title: Energetics of nucleotide-induced DnaK conformational states.
abstract: 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.
authors.affiliation: Unidad de Biofsica (CSIC/UPV-EHU) y Departamento de Bioquímica y Biología Molecular, Facultad de Ciencia y Tecnología, Universidad del País Vasco, Apartado 644, 48080 Bilbao, Spain.
authors.forename: Stefka G
authors.initials: SG
authors.lastname: Taneva
authors.forename: Fernando
authors.initials: F
authors.lastname: Moro
authors.forename: Adrián
authors.initials: A
authors.lastname: Velázquez-Campoy
authors.forename: Arturo
authors.initials: A
authors.lastname: Muga
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D003198:Computer Simulation; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013696:Temperature; D013816:Thermodynamics
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D009711:Nucleotides; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli