Functional Status of Neuronal Calcium Sensor-1 Is Modulated by Zinc Binding

Neuronal calcium sensor-1 (NCS-1) protein is abundantly expressed in the central nervous system and retinal neurons, where it regulates many vital processes such as synaptic transmission. It coordinates three calcium ions by EF-hands 2-4, thereby transducing Ca(2+) signals to a wide range of protein targets, including G protein-coupled receptors and their kinases. Here, we demonstrate that NCS-1 also has Zn(2+)-binding sites, which affect its structural and functional properties upon filling. Fluorescence and circular dichroism experiments reveal the impact of Zn(2+) binding on NCS-1 secondary and tertiary structure. According to atomic absorption spectroscopy and isothermal titration calorimetry studies, apo-NCS-1 has two high-affinity (4 × 10(6) M(-1)) and one low-affinity (2 × 10(5) M(-1)) Zn(2+)-binding sites, whereas Mg(2+)-loaded and Ca(2+)-loaded forms (which dominate under physiological conditions) bind two zinc ions with submicromolar affinity. Metal competition analysis and circular dichroism studies suggest that Zn(2+)-binding sites of apo- and Mg(2+)-loaded NCS-1 overlap with functional EF-hands of the protein. Consistently, high Zn(2+) concentrations displace Mg(2+) from the EF-hands and decrease the stoichiometry of Ca(2+) binding. Meanwhile, one of the EF-hands of Zn(2+)-saturated NCS-1 exhibits a 14-fold higher calcium affinity, which increases the overall calcium sensitivity of the protein. Based on QM/MM molecular dynamics simulations, Zn(2+) binding to Ca(2+)-loaded NCS-1 could occur at EF-hands 2 and 4. The high-affinity zinc binding increases the thermal stability of Ca(2+)-free NCS-1 and favours the interaction of its Ca(2+)-loaded form with target proteins, such as dopamine receptor D2R and GRK1. In contrast, low-affinity zinc binding promotes NCS-1 aggregation accompanied by the formation of twisted rope-like structures. Altogether, our findings suggest a complex interplay between magnesium, calcium and zinc binding to NCS-1, leading to the appearance of multiple conformations of the protein, in turn modulating its functional status.