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首页> 外文期刊>Journal of Molecular Biology >X-ray crystallographic and MD simulation studies on the mechanism of interfacial activation of a family I.3 lipase with two lids.
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X-ray crystallographic and MD simulation studies on the mechanism of interfacial activation of a family I.3 lipase with two lids.

机译：X射线晶体学和MD模拟研究了具有两个盖子的I.3家族脂肪酶的界面活化机理。

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摘要

The interfacial activation mechanism of family I.3 lipase from Pseudomonas sp. MIS38 (PML), which has two alpha-helical lids (lid1 and lid2), was investigated using a combination of X-ray crystallography and molecular dynamics (MD) simulation. The crystal structure of PML in an open conformation was determined at 2.1 A resolution in the presence of Ca(2+) and Triton X-100. Comparison of this structure with that in the closed conformation indicates that both lids greatly change their positions and lid1 is anchored by the calcium ion (Ca1) in the open conformation. This structure was not seriously changed even when the protein was dialyzed extensively against the Ca(2+)-free buffer containing Triton X-100 before crystallization, indicating that the open conformation is fairly stable unless a micellar substance is removed. The crystal structure of the PML derivative, in which the active site serine residue (Ser207) is diethylphosphorylated by soaking the crystal of PML in the open conformation in a solution containing diethyl p-nitrophenyl phosphate, was also determined. This structure greatly resembles that in the open conformation, indicating that PML structure in the open conformation represents that in the active form. MD simulation of PML in the open conformation in the absence of micelles showed that lid2 closes first, while lid1 maintains its open conformation. Likewise, MD simulation of PML in the closed conformation in the absence of Ca(2+) and in the presence of octane or trilaurin micelles showed that lid1 opens, while lid2 remains closed. These results suggest that Ca1 functions as a hook for stabilization of a fully opened conformation of lid1 and for initiation of subsequent opening of lid2.
机译：假单胞菌属的I.3族脂肪酶的界面活化机制。使用X射线晶体学和分子动力学（MD）模拟相结合的方法研究了具有两个α螺旋盖（lid1和lid2）的MIS38（PML）。在存在Ca（2+）和Triton X-100的情况下，以2.1 A的分辨率确定了呈开放构型的PML的晶体结构。将该结构与闭合构型的结构进行比较表明，两个盖都大大改变了它们的位置，并且盖1被钙离子（Ca1）锚定在开放构型中。即使该蛋白质在结晶前对含有Triton X-100的不含Ca（2+）的缓冲液进行了充分的透析，该结构也没有发生严重改变，这表明除非除去胶束物质，否则开放构象是相当稳定的。还确定了PML衍生物的晶体结构，其中通过将PML的晶体以开放构象浸泡在含磷酸二硝基苯酯的溶液中而使活性位丝氨酸残基（Ser207）被二乙基磷酸化。该结构非常类似于开放构象中的结构，表明开放构象中的PML结构代表活性形式的PML结构。在不存在胶束的情况下，呈开放构象的PML的MD模拟表明，lid2首先关闭，而lid1保持其开放构象。同样，在不存在Ca（2+）且存在辛烷或三月桂酸酯胶束的情况下，MDL在闭合构象下的MD模拟表明，lid1打开，而lid2保持关闭。这些结果表明，Ca1起钩子的作用，以稳定盖子1的完全打开的构型并引发随后盖子2的打开。

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