title: Mapping the conformation of a client protein through the Hsp70 functional cycle.
abstract: The 70 kDa heat shock protein (Hsp70) chaperone system is ubiquitous, highly conserved, and involved in a myriad of diverse cellular processes. Its function relies on nucleotide-dependent interactions with client proteins, yet the structural features of folding-competent substrates in their Hsp70-bound state remain poorly understood. Here we use NMR spectroscopy to study the human telomere repeat binding factor 1 (hTRF1) in complex with Escherichia coli Hsp70 (DnaK). In the complex, hTRF1 is globally unfolded with up to 40% helical secondary structure in regions distal to the binding site. Very similar conformational ensembles are observed for hTRF1 bound to ATP-, ADP- and nucleotide-free DnaK. The patterns in substrate helicity mirror those found in the unfolded state in the absence of denaturants except near the site of chaperone binding, demonstrating that DnaK-bound hTRF1 retains its intrinsic structural preferences. To our knowledge, our study presents the first atomic resolution structural characterization of a client protein bound to each of the three nucleotide states of DnaK and establishes that the large structural changes in DnaK and the associated energy that accompanies ATP binding and hydrolysis do not affect the overall conformation of the bound substrate protein.
authors.affiliation: Departments of Molecular Genetics, Biochemistry, and Chemistry, The University of Toronto, Toronto, ON, Canada M5S 1A8; ashok.sekhar@utoronto.ca rina.rosenzweig@utoronto.ca kay@pound.med.utoronto.ca.
authors.forename: Ashok
authors.initials: A
authors.lastname: Sekhar
authors.affiliation: Departments of Molecular Genetics, Biochemistry, and Chemistry, The University of Toronto, Toronto, ON, Canada M5S 1A8; ashok.sekhar@utoronto.ca rina.rosenzweig@utoronto.ca kay@pound.med.utoronto.ca.
authors.forename: Rina
authors.initials: R
authors.lastname: Rosenzweig
authors.affiliation: Université Grenoble Alpes, Centre National de la Recherche Scientifique, and Commissariat à l'Énergie Atomique et aux Énergies Alternatives, Institut de Biologie Structurale, F-38044 Grenoble, France;
authors.forename: Guillaume
authors.initials: G
authors.lastname: Bouvignies
authors.affiliation: Departments of Molecular Genetics, Biochemistry, and Chemistry, The University of Toronto, Toronto, ON, Canada M5S 1A8; Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8 ashok.sekhar@utoronto.ca rina.rosenzweig@utoronto.ca kay@pound.med.utoronto.ca.
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D001665:Binding Sites; D004058:Diffusion; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D018832:Molecular Chaperones; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D035321:Telomeric Repeat Binding Protein 1 / Q000737:chemistry*
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D035321:Telomeric Repeat Binding Protein 1; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C063842:dnaK protein, E coli
keywords: CEST; Hsp70; NMR; molecular chaperones; protein folding