Crystal structure and calcium-induced conformational changes of diacylglycerol kinase α EF-hand domains
Crystal structure and calcium-induced conformational changes of diacylglycerol kinase α EF-hand domains
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二酰甘油激酶α EF-手结构域的晶体结构和钙诱导的构象变化
DOI：
10.1002/pro.3572
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发表时间：
2019
期刊：
影响因子：
8
通讯作者：
Sakane Fumio
中科院分区：
文献类型：
--
作者：
Takahashi Daisuke;Suzuki Kano;Sakamoto Taiichi;Iwamoto Takeo;Murata Takeshi;Sakane Fumio
二酰基甘油激酶（DGK）是一种多结构域脂质激酶，可将二酰基甘油磷酸化为磷脂酸，调节这些关键信号脂质的水平。近年来，DGKα同工酶作为肿瘤免疫治疗的一个潜在靶点受到越来越多的关注。我们之前已经证明，DGKα是由Ca 2+结合到其N-末端EF-手结构域（DGKα-EF）的正调控。然而，哺乳动物DGKs的结构生物学研究进展甚微，Ca 2+触发激活的分子机制仍不清楚。在这里，我们报告了Ca 2+结合DGKα-EF的第一个晶体结构，并分析了与Ca 2+结合后的结构变化。DGKα-EF采用典型的EF-手折叠，但出乎意料的是，具有额外的α-螺旋（通常称为配体模拟[LM]螺旋），其被包装到疏水核心中。生物物理和生物化学分析表明，DGKα-EF采用蛋白酶敏感的“开放”构象，没有Ca 2+，倾向于形成二聚体。两个Ca 2+离子的协同结合将二聚体解离成良好折叠的单体，其抵抗蛋白水解。综上所述，我们的研究结果提供了实验证据，表明Ca 2+结合诱导DGKα-EF的大量构象变化，这可能调节负责DGKα激活的分子内相互作用，并表明LM螺旋在Ca 2+诱导的构象变化中可能发挥作用。显著性声明二酰基甘油激酶（DGKs），调节两种脂质第二信使，二酰基甘油和磷脂酸的水平，自1959年首次鉴定以来，它在结构上仍然是一个谜。我们在此展示了二酰甘油激酶α EF-手结构域的第一个晶体结构，其钙离子结合形式，并表征了钙离子诱导的构象变化，这可能调节分子内相互作用。我们的研究为进一步了解DGK同工酶的结构基础奠定了基础。
Diacylglycerol kinases (DGKs) are multi‐domain lipid kinases that phosphorylate diacylglycerol into phosphatidic acid, modulating the levels of these key signaling lipids. Recently, increasing attention has been paid to DGKα isozyme as a potential target for cancer immunotherapy. We have previously shown that DGKα is positively regulated by Ca2+binding to its N‐terminal EF‐hand domains (DGKα‐EF). However, little progress has been made for the structural biology of mammalian DGKs and the molecular mechanism underlying the Ca2+‐triggered activation remains unclear. Here we report the first crystal structure of Ca2+‐bound DGKα‐EF and analyze the structural changes upon binding to Ca2+. DGKα‐EF adopts a canonical EF‐hand fold, but unexpectedly, has an additional α‐helix (often called a ligand mimic [LM] helix), which is packed into the hydrophobic core. Biophysical and biochemical analyses reveal that DGKα‐EF adopts a protease‐susceptible “open” conformation without Ca2+that tends to form a dimer. Cooperative binding of two Ca2+ions dissociates the dimer into a well‐folded monomer, which resists to proteolysis. Taken together, our results provide experimental evidence that Ca2+binding induces substantial conformational changes in DGKα‐EF, which likely regulates intra‐molecular interactions responsible for the activation of DGKα and suggest a possible role of the LM helix for the Ca2+‐induced conformational changes.Significance statementDiacylglycerol kinases (DGKs), which modulates the levels of two lipid second messengers, diacylglycerol and phosphatidic acid, is still structurally enigmatic enzymes since its first identification in 1959. We here present the first crystal structure of EF‐hand domains of diacylglycerol kinase α in its Ca2+bound form and characterize Ca2+‐induced conformational changes, which likely regulates intra‐molecular interactions. Our study paves the way for future studies to understand the structural basis of DGK isozymes.