Mg2+ binding to coenzyme A.

Magnesium (Mg²⁺), the second most abundant intracellular cation, plays a crucial role in cellular functions. In this study, we investigate the interaction between Mg²⁺ and coenzyme A (CoA), a thiol-containing cofactor central to cellular metabolism also involved in protein modifications. Isothermal titration calorimetry revealed a 1:1 binding stoichiometry between Mg²⁺ and free CoA under biologically relevant conditions. Association constants of (537 ± 20) M^-1 and (312 ± 7) M^-1 were determined at 25 °C and pH 7.2 and 7.8, respectively, suggesting that a significant fraction of CoA is likely bound to Mg²⁺ both in the cytosol and in the mitochondrial matrix. Additionally, the process is entropically-driven, and our results support that the origin of the entropy gain is solvent-related. On the other hand, the combination of 1- and 2-dimensional nuclear magnetic resonance spectroscopy with molecular dynamics simulations and unsupervised learning demonstrate a direct coordination between Mg²⁺ and the phosphate groups of the 4-phosphopantothenate unit and bound to position 5' of the adenosine ring. Interestingly, the phosphate in position 3' only indirectly contributes to Mg²⁺ coordination. Finally, we discuss how the binding of Mg²⁺ to CoA perturbates the chemical environment of different CoA atoms, regardless of their apparent proximity to the coordination site, through the modulation of the CoA conformational landscape. This insight holds implications for understanding the impact on both CoA and Mg²⁺ functions in physiological and pathological processes.