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 Frontiers in Molecular Neuroscience | www.frontiersin.org 1