Kovalevsky Andrey Y, Johnson Hanna, Hanson B Leif, Waltman Mary Jo, Fisher S Zoe, Taylor Susan, Langan Paul
Bioscience Division, Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545, USA.
Acta Crystallogr D Biol Crystallogr. 2012 Jul;68(Pt 7):854-60. doi: 10.1107/S0907444912014886. Epub 2012 Jun 15.
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.
蛋白激酶A（PKA）介导的翻译后蛋白质磷酸化是一种普遍存在的信号传导机制，可调节许多细胞过程。在生理镁离子浓度约为0.5 mM的条件下，PKA催化亚基（PKAc）与ATP及一种20个残基的肽类抑制剂（IP20）形成的三元复合物的低温X射线结构（LT PKA-MgATP-IP20）显示，活性位点中有单个金属离子。在LT PKA-MgATP-IP20中缺乏第二个金属离子，使得ATP的β和γ磷酰基团非常灵活，具有较高的热B因子。因此，通过将前一种结构与在高镁离子浓度下获得的LT PKA-Mg(2)ATP-IP20复合物的结构进行比较可知，第二个金属离子对于将末端磷酰基团紧密定位以转移至底物至关重要。除了其激酶活性外，PKAc还能够以水分子为底物缓慢催化ATP的水解。研究发现，在室温下通过X射线照射，三元复合物PKA-Mg(2)ATP-IP20的晶体中ATP能够迅速且完全水解为ADP和游离磷酸根离子。ATP的裂解可能借助于X射线产生的自由羟基自由基，这是一种极具反应性的化学物质，在室温下能迅速穿过晶体。磷酸根阴离子在电子密度图中清晰可见；它保留在活性位点，但从其在ATP中的位置向IP20的Ala21滑动约2 Å，IP20模拟磷酸化位点。因此，磷酸根将肽类抑制剂从产物ADP推开，同时导致IP20末端残基24和25发生显著构象变化。PKAc与不可水解的ATP类似物AMP-PNP在室温和低温下形成的复合物的X射线结构表明，温度对IP20的构象和位置没有影响。
