The functional cycle and regulation of the Thermus thermophilus DnaK chaperone system

摘要: The Escherichia coli DnaK (DnaKEco) chaperone cycle is tightly regulated by the cochaperones DnaJ, which stimulates ATP hydrolysis, and GrpE, acts as a nucleotide exchange factor. Thermus thermophilus (DnaKTth) system additionally comprises DnaK-DnaJ assembly factor (DafATth) that mediating formation of 300 kDa DnaKTth. DnaJTth.DafATth complex.A model peptide derived from tumor suppressor protein p53 was used to dissect regulation individual kinetic key steps DnaKTth nucleotide/chaperone cycle. As with DnaKEco DnaKTth.ATP complex binds substrates reduced affinity large rates compared DnaKTth.ADP.Pi state. In contrast DnaKEco, ADP-Pi release slow rate reversing balance two functional states. Whereas GrpETth DnaKTth, DnaJTth does not accelerate hydrolysis under various experimental conditions. However, it exerts influence on interaction substrates: in presence DafATth, inhibits substrate binding, already bound displaced indicating competitive binding DnaJTth/DafATth substrate. It thus appears isolated T. represent active species Isothermal titration calorimetry showed ternary DafATth assembling high affinity, whereas binary complexes or were detectable, highly synergistic complex. Based these results, describing its proposed where constitutes resting state, substrate.DnaJTth species. novel mediates would serve "template" stabilise DnaKTth.DafATth.DnaJTth until replaced proteins heat shock