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

Noa Stern, Dan Thomas Major, Hugo Emilio Gottlieb, Daniel Weizman, Alon Haim Sayer, Eliav Blum, Bilha Fischer

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4 Scopus citations


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.]

Original languageEnglish
Pages (from-to)861-879
Number of pages19
JournalJournal of Biological Inorganic Chemistry
Issue number6
StatePublished - Aug 2012

Bibliographical note

Funding Information:
31P NMR shifts can be used to determine whether a phosphate-containing ligand is coordinated to a metal ion and with what stoichiometry [55]. Here, we determined, by NMR-monitored Mg2?/Ca2? titrations of dinucleotides, that NpnN′s form complexes with Mg2?/Ca2? with a 1:1 stoichiometry for all the complexes, except for Ap5A and Gp5G, which may also bind two ions. Indeed, during the MD simulation, Ap4A preferred coordination to only one Mg2? ion (see the electronic supplementary material). A 2:1 stoichiometry for Ap5A complexes is supported by previous ITC data for ApnAs (n = 3–5) [18].


  • Conformational analysis
  • Dinucleoside polyphosphate
  • Molecular dynamics simulation
  • Potential of mean force
  • Potentiometric titration


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