Low- and room-temperature X-ray structures of protein kinase A ternary complexes shed new light on its activity

Abstract

Post-translational protein phosphorylation by protein kinase A (PKA) is a ubiquitous signalling mechanism which regulates many cellular processes. A low-temperature X-ray structure of the ternary complex of the PKA catalytic subunit (PKAc) with ATP and a 20-residue peptidic inhibitor (IP20) at the physiological Mg(2+) concentration of ∼0.5 mM (LT PKA-MgATP-IP20) revealed a single metal ion in the active site. The lack of a second metal in LT PKA-MgATP-IP20 renders the β- and γ-phosphoryl groups of ATP very flexible, with high thermal B factors. Thus, the second metal is crucial for tight positioning of the terminal phosphoryl group for transfer to a substrate, as demonstrated by comparison of the former structure with that of the LT PKA-Mg(2)ATP-IP20 complex obtained at high Mg(2+) concentration. In addition to its kinase activity, PKAc is also able to slowly catalyze the hydrolysis of ATP using a water molecule as a substrate. It was found that ATP can be readily and completely hydrolyzed to ADP and a free phosphate ion in the crystals of the ternary complex PKA-Mg(2)ATP-IP20 by X-ray irradiation at room temperature. The cleavage of ATP may be aided by X-ray-generated free hydroxyl radicals, a very reactive chemical species, which move rapidly through the crystal at room temperature. The phosphate anion is clearly visible in the electron-density maps; it remains in the active site but slides about 2 Å from its position in ATP towards Ala21 of IP20, which mimics the phosphorylation site. The phosphate thus pushes the peptidic inhibitor away from the product ADP, while resulting in dramatic conformational changes of the terminal residues 24 and 25 of IP20. X-ray structures of PKAc in complex with the nonhydrolysable ATP analogue AMP-PNP at both room and low temperature demonstrated no temperature effects on the conformation and position of IP20.

Figure 1

(a) Overall structure of PKAc in cartoon representation. The small lobe is colored green, the large lobe is colored magenta and IP20 is colored blue. The two Mg²⁺ ions are represented by cyan spheres, while ATP is shown in stick representation colored by atom type. (b) A close-up view of the active site. Water molecules are represented by red spheres. Metal coordination is shown as black solid lines, whereas hydrogen bonding is represented by orange dashed lines.

Figure 2

(a) Electron density for the active-site components ATP, Mg²⁺, water molecules and IP20 in LT PKA–MgATP–IP20 contoured at the 1.0σ level (2.0σ for the metal). (b) Electron density for the active-site components ATP, Mg²⁺, water molecules and IP20 in LT PKA–Mg₂ATP–IP20 contoured at the 1.6σ level. (c) Superposition of low-Mg²⁺ (purple; metal in magenta) and high-Mg²⁺ (colored by atom type) structures; the coordination of the metal ions is shown by orange dashed and black solid lines, respectively.

Figure 3

(a) Active site of PKAc in the room-temperature structure RT PKA–Mg₂ADP·PO₄–IP20. Mg²⁺ coordination with the enzyme residues and water molecules is shown as black solid lines. (b) Electron density for ADP, phosphate, Mg²⁺ and water molecules in RT PKA–Mg₂ADP·PO₄–IP20 contoured at the 1.4σ level. (c, d) Superposition of the room- and low-temperature structures RT PKA–Mg₂ADP·PO₄–IP20 (yellow C atoms, cyan Mg²⁺ ions) and LT PKA–Mg₂ATP–IP20 (purple C atoms and Mg²⁺ ions), respectively. Hydrogen bonds are shown as orange dashed lines. C—H⋯O interactions are shown as brown dashed lines. All distances are in Å.

Figure 4

Superposition of LT PKA–Mg₂ATP–IP20 (yellow C atoms), LT PKA–Mg₂AMPPNP–IP20 (dark cyan C atoms) and RT PKA–Mg₂AMPPNP–IP20 (light brown C atoms).