The Roles of the Two Zinc Binding Sites in DnaJ

Katrin Linke,Tobias Wolfram,Johanna Bussemer,Ursula Jakob

doi:10.1074/jbc.m307491200

Katrin Linke, Tobias Wolfram + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m307491200

Copy DOI

Abstract

All type I DnaJ (Hsp40) homologues share the presence of two highly conserved zinc centers. To elucidate their function, we constructed DnaJ mutants that separately replaced cysteines of either zinc center I or zinc center II with serine residues. We found that in the absence of zinc center I, the autonomous, DnaK-independent chaperone activity of DnaJ is dramatically reduced. Surprisingly, this only slightly impaired the in vivo function of DnaJ, and its ability to function as a co-chaperone in the DnaK/DnaJ/GrpE foldase machine. The DnaJ zinc center II, on the other hand, was found to be absolutely essential for the in vivo and in vitro function of DnaJ. This did not seem to be caused by a lack of substrate binding affinity or an inability to work as an ATPase-stimulating factor. Rather it appears that zinc center II mutant proteins lack a necessary additional interaction site with DnaK, which seems to be crucial for locking-in substrate proteins onto DnaK. These findings led us to a model in which ATP hydrolysis in DnaK is only the first step in converting DnaK into its high affinity binding state. Additional interactions between DnaK and DnaJ are required to make the DnaK/DnaJ/GrpE foldase machinery catalytically active.

Highlights

The heat shock proteins DnaJ (Hsp40), DnaK (Hsp70), and GrpE form a highly efficient and conserved molecular chaperone machinery
To determine the amount of zinc that is cysteine-coordinated in wild-type DnaJ and mutant proteins, 2-␮l aliquots of a 1 mM p-hydroxymercuriphenylsulfonic acid (PMPS) (Sigma) titration solution were added, and changes in A500 nm were monitored after each addition, until no further changes were detected [24]
To test the in vivo function of the two mutant proteins, plasmids expressing either the mutated dnaJ genes or wild-type dnaJ were transformed into the dnaJ deletion strain PK11. dnaJ deletion strains such as PK11 are unable to propagate ␭-growth [27], do not grow at heat shock temperatures (Ն43 °C) [28], and are unable to swarm [29]

Summary

The Roles of the Two Zinc Binding Sites in DnaJ*

The DnaJ zinc center II, on the other hand, was found to be absolutely essential for the in vivo and in vitro function of DnaJ This did not seem to be caused by a lack of substrate binding affinity or an inability to work as an ATPase-stimulating factor. Class II DnaJ/Hsp homologues lack the central cysteinerich zinc binding domain but have two C-terminal domains, CTD1 and CTD2, instead [16] They have not been found to exert any autonomous, Hsp70-independent ability to interact with unfolded substrate proteins. 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]. Prior to the solution of the structure, it was assumed that the four imme-

Functional Analysis of Conserved Zinc Centers in DnaJ

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

DnaJ WT