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. 2009 Feb 5;457(7230):687-93.
doi: 10.1038/nature07661. Epub 2008 Dec 14.

The unfolded protein response signals through high-order assembly of Ire1

Alexei V Korennykh  1 , Pascal F Egea, Andrei A Korostelev, Janet Finer-Moore, Chao Zhang, Kevan M Shokat, Robert M Stroud, Peter Walter
Affiliations

The unfolded protein response signals through high-order assembly of Ire1

Alexei V Korennykh et al. Nature. .

Abstract

Aberrant folding of proteins in the endoplasmic reticulum activates the bifunctional transmembrane kinase/endoribonuclease Ire1. Ire1 excises an intron from HAC1 messenger RNA in yeasts and Xbp1 messenger RNA in metozoans encoding homologous transcription factors. This non-conventional mRNA splicing event initiates the unfolded protein response, a transcriptional program that relieves the endoplasmic reticulum stress. Here we show that oligomerization is central to Ire1 function and is an intrinsic attribute of its cytosolic domains. We obtained the 3.2-A crystal structure of the oligomer of the Ire1 cytosolic domains in complex with a kinase inhibitor that acts as a potent activator of the Ire1 RNase. The structure reveals a rod-shaped assembly that has no known precedence among kinases. This assembly positions the kinase domain for trans-autophosphorylation, orders the RNase domain, and creates an interaction surface for binding of the mRNA substrate. Activation of Ire1 through oligomerization expands the mechanistic repertoire of kinase-based signalling receptors.

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Figures

Figure 1
Figure 1. Activation of Ire1 by self-association
a, A general scheme of Ire1 activation during the UPR summarizing the key events. The kinase domain of Ire1 is coloured light brown; the RNase domain is coloured purple. TM, transmembrane. b, Schematic representation of RNA substrates used in this work. Triangles mark sites of specific cleavage by Ire1. c, Ire1 constructs used for cleavage assays and structure determination. d, Cooperative activation profiles for Ire1KR, Ire1KR24 and Ire1KR32 obtained using 5′-32P-HP21, with (filled circles) and without (open circles) cofactor. E, enzyme. Asterisks are used for reference in Fig. 2. Assay details are provided in Methods.
Figure 2
Figure 2. Linker controls the oligomerization and activation of Ire1
a, Observation of visible self-association of Ire1KR32 that can be reversed by salt (NaCl). b, Analytical ultracentrifugation reveals monomers and dimers for Ire1KR as well as dimers and higher-order assemblies for Ire1KR32. Conditions were as in Fig. 1d; open symbols, 13.5 μM Ire1 (20 °C). c, Salt inhibits the RNase activity of Ire1KR32. d, Ire1KR32 has higher RNase activity against HP21 and Xbp1 RNA compared to Ire1KR and Ire1KR24 (Supplementary Fig. 1b; 2). Error bars show variability between single-exponential fits from two to five independent measurements. Conditions similar to those used in Fig. 1d are marked * and **.
Figure 3
Figure 3. Kinase inhibitors activate the RNase of wild-type Ire1
a, Activation of Ire1KR32 (3 μM) in the presence of different kinase inhibitors. b, Inhibitor structures, with probable hydrogen bonds shown by dashed lines. c, σA-weighted 3Fobs–2Fcalc map for APY29 bound to Ire1KR32Δ28 contoured at 1.5σ. d, The network of probable hydrogen bonds between APY29 and Ire1. e, The network of interactions between ADP•Mg and Ire1 (PDB ID 2RIO). f, Chelation of magnesium inhibits Ire1 RNase in the presence of ADP, but not of APY29. Error bars show variability between single-exponential fits from two independent measurements. Reactions contained 2 mM ADP or 100 μM APY29.
Figure 4
Figure 4. Structure of the Ire1 oligomer
a, Assembly of Ire1KR32Δ28•APY29. A parallel filament in the crystal packing is shown above the main filament. Domains are coloured as in Fig. 1a. The inset shows crystal packing of Ire1 dimers (PDB ID 2RIO). Letters A–N below the structure refer to individual monomers in the asymmetric unit. K, kinase; R, ribonuclease. b, Three intermolecular interfaces of Ire1KR32Δ28 in the oligomer. c, Dimer formed via interface IF1c. d, Dimer formed via interface IF2c. e, Dimer formed via interface IF3c (left). Close view of the activation loop (right). Phosphates are shown in ball representation. Arrows in b and e show the direction of the activation loop donation. Regions with previously unknown structures are coloured green.
Figure 5
Figure 5. Three interfaces of Ire1 contribute to the RNase activity
a, Schematic representation of the Ire1 oligomer packing. Stars mark the ATP-binding pocket of the Ire1 kinase. Lower panel shows a single monomer; upper panel shows packing of four monomers. b, Contacts at the intermolecular interfaces of the oligomer. c, Mutations of the predicted interface residues designed to weaken IF1c, IF2c and IF3c inhibit the RNase activity of Ire1KR32. Reactions contained 5′-32P-HP21, 3 μM Ire1KR32 and 2 mM ADP. WT, wild type. Error bars show variability between single-exponential fits from two independent measurements. The colour of the bars matches that in a and b.
Figure 6
Figure 6. The mechanism of Ire1 activation
a, Time courses for cleavage of Xbp1 (lanes 1–6) and of Xbp1/PstI (lanes 7–12) with 5 nM Ire1KR32. b, Quantification of the gel in a. c, Cleavage of RNA with one and two splice sites by Ire1KR32. Error bars show variability between single-exponential fits from two to five independent measurements. nt, nucleotide. d, Position of the HLE at the RNase–RNase interface IF2c. e, Molecular surface representation of the RNase domain in the oligomer and in the dimer structures. Putative catalytic residues in d and e are coloured orange; HLE is coloured green. f, Revised model of Ire1 activation during the UPR.
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