Cm3+/Eu3+ induced structural, mechanistic and functional implications for calmodulin.

Trivalent actinides and their lanthanide homologues are being scrutinized for their potential health risk when ingested as a result of a range of industrial activities such as mining. Importantly, these ions are known to exhibit high affinity towards calmodulin (CaM). In case of their inadvertent uptake, the holoproteins that are occupied by these cations may block signal transduction pathways or increase the concentration of these ions in intact cells, which could lead to accumulation in human organs. Accordingly, this investigation employed spectroscopy, computational chemistry, calorimetry, and biochemistry to study the results of metal ion substitution on the protein structure, enzymatic activity and chemo- and cytotoxicity of An3+/Ln3+ ions. As will be demonstrated herein, our data confirm the higher affinity of Cm3+ and Eu3+ compared to Ca2+ to all 4 binding sites of CaM, with one site differing from the remaining three. This higher-affinity site will complex Eu3+ in an exothermic fashion; in contrast, ion binding to the three lower-affinity EF-hands was found to be endothermic. The overall endothermic binding process is ascribed to the loss of the hydration shells of the trivalent ions upon protein binding. These findings are supported by extensive quantum chemical calculations of full holo-CaM, which were performed at the MP2 level using the fragment molecular orbital method. The exceptional binding site (EF-hand 3) features fewer negatively charged residues compared to the other EF-hands, thereby allowing Eu3+ and Cm3+ to carry one or two additional waters compared to Ca2+-CaM, while also causing the structure of Cm3+/Eu3+-CaM to become slightly disordered. Moreover, the enzymatic activity decreases somewhat in comparison to Ca2+-CaM. By utilizing a combination of techniques, we were able to generate a comprehensive picture of the CaM-actinide/lanthanide system from the molecular level to its functional impact. Such knowledge could also be applied to other metal-binding proteins.

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

Better understanding of uranyl-protein interactions is a prerequisite to predict uranium chemical toxicity in cells. The EF-hand motif of the calmodulin site I is about thousand times more affine for uranyl than for calcium, and threonine phosphorylation increases the uranyl affinity by two orders of magnitude at pH 7. In this study, we confront X-ray absorption spectroscopy with Fourier transform infrared (FTIR) spectroscopy, time-resolved laser-induced fluorescence spectroscopy (TRLFS), and structural models obtained by molecular dynamics simulations to analyze the uranyl coordination in the native and phosphorylated calmodulin site I. For the native site I, extended X-ray absorption fine structure (EXAFS) data evidence a short U-Oeq distance, in addition to distances compatible with mono- and bidentate coordination by carboxylate groups. Further analysis of uranyl speciation by TRLFS and thorough investigation of the fluorescence decay kinetics strongly support the presence of a hydroxide uranyl ligand. For a phosphorylated site I, the EXAFS and FTIR data support a monodentate uranyl coordination by the phosphoryl group and strong interaction with mono- and bidentate carboxylate ligands. This study confirms the important role of a phosphoryl ligand in the stability of uranyl-protein interactions. By evidencing a hydroxide uranyl ligand in calmodulin site I, this study also highlights the possible role of less studied ligands as water or hydroxide ions in the stability of protein-uranyl complexes.

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

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.

Fetuin exhibits a strong affinity for plutonium and may facilitate its accumulation in the skeleton.

After entering the blood, plutonium accumulates mainly in the liver and the bones. The mechanisms leading to its accumulation in bone are, however, completely unknown. We already know that another uptake pathway not involving the transferrin-mediated pathways is suspected to intervene in the case of the liver. Fetuin, a protein playing an important role in bone metabolism, is proposed as a potential transporter of Pu from serum to bone. For the first time, the binding constants of these two proteins (transferrin and fetuin) with tetravalent plutonium at physiological pH (pH 7.0) were determined by using capillary electrophoresis (CE) coupled with inductively coupled plasma mass spectrometry (ICP-MS). Their very close values (log10 KPuTf = 26.44 ± 0.28 and log10 KPuFet = 26.20 ± 0.24, respectively) suggest that transferrin and fetuin could compete to chelate plutonium, either in the blood or directly at bone surfaces in the case of Pu deposits. We performed competition reaction studies demonstrating that the relative distribution of Pu-protein complexes is fully explained by thermodynamics. Furthermore, considering the average concentrations of transferrin and fetuin in the blood, our calculation is consistent with the bio-distribution of Pu observed in humans.

Topological speciation of actinide-transferrin complexes by capillary isoelectric focusing coupled with inductively coupled plasma mass spectrometry: evidence of the non-closure of the lobes.

The determination of the binding constant between transferrin and thorium and the conformational changes of the protein upon metal complexation (thorium and plutonium) have been studied by both capillary electrophoresis (CE) and capillary isoelectric focusing (cIEF) coupled with inductively coupled plasma mass spectrometry (ICP-MS). This method allows the use of both the separation power of the cIEF and the low detection limit of ICP-MS which is critical when working with highly radioactive elements. To our knowledge, this is the first time a method coupling cIEF and ICP-MS is reported in the literature. Nitrilotriacetate was used to prevent from actinide hydrolysis and as a competitive ligand with transferrin. The binding constant for the complexation of transferrin and thorium, in the absence of bicarbonate at pH 7, was found to be log K = 18.65 ± 0.19. This value, close to that of transferrin with iron, evidenced the high affinity of the protein for thorium. The results obtained by the newly developed method, cIEF-ICPMS, showed no pI change after the addition of thorium or plutonium, whereas a pI shift (linked to conformational changes) occurred for the transferrin-indium complex. This suggests that, despite the high affinity towards the actinides, the protein does not undergo a significant structure change upon complexation. The important ionic radius of the cations Th4+ (1.05 Å, CN = 8) and Pu4+ (0.96 Å, CN = 8) with respect to Fe3+ (0.645 Å, CN = 6) and to a lesser extent to In3+ (0.800 Å, CN = 6) suggests that the transferrin lobe does not close completely after complexation. However, mixed indium-actinide complexes showed structural changes even at high concentrations of apotransferrin. The conformational change is not governed by the actinide but by the other metals present.

Enzymatic activity of the CaM-PDE1 system upon addition of actinyl ions.

The threat of a dirty bomb which could cause internal contamination has been of major concern for the past decades. Because of their high chemical toxicity and their presence in the nuclear fuel cycle, uranium and neptunium are two actinides of high interest. Calmodulin (CaM) which is a ubiquitous protein present in all eukaryotic cells and is involved in calcium-dependent signaling pathways has a known affinity for uranyl and neptunyl ions. The impact of the complexation of these actinides on the physiological response of the protein remains, however, largely unknown. An isothermal titration calorimetry (ITC) was developed to monitor in vitro the enzymatic activity of the phosphodiesterase enzyme which is known to be activated by CaM and calcium. This approach showed that addition of actinyl ions (AnO2n+), uranyl (UO22+) and neptunyl (NpO2+), resulted in a decrease of the enzymatic activity, due to the formation of CaM-actinide complexes, which inhibit the enzyme and alter its interaction with the substrate by direct interaction. Results from dynamic light scattering rationalized this result by showing that the CaM-actinyl complexes adopted a specific conformation different from that of the CaM-Ca2+ complex. The effect of actinides could be reversed using a hydroxypyridonate actinide decorporation agent (5-LIO(Me-3,2-HOPO)) in the experimental medium demonstrating its capacity to efficiently bind the actinides and restore the calcium-dependent enzyme activation.