title: Nucleotides regulate the mechanical hierarchy between subdomains of the nucleotide binding domain of the Hsp70 chaperone DnaK.
abstract: The regulation of protein function through ligand-induced conformational changes is crucial for many signal transduction processes. The binding of a ligand alters the delicate energy balance within the protein structure, eventually leading to such conformational changes. In this study, we elucidate the energetic and mechanical changes within the subdomains of the nucleotide binding domain (NBD) of the heat shock protein of 70 kDa (Hsp70) chaperone DnaK upon nucleotide binding. In an integrated approach using single molecule optical tweezer experiments, loop insertions, and steered coarse-grained molecular simulations, we find that the C-terminal helix of the NBD is the major determinant of mechanical stability, acting as a glue between the two lobes. After helix unraveling, the relative stability of the two separated lobes is regulated by ATP/ADP binding. We find that the nucleotide stays strongly bound to lobe II, thus reversing the mechanical hierarchy between the two lobes. Our results offer general insights into the nucleotide-induced signal transduction within members of the actin/sugar kinase superfamily.
authors.affiliation: Physik Department E22, Technische Universität München, 85748 Garching, Germany;
authors.forename: Daniela
authors.initials: D
authors.lastname: Bauer
authors.affiliation: Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221;
authors.forename: Dale R
authors.initials: DR
authors.lastname: Merz
authors.affiliation: Physik Department E22, Technische Universität München, 85748 Garching, Germany;
authors.forename: Benjamin
authors.initials: B
authors.lastname: Pelz
authors.affiliation: Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221;
authors.forename: Kelly E
authors.initials: KE
authors.lastname: Theisen
authors.affiliation: Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221;
authors.forename: Gail
authors.initials: G
authors.lastname: Yacyshyn
authors.affiliation: Department of Physiological Chemistry, Medical Faculty, University of Munich, 81377 Munich, Germany;
authors.forename: Dejana
authors.initials: D
authors.lastname: Mokranjac
authors.affiliation: Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221; gabriel.zoldak@tum.de dimari@ucmail.uc.edu mrief@ph.tum.de.
authors.forename: Ruxandra I
authors.initials: RI
authors.lastname: Dima
authors.affiliation: Physik Department E22, Technische Universität München, 85748 Garching, Germany; Munich Center for Integrated Protein Science, 81377 München, Germany gabriel.zoldak@tum.de dimari@ucmail.uc.edu mrief@ph.tum.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Rief
authors.affiliation: Physik Department E22, Technische Universität München, 85748 Garching, Germany; gabriel.zoldak@tum.de dimari@ucmail.uc.edu mrief@ph.tum.de.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Žoldák
mesh_terms: D000199:Actins / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000595:Amino Acid Sequence; D003198:Computer Simulation; D004548:Elasticity; D029968:Escherichia coli Proteins / Q000737:chemistry*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry*; D007834:Lasers; D008928:Mitochondria / Q000378:metabolism; D008958:Models, Molecular; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry*; D010802:Phylogeny; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D015398:Signal Transduction
chemical_list: D000199:Actins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D009711:Nucleotides; C090869:SSA3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli
keywords: ATPase; elasticity; force; laser trapping; protein extension