Lasso Peptide Syanodin I: Loop-Pulling Two-Step Thermal Unthreading Mechanism

Area of Science:

• Biochemistry
• Structural Biology
• Chemical Physics

Background:

• Lasso peptides are ribosomally synthesized and post-translationally modified peptides (RiPPs).
• They feature a C-terminal tail threaded through an N-terminal macrolactam ring, forming a unique topology.
• Understanding their structural dynamics, especially thermal stability, is crucial for their function.

Purpose of the Study:

• To characterize the thermal unthreading process of the lasso peptide syanodin I.
• To identify kinetic intermediates and elucidate the unfolding mechanism.
• To investigate the role of specific residues in maintaining the lasso structure.

Main Methods:

• Liquid chromatography-trapped ion mobility spectrometry-electron-capture dissociation-tandem mass spectrometry (LC-TIMS-q-ECD-ToF MS/MS).
• Steered molecular dynamics (SMD) simulations.
• Site-directed mutagenesis studies.

Main Results:

• Identified kinetic intermediates of syanodin I during thermal unfolding using LC-TIMS-q-ECD-ToF MS/MS.
• Mutagenesis confirmed Leu15 as a key residue in an intermediate lasso structure.
• Observed a two-step thermal unfolding mechanism involving loop-pulling and potentially tail-pulling, supported by SMD simulations.

Conclusions:

• Syanodin I exhibits a two-step thermal unfolding mechanism driven by steric constraints.
• The process involves loop-pulling of the lasso tail, with potential contributions from tail-pulling.
• This study provides the first report of such a mechanism in lasso peptides.