Kinetic analysis of interdomain coupling in a lidless variant of the molecular chaperone DnaK: DnaK's lid inhibits transition to the low affinity state.

摘要: DnaK, the Escherichia coli Hsp70, possesses two functional domains, N- and C-terminal ATPase peptide-binding respectively. Elucidation of mechanism allosteric coupling between domains is key to understanding how Hsp70 chaperones interact with their substrates. We previously reported that ATP reacts wild-type DnaK-peptide complexes according two-step reaction, + DnaK-P if ATP-DnaK-P ATP-DnaK P, where binds in first step, a conformational change quenches DnaK's tryptophan fluorescence (denoted by asterisk) expels bound peptide occurs second step. Here we report DnaK(2-517), lidless variant, also this mechanism. Compared found that, depending on sequence temperature, deletion lid produces 27- 66-fold increase rate constant (k(2)) for ATP-triggered (ATP-DnaK-P --> ATP-DnaK+P) but only approximately 2-fold (k(-)(2)) reverse reaction (ATP-DnaK+P ATP-DnaK-P). A model proposed which regulates interdomain communication retarding motions within beta-sandwich occur as consequence binding. New evidence support reversible, switch presented.