title: Crowding activates ClpB and enhances its association with DnaK for efficient protein aggregate reactivation.
abstract: Reactivation of intracellular protein aggregates after a severe stress is mandatory for cell survival. In bacteria, this activity depends on the collaboration between the DnaK system and ClpB, which in vivo occurs in a highly crowded environment. The reactivation reaction includes two steps: extraction of unfolded monomers from the aggregate and their subsequent refolding into the native conformation. Both steps might be compromised by excluded volume conditions that would favor aggregation of unstable protein folding intermediates. Here, we have investigated whether ClpB and the DnaK system are able to compensate this unproductive effect and efficiently reactivate aggregates of three different substrate proteins under crowding conditions. To this aim, we have compared the association equilibrium, biochemical properties, stability, and chaperone activity of the disaggregase ClpB in the absence and presence of an inert macromolecular crowding agent. Our data show that crowding i), increases three to four orders of magnitude the association constant of the functional hexamer; ii), shifts the conformational equilibrium of the protein monomer toward a compact state; iii), stimulates its ATPase activity; and iv), favors association of the chaperone with substrate proteins and with aggregate-bound DnaK. These effects strongly enhance protein aggregate reactivation by the DnaK-ClpB network, highlighting the importance of volume exclusion in complex processes in which several proteins have to work in a sequential manner.
authors.affiliation: Unidad de Biofísica (Consejo Superior de Investigaciones Científicas/Universidad del País Vasco-Euskal Herriko Unibertsitatea) and Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco, Apartado 644, Bilbao 48080, Spain.
authors.forename: Ianire
authors.initials: I
authors.lastname: Martín
authors.affiliation: Unidad de Biofísica (Consejo Superior de Investigaciones Científicas/Universidad del País Vasco-Euskal Herriko Unibertsitatea) and Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco, Apartado 644, Bilbao 48080, Spain.
authors.forename: Garbiñe
authors.initials: G
authors.lastname: Celaya
authors.affiliation: Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas), Ramiro de Maeztu 9, Madrid 28040, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Alfonso
authors.affiliation: Unidad de Biofísica (Consejo Superior de Investigaciones Científicas/Universidad del País Vasco-Euskal Herriko Unibertsitatea) and Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco, Apartado 644, Bilbao 48080, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: Moro
authors.affiliation: Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas), Ramiro de Maeztu 9, Madrid 28040, Spain.
authors.forename: Germán
authors.initials: G
authors.lastname: Rivas
authors.affiliation: Unidad de Biofísica (Consejo Superior de Investigaciones Científicas/Universidad del País Vasco-Euskal Herriko Unibertsitatea) and Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco, Apartado 644, Bilbao 48080, Spain. Electronic address: arturo.muga@ehu.es.
authors.forename: Arturo
authors.initials: A
authors.lastname: Muga
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D066329:Protein Aggregates; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary
chemical_list: D018840:HSP70 Heat-Shock Proteins; D066329:Protein Aggregates; D000251:Adenosine Triphosphatases