Protein kinases are dynamic enzymes that couple catalysis to pronounced conformational changes. Studies of their dynamics are limited by the intrinsic difficulty of uncoupling enzymatic phosphoryl transfer from conformational properties. In the present study, we exploited the unique facets of Ire1 biology to overcome this shortcoming. The approach was made possible by Ire1's unique property to form easily quantified high-order homo-oligomers upon activation and by Ire1's RNase domain, which provides a natural built-in reporter of the kinase domain conformation. Conformational changes in Ire1's kinase domain modulate the enzyme's predisposition to form oligomers, in which Ire1-Ire1 contacts across multiple interfaces activate Ire1's RNase activity.
The propensity of Ire1 for activation is modulated by three parameters: (1) its local concentration, (2) its phosphorylation state and (3) its interactions with cofactors in the ATP binding site of Ire1's kinase module. In its physiological setting, the local concentration of the cytoplasmic Ire1 kinase/RNase module is responsive to the protein-folding conditions inside the ER lumen. As unfolded proteins accumulate there, the Ire1's luminal domain oligomerizes, thereby concentrating the covalently tethered kinase/RNase domains on the other side of the membrane. This concentration event is thought to provide the primary switch leading to Ire1 activation. In this view, trans
-autophosphorylation would ensue following juxtaposition of the kinase domains. The phosphorylation state of Ire1 is a dynamic parameter that changes over time and is thought to contribute to a molecular timer. Initial phosphorylation events on the activation loop lead to phosphorylated side chains that form stabilizing salt bridges to neighboring Ire1 molecules in the active oligomer [8
], whereas subsequent hyperphosphorylation events appear to promote Ire1 turnoff, perhaps because of oligomer-destabilizing charge repulsion effects [20
Cofactor binding to the ATP pocket of the kinase domain is expected to provide another regulatory input that strongly modulates the sensitivity of the switch to integrate UPR signaling with the physiological state in the cell's cytoplasm. In particular, it is intriguing to speculate that UPR may be modulated by the ADP/(ATP+AMP) ratio due to the 20-fold higher Pcof
of ADP compared to ATP and AMP. Up to an order of magnitude change in the ADP/ATP ratio has been reported during glucose starvation in pancreatic β cells and during apoptosis [30
]. If this were also the case during the UPR, an increase in the relative ADP level would sensitize Ire1, requiring lower concentrations of unfolded proteins to activate the UPR. An alternative possibility could be that an unknown stress-induced small-molecule metabolite with high Pcof
and low Kcof
, other than nucleotides, could serve biological roles in modulating Ire1 activity via binding in the ATP pocket. Work in mammalian systems suggests that Ire1 can be activated independently of an accumulation of unfolded proteins in the ER lumen, perhaps by utilizing such a mechanism to shift the oligomerization activation threshold [32
]. It has been shown that, in addition to the ATP pocket, Ire1 has a different pocket in the RNase domain and that binding of quercetin to this pocket stimulates Ire1 RNase [33
]. This finding opens up a possibility that, in addition to the kinase-binding ligands, ligands binding to the RNase domain of Ire1 contribute to modulation of Ire1 signaling.
Phosphorylation of Ire1 considerably affects its responsiveness to cofactors. In the presence of ADP, fully phosphorylated Ire1KR32 exhibits a 40-fold stronger Pcof
(approximately 200) compared to Ire1KR32(D797N, K799N) lacking phosphates (Pcof
approximately 5) (data not shown). Therefore, phosphorylation not only promotes Ire1 oligomerization but also "primes" the receptor for sensing ATP pocket-binding cofactors. Notably, unlike Pcof
, binding of cofactors to Ire1 does not appear to depend on Ire1 phosphorylation, as both Ire1KR32 and Ire1KR32(D797N, K799N) exhibit similar Kcof
values with ADP (data not shown). The effect of phosphorylation on Ire1 sensitivity to cofactors has also been observed during the study of the quercetin pocket [33
]. These observations suggest that modulation of Ire1 signaling by cofactors may be physiologically most important after the UPR has been initiated, amplifying the activity of Ire1 molecules that have already been phosphorylated.
By measuring the Ire1 RNase activity in response to kinase-bound small molecules, we resolved two independent steps in cofactor-Ire1 interactions: cofactor binding and the conformational response to a bound cofactor. Noting the wide spectrum of potencies of different cofactors at saturation, Pcof, we propose a simple model in which cofactors bound in the ATP pocket shift an equilibrium between an inactive and an active conformation of Ire1 (Figure ). In the model, the inactive conformation "O" corresponds to that of a free Ire1 monomer that has the αC-helix in the inactive, "out" position also observed for other protein kinases. The active conformation "I" corresponds to that of Ire1 oligomers with the αC-helix in the active, "in" position. The conformational equilibrium between the "O" and "I" states depends strongly on the chemical nature of the bound cofactor. Whereas some cofactors, such as ADPβS, bind without noticeably perturbing this equilibrium, other natural and synthetic cofactors effect an equilibrium shift of over two orders of magnitude. The effect of the cofactors on the oligomerization equilibria (Figures versus ) is summarized in the free energy diagram shown in Figure . According to our model shown in Figure , cofactors lower the free energy of the "monomer I" state (compare the "monomer I" states on the solid and the dashed lines), ultimately resulting in an equilibrium shift toward oligomerization (compare the "oligomer I" states on the solid and the dashed lines).
Figure 6 Model of cofactor action during Ire1 oligomerization and activation. (a) Two-step model for oligomerization and activation of apo-Ire1. (b) Three-step model for cofactor-stimulated Ire1 activation involving equilibrium of two distinct cofactor-bound Ire1 (more ...)
Quantitative manifestations of the two steps, apparent cofactor affinity Kcof
and cofactor potency Pcof
, do not correlate (Figure vs. Figure ), suggesting that they arise from distinct (perhaps partially overlapping) subsets of molecular interactions. The ADP/ADPβS comparison (Figure ) indicates that the kinase hinge-contacting face [8
] of the cofactors serves for binding, whereas the K702/E715-contacting face (Figure and Additional file 1
Figure S4) modulates the kinase conformation and could be exploited for designing potent synthetic Ire1 activators. Our findings show that Ire1, as already known for other protein kinases, can exist with the ATP pocket filled by cofactors while remaining in the inactive conformation. With certain cofactors such as ADPβS, this functional state could dominate in solution, a notion previously unappreciated in studies of Ire1.
The macroscopically measured parameters Kcof
are composite derivatives of the microscopic constants
. (Figure and Additional file 1
, Supplementary Analysis 1). The Kcof
value depends largely on (and is equal to, when
is large) the true microscopic binding constant
. The Pcof
value depends only on
and not on the binding constant
. Therefore, Pcof
is indeed a quantitative metric of conformational responsiveness to bound cofactor, uncoupled from the cofactor binding step, and describes the apparent pressure pushing the kinase domain toward the active conformation with the αC-helix "in" position. For ADP, Pcof
arises predominantly because of the β-phosphate-magnesium insertion into the kinase domain (β-phosphate latch), which is functionally defined herein using the metal specificity switch approach.
Synthetic molecules lacking a β-phosphate equivalent altogether can achieve the same goal in the absence of Mg2+
ions by inserting bulky moieties in the β-phosphate position. In agreement with this model, sunitinib (Additional file 1
, Figure S4), which is predicted to occupy the β-phosphate position only partially [8
], has a relatively low Pcof
(approximately 30) (Figure ).
In CDK2, productive docking of the ATP β-phosphate in the latched position is facilitated by an external effector, cyclin, that stabilizes the αC-helix "in" conformation extrinsically [12
]. A conceptually similar mechanism is employed by EGFR, which dimerizes using one EGFR monomer to stabilize another EGFR monomer in the αC-helix "in" conformation [12
]. Ire1 does not require a cyclin equivalent, but instead uses homo-oligomerization to stabilize the αC-helix "in" conformation.