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

作者：Liao Jiahn Haur; Lin Yu Ching; Hsu Jowey; Lee Alan Yueh Luen; Chen Tse An; Hsu Chun Hua; Chir Jiun Ly; Hua Kuo Feng; Wu Tzu Hua; Hong Li Jenn; Yen Pei Wen; Chiou Arthur; Wu Shih Hsiung^*

来源：Biophysical Journal, 2010, 98(1): 129-137.

DOI：10.1016/j.bpj.2009.09.052

The Escherichia coli Lon protease degrades the E. coli DNA-binding protein HU beta, but not the related protein HU alpha. Here we show that the Lon protease binds to both HU beta and HU alpha, but selectively degrades only HU beta in the presence of ATP. Mass spectrometry of HU beta 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 beta-A20Q and HU beta-A20D more slowly than HU beta. We used optical tweezers to measure the rupture force between HU proteins and Lon; HU alpha, HU beta, and HU beta-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 beta, HU alpha, or HU beta-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 beta.

• 出版日期2010-1-6