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

ABSTRACT: VhCBP is a periplasmic chitooligo saccharide-specific binding protein mainly responsible for translocation of the chitooligosaccharide (GlcNAc)₂ across the double membranes of marine bacteria Vibrio harveyi. However, structural and thermodynamic understanding of the sugar binding/release processes of VhCBP are relatively unknown. VhCBP displayed the greatest affinity towards (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 towards 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 periplasm to the inner membrane-transporting components. Isothermal calorimetry data suggested that VhCBP adopts the high affinity conformation to bind (GlcNAc)₂, whilst 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.

SUBMITTER: Kitaoku Y

PROVIDER: S-EPMC8449061 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Vibrio fischeri, a marine bacterium that forms a bioluminescent symbiosis with certain fish and squids, exhibits the unusual attribute of growth on 3':5'-cyclic AMP (cAMP), apparently through the activity of a 3':5'-cyclic nucleotide phosphodiesterase (3':5'-CNP) with exceptionally high activity. The V. fischeri 3':5'-CNP is located in the periplasm, a novel cellular location for this enzyme in bacteria. To gain insight into the physiological function of this enzyme, we cloned the gene (designated cpdP) encoding it from V. fischeri MJ-1. This is the first bacterial 3':5'-CNP gene to be cloned. Sequencing and analysis of the 1.26-kb cpdP locus revealed a single open reading frame specifying a protein of 330 amino acid residues, including a 22-amino-acid leader peptide. The putative cpdP promoter contained a reasonable -10 promoter region (TATTAT) but contained no obvious -35 region; instead, a 12-bp inverted repeat (TTAAATATTTAA) occurred just upstream of this location. A possible rho-independent transcriptional terminator with a calculated free energy of -21.2 kcal.mol-1 (ca. -88.7 kJ.mol-1) followed the CpdP protein coding sequence. The predicted subunit molecular weight of 33,636 for the mature CpdP protein (36,087 less 2,451 for the leader peptide) was consistent with the molecular weight of 34,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the CpdP protein exhibited 30.3% identity with that of the low-affinity 3':5'-CNP (PDE1) of Saccharomyces cerevisiae and 33.6% identity with that of the extracellular 3':5'-CNP of Dictyostelium discoideum. The residue identities clustered in two regions, residues 100 to 146 and 238 to 269, which contained 30 of the 33 amino acids conserved in all three proteins, 4 of which were histidines. A gene replacement mutant of V. fischeri MJ-1 containing a 0.45-kb BglII deletion within the cpdP gene lacked periplasmic 3':5'-CNP activity and did not grow on cAMP, confirming for V. fischeri the relationship among cpdP, synthesis of the periplasmic 3':5'-CNP, and growth on cAMP. The mutant exhibited no obvious sensitivity to high extracellular concentrations of cAMP (5 and 10 mM), suggesting that the enzyme does not play a role in defense against extracellular cAMP.

Project description:Based on the crystal structure of lactose permease (LacY) open to the cytoplasm, a hybrid molecular simulation approach with self-guided Langevin dynamics is used to describe conformational changes that lead to a periplasmic-open state. This hybrid approach consists of implicit (IM) and explicit (EX) membrane simulations and requires self-guided Langevin dynamics to enhance protein motions during the IM simulations. The pore radius of the lumen increases by 3.5 Å on the periplasmic side and decreases by 2.5 Å on the cytoplasmic side (relative to the crystal structure), suggesting a lumen that is fully open to the periplasm to allow for extracellular sugar transport and closed to the cytoplasm. Based on our simulations, the mechanism that triggers this conformational change to the periplasmic-open state is the protonation of Glu269 and binding of the disaccharide. Then, helix packing is destabilized by breaking of several side chains involved in hydrogen bonding (Asn245, Ser41, Glu374, Lys42, and Gln242). For the periplasmic-open conformations obtained from our simulations, helix-helix distances agree well with experimental measurements using double electron-electron resonance, fluorescence resonance energy transfer, and varying sized cross-linkers. The periplasmic-open conformations are also in compliance with various substrate accessibility/reactivity measurements that indicate an opening of the protein lumen on the periplasmic side on sugar binding. The comparison with these measurements suggests a possible incomplete closure of the cytoplasmic half in our simulations. However, the closure is sufficient to prevent the disaccharide from transporting to the cytoplasm, which is in accordance with the well-established alternating access model. Ser53, Gln60, and Phe354 are determined to be important in sugar transport during the periplasmic-open stage of the sugar transport cycle and the sugar is found to undergo an orientational change in order to escape the protein lumen.

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

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