title: A conserved histidine in human DNLZ/HEP is required for stimulation of HSPA9 ATPase activity.
abstract: The DNL-type zinc-finger protein DNLZ regulates the activity and solubility of the human mitochondrial chaperone HSPA9. To identify DNLZ residues that are critical for chaperone regulation, we carried out an alanine mutagenesis scan of charged residues in a W115I mutant of human DNLZ and assessed the effect of each mutation on interactions with HSPA9. All mutants analyzed promote the solubility of HSPA9 upon expression in Escherichia coli. However, binding studies examining the effect of DNLZ mutants on chaperone tryptophan fluorescence identified three mutations (R81A, H107A, and D111A) that decrease DNLZ binding affinity for nucleotide-free chaperone. In addition, ATPase measurements revealed that DNLZ-R81A and DNLZ-D111A both stimulate the catalytic activity HSPA9, whereas DNLZ-H107A does not elicit an increase in activity even when present at a concentration that is 10-fold higher than the level required for half-maximal stimulation by DNLZ. These findings implicate a conserved histidine as critical for DNLZ regulation of mitochondrial HSPA9 catalytic activity.
authors.affiliation: Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhai
authors.forename: Michael T
authors.initials: MT
authors.lastname: Vu
authors.forename: Kevin G
authors.initials: KG
authors.lastname: Hoff
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Silberg
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D017124:Conserved Sequence; D005453:Fluorescence; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D024101:Mitochondrial Proteins / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D014364:Tryptophan / Q000235:genetics; D015032:Zinc / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: C559905:DNLZ protein, human; D018840:HSP70 Heat-Shock Proteins; C492001:HSPA9 protein, human; D024101:Mitochondrial Proteins; D018832:Molecular Chaperones; D006639:Histidine; D014364:Tryptophan; D000251:Adenosine Triphosphatases; D015032:Zinc