Mechanism of Coupled Folding of Disordered Colicin E3 rRNase Domain Upon Binding with Im3

Abstract

Intrinsically disordered proteins (IDPs) are universally observed in all domains of life. They play critial roles in many cellular processes, including signal transduction, transcription and translation regulation, cell division, and cell death. IDPs are also involved in various diseases, such as cancer and Alzheimer's disease. Therefore, it is important to understand the mechanisms of association of IDPs with their targets. Colicin E3 is a ribosomal RNase toxin released by Escherichia coli. Although the colicin E3 rRNase domain is folded, alanine mutant of Tyr507 within the hydrophobic core unfolded the protein. Interestingly, the unfolded E3 rRNaseY507A is still able to bind to Im3 and forms a complex identical to the wild-type protein. In this work, pre-steady-state kinetic analysis, thermodynamic analysis and Φ value analysis were performed to investigate the coupled folding-binding mechanism between E3 rRNaseY507A and Im3, and the binding mechanism of the E3 rRNaseY507A was compared with the WT folded protein. We found that the encounter process was steered by complementary electrostatic interactions between the two molecules. However, the effect of electrostatic steering is much weaker for the unfolded E3 rRNaseY507A than that for the WT E3 rRNase. Φ value analysis revealed that the E3 rRNaseY507A is folded and forms many native intermolecular contacts in the transition state. Our results provide clues to explain why introducing disorder affects the association rate but not the dissociation rate for the E3 rRNase/Im3 interaction.