Scott John W, Norman David G, Hawley Simon A, Kontogiannis Luke, Hardie D Grahame
Division of Molecular Physiology, School of Life Sciences and Wellcome Trust Biocentre, Dundee University, Scotland, UK.
J Mol Biol. 2002 Mar 22;317(2):309-23. doi: 10.1006/jmbi.2001.5316.
We have expressed a truncated form of the alpha1 kinase domain of AMP-activated protein kinase (AMPK) in Escherichia coli as a glutathione-S-transferase fusion (GST-KD). A T172D mutant version did not require prior phosphorylation and was utilized for most subsequent studies. We have also created a recombinant substrate (GST-ACC) by expressing 34 residues around the major phosphorylation site (Ser79) on rat acetyl-CoA carboxylase-1/alpha (ACC1) as a GST fusion. This was an excellent substrate that was phosphorylated with similar kinetic parameters to ACC1 by both native AMPK and the bacterially expressed kinase domain. We also constructed a structural model for the binding of the ACC1 sequence to the kinase domain, based on crystal structures for related protein kinases. The model was tested by making a total of 25 mutants of GST-ACC and seven mutants of GST-KD, and measuring kinetic parameters with different combinations. The results reveal that AMPK and ACC1 interact over a much wider region than previously realized (>20 residues). The features of the interaction can be summarised as follows: (i) an amphipathic helix from P-16 to P-5 on the substrate binds in a hydrophobic groove on the large lobe of the kinase; (ii) basic residues at P-6 and P-4 bind to two acidic patches (D215/D216/D217 and E103/D100/E143, respectively), on the large lobe; (iii) a histidine at P+3 interacts with D56 on the small lobe; (iv) the side-chain of P+4 leucine could not be precisely positioned, but a new finding was that asparagine or glutamine could replace a hydrophobic residue at this position. These interactions position the serine residue to be phosphorylated in close proximity to the gamma-phosphate group of ATP. Although based on modelling rather than a determined structure, this represents one of the most detailed studies of the interaction between a kinase and its substrate achieved to date.
我们在大肠杆菌中表达了一种截短形式的AMP激活蛋白激酶（AMPK）的α1激酶结构域，作为谷胱甘肽-S-转移酶融合蛋白（GST-KD）。T172D突变体形式不需要预先磷酸化，并且在大多数后续研究中被使用。我们还通过将大鼠乙酰辅酶A羧化酶-1/α（ACC1）主要磷酸化位点（Ser79）周围的34个残基作为GST融合蛋白表达，创建了一种重组底物（GST-ACC）。这是一种极好的底物，天然AMPK和细菌表达的激酶结构域都能以与ACC1相似的动力学参数对其进行磷酸化。我们还基于相关蛋白激酶的晶体结构构建了ACC1序列与激酶结构域结合的结构模型。通过对GST-ACC总共进行25个突变体和GST-KD的7个突变体，并测量不同组合的动力学参数来测试该模型。结果表明，AMPK和ACC1相互作用的区域比之前认识到的要广泛得多（超过20个残基）。相互作用的特征可以总结如下：（i）底物上从P-16到P-5的一个两亲性螺旋结合在激酶大亚基的一个疏水凹槽中；（ii）P-6和P-4处的碱性残基分别与大亚基上的两个酸性区域（D215/D216/D217和E103/D100/E143）结合；（iii）P+3处的组氨酸与小亚基上的D56相互作用；（iv）P+4亮氨酸的侧链无法精确定位，但一个新发现是天冬酰胺或谷氨酰胺可以取代该位置的疏水残基。这些相互作用将待磷酸化的丝氨酸残基定位在靠近ATP的γ-磷酸基团的位置。尽管基于建模而非确定的结构，但这是迄今为止对激酶与其底物之间相互作用进行的最详细研究之一。
