Structural Environment and Stability of the Complexes Formed Between Calmodulin and Actinyl Ions

Abstract

Because of their presence in the nuclear fuel cycle, neptunium and uranium are two actinides of main interest in case of internal contamination. Complexation of U(VI) and Np(V) by the target protein calmodulin (CaM(WT)) was therefore studied herein. Both actinides have two axial oxygen atoms, which, charge aside, makes them very similar structurally wise. This work combines spectroscopy and theoretical density functional theory (DFT) calculations. Structural characterization was performed by extended X-ray absorption fine structure (EXAFS) at the L(III)-edge for each studied actinide. Models for the binding site of the protein were developed and then refined by using DFT to fit the obtained experimental EXAFS data. The effect of hydrolysis was also considered for both actinides (the uranyl experiment was performed at pH 3 and 6, while the neptunyl experiment was conducted at pH 7 and 9). The effect of the pH variation was apparent on the coordination sphere of the uranyl complexes, while the neptunyl complex characteristics remained stable under both studied conditions. The DFT calculations showed that at near physiological pH the complex formed by CaM(WT) with the neptunium ion is more stable than the one formed with uranyl.

Structural Analysis of Uranyl Complexation by the EF‐Hand Motif of Calmodulin: Effect of Phosphorylation

“…The conclusions from the EXAFS experiments for the phosphorylated peptides at pH 6 and 7 are in agreement with a structural model involving three short U−O eq distances and two longer U−O eq distances, which could correspond to the presence of a monodentate phosphoryl group and two monodentate and one bidentate carboxylate ligands, which is in agreement with the FTIR data and the MD simulations. For the CaM1–U complex, the EXAFS data evidenced a short U−O eq distance of approximately 2.2 Å, which was similar to that observed for the CaMWT–U complex . Distinction between the contribution at the uranyl–oxygen distance of approximately 2,2 Å of either a monodentate carboxylate with strong interaction with the uranyl or that of a hydroxide ligand is however difficult by solely using the EXAFS data.…”

“…Alternately, this short distance could indicate the presence of another type of ligand, as a hydroxide ligand . Actually, previous EXAFS analysis of the uranyl complex formed at pH 7 with the CaMWT peptide, which presents high sequence similarity with CaM1 (Table S1 in the Supporting Information) concluded to the presence of a hydroxide uranyl ligand …”

“…Structural data are missing to assess the uranyl coordination sphere and the structural origin of the strong affinity of CaM1P for uranyl as compared to non‐phosphorylated CaM1. EXAFS data of the CaMWT–U complex revealed an uranyl–ligand distance too short to correspond to a monodentate carboxylate ligand, which could be modeled by the presence of a hydroxide ligand in the uranyl equatorial plane . In the present study, we analyze the coordination sphere of uranyl in complexes formed with the non‐phosphorylated CaM1 and the phosphorylated CaM1P peptides in solution to better assess the role of the phosphoryl group and of exogenous ligands as hydroxide on the stability of the uranyl–protein interactions.…”

“…In addition, there are only few three‐dimensional structures related to characterized uranyl binding sites in proteins . To unravel structure–properties relationships in uranium–protein complexes, structural information is often obtained by using complementary spectroscopic techniques, such as X‐ray absorption spectroscopy (XAS), vibrational spectroscopy, or time‐resolved laser‐induced fluorescence spectroscopy (TRLFS), and the use of theoretical models ,,. In particular, extended X‐ray absorption fine structure (EXAFS) spectroscopy at the uranium L III edge has been successfully applied to analyze the uranium coordination in various compounds, including proteins and peptides,, and is particularly useful to analyze properties of metal sites in solution.…”

“…Important information on the molecular determinants of the affinity of proteins for uranyl has also been obtained by engineering of proteins or peptides ,–,,. In this respect, we targeted the EF‐hand calcium binding motif of calmodulin to engineer variants with an enhanced affinity for uranyl.…”