Abstract: To elucidate the structural characteristics and transport mechanism of the signaling molecule auto inducer-2 (AI-2) transporter GXS4 from Limosilactobacillus fermentum, this study utilized multiple bioinformatics methods to systematically analyze its physicochemical properties and structural features. Molecular docking and molecular dynamics simulations were conducted to investigate the binding sites and interaction mechanism between GXS4 and AI-2. The results showed that GXS4 consisted of 366 amino acid residues with a molecular mass of 40.0 kDa. It was a membrane protein lacking a signal peptide and possessed 8 transmembrane helices. The secondary structure consisted of up to 77.32% α-helix, contributing to maintaining the stability of the protein backbone and facilitating transmembrane transport. The three-dimensional structure presented a typical “half-moon” conformation, suggesting that AI-2 transport occurs through an “alternating access” mechanism. Molecular docking revealed that GXS4 preferentially binds to the (R)-2,3,3,4-tetrahydroxytetrahydrofuran borate (R-THMF) configuration of the AI-2 molecule, with key residues Val233 and Ala263 forming a stable complex with AI-2 through hydrogen bonds. Molecular dynamics simulations further verified the structural stability of the complex and found that a mutation at Ala263 significantly reduced the complex’s stability, identifying Ala263 as a critical target site of GXS4. This study provides a theoretical basis and new strategies for targeted regulation of the quorum sensing system in L. fermentum.

Key words: Limosilactobacillus fermentum; autoinducer-2 transporter protein; structural characteristics; bioinformatics; molecular dynamics simulations