ATP hydrolysis is required for the DnaJ-dependent activation of DnaK chaperone for binding to both native and denatured protein substrates.
Using two independent experimental approaches to monitor protein-protein interactions (enzyme-linked immunosorbent assay and size exclusion high performance liquid chromatography) we describe a general mechanism by which DnaJ modulates the binding of the DnaK chaperone to various native protein substrates, e.g. lambda P, lambda O, delta 32, P1, RepA, as well as permanently denatured alpha-carboxymethylated lactalbumin. The presence of DnaJ promotes the DnaK for efficient DnaK-substrate complex formation. ATP hydrolysis is absolutely required for such DnaJ-dependent activation of DnaK for binding to both native and denatured protein substrates. Although ADP can stabilize such as an activated DnaK-protein complex, it cannot substitute for ATP in the activation reaction. In the presence of DnaJ and ATP, DnaK possesses the affinity to different substrates which correlates well with the affinity of DnaJ alone for these protein substrates. Only when the affinity of the DnaJ chaperone for its protein substrate is relatively high (e.g. delta 32, RepA) can a tertiary complex DnaK-substrate-DnaJ be detected. In the case that DnaJ binds weakly to its substrate (lambda P, alpha-carboxymethylated lactalbumin), DnaJ is only transiently associated with the DnaK-substrate complex, but the DnaK activation reaction still occurs, albeit less efficiently.
Wawrzynów
A
A
Department of Molecular Biology, University of Gdansk, Poland.
Banecki
B
B
Wall
D
D
Liberek
K
K
Georgopoulos
C
C
Zylicz
M
M
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006868:Hydrolysis; D018832:Molecular Chaperones / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation
C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C063842:dnaK protein, E coli