Structural and functional insights into alpha-kinases and their regulation

蛋白质(去)磷酸化(即磷酸基团的添加/移除)是生物体中一个关键和基本的过程，几乎是所有细胞内信号转导途径的调控基础。非典型激酶的一个小亚类，α-激酶，最初是因为它们识别α-螺旋结构而不是特定的序列而被发现，但关于它们的结构和功能却知之甚少。这项建议的目的是深入了解非典型激酶的机制和调控。已经确定了两种α-激酶结构：TRMP7(一种对镁离子敏感的通道激酶)和MHCK(一种作用于肌球蛋白II的苏氨酸特异性激酶)。我们对MHCK激酶结构域结构的测定显示了一个磷酸化的天冬氨酸残基，这表明与大多数激酶的直接磷酸化不同，通过磷酸天冬氨酸中间体进行的反应是一种新的两步反应机制。在保守的富含甘氨酸的N/D环(以催化关键的AsN/Asp残基命名)中，额外的镁离子可能作为镁离子敏感的构象开关，如果得到证实，这将是一种新的调控机制。虽然类似的机制可能是TRPM7功能的基础，但尚不清楚的是TRMP7的S底物。底物鉴定，使用肽阵列和其他方法，将允许进一步分析其活性的额外的镁依赖关系。另一种结构未知的α-激酶，eEF2K，是蛋白质合成的“主控制器”，其独特之处在于它受钙调蛋白的调节并依赖于钙调蛋白来激活。开发特定的抑制剂和随后确定酶-抑制剂复合体将有助于破译这些非典型激酶的作用模式，同时优化进一步的抑制剂设计。学员将利用生化、生物物理和结构技术的组合来揭示控制底物专一性、催化活性和调节的结构基础和分子机制。我们的合作努力使我们在“过度拥挤”的哺乳动物激酶领域获得了一个“利基”，我们可以在那里做出新的贡献，而我们的MHCK工作则给了我们一个“竞争优势”。

Protein (de)phosphorylation (i.e. the addition/removal of phosphate groups) is a crucial and fundamental process in living organisms, underlying the regulatory control of virtually all intracellular signal transduction pathways. A small subclass of atypical kinases, the alpha-kinases, was originally discovered as they recognized an alpha-helical structure rather than a particular sequence, yet very little is known with respect to their structure and function. The object of this proposal is to gain an in-depth understanding into the mechanism and regulation of the atypical kinases. Two alpha-kinase structures have been determined: TRMP7 (an Mg2+-sensing channel kinase) and MHCK (a threonine-specific kinase which acts on myosin II). Our determination of the MHCK kinase domain structure revealed a phosphorylated aspartyl residue, suggesting a novel 2-step reaction mechanism via a phosphoaspartyl intermediate in contrast to the direct phosphorylation of most kinases. An additional Mg2+ ion, in a conserved Gly-rich N/D loop (named for a catalytically critical Asn/Asp residue) may act as Mg2+-sensing conformational switch which, if confirmed, would be a novel, regulatory mechanism. While similar mechanisms presumably underlie TRPM7 function, what is unknown is TRMP7's substrate. Substrate identification, using peptide arrays and other approaches, will allow further analyses regarding the additional Mg2+-dependence of its activity. Another alpha-kinase of unknown structure, eEF2K, is a "master controller" of protein synthesis, and is unique in its regulation by and dependence upon calmodulin for activation. Development of specific inhibitors and the subsequent determination of enzyme-inhibitor complexes will help decipher the mode of action of these atypical kinases while optimizing further inhibitor design. Trainees will utilize a combination of biochemical, biophysical and structural techniques to uncover the structural basis and molecular mechanisms governing substrate specificity, catalytic activity and regulation. Our collaborative efforts gave us a "niche" in the "overcrowded" mammalian kinase field where we can make novel contributions, while our MHCK work gives us a "competitive edge".