title: The protein-free IANUS peptide array uncovers interaction sites between Escherichia coli parvulin 10 and alkyl hydroperoxide reductase.
abstract: The reliable identification of interacting structural elements without prior isolation of interacting proteins can be achieved by using the novel fluorescence resonance energy transfer-coupled IANUS (Induced orgANization of strUcture by matrix-assisted togethernesS) peptide array. Here we report that parvulin 10 (Par10), an abundant Escherichia coli peptidyl prolyl cis/trans isomerase (PPIase), physically interacts with the alkyl hydroperoxide reductase subunit C (AhpC) in bacterial cell extracts, as determined by affinity chromatography and chemical cross-linking experiments. A Par10-negative E. coli strain showed increased sensitivity toward hydrogen peroxide compared to the wild-type strain. The IANUS experiment revealed three segments of the peroxiredoxin AhpC chain as potential Par10 binding partners. Inhibition of the Par10 PPIase activity by the corresponding AhpC-derived peptides as well as NMR data of (15)N-labeled Par10 in the presence of the AhpC(115-132) peptide or full-length AhpC confirmed that the putative Par10 active site is involved in the Par10-AhpC interaction. Moreover, NMR-based docking calculations as well as NOESY exchange peaks between the proline cis and trans isomers revealed the Asp125-Pro126 moiety of the AhpC segment G115-A132 as a substrate for Par10 enzymatic action. On the basis of these data, we conclude that Par10 catalytic activity is involved in the cellular protection against oxidative stress.
authors.affiliation: Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle/Saale, Germany.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Malesević
authors.forename: Angela
authors.initials: A
authors.lastname: Poehlmann
authors.forename: Birte
authors.initials: B
authors.lastname: Hernandez Alvarez
authors.forename: André
authors.initials: A
authors.lastname: Diessner
authors.forename: Mario
authors.initials: M
authors.lastname: Träger
authors.forename: Jens-Ulrich
authors.initials: JU
authors.lastname: Rahfeld
authors.forename: Günther
authors.initials: G
authors.lastname: Jahreis
authors.forename: Sandra
authors.initials: S
authors.lastname: Liebscher
authors.forename: Frank
authors.initials: F
authors.lastname: Bordusa
authors.forename: Gunter
authors.initials: G
authors.lastname: Fischer
authors.forename: Christian
authors.initials: C
authors.lastname: Lücke
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D018384:Oxidative Stress; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000378:metabolism*; D054464:Peroxiredoxins / Q000737:chemistry / Q000378:metabolism*; D040081:Protein Array Analysis / Q000379:methods; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D054464:Peroxiredoxins; C515937:ahpC protein, E coli; C499433:Par10 protein, E coli; D019696:Peptidylprolyl Isomerase