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

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.

The Roles of the Two Zinc Binding Sites in DnaJ

“…A number of studies have been conducted to study the involvement of this region in the autonomous, DnaK-independent substrate binding activity as well as in the DnaK-dependent co-chaperone activity of class I DnaJ homologues (8,18,19). However, most of these studies were based on the presumption that the four N-terminal cysteines are involved in forming zinc center I and that the four C-terminal cysteines are involved in forming zinc center II.…”

“…The cysteine-rich zinc binding region of class I DnaJ homologues is thought to be involved in the DnaK-dependent chaperone activity based on domain truncation studies and cysteine mutagenesis (9,18,19). However, conflicting results have been reported concerning the role of the entire zinc binding region in the autonomous DnaK-independent chaperone activity (8,9,19).…”

“…DnaJ cysteine-rich domain forms two zinc centers, each of which is composed of four conserved cysteines (18,19,21) (Fig. 1).…”

“…The cysteine-rich CR-domain contains four repeated CXXCXGXG motifs, which are invariable to all class I DnaJ proteins (20,21). It has long been known that the two sulfur atoms of each consensus motif are involved in zinc coordination, resulting in the tetrahedral coordination of two zinc ions per DnaJ monomer (18,19). However, the identity of the specific residues involved in each zinc coordination remained unclear until the NMR structure of the isolated cysteine-rich domain was solved in 2000 ( Fig.…”

Differential expression of three Paralichthys olivaceus Hsp40 genes in responses to virus infection and heat shock☆

“…One categorization proposes the division of Hsp40 family into three subgroups [13]. Type I Hsp40 proteins possess all four domains similar to that of DnaJ, including the J domain with a conserved HPD tripeptide, which is responsible for regulation of the ATP hydrolytic cycle of Hsp70 [18e20], the glycine/phenylalanine-rich (G/F) domain, which appears to function as a spacer between the J-domain and other regions of Hsp40 proteins [21,22], a cysteine-rich Zn 2þ binding domain (CR) (containing four repeats of the motif CXXCXGXG where X is any amino acid), which plays a role in polypeptide binding by Hsp40 family members [23,24], and a poorly conserved C-terminal domain (CTD), which can interact with nonnative substrates [20]. Type II Hsp40 proteins lack the CR domain, and Type III proteins possess only the J domain [13].…”