Insights into the molecular organization of Ire1

The crystal structure of Ire1’s ER-lumenal domain from S. cerevisiae has been solved in an oligomeric state (Credle et al., 2005). The structure provided valuable structural information promoting the characterization of Ire1’s activation mechanism. It revealed interfaces for oligomerization and the presence of a putative binding site for unfolded proteins (Credle et al., 2005). Systematic deletion studies have helped to divide the lumenal domain of Ire1 into five functionally distinct subregions (Fig.13, A and B). Subregion I (AA 25-104) has a regulatory function that serves to inhibit Ire1’s self-association in the absence of ER-stress (Oikawa et al., 2007). Subregion V contains a binding site for the ER chaperone Kar2 (BiP), which is known to bind Ire1 in the absence of ER-stress, and is thought to keep it in a monomeric inactive state (Kimata et al., 2004). Simultaneous deletion of these two regulatory subregions results in self-association of Ire1 molecules and renders Ire1 constitutively active (Oikawa et al., 2007; Promlek et al., 2011). The subregions II (AA 105-235), III (AA 236-265) and IV (AA 266-447) form the core lumenal domain (cLD), the protein folding stress-sensing region of Ire1. These subregions mostly form β-sheets, and subregions II and III form the peptide binding groove within the cLD of Ire1. The cLD is conserved among species and contains a peptide binding groove for unfolded proteins and tends to oligomerize (Fig. 13 D). The peptide binding groove shows striking similarities to the geometry of the central peptide binding groove in major-histocompatibility complex I (MHC-I) molecules (Bjorkman et al., 1987). The cLD consists of β-sheets forming a flat bottom surface and two parallel α-helices, which lay on top

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of the flat structure formed by the β-sheets. Thus, the two alpha helices form the outside border of a peptide binding groove (Fig. 13 D). When Ire1 dimerizes, the peptide binding groove fully assembles and its hydrophobic nature allows for a direct interaction of Ire1 with unfolded proteins, which has been proposed to drive the oligomerization of Ire1 upon binding unfolded proteins (Credle et al., 2005; Gardner and Walter, 2011; Oikawa et al., 2007).

Figure 13 | Schematic representation, and structural organization of Ire1's ER-lumenal sensor domain.

(A) Schematic representation of full-length Ire1. The ER-lumenal domain consists of subregions I and V (I; V) and the core lumenal domain (cLD). The lumenal domain is connected to the cytosolic kinase (K) and RNase (R) domain by a TMH, followed by a large loop region. (B) Organization of the ER-lumenal domain. Subregions I to V and their respective amino acid regions are depicted. The cLD and localization of the Kar2 binding domain are labeled. (C) Schematic representation of IFL1 and IFL2 localization in Ire1 oligomers and (D) x-ray structure of the dimeric lumenal domain of Ire1 (pdb: 2BE1; (Credle et al., 2005).

Mutations destroying the IFL1 (T226A, F247A; red) and IFL2 (W426A; yellow) are depicted. Individual figures are adapted from Halbleib et al. (Halbleib et al., 2017).

Oligomerization of Ire1’s lumenal domain is crucial for the activation of Ire1. During this process, two interaction interfaces are established. The lumenal interface 1 (IFL1) is located at the interface of two Ire1 monomers (Fig. 13, C and D). The mutation of two residues in the cLD domain (T226, F247) destroys the formation of this interface, resulting in a diminished formation of Ire1 dimers and consequently, impaired Ire1 functionality. The lumenal interface 2 (IFL2) is located at the interface of two Ire1 dimers (Fig. 13, C and D). Mutation of the W426 residue in the cLD destroys this interface, hence inhibits the formation of high-order Ire1 oligomers, which reduces the functionality of Ire1 to the same extend (Credle et al., 2005).

Dimerization of Ire1’s lumenal domain leads to a simultaneous dimerization of the cytosolic

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part of the protein in a back-to-back fashion (Lee et al., 2008). The dimerization is followed by the formation of higher order oligomers by the cytosolic portion of Ire1. This oligomerization renders Ire1’s kinase and RNase domains active by providing an interaction of cytosolic domains of neighboring Ire1 molecules. The interaction is a prerequisite for trans-autophosphorylation of the kinase domains at specific serine and threonine residues (S840, S841 and T844), and for stabilizing the active sites of the RNase domains of neighboring Ire1 molecules, which collectively form an RNA-substrate binding cavity (Korennykh et al., 2009;

Walter and Ron, 2011).

Figure 14 | Schematic representation, and structural organization of Ire1.

(A) Schematic representation of full-length Ire1. The ER-lumenal domain consists of subregions I and V (I; V) and the core lumenal domain (cLD). The lumenal domain is connected to the cytosolic kinase (K) and RNase (R) domain by a TMH, followed by a large loop region. The activation loop in the kinase domain is illustrated in green. (B) Organization of Ire1 monomers within high-order oligomers of Ire1. This illustration is based on the x-ray structure (pdb: 2RIO) by Korennykh et al. (Korennykh et al., 2009). The interaction interfaces are highlighted by the illustrated color code. (C) 3 -dimensional model of the organization of Ire1 monomers within high-order oligomers as described in (B). Figures were adapted from Van Anken et al. (van Anken et al., 2014)

As previously described for the lumenal domain of Ire1, the cytosolic domain also establishes interaction interfaces, which are pivotal for Ire1 function. Three cytosolic interfaces have been identified, and Ire1’s functionality is impaired, when residues forming these interfaces are mutated (Fig. 14) (van Anken et al., 2014; Korennykh et al., 2009): i) IFC1 establishes RNase-RNase contacts of two neighboring Ire1 molecules in the back-to-back dimer. ii) IFC2 establishes interactions of RNase and kinase domains of inter-dimeric Ire1 molecules within higher oligomers. iii) IFC3 establishes a kinase-kinase interaction of inter-dimeric Ire1 molecules within higher oligomers (Korennykh et al., 2009).

While active RNase domains of Ire1 are required for processing of HAC1 mRNA and therefore essential for Ire1 function, it has been reported that kinase activity of Ire1 is not required for

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Ire1 activation. It rather serves as a regulator in the process of Ire1 deactivation by changing the position of an activation loop, located in the kinase domain of Ire1 (Fig.14 B and C) (Korennykh et al., 2009; Rubio et al., 2011). These and other observations emphasize that the intermolecular interactions of Ire1 molecules are extremely complex, which in turn highlights the importance of the formation of inter- and intradimer interfaces to stabilize the formation of high-order Ire1 oligomers to reach full Ire1 signaling activity.

The transmembrane helix and juxta-membrane regions of Ire1 (collectively referred to as transmembrane region; TMR) connects the ER-lumenal domain of Ire1 with the cytosolic effector domains. The underlying hypothesis of this thesis is that the TMH and/ or juxta-membrane region of Ire1 is involved in the sensing and activation mechanism of Ire1 by lipid bilayer stress.

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