In eukaryotic cells, most secreted and transmembrane proteins are modified and refold in the lumen of the endoplasmic reticulum(ER). The mature proteins which could fold into correct structure are coated by transport vesicles and delivered to the secretion pathway of the Golgi apparatus.(Refer to the PDB:2PM7 for the details).On the other hand, misfolded proteins which could not fold into correct structure are accumulated as defectives in the ER lumen. This situation is termed the ER stress. ER shows mainly three responses to the ER stress. (Fig.1).
So far, three different classes of ER stress transducer protein have been identified in (2)UPR pathway.
Each of them are ER-transmembrane protein that consists of a ER lumen part and a cytoplasm part: each part is located at inside and outside of the ER-membrane respectively. Although these three types of transducer protein recognize the misfold protein as a common input, their output signals are different. For instance, Ire1 outputs a signal which promotes splicing reaction of XBP1 mRNA. The crystal structure of Ire1 ER lumen part has already been determined. And it was turned out that Ire1 dimerizes into the active state in response to the misfold proteins. However, how Ire1 cytoplasm part dimerizes or the manner in which Ire1 removes intron remains unclear. For understanding these mechanisms, the crystal structure of Ire1 cytoplasm part has been determined.
The crystal structure of Ire1 dimer was determined at 2.4Å resolution. (Fig.3). Each protomer is consists of an N-terminal kinase domain and a C-terminal KEN domain(Kinase-Extension Nuclease domain). Furthermore, kinase domain is divided into two subdomains: a smaller N-terminal N-lobe and a larger C-terminal C-lobe. There is an ADP bounded to the cleft between N-lobe and C-lobe.
The dimer is bound together with some interactions which resident in two interfaces: N-lobe/N-lobe interface (Fig.4), and KEN domain/KEN domain interface (Fig.5). The amino acid residues forming these interactions are essential to the structure stability of Ire1. It was confirmed that Ire1 decreases significantly its functional ability by mutating these residues.
A set of experiments conducted by Lee,K et al revealed that Ire1 dimerize through multi-processes. The dimerization mechanism of Ire1 proposed by them was described below. (Fig.6).
The ribonuclease catalytic site of Ire1 is expected to be located near four residues(Y1049、R1056、N1057、H1061). (Fig.7). The arrangement of these four residues shows similarity to it of four ribonuclease catalytic residues in the tRNA-splicing endonuclease(xPSSS:2GJW). This fact indicates that the region is a candidate of the ribonuclease catalytic site. Further understanding of Ire1's ribonuclease catalytic mechanism awaits the crystal structure of Ire1 KEN domain in complex with RNA.
Protein Data Bank (PDB)
Lee, K.P. Dey, M. Neculai, D. Cao, C. Dever, T.E. Sicheri, F.; "Structure of the dual enzyme ire1 reveals the basis for catalysis and regulation in nonconventional RNA splicing."; Cell(Cambridge,Mass.); (2008) 132:89-100 PubMed:18191223.
author: Jun-ichi Ito