Structure-Function Analysis of the Zinc Finger Region of the DnaJ Molecular Chaperone

• Published in: The Journal of biological chemistry 1996-06, Vol.271 (25), p.14840-14848
• Main Authors: Banecki, B, Liberek, K, Wall, D, Wawrzynów, A, Georgopoulos, C, Bertoli, E, Tanfani, F, Zylicz, M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Base Sequence; Binding Sites; Calorimetry; Calorimetry, Differential Scanning; Cysteine; DNA Primers; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli - metabolism; Escherichia coli Proteins; Heat-Shock Proteins - biosynthesis; Heat-Shock Proteins - chemistry; Heat-Shock Proteins - isolation & purification; HSP40 Heat-Shock Proteins; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Receptors, Steroid - chemistry; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Deletion; Sequence Homology, Amino Acid; Spectrophotometry; Zinc - analysis; Zinc Fingers
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.25.14840

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, DnaJΔ144-200, to bind DnaK nor its ability to stimulate DnaK's ATPase activity. However, the DnaJΔ144-200 was unable to induce DnaK to a conformation required for the stabilization of the DnaK-substrate complex. Additionally, the DnaJΔ144-200 mutant protein alone was unimpaired in its ability to interact with its σ 32 transcription factor substrate, but exhibited reduced affinity toward its P1 RepA and λP substrates. Finally, these in vitro results correlate well with the in vivo observed partial inhibition of bacteriophage λ growth in a DnaJΔ144-200 mutant background.