Stretching the chains: the destabilizing impact of Cu2+ and Zn2+ ions on K48-linked diubiquitin

Ubiquitin signalling and metal homeostasis play key roles in controlling several physiological cellular activities, including protein trafficking and degradation. While some relationships between these two biochemical pathways have started to surface, our knowledge of their interplay remains limited. Here, we employ a variety of techniques, such as circular dichroism, differential scanning calorimetry, pressure perturbation calorimetry, fluorescence emission, SDS-PAGE, and small-angle X-ray scattering (SAXS) to evaluate the impact of Cu2+ and Zn2+ ions on the structure and stability of K48 linked diubiquitin (K48-Ub(2)), a simple model for polyubiquitin chains. The SAXS analysis results show that the structure of the metal-free protein is similar to that observed when the protein is bound to the E2 conjugating enzyme, lending support to the idea that the structure of unanchored K48-linked ubiquitin chains is sufficient for identification by conjugating enzymes without the need for an induced fit mechanism. Our results indicate that K48-Ub(2) can coordinate up to four metal ions with both copper and zinc ions inducing slight changes to the secondary structure of the protein. However, we noted significant distinctions in their impacts on protein stability and overall architecture. Specifically, Cu2+ ions resulted in a destabilization of the protein structure, which facilitated the formation of dimer aggregates. Next, we observed a shift in the conformational dynamics of K48-Ub(2) toward less compact and more flexible states upon metal ion binding, with Zn2+ inducing a more significant effect than Cu2+ ions. Our structural modelling study demonstrates that both metal ions induced perturbations in the K48-Ub(2) structure, leading to the separation of the two monomers thus inhibiting interactions with E2 enzymes. In conclusion, the findings from this study enhance our comprehension of the mechanisms underlying Ub chains recognition. Moreover, they strengthen the notion that drug discovery initiatives aimed at targeting metal-mediated disruptions in Ub signaling hold great potential for treating a wide range of diseases that stem from abnormal protein accumulation.