Solution Structure of the Broad-Spectrum Bacteriocin Garvicin Q

Class IId bacteriocins are linear, unmodified antimicrobial peptides produced by Gram-positive bacteria, and often display potent, narrow-spectrum inhibition spectra. Garvicin Q (GarQ) is a class IId bacteriocin produced by the lactic acid bacterium <i>Lactococcus garvieae</i>. It stands out for its unusual broad-spectrum antimicrobial activity against various bacterial species, including <i>Listeria monocytogenes</i>, <i>Pediococcus pentosaceus</i>, <i>Carnobacterium maltaromaticum</i>, <i>Enterococcus faecalis</i>, and <i>Lactococcus</i> spp. Its protein target is the mannose phosphotransferase system (Man-PTS) of susceptible bacterial strains, though little is known about the precise molecular mechanism behind GarQ's unusual broad spectrum of activity. In this work, ¹³C- and ¹⁵N-labelled GarQ was recombinantly produced using our previously described "sandwiched" protein expression system in <i>Escherichia coli</i>. We also developed a protocol to purify a uniformly labelled sample of the small ubiquitin-like modifier His₆-SUMO, which is produced as a byproduct of the expression procedure. We demonstrated its use as a "free" protein standard for 3D NMR experiment calibrations. The GarQ solution structure was solved using triple-resonance nuclear magnetic resonance (NMR) spectroscopy and was compared with the structures of other Man-PTS-targeting bacteriocins. GarQ adopts a helix-hinge-helix fold, which is contrary to its structural predictions according to AlphaFold 3.