Binding and Cleavage of E. coli HU β by the E. coli Lon Protease

The Escherichia coli Lon protease degrades the E. coli DNA-binding protein HU β, but not the related protein HU α. Here we show that the Lon protease binds to both HU β and HU α, but selectively degrades only HU β in the presence of ATP. Mass spectrometry of HU β peptide fragments revealed that region K18-G22 is the preferred cleavage site, followed in preference by L36-K37. The preferred cleavage site was further refined to A20-A21 by constructing and testing mutant proteins; Lon degraded HU β-A20Q and HU β-A20D more slowly than HU β. We used optical tweezers to measure the rupture force between HU proteins and Lon; HU α, HU β, and HU β-A20D can bind to Lon, and in the presence of ATP, the rupture force between each of these proteins and Lon became weaker. Our results support a mechanism of Lon protease cleavage of HU proteins in at least three stages: binding of Lon with the HU protein (HU β, HU α, or HU β-A20D); hydrolysis of ATP by Lon to provide energy to loosen the binding to the HU protein and to allow an induced-fit conformational change; and specific cleavage of only HU β.