title: Crowding Modulates the Conformation, Affinity, and Activity of the Components of the Bacterial Disaggregase Machinery.
abstract: Chaperone-mediated protein aggregate reactivation is a complex reaction that depends on the sequential association of molecular chaperones on their interaction with protein aggregates and on substrate refolding. This process could be modulated by the highly crowded intracellular environment, which is known to affect protein conformational change, enzymatic activity, and protein-protein interactions. Here, we report that molecular crowding shapes the chaperone activity of bacterial disaggregase composed of the DnaK system (DnaK, DnaJ, and GrpE) and the molecular motor ClpB. A combination of biophysical and biochemical methods shows that the excluded volume conditions modify the conformation of DnaK and DnaJ without affecting that of GrpE. These crowding-induced conformational rearrangements activate DnaK, enhance the affinity of DnaK for DnaJ, but not for GrpE, and increase the sensitivity of the chaperone activity to cochaperone concentration, explaining the tight control of their relative intracellular amounts. Furthermore, crowding-mediated disordering of the G/F domain of DnaJ facilitates the reversible formation of intermolecular DnaJ conglomerates. These assemblies could drive the formation of Hsp70 clusters at the aggregate surface with the consequent enhancement of the disaggregation efficiency through their coordinated action via entropic pulling. Finally, crowding helps ClpB to outcompete GrpE for DnaK binding, a key aspect of DnaK/ClpB cooperation given the low affinity of the disaggregase for DnaK. Excluded volume conditions promote the formation of the bichaperone complex that disentangles aggregates, enhancing the efficiency of the disaggregation reaction.
authors.affiliation: Unidad de Biofisica (CSIC-UPV/EHU) and Departmento de Bioquímica y Biología Molecular, Facultad de Ciencia y Tecnología, Universidad del País Vasco (UPV/EHU), Aptdo. 644, 48080 Bilbao, Spain.
authors.forename: Garbiñe
authors.initials: G
authors.lastname: Celaya
authors.affiliation: Unidad de Biofisica (CSIC-UPV/EHU) and Departmento de Bioquímica y Biología Molecular, Facultad de Ciencia y Tecnología, Universidad del País Vasco (UPV/EHU), Aptdo. 644, 48080 Bilbao, Spain.
authors.forename: José Angel
authors.initials: JA
authors.lastname: Fernández-Higuero
authors.affiliation: Unidad de Biofisica (CSIC-UPV/EHU) and Departmento de Bioquímica y Biología Molecular, Facultad de Ciencia y Tecnología, Universidad del País Vasco (UPV/EHU), Aptdo. 644, 48080 Bilbao, Spain.
authors.forename: Ianire
authors.initials: I
authors.lastname: Martin
authors.affiliation: Centro de Investigaciones Biológicas, CSIC, C/ Ramiro de Maeztu 9, E-40 28040 Madrid, Spain.
authors.forename: Germán
authors.initials: G
authors.lastname: Rivas
authors.affiliation: Unidad de Biofisica (CSIC-UPV/EHU) and Departmento de Bioquímica y Biología Molecular, Facultad de Ciencia y Tecnología, Universidad del País Vasco (UPV/EHU), Aptdo. 644, 48080 Bilbao, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: Moro
authors.affiliation: Unidad de Biofisica (CSIC-UPV/EHU) and Departmento de Bioquímica y Biología Molecular, Facultad de Ciencia y Tecnología, Universidad del País Vasco (UPV/EHU), Aptdo. 644, 48080 Bilbao, Spain. Electronic address: arturo.muga@ehu.es.
authors.forename: Arturo
authors.initials: A
authors.lastname: Muga
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D008968:Molecular Conformation; D066329:Protein Aggregates / Q000502:physiology*; D011485:Protein Binding / Q000502:physiology; D000072417:Protein Domains / Q000502:physiology; D017510:Protein Folding; D060066:Protein Interaction Maps / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D066329:Protein Aggregates; D000251:Adenosine Triphosphatases
keywords: ClpB; DnaK; aggregate reactivation; chaperone; macromolecular crowding