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

Yoshihito Kitaoku,Tamo Fukamizo,Sawitree Kumsaoad,Prakayfun Ubonbal,Robert C Robinson,Wipa Suginta

doi:10.1016/j.jbc.2021.101071

Abstract

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.

Highlights

Vibrio spp. are Gram-negative, ubiquitous bacteria that live in oceanic, estuarine, and fresh waters
Chitooligosaccharide binding to Vibrio chitooligosaccharide-binding protein (CBP) has been proposed to trigger translocation of the bound chitooligosaccharides through the inner membrane by the (GlcNAc)2-specific ABC transporter and activation of the gene cluster involved in the chitinolytic cascade, releasing CBPs from chitin-sensor kinase, CBP from a membrane-bound histidine kinase sensor (ChiS), localized to
The structure and mechanism of a ChiP from Vibrio campbellii type strain ATCC BAA-1116 were intensively studied by crystallography in combination with physicochemical and electrochemical techniques [10, 12, 18,19,20], the molecular mechanism of the periplasmic CBP component involved in sugar translocation and control of gene activation is unknown

Summary

Introduction

Vibrio spp. are Gram-negative, ubiquitous bacteria that live in oceanic, estuarine, and fresh waters. To further analyze the sugar residue interactions in the groove extending beyond the (GlcNAc)2-binding site, we solved the crystal structures of VhCBP in complex with (GlcNAc)3 and (GlcNAc)4.

Results

Conclusion