Binding and cleavage of E. coli HUβ by the E. coli Lon protease
Journal
Biophysical Journal
Journal Volume
98
Journal Volume
98
Journal Issue
1
Journal Issue
1
Pages
129-137
Start Page
129
End Page
137
ISSN
00063495
Date Issued
2010-01-06
Author(s)
Liao, Jiahn-Haur
Lin, Yu-Ching
Hsu, Jowey
Lee, Alan Yueh-Luen
Chen, Tse-An
Chir, Jiun-Ly
Hua, Kuo-Feng
Wu, Tzu-Hua
Hong, Li-Jenn
Yen, Pei-Wen
Chiou, Arthur
Wu, Shih-Hsiung
Abstract
The Escherichia coli Lon protease degrades the E. coli DNA-binding protein HUβ, but not the related protein HUα. Here we show that the Lon protease binds to both HUβ and HUα, but selectively degrades only HUβ in the presence of ATP. Mass spectrometry of HUβ 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 HUβ-A20Q and HUβ-A20D more slowly than HUβ. We used optical tweezers to measure the rupture force between HU proteins and Lon; HUα, HUβ, and HUβ-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 (HUβ, HUα, or HUβ-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 HUβ.
Publisher
Biophysical Society
Type
journal article