GrpE accelerates nucleotide exchange of the molecular chaperone DnaK with an associative displacement mechanism.
The ATP hydrolysis and protein-binding and release cycle of the molecular chaperone DnaK is regulated by the accessory proteins GrpE and DnaJ. Here we describe a study of the formation of complexes between the molecular chaperone DnaK, its nucleotide exchange factor GrpE, and the fluorescent ADP analog N8-[4-[(N'-methylanthraniloyl)amino]butyl]-8-aminoadenosine 5'-diphosphate (MABA-ADP) by equilibrium and stopped flow kinetic experiments. The catalytic cycle of the GrpE-stimulated nucleotide exchange involves a ternary DnaK x GrpE x ADP complex as well as the binary DnaK x GrpE and DnaK x ADP complexes. The equilibrium data of the interaction of GrpE with DnaK x ADP and the nucleotide-free DnaK can be described by a simple equilibrium system where GrpE reduces the affinity of ADP for DnaK 200-fold. However, transient kinetic studies revealed that the functional cycle of GrpE in addition includes at least two distinct ternary DnaK x GrpE x ADP complexes. Our data indicate that the initial weak binding of GrpE to DnaK x ADP is followed by an isomerization of the ternary complex which leads to weakening of nucleotide binding and finally to its rapid dissociation. The maximal stimulation for nucleotide exchange brought about by GrpE was found to be 5000-fold. We propose that this kinetically observed isomerization represents a structural change (opening) of the nucleotide binding pocket of DnaK that allows for fast nucleotide exchange.
Packschies
L
L
Abteilung Physikalische Biochemie, Max-Planck-Institut für Molekulare Physiologie, Dortmund, Germay.
Theyssen
H
H
Buchberger
A
A
Bukau
B
B
Goody
R S
RS
Reinstein
J
J
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D018832:Molecular Chaperones / Q000378:metabolism*; D011487:Protein Conformation; D062367:ortho-Aminobenzoates / Q000378:metabolism
D001426:Bacterial Proteins; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C068113:GrpE protein, Bacteria; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; C105264:N(8)-(4-((N'-methylanthraniloyl)amino)butyl)-8-aminoadenosine 5'-diphosphate; D062367:ortho-Aminobenzoates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli