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.

Conformational Selection of Ubiquitin Quaternary Structures Driven by Zinc Ions

“…In our opinion, the chemical‐shift perturbations identified by Arena et al 16. could be due, at least in part, to a zinc‐induced association of hUb molecules in dimers and trimers.…”

“… Mapping, on chain A of orthorhombic 25 m M Zn–hUb crystal structure, of the three regions (residues 1–4, 12, 15–18, and 61–62: purple; 23–24, 27–28, 49, 51–52, and 54–55: light blue; 8, 42–46, 67–68, and 70–73: blue) undergoing chemical‐shift perturbation after equimolar addition of zinc ions to the hUb solution, identified by Arena et al 16…”

“…The second, lower affinity, binding site was centered on His68. Changes in the diffusion coefficient of hUb (0.5 m M ) after Zn 2+ addition (0.25 m M ), measured by DOSY (diffusion ordered spectroscopy) NMR spectroscopic experiments, corresponded to an increase of the protein hydrodynamic radius consequent to formation of zinc‐promoted protein assemblies 16…”

“…Changes in the diffusion coefficient of hUb (0.5 mm) after Zn 2 + addition (0.25 mm), measured by DOSY (diffusion ordered spectroscopy) NMR spectroscopic experiments, corresponded to an increase of the protein hydrodynamic radius consequent to formation of zinc-promoted protein assemblies. [16] The aim of the present study was to investigate how metal-ion coordination may influence the molecular assembly of hUb in solution as well as in the solid state. It is found that Zn 2 + ions bridging hUb molecules can select a subset of solution conformers that translate into different 3D architectures in the solid state, just following the rules used for designing fusion proteins that yield nanohedral materials by self-assembling oligomerization domains.…”

The prokaryotic zinc‐finger: structure, function and comparison with the eukaryotic counterpart

“…These findings have outlined how the metal cofactors can drastically alter the folding pathway of a protein even when the native structure itself does not a priori require such a cofactor, and one could easily imagine that the opposite holds true. It is noted that many proteins involved in neurodegenerative disorders have metal‐binding capabilities , and that if this results in the type of behaviour observed in the case of Ros87 and its homologue, this may facilitate the intermolecular assembly of these proteins into pathological aggregates. The very versatile ZF domain, originally described in eukaryotes, present in its prokaryotic counterpart, appears to be extremely adaptable.…”