title: Disruption of ionic interactions between the nucleotide binding domain 1 (NBD1) and middle (M) domain in Hsp100 disaggregase unleashes toxic hyperactivity and partial independence from Hsp70.
abstract: Hsp100 chaperones cooperate with the Hsp70 chaperone system to disaggregate and reactivate heat-denatured aggregated proteins to promote cell survival after heat stress. The homology models of Hsp100 disaggregases suggest the presence of a conserved network of ionic interactions between the first nucleotide binding domain (NBD1) and the coiled-coil middle subdomain, the signature domain of disaggregating chaperones. Mutations intended to disrupt the putative ionic interactions in yeast Hsp104 and bacterial ClpB disaggregases resulted in remarkable changes of their biochemical properties. These included an increase in ATPase activity, a significant increase in the rate of in vitro substrate renaturation, and partial independence from the Hsp70 chaperone in disaggregation. Paradoxically, the increased activities resulted in serious growth impediments in yeast and bacterial cells instead of improvement of their thermotolerance. Our results suggest that this toxic activity is due to the ability of the mutated disaggregases to unfold independently from Hsp70, native folded proteins. Complementary changes that restore particular salt bridges within the suggested network suppressed the toxic effects. We propose a novel structural aspect of Hsp100 chaperones crucial for specificity and efficiency of the disaggregation reaction.
authors.affiliation: Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology, University of Gdańsk, 80-822 Gdansk, Kladki 24, Poland.
authors.forename: Natalia
authors.initials: N
authors.lastname: Lipińska
authors.forename: Szymon
authors.initials: S
authors.lastname: Ziętkiewicz
authors.forename: Alicja
authors.initials: A
authors.lastname: Sobczak
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Jurczyk
authors.forename: Wojciech
authors.initials: W
authors.lastname: Potocki
authors.forename: Ewa
authors.initials: E
authors.lastname: Morawiec
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Wawrzycka
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Gumowski
authors.forename: Magdalena
authors.initials: M
authors.lastname: Ślusarz
authors.forename: Sylwia
authors.initials: S
authors.lastname: Rodziewicz-Motowidło
authors.forename: Elżbieta
authors.initials: E
authors.lastname: Chruściel
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Liberek
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D000595:Amino Acid Sequence; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry*; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D007477:Ions; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D007477:Ions; D029701:Saccharomyces cerevisiae Proteins; C070972:HsP104 protein, S cerevisiae; D049452:Green Fluorescent Proteins; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C069775:ClpB protein, E coli