Yde Christina W, Ermakova Inessa, Issinger Olaf-Georg, Niefind Karsten
Syddansk Universitet, Institut for Biokemi og Molekylaerbiologi, Campusvej 55, DK-5230 Odense, Denmark.
J Mol Biol. 2005 Mar 25;347(2):399-414. doi: 10.1016/j.jmb.2005.01.003. Epub 2005 Jan 18.
Protein kinase CK2 (casein kinase 2) is a highly conserved and ubiquitously found eukaryotic serine/threonine kinase that plays a role in various cellular key processes like proliferation, apoptosis and circadian rhythm. One of its prominent biochemical properties is its ability to use GTP as well as ATP as a cosubstrate (dual-cosubstrate specificity). This feature is exceptional among eukaryotic protein kinases, and its biological significance is unknown. We describe here a mutant of the catalytic subunit of protein kinase CK2 (CK2alpha) from Homo sapiens (hsCK2alpha) with a clear and CK2-atypical preference for ATP compared to GTP. This mutant was designed on the basis of several structures of CK2alpha from Zea mays (zmCK2alpha) in complex with various ATP-competitive ligands. A structural overlay revealed the existence of a "purine base binding plane" harbouring the planar moiety of the respective ligand like the purine base of ATP and GTP. This purine base binding plane is sandwiched between the side-chains of Ile66 (Val66 in hsCK2alpha) and Met163, and it adopts a significantly different orientation than in prominent homologues like cAMP-dependent protein kinase (CAPK). By exchanging these two flanking amino acids (Val66Ala, Met163Leu) in hsCK2alpha(1-335), a C-terminally truncated variant of hsCK2alpha, the cosubstrate specificity shifted in the expected direction so that the mutant strongly favours ATP. A structure determination of the mutant in complex with an ATP-analogue confirmed the predicted change of the purine base binding plane orientation. An unexpected but in retrospect plausible consequence of the mutagenesis was, that the helix alpha D region, which is in the direct neighbourhood of the ATP-binding site, has adopted a conformation that is more similar to CAPK and less favourable for binding of GTP. These findings demonstrate that CK2alpha possesses sophisticated structural adaptations in favour of dual-cosubstrate specificity, suggesting that this property could be of biological significance.
蛋白激酶CK2（酪蛋白激酶2）是一种高度保守且广泛存在的真核丝氨酸/苏氨酸激酶，在细胞增殖、凋亡和昼夜节律等各种细胞关键过程中发挥作用。其突出的生化特性之一是能够将GTP以及ATP用作共底物（双共底物特异性）。这一特性在真核蛋白激酶中是例外，其生物学意义尚不清楚。我们在此描述了一种来自智人的蛋白激酶CK2（CK2α）催化亚基的突变体（hsCK2α），与GTP相比，它对ATP具有明显且非典型的偏好。该突变体是基于玉米（zmCK2α）的CK2α与各种ATP竞争性配体复合物的几种结构设计的。结构叠加显示存在一个“嘌呤碱基结合平面”，该平面容纳了各自配体的平面部分，如ATP和GTP的嘌呤碱基。这个嘌呤碱基结合平面夹在Ile66（hsCK2α中的Val66）和Met163的侧链之间，其取向与环磷酸腺苷依赖性蛋白激酶（CAPK）等主要同源物相比有显著不同。通过在hsCK2α（1 - 335）（hsCK2α的C末端截短变体）中交换这两个侧翼氨基酸（Val66Ala，Met163Leu），共底物特异性朝着预期方向转变，使得突变体强烈偏好ATP。与ATP类似物复合物的突变体结构测定证实了嘌呤碱基结合平面取向的预测变化。诱变产生的一个意外但事后看来合理的结果是，位于ATP结合位点直接附近的αD螺旋区域采用了一种与CAPK更相似且不利于GTP结合的构象。这些发现表明CK2α具有有利于双共底物特异性的复杂结构适应性，这表明该特性可能具有生物学意义。
