title: Substrate binding to the molecular chaperone Hsp104 and its regulation by nucleotides.
abstract: The Hsp104 protein from Saccharomyces cerevisiae is a member of the Hsp100/Clp family of molecular chaperones. It mediates the solubilization of aggregated proteins in an ATP-dependent process assisted by the Hsp70/40 system. Although the principal function of Hsp104 is well established, the mechanistic details of this catalyzed disaggregation are poorly understood. In this work, we have investigated the interaction of Hsp104 with reduced, carboxymethylated alpha-lactalbumin (RCMLa), a permanently unfolded model substrate. Our results demonstrate that the affinity of Hsp104 toward polypeptides is regulated by nucleotides. In the presence of ATP or adenosine-5' -O-(3-thiotriphosphate), the chaperone formed complexes with RCMLa, whereas no binding was observed in the presence of ADP. In particular, the occupation of the N-terminally located nucleotide-binding domain with ATP seems to be crucial for substrate interaction. When ATP binding to this domain was impaired by mutation, Hsp104 lost its ability to interact with RCMLa. Our results also indicate that upon association with a polypeptide, a conformational change occurs within Hsp104 that strongly reduces the dynamics of nucleotide exchange and commits the bound polypeptide to ATP hydrolysis.
authors.affiliation: Department Chemie, Technische Universität München, 85747 Garching, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Bösl
authors.forename: Valerie
authors.initials: V
authors.lastname: Grimminger
authors.forename: Stefan
authors.initials: S
authors.lastname: Walter
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D016880:Anisotropy; D002244:Carbon / Q000737:chemistry; D002384:Catalysis; D002845:Chromatography; D003001:Cloning, Molecular; D004058:Diffusion; D019793:Fluorescein / Q000494:pharmacology; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry; D008027:Light; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D012542:Scattering, Radiation; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D014461:Ultracentrifugation
chemical_list: D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009711:Nucleotides; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; C070972:HsP104 protein, S cerevisiae; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D002244:Carbon; D000255:Adenosine Triphosphate; D007768:Lactalbumin; D019793:Fluorescein