Allosteric modulation of the Lon protease by effector binding and local charges.

Abstract

ssDNA binding allosterically activates Lon ATP hydrolysisNegative charge at DNA binding site is sufficient for Lon activationNeutralization of charge at DNA binding site inhibits Lon ATP hydrolysisLon activity is linked to formation of stable Lon hexamers. The energy-dependent protease Lon is integral in both eukaryotic and prokaryotic physiology, contributing to protein quality control, stress management, developmental regulation, and pathogenicity. The ability to precisely regulate protein levels through targeted degradation underscores a need for tunability. We find that single-stranded DNA (ssDNA) acts as an allosteric regulator of Lon, leading to enhanced enzymatic activity. Mutations in basic residues crucial for DNA binding were found to affect Lon activity in a charge-specific manner highlighting the importance of electrostatic interactions regulating Lon's function. Changes in Lon activity due to ssDNA binding or mutations were correlated with its oligomerization state. Our findings provide insights into the activation strategies of Lon, emphasizing the role of electrostatic contribution that modulate nucleotide affinity, oligomerization and proteolysis to advance our understanding of the complex regulatory mechanisms of the Lon protease.