Howard Hughes Medical Institute, University of California, San Francisco, 600 16th Street, Room S272, Box 0724, San Francisco, CA 94158, USA.
BMC Biol. 2011 Jul 6;9:48. doi: 10.1186/1741-7007-9-48.
Ire1 is a signal transduction protein in the endoplasmic reticulum (ER) membrane that serves to adjust the protein-folding capacity of the ER according to the needs of the cell. Ire1 signals, in a transcriptional program, the unfolded protein response (UPR) via the coordinated action of its protein kinase and RNase domains. In this study, we investigated how the binding of cofactors to the kinase domain of Ire1 modulates its RNase activity.
Our results suggest that the kinase domain of Ire1 initially binds cofactors without activation of the RNase domain. RNase is activated upon a subsequent conformational rearrangement of Ire1 governed by the chemical properties of bound cofactors. The conformational step can be selectively inhibited by chemical perturbations of cofactors. Substitution of a single oxygen atom in the terminal β-phosphate group of a potent cofactor ADP by sulfur results in ADPβS, a cofactor that binds to Ire1 as well as to ADP but does not activate RNase. RNase activity can be rescued by thiophilic metal ions such as Mn2+ and Cd2+, revealing a functional metal ion-phosphate interaction which controls the conformation and RNase activity of the Ire1 ADP complex. Mutagenesis of the kinase domain suggests that this rearrangement involves movement of the αC-helix, which is generally conserved among protein kinases. Using X-ray crystallography, we show that oligomerization of Ire1 is sufficient for placing the αC-helix in the active, cofactor-bound-like conformation, even in the absence of cofactors.
Our structural and biochemical evidence converges on a model that the cofactor-induced conformational change in Ire1 is coupled to oligomerization of the receptor, which, in turn, activates RNase. The data reveal that cofactor-Ire1 interactions occur in two independent steps: binding of a cofactor to Ire1 and subsequent rearrangement of Ire1 resulting in its self-association. The pronounced allosteric effect of cofactors on protein-protein interactions involving Ire1's kinase domain suggests that protein kinases and pseudokinases encoded in metazoan genomes may use ATP pocket-binding ligands similarly to exert signaling roles other than phosphoryl transfer.
IRE1 是内质网 (ER) 膜中的一种信号转导蛋白，它根据细胞的需要调整 ER 的蛋白质折叠能力。IRE1 通过其蛋白激酶和核糖核酸酶结构域的协同作用，在转录程序中发出未折叠蛋白反应 (UPR) 的信号。在这项研究中，我们研究了辅助因子与 IRE1 的激酶结构域的结合如何调节其核糖核酸酶活性。
我们的结果表明，IRE1 的激酶结构域最初在没有激活核糖核酸酶结构域的情况下结合辅助因子。在受结合辅助因子化学性质控制的 IRE1 的后续构象重排之后，核糖核酸酶被激活。该构象步骤可以通过化学干扰辅助因子来选择性抑制。在有效的辅助因子 ADP 的末端β-磷酸基团中的单个氧原子被硫取代，导致 ADPβS，这是一种与 ADP 结合但不激活核糖核酸酶的辅助因子。硫亲核金属离子（如 Mn2+ 和 Cd2+）可以挽救核糖核酸酶活性，揭示了一种功能金属离子-磷酸相互作用，控制 Ire1 ADP 复合物的构象和核糖核酸酶活性。激酶结构域的突变表明，这种重排涉及αC-螺旋的移动，αC-螺旋在一般蛋白激酶中是保守的。使用 X 射线晶体学，我们表明 Ire1 的寡聚化足以将αC-螺旋置于活性、辅助因子结合样构象中，即使在没有辅助因子的情况下也是如此。
我们的结构和生化证据集中在一个模型上，即辅助因子诱导的 Ire1 构象变化与受体的寡聚化相关联，而受体的寡聚化又激活了核糖核酸酶。数据表明，辅助因子与 Ire1 的相互作用发生在两个独立的步骤中：辅助因子与 Ire1 的结合和随后的 Ire1 重排导致其自身缔合。辅助因子对涉及 Ire1 的激酶结构域的蛋白质-蛋白质相互作用的显著变构效应表明，后生动物基因组中编码的蛋白激酶和假激酶可能类似地使用 ATP 口袋结合配体来发挥除磷酸转移以外的信号作用。
