonance intensity as a function of sequence position for micelle-bound full-length and truncated aS in the presence of increasing concentrations of CuCl2. As expected, noticeable resonance broadening occurred at somewhat lower copper concentrations than in the case of the free protein. In the C-terminal region of the full-length protein, the broadening pattern is similar to that observed for the free protein, consistent with the fact that the acidic C-terminal tail of the protein does not bind to the micelle surface [16]. In the N-terminal lipid-binding region, the data still show broadening both at the N-terminus and around position 50, suggesting that these sites can coordinate copper in the micelle-bound form of the protein as well. However, the broadening surrounding these sites is qualitatively different from that seen in the free protein, having more of a Fsquare well_ shape, with sharply defined boundaries. This likely results from the less flexible structure of the micelle-bound protein, which prevents residues past a certain distance in the sequence from the binding site from closely approaching the bound copper ion. In addition to this difference, the site at position 98 is more easily broadened than in the free state of the protein, while other sites in the N-terminal domain, such as positions 21, 35 and 66, are hardly apparent in the data from the full-length protein, and only weakly apparent for the truncated protein, suggesting that the helical micelle-bound structure of the protein interferes with copper binding to the latter sites. Studies of the topology of the highly helical micelle-bound conformation of aS rule out tertiary contacts between the Nterminus of the protein and His 50 [34,35], and an absence of significant changes (beyond the aforementioned resonance broadening) in the 2D NMR spectra of the protein upon copper binding indicate that no large-scale conformational changes occur. Therefore, in micelle-bound aS, it is highly unlikely that
the two distant locations participate in the same copper-binding site. This again indicates that the N-terminus of aS and the site involving His 50 are capable of binding copper independently of one another. In addition, binding of copper to these two sites in the micelle-bound state indicates that in the copper-bound conformation of these sites is not precluded by the helical conformation of the micelle-bound protein. Our results indicate that aS binds to copper through at large number of independent local binding sites which are formed by groups that would be expected to bind to copper in any largely unstructured polypeptide, namely negatively charged side chains, the one histidine in the protein, and the primary amino group of the protein. The presence of a large number of sites is consistent with a previous study of copper induced aS aggregation that concluded that full-length aS bound to 10.4 copper ions [10], although our results suggest that aS contains as many as 16 different sites capable of binding copper. Our results also suggest that these sites are unlikely to be specific to copper beyond any copper preference inherent in the individual groups involved, since those groups (Asp, Glu and His side chains and the primary amino group) are capable of coordinating a number of other metals, including zinc, nickel, iron and calcium [36]. In contrast to a recent report [27], we show that copper-binding by aS does not involve long-range interactions. Our results suggest that it is unlikely that copper binding plays an important role in the normal function of alpha-synuclein. This is a contrast with the prion protein, where copper binding appears to be highly specific and is thought to be important in the protein’s normal function. Nevertheless, previous results strongly suggest that copper binding could be important in the pathological role of aS in PD. We also show that copper binding by aS can occur in the lipid-bound conformation as well as in the free state. Because it is still unclear which of these two states of the protein is responsible for its aggregation in vivo, this is an important point to consider. The identification of specific residues involved in copper binding by aS should facilitate studies of how and whether such binding influences the pathogenic behavior of the protein. For example, specific mutants can now be designed to
Fig. 7. Normalized NMR resonance intensities as a function of aS sequence position from 2D proton – nitrogen correlation spectra collected at Cu:aS ratios of 1:10 (triangles), 1:4 (open circles), 2:4 (filled circles), and 3:4 (crosses) for full-length (top panel) and truncated (bottom panel) aS bound to SDS micelles.
Y. Sung et al. / Biochimica et Biophysica Acta 1764 (2006) 5 – 12
perturb each different site of copper binding by aS and to study the consequent effects on the copper-induced aggregation the protein in vitro, as well as on its functional and toxic properties in situ and in vivo.
[13] [14]
Acknowledgements This work was supported in part by the NIA, NIH, grant AG019391 (to D.E.) and by a gift from Herbert and Ann Siegel (to D.E.).
[15]
[16]
Appendix A. Supplementary data [17]
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.bbapap.2005.11.003. References [1] M.H. Polymeropoulos, C. Lavedan, E. Leroy, S.E. Ide, A. Dehejia, A. Dutra, B. Pike, H. Root, J. Rubenstein, R. Boyer, E.S. Stenroos, S. Chandrasekharappa, A. Athanassiadou, T. Papapetropoulos, W.G. Johnson, A.M. Lazzarini, R.C. Duvoisin, G. Di Iorio, L.I. Golbe, R.L. Nussbaum, Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease, Science 276 (1997) 2045 – 2047. [2] R. Kruger, W. Kuhn, T. Muller, D. Woitalla, M. Graeber, S. Kosel, H. Przuntek, J.T. Epplen, L. Schols, O. Riess, Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson’s disease, Nat. Genet. 18 (1998) 106 – 108. [3] J.J. Zarranz, J. Alegre, J.C. Gomez-Esteban, E. Lezcano, R. Ros, I. Ampuero, L. Vidal, J. Hoenicka, O. Rodriguez, B. Atares, V. Llorens, E. Gomez Tortosa, T. del Ser, D.G. Munoz, J.G. de Yebenes, The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia, Ann. Neurol. 55 (2004) 164 – 173. [4] A.B. Singleton, M. Farrer, J. Johnson, A. Singleton, S. Hague, J. Kachergus, M. Hulihan, T. Peuralinna, A. Dutra, R. Nussbaum, S. Lincoln, A. Crawley, M. Hanson, D. Maraganore, C. Adler, M.R. Cookson, M. Muenter, M. Baptista, D. Miller, J. Blancato, J. Hardy, K. Gwinn-Hardy, alpha-Synuclein locus triplication causes Parkinson’s disease, Science 302 (2003) 841. [5] M.G. Spillantini, M.L. Schmidt, V.M. Lee, J.Q. Trojanowski, R. Jakes, M. Goedert, Alpha-synuclein in Lewy bodies, Nature 388 (1997) 839 – 840. [6] M.G. Spillantini, J.R. Murrell, M. Goedert, M.R. Farlow, A. Klug, B. Ghetti, Mutation in the tau gene in familial multiple system tauopathy with presenile dementia, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 7737 – 7741. [7] M. Hashimoto, L.J. Hsu, A. Sisk, Y. Xia, A. Takeda, M. Sundsmo, E. Masliah, Human recombinant NACP/alpha-synuclein is aggregated and fibrillated in vitro: relevance for Lewy body disease, Brain Res. 799 (1998) 301 – 306. [8] L.C. Serpell, J. Berriman, R. Jakes, M. Goedert, R.A. Crowther, Fiber diffraction of synthetic alpha-synuclein filaments shows amyloid-like cross-beta conformation, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 4897 – 4902. [9] K.A. Conway, J.D. Harper, P.T. Lansbury Jr., Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson’s disease are typical amyloid, Biochemistry 39 (2000) 2552 – 2563. [10] S.R. Paik, H.J. Shin, J.H. Lee, C.S. Chang, J. Kim, Copper(II)-induced self-oligomerization of alpha-synuclein, Biochem. J. 340 (Pt. 3) (1999) 821 – 828. [11] V.N. Uversky, J. Li, A.L. Fink, Metal-triggered structural transformations, aggregation, and fibrillation of human alpha-synuclein. A possible molecular link between Parkinson’s disease and heavy metal exposure, J. Biol. Chem. 276 (2001) 44284 – 44496. [12] D.R. Brown, K. Qin, J.W. Herms, A. Madlung, J. Manson, R. Strome, P.E.
[18]
[19]
[20]
[21]
[22]
[23] [24]
[25]
[26]
[27]
[28] [29]
[30]
[31]
11
Fraser, T. Kruck, A. von Bohlen, W. Schulz-Schaeffer, A. Giese, D. Westaway, H. Kretzschmar, The cellular prion protein binds copper in vivo, Nature 390 (1997) 684 – 687. J. Stockel, J. Safar, A.C. Wallace, F.E. Cohen, S.B. Prusiner, Prion protein selectively binds copper(II) ions, Biochemistry 37 (1998) 7185 – 7193. C.S. Atwood, R.D. Moir, X. Huang, R.C. Scarpa, N.M. Bacarra, D.M. Romano, M.A. Hartshorn, R.E. Tanzi, A.I. Bush, Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis, J. Biol. Chem. 273 (1998) 12817 – 12826. W. Garzon-Rodriguez, A.K. Yatsimirsky, C.G. Glabe, Binding of Zn(II), Cu(II), and Fe(II) ions to Alzheimer’s A beta peptide studied by fluorescence, Bioorg. Med. Chem. Lett. 9 (1999) 2243 – 2248. D. Eliezer, E. Kutluay, R. Bussell Jr., G. Browne, Conformational properties of alpha-synuclein in its free and lipid-associated states, J. Mol. Biol. 307 (2001) 1061 – 1073. J.H. Viles, F.E. Cohen, S.B. Prusiner, D.B. Goodin, P.E. Wright, H.J. Dyson, Copper binding to the prion protein: structural implications of four identical cooperative binding sites, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 2042 – 2047. G.S. Jackson, I. Murray, L.L. Hosszu, N. Gibbs, J.P. Waltho, A.R. Clarke, J. Collinge, Location and properties of metal-binding sites on the human prion protein, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 8531 – 8535. C.D. Syme, R.C. Nadal, S.E. Rigby, J.H. Viles, Copper binding to the amyloid-beta (Abeta) peptide associated with Alzheimer’s disease: folding, coordination geometry, pH dependence, stoichiometry, and affinity of Abeta-(1 – 28): insights from a range of complementary spectroscopic techniques, J. Biol. Chem. 279 (2004) 18169 – 18177. R. Lowe, D.L. Pountney, P.H. Jensen, W.P. Gai, N.H. Voelcker, Calcium(II) selectively induces alpha-synuclein annular oligomers via interaction with the C-terminal domain, Protein Sci. 13 (2004) 3245 – 3252. C.O. Fernandez, W. Hoyer, M. Zweckstetter, E.A. Jares-Erijman, V. Subramaniam, C. Griesinger, T.M. Jovin, NMR of alpha-synuclein – polyamine complexes elucidates the mechanism and kinetics of induced aggregation, EMBO J. 23 (2004) 2039 – 2046. E.I. Solomon, R.K. Szilagyi, S. DeBeer George, L. Basumallick, Electronic structures of metal sites in proteins and models: contributions to function in blue copper proteins, Chem. Rev. 104 (2004) 419 – 458. M.R. Eftink, Fluorescence methods for studying equilibrium macromolecule – ligand interactions, Methods Enzymol. 278 (1997) 221 – 257. M.M. Dedmon, K. Lindorff-Larsen, J. Christodoulou, M. Vendruscolo, C.M. Dobson, Mapping long-range interactions in alpha-synuclein using spin-label NMR and ensemble molecular dynamics simulations, J. Am. Chem. Soc. 127 (2005) 476 – 477. C.W. Bertoncini, Y.S. Jung, C.O. Fernandez, W. Hoyer, C. Griesinger, T.M. Jovin, M. Zweckstetter, From The Cover: release of long-range tertiary interactions potentiates aggregation of natively unstructured {alpha}-synuclein, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 1430 – 1435. R.A. Crowther, R. Jakes, M.G. Spillantini, M. Goedert, Synthetic filaments assembled from C-terminally truncated alpha-synuclein, FEBS Lett. 436 (1998) 309 – 312. R.M. Rasia, C.W. Bertoncini, D. Marsh, W. Hoyer, D. Cherny, M. Zweckstetter, C. Griesinger, T.M. Jovin, C.O. Fernandez, Structural characterization of copper(II) binding to alpha-synuclein: Insights into the bioinorganic chemistry of Parkinson’s disease, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 4294 – 4299. E.W. Miles, Modification of histidyl residues in proteins by diethylpyrocarbonate, Methods Enzymol. 47 (1977) 431 – 442. R. Bussell Jr., D. Eliezer, Residual structure and dynamics in Parkinson’s disease-associated mutants of alpha-synuclein, J. Biol. Chem. 276 (2001) 45996 – 46003. W.S. Davidson, A. Jonas, D.F. Clayton, J.M. George, Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes, J. Biol. Chem. 273 (1998) 9443 – 9449. R. Bussell Jr., D. Eliezer, A structural and functional role for 11-mer repeats in alpha-synuclein and other exchangeable lipid binding proteins, J. Mol. Biol. 329 (2003) 763 – 778.
12
Y. Sung et al. / Biochimica et Biophysica Acta 1764 (2006) 5 – 12
[32] S. Chandra, X. Chen, J. Rizo, R. Jahn, T.C. Sudhof, A broken alpha-helix in folded alpha-Synuclein, J. Biol. Chem. 278 (2003) 15313 – 15318. [33] E.N. Lee, S.Y. Lee, D. Lee, J. Kim, S.R. Paik, Lipid interaction of alphasynuclein during the metal-catalyzed oxidation in the presence of Cu2+ and H2O2, J. Neurochem. 84 (2003) 1128 – 1142. [34] R. Bussell Jr., T.F. Ramlall, D. Eliezer, Helix periodicity, topology, and
dynamics of membrane-associated alpha-synuclein, Protein Sci. 14 (2005) 862 – 872. [35] T.S. Ulmer, A. Bax, N.B. Cole, R.L. Nussbaum, Structure and dynamics of micelle-bound human alpha-synuclein, J. Biol. Chem. 280 (2005) 9595 – 9603. [36] M.M. Harding, The architecture of metal coordination groups in proteins, Acta Crystallogr., D Biol. Crystallogr. 60 (2004) 849 – 859.
Rapid report
NMR mapping of copper binding sites in alpha-synuclein Yoon-hui Sung, Carla Rospigliosi, David Eliezer * Department of Biochemistry and Program in Structural Biology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA Received 7 February 2005; received in revised form 18 October 2005; accepted 1 November 2005 Available online 28 November 2005
Abstract Copper binding to the Parkinson disease-linked protein alpha-synuclein (aS) has been shown to accelerate its oligomerization in vitro and may therefore play a role in aS-mediated pathology in vivo. We use NMR spectroscopy to identify a number of independent copper binding sites in both the lipid-binding N-terminal domain and the highly acidic C-terminal domain of aS. Most of the sites appear to involve negatively charged amino acid side chains, but binding is also observed to the sole histidine residue located at position 50 and to the N-terminal amino group. Both the N-terminal and the histidine sites, as well as the sites in the C-terminal tail, can also bind copper in the more highly structured conformation adopted by aS upon binding to detergent micelles or lipid vesicles. There is no evidence for the formation of any sites requiring long-range order in the protein. D 2005 Elsevier B.V. All rights reserved. Keywords: Synuclein; Parkinson’s disease; Copper binding; Protein aggregation
Alpha-synuclein (aS) is the first of a number of recently identified proteins genetically linked to Parkinson’s disease, with three different aS mutations now associated with familial PD [1,2] or a closely related disorder [3]. In addition, triplication of the wild type aS gene also results in early onset PD [4] and aS is a primary component of the Lewy body deposits that are a pathological hallmark of both familial and sporadic PD [5]. Within Lewy bodies, aS is found in an aggregated fibrillar state [6] and similar aS fibrils formed in vitro possess all of the characteristics of amyloid fibrils found in other amyloid disorders [7 –9]. Therefore, aggregation of aS is thought to play a role in the pathogenesis of PD. Various metals, including copper, have long been implicated in the pathogenesis of neurodegenerative syndromes such as Alzheimer’s disease and the Prion disorders, and a number of amyloidogenic proteins, including aS [10,11], the human prion protein [12,13] and the amyloid-h peptide of Alzheimer’s disease [14,15] have been shown to bind copper in vitro. In the case of aS, copper binding has been demonstrated to increase the rate at which the protein polymerizes in vitro [10,11], suggesting a possible role for copper binding in aS toxicity. Here we use NMR spectros* Corresponding author. Tel.: +1 212 746 6557; fax: +1 212 746 4843. E-mail address: [email protected] (D. Eliezer). 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.11.003
copy to identify copper binding sites in aS. The negatively charged C-terminal tail of the protein appears to contain a number of copper-binding sites associated with negatively charged side chains, while the N-terminal domain also features several binding sites, including two independent binding sites involving the N-terminus of the protein and the only histidine in the protein sequence, His 50. Based on prior reports of copper binding to aS and other amyloid proteins, we examined the effects of CuCl2 on the 600 MHz proton-nitrogen NMR spectrum of free aS at pH 7.4, 10 -C, for which we have previously obtained resonance assignments [16]. Because of the low solubility product constant of copper and phosphate, we worked in 20 mM MOPS, 20 mM NaCl instead of PBS. Broadening of NMR resonances by paramagnetic Copper II has been used to identify copper binding sites in the prion protein [17,18] and amyloid-h peptide [19]. We found that a number of specific backbone resonances of aS were significantly broadened in the presence of a stoichiometric excess of CuCl2, as illustrated in Fig. 1 for one particular region of the aS spectrum. Although copper can induce aS aggregation, under our conditions, no aS aggregation occurred over period of at several days as judged by a lack of light scattering at 300 nm (data now shown) and the absence of any changes in the location or intensity of resonances in our NMR spectra. This may be attributed to the lower copper
6
Y. Sung et al. / Biochimica et Biophysica Acta 1764 (2006) 5 – 12
Fig. 1. One region of a 2D proton – nitrogen correlation spectrum of aS in the absence (solid lines) and presence (dashed lines) of a stoichiometric excess of CuCl2, showing the disappearance of resonances corresponding to positions 48 and 52.
concentrations used in our studies, to the lower sample temperature, and to the lack of agitation. Because of the crowded nature of 2D NMR spectra of free aS, we collected 3D HNCO NMR spectra of matched aS samples with and without a CuCl2. HNCO resonance intensities in the presence of a 3:4 ratio of CuCl2:aS were normalized to the corresponding HNCO resonance intensities from a copper free sample and are shown as a function of sequence position in Fig. 2. A decrease in resonance intensity is visible at many locations in the acidic Cterminal tail of aS, as might be anticipated based on reports of electrostatic interactions between this region of the protein and calcium ions [20] as well as various polycations
[21]. Minima in the intensity data in the C-terminal tail correspond closely to the presence of negatively charged side chains in the protein sequence, as seen for E104E105, E110, E114D115, D119P120D121N122E123, E130E131, and D135, suggesting that these locations constitute copper binding sites (the sequence of aS is shown in supplementary material figure S1). Although a number of copper-binding sites are apparent as intensity minima in the C-terminal tail, the most severe broadening of NMR resonances is observed in the N-terminal region of the protein. There are deep and broad minima at the very N-terminus, of the protein and at position 50, and a deep minimum at position 20. In addition, there is a slightly less deep minimum at position 42, and distinct but lesser minima at positions 14, 27, 35 and 60. With the exception of the attenuated signal at position 42, each of these minima corresponds to an expected favorable interaction between a nearby sidechain and a copper ion. Specifically, E13, E20, E28, E35 and E61 carry a negative charge, while the primary alpha amino and imidazole groups present at the N-terminus and at residue H50, respectively, are known to readily coordinate copper [22]. There is also a negative charge at residue D2 that could contribute to binding at the N-terminus. To determine the concentration dependence of copper binding to the different sites aS, we used 2D NMR spectra to monitor resonance intensities in a copper titration (Fig. 3). Copper was added as aliquots of a 5-mM stock solution to achieve a final aS concentration of 35 AM. The lower resolution of the 2D spectra resulted in a number of overlapped resonances, the intensity of which could not be reliably determined. Nevertheless, essentially all of the features present in the HNCO-based data of Fig. 2 are visible in Fig. 3 as well. In addition, from the copper concentration series, intensity minima were also evident at
Fig. 2. Normalized NMR resonance intensities as a function of aS sequence position from matched HNCO spectra collected in the absence and presence of a stoichiometric amount of CuCl2. aS was prepared at 35 AM concentration in 20 mM MOPS, 20 mM NaCl, pH 7.4 and CuCl2 was added to a final Cu:aS ratio of 3:4. Spectra were collected on a Varian 600 MHz NMR spectrometer as previously described [16]. Resonances from positions 1 and 41 were not observed reliably. Resonances from positions 107, 116, 119, 127, and 137 (marked with filled diamonds) are absent because they are proline-preceding residues.
Y. Sung et al. / Biochimica et Biophysica Acta 1764 (2006) 5 – 12
7
Fig. 3. NMR resonance intensities as a function of aS sequence position from 2D proton – nitrogen correlation spectra collected at Cu:aS ratios of 1:2 (triangles), 3:4 (open circles), 1:1 (filled circles), and 2:1 (crosses) normalized by the intensities observed in the absence of copper, for the full-length protein (top panel), the truncated protein (middle panel) and the full-length H50A mutant (bottom panel).
sites corresponding to residues E83 and D98, which were not clearly seen in the HNCO data. Fig. 4 shows plots of resonance intensity versus copper concentration for a number of individual residues in aS. These plots show that at the protein concentration used, the intensity decrease for sites in the N-terminal region of the protein is essentially linear until saturation. This implies that the effective dissociation constants associated with copper binding to these sites is considerably lower than the protein concentration used (i.e., all copper is bound as it is added). In contrast, sites in the Cterminal tail of the protein show a less linear response and
require higher concentrations of copper to achieve complete broadening, suggesting a higher dissociation constant. Each curve was fit to equation 1, which provides an analytical expression for the occupancy of a single binding site in a simple two-component interaction [23]. F ¼ 1=ð1 þ ½ L=Kd Þ
ð1Þ
F is the fraction of bound protein, K d is the dissociation constant, [L]={-([P]0-[L]0 + K d)+sqrt(([ P]0 [L]0 + K d)2 + 4[L]0K d)}/2 is the concentration of free ligand (copper in this case) and [P]0 and [L]0 are the total concentrations of protein and ligand (copper)
Fig. 4. Normalized intensities of individual resonances from 2D HSQC spectra collected as a function of increasing copper concentration for full-length wild type (open circles) and H50A mutant (filled squares) aS. Fits to an independent binding sites model, as described in the text, are shown in solid lines for the wild type and hashed lines for the H50A mutant data.
8
Y. Sung et al. / Biochimica et Biophysica Acta 1764 (2006) 5 – 12
respectively. Although the presence of multiple binding sites in synuclein means that applying this equation is an oversimplification [23], reasonable fits were obtained, as shown in the figure, and allowed for the extraction of apparent binding constants. However, in addition to the inaccuracy introduced by ignoring the presence of multiple binding sites, it is important to note that the intensity of each NMR resonance is a function not only of the copper-bound and copper-free populations, but also of the rate of exchange between the two populations. Thus, in the absence of information regarding the dynamics of copper binding and release, it is not possible to extract quantitatively accurate binding constant information. Therefore, it must be emphasized that the fits obtained cannot be directly related to the true binding constants, and we consider them only in qualitative terms. To establish that copper binding is indeed reversible, 2D NMR spectra were collected from copper-containing samples to which EDTA had been added. No resonance broadening was observed in these spectra. A number of recent reports have suggested that long-range interactions may exist between the C- and N-terminal regions of aS [24,25]. To determine if any of the N-terminal copperbinding sites depended on such an interaction, we repeated the 2D NMR monitored copper titration using a truncated form of aS consisting of residues 1– 102. Despite the fact that Cterminally truncated aS has been reported to aggregate more rapidly than the full-length protein [26], both light scattering and NMR data again showed no indications of aggregation in our samples over a period of days. Fig. 3 shows that removal of the C-terminal tail did not eliminate any of the major binding sites in the N-terminal region, although broadening at positions 83 and 98 was diminished. Instead, the primary sites in the truncated protein appear to become more sensitive, with a greater degree of broadening apparent at the lowest copper concentration. This may be a result of reduced competition for copper ions in the absence of the C-terminal sites. The broad copper binding-induced intensity minima around position 50 and at the N-terminus of the protein suggested that these sites might feature a somewhat compact structure that would bring several backbone amide sites within the range of the broadening influence of the bound copper ions. In light of the similar binding curves obtained for positions 5 and 50 (Fig. 4), we also considered that these two sites could potentially comprise a single binding site involving residue H50 and Nterminal amino group. To test this idea, we produced an aS mutant in which residue H50 was replaced by an alanine. Fig. 3 shows the 2D NMR monitored copper titration for the H50A mutant. Broadening at the N-terminus of the protein, as well as at positions 20 and 28, is not abolished for the mutant protein and instead appears to be slightly enhanced. Clearly, the removal of the imidazole group at position 50 does not preclude copper binding to the N-terminus of the protein, nor to the sites at positions 21 and 28. Therefore, it is highly unlikely that H50 and the N-terminal amino group form a single longrange copper binding site in aS. Binding to the C-terminal sites is also essentially unaltered or perhaps slightly enhanced. Surprisingly, while this paper was under review a similar study appeared concluding, in contradiction to our own results,
that a long-range copper-binding site involving residue H50 and the N-terminal amino group is in fact present in aS [27]. In drawing this conclusion, this report relied heavily on the observation that DEPC treatment of aS resulted in elimination of copper binding both at position 50 and at the N-terminus of the protein. DEPC is known to modify histidine sidechains and prevent them from being able to coordinate copper. Therefore, it was reasoned that DEPC treatment would selectively inhibit copper binding to H50, and should not affect binding to the Nterminus unless such binding required the participation of H50. However, besides histidines, DEPC is known to modify several other sidechain groups, as well at the N-terminal primary amino group of proteins [28]. Therefore, we deemed it likely that the elimination of copper binding at both position 50 and at the N-terminus of aS by DEPC treatment was caused by modification of the protein at both of these sites. To test this, we incubated our aS constructs with DEPC and analyzed the samples using mass spectrometry. The results clearly demonstrated that DEPC modifies both full length and C-terminally truncated aS at multiple sites (data not shown), and is therefore not specific to H50. To further show that the N-terminal copper-binding site is directly targeted by DEPC, we incubated wild type full length, wild type C-terminally truncated, and H50A full-length aS with DEPC prior to the addition of copper and monitored copper binding by 2D NMR. Fig. 5 shows that DEPC does indeed eliminate copper binding to both the site around position 50 and to the N-terminal site in both full-length and truncated wild type aS, but that it also eliminates copperbinding to the N-terminal site in the H50A mutant, where modification of H50 is not possible. In addition, we note that DEPC treatment eliminated copper binding at other sites, both in the N-terminal and C-terminal region of the protein, consistent with the mass spectrometry data and with the ability of this reagent to modify a number of different groups. Interestingly, copper binding to the site around position 121 is greatly enhanced by DEPC modification in both the wild type and H50A proteins. This site is therefore clearly not blocked by DEPC, and as in the case of C-terminal truncation or the H50A substitution we attribute the enhanced copper sensitivity to the elimination of competition from other sites, in this case from those blocked by DEPC. After showing that copper binding to the N-terminal site in aS does not require the participation of H50, we also investigated the idea that H50 and the N-terminus could be involved in a long range contact even in the absence of copper. This was suggested to be the case based on the observation that DEPC modification leads to chemical shift changes both around position 50 and at the N-terminus of aS [27]. Our results show that the N-terminus of aS is also modified by DEPC, providing a more direct explanation for the chemical shift changes at this site. Nevertheless, we constructed an H50C mutant of truncated aS and produced samples in which the Cys residue at position 50 was modified with the paramagnetic spin label MTSL (methanethiosulfonate, Toronto Research Chemicals). The presence of such a spin label leads to severe broadening of NMR resonances from sites that are ˚ of the labeled site. Fig. 6 shows the intensity within 15– 20 A
Y. Sung et al. / Biochimica et Biophysica Acta 1764 (2006) 5 – 12
9
Fig. 5. Normalized NMR resonance intensities as a function of aS sequence position from 2D proton – nitrogen correlation spectra collected at a Cu:aS ratio of 3:4 for unmodified (filled circles) and DEPC-modified (open squares) full-length (top panel), truncated (middle panel) and full-length H50A mutant (bottom panel) aS.
of resonances in spin-labeled H50C truncated aS normalized to the intensity observed for concentration matched samples in which spin labeling was prevented by an excess of the reducing agent DTT. Although there is dramatic broadening of resonances that are close in sequence to position 50, resonances at the N-terminus are not dramatically broadened, confirming that there is no persistent long-range contact between position 50 and the N-terminus of aS. Instead, a weak broadening is observed throughout the regions both N- and C-terminal to those that are dramatically broadened by labeling at position 50. This likely results from an overall preference for compact
conformations within the ensemble sampled by the N-terminal lipid-binding domain of aS [16,29], and is consistent with early reports of residual structure within this region [29], as well as more recent constrained simulations of the behavior of the fulllength protein [24]. The N-terminal 100 residues of aS adopt a more ordered highly helical structure upon binding to lipid surfaces or detergent micelles [30 – 32], which is likely to mediate the normal function(s) of the protein. To explore whether copper bound to this folded conformation of the protein as well, we compared 2D NMR spectra of the micelle-bound protein in the
Fig. 6. NMR resonance intensities as a function of aS sequence position from proton – nitrogen correlation spectra collected from truncated MTSL spin-labeled H50C aS normalized by the intensities observed for an equivalent reduced sample.
10
Y. Sung et al. / Biochimica et Biophysica Acta 1764 (2006) 5 – 12
absence and presence of copper. Although the negatively charged surfaces of detergent micelles are likely to attract copper ions, increasing the effective copper concentration in the vicinity of micelle-bound aS [33], this is unlikely to result in any residue-specific broadening effects in the spectrum of micelle-bound aS. Fig. 7 shows the normalized resonance intensity as a function of sequence position for micelle-bound full-length and truncated aS in the presence of increasing concentrations of CuCl2. As expected, noticeable resonance broadening occurred at somewhat lower copper concentrations than in the case of the free protein. In the C-terminal region of the full-length protein, the broadening pattern is similar to that observed for the free protein, consistent with the fact that the acidic C-terminal tail of the protein does not bind to the micelle surface [16]. In the N-terminal lipid-binding region, the data still show broadening both at the N-terminus and around position 50, suggesting that these sites can coordinate copper in the micelle-bound form of the protein as well. However, the broadening surrounding these sites is qualitatively different from that seen in the free protein, having more of a Fsquare well_ shape, with sharply defined boundaries. This likely results from the less flexible structure of the micelle-bound protein, which prevents residues past a certain distance in the sequence from the binding site from closely approaching the bound copper ion. In addition to this difference, the site at position 98 is more easily broadened than in the free state of the protein, while other sites in the N-terminal domain, such as positions 21, 35 and 66, are hardly apparent in the data from the full-length protein, and only weakly apparent for the truncated protein, suggesting that the helical micelle-bound structure of the protein interferes with copper binding to the latter sites. Studies of the topology of the highly helical micelle-bound conformation of aS rule out tertiary contacts between the Nterminus of the protein and His 50 [34,35], and an absence of significant changes (beyond the aforementioned resonance broadening) in the 2D NMR spectra of the protein upon copper binding indicate that no large-scale conformational changes occur. Therefore, in micelle-bound aS, it is highly unlikely that
the two distant locations participate in the same copper-binding site. This again indicates that the N-terminus of aS and the site involving His 50 are capable of binding copper independently of one another. In addition, binding of copper to these two sites in the micelle-bound state indicates that in the copper-bound conformation of these sites is not precluded by the helical conformation of the micelle-bound protein. Our results indicate that aS binds to copper through at large number of independent local binding sites which are formed by groups that would be expected to bind to copper in any largely unstructured polypeptide, namely negatively charged side chains, the one histidine in the protein, and the primary amino group of the protein. The presence of a large number of sites is consistent with a previous study of copper induced aS aggregation that concluded that full-length aS bound to 10.4 copper ions [10], although our results suggest that aS contains as many as 16 different sites capable of binding copper. Our results also suggest that these sites are unlikely to be specific to copper beyond any copper preference inherent in the individual groups involved, since those groups (Asp, Glu and His side chains and the primary amino group) are capable of coordinating a number of other metals, including zinc, nickel, iron and calcium [36]. In contrast to a recent report [27], we show that copper-binding by aS does not involve long-range interactions. Our results suggest that it is unlikely that copper binding plays an important role in the normal function of alpha-synuclein. This is a contrast with the prion protein, where copper binding appears to be highly specific and is thought to be important in the protein’s normal function. Nevertheless, previous results strongly suggest that copper binding could be important in the pathological role of aS in PD. We also show that copper binding by aS can occur in the lipid-bound conformation as well as in the free state. Because it is still unclear which of these two states of the protein is responsible for its aggregation in vivo, this is an important point to consider. The identification of specific residues involved in copper binding by aS should facilitate studies of how and whether such binding influences the pathogenic behavior of the protein. For example, specific mutants can now be designed to
Fig. 7. Normalized NMR resonance intensities as a function of aS sequence position from 2D proton – nitrogen correlation spectra collected at Cu:aS ratios of 1:10 (triangles), 1:4 (open circles), 2:4 (filled circles), and 3:4 (crosses) for full-length (top panel) and truncated (bottom panel) aS bound to SDS micelles.
Y. Sung et al. / Biochimica et Biophysica Acta 1764 (2006) 5 – 12
perturb each different site of copper binding by aS and to study the consequent effects on the copper-induced aggregation the protein in vitro, as well as on its functional and toxic properties in situ and in vivo.
[13] [14]
Acknowledgements This work was supported in part by the NIA, NIH, grant AG019391 (to D.E.) and by a gift from Herbert and Ann Siegel (to D.E.).
[15]
[16]
Appendix A. Supplementary data [17]
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.bbapap.2005.11.003. References [1] M.H. Polymeropoulos, C. Lavedan, E. Leroy, S.E. Ide, A. Dehejia, A. Dutra, B. Pike, H. Root, J. Rubenstein, R. Boyer, E.S. Stenroos, S. Chandrasekharappa, A. Athanassiadou, T. Papapetropoulos, W.G. Johnson, A.M. Lazzarini, R.C. Duvoisin, G. Di Iorio, L.I. Golbe, R.L. Nussbaum, Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease, Science 276 (1997) 2045 – 2047. [2] R. Kruger, W. Kuhn, T. Muller, D. Woitalla, M. Graeber, S. Kosel, H. Przuntek, J.T. Epplen, L. Schols, O. Riess, Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson’s disease, Nat. Genet. 18 (1998) 106 – 108. [3] J.J. Zarranz, J. Alegre, J.C. Gomez-Esteban, E. Lezcano, R. Ros, I. Ampuero, L. Vidal, J. Hoenicka, O. Rodriguez, B. Atares, V. Llorens, E. Gomez Tortosa, T. del Ser, D.G. Munoz, J.G. de Yebenes, The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia, Ann. Neurol. 55 (2004) 164 – 173. [4] A.B. Singleton, M. Farrer, J. Johnson, A. Singleton, S. Hague, J. Kachergus, M. Hulihan, T. Peuralinna, A. Dutra, R. Nussbaum, S. Lincoln, A. Crawley, M. Hanson, D. Maraganore, C. Adler, M.R. Cookson, M. Muenter, M. Baptista, D. Miller, J. Blancato, J. Hardy, K. Gwinn-Hardy, alpha-Synuclein locus triplication causes Parkinson’s disease, Science 302 (2003) 841. [5] M.G. Spillantini, M.L. Schmidt, V.M. Lee, J.Q. Trojanowski, R. Jakes, M. Goedert, Alpha-synuclein in Lewy bodies, Nature 388 (1997) 839 – 840. [6] M.G. Spillantini, J.R. Murrell, M. Goedert, M.R. Farlow, A. Klug, B. Ghetti, Mutation in the tau gene in familial multiple system tauopathy with presenile dementia, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 7737 – 7741. [7] M. Hashimoto, L.J. Hsu, A. Sisk, Y. Xia, A. Takeda, M. Sundsmo, E. Masliah, Human recombinant NACP/alpha-synuclein is aggregated and fibrillated in vitro: relevance for Lewy body disease, Brain Res. 799 (1998) 301 – 306. [8] L.C. Serpell, J. Berriman, R. Jakes, M. Goedert, R.A. Crowther, Fiber diffraction of synthetic alpha-synuclein filaments shows amyloid-like cross-beta conformation, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 4897 – 4902. [9] K.A. Conway, J.D. Harper, P.T. Lansbury Jr., Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson’s disease are typical amyloid, Biochemistry 39 (2000) 2552 – 2563. [10] S.R. Paik, H.J. Shin, J.H. Lee, C.S. Chang, J. Kim, Copper(II)-induced self-oligomerization of alpha-synuclein, Biochem. J. 340 (Pt. 3) (1999) 821 – 828. [11] V.N. Uversky, J. Li, A.L. Fink, Metal-triggered structural transformations, aggregation, and fibrillation of human alpha-synuclein. A possible molecular link between Parkinson’s disease and heavy metal exposure, J. Biol. Chem. 276 (2001) 44284 – 44496. [12] D.R. Brown, K. Qin, J.W. Herms, A. Madlung, J. Manson, R. Strome, P.E.
[18]
[19]
[20]
[21]
[22]
[23] [24]
[25]
[26]
[27]
[28] [29]
[30]
[31]
11
Fraser, T. Kruck, A. von Bohlen, W. Schulz-Schaeffer, A. Giese, D. Westaway, H. Kretzschmar, The cellular prion protein binds copper in vivo, Nature 390 (1997) 684 – 687. J. Stockel, J. Safar, A.C. Wallace, F.E. Cohen, S.B. Prusiner, Prion protein selectively binds copper(II) ions, Biochemistry 37 (1998) 7185 – 7193. C.S. Atwood, R.D. Moir, X. Huang, R.C. Scarpa, N.M. Bacarra, D.M. Romano, M.A. Hartshorn, R.E. Tanzi, A.I. Bush, Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis, J. Biol. Chem. 273 (1998) 12817 – 12826. W. Garzon-Rodriguez, A.K. Yatsimirsky, C.G. Glabe, Binding of Zn(II), Cu(II), and Fe(II) ions to Alzheimer’s A beta peptide studied by fluorescence, Bioorg. Med. Chem. Lett. 9 (1999) 2243 – 2248. D. Eliezer, E. Kutluay, R. Bussell Jr., G. Browne, Conformational properties of alpha-synuclein in its free and lipid-associated states, J. Mol. Biol. 307 (2001) 1061 – 1073. J.H. Viles, F.E. Cohen, S.B. Prusiner, D.B. Goodin, P.E. Wright, H.J. Dyson, Copper binding to the prion protein: structural implications of four identical cooperative binding sites, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 2042 – 2047. G.S. Jackson, I. Murray, L.L. Hosszu, N. Gibbs, J.P. Waltho, A.R. Clarke, J. Collinge, Location and properties of metal-binding sites on the human prion protein, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 8531 – 8535. C.D. Syme, R.C. Nadal, S.E. Rigby, J.H. Viles, Copper binding to the amyloid-beta (Abeta) peptide associated with Alzheimer’s disease: folding, coordination geometry, pH dependence, stoichiometry, and affinity of Abeta-(1 – 28): insights from a range of complementary spectroscopic techniques, J. Biol. Chem. 279 (2004) 18169 – 18177. R. Lowe, D.L. Pountney, P.H. Jensen, W.P. Gai, N.H. Voelcker, Calcium(II) selectively induces alpha-synuclein annular oligomers via interaction with the C-terminal domain, Protein Sci. 13 (2004) 3245 – 3252. C.O. Fernandez, W. Hoyer, M. Zweckstetter, E.A. Jares-Erijman, V. Subramaniam, C. Griesinger, T.M. Jovin, NMR of alpha-synuclein – polyamine complexes elucidates the mechanism and kinetics of induced aggregation, EMBO J. 23 (2004) 2039 – 2046. E.I. Solomon, R.K. Szilagyi, S. DeBeer George, L. Basumallick, Electronic structures of metal sites in proteins and models: contributions to function in blue copper proteins, Chem. Rev. 104 (2004) 419 – 458. M.R. Eftink, Fluorescence methods for studying equilibrium macromolecule – ligand interactions, Methods Enzymol. 278 (1997) 221 – 257. M.M. Dedmon, K. Lindorff-Larsen, J. Christodoulou, M. Vendruscolo, C.M. Dobson, Mapping long-range interactions in alpha-synuclein using spin-label NMR and ensemble molecular dynamics simulations, J. Am. Chem. Soc. 127 (2005) 476 – 477. C.W. Bertoncini, Y.S. Jung, C.O. Fernandez, W. Hoyer, C. Griesinger, T.M. Jovin, M. Zweckstetter, From The Cover: release of long-range tertiary interactions potentiates aggregation of natively unstructured {alpha}-synuclein, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 1430 – 1435. R.A. Crowther, R. Jakes, M.G. Spillantini, M. Goedert, Synthetic filaments assembled from C-terminally truncated alpha-synuclein, FEBS Lett. 436 (1998) 309 – 312. R.M. Rasia, C.W. Bertoncini, D. Marsh, W. Hoyer, D. Cherny, M. Zweckstetter, C. Griesinger, T.M. Jovin, C.O. Fernandez, Structural characterization of copper(II) binding to alpha-synuclein: Insights into the bioinorganic chemistry of Parkinson’s disease, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 4294 – 4299. E.W. Miles, Modification of histidyl residues in proteins by diethylpyrocarbonate, Methods Enzymol. 47 (1977) 431 – 442. R. Bussell Jr., D. Eliezer, Residual structure and dynamics in Parkinson’s disease-associated mutants of alpha-synuclein, J. Biol. Chem. 276 (2001) 45996 – 46003. W.S. Davidson, A. Jonas, D.F. Clayton, J.M. George, Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes, J. Biol. Chem. 273 (1998) 9443 – 9449. R. Bussell Jr., D. Eliezer, A structural and functional role for 11-mer repeats in alpha-synuclein and other exchangeable lipid binding proteins, J. Mol. Biol. 329 (2003) 763 – 778.
12
Y. Sung et al. / Biochimica et Biophysica Acta 1764 (2006) 5 – 12
[32] S. Chandra, X. Chen, J. Rizo, R. Jahn, T.C. Sudhof, A broken alpha-helix in folded alpha-Synuclein, J. Biol. Chem. 278 (2003) 15313 – 15318. [33] E.N. Lee, S.Y. Lee, D. Lee, J. Kim, S.R. Paik, Lipid interaction of alphasynuclein during the metal-catalyzed oxidation in the presence of Cu2+ and H2O2, J. Neurochem. 84 (2003) 1128 – 1142. [34] R. Bussell Jr., T.F. Ramlall, D. Eliezer, Helix periodicity, topology, and
dynamics of membrane-associated alpha-synuclein, Protein Sci. 14 (2005) 862 – 872. [35] T.S. Ulmer, A. Bax, N.B. Cole, R.L. Nussbaum, Structure and dynamics of micelle-bound human alpha-synuclein, J. Biol. Chem. 280 (2005) 9595 – 9603. [36] M.M. Harding, The architecture of metal coordination groups in proteins, Acta Crystallogr., D Biol. Crystallogr. 60 (2004) 849 – 859.