title: Visualizing the ATPase cycle in a protein disaggregating machine: structural basis for substrate binding by ClpB.
abstract: ClpB is a ring-shaped molecular chaperone that has the remarkable ability to disaggregate stress-damaged proteins. Here we present the electron cryomicroscopy reconstruction of an ATP-activated ClpB trap mutant, along with reconstructions of ClpB in the AMPPNP, ADP, and in the nucleotide-free state. We show that motif 2 of the ClpB M domain is positioned between the D1-large domains of neighboring subunits and could facilitate a concerted, ATP-driven conformational change in the AAA-1 ring. We further demonstrate biochemically that ATP is essential for high-affinity substrate binding to ClpB and cannot be substituted with AMPPNP. Our structures show that in the ATP-activated state, the D1 loops are stabilized at the central pore, providing the structural basis for high-affinity substrate binding. Taken together, our results support a mechanism by which ClpB captures substrates on the upper surface of the AAA-1 ring before threading them through the ClpB hexamer in an ATP hydrolysis-driven step.
authors.affiliation: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
authors.forename: Sukyeong
authors.initials: S
authors.lastname: Lee
authors.forename: Jae-Mun
authors.initials: JM
authors.lastname: Choi
authors.forename: Francis T F
authors.initials: FT
authors.lastname: Tsai
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D049071:Endopeptidase Clp; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D007091:Image Processing, Computer-Assisted; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism*
chemical_list: D000227:Adenine Nucleotides; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C069775:ClpB protein, E coli