Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1α Endoribonuclease Activity
Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1α Endoribonuclease Activity
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DOI：
10.1124/mol.115.100917
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发表时间：
2015-12-01
影响因子：
3.6
通讯作者：
DeYoung, M. Phillip
中科院分区：
文献类型：
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作者：
Concha, Nestor O.;Smallwood, Angela;DeYoung, M. Phillip
内质网应激引起的肌醇需要酶-1α(IRE1α)蛋白的激活导致N-末端内质网腔结构域的同源二聚化，胞浆内激酶区的自磷酸化，以及胞浆内切核酸酶(RNase)结构域的构象变化，这些变化使它们具有功能，并可导致X-box结合蛋白1(XBP 1)mRNA的剪接。在这里，我们报道了人的磷酸化的IRE1a二聚体与(R)-2-(3，4-dichlorobenzyl)-N-(4-methylbenzyl)2，7-重氮螺-(4，5)癸烷-7-甲酰胺，一种新型的IRE1α选择性激酶抑制剂和一种广谱的激酶抑制剂星形孢子素的复合体的第一个晶体结构。(R)-2-(3，4-dichlorobenzyl)-N-(4-methylbenzyl)2，7-重氮螺-7-甲酰胺对IRE1α的激酶和核糖核酸酶活性均有抑制作用。该抑制剂与催化残基Lys599和Glu612相互作用，并将激酶激活环置换为DFG-out构象。IRE1α核糖核酸酶活性的失活似乎是由构象变化引起的，由此αC螺旋被移位，导致激酶结构域-二聚体界面的重新排列和核糖核酸酶结构域彼此远离的旋转。相反，星孢菌素结合在IRE1α的ATP结合部位，导致与核糖核酸酶活性酵母IRE1二聚体一致的二聚体。IRE1αRNase活性的激活似乎是由RNase二聚体界面上高度保守的残基之间的氢键相互作用网络促进的，这些氢键作用使关键的催化残基为反应做好了准备。这些数据表明，RNase结构域中保守残基之间的分子间相互作用是活性所必需的，这些相互作用的破坏可以通过小分子激酶域抑制剂在药理学上实现。
Activation of the inositol-requiring enzyme-1 alpha (IRE1 alpha) protein caused by endoplasmic reticulum stress results in the homodimerization of the N-terminal endoplasmic reticulum luminal domains, autophosphorylation of the cytoplasmic kinase domains, and conformational changes to the cytoplasmic endoribonuclease (RNase) domains, which render them functional and can lead to the splicing of X-box binding protein 1 (XBP 1) mRNA. Herein, we report the first crystal structures of the cytoplasmic portion of a human phosphorylated IRE1a dimer in complex with (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)2,7- diazaspiro(4.5) decane-7-carboxamide, a novel, IRE1 alpha-selective kinase inhibitor, and staurosporine, a broad spectrum kinase inhibitor. (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)2,7- diazaspiro(4.5) decane-7-carboxamide inhibits both the kinase and RNase activities of IRE1 alpha. The inhibitor interacts with the catalytic residues Lys599 and Glu612 and displaces the kinase activation loop to the DFG-out conformation. Inactivation of IRE1 alpha RNase activity appears to be caused by a conformational change, whereby the alpha C helix is displaced, resulting in the rearrangement of the kinase domain-dimer interface and a rotation of the RNase domains away from each other. In contrast, staurosporine binds at the ATP-binding site of IRE1 alpha, resulting in a dimer consistent with RNase active yeast Ire1 dimers. Activation of IRE1 alpha RNase activity appears to be promoted by a network of hydrogen bond interactions between highly conserved residues across the RNase dimer interface that place key catalytic residues poised for reaction. These data implicate that the intermolecular interactions between conserved residues in the RNase domain are required for activity, and that the disruption of these interactions can be achieved pharmacologically by small molecule kinase domain inhibitors.