Molecular basis of peptide recognition in the truncated Vibrio harveyi chitoporin

Nanopore-forming outer membrane proteins mediate the selective transport of peptides and small molecules in Gram-negative bacteria. In this work, we investigate how peptides are recognized by a truncated form of Vibrio harveyi chitoporin (VhChiPΔN-plug), which lacks the native N-terminal plug. Using electrophysiology black lipid membrane, we show that a synthetic peptide mimicking the N-plug binds selectively and directionally to VhChiPΔN-plug pores when applied from the trans (periplasmic) side, consistent with the native extracellular orientation of the plug. These interactions are voltage dependent with prolonged dwell times and submicromolar affinity, indicating strong and specific binding. The cocrystal structures of the N-plug peptide-VhChiPΔN-plug complex (PDB: 8ZXI) reveal that the conserved Asp¹ residue at the N-terminus of the peptide forms salt bridges with arginine residues in the pore constriction region. The positively charged arginines create an electrostatic environment that stabilizes the negatively charged peptide end, providing a molecular basis for the observed binding directionality and specificity. Together, electrophysiological and structural data demonstrate that the synthetic peptide effectively mimics the natural occlusion mechanism of the N-plug. These findings validate that VhChiPΔN-plug is a useful model for studying peptide recognition and offer insights for designing nanopore-based sensing applications.