Characterization of Cu²⁺ and Zn²⁺ binding sites in SUMO1 and its impact on protein stability

Metal ions like Cu²⁺ and Zn²⁺ have been shown to impact protein misfolding pathways in neurodegenerative proteinopathies like Alzheimer's and Parkinson's. Also, due to their strong interaction with Ubiquitin, they interfere in degradation of misfolded proteins by impairing the ubiquitin-proteasome system (UPS). In this work, we have studied the interaction of these metal ions with a small Ubiquitin like post-translation modifier SUMO1, which is known to work co-operatively with Ubiquitin to regulate UPS system. Between Cu²⁺ and Zn²⁺, the former binds more strongly with SUMO1 as determined using fluorescence spectroscopy. SUMO1 aggregates, forming trimer and higher oligomers in presence of Cu²⁺ ions which were characterized using gel electrophoresis, Bradford assay, and transmission electron microscopy. Chemical shift analysis using ¹⁵N/¹H based NMR spectroscopy revealed that SUMO1 retains its structural fold in its trimeric state. Cu²⁺ induced paramagnetic quenching and Zn²⁺ induced chemical shift perturbation of ¹⁵N-¹H cross-peaks were used to identify their respective binding sites in SUMO1. Binding sites so obtained were further validated with molecular dynamics studies. Our findings provide structural insights into the SUMO1-Cu²⁺/Zn²⁺ interaction, and its impact on aggregation of SUMO1 which might affect its ability to modify functions of target proteins.