Structure and function of a novel periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria

Wipa Suginta,Natchanok Sritho,Araya Ranok,David Michael Bulmer,Yoshihito Kitaoku,Bert Van Den Berg,Tamo Fukamizo

doi:10.1074/jbc.ra117.001012

Abstract

Periplasmic solute-binding proteins in bacteria are involved in the active transport of nutrients into the cytoplasm. In marine bacteria of the genus Vibrio, a chitooligosaccharide-binding protein (CBP) is thought to be the major solute-binding protein controlling the rate of chitin uptake in these bacteria. However, the molecular mechanism of the CBP involvement in chitin metabolism has not been elucidated. Here, we report the structure and function of a recombinant chitooligosaccharide-binding protein from Vibrio harveyi, namely VhCBP, expressed in Escherichia coli Isothermal titration calorimetry revealed that VhCBP strongly binds shorter chitooligosaccharides ((GlcNAc) n , where n = 2, 3, and 4) with affinities that are considerably greater than those for glycoside hydrolase family 18 and 19 chitinases but does not bind longer ones, including insoluble chitin polysaccharides. We also found that VhCBP comprises two domains with flexible linkers and that the domain-domain interface forms the sugar-binding cleft, which is not long extended but forms a small cavity. (GlcNAc)2 bound to this cavity, apparently triggering a closed conformation of VhCBP. Trp-363 and Trp-513, which stack against the two individual GlcNAc rings, likely make a major contribution to the high affinity of VhCBP for (GlcNAc)2 The strong chitobiose binding, followed by the conformational change of VhCBP, may facilitate its interaction with an active-transport system in the inner membrane of Vibrio species.

Highlights

Periplasmic solute-binding proteins in bacteria are involved in the active transport of nutrients into the cytoplasm
The two lobes are separated; the solute binds to the cleft formed between the two domains, 2 The abbreviations used are: GH, glycoside hydrolase; SBP, solute-binding protein; CBP, chitooligosaccharide-binding protein; VhCBP, CBP from V. harveyi; VcCBP, CBP from V. cholerae; (GlcNAc)n, ␤-1,4-linked oligosaccharide of GlcNAc with a polymerization degree of n; ATP-binding cassette-type (ABC), ATP-binding cassette; ITC, isothermal titration calorimetry; PDB, Protein Data Bank; Root-mean-square deviation (RMSD), root mean square deviation
Production of recombinant VhCBP protein by Escherichia coli expression system Using E. coli strain Origami (DE3) and the pET23a expression plasmid, we successfully produced the recombinant VhCBP protein, which was purified by Ni2ϩ-affinity chro matography followed by anion exchange chromatography (HiTrap Q) and gel filtration on 16/60 Superdex 200

Summary

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Cean and zooplankton [1]. Chitin, a ␤-1,4-linked polysaccharide of GlcNAc, is an important marine biomass, which is efficiently converted by Vibrios. V. harveyi has an efficient uptake system of GlcNAc or (GlcNAc)n, including an ATP-binding cassette-type (ABC) transporter for GlcNAc or (GlcNAc)2 [11] and a chitoporin for the transport of chitin oligosaccharides (GlcNAc)n (n ϭ 3, 4, 5, and 6) [12,13,14]. It has been recognized that solute-binding proteins (SBPs) localized to the periplasmic space of Gramnegative bacteria are involved in nutrient import as components of ABC transporters [15]. Resulting in closure of the two lobes [15] This closed conformation of SBP has been regarded as important for interaction with the transporter; for the active transport of the solute [17]. The crystal structure revealed the molecular basis of the strong binding of chitooligosaccharide to VhCBP followed by the drastic conformational change, which may be important for the active transport

Results

Discussion

Experimental procedures