4DH5

Room temperature X-ray structure of cAMP dependent Protein Kinase A catalytic subunit with high Mg2+, ADP, Phosphate, and IP20

Summary for 4DH5

• Entry DOI: 10.2210/pdb4dh5/pdb
• Related: 4DH1 4DH3 4DH7 4DH8
• Descriptor: cAMP-dependent protein kinase catalytic subunit alpha, cAMP-dependent protein kinase inhibitor alpha, MAGNESIUM ION, ... (6 entities in total)
• Functional Keywords: protein kinase, phosphotransfer, peptidic inhibitor, transferase-transferase inhibitor complex, transferase/transferase inhibitor
• Biological source: Mus musculus (mouse); More
• Cellular location: Cytoplasm: P05132
• Total number of polymer chains: 2
• Total formula weight: 43534.49

Authors

Kovalevsky, A.Y.,Langan, P. (deposition date: 2012-01-27, release date: 2012-06-27, Last modification date: 2024-11-27)

Primary citation

Kovalevsky, A.Y.,Johnson, H.,Hanson, B.L.,Waltman, M.J.,Fisher, S.Z.,Taylor, S.,Langan, P.
Low- and room-temperature X-ray structures of protein kinase A ternary complexes shed new light on its activity.
Acta Crystallogr.,Sect.D, 68:854-860, 2012

PubMed 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.

PubMed: 22751671
DOI: 10.1107/S0907444912014886

Experimental method

X-RAY DIFFRACTION (2.2 Å)