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Title: The ClpP N-Terminus Coordinates Substrate Access with Protease Active Site Reactivity

Abstract

Energy-dependent protein degradation machines, such as the Escherichia coli protease ClpAP, require regulated interactions between the ATPase component (ClpA) and the protease component (ClpP) for function. Recent studies indicate that the ClpP N-terminus is essential in these interactions, yet the dynamics of this region remain unclear. Here, we use synchrotron hydroxyl radical footprinting and kinetic studies to characterize functionally important conformational changes of the ClpP N-terminus. Footprinting experiments show that the ClpP N-terminus becomes more solvent-exposed upon interaction with ClpA. In the absence of ClpA, deletion of the ClpP N-terminus increases the initial degradation rate of large peptide substrates 5-15-fold. Unlike ClpAP, ClpP?N exhibits a distinct slow phase of product formation that is eliminated by the addition of hydroxylamine, suggesting that truncation of the N-terminus leads to stabilization of the acyl-enzyme intermediate. These results indicate that (1) the ClpP N-terminus acts as a 'gate' controlling substrate access to the active sites, (2) binding of ClpA opens this 'gate', allowing substrate entry and formation of the acyl-enzyme intermediate, and (3) closing of the N-terminal 'gate' stimulates acyl-enzyme hydrolysis.

Authors:
; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
980622
Report Number(s):
BNL-93540-2010-JA
TRN: US1005531
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 47
Country of Publication:
United States
Language:
English
Subject:
43 PARTICLE ACCELERATORS; CONFORMATIONAL CHANGES; COORDINATES; DYNAMICS; ESCHERICHIA COLI; HYDROLYSIS; HYDROXYL RADICALS; HYDROXYLAMINE; INTERACTIONS; KINETICS; PEPTIDES; PROTEINS; REACTIVITY; STABILIZATION; SUBSTRATES; SYNCHROTRONS; USES; national synchrotron light source

Citation Formats

Jennings, L., Bohon, J, Chance, M, and Licht, S. The ClpP N-Terminus Coordinates Substrate Access with Protease Active Site Reactivity. United States: N. p., 2008. Web. doi:10.1021/bi8010169.
Jennings, L., Bohon, J, Chance, M, & Licht, S. The ClpP N-Terminus Coordinates Substrate Access with Protease Active Site Reactivity. United States. doi:10.1021/bi8010169.
Jennings, L., Bohon, J, Chance, M, and Licht, S. Tue . "The ClpP N-Terminus Coordinates Substrate Access with Protease Active Site Reactivity". United States. doi:10.1021/bi8010169.
@article{osti_980622,
title = {The ClpP N-Terminus Coordinates Substrate Access with Protease Active Site Reactivity},
author = {Jennings, L. and Bohon, J and Chance, M and Licht, S},
abstractNote = {Energy-dependent protein degradation machines, such as the Escherichia coli protease ClpAP, require regulated interactions between the ATPase component (ClpA) and the protease component (ClpP) for function. Recent studies indicate that the ClpP N-terminus is essential in these interactions, yet the dynamics of this region remain unclear. Here, we use synchrotron hydroxyl radical footprinting and kinetic studies to characterize functionally important conformational changes of the ClpP N-terminus. Footprinting experiments show that the ClpP N-terminus becomes more solvent-exposed upon interaction with ClpA. In the absence of ClpA, deletion of the ClpP N-terminus increases the initial degradation rate of large peptide substrates 5-15-fold. Unlike ClpAP, ClpP?N exhibits a distinct slow phase of product formation that is eliminated by the addition of hydroxylamine, suggesting that truncation of the N-terminus leads to stabilization of the acyl-enzyme intermediate. These results indicate that (1) the ClpP N-terminus acts as a 'gate' controlling substrate access to the active sites, (2) binding of ClpA opens this 'gate', allowing substrate entry and formation of the acyl-enzyme intermediate, and (3) closing of the N-terminal 'gate' stimulates acyl-enzyme hydrolysis.},
doi = {10.1021/bi8010169},
journal = {Biochemistry},
number = ,
volume = 47,
place = {United States},
year = {2008},
month = {1}
}