The ClpP N-terminus coordinates substrate access with protease active site reactivity.
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, ClpPDeltaN 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.
Jennings
Laura D
LD
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Bohon
Jen
J
Chance
Mark R
MR
Licht
Stuart
S
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017665:Hydroxyl Radical / Q000737:chemistry; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D023302:Protein Footprinting; D017384:Sequence Deletion; D013379:Substrate Specificity; D017356:Synchrotrons
D029968:Escherichia coli Proteins; D007328:Insulin; D010446:Peptide Fragments; D017665:Hydroxyl Radical; C483568:ClpA protease, E coli; C483572:ClpP protease, E coli; D049071:Endopeptidase Clp