Low- and room-temperature X-ray structures of protein kinase A ternary complexes shed new light on its activity
- Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States)
- University of Toledo, 2801 West Bancroft Street, Toledo, OH 43606 (United States)
- University of San Diego, 9500 Gilman Drive, Leichtag 415, La Jolla, CA 92093 (United States)
- Oak Ridge National Laboratory, PO Box 2008, MS 6475, Oak Ridge, TN 37831 (United States)
ATP bound in the active site of protein kinase A is readily hydrolysed to ADP and free phosphate by X-ray irradiation at room temperature. The phosphate ion observed in the active site causes a dramatic conformational change of the bound peptide inhibitor. 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{sup 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{sub 2}ATP–IP20 complex obtained at high Mg{sup 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{sub 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.
- OSTI ID:
- 22351262
- Journal Information:
- Acta Crystallographica. Section D: Biological Crystallography, Vol. 68, Issue Pt 7; Other Information: PMCID: PMC3388813; PMID: 22751671; PUBLISHER-ID: mn5009; OAI: oai:pubmedcentral.nih.gov:3388813; Copyright (c) International Union of Crystallography 2012; Country of input: International Atomic Energy Agency (IAEA); ISSN 0907-4449
- Country of Publication:
- Denmark
- Language:
- English
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