Abstract
Nucleobindin-2 (Nucb2) is a protein that has been suggested to play roles in a variety of biological processes. Nucb2 contains two Ca(2+)/Mg(2+)-binding EF-hand domains separated by an acidic amino acid residue-rich region and a leucine zipper. All of these domains are located within the C-terminal half of the protein. At the N-terminal half, Nucb2 also possesses a putative Zn(2+)-binding motif. In our recent studies, we observed that Nucb2 underwent Ca(2+)-dependent compaction and formed a mosaic-like structure consisting of intertwined disordered and ordered regions at its C-terminal half. The aim of this study was to investigate the impact of two other potential ligands: Mg(2+), which possesses chemical properties similar to those of Ca(2+), and Zn(2+), for which a putative binding motif was identified. In this study, we demonstrated that the binding of Mg(2+) led to oligomerization state changes with no significant secondary or tertiary structural alterations of Nucb2. In contrast, Zn(2+) binding had a more pronounced effect on the structure of Nucb2, leading to the local destabilization of its N-terminal half while also inducing changes within its C-terminal half. These structural rearrangements resulted in the oligomerization and/or aggregation of Nucb2 molecules. Taken together, the results of our previous and current research help to elucidate the structure of the Nucb2, which can be divided into two parts: the Zn(2+)-sensitive N-terminal half (consisting of nesfatin-1 and -2) and the Ca(2+)-sensitive C-terminal half (consisting of nesfatin-3). These results may also help to open a new discussion regarding the diverse roles that metal cations play in regulating the structure of Nucb2 and the various physiological functions of this protein.