Keywords: Aggregation, Metal-Protein Interactions, Ubiquitin, Zinc, Affinity Constants, Binding Modes, Copper Binding, Dynamic Equilibria, Equimolar Addition, In-Line, Neurodegeneration, Neutral Ph, Potentiometric Titrations, Preferential Binding, Protein Assembly, Protein Stability, Protein Thermal Stability, Ubiquitin-Proteasome System, Ubiquitinated Proteins, Zinc Complex, Agglomeration, Binding Energy, Binding Sites, Complexation, Differential Scanning Calorimetry, Metal Ions, Zinc Compounds, Metalloprotein, Article, Chemistry, Metabolism, Nuclear Magnetic Resonance Spectroscopy, Protein Binding,
Affiliations: *** IBB - CNR ***
Dipartimento di Scienze Chimiche, Universita degli Studi di Catania, Viale A. Doria 6, 95125 Catania, Italy.
Istituto di Biostrutture e Bioimmagini-UOS CT, Consiglio Nazionale Delle Ricerche, V.le A. Doria 6, 95125 Catania, Italy
References: Glickman, M.H., Ciechanover, A., (2002) Physiol. Rev., 82, pp. 373-42
Linkous, D.H., Flinn, J.M., Koh, J.Y., Lanzirotti, A., Bertsch, P.M., Jones, B.F., Giblin, L.J., Frederickson, C.J., (2008) J. Histochem. Cytochem., 56, pp. 3-6
Danscher, G., Jensen, K.B., Frederickson, C.J., Kemp, K., Andreasen, A., Juhl, S., Stoltenberg, M., Ravid, R.J., (1997) J. Neurosci. Methods, 76, pp. 53-59
Lovell, M.A., Robertson, J.D., Teesdale, W.J., Campbell, J.L., Markesbery, W.R., (1998) J. Neurol. Sci., 158, pp. 47-52
Opazo, C., Luza, S., Villemagne, V.L., Volitakis, I., Rowe, C., Barnham, K.J., Strozyk, D., Bush, A.I., (2006) Aging Cell, 5, pp. 69-79
Religa, D., Strozyk, D., Cherny, R.A., Volitakis, I., Haroutunian, V., Winblad, B., Naslund, J., Bush, A.I., (2006) Neurology, 67, pp. 69-75
Sensi, S.L., Paoletti, P., Bush, A.I., Sekler, I., (2009) Nat. Rev. Neurosci., 10, pp. 780-792
Manetto, G.D., Rosa, C.L., Grasso, D.M., Milardi, D., (2005) J. Therm. Anal. Calorim., 80, pp. 263-270
Grasso, D., La Rosa, C., Milardi, D., Fasone, S., (1995) Thermochim. Acta, 265, pp. 163-175
Russo, L., Palmieri, M., Baglivo, I., Esposito, S., Isernia, C., Malgieri, G., Pedone, P.V., Fattorusso, R., (2009) Biomol. NMR Assign., 4, pp. 55-56
Stejskal, E.O., Tanner, J.E., (1965) J. Chem. Phys., 42, pp. 288-292
Bartels, C., X, T.-H., Billeter, M., Wuthrich, K., (1995) J. Biomol. NMR, 5, pp. 1-10
Glickman, M. H., Ciechanover, A., (2002) Physiol. Rev., 82, pp. 373-42
Ulrich, H. D., (2002) Eukaryotic Cell, 1, pp. 1-10
Sun, L., Chen, Z. J., (2004) Curr. Opin. Cell Biol., 16, pp. 119-126
Cummings, C. J., Mancini, M. A., Antalffy, B., De Franco, D. B., Orr, H. T., Zoghbi, H. Y., (1998) Nat. Genet., 19, pp. 148-154
Sherman, M. Y., Goldberg, A. L., (2001) Neuron, 29, pp. 15-32
Daniel, K. G., Chen, D., B, B. Y., Dou, Q. P., (2007) Front. Biosci., 12, pp. 135-144
Daniel, K. G., Kuhn, D. J., Kazi, A., Dou, Q. P., (2005) Curr. Cancer Drug Targets, 5, pp. 529-541
Daniel, K. G., Gupta, P., Harbach, R. H., Guida, W. C., Dou, Q. P., (2004) Biochem. Pharmacol., 67, pp. 1139-1151
Bush, A. I., (2003) Trends Neurosci., 26, pp. 207-214
Barnham, K. J., Bush, A. I., (2008) Curr. Opin. Chem. Biol., 12, pp. 222-228
Jackson, S. E., (2006) Org. Biomol. Chem., 4, pp. 1845-1853
Schweiker, K. L., Fitz, V. W., Makhatadze, G. I., (2009) Biochemistry, 48, pp. 10846-10851
Di Stefano, D. L., Wand, A. J., (1987) Biochemistry, 26, pp. 7272-7281
Walters, K. J., Goh, A. M., Wang, Q., Wagner, G., Howley, P. M., (2004) Biochim. Biophys. Acta Mol. Cell Res., 1695, pp. 73-87
Boal, A. K., Rosenzweig, A. C., (2009) Chem. Rev., 109, pp. 4760-4779
Linkous, D. H., Flinn, J. M., Koh, J. Y., Lanzirotti, A., Bertsch, P. M., Jones, B. F., Giblin, L. J., Frederickson, C. J., (2008) J. Histochem. Cytochem., 56, pp. 3-6
Lovell, M. A., Robertson, J. D., Teesdale, W. J., Campbell, J. L., Markesbery, W. R., (1998) J. Neurol. Sci., 158, pp. 47-52
Sensi, S. L., Paoletti, P., Bush, A. I., Sekler, I., (2009) Nat. Rev. Neurosci., 10, pp. 780-792
Koh, J. Y., Suh, S. W., Gwag, B. J., He, Y. Y., Hsu, C. Y., Choi, D. W., (1996) Science, 272, pp. 1013-1016
Suh, S. W., Chen, J. W., Motamedi, M., Bell, B., Listiak, K., Pons, N. F., Danscher, G., Frederickson, C. J., (2000) Brain Res., 852, pp. 268-273
Koh, J. -Y., Choi, D. W., (1994) Neuroscience, 60, pp. 1049-1057
Cary, P. D., King, D. S., Crane-Robinson, C., Bradbury, E. M., Rabbani, A., Roodwin, G. H., Johns, E. W., (1980) Eur. J. Biochem., 112, pp. 577-580
Edgcomb, S. P., Murphy, K. P., (2002) Proteins: Struct., Funct., Bioinf., 49, pp. 1-6
Di Marco, V. B., Bombi, G. G., (2006) Mass Spectrom. Rev., 25, pp. 347-379
K llay, C., Asz, K., D vid, A., Valasty n, Z., Malandrinos, G., Hadjiliadis, N., S v g, I., (2007) Dalton Trans., 36, pp. 4040-4047
Privalov, P. L., Khechinashvili, N. N., (1974) J. Mol. Biol., 86, pp. 665-684
Wintrode, P. L., Makhatadze, G. I., Privalov, P. L., (1994) Proteins Struct. Funct. Bioinf., 18, pp. 246-253
Jain, N. K., Roy, I., (2009) Protein Sci., 18, pp. 24-36
Hu, C. Q., Sturtevant, J. M., Thomson, J. A., Erickson, R. E., Pace, C. N., (1992) Biochemistry, 31, pp. 4876-4882
Manetto, G. D., Rosa, C. L., Grasso, D. M., Milardi, D., (2005) J. Therm. Anal. Calorim., 80, pp. 263-270
Stejskal, E. O., Tanner, J. E., (1965) J. Chem. Phys., 42, pp. 288-292
Bartels, C., X, T. -H., Billeter, M., Wuthrich, K., (1995) J. Biomol. NMR, 5, pp. 1-10
Zinc(II) complexes of ubiquitin: speciation, affinity and binding features
Intraneuronal inclusions consisting of hypermetallated, (poly-)ubiquitinated proteins are a hallmark of neurodegeneration. To highlight the possible role played by metal ions in the dysfunction of the ubiquitin-proteasome system, here we report on zinc(II)/ubiquitin binding in terms of affinity constants, speciation, preferential binding sites and effects on protein stability and self-assembly. Potentiometric titrations allowed us to establish that at neutral pH only two species, ZnUb and Zn(2)Ub, are present in solution, in line with ESI-MS data. A change in the diffusion coefficient of ubiquitin was observed by NMR DOSY experiments after addition of Zn(II) ions, and thus indicates metal-promoted formation of protein assemblies. Analysis of (1)H, (15)N, (13)Calpha and (13)CO chemical-shift perturbation after equimolar addition of Zn(II) ions to ubiquitin outlined two different metal-binding modes. The first involves a dynamic equilibrium in which zinc(II) is shared between a region including Met1, Gln2, Ile3, Phe4, Thr12, Leu15, Glu16, Val17, Glu18, Ile61 and Gln62 residues, which represent a site already described for copper binding, and a domain comprising Ile23, Glu24, Lys27, Ala28, Gln49, Glu51, Asp52, Arg54 and Thr55 residues. A second looser binding mode is centred on His68. Differential scanning calorimetry evidenced that addition of increasing amounts of Zn(II) ions does not affect protein thermal stability; rather it influences the shape of thermograms because of the increased propensity of ubiquitin to self-associate. The results presented here indicate that Zn(II) ions may interact with specific regions of ubiquitin and promote protein-protein contacts. Copyright 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Zinc(II) complexes of ubiquitin: speciation, affinity and binding features