Solution Structure of the Broad-Spectrum Bacteriocin Garvicin Q.

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Class IId bacteriocins are linear antimicrobial peptides with narrow-spectrum activity.
• Garvicin Q (GarQ) from *Lactococcus garvieae* displays unusual broad-spectrum inhibition.
• The precise mechanism of GarQ's broad activity and its protein target, the mannose phosphotransferase system (Man-PTS), remain largely uncharacterized.

Purpose of the Study:

• To elucidate the molecular mechanism behind GarQ's broad-spectrum antimicrobial activity.
• To determine the solution structure of GarQ using NMR spectroscopy.
• To establish a protocol for recombinant production and purification of labeled GarQ and a His6-SUMO protein standard.

Main Methods:

• Recombinant production of 13C- and 15N-labeled GarQ in *Escherichia coli* using a "sandwiched" expression system.
• Development of a purification protocol for uniformly labeled His6-SUMO as a protein standard.
• Triple-resonance nuclear magnetic resonance (NMR) spectroscopy for solving the GarQ solution structure.

Main Results:

• The solution structure of GarQ was determined, revealing a helix-hinge-helix fold.
• GarQ's structure contradicts predictions made by AlphaFold 3.
• A protocol for producing labeled GarQ and His6-SUMO was established, with the latter serving as a standard for NMR calibrations.

Conclusions:

• GarQ possesses a unique helix-hinge-helix structure distinct from other Man-PTS-targeting bacteriocins.
• The structural determination provides insights into GarQ's broad-spectrum activity.
• The developed recombinant expression and purification methods facilitate further structural and functional studies of bacteriocins.