title: Binding and cleavage of E. coli HUbeta by the E. coli Lon protease.
abstract: The Escherichia coli Lon protease degrades the E. coli DNA-binding protein HUbeta, but not the related protein HUalpha. Here we show that the Lon protease binds to both HUbeta and HUalpha, but selectively degrades only HUbeta in the presence of ATP. Mass spectrometry of HUbeta 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 HUbeta-A20Q and HUbeta-A20D more slowly than HUbeta. We used optical tweezers to measure the rupture force between HU proteins and Lon; HUalpha, HUbeta, and HUbeta-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 (HUbeta, HUalpha, or HUbeta-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 HUbeta.
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Jiahn-Haur
authors.initials: JH
authors.lastname: Liao
authors.forename: Yu-Ching
authors.initials: YC
authors.lastname: Lin
authors.forename: Jowey
authors.initials: J
authors.lastname: Hsu
authors.forename: Alan Yueh-Luen
authors.initials: AY
authors.lastname: Lee
authors.forename: Tse-An
authors.initials: TA
authors.lastname: Chen
authors.forename: Chun-Hua
authors.initials: CH
authors.lastname: Hsu
authors.forename: Jiun-Ly
authors.initials: JL
authors.lastname: Chir
authors.forename: Kuo-Feng
authors.initials: KF
authors.lastname: Hua
authors.forename: Tzu-Hua
authors.initials: TH
authors.lastname: Wu
authors.forename: Li-Jenn
authors.initials: LJ
authors.lastname: Hong
authors.forename: Pei-Wen
authors.initials: PW
authors.lastname: Yen
authors.forename: Arthur
authors.initials: A
authors.lastname: Chiou
authors.forename: Shih-Hsiung
authors.initials: SH
authors.lastname: Wu
mesh_terms: D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D008956:Models, Chemical; D049070:Protease La / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C483567:Lon protein, E coli; D049070:Protease La