title: The roles of the two zinc binding sites in DnaJ.
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.
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1048, USA.
authors.forename: Katrin
authors.initials: K
authors.lastname: Linke
authors.forename: Tobias
authors.initials: T
authors.lastname: Wolfram
authors.forename: Johanna
authors.initials: J
authors.lastname: Bussemer
authors.forename: Ursula
authors.initials: U
authors.lastname: Jakob
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine; D004347:Drug Interactions; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D015870:Gene Expression; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006358:Hot Temperature; D006868:Hydrolysis; D008156:Luciferases / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000502:physiology; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D012694:Serine; D013329:Structure-Activity Relationship; D014162:Transfection; D015032:Zinc / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D012694:Serine; D000255:Adenosine Triphosphate; D008156:Luciferases; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli; D015032:Zinc; D003545:Cysteine