Probing the role of H1061 in catalysis. (a) Effect of H1061N mutation on the RNase activity of Ire1. Error bars show standard error of single-exponential fitting. (b) Oligomerization profiles of Ire1KR32 and three mutants, Ire1KR32(H1061N), Ire1KR32(Y1043F) and Ire1KR32(R1039A) measured using light absorbance at 500 nM (see Methods). Measurements were conducted at room temperature using the same reaction buffers as in Figure 1c. (c) Comparison of Ire1 structures from oligomers formed by wild-type Ire1 (PDB ID 3fbv, space group P21212) and by H1061N mutant (new 3.65Å crystal structure, also in space group P21212). Shown are overall oligomer architectures (left), 2Fo-Fc electron density map for the H1061N RNase domain contoured at 1.3σ (middle) and superposition of the RNase domains (right). Simulated annealing Fo-Fc omit map for H1061N calculated without non-crystallographic symmetry (NCS) is shown in Additional file 1, Figure S2. (d) In trans activation of Ire1KR32 by the non-catalytic mutant H1061N. Concentration of wild-type Ire1KR32 was 1.5 μM, concentration of the H1061N Ire1 varied between 0.1-15 μM. Arrow marks the point of equivalent concentrations. Reactions were conducted under single-turnover conditions using the same reaction buffer as in Figure 1c.
Korennykh et al. BMC Biology 2011 9:47 doi:10.1186/1741-7007-9-47