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Figure 2 | BMC Biology

Figure 2

From: Cofactor-mediated conformational control in the bifunctional kinase/RNase Ire1

Figure 2

Allosteric activation of Ire1 RNase by ligands binding to the kinase domain. (a) Activation of Ire1KR32 by AMP, ADP and ATP. The kobs values of RNase activity were determined as a function of nucleotide Mg2+ concentration (see Methods). (b) and (c) Characterization of Ire1 activity with a panel of nucleotides and synthetic ligands. Maximum Ire1 RNase stimulation achieved with cofactors at saturation (b) and apparent cofactor binding constants (c) are shown. The scale of cofactor potency (Pcof) is indicated in (b). SUNT = sunitinib. Cofactor structures are shown in Additional file 1, Figure S4. (d) Measurement of binding constants for ADPβS and ATP via inhibition of a reaction stimulated with 0.2 mmol ADP. Ki values of 0.20 ± 0.07 mmol and 0.10 ± 0.04 mmol were determined for ADPβS and ATP, respectively. The data were fit to the binding isotherm and corrected for the 0.2 mmol ADP background. All reactions contained 3 μmol Ire1KR32 and 2 mmol Mg2+. Error bars show standard errors of binding isotherm fitting.

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