title: ClpB N-terminal domain plays a regulatory role in protein disaggregation.
abstract: ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. The ClpB-substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD-substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD-substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD-substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation.
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; rina.rosenzweig@utoronto.ca.
authors.forename: Rina
authors.initials: R
authors.lastname: Rosenzweig
authors.affiliation: Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8;
authors.forename: Patrick
authors.initials: P
authors.lastname: Farber
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 1A8;
authors.forename: Algirdas
authors.initials: A
authors.lastname: Velyvis
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 1A8;
authors.forename: Enrico
authors.initials: E
authors.lastname: Rennella
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Latham
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8;
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
mesh_terms: D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000502:physiology*; D057927:Hydrophobic and Hydrophilic Interactions; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding
chemical_list: D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D049071:Endopeptidase Clp; C069775:ClpB protein, E coli
keywords: ClpB; Hsp104; chaperones; methyl-TROSY NMR; protein disaggregation