Distinct mechanism of Tb³⁺ and Eu³⁺ binding to NCS1

Lanthanides have been frequently used as biomimetic compounds for NMR and fluorescence studies of Ca²⁺ binding proteins due to having similar physical properties and coordination geometry to Ca²⁺ ions. Here we report that a member of the neuronal calcium sensor family, neuronal calcium sensor 1, complexes with two lanthanide ions Tb³⁺ and Eu³⁺. The affinity for Tb³⁺ is nearly 50 times higher than that for Ca²⁺ (K_d,Tb = 0.002 ± 0.0001 μM and K_{d, Ca} = 91 nM) whereas Eu³⁺ binding is notably weaker, K_d,Eu = 26 ± 1 μM. Interestingly, despite having identical charge and similar ionic radii, Tb³⁺ and Eu³⁺ ions exhibit a distinct binding stoichiometry for NCS1 with one Eu³⁺ and two Tb³⁺ ions bound per NCS1 monomer, as demonstrated in fluorescence titration and mass spectrometry studies. These results suggest that the lanthanides' affinity for the individual EF hands is fine-tuned by a small variation in the ion charge density as well as EF hand binding loop amino acid sequence. As observed previously for other lanthanide:protein complexes, the emission intensity of Ln³⁺ is enhanced upon complexation with the protein, likely due to the displacement of water molecules by oxygen atoms from the coordinating amino acid residues. The overall shape of the Tb³⁺NCS1 and Eu³⁺NCS1 monomer shows high levels of similarity compared to the Ca²⁺ bound protein based on their collision cross section. However, the distinct occupation of EF hands impacts NCS1 oligomerization and affinity for the D2R peptide that mimics the NCS1 binding site on the D2R receptor. Specifically, the Tb³⁺NCS1 complex populates the dimer and has comparable affinity for the D2R peptide, whereas Eu³⁺ bound NCS1 remains in the monomeric form with a negligible affinity for the D2R peptide.