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首页> 外文期刊>Biochemistry >Interaction between CKαalpha and CKβ beta, the Subunits of Protein Kinase CK2: Thermodynamic Contributions of Key Residues on the CKα alpha Surface
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Interaction between CKαalpha and CKβ beta, the Subunits of Protein Kinase CK2: Thermodynamic Contributions of Key Residues on the CKα alpha Surface

机译：CKαalpha和CKβbeta，蛋白激酶CK2的亚基之间的相互作用：CKαalpha表面上关键残基的热力学贡献

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The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2 alpha) bound to a dimer of noncatalytic subunits (CK2 beta). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2 alpha and CK2 beta as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2 alpha interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2 beta. Additionally, these CK2 alpha mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2 alpha, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2 beta-bound CK2 alpha. Identifying residues on CK2 alpha that play a key role in CK2 alpha/CK2 beta interactions is important for the future generation of small molecule drug design.
机译：蛋白质Ser / Thr激酶CK2（以前的名称：酪蛋白激酶II）主要以异四聚体全酶的形式存在，该酶由两个催化亚基（CK2 alpha）和一个非催化亚基的二聚体（CK2 beta）结合而成。我们进行了一项研究，以进一步了解这些亚基如何相互作用形成四聚体。为此，我们使用了能够形成全酶的人CK2亚基的重组C端截短形式。我们使用等温滴定量热法（ITC）分析了CK2 alpha和CK2 beta结合之间的相互作用热力学以及温度，pH和缓冲液电离焓的变化的影响。通过结构引导的丙氨酸扫描诱变，我们截断了位于CK2 alpha界面内的疏水性氨基酸簇中的各个侧链，从而实验性地确定了主导亲和力的氨基酸。 ITC结果表明Leu41或Phe54单一突变对CK2 beta的结合最具破坏性。此外，这些CK2 alpha突变体保留了其激酶活性。此外，Leu41与Phe54的组合替代显示单个突变不是累加的，表明两个残基的协同作用都起作用。有趣的是，在CK2 alpha相互作用表面上占据中心位置的Ile69的替换仅产生了适度的作用。相互作用相关性中Leu41，Phe54和Ile69之间的差异与从未结合到CK2 beta结合的CK2α过渡过程中溶剂可及性的变化相关。鉴定在CK2α/CK2β相互作用中起关键作用的CK2α残基对于下一代小分子药物设计至关重要。

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