title: Zinc(II) complexes of ubiquitin: speciation, affinity and binding features.
abstract: 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)Cα 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.
authors.affiliation: Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Viale A. Doria 6, 95125 Catania, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Arena
authors.forename: Roberto
authors.initials: R
authors.lastname: Fattorusso
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Grasso
authors.forename: Giuseppa Ida
authors.initials: GI
authors.lastname: Grasso
authors.forename: Carla
authors.initials: C
authors.lastname: Isernia
authors.forename: Gaetano
authors.initials: G
authors.lastname: Malgieri
authors.forename: Danilo
authors.initials: D
authors.lastname: Milardi
authors.forename: Enrico
authors.initials: E
authors.lastname: Rizzarelli
mesh_terms: D001665:Binding Sites; D003300:Copper / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism; D008670:Metals / Q000737:chemistry*; D011485:Protein Binding; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D015032:Zinc / Q000737:chemistry*
chemical_list: D008667:Metalloproteins; D008670:Metals; D025801:Ubiquitin; D003300:Copper; D015032:Zinc