Isothermal titration calorimetry reveals a zinc ion as an atomic switch in the diadenosine polyphosphates

Abstract

Diadenosine polyphosphates (diadenosine 5′,5‴-P1,Pn-polyphosphate (ApnA)) are 5′-5‴-phosphate-bridged dinucleosides that have been proposed to act as signaling molecules in a variety of biological systems. Isothermal titration calorimetry was used to measure the affinities of a variety of metal cations for ATP, diadenosine 5′,5‴,P1,P3-triphosphate (Ap3A), diadenosine 5′,5′-P1,P4-tetraphosphate (Ap4A), and diadenosine 5′,5‴-P1,P5-pentaphosphate (Ap5A). The binding of Mg2+, Ca2+, and Mn2+ to ATP is shown to take place with the β, γ-phosphates (primary site) and be endothermic in character. The binding of Ni2+, Cd2+, and Zn2+ to ATP is found to take place at both the primary site and at a secondary site identified as N-7 of the adenine ring. Binding to this second site is exothermic in character. Generally, the binding of metal cations to diadenosine polyphosphates involves a similar primary site to ATP. No exothermic binding events are identified. Critically, the binding of Zn2+ to diadenosine polyphosphates proves to be exceptional. This appears to involve a very high affinity association involving the N-7 atoms of both adenine rings in each ApnA, as well as the more usual endothermic association with the phosphate chain. The high affinity association is also endothermic in character. A combination of NMR and CD evidence is provided in support of the calorimetry data demonstrating chemical shift changes and base stacking disruptions entirely consistent with N-7 bridging interactions. N-7 bridging interactions are entirely reversible, as demonstrated by EDTA titration. Considering the effects of Zn2+ on a wide variety of dinucleoside polyphosphate-metabolizing enzymes, we examine the possibility of Zn2+ acting as an atomic switch to control the biological function of the diadenosine polyphosphates.