Structural determinants stabilizing the axial channel of ClpP for substrate translocation.
Acyldepsipeptides (ADEPs) antibiotics bind to Escherichia coli ClpP mimicking the interactions that the IGL/F loops in ClpA or ClpX ATPases establish with the hydrophobic pockets surrounding the axial pore of the tetradecamer that the protease forms. ADEP binding induces opening of the gates blocking the axial channel of ClpP and allowing protein substrates to be translocated and hydrolysed in the degradation chamber. To identify the structural determinants stabilizing the open conformation of the axial channel for efficient substrate translocation, we constructed ClpP variants with amino acid substitutions in the N-terminal region that forms the axial gates. We found that adoption of a β-hairpin loop by this region and the integrity of the hydrophobic cluster at the base of this loop are necessary elements for the axial gate to efficiently translocate protein substrates. Analysis of ClpP variants from Bacillus subtilis suggested that the identified structural requirements of the axial channel for efficient translocation are conserved between Gram-positive and Gram-negative bacteria. These findings provide mechanistic insights into the activation of ClpP by ADEPs as well as the gating mechanism of the protease in the context of the ClpAP and ClpXP complexes.
Department of Biochemistry and Biomedical Sciences and M. G. DeGroote Institute for Infectious Diseases Research, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S4K1, Canada.
John
J
Alexopoulos
Bilal
B
Ahsan
Lopamudra
L
Homchaudhuri
Nabiha
N
Husain
Yi-Qiang
YQ
Cheng
Joaquin
J
Ortega
D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology*; D020285:Cryoelectron Microscopy; D004252:DNA Mutational Analysis; D047630:Depsipeptides / Q000737:chemistry* / Q000378:metabolism*; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007091:Image Processing, Computer-Assisted; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization
D047630:Depsipeptides; D050505:Mutant Proteins; D049071:Endopeptidase Clp