Functional defects of the DnaK756 mutant chaperone of Escherichia coli indicate distinct roles for amino- and carboxyl-terminal residues in substrateand co-chaperone interaction and interdomain communication

The first discovery of an Hsp70 chaperone gene was the isolation of an Esch erichia coil mutant, dnaK756, which rendered the cells resistant to lytic i nfection with bacteriophage lambda, The DnaK756 mutant protein has since be en used to establish many of the cellular roles and biochemical properties of DnaK, DnaK756 has three glycine-to-aspartate substitutions at residues 3 2, 455, and 468, which were reported to result in defects in intrinsic and GrpE-stimulated ATPase activities, substrate binding, stability of the subs trate-binding domain, inrerdomain communication, and, consequently, defects in chaperone activity, To dissect the effects of the different amino acid substitutions in DnaK756, we analyzed two DnaK variants carrying only the a mino-terminal (residue 32) or the two carboxyl-terminal (residues 455 and 4 68) substitutions, The amino-terminal substitution interfered with the GrpE -stimulated ATPase activity. The carboxyl-terminal mutations (i) affected s tability and function of the substrate-binding domain, (ii) caused a 10-fol d elevated ATP hydrolysis rate, but (iii) did not severely affect domain co upling. Surprisingly, DnaK chaperone activity was more severely compromised by the amino-terminal than by the carboxyl-terminal amino acid substitutio ns both in vivo and in vitro, In the in vitro refolding of denatured firefl y luciferase, the defect of the DnaK variant carrying the amino-terminal su bstitution results from its inability to release, upon GrpE-mediated nucleo tide exchange, bound luciferase in a folding competent state. Our results i ndicate that the DnaK-DnaJ-GrpE chaperone system can tolerate suboptimal su bstrate binding, whereas the tight kinetic control of substrate dissociatio n by GrpE is essential.