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Improving coiled coil stability while maintaining specificity by a bacterial hitchhiker selection system.

Tim Kükenshöner1, Daniel Wohlwend2, Christoph Niemöller3

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Summary

We developed a novel bacterial selection system, hitchhiker translocation (HiT) selection, to design peptides targeting cellular proteins. This method successfully identified a highly stable and specific peptide (iM10) with nanomolar affinity for the microphthalmia-associated transcription factor.

Keywords:
Basic helix-loop-helix leucine zipperCoiled coilsMicrophthalmia associated transcription factorSelection and designTwin arginine translocation pathway

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Area of Science:

  • Molecular Biology
  • Protein Engineering
  • Biochemistry

Background:

  • Designing peptides that specifically target cellular proteins is complex, often resulting in candidates with suboptimal characteristics.
  • The twin-arginine translocase (TAT) pathway in Escherichia coli offers a potential platform for novel peptide selection systems.

Purpose of the Study:

  • To introduce and validate a new bacterial selection system, hitchhiker translocation (HiT) selection, for generating targeted peptides.
  • To select and characterize peptides that interfere with the microphthalmia-associated transcription factor (MITF), a key protein in melanoma.

Main Methods:

  • Utilized the Escherichia coli twin-arginine translocase (TAT) pathway for the hitchhiker translocation (HiT) selection system.
  • Designed and selected α-helix encoding sequences for interference with the coiled coil (CC) domain of the microphthalmia-associated transcription factor (MITF).
  • Determined the crystal structure of the selected peptide (iM10) in complex with the MITF-CC heterodimer.

Main Results:

  • The HiT selection enriched a predominant sequence (iM10) with high protease resistance, solubility, and thermal stability.
  • The selected peptide iM10 demonstrated nanomolar affinity for the target MITF peptide and maintained specificity.
  • Structural analysis confirmed the iM10/MITF-CC heterodimer formation and revealed binding to both the coiled coil and HLHLZ domains of MITF.

Conclusions:

  • The hitchhiker translocation (HiT) selection system is an effective approach for designing functional peptides with desired properties.
  • The identified peptide iM10 represents a promising candidate for targeting MITF, with potential therapeutic implications.
  • The study validates the utility of bacterial selection systems for advancing protein engineering and drug discovery efforts.