Structural studies of stress-responsive sensor protein kinases

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The accumulation of unfolded proteins in the lumen of the endoplasmic reticulum induces an unfolded protein response. The response alleviates stress by upregulating protein folding and degradation pathways in the endoplasmic reticulum and inhibiting protein synthesis. This response is mediated by a novel membrane-integrate protein serine/threonine kinase, IRE. IRE consists of a sensor domain in the lumen, a transmembrane helix region, and a cytoplasmic domain. The cytoplasmic domain has a linker at the juxtamembrane region, which is followed by protein kinase domain and nuclease domain. Interestingly, the nuclease activity was regulated by the kinase that is activated by the oligomerization of the protein through the sensor domain in the lumen. This nuclease domain resembles to that of RNase L, which is known to be a component of the interferon response pathway, and, again, the organization of the kinase-nuclease in one polypeptide chain is also common in these proteins. In this study, we tried to establish the protein preparation of IREs, human IREα,IREβ and yeast Ire1p, for the three-dimensional structural studies. We produced several kinds of constructs for protein expression in both E.coli cells and insect cells and tried to purify the expressed proteins. We found high protein expression and relatively efficient purification procedure of a protein using a construct of the cytoplasmic domain from Ire1p. We also found that constructs of the sensor domain of Ire1p provide proteins that can be efficiently purified. These advances in protein preparation enable us to prepare protein crystals for X-ray diffraction studies or to measure NMR spectra for structural analyses and determination.