Chem. Res. Toxicol. 2004, 17, 471-475
471
A Theoretical Study on Cu(II) Binding Modes and Antioxidant Activity of Mammalian Normal Prion Protein Hong-Fang Ji and Hong-Yu Zhang* Laboratory for Computational Biology and Shandong Provincial Research Center for Bioinformatic Engineering and Technique, Shandong University of Technology, Zibo 255049, People’s Republic of China Received November 10, 2003
In this paper, the density functional theory (DFT) method B3LYP/LANL2DZ was used to calculate binding energies and electron affinities for various Cu(II) binding modes of mammalian normal prion protein (PrPc). The calculation results not only provide solid evidence to support one of the experimentally determined Cu(II) binding modes of PrPc but also shed new light on the normal function of the elusive protein; that is, PrPc is rather a Cu(II) transporter than an antioxidant. In addition, the employed theoretical methodology is also useful to investigate the metal chelating properties of other proteins and to rationally design Cu,Zn-superoxide dismutase mimics.
Introduction Fatal neurodegenerative diseases induced by prion are thought to arise from conformational transition of normal cellular prion protein (PrPc) to its scrapie isoform (PrPSc) (1, 2). Hence, the possible pathogenesis of the diseases involves not only the gain of toxicity of PrPSc but also the loss of normal function of PrPc, which is the reason much effort has been devoted to elucidating the normal function of PrPc (3-5). A major breakthrough of recent studies is the finding that PrPc binds Cu(II) with a considerably high affinity (6-9), and the chelate holds superoxide dismutase (SOD)-like activity (10). Accordingly, it has been proposed that PrPc participates in the regulation of copper and possibly modulates the concentration of reactive oxygen species (11-18). However, although the octapeptide repeats (PHGGGWGQ) at the N-terminal domain of PrPc have been recognized as the major Cu(II) binding sites (6, 7, 19-21), the detailed binding modes are still in controversy, based on the different experimental investigations (22-31). On the other hand, up to now, there is no quantitative evaluation on the antioxidant activity of PrPc-Cu(II). Considering the successful use of quantum chemical methods in predicting the metal binding sites of metalloproteins (32, 33) and in elucidating the radical scavenging activity difference of various antioxidants (34-36), we attempt to explore the Cu(II) binding modes and normal function of mammalian PrPc by calculating the binding energy and other physicochemical parameters as well (37).
Materials and Methods The calculation details are as follows. The full geometry optimization for each molecule was performed in a vacuum using hybrid density functional theory (DFT) method B3LYP. For open shell systems, unrestricted DFT was used. The standard * To whom correspondence can be addressed. Tel: 86-533-2780271. Fax: 86-533-2780271. E-mail:
[email protected].
double-ζ basis set was used for all light elements, while for metals, nonrelativistic effective core potential (ECP) was employed. The valence basis set used in connection with the ECP is essentially of double-ζ quality (the LANL2DZ basis set). All of the calculations were performed with the GAUSSIAN 98 package of programs (38). As the molecules are rather large, the B3LYP/LANL2DZ method failed to give zero point vibrational energy and thermal correction to energy. However, according to the previous studies, physicochemical parameters derived from total electronic energy (TE) are fairly accurate in a relative sense (39-41). To evaluate the solvent effect on the physicochemical parameters, the self-consistent reaction field method with polarized continuum model was employed. As indicated in the following section, solvent effect has little influence on the relative order of physicochemical parameters. Hence, in this paper, the TE-derived in-vacuum results are applicable to reach a qualitative conclusion.
Results and Discussion Up to now, at least four Cu(II) binding modes have been proposed for the octapeptide regions (Scheme 1; I-IV) (22-31). To determine which one is the most probable mode, we calculated the binding energy for each chelate. As shown in Table 1, the order for the Cu(II) binding energy is as follows: I < II ∼ III < IV, with IV being much more stable than the others. Therefore, IV is the most probable mode. In fact, this mode gains the most experimental supports and is the only crystal structure of a single Cu(II) octarepeat (25-31). Moreover, it is very interesting to notice that the binding energy for ceruloplasmin, a primary metalloprotein in blood to transport Cu(II), is only 406.29 kcal/mol (Table 1), much lower than that of PrPc-Cu(II), suggesting that it is favorable in energy for PrPc to accept Cu(II) from ceruloplasmin. This provides solid evidence to support the idea that the normal function of PrPc is to transport Cu(II) across membrane. To evaluate the antioxidant activity of PrPc-Cu(II) theoretically, we have to select a theoretical parameter to measure the radical scavenging activity at first. As is
10.1021/tx034232y CCC: $27.50 © 2004 American Chemical Society Published on Web 03/09/2004
472
Chem. Res. Toxicol., Vol. 17, No. 4, 2004
Ji and Zhang
Scheme 1. Structures of Four PrPc-Cu(II) Modes, in Which Nitrogen Is in Blue, Oxygen Is in Red, Carbon Is in Gray, Hydrogen Is in Light Gray, and Copper Is in Orange
Table 1. B3LYP/LANL2DZ Calculated Binding Energies of Cu(II) Chelates TEca (hartree) TEsb (hartree) BEc (kcal/mol) mode I (22) mode II (23) mode III (24) mode IV (25-31) ceruloplasmin Aβ peptided (44)
-1560.914129 -2978.890992 -1368.794162 -1236.052940 -1737.081458 -2170.472707
-1560.075625 -2974.376564 -1367.667054 -1234.834410 -1736.434112 -2169.273431
526.17 708.21 707.27 764.64 406.29 752.56
a TE of chelate. b Sum of TE for each group and metal ion. Binding energy: TEs - TEc. d Cu(II) chelating center of Aβ peptide.
c
well-known, Cu,Zn-SOD and other mimics scavenge superoxide anion through two coupled steps:
E-Cu(II) + O2•- f E-Cu(I) + O2
(1)
E-Cu(I) + O2•- + 2H+ f E-Cu(II) + H2O2
(2)
The first one is the rate limiting step, and an appropriate theoretical index to characterize the electron transfer rate is electron affinity (EA), which is defined as EA ) TEE-Cu(I) - TEE-Cu(II), in which TE E-Cu(I) is the TE of E-Cu(I) and TE E-Cu(II) is that for E-Cu(II). The lower the EA is, the higher the electron transfer rate is. To verify the effectiveness of the parameter, we calculated EAs for three Cu,Zn-SOD mimics (APEN, APPN, and APTN) and the active center of Cu,Zn-SOD (Scheme 2) (Table 2) (42). It is very interesting to find that there exists a very good linear correlation between -log(1/EC50) and EA (r ) 0.996; Figure 1), indicating that EA is indeed a good theoretical parameter to characterize the superoxide anion scavenging activity. More interestingly, when the solvent effect was considered, the EAs for APEN, APPN, and APTN were calculated to be -89.05, -91.26, and -92.59 kcal/mol, respectively. Although the values are much higher than those in a vacuum, they still correlate well with -log(1/EC50) (r ) 0.993), supporting the idea that solvent effect has little influence on the relative order of physicochemical parameters. In addition, the previous study revealed that the different SOD-like activities of APEN, APPN, and APTN resulted from the distinct geometries of the chelates, which distorted from nearly square planar to tetrahedron (42). The dihedral angles for the four nitrogen atoms in the optimized structures of three Cu,Zn-SOD mimics are listed in Table 2, which manifests that the larger the dihedral angle is, the lower the EA is. Furthermore, obviously, the dihedral angles are related to the length of the side chains, i.e., ethylene, propylene, and butylenes. That is, the longer the side chain is, the higher the dihedral angle, which offers an opportunity to design better Cu,Zn-SOD mimics on the basis of APTN. The EA of mode IV was calculated to be -50.99 kcal/ mol (Table 1). According to the linear correlation equation (Figure 1), the EC50 for PrPc-Cu(II) will be extremely higher than that of Cu,Zn-SOD, suggesting that PrPcCu(II) is a much less efficient antioxidant than Cu,ZnSOD. Apparently, the present methodology will also be useful to investigate the metal chelating properties for other proteins, such as amyloid-β (Aβ) peptide, a pathogenic factor in Alzheimer’s disease, where transition metals were also found to play a crucial role in the pathogenesis (16, 43), and Aβ can bind Cu(II) with ordered structure (Scheme 2) (44). The Cu(II) binding energy for Aβ was calculated to be 752.56 kcal/mol (Table 1), a little lower than that of PrPc, while the EA for Aβ-Cu(II) was calculated to be -105.52 kcal/mol (Table 2), much lower than that of PrPc-Cu(II), implying that Aβ is also a Cu(II) chelator similar to PrPc but holds more potential than PrPc as an antioxidant. In addition, the effectiveness
Mammalian Normal Prion Protein
Chem. Res. Toxicol., Vol. 17, No. 4, 2004 473
Scheme 2. Structures of Four Cu,Zn-SOD Mimics, Active Center of Cu,Zn-SOD and Cu(II) Chelating Center of Aβ Peptide, in Which Nitrogen Is in Blue, Oxygen Is in Red, Carbon Is in Gray, Hydrogen Is in Light Gray, and Copper Is in Orangea
a
APPTN is N,N′-pentene bis-(2-acetylpyridine iminato) copper(II). Table 2. B3LYP/LANL2DZ Calculated Electron Affinities (EAs) and Dihedral Angles for Cu(II) Chelates
APEN APPN APTN Cu,Zn-SODf mode IV Aβ peptideg APPTNh
dihedral anglee (degree)
TECu(II)a (hartree)
TECu(I)b (hartree)
EAc (kcal/mol)
EC50d (µM)
-1111.558268 -1150.865563 -1190.174906 -2165.626412 -1236.052940 -2170.472707 -1229.475637
-1111.857429 -1151.169268 -1190.482402 -2165.938423 -1236.134196 -2170.640868 -1229.785184
-187.73 -190.58 -192.96 -195.79 -50.99 -105.52 -194.25
11.04 2.33 0.56 0.06
11.51 31.62 42.20
0.14i
44.21
TE of Cu(II) chelate. b TE of Cu(I) chelate. c EA: TECu(I) - TECu(II). d Data from ref 42. e Defined by N1-N2-N3-N4. f Active center of Cu,Zn-SOD. g Cu(II) chelating center of Aβ peptide. h N,N′-pentene bis-(2-acetylpyridine iminato) copper(II). i Extrapolated from the linear equation between -log(1/EC50) and EA. a
of EA in characterizing the superoxide anion scavenging activity suggests that this parameter is an appropriate theoretical index in quantitative structure-activity relationship study and rational design of SOD mimics. According to the above-mentioned SOD mimic design strategy, we could use pentene (a longer side chain) to substitute the butylene in APTN to reach a higher dihedral angle. As expected, as compared with APTN, the dihedral angle of the novel SOD mimic (APPTN; Scheme 2) is enhanced and the corresponding EA is
reduced (Table 2). The predicted EC50 suggests that APPTN is a better SOD mimic than APTN (Table 2).
Conclusion By means of theoretical calculations, we not only provided new evidence to support one of the experimentally determined Cu(II) binding modes for PrPc (mode IV) but also shed new light on the normal function of the elusive protein; that is, PrPc is rather a Cu(II) transporter
474
Chem. Res. Toxicol., Vol. 17, No. 4, 2004
Ji and Zhang
(9)
(10)
(11) (12) (13)
(14)
Figure 1. Correlation between -log(1/EC50) and EA. Linear equation: -log(1/EC50) ) 53.54 + 0.28 × EA (r ) 0.996). (a) N,N′-Ethylene bis-(2-acetylpyridine iminato) copper(II) (APEN); (b) N,N′-propylene bis-(2-acetylpyridine iminato) copper(II) (APPN); (c) N,N′-butylene bis-(2-acetylpyridine iminato) copper(II) (APTN); and (d) active center of Cu,Zn-SOD.
(15)
(16) (17)
than an antioxidant. The present theoretical methodology will also be helpful to investigate the metal chelating properties of other proteins and to rationally design novel SOD mimics as well.
(18)
(19)
Acknowledgment. We are grateful to Prof. Qin-Hui Luo, Ms. Sheng-Juan Jiang, and Mr. Liang Shen for their helpful assistance in the calculations. This work was partially supported by National Natural Science Foundation of China (Grant No. 30100035).
(20)
(21)
Supporting Information Available: Optimized structures and total energies for all calculated molecules. This material is available free of charge via the Internet at http:// pubs.acs.org.
Note Added in Proof: Following the acceptance of the paper, a critical review appeared (45) in which the author presented a similar opinion to ours that according to the existing Cu(II)-chelating mode, PrPc seems more like a copper buffer or a copper transporter than an antioxidant.
References (1) Prusiner, S. B. (1997) Prion diseases and the BSE crisis. Science 278, 245-251. (2) Prusiner, S. B. (1998) Prions. Proc. Natl. Acad. Sci. U.S.A. 95, 13363-13383. (3) Martins, V. R., Linden, R., Prado, M. A. M., Walz, R., Sakamoto, A. C., Izquierdo, I., and Brentani, R. R. (2002) Cellular prion protein: on the road for functions. FEBS Lett. 512, 25-28. (4) Hetz, C., Maundrell, K., and Soto, C. (2003) Is loss of function of the prion protein the cause of prion disorders? Trends Mol. Med. 9, 237-243. (5) Martins, V. R., and Brentani, R. R. (2002) The biology of the cellular prion protein. Neurochem. Int. 41, 353-355. (6) Hornshaw, M. P., McDermott, J. R., and Candy, J. M. (1995) Copper binding to the N-terminal tandem repeat regions of mammalian and avian prion protein. Biochem. Biophys. Res. Commun. 207, 621-629. (7) Hornshaw, M. P., McDermott, J. R., Candy, J. M., and Lakey, J. H. (1995) Copper binding to N-terminal tandem repeat region of mammalian and avian prion protein: structural studies using synthetic peptides. Biochem. Biophys. Res. Commun. 214, 993999. (8) Kramer, M. L., Kratzin, H. D., Schmidt, B., Ro¨mer, A., Windl, O., Liemann, S., Hornemann, S., and Kretzschmar, H. (2001)
(22)
(23)
(24)
(25)
(26)
(27)
(28)
(29)
(30)
Prion protein binds copper within the physiological concentration range. J. Biol. Chem. 276, 16711-16719. Brown, D. R., Qin, K. F., Herms, J. W., Madlung, A., Manson, J., Strome, R., Fraser, P. E., Kruck, T., Bohlen, A. V., SchulzSchaeffer, W., Giese, A., Westaway, D., and Kretzschmar, H. (1997) The cellular prion protein binds copper in vivo. Nature 390, 684-687. Brown, D. R., Wong, B. S., Hafiz, F., Clive, C., Haswell, S. J., and Jones, I. M. (1999) Normal prion protein has an activity like that of superoxide dismutase. Biochem. J. 344, 1-5. Although this proposal was supported by plenty of studies (1217), there are still a few negative reports (18). Pauly, P. C., and Harris, D. A. (1998) Copper stimulates endocytosis of the prion protein. J. Biol. Chem. 273, 33107-33110. Sumudhu, W., Perera, S., and Hooper, N. M. (2001) Ablation of the metal ion-induced endocytosis of the prion protein by diseaseassociated mutation of the octarepeat region. Curr. Biol. 11, 519523. Milhavet, O., McMahon, H. E. M., Rachidi, W., Nishida, N., Katamine, S., Mange, A., Arlotto, M., Casanova, D., Riondel, J., Favier, A., and Lehmann, S. (2000) Prion infection impairs the cellular response to oxidative stress. Proc. Natl. Acad. Sci. U.S.A. 97, 13937-13942. Klamt, F., Dal-Pizzol, F., Da Frota, M. L. C., Jr., Walz, R., Andrades, E. M., Da Silva, G. E., Brentani, R. R., Izquierdo, I., and Moreira, J. C. F. (2001) Imbalance of antioxidant defense in mice lacking cellular prion protein. Free Radical Biol. Med. 30, 1137-1144. Bush, A. I. (2000) Metals and neuroscience. Curr. Opin. Chem. Biol. 4, 184-191. Lehmann, S. (2002) Metal ions and prion diseases. Curr. Opin. Chem. Biol. 6, 187-192. Waggoner, D. J., Drisaldi, B., Bartnikas, T. B., Casareno, R. L. B., Gitlin, J. D., and Harris, D. A. (2000) Brain copper content and cuproenzyme activity do not vary with prion protein expression level. J. Biol. Chem. 275, 7455-7458. The C-terminal region and the flexible region between the octapeptides and the C-terminal domain were also suggested to be Cu(II)-binding sites (20, 21). Doorslaer, S. V., Cereghtti, G. M., Glockshuber, R., and Schweiger, A. (2001) Unraveling the Cu2+ binding sites in the C-terminal domain of the murine prion protein: A pulse EPR and ENDOR study. J. Phys. Chem. B 105, 1631-1639. Hasnain, S. S., Murphy, L. M., Strange, R. W., Grossmann, J. G., Clarke, A. R., Jackson, G. S., and Collinge, J. (2001) XAFS Study of the high-affinity copper-binding site of human PrP91231 and its low-resolution structure. J. Mol. Biol. 311, 467-473. Stockel, J., Safar, J., Wallace, A. C., Cohen, F. E., and Prusiner, S. B. (1998) Prion protein selectively binds copper ions. Biochemistry 37, 7185-7193. Viles, J. H., Cohen, F. E., Prusiner, S. B., Goodin, D. B., and Wright, P. E. (1999) Copper binding to the prion protein: Structural implications of four identical cooperative binding sites. Proc. Natl. Acad. Sci. U.S.A. 96, 2042-2047. Miura, T., Hori, I. A., Mototani, H., and Takeuchi, H. (1999) Raman spectroscopic study on the copper(II) binding mode of prion octapeptide and its pH dependence. Biochemistry 38, 11560-11569. Burns, C. S., Aronoff-Spencer, E., Dunham, C. M., Lario, P., Avdievich, N. I., Antholine, W. E., Olmstead, M. M., Vrielink, A., Gerfen, G. J., Peisach, J., Scott, W. G., and Millhauser, G. L. (2002) Molecular features of the copper binding sites in the octarepeat domain of the prion protein. Biochemistry 41, 39914001. Burns, C. S., Aronoff-Spencer, E., Legname, G., Prusiner, S. B., Antholine, W. E., Gerfen, G. J., Peisach, J., and Millhauser, G. L. (2003) Copper coordination in the full-length recombinant prion protein. Biochemistry 42, 6794-6803. Jackson, G. S., Murray, I., Hosszu, L. L. P., Gibbs, N., Waltho, J. P., Clarke, A. R., and Collinge, J. (2001) Location and properties of metal-binding sites on the human prion protein. Proc. Natl. Acad. Sci. U.S.A. 98, 8531-8535. Whittal, R. M., Ball, H. L., Cohen, F. E., Burlingame, A. L., Prusiner, S. B., and Baldwin, M. A. (2000) Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry. Protein Sci. 9, 332-343. Qin, K. F., Yang, Y., Mastrangelo, P., and Westaway, D. (2002) Mapping Cu(II) binding sites in prion proteins by diethyl pyrocarbonate modification and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometric footprinting. J. Biol. Chem. 277, 1981-1990. Garnett, A. P., and Viles, J. H. (2003) Copper binding to the octarepeats of the prion protein. Affinity, specificity, folding, and
Mammalian Normal Prion Protein
(31)
(32) (33) (34) (35)
(36) (37) (38)
cooperativity: insights from circular dichroism. J. Biol. Chem. 278, 6795-6802. Leczkowski, M., Kozlowski, H., Legowska, A., Rolka, K., and Remelli, M. (2003) The possible role of Gly residues in the prion octarepeat region in the coordination of Cu2+ ions. J. Chem. Soc., Dalton Trans. 619-624. Rulı´ek, L., and Havlas, Z. (2003) Using DFT methods for the prediction of the structure and energetics of metal-binding sites in metalloproteins. Int. J. Quantum Chem. 91, 504-510. Siegbahn, P. E. M. (2003) Quantum chemical studies of redoxactive enzymes. Faraday Discuss. 124, 289-296. Zhang, H. Y. (1999) Theoretical methods used in elucidating activity differences of phenolic antioxidants. J. Am. Oil. Chem. Soc. 76, 745-748. Wright, J. S., Johnson, E. R., and DiLabio, G. A. (2001) Predicting the activity of phenolic antioxidants: Theoretical method, analysis of substituent effects, and application to major families of antioxidants. J. Am. Chem. Soc. 123, 1173-1183. Zhang, H. Y., Wang, L. F., and Sun, Y. M. (2003) Why B-ring is the active center for genistein to scavenge peroxyl radical. A DFT study. Bioorg. Med. Chem. Lett. 13, 909-911. As the N-terminal region of avian PrPc is much different from that of mammalian PrPc, we only concentrated the study on the latter in this paper. Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R., Zakrzewski, V. G., Montgomery, J. A., Jr., Stratmann, R. E., Burant, J. C., Dapprich, S., Millam, J. M., Daniels, A. D., Kudin, K. N., Strain, M. C., Farkas, O., Tomasi, J., Barone, V., Cossi, M., Cammi, R., Mennucci, B., Pomelli, C., Adamo, C., Clifford, S., Ochterski, J., Petersson, G. A., Ayala, P. Y., Cui, Q., Morokuma, K., Malick, D. K., Rabuck, A. D., Raghavachari, K., Foresman, J. B., Cioslowski, J., Ortiz, J. V., Baboul, A. G., Stefanov, B. B., Liu, G., Liashenko, A., Piskorz, P., Komaromi, I., Gomperts, R., Martin, R. L., Fox, D. J., Keith, T., Al-Laham, M. A., Peng, C. Y., Nanayakkara, A., Gonzalez,
Chem. Res. Toxicol., Vol. 17, No. 4, 2004 475
(39)
(40) (41) (42) (43)
(44)
(45)
C., Challacombe, M., Gill, P. M. W., Johnson, B., Chen, W., Wong, M. W., Andres, J. L., Gonzalez, C., Head-Gordon, M., Replogle, E. S., and Pople, J. A. (1998) Gaussian 98 (Revision A.7), Gaussian, Inc., Pittsburgh, PA. Wu, Y. D., and Lai, D. K. W. (1996) Density functional study of the substituent effect on the OH and O-CH3 bond dissociation energies in para-substituted phenols and anisoles. J. Org. Chem. 61, 7904-7910. Zhang, H. Y., Sun, Y. M., and Wang, X. L. (2002) Electronic effects on O-H proton dissociation energies of phenolic cation radicals. A DFT study. J. Org. Chem. 67, 2709-2712. Zielonka, J., Gebicki, J., and Grynkiewicz, G. (2003) Radical scavenging properties of genistein. Free Radical Biol. Med. 35, 958-965. Lu, Q., Shen, C. Y., and Luo, Q. H. (1993) A study on the Schiff base Cu2Zn2SOD model complexessthe relationship between structure and activity. Polyhedron 12, 2005-2008. Huang, X. D., Cuajungco, M. P., Atwood, C. S., Hartshorn, M. A., Tyndall, J. D. A., Hanson, G. R., Stokes, K. C., Leopold, M., Multhaup, G., Goldstein, L. E., Scarpa, R. C., Saunders, A. J., Lim, J., Moir, R. D., Glabe, C., Bowden, E. F., Masters, C. L., Fairlie, D. P., Tanzi, R. E., and Bush, A. I. (1999) Cu(II) potentiation of Alzheimer Aβ neurotoxicity. Correlation with cellfree hydrogen peroxide production and metal reduction. J. Biol. Chem. 274, 37111-37116. Curtain, C. C., Ali, F., Volitakis, I., Cherny, R. A., Norton, R. S., Beyreuther, K., Barrow, C. J., Masters, C. L., Bush, A. I., and Barnham, K. J. (2001) Alzheimer’s disease Amyloid-β binds copper and zinc to generate an allosterically ordered membranepenetrating structure containing superoxide dismutase-like subunits. J. Biol. Chem. 276, 20466-20473. Millhauser, G. L. (2004) Copper binding in the prion protein. Acc. Chem. Res. 37, 79-85.
TX034232Y
