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. 2018 Dec 14:11:459.
doi: 10.3389/fnmol.2018.00459. eCollection 2018.

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

Philipp O Tsvetkov  1 , Andrei Yu Roman  2 , Viktoriia E Baksheeva  3 , Aliya A Nazipova  4 , Marina P Shevelyova  4 , Vasiliy I Vladimirov  5 , Michelle F Buyanova  6 , Dmitry V Zinchenko  5 , Andrey A Zamyatnin Jr  3   7 , François Devred  1 , Andrey V Golovin  3   6   7   8 , Sergei E Permyakov  4 , Evgeni Yu Zernii  3   7
Affiliations

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

Philipp O Tsvetkov et al. Front Mol Neurosci. .

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.

Keywords: EF-hand motif; GRK1; calcium; dopamine receptor D2R; magnesium; neuronal calcium sensor-1; protein aggregation; zinc.

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Figures

FIGURE 1
FIGURE 1
Zn2+ binding to NCS-1 according to equilibrium dialysis experiments. The NCS-1 (5 μM) sample was pre-equilibrated with (2–150) μM Zn2+ by equilibrium dialysis (3.5 kDa MWCO) at 25°C. The concentrations of NCS-1-bound zinc ([Zn2+]bound) and free zinc ([Zn2+]free) in the resulting solutions were measured by electrothermal atomization AAS, using the absorption bands at 213.9 nm or 307.6 nm. The solid curve approximates the experimental data by Hill equation.
FIGURE 2
FIGURE 2
Thermodynamics of calcium, zinc and magnesium binding to NCS-1. Typical ITC curves (upper panels) and binding isotherms (lower panels) representing titration of NCS-1 (25 μM) by different cations. (A) Binding of Ca2+. (B) Binding of Zn2+. (C) Binding of Mg2+. (D) Binding of Ca2+ in the presence of 250 μM Zn2+. (E) Binding of Zn2+ in the presence of 1 mM Ca2+. (F) Binding of Mg2+ in the presence of 250 μM Zn2+. (G) Binding of Ca2+ in the presence of 5 mM Mg2+. (H) Binding of Zn2+ in the presence of 5 mM Mg2+. (I) Binding of Mg2+ in the presence of 1 mM Ca2+. Best fits are shown as solid curves (see Table 1).
FIGURE 3
FIGURE 3
Conformational properties of NCS-1 under saturating concentrations of calcium, magnesium and zinc. (A) Representative tryptophan fluorescence spectra of 14 μM NCS-1 under metal-free conditions (1 mM EDTA) or in the presence of Ca2+ (100 μM CaCl2), Mg2+ (1 mM MgCl2), Zn2+ (100 μM ZnCl2) or their combinations. (B) The histogram demonstrating maximum position λmax (left bar) and maximal intensity Imax (right bar) of fluorescence spectrum for the data shown in panel (A). (C) Representative CD spectra of 8 μM NCS-1 under metal-free conditions or in the presence of Ca2+ (100 μM CaCl2), Mg2+ (1 mM MgCl2), Zn2+ (100 μM ZnCl2) or their combinations. (D) The histogram representing secondary structure contents (in %) for each NCS-1 spectrum shown in panel (C).
FIGURE 4
FIGURE 4
Calcium, magnesium and zinc dependences of conformational properties of NCS-1. Tryptophan fluorescence intensity at 350/330 nm (I350/I330) for apo (A), Mg2+-saturated (B), Ca2+-saturated (C), or Zn2+-saturated (D) NCS-1 (25 μM) in the presence of increasing concentrations of the other cations. Standard deviation of I350/I330 values did not exceed 0.02.
FIGURE 5
FIGURE 5
Thermal stability of NCS-1 (14 μM) under saturating levels of calcium, magnesium and zinc, monitored by tryptophan fluorescence. (A) Denaturation profiles of Ca2+-free NCS-1 in the presence of 1 mM EDTA, 1 mM Mg2+, 100 μM Zn2+, or 1 mM Mg2+ and 100 μM Zn2+. (B) Denaturation profiles of Ca2+-bound NCS-1 (100 μM Ca2+) in the absence, and in the presence of 100 μM Zn2+. Standard deviation of λmax values did not exceed 0.3 nm.
FIGURE 6
FIGURE 6
Calcium, magnesium and zinc dependences of thermal stability of NCS-1. Mid-transition temperatures of NCS-1 (25 μM) denaturation determined form tryptophan fluorescence at 350/330 nm (I350/I330) for apo (A), Mg2+-saturated (B), Ca2+-saturated (E), or Zn2+-saturated (F) protein in the presence of increasing concentrations of the alternative cations. Mid-transition temperatures of NCS-1 (25 μM) aggregation determined form light scattering at 350 nm for apo (C), Mg2+-saturated (D), Ca2+-saturated (G), or Zn2+-saturated (H) protein in the presence of increasing concentrations of the other cations.
FIGURE 7
FIGURE 7
Prediction of zinc-binding sites in Ca2+-saturated NCS-1 using molecular modeling. (A) Density of putative Zn2+ positions in NCS-1 from geometry search visulated as volumetric data, from low (yellow) to high (black) values. (B–D) Positions of chelators for zinc and calcium in Ca2+-binding loops of EF2 (B), EF3 (C) and EF4 (D) according to QM/MM molecular dynamics simulations.
FIGURE 8
FIGURE 8
Target-binding properties of NCS-1 in the presence of zinc. (A,B) Typical ITC curves (upper panels) and binding isotherms (lower panels) representing titration of 50 μM D2R peptide with 5–150 μM NCS-1 in the presence of 5 mM Ca2+ (A) or 5 mM Ca2+ and 100 μM Zn2+ (B). (C) Binding of 25 μM NCS-1 to GST-N-GRK1 at 1 mM Ca2+ in the presence of 0, 25, 50, or 75 μM Zn2+ (i.e., at [Zn2+]/[NCS-1] ratio of 0-3), monitored by pull-down assay. (D) Representative fluorescence spectra of bis-ANS (1.2 μM) and NCS-1 (5 μM) complexes formed in the presence of either 1 mM Mg2+ or 100 μM Ca2+ with or without addition of 100 μM Zn2+.
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