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

Area of Science:

• Molecular Biology
• Biochemistry
• Cellular Biology

Background:

• Inositol-requiring enzyme 1 (Ire1) is a key endoplasmic reticulum (ER) transmembrane protein.
• Ire1 regulates the unfolded protein response (UPR) through its kinase and RNase domains.
• This study investigates how cofactor binding to Ire1's kinase domain affects its RNase activity.

Purpose of the Study:

• To elucidate the mechanism by which cofactor binding to Ire1's kinase domain modulates RNase activity.
• To understand the role of conformational changes and cofactor properties in Ire1 activation.
• To explore the structural basis of Ire1 oligomerization and its impact on RNase function.

Main Methods:

• Biochemical assays to measure Ire1 RNase activity.
• Cofactor binding studies using wild-type and mutant Ire1.
• X-ray crystallography to determine the structure of Ire1 complexes.
• Site-directed mutagenesis of the Ire1 kinase domain.

Main Results:

• Cofactor binding to Ire1's kinase domain occurs in two steps: initial binding followed by a conformational rearrangement.
• This conformational change, governed by cofactor chemical properties, activates the RNase domain.
• Substitution of oxygen with sulfur in ADP (ADPβS) prevents RNase activation, but this can be rescued by metal ions (Mn2+, Cd2+).
• Ire1 oligomerization, visualized by X-ray crystallography, is sufficient to induce an active conformation, even without cofactors.

Conclusions:

• Cofactor-induced Ire1 activation is coupled to receptor oligomerization, which triggers RNase activity.
• Ire1 activation involves distinct steps of cofactor binding and subsequent conformational rearrangement leading to self-association.
• Cofactors allosterically regulate protein-protein interactions in Ire1's kinase domain, suggesting broader roles for kinase-binding ligands in signaling.