Structural basis for inhibition of mammalian adenylyl cyclase by calcium

Abstract

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) = approximately 0.4 microM) and "low-affinity" (K(i) = approximately 100 microM) 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 microM Ca(2+), inhibition appears to be exclusive with respect to PP(i). The 3.0 A resolution structure of Galphas.GTPgammaS/forskolin-activated VC1:IIC2 crystals soaked in the presence of ATPalphaS and 8 microM 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 A 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 A) or 2 mM Ca(2+) (2.7 A) 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.

Figure 1

Biphasic inhibition of VC1:IIC2 activity in the presence and absence of pyrophosphate. (A) Activity of VC1 (50 nM), IIC2 (250 nM) in the presence of 500 nM Gαs•GTPγS, and 10 μM FSK was determined in the absence (○; upper panel) or presence (lower panel) of a concentration of PPi sufficient to inhibit AC activity by 25%, 50% or 75% (47 μM; (■), 115 μM; (△), or 235 μM; (◆), respectively). Data represent the mean ± S.D. of at least 3 independent experiments, and were fit to a two-site competition model. (B) Dixon plot to show the relation between the sub-micromolar (upper panel) or surpa-micromolar concentration (lower panel) of Ca²⁺ and PPi. The data were recast from the Ca²⁺ inhibition of AC activity that were determined in the absence or presence of a concentration of PPi. ●, no added calcium; ▲, [Ca²⁺]_free=200nM; ▼, [Ca²⁺]_free=500nM; ◆, [Ca²⁺]_free=684nM; +, [Ca²⁺]_free=1.40μM; ○, [Ca²⁺]_free=30μM ; □, [Ca²⁺]_free=90μM; △, [Ca²⁺]_free=180μM ; ▽, [Ca²⁺]_free=300μM. For some data points, the width of the error bars is smaller than the symbol.

Figure 2

Ca²⁺ inhibition of ATP synthesis. Double reciprocal plots for inhibition by Ca²⁺ of ATP synthesis by VC1 (0.4 μM), IIC2 (2 μM) and 1 μM Gαs•GTPγS; Reactions were conducted in the absence (∎) of Ca²⁺ or in the presence of 2 μM (○), or 5 μM (▲) free Ca²⁺.

Figure 3

Global views of Gαs•GTPγS/FSK-activated VC1:IIC2 substrate complexes with Ca²⁺ or Mg²⁺. (A) The structure of VC1 and IIC2 domains in the ATPγS•Ca²⁺ complex shown in mauve and tan, respectively (26). Shown in superposition are the secondary structure elements β1—α1—α2 and α3—β4 of VC1 and β7′—β8′ of IIC2 in the open-state VC1:IIC2 apoenzyme (PDB code:1AZS), gray, and those of the closed-state ATPαS complex with Mn²⁺ and Mg²⁺ (PDB code:1CJK), yellow (26,27). The switch II helix of GTPγS-activated Gαs subunit is shown as a red cylinder. Ligands are drawn as stick models; for ATPαS and FSK, carbon atoms are gray, nitrogens blue, oxygens red, and phosphorus green. Metal ions are shown as metallic spheres; Ca²⁺ ion is violet, Mg²⁺ light-yellow, and Mn²⁺ orange. This coloring scheme for atoms is retained in all figures unless otherwise noted. (B) Superposition of the secondary structure elements β1—α1—α2 and α3—β4 of VC1 and β7′—β8′ of IIC2 of the ATP•2Ca complex with two Ca²⁺ ions cyan with the VC1 and IIC2 domains structure of ATPαS•Ca, using atomic coloring scheme as in panel A. The two structures are similar to each other except that the α1 helix of ATPaS•Ca complex is rotated ∼10° toward the IIC2 domain relative to the α1 helix of ATP•2Ca complex [as shown by the lines indicating α1 helix axis in two structures].

Figure 4

Binding of ATP and the inhibitor ATPαS to AC in the presence of high (1.5 mM) or low (8 μM) Ca²⁺. Gαs•GTPγS/FSK-activated VC1:IIC2 bound to (A) ATPαS and Ca²⁺, or (C) ATP and two Ca²⁺ ions. The nucleotide, Ca²⁺ ions, and water molecules are shown as stick models or spheres. The 3.0 Å or 2.9 Å |F_o| — | F_c| electron density maps, computed from the refined model from which coordinates for the Ca²⁺ and nucleotide in the ATPαs•Ca or ATP•2Ca complexes, respectively, are omitted and contoured at the 2.5σ level, is shown as lime-green wire cages. Side chains of protein residues in the catalytic site are shown as sticks. The network of non-bonded interactions among (B) ATPαs and Ca²⁺, or (D) ATP, the two Ca²⁺ ions, water molecules, and side chains of protein residues are shown. Individual calcium ion(s) and water molecules are positioned according to the |F_o| — | F_c| difference electron density maps calculated with the respective atoms omitted from the phasing model or Ca replaced with water . The difference maps for Ca²⁺ ion(s) and waters are contoured at the 3.0σ and 2.5σ level and shown as blue and cyan wire cages, respectively. The gray dashed lines depict hydrogen bonds (<3.2 Å) and metal coordination contacts (<3.0 Å) between ligand atoms, metal ions, water molecules, and protein residues. The hydrogen bonds, metal coordination contacts, single letter amino acid code, and residue ID numbers are shown for active site residues in this figure and in Figure 5 and 6.

Figure 5

ATP or ATPαS coordination with Mg²⁺ or Ca²⁺ in the catalytic site of VC1:IIC2. (A) ATPαS:Mg²⁺/Mn²⁺ (PDB ID 1CJK); (B) ATPαS•Ca; (C) ATP•2Ca. The main chain of the phosphate-binding loop (Ile-397—Thr-401 of VC1) and the side chains of substrate and metal ion interacting residues are shown, and colored according to the scheme in Figure 4, except that carbon atoms are gray, yellow, and white for ATPαS with Mg²⁺, ATPαS with Ca²⁺, and ATP, respectively.

Figure 6

VC1:IIC2 complexes with PPi•Ca²⁺ and PPi•Mg²⁺. Electron density |F_o| — | F_c| omit maps are shown as lime-green wire cages that were calculated at the respective final resolution and contoured at the 2.5σ level for (A) the PPi•Mg²⁺ and (B) the PPi•Ca²⁺ complex. Models of PPi with Mg²⁺ or Ca²⁺ at the B site are shown as stick models and spheres. A water molecule is observed adjacent to the Mg²⁺ at the B site, but no significant electron density is observed at the metal A site in either structures. Stick models of side chains of protein residues from the open conformation (1AZS) of VC1:IIC2 were superimposed onto both structures and depicted as gray-colored side chains. Interactions of PPi•Mg²⁺ and PPi•Ca²⁺ with the AC catalytic site residues are shown in panels (C) and (D), respectively. The 2.8 Å or 2.7 Å |F_o| — | F_c| electron density maps for the PPi•Mg²⁺ or PPi•Ca²⁺ complexes, respectively, are contoured at the 2.5σ level and shown as lime-green wire cages. In panel D, the blue wire cage represents electron density contoured at 3.0σ for the 2.8Å |Fo_Ca|—|Fo_Mg| omit map, indicating that Ca²⁺ binds at site B. The VC1:IIC2 product complex with pyrophosphate and Mg²⁺ is, like that with Ca²⁺, in the open conformation. Ligand and side chain atoms of protein residues are drawn as stick models with carbon atoms colored in cyan or orange for the PPi•Mg²⁺ or PPi•Ca²⁺ complexes, respectively.

Figure 7

A detailed view of 2′-d-3′-AMP•PPi•Mg²⁺ and PPi•Ca²⁺ complexes in the catalytic site of the superimposed complexes. The PPi is rotated ∼35° toward to the β1-α1 loop of VC1 relative to the pyrophosphate moiety of 2′-d-3′-AMP•PPi•Mg²⁺ complex (PDB code:1CS4). Calcium ion in the complex with PPi occupy the B site as does Mg²⁺ in the complex with 2′-d-3′-AMP•PPi (shown in blue).