title: Ionic contacts at DnaK substrate binding domain involved in the allosteric regulation of lid dynamics.
abstract: To gain further insight into the interactions involved in the allosteric transition of DnaK we have characterized wild-type (wt) protein and three mutants in which ionic interactions at the interface between the two subdomains of the substrate binding domain, and within the lid subdomain have been disrupted. Our data show that ionic contacts, most likely forming an electrically charged network, between the N-terminal region of helix B and an inner loop of the beta-sandwich are involved in maintaining the position of the lid relative to the beta-subdomain in the ADP state but not in the ATP state of the protein. Disruption of the ionic interactions between the C-terminal region of helix B and the outer loops of the beta-sandwich, known as the latch, does not have the same conformational consequences but results equally in an inactive protein. This indicates that a variety of mechanisms can inactivate this complex allosteric machine. Our results identify the ionic contacts at the subdomain and interdomain interfaces that are part of the hinge region involved in the ATP-induced allosteric displacement of the lid away from the peptide binding site. These interactions also stabilize peptide-Hsp70 complexes at physiological (37 degrees C) and stress (42 degrees C) temperatures, a requirement for productive substrate (re)folding.
authors.affiliation: Unidad de Biofísica (Consejo Superior de Investigaciones Científicas-Universidad del País Vasco, Euskal Herriko Unibertsitatea), 48080 Bilbao, Spain.
authors.forename: Vanesa
authors.initials: V
authors.lastname: Fernández-Sáiz
authors.forename: Fernando
authors.initials: F
authors.lastname: Moro
authors.forename: Jesus M
authors.initials: JM
authors.lastname: Arizmendi
authors.forename: Sergio P
authors.initials: SP
authors.lastname: Acebrón
authors.forename: Arturo
authors.initials: A
authors.lastname: Muga
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D016880:Anisotropy; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D004926:Escherichia coli / Q000378:metabolism*; D006358:Hot Temperature; D007477:Ions; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D018832:Molecular Chaperones / Q000737:chemistry*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D007477:Ions; D018832:Molecular Chaperones; D010455:Peptides; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008156:Luciferases; D014357:Trypsin; D000251:Adenosine Triphosphatases; C115251:DnaK protein, Brevibacillus choshinensis