Proton and phosphorus-31 NMR study of the dependence of diadenosine tetraphosphate conformation on metal ions

Adenosine 5'-tetraphospho-5'-adenosine (Ap ₄A) plays a role in cellular metabolism in a wide variety of organisms. Because the divalent cations Mg ²⁺ and Zn ²⁺ are involved in the synthesis and function of Ap ₄A, the effect of divalent cations on the dinucleotide's conformation is of interest. ¹H and ³¹P chemical shift experiments were carried out as a function of Mg ²⁺ concentration and pH. We propose that Mg ²⁺ stabilizes the unusual ring-stacked conformation of Ap ₄A at pH > 2 by interacting with the β-phosphates. To further probe conformational effects, stable complexes of Ap ₄A with Co ³⁺ were studied using ¹H and ³¹P NMR. Co ³⁺ forms two different bidentate complexes with Ap ₄A, independent of whether the other four octahedral coordination sites are occupied by ammonia or trimethylenediamine. NMR results suggest that in one complex the Co ³⁺ is coordinated to two β-phosphates and ring stacking is stabilized. In the other complex, Co ³⁺ is coordinated to an α-phosphate and its neighboring β-phosphate and ring stacking is destabilized. These results further support the hypothesis that Mg ²⁺ stabilizes the ring-stacked conformation by interacting symmetrically with the two β-phosphate groups.