title: A quantitative analysis of the effect of nucleotides and the M domain on the association equilibrium of ClpB.
abstract: ClpB is a hexameric molecular chaperone that, together with the DnaK system, has the ability to disaggregate stress-denatured proteins. The hexamer is a highly dynamic complex, able to reshuffle subunits. To further characterize the biological implications of the ClpB oligomerization state, the association equilibrium of the wild-type (wt) protein and of two deletion mutants, which lack part or the whole M domain, was quantitatively analyzed under different experimental conditions, using several biophysical [analytical ultracentrifugation, composition-gradient (CG) static light scattering, and circular dichroism] and biochemical (ATPase and chaperone activity) methods. We have found that (i) ClpB self-associates from monomers to form hexamers and higher-order oligomers that have been tentatively assigned to dodecamers, (ii) oligomer dissociation is not accompanied by modifications of the protein secondary structure, (iii) the M domain is engaged in intersubunit interactions that stabilize the protein hexamer, and (iv) the nucleotide-induced rearrangement of ClpB affects the protein oligomeric core, in addition to the proposed radial extension of the M domain. The difference in the stability of the ATP- and ADP-bound states [ΔΔG(ATP-ADP) = -10 kJ/mol] might explain how nucleotide exchange promotes the conformational change of the protein particle that drives its functional cycle.
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, Bilbao 48080, Spain.
authors.forename: Urko
authors.initials: U
authors.lastname: del Castillo
authors.forename: Carlos
authors.initials: C
authors.lastname: Alfonso
authors.forename: Sergio P
authors.initials: SP
authors.lastname: Acebrón
authors.forename: Ariadna
authors.initials: A
authors.lastname: Martos
authors.forename: Fernando
authors.initials: F
authors.lastname: Moro
authors.forename: Germn
authors.initials: G
authors.lastname: Rivas
authors.forename: Arturo
authors.initials: A
authors.lastname: Muga
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D057446:Hydrodynamics; D008958:Models, Molecular; D009711:Nucleotides / Q000494:pharmacology*; D055503:Protein Multimerization / Q000187:drug effects*; D055550:Protein Stability / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D006360:Heat-Shock Proteins; D009711:Nucleotides; D021122:Protein Subunits; D000255:Adenosine Triphosphate