Abstract
DnaJ is a molecular chaperone, which not only binds to its various protein substrates, but can also activate the DnaK cochaperone to bind to its various protein substrates as well. DnaJ is a modular protein, which contains a putative zinc finger motif of unknown function. Quantitation of the released Zn(II) ions, upon challenge with p-hydroxymercuriphenylsulfonic acid, and by atomic absorption showed that two Zn(II) ions interact with each monomer of DnaJ. Following the release of Zn(II) ions, the free cysteine residues probably form disulfide bridge(s), which contribute to overcoming the destabilizing effect of losing Zn(II). Supporting this view, infrared and circular dichroism studies show that the DnaJ secondary structure is largely unaffected by the release of Zn(II). Moreover, infrared spectra recorded at different temperatures, as well as scanning calorimetry, show that the Zn(II) ions help to stabilize DnaJ's tertiary structure. An internal 57-amino acid deletion of the cysteine-reach region did not noticeably affect the affinity of this mutant protein, DnaJDelta144-200, to bind DnaK nor its ability to stimulate DnaK's ATPase activity. However, the DnaJDelta144-200 was unable to induce DnaK to a conformation required for the stabilization of the DnaK-substrate complex. Additionally, the DnaJDelta144-200 mutant protein alone was unimpaired in its ability to interact with its final sigma32 transcription factor substrate, but exhibited reduced affinity toward its P1 RepA and lambdaP substrates. Finally, these in vitro results correlate well with the in vivo observed partial inhibition of bacteriophage lambda growth in a DnaJDelta144-200 mutant background.
Highlights
The Hsp[70] family of proteins, the DnaK for Escherichia coli being the prototype, participate in a variety of cellular functions, such as protein folding, proteolysis, protein transport, the activation of various transcriptional and replication factors to bind to specific DNA sequences, as well as the protection and renaturation of some heat-labile proteins
Following ATP hydrolysis, DnaK changes its conformation to the DnaK*-ADP form, which destabilizes its complex with protein substrates
Sults not shown), suggesting that the presence of ATP is required for the DnaJ-DnaK interaction. In support of these conclusions, we showed that ATP hydrolysis is important for the DnaJ-DnaK-ADP complex formation, and such a complex can only be detected when ATP was present during both the preincubation and mobile phase of size high pressure liquid chromatography (Wawrzynow and Zylicz, 1995)
Summary
The Hsp[70] family of proteins, the DnaK for Escherichia coli being the prototype, participate in a variety of cellular functions, such as protein folding, proteolysis, protein transport, the activation of various transcriptional and replication factors to bind to specific DNA sequences, as well as the protection and renaturation of some heat-labile proteins These observations have led to their classification as molecular chaperones (for review, see Georgopoulos et al (1994) and Hendrick and Hartl (1993)). The DnaK chaperone, in a GrpE/ATP-dependent reaction, dissociates from the substrate complex and is converted back to the DnaK*-ADP conformation, which is ready to rebind (in a DnaJdependent reaction) to its protein substrates (Banecki and Zylicz, 1996) Both genetic and biochemical studies of various eukaryotic DnaJ-like proteins indicate that most, if not all, of the activities of E. coli DnaJ have been functionally conserved throughout evolution (Caplan et al, 1993; Silver and Way, 1993; Cyr et al, 1994).
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