NMR and Fluorescence Spectroscopies Reveal the Preorganized Binding Site in Family 14 Carbohydrate-Binding Module from Human Chitotriosidase

Abstract

Carbohydrate-binding modules (CBM) play important roles in targeting and increasing the concentration of carbohydrate active enzymes on their substrates. Using NMR to get the solution structure of CBM14, we can gain insight into secondary structure elements and intramolecular interactions with our assigned nuclear overhauser effect peaks. This reveals that two conserved aromatic residues (Phe437 and Phe456) make up the hydrophobic core of the CBM. These residues are also responsible for connecting the two β-sheets together, by being part of β2 and β4, respectively, and together with disulfide bridges, they create CBM14’s characteristic “hevein-like” fold. Most CBMs rely on aromatic residues for substrate binding; however, CBM14 contains just a single tryptophan (Trp465) that together with Asn466 enables substrate binding. Interestingly, an alanine mutation of a single residue (Leu454) located behind Trp465 renders the CBM incapable of binding. Fluorescence spectroscopy performed on this mutant reveals a significant blue shift, as well as a minor blue shift for its neighbor Val455. The reduction in steric hindrance causes the tryptophan to be buried into the hydrophobic core of the structure and therefore suggests a preorganized binding site for this CBM. Our results show that both Trp465 and Asn466 are affected when CBM14 interacts with both (GlcNAc)3 and β-chitin, that the binding interactions are weak, and that CBM14 displays a slightly higher affinity toward β-chitin.

Structural basis of EHEP-mediated offense against phlorotannin-induced defense from brown algae to protectakuBGL activity

“…Although these proteins share a highly conserved core structure, they have different biochemical characteristics. For example, the chitin-binding domain of human chitotriosidase, Avr4 and tachycitin possess chitin-binding activity, but the critical residues for chitin-binding are not conserved 16, 18, 21 , indicating that they employ different binding mechanisms. In contrast, EHEP and allergen Der p 23 do not possess chitin-binding activity 4, 19 .…”

Structural basis of EHEP-mediated offense against phlorotannin-induced defense from brown algae to protect akuBGL activity

“…Although these proteins share a highly conserved core structure, they have different biochemical characteristics. For example, the chitin-binding domain of human chitotriosidase, Avr4, and tachycitin possess chitin-binding activity, but the critical residues for chitin binding are not conserved ( Fadel et al, 2016 ; Hurlburt et al, 2018 ; Madland et al, 2019 ), indicating that they employ different binding mechanisms. In contrast, EHEP and allergen Der p 23 do not possess chitin-binding activity ( Tsuji et al, 2017 ; Mueller et al, 2016 ).…”

Material-specific binding peptides empower sustainable innovations in plant health, biocatalysis, medicine and microplastic quantification

“…50,126 In general, a productive conformation and precise positioning of aromatic residues that align with the conformation of carbohydrate moieties (cellulose and chitin moieties are planar, and three-fold screw xylan moieties rotate 120°) is crucial for face-to-face stacking interactions. 81,123,124,190 Starch-binding domains utilize a cooperative mechanism involving two binding sites—one for initial recognition and the second one for tight/precise binding. 103…”

“…(c) Type C chitin-binding module that bind to (GlcNAc) 3 and b-chitin. Representative CBM is CBM14 from Homo sapiens chitotriosidase-1, 81 which has a small binding pocket. Key residues in CBMs for chitin binding interactions are highlighted in red.…”

“…50,126 In general, a productive conformation and precise positioning of aromatic residues that align with the conformation of carbohydrate moieties (cellulose and chitin moieties are planar, and three-fold screw xylan moieties rotate 1201) is crucial for face-to-face stacking interactions. 81,123,124,190 Starch-binding domains utilize a cooperative mechanism involving two binding sites-one for initial recognition and the second one for tight/precise binding. 103 In the case of the positively charged chitosan, and the negatively charged polysaccharides HA, alginate, PSA, and heparin, CH/p interactions, hydrogen bonding, and additionally electrostatic interactions are involved in carbohydrate-specific binding.…”