A structural model for (GlcNAc)₂ translocation via a periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria

VhCBP is a periplasmic chitooligosaccharide-binding protein mainly responsible for translocation of the chitooligosaccharide (GlcNAc)₂ across the double membranes of marine bacteria. However, structural and thermodynamic understanding of the sugar-binding/-release processes of VhCBP is relatively less. VhCBP displayed the greatest affinity toward (GlcNAc)₂, with lower affinity for longer-chain chitooligosaccharides [(GlcNAc)_3–4]. (GlcNAc)₄ partially occupied the closed sugar-binding groove, with two reducing-end GlcNAc units extending beyond the sugar-binding groove and barely characterized by weak electron density. Mutation of three conserved residues (Trp³⁶³, Asp³⁶⁵, and Trp⁵¹³) to Ala resulted in drastic decreases in the binding affinity toward the preferred substrate (GlcNAc)₂, indicating their significant contributions to sugar binding. The structure of the W513A–(GlcNAc)₂ complex in a ‘half-open’ conformation unveiled the intermediary step of the (GlcNAc)₂ translocation from the soluble CBP in the periplasm to the inner membrane–transporting components. Isothermal calorimetry data suggested that VhCBP adopts the high-affinity conformation to bind (GlcNAc)₂, while its low-affinity conformation facilitated sugar release. Thus, chitooligosaccharide translocation, conferred by periplasmic VhCBP, is a crucial step in the chitin catabolic pathway, allowing Vibrio bacteria to thrive in oceans where chitin is their major source of nutrients.