Abstract
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 Ca2+ signals to a wide range of protein targets, including G protein-coupled receptors and their kinases. Here, we demonstrate that NCS-1 also has Zn2+-binding sites, which affect its structural and functional properties upon filling. Fluorescence and circular dichroism experiments reveal the impact of Zn2+ 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 × 106 M-1) and one low-affinity (2 × 105 M-1) Zn2+-binding sites, whereas Mg2+-loaded and Ca2+-loaded forms (which dominate under physiological conditions) bind two zinc ions with submicromolar affinity. Metal competition analysis and circular dichroism studies suggest that Zn2+-binding sites of apo- and Mg2+-loaded NCS-1 overlap with functional EF-hands of the protein. Consistently, high Zn2+ concentrations displace Mg2+ from the EF-hands and decrease the stoichiometry of Ca2+ binding. Meanwhile, one of the EF-hands of Zn2+-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, Zn2+ binding to Ca2+-loaded NCS-1 could occur at EF-hands 2 and 4. The high-affinity zinc binding increases the thermal stability of Ca2+-free NCS-1 and favours the interaction of its Ca2+-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.
Highlights
Divalent metal ions play a vital role in the vast majority of cellular processes
To test the hypothesis of the zinc interaction with neuronal calcium sensor (NCS)-1, we directly assessed the amount of zinc ions that can be bound per protein molecule using a micro-equilibrium dialysis system
Since atomic absorption spectroscopy (AAS) analysis of the Neuronal calcium sensor-1 (NCS-1) sample revealed a calciumto-protein molar ratio of 0.42, we suggest that at least one of zinc-binding sites of NCS-1 overlapped with its active EF-hands
Summary
Two alkaline earth metals, magnesium and calcium, as well as the transition metal zinc, are important, since they are the most abundant ones in the human body. These ions are significantly different in intracellular levels and play different physiological roles. At high concentrations, zinc can compete with other metals or bind to nonspecific sites in proteins, which may interfere with their structural integrity and normal function, contributing to pathology (Choi and Koh, 1998;Barwinska-Sendra and Waldron, 2017)
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