Characterisation of a GroEL single-ring mutant that supports growth of Escherichia coli and has GroES-dependent ATPase activity.
Binding and folding of substrate proteins by the molecular chaperone GroEL alternates between its two seven-membered rings in an ATP-regulated manner. The association of ATP and GroES to a polypeptide-bound ring of GroEL encapsulates the folding proteins in the central cavity of that ring (cis ring) and allows it to fold in a protected environment where the risk of aggregation is reduced. ATP hydrolysis in the cis ring changes the potentials within the system such that ATP binding to the opposite (trans) ring triggers the release of all ligands from the cis ring of GroEL through a complex network of allosteric communication between the rings. Inter-ring allosteric communication thus appears indispensable for the function of GroEL, and an engineered single-ring version (SR1) cannot substitute for GroEL in vivo. We describe here the isolation and characterisation of an active single-ring form of the GroEL protein (SR-A92T), which has an exceptionally low ATPase activity that is strongly stimulated by the addition of GroES. Dissection of the kinetic pathway of the ATP-induced structural changes in this active single ring can be explained by the fact that the mutation effectively blocks progression through the full allosteric pathway of the GroEL reaction cycle, thus trapping an early allosteric intermediate. Addition of GroES is able to overcome this block by binding this intermediate and pulling the allosteric pathway to completion via mass action, explaining how bacterial cells expressing this protein as their only chaperonin are viable.
Kovács
Eszter
E
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Sun
Zhe
Z
Liu
Han
H
Scott
David J
DJ
Karsisiotis
Andreas I
AI
Clarke
Anthony R
AR
Burston
Steven G
SG
Lund
Peter A
PA
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D018835:Chaperonin 10 / Q000378:metabolism*; D018834:Chaperonin 60 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D005798:Genes, Bacterial; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018835:Chaperonin 10; D018834:Chaperonin 60; D021122:Protein Subunits; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases