Structural Basis for Inhibition of Mammalian Adenylyl Cyclase by Calcium

Type V and VI mammalian adenylyl cyclases (AC5, AC6) are inhibited by Ca2+ at both suband supramicromolar concentration. This inhibition may provide feedback in situations where cAMP promotes opening of Ca2+ channels, allowing fine control of cardiac contraction and rhythmicity in cardiac tissue where AC5 and AC6 predominate. Ca2+ 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 = similar to 0.4 mu M) and low-affinity (K-i = similar to 100 mu M) modes of inhibition. At micromolar concentration, Ca2+ inhibition is nonexclusive with respect to pyrophosphate (PPi), a noncompetitive inhibitor with respect to ATP, but at > 100 mu M Ca2+, inhibition appears to be exclusive with respect to PPi. The 3.0 angstrom resolution structure of G alpha s-GTP gamma S/forskolin-activated VC1:IIC2 crystals soaked in the presence of ATP alpha S and 8 mu M free Ca2+ contains a single, loosely coordinated metal ion. ATP soaked into VC1:IIC2 crystals in the presence of 1.5 mM Ca2+ is not cyclized, and two calcium ions are observed in the 2.9 angstrom 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 Mg2+ or Mn2+. Structures of the pyrophosphate (PPi) complex with 10 mM Mg2+ (2.8 angstrom) or 2 mM Ca2+ (2.7 angstrom) also adopt the open conformation, indicating that the closed to open transition occurs after cAMP release. In the latter complexes, Ca2+ and Mg2+ bind only to the high-affinity B metal site associated with substrate/product stabilization. Ca2+ thus stabilizes the inactive conformation in both ATP- and PPi-bound states.