Abstract
Posttranslational protein phosphorylation by protein kinase A (PKA) is a ubiquitous signaling mechanism which regulates many cellular processes. A low temperature X-ray structure of the PKA catalytic subunit (PKAc) ternary complex with ATP and a 20-residue peptidic inhibitor (IP20) at the physiological Mg2+ concentration of < 0.5mM revealed a single metal ion in the active site. The lack of a second metal in the low-temperature LT-PKAc-MgATP-IP20 renders the and phosphoryl groups of ATP to be very flexibile, with high thermal B-factors. Thus, the second metal is crucial for tight positioning of the terminal phosphoryl for transfer to a substrate, as demonstrated by comparison of the former structure with LT-PKAc- Mg2ATP-IP20 complex. In addition to the kinase activity, PKAc is also able to slowly catalyze the hydrolysis of ATP using a water molecule as a substrate. We found that at room temperature under X-ray irradiation ATP can be readily and completely hydrolyzed into ATP and a free phosphate ion in the crystals of the ternary complex LT-PKAc- Mg2ATP-IP20. The cleavage of ATP may be aided by X-ray-born free hydroxyl radicals, a very reactive chemical species, that move quickly 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 2Å from its position in ATP toward Ala21 of IP20 that mimics the phosphorylation site. The phosphate, thus, pushes the peptidic inhibitor away from the product ADP, while resulting in dramatic conformational changes of IP20 terminal residues 24 and 25. X-ray structures of PKAc in complex with non-hydrolyzable ATP analog, AMPPNP, at both room and low temperatures demonstrated no temperature effects on the conformation and position of IP20.
