Mallett Tyler, Lamer Tess, Aleksandrzak-Piekarczyk Tamara, McKay Ryan T, Catenza Karizza, Sit Clarissa, Rainey Jan K, Towle-Straub Kaitlyn M, Vederas John C, van Belkum Marco J
Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada.
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland.
Int J Mol Sci. 2025 Aug 14;26(16):7846. doi: 10.3390/ijms26167846.
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 . It stands out for its unusual broad-spectrum antimicrobial activity against various bacterial species, including , , , , and 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, C- and N-labelled GarQ was recombinantly produced using our previously described "sandwiched" protein expression system in . We also developed a protocol to purify a uniformly labelled sample of the small ubiquitin-like modifier His-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.
II类细菌素是由革兰氏阳性菌产生的线性、未修饰的抗菌肽，通常表现出强大的窄谱抑制谱。加维菌素Q（GarQ）是一种由乳酸菌产生的II类细菌素。它以其对包括[具体细菌种类1]、[具体细菌种类2]、[具体细菌种类3]、[具体细菌种类4]和[具体细菌种类5]等多种细菌的异常广谱抗菌活性而脱颖而出。其蛋白质靶点是敏感细菌菌株的甘露糖磷酸转移酶系统（Man-PTS），尽管对于GarQ异常广谱活性背后的确切分子机制知之甚少。在这项工作中，使用我们之前描述的“夹心”蛋白质表达系统在[具体表达宿主]中重组生产了C和N标记的GarQ。我们还开发了一种方案来纯化作为表达过程副产物产生的小泛素样修饰物His-SUMO的均匀标记样品。我们展示了其作为3D NMR实验校准的“游离”蛋白质标准品的用途。使用三重共振核磁共振（NMR）光谱解析了GarQ溶液结构，并与其他靶向Man-PTS的细菌素结构进行了比较。GarQ采用螺旋-铰链-螺旋折叠，这与其根据AlphaFold 3的结构预测相反。
