The effect of mutating arginine-469 on the substrate binding and refolding activities of 70-kDa heat shock cognate protein.

On the basis of the X-ray structure of DnaK, we obtained an energy-minimized model for the C-terminal domain of rat 70-kDa heat shock cognate protein (hsc70). The model suggests that Arg-469 may play an important role in maintaining the substrate-bound conformation of hsc70. To verify this hypothesis, we substituted cysteine for Arg-469 and generated the hsc70(R469C) mutant. Compared to the wild-type hsc70, the mutant was more accessible to cleavage by endopeptidase Lys-C, implying that the overall structure of hsc70(R469C) is relatively loose. Moreover, hsc70(R469C) did not form tightly associated complexes with S-carboxymethyl-alpha-lactalbumin, an unfolded protein. The amount of heptapeptide FYQLALT bound to hsc70(R469C) was also decreased as determined by gel filtration. Thus, the affinity of hsc70(R469C) for polypeptide substrates is reduced. In the presence of DnaJ, the capability of hsc70(R469C) to refold the denatured luciferase was decreased by 50%. Therefore, for hsc70, reduction in affinity for substrates may affect its DnaJ-dependent refolding activity.