Nanomolar affinity of EF-hands in neuronal calcium sensor 1 for bivalent cations Pb2+, Mn2+, and Hg2

Abstract

Abiogenic metals Pb and Hg are highly toxic since chronic and/or acute exposure often leads to severe neuropathologies. Mn2+ is an essential metal ion but in excess can impair neuronal function. In this study, we address in vitro the interactions between neuronal calcium sensor 1 (NCS1) and divalent cations. Results showed that non-physiological ions (Pb2+ and Mn2+) bind to EF-hands in NCS1 with nanomolar affinity and lower equilibrium dissociation constant than the physiological Ca2+ ion. (Kd, Pb2+ = 7.0 ± 1.0 nM; Kd, Mn2+ = 34.0 ± 6.0 nM; K). Native ultra-high resolution mass spectrometry (FT-ICR MS) and trapped ion mobility spectrometry-mass spectrometry (nESI-TIMS-MS) studies provided the NCS1-metal complex compositions-up to four Ca2+ or Mn2+ ions and three Pb2+ ions (M⋅Pb1-3Ca1-3, M⋅Mn1-4Ca1-2, and M⋅Ca1-4) were observed in complex-and similarity across the mobility profiles suggests that the overall native structure is preserved regardless of the number and type of cations. However, the non-physiological metal ions (Pb2+, Mn2+, and Hg2+) binding to NCS1 leads to more efficient quenching of Trp emission and a decrease in W30 and W103 solvent exposure compared to the apo and Ca2+ bound form, although the secondary structural rearrangement and exposure of hydrophobic sites are analogous to those for Ca2+ bound protein. Only Pb2+ and Hg2+ binding to EF-hands leads to the NCS1 dimerization whereas Mn2+ bound NCS1 remains in the monomeric form, suggesting that other factors in addition to metal ion coordination, are required for protein dimerization.

Keywords: EF-hands; calcium; fluorescence; metal binding; neuronal calcium sensor 1; non-physiological metals.

Graphical Abstract

Association of Pb²⁺ and Hg²⁺ to EF hands stabilizes a NCS1 dimer whereas Mn²⁺ bound NCS1 favors a monomeric form.

Fig. 1

Left: Cartoon presentation of the NCS1 structure (PDB entry 2LCP). Non-functional EF-hand 1 is colored in green, Mg²⁺/Ca²⁺ binding EF-hand 2 is shown in red, and Ca²⁺ binding EF-hand 3 and 4 are shown in yellow and cyan, respectively. Ca²⁺ ions are shown as green spheres and the sidechains of W30 and W103 are colored in green and yellow, respectively. Right top: Sequence alignment for NCS1 EF-hand 2, 3, and 4. Positions of amino acid residues that coordinate Ca²⁺ ions are labeled using arrows. Right bottom: Structure of Ca²⁺ bound EF-hand 4 in NCS1 with the Ca²⁺ coordinating residues shown as sticks (PDB entry 2LCP).

Fig. 2

Intrinsic fluorescence emission of 10 μM NCS1 in the apo form (1 mM EDTA) and metal bound form (1 mM Mg²⁺and 1 mM EGTA, 1 mM Ca²⁺ and/or 120 μM Pb²⁺, 1 mM Mn²⁺, 1 mM Hg²⁺). Conditions: 10 μM NCS1 solubilized in 20 mM Tris buffer, pH 7.40, λ_exc = 295 nm and temperature of 20°C.

Fig. 3

Titration curves for Pb²⁺ left, Mn²⁺ (middle), Hg²⁺ (right) binding to apoNCS1 (in red), and Ca²⁺NCS1 (in black). Conditions: 10 μM apoNCS1 or 10 μM NCS1 and 1 mM Ca²⁺ in 20 mM Tris buffer, pH 7.40. The solid line represents the fit of the experimental data according to Equation 2 for Pb²⁺ and Mn²⁺ titration curve and Equation 3 for Hg²⁺ association to NCS1.

Fig. 4

Far-UV CD spectra of 10 μM NCS1 protein in apo and metal bound form. Experimental conditions as in the Fig. 2.

Fig. 5

Stern–Volmer plot for acrylamide quenching of Trp emission in apo and metal bound NCS1 protein. Conditions: 10 μM NCS1, 1 mM EDTA, 1 mM Mg²⁺, 1 mM Ca²⁺ and/or 120 μM Pb²⁺, 1 mM Mn²⁺, or 1 mM Hg² in 20 mM Tris buffer, pH 7.40. Solid lines correspond to the linear fit of the experimental data, and the error bar represents the standard deviations from three independent measurements.

Fig. 6

Time resolved anisotropy data for apo and metal bound NCS1. Solid and open symbols represent Δ phase shift and amplitude ratio, respectively, measured as a function of modulation frequency. Solid line represents the non-linear fit of the experimental data using a two exponential decay model. Conditions: 10 μM NCS1, 20 mM Tris buffer, pH 7.40, 1 mM EDTA, 1 mM Ca²⁺, 120 μM Pb²⁺, 1 mM Mn²⁺ or 1 mM Hg²⁺. Temperature 20 °C.

Fig. 7

Emission spectra of NCS1:1,8-ANS complexes in absence and presence of metal ions. Samples were excited at 350 nm. The experimental conditions as in Fig. 2.

Fig. 8

Expanded view of the 8 + charge state with annotated adduct species and the isotopic pattern of the ion [M•2Pb•2Ca]⁸⁺ shown in the inset (A). Typical broadband nESI-FT-ICR MS spectrum of NCS1 in the presence of Pb²⁺ (B). Deconvoluted spectrum showing the neutral species. The isotopic pattern shown in the inset confirms the theoretical isotopic average mass of the NCS1 protein 22.976 kDa (C).