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.