*Article* **Structure of Lacticaseicin 30 and Its Engineered Variants Revealed an Interplay between the N-Terminal and C-Terminal Regions in the Activity against Gram-Negative Bacteria**

**Désiré Madi-Moussa 1, Barbara Deracinois 1, Radja Teiar 1, Yanyan Li 2, Marius Mihasan 3, Christophe Flahaut 1, Sylvie Rebuffat 2, Françoise Coucheney 1,\* and Djamel Drider 1,\***


**Abstract:** Lacticaseicin 30 is one of the five bacteriocins produced by the Gram-positive *Lacticaseibacillus paracasei* CNCM I-5369. This 111 amino acid bacteriocin is noteworthy for being active against Gram-negative bacilli including *Escherichia coli* strains resistant to colistin. Prediction of the lacticaseicin 30 structure using the Alphafold2 pipeline revealed a largely helical structure including five helix segments, which was confirmed by circular dichroism. To identify the structural requirements of the lacticaseicin 30 activity directed against Gram-negative bacilli, a series of variants, either shortened or containing point mutations, was heterologously produced in *Escherichia coli* and assayed for their antibacterial activity against a panel of target strains including Gram-negative bacteria and the Gram-positive *Listeria innocua*. Lacticaseicin 30 variants comprising either the N-terminal region (amino acids 1 to 39) or the central and C-terminal regions (amino acids 40 to 111) were prepared. Furthermore, mutations were introduced by site-directed mutagenesis to obtain ten bacteriocin variants E6G, T7P, E32G, T33P, T52P, D57G, A74P, Y78S, Y93S and A97P. Compared to lacticaseicin 30, the anti-Gram-negative activity of the N-terminal peptide and variants E32G, T33P and D57G remained almost unchanged, while that of the C-terminal peptide and variants E6G, T7P, T52P, A74P, Y78S, Y93S and A97P was significantly altered. Finally, the N-terminal region was further shortened to keep only the first 20 amino acid part that was predicted to include the first helix. The anti-Gram-negative activity of this truncated peptide was completely abolished. Overall, this study shows that activity of lacticaseicin 30, one of the rare Gram-positive bacteriocins inhibiting Gram-negative bacteria, requires at least two helices in the N-terminal region and that the C-terminal region carries amino acids playing a role in modulation of the activity. Taken together, these data will help to design forthcoming variants of lacticaseicin 30 as promising therapeutic agents to treat infections caused by Gram-negative bacilli.

**Keywords:** *Escherichia coli*; antimicrobial activity; structure-activity relationship; helical conformation
