Identification of Api88 Binding Partners in Escherichia coli Using a Photoaffinity-Cross-Link Strategy and Label-Free
Daniela Volke1, Andor Krizsan1, Nicole Berthold1
1†Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and ‡Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, Leipzig, Germany.
Abstract:
Gene-encoded antimicrobial peptides (AMPs) kill bacteria very efficiently by either lytic mechanisms or inhibition of specific bacterial targets. Proline-rich AMPs (PrAMPs), for example, produced in insects and mammals rely on the second mechanism. They bind to the 70 kDa bacterial heat shock protein DnaK and the 60 kDa chaperonin GroEL and interfere with protein folding, but this does not explain their strong bactericidal effects. Thus, we looked for further binding partners of apidaecin 1b, originally identified in honey bees, and two rationally optimized analogues (Api88 and Api137). Because affinity chromatography using Api88 as an immobilized ligand enriched only a few proteins at low levels besides DnaK, we synthesized Api88 analogues substituting Tyr7 with p-benzoyl-phenylalanine (Bpa), which can cross-link the peptide to binding partners after UV irradiation. Escherichia coli was incubated with biotinylated Api88 Tyr7Bpa or the corresponding all-d-peptide, irradiated, and lysed. The protein extract was enriched by streptavidin, separated by SDS-PAGE, digested with trypsin, and analyzed by nanoRP-UPLC-ESI-QqTOF-MS/MS. Among the 41 proteins identified, 34 were detected only in the l-Api88 Tyr7Bpa sample, including five 70S ribosomal proteins, DNA-directed RNA polymerase, and pyruvate dehydrogenase, indicating that PrAMPs might interfere with protein translation and energy metabolism.
Insights
Proline-rich antimicrobial peptides (PrAMPs) kill bacteria by targeting essential proteins. This study identified new bacterial targets, including those involved in protein translation and energy metabolism, revealing novel mechanisms of action for PrAMPs.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Gene-encoded antimicrobial peptides (AMPs) are crucial in innate immunity, exhibiting potent bactericidal activity through various mechanisms.
- Proline-rich AMPs (PrAMPs) are known to inhibit bacterial protein folding by interacting with heat shock proteins like DnaK and GroEL, but their full spectrum of targets remains unclear.
- Understanding the precise molecular targets of PrAMPs is essential for developing novel antibacterial strategies.
Purpose of the Study:
- To identify novel binding partners of apidaecin 1b analogues (Api88 and Api137) beyond known protein folding chaperones.
- To elucidate the complete mechanism of action of proline-rich antimicrobial peptides (PrAMPs) by uncovering their interactions with essential bacterial proteins.
- To explore potential new targets for the development of next-generation antibacterial agents.
Main Methods:
- Synthesis of photo-crosslinkable analogues of apidaecin 1b (Api88) incorporating p-benzoyl-phenylalanine (Bpa).
- Affinity-based enrichment of peptide-protein complexes from UV-irradiated Escherichia coli lysates using streptavidin.
- Proteomic analysis employing nanoRP-UPLC-ESI-QqTOF-MS/MS to identify cross-linked proteins.
Main Results:
- Identification of 41 unique proteins interacting with the photo-crosslinkable Api88 analogue.
- Significant enrichment of proteins involved in crucial cellular processes, including five 70S ribosomal proteins, DNA-directed RNA polymerase, and pyruvate dehydrogenase.
- Demonstration that PrAMPs interact with a broader range of essential bacterial targets than previously understood, extending beyond protein folding chaperones.
Conclusions:
- Proline-rich antimicrobial peptides (PrAMPs) exert potent bactericidal effects by interfering with fundamental bacterial processes, including protein translation and energy metabolism.
- The identified novel targets suggest that PrAMPs represent a promising class of antimicrobials with multifaceted mechanisms of action.
- Further investigation into these interactions could pave the way for the rational design of highly effective antibacterial therapies.
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