Distinct mechanism of Tb3+ and Eu3+ binding to NCS1.

Abstract

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+ (Kd,Tb3+ = 0.002 ± 0.0001 μM and Kd, Ca2+ = 91 nM) whereas Eu3+ binding is notably weaker, Kd,Eu3+ = 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.