Journal
CHEMISTRY-A EUROPEAN JOURNAL
Volume 17, Issue 41, Pages 11596-11603Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/chem.201101364
Keywords
aggregation; metal-protein interactions; proteins; ubiquitin; zinc
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Funding
- Consiglio Nazionale delle Ricerche [RSTL. 620]
- Universita' di Catania (Progetti di Ateneo)
- MIUR [PRIN2008 R23Z7K, PRIN 2008F5A3AF_005]
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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 H-1, N-15, C-13 alpha and (CO)-C-13 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.
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