The Protein-Free IANUS Peptide Array Uncovers Interaction Sites between Escherichia coli Parvulin 10 and Alkyl Hydroperoxide Reductase

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• 作者：Miroslav Maleevi ; Angela Poehlmann ; Birte Hernandez Alvarez ; Andr Diessner ; Mario Trger ; Jens-Ulrich Rahfeld ; Gnther Jahreis ; Sandra Liebscher ; Frank Bordusa ; Gunter Fischer ; Christian Lcke
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：October 5, 2010
• 年：2010
• 卷：49
• 期：39
• 页码：8626-8635
• 全文大小：1061K
• 年卷期：v.49,no.39(October 5, 2010)
• ISSN：1520-4995

文摘

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 ¹⁵N-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.