Peptide-Induced Conformational Changes in the Molecular Chaperone DnaK

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Abstract
DnaK, the 70 kDa molecular chaperone of Escherichia coli, adopts a high-affinity state in the presence of ADP that tightly binds its target peptide, whereas replacement of ADP by ATP induces a structural switch to a low-affinity chaperone state that weakly binds its target. An ∼15% decrease in tryptophan fluorescence of DnaK occurs in concert with this switch from the high- to low-affinity state. The reversibility of this structural transition in DnaK was investigated using rapid mixing and equilibrium fluorescence methods. The Cro peptide (MQERITLKDYAM) was used to mimic an unfolded substrate. When the Cro peptide is rapidly mixed with preformed low-affinity DnaK complexes (DnaK*−ATP), a rapid increase (kobs = 3−30 s-1) in the tryptophan fluorescence of DnaK occurs. We suggest that the Cro peptide induces the transition of the low-affinity state of DnaK back to the high-affinity state, without ATP hydrolysis. The combined results in this report are consistent with the minimal mechanism ATP + EP ⇔ ATP−EP ⇔ ATP−E* + P, where ATP binding (K1) induces a conformational change and concerted peptide release (koff), and peptide binding (kon) to the low-affinity state (ATP−E*) induces the transition back to ATP−EP, a high-affinity state. At 25 °C, in the presence of the Cro peptide, values for K1, koff, and kon are 22 μM, 3.3 s-1, and 2.4 × 104 M-1 s-1, respectively. Evidence for an equilibrium between closed and open forms of DnaK in the absence of ATP and peptide is also presented.