Structural and functional insights into alpha-kinases and their regulation

α-激酶及其调节的结构和功能见解

• 批准号: 203705-2013
• 负责人: Jia, Zongchao
• 金额: $ 5.03万
• 依托单位: Queen's University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2013
• 资助国家: 加拿大
• 起止时间: 2013-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=523520
• 关键词: Structural; functional; insights; into; alpha

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".

蛋白质磷酸化（去磷酸化）（即磷酸基团的添加/去除）是活生物体中的关键和基本过程，是几乎所有细胞内信号转导途径的调节控制的基础。非典型激酶的一个小亚类，α-激酶，最初被发现，因为它们识别α-螺旋结构，而不是特定的序列，但对它们的结构和功能知之甚少。本研究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：TRMP 7（Mg 2+敏感通道激酶）和MHCK（作用于肌球蛋白II的苏氨酸特异性激酶）。我们的测定的MHCK激酶结构域的结构揭示了磷酸化的乙酰基残基，这表明了一种新的2步反应机制，通过磷酸化的乙酰基中间体相比，大多数激酶的直接磷酸化。一个额外的Mg 2+离子，在一个保守的富含Gly的N/D环（命名为催化关键的Asn/Asp残基）可以作为Mg 2+传感构象开关，如果证实，这将是一个新的，监管机制。虽然类似的机制可能是TRPM 7功能的基础，但TRMP 7的底物是未知的。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Mg 2+依赖性。另一种未知结构的α-激酶eEF 2K是蛋白质合成的“主控制器”，并且在其通过钙调蛋白调节和依赖钙调蛋白激活方面是独特的。特异性抑制剂的开发和随后的酶抑制剂复合物的测定将有助于解释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。学员将利用生物化学，生物物理和结构技术的组合，以揭示结构基础和分子机制，底物特异性，催化活性和调节。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”，在那里我们可以做出新的贡献，而我们的MHCK工作使我们具有“竞争优势”。