Studies of Mg ²⁺/Ca ²⁺ complexes of naturally occurring dinucleotides: Potentiometric titrations, NMR, and molecular dynamics

Dinucleotides (Np _n N′; N and N′ are A, U, G, or C, n = 2-7) are naturally occurring physiologically active compounds. Despite the interest in dinucleotides, the composition of their complexes with metal ions as well as their conformations and species distribution in living systems are understudied. Therefore, we investigated a series of Mg ²⁺ and Ca ²⁺ complexes of Np _n N′s. Potentiometric titrations indicated that a longer dinucleotide polyphosphate (N is A or G, n = 3-5) linker yields more stable complexes (e.g., log K of 2.70, 3.27, and 3.73 for Ap _n A-Mg ²⁺, n = 3, 4, 5, respectively). The base (A or G) or ion (Mg ²⁺ or Ca ²⁺) has a minor effect on log K ^M _ML values. In a physiological medium, the longer Ap _n As (n = 4, 5) are predicted to occur mostly as the Mg ²⁺/Ca ²⁺ complexes. ³¹P NMR monitored titrations of Np _n N′s with Mg ²⁺/Ca ²⁺ ions showed that the middle phosphates of the dinucleotides coordinate with Mg ²⁺/Ca ²⁺. Multidimensional potential of mean force (PMF) molecular dynamics (MD) simulations suggest that Ap ₂A and Ap ₄A coordinate Mg ²⁺ and Ca ²⁺ ions in both inner-sphere and outer-sphere modes. The PMF MD simulations additionally provide a detailed picture of the possible coordination sites, as well as the cation binding process. Moreover, both NMR and MD simulations showed that the conformation of the nucleoside moieties in Np _n N′-Mg ²⁺/Ca ²⁺ complexes remains the same as that of free mononucleotides. Graphical abstract: Dinucleotides form complexes with Mg ²⁺/Ca ²⁺ ions which coordinate to the middle phosphate groups in the polyphosphate chain. Mg ²⁺/Ca ²⁺ coordination slightly increases intramolecular π-stacking interactions in the complexes, whereas the conformation of nucleoside moieties remains almost the same as in the corresponding Na ⁺ salts. Considering physiological Mg ²⁺/Ca ²⁺ concentrations, the Ap ₄A-Mg ²⁺/Ap ₅A-Mg ²⁺ and Ap ₄A-Ca ²⁺/Ap ₅A-Ca ²⁺ species are expected to be dominant (approximately 70-90 %) in living systems versus the free dinucleotide.[Figure not available: see fulltext.]