NMR backbone assignment of a protein kinase catalytic domain by a combination of several approaches: Application to the catalytic subunit of cAMP-dependent protein kinase
NMR backbone assignment of a protein kinase catalytic domain by a combination of several approaches: Application to the catalytic subunit of cAMP-dependent protein kinase
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DOI：
10.1002/cbic.200400129
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发表时间：
2004-11-05
期刊：
影响因子：
3.2
通讯作者：
Schwalbe, H
中科院分区：
文献类型：
--
作者：
Langer, T;Vogtherr, M;Schwalbe, H
蛋白磷酸化是真核细胞胞内调控的重要机制之一。目前，最具特征的蛋白激酶之一是camp依赖性蛋白激酶或蛋白激酶A （PKA）的催化亚基。PKA具有典型的激酶小叶结构，活性位点由两个结构裂片之间的间隙组成。为了充分发挥激酶的活性，催化亚基必须被磷酸化。PKA催化亚基有两个主要的磷酸化位点：Thr197和Ser338。ATP或抑制剂与ATP位点的结合会引起大的结构变化。在这里，我们通过核磁共振光谱描述了PKA催化结构域的部分骨架分配，这是任何蛋白激酶催化结构域的第一次核磁共振分配。42 kDa蛋白的主链共振分配是通过以下方法完成的：1)三重（H-2, C-13, N-15）标记蛋白和经典核磁共振分配实验；2)从已知的x射线结构反向计算化学位移；3)使用顺磁腺苷衍生物作为自旋标记；4)选择性氨基酸标记。化学位移扰动的解释允许绘制与蛋白激酶抑制剂H7的相互作用表面。此外，通过2D， H-1， N-15 TROSY获得的无活性Thr197Ala突变体与野生型酶的主干酰胺移位比较，观察到结构构象的变化。
Protein phosphorylation is one of the most important mechanisms used for intracellular regulation in eukaryotic cells. Currently, one of the best-characterized protein kinases is the catalytic subunit of cAMP-dependent protein kinase or protein kinase A (PKA). PKA has the typical bilobular structure of kinases, with the active site consisting of a cleft between the two structural lobes. For full kinase activity, the catalytic subunit has to be phosphorylated. The catalytic subunit of PKA has two main phosphorylation sites: Thr197 and Ser338. Binding of ATP or inhibitors to the ATP site induces large structural changes. Here we describe the partial backbone assignment of the PKA catalytic domain by NMR spectroscopy, which represent the first NMR assignment of any protein kinase catalytic domain. Backbone resonance assignment for the 42 kDa protein was accomplished by an approach employing 1) triply (H-2, C-13, N-15) labeled protein and classical NMR assignment experiments, 2)back-calculation of chemical shifts from known x-ray structures, 3) use of paramagnetic adenosine derivatives as spin-labels, and 4) selective amino acid labeling. Interpretation of chemical-shift perturbations allowed mapping of the interaction surface with the protein kinase inhibitor H7. Furthermore, structural conformational changes were observed by comparison of backbone amide shifts obtained by 2D, H-1, N-15 TROSY of an inactive Thr197Ala mutant with the wild-type enzyme.