Structural basis for inhibition of mammalian adenylyl cyclase by calcium

Type V and VI mammalian adenylyl cyclases (AC5, AC6) are inhibited by Ca ²⁺ at both sub-and supramicromolar concentration. This inhibition may provide feedback in situations where cAMP promotes opening of Ca ²⁺ channels, allowing fine control of cardiac contraction and rhythmicity in cardiac tissue where AC5 and AC6 predominate. Ca ²⁺ inhibits the soluble AC core composed of the C1 domain of AC5 (VC1) and the C2 domain of AC2 (IIC2). As observed for holo-AC5, inhibition is biphasic, showing "high-affinity" (K _i = ∼0.4 μM) and "low-affinity" (K _i = ∼100 μM) modes of inhibition. At micromolar concentration, Ca ²⁺ inhibition is nonexclusive with respect to pyrophosphate (PP _i), a noncompetitive inhibitor with respect to ATP, but at >100 μM Ca ²⁺, inhibition appears to be exclusive with respect to PP _i. The 3.0 Å resolution structure of Gαs GTPγS/forskolin-activated VC1:IIC2 crystals soaked in the presence of ATPαS and 8 μM free Ca ²⁺ contains a single, loosely coordinated metal ion. ATP soaked into VC1:IIC2 crystals in the presence of 1.5 mM Ca ²⁺ is not cyclized, and two calcium ions are observed in the 2.9 Å resolution structure of the complex. In both of the latter complexes VC1:IIC2 adopts the "open", catalytically inactive conformation characteristic of the apoenzyme, in contrast to the "closed", active conformation seen in the presence of ATP analogues and Mg ²⁺ or Mn ²⁺. Structures of the pyrophosphate (PP _i) complex with 10 mM Mg ²⁺ (2.8 Å) or 2 mM Ca ²⁺ (2.7 Å) also adopt the open conformation, indicating that the closed to open transition occurs after cAMP release. In the latter complexes, Ca ²⁺ and Mg ²⁺ bind only to the high-affinity "B" metal site associated with substrate/product stabilization. Ca ²⁺ thus stabilizes the inactive conformation in both ATP- and PP _i-bound states.