Binding and cleavage of E. coli HUbeta by the E. coli Lon protease.
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.
Liao
Jiahn-Haur
JH
Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.
Lin
Yu-Ching
YC
Hsu
Jowey
J
Lee
Alan Yueh-Luen
AY
Chen
Tse-An
TA
Hsu
Chun-Hua
CH
Chir
Jiun-Ly
JL
Hua
Kuo-Feng
KF
Wu
Tzu-Hua
TH
Hong
Li-Jenn
LJ
Yen
Pei-Wen
PW
Chiou
Arthur
A
Wu
Shih-Hsiung
SH
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
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C483567:Lon protein, E coli; D049070:Protease La