The conserved G/F motif of the DnaJ chaperone is necessary for the activation of the substrate binding properties of the DnaK chaperone

Abstract

The universally conserved DnaK and DnaJ molecular chaperone proteins bind in a coordinate manner to protein substrates to prevent aggregation, to disaggregate proteins, or to regulate proper protein function. To further examine their synergistic mechanism of action, we constructed and characterized two DnaJ deletion proteins. One has an 11-amino-acid internal deletion that spans amino acid residues 77-87 (DnaJΔ77-87) and the other amino acids 77-107 (DnaJΔ77-107). The DnaJΔ77-87 mutant protein, was normal in all respects analyzed. The DnaJΔ77-107 mutant protein has its entire G/F (Gly/Phe) motif deleted. This motif is found in most, but not all DnaJ family members. In vivo, DnaJΔ77-107 supported bacteriophage λ growth, albeit at reduced levels, demonstrating that at least some protein function was retained. However, DnaJΔ77-107 did not exhibit other wild type properties, such as proper down-regulation of the heat-shock response, and had an overall poisoning effect of cell growth. The purified DnaJΔ77-107 protein was shown to physically interact and stimulate DnaK's ATPase activity at wild type levels, unlike the previously characterized DnaJ259 point mutant (DnaJH33Q). Moreover, both DnaJΔ77-107 and DnaJ259 bound to substrate proteins, such as σ32, at similar affinities as DnaJ. However, DnaJΔ77-107 was found to be largely defective in activating the ATP-dependent substrate binding mode of DnaK. In vivo, the ability of the mutant DnaJ proteins to down-regulate the heat-shock response was correlated only with their in vitro ability to activate DnaK to bind σ32, in an ATP-dependent manner, and not with their ability to bind σ32. We conclude, that although the G/F motif of DnaJ does not directly participate in the stimulation of DnaK's ATPase activity, nevertheless, it is involved in an important manner in modulating DnaK's substrate binding activity.