Hsp70 chaperones are molecular switches that use the free energy of ATP binding and hydrolysis to modulate their affinity for protein substrates and, most likely, to remodel non-native interactions allowing proper substrate folding. By means of isothermal titration calorimetry, we have measured the thermodynamics of ATP and ADP binding to (i) wild-type DnaK, the main bacterial Hsp70; (ii) two single-point mutants, DnaK(T199A), which lacks ATPase activity but maintains conformational changes similar to those observed in the wild-type protein, and DnaK(R151A), defective in interdomain communication; and iii) two deletion mutants, the isolated nucleotide binding domain (K-NBD) and a DeltaLid construct [DnaK(1-507)]. At 25 degrees C, ATP binding to DnaK results in a fast endothermic and a slow exothermic process due to ATP hydrolysis. We demonstrate that the endothermic event is due to the allosteric coupling between ATP binding to the nucleotide binding domain and the conformational rearrangement of the substrate binding domain. The interpretation of our data is compatible with domain docking upon ATP binding and shows that this conformational change carries an energy penalty of ca. 1 kcal/mol. The conformational energy stored in the ATP-bound DnaK state, together with the free energy of ATP hydrolysis, can be used in remodeling bound substrates.