Rational mutagenesis of a 40 kDa heat shock protein from Agrobacterium tumefaciens identifies amino acid residues critical to its in vivo function.

Prokaryotic DnaJ and DnaK, homologous to the eukaryotic 40 and 70kDa heat shock proteins (Hsp40 and Hsp70) respectively, play an important role as molecular chaperones in assisted protein folding under both normal and stressed conditions. DnaJ-like proteins are defined by the presence of a 70 amino acid domain termed the J domain, similar to the initial 73 amino acids of the Escherichia coli protein DnaJ. The J domain comprises four alpha-helices and a loop region containing the invariant tripeptide of histidine, proline and aspartic acid (HPD motif). This motif and Helix II have been shown previously to be important for the interaction with partner Hsp70s. Conserved amino acid residues present in the J domain were identified, and substitutions of these residues were performed to examine their effect on the in vivo functioning of the J domain of Agrobacterium tumefaciens DnaJ. Three conserved, charged residues, and three conserved, hydrophobic residues, in addition to the HPD motif, were shown to be important for the correct functioning of A. tumefaciens DnaJ. These included Arg26 located on Helix II, Arg63 and Asp59 located on Helix IV, Tyr7 and Leu10 located on Helix I, and Leu57 located on Helix III. This study has identified charged and hydrophobic residues on all the structural elements of the J domain that were critical to the structure and function of DnaJ, and in particular shown that Helix IV may have an important role in the structure and function of DnaJs in general.