The protein-free IANUS peptide array uncovers interaction sites between Escherichia coli parvulin 10 and alkyl hydroperoxide reductase.
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.
Malesević
Miroslav
M
Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle/Saale, Germany.
Poehlmann
Angela
A
Hernandez Alvarez
Birte
B
Diessner
André
A
Träger
Mario
M
Rahfeld
Jens-Ulrich
JU
Jahreis
Günther
G
Liebscher
Sandra
S
Bordusa
Frank
F
Fischer
Gunter
G
Lücke
Christian
C
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*
D029968:Escherichia coli Proteins; D054464:Peroxiredoxins; C515937:ahpC protein, E coli; C499433:Par10 protein, E coli; D019696:Peptidylprolyl Isomerase