To establish the mechanism of Ire1 oligomerization and activation, we used X-ray crystallography. Because efforts to crystallize Ire1KR32 with ADP produced crystals unsuitable for X-ray data collection, we attempted to co-crystallize Ire1KR32 with structurally diverse protein kinase inhibitors. Remarkably, several kinase inhibitors activated the RNase function revealing synthetic activators of wild-type Ire1 ( and Supplementary Fig. 3
). These results have profound implications for therapeutic uses of kinase inhibitors (see Conclusions).
Kinase inhibitors activate the RNase of wild-type Ire1
Crystals obtained with the inhibitor APY29 allowed determination of the structure of the Ire1KR32•APY29 complex at 3.9 Å resolution. The resolution improved to 3.2 Å with a mutant version of Ire1KR32, Ire1KR32Δ28•APY29, in which we deleted the αF–αEF loop (28 amino acids, 865–892). The αF–αEF loop is not evolutionary conserved and was disordered in the 3.9-Å structure. Its deletion had no effect on the RNase activity of Ire1 (Supplementary Fig. 4
). Electron density for the APY29 molecule was found in the ATP-binding pocket of the Ire1 kinase domain (). The position of APY29 indicates that it could form three hydrogen bonds with residues Glu 746 and Cys 748 of the main chain and two additional hydrogen bonds or van der Waals contacts with the side chains Asn 751 and Asp 828 at the active site ().
Although all tested compounds can potentially form hydrogen bonds with the protein backbone (), the most potent activators, APY29 and APY24, also interact with the side chain Asn 751 and insert bulky aromatic rings in place of the ribose-phosphate moiety of ADP. Manual fitting of the FDA-approved anti-cancer drug Sunitinib guided by known structures of kinase•inhibitor complexes (Protein Data Bank (PDB) IDs 2G9X and 2F4J) predicts that the compound fills the adenine-binding site, but not the ribose and the phosphate subsites. Such partial occupancy could explain the fairly good binding of Sunitinib to Ire1 accompanied by partial activation of the enzyme (). AIN54 could not be fit to the ATP pocket owing to steric clashes with the β1 strand.
The interactions of APY29 with the nucleotide-binding pocket closely mimic those of ADP except that APY29 does not use a divalent metal ion for docking (). Accordingly, addition of EDTA inhibits the RNase activity of Ire1 for reactions stimulated by ADP but not APY29 (). These findings support a model first proposed based on Ire1 mutants16
: that ADP and kinase inhibitors activate Ire1 RNase by filling the ATP pocket. For maximum activity the adenine and the ribose subsites should be occupied, apparently to stabilize the active open conformation of the kinase that favours self-association of Ire1. Electrostatic interactions due to coordination of the magnesium ion and the phosphate groups of ADP do not have an indispensable role as the charged moieties can be replaced with neutral space-filling groups.
In contrast to Ire1, which lacks the oligomerization-inducing N-terminal segment and crystallizes as a back-to-back dimer14
, Ire1KR32 and Ire1KR32Δ28 crystallize as a symmetric high-order assembly (). Fourteen Ire1 molecules constitute the asymmetric unit in the crystal lattice. Formation of the oligomer can be described by incremental addition of symmetric back-to-back Ire1 dimers to an end of a growing filament, with a simultaneous clockwise turn of 51.4° per dimer, with a complete 360° turn every 14 molecules.
The use of 14-fold non-crystallographic symmetry (NCS) improved the quality of averaged electron density maps and helped the modelling of all of the regions missing from the starting model (Supplementary Fig. 5
). The structure of the kinase/RNase domain in the oligomer is similar to that in the Ire1•ADP dimer14
. However, tight packing of Ire1 in the oligomer compared to the crystal packing of the Ire1•ADP dimers (, inset) orders several fragments of Ire1 absent in the previous model (coloured green in ). None of the new elements belong to the interface IF1c
defined previously in the back-to-back dimer14
(). Two new interfaces, IF2c
, form in addition to the interface IF1c
in the oligomer. Interface IF2c
has a two-fold symmetry and forms by contacts between the RNase domains of monomers A–D, C–F, and so on (). Interface IF3c
creates a linear side-to-side arrangement of monomers into filaments (B→D→F and, with opposite polarity, A←C←E). Interface IF3c
is formed by contacts between the kinase domains and involves two new elements, the αD′ helix and the activation loop (). The oligomerization-inducing N-terminal extension (residues 641–662) was disordered. Its structure and the mechanism of facilitating Ire1 oligomerization remain to be determined. It is possible that part of the N-tail contacts a dimerization interface, as proposed recently for the arginine-rich linker extension of epidermal growth factor receptor20
Architecturally, the oligomer resembles the double helix of DNA ( and Supplementary Fig. 6
), where interface IF1c
parallels the interaction between nucleobases of opposing strands and interface IF3c
parallels phosphodiester linkages between nucleotides of the same strand.