Carbohydrate Binding Module Family 102

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• Author: Marie-Katherin Zühlke
• Responsible Curator: Elizabeth Ficko-Blean

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CAZy DB link

https://www.cazy.org/CBM102.html

Ligand specificities

The dissected C-terminal CBM102_IV of a surface glycan-binding protein (SGBP; containing four CBM102s I-IV, Figure 1a) of Christiangramia forsetii KT0803^T binds laminarin (from Laminaria digitata) and laminarin-derived oligosaccharides as determined by affinity gel electrophoresis and/or isothermal titration calorimetry [1]. CBM102_IV has a higher affinity to laminarin (K_a ~1.04 x 10⁵ M^-1) than to laminariheptaose (K_a ~1.87 x 10⁴ M^-1) and laminaripentaose (K_a ~1.39 x 10⁴ M^-1). Laminaribiose did not bind the CBM102_IV. In addition, the dissected C-terminal CBM102_IV has a higher affinity to laminarin than two CBM102 tandem constructs of the SGBP (K_a ~2.76 x 10⁴ M^-1 or ~3.63 x 10⁴ M^-1) and the full length SGBP (K_a ~3.09 x 10⁴ M^-1) [1].

Structural Features

The CBM102 displays a β-sandwich fold established by two antiparallel β-sheets forming a convex and a concave face. The 3D X-ray crystal structure of the C-terminal CBM102_IV of the C. forsetii SGBP showed that, together with two expansive loops, the concave face provides a narrow cleft to accommodate laminaritriose [1] (Figure 1b). CBM102 thus represents a type B CBM. Three residues located in the concave face mediate polar interactions with laminaritriose (Lys825, Glu827 and Glu837), while one aromatic residue of each loop (Tyr790, Phe861) is involved in CH-π interaction with the ligand (Figure 1c). However, the C. forsetii protein contains three additional CBM102s. Alphafold2 [2] predictions showed that two of them are highly similar to the 3D crystal structure of the C-terminal CBM102_IV, but that one CBM102 is missing one of the loops shaping the pocket, which results in a very shallow cleft (Figure 2). This was speculated to represent an adaptation to highly branched laminarins [1].

Figure 1. a The Alphafold2-predicted structure of the CBM102-containing SGBP of C. forsetii [1]. The four CBM102s (I-IV, red) follow two N-terminal Ig-like domains (grey). b 3D crystal structure of the C-terminal CBM102_IV of the C. forsetii SGBP with laminaritriose (PDB ID 8QX6) [1]. c Zooming into the binding site: residues mediating polar and CH-π interactions with laminaritriose.

Figure 2. Surfaces of the four CBM102s of the C. forsetii SGBP [1]. Left: 3D crystal structure of the C-terminal CBM102_IV with laminaritriose (PDB ID 8QX6). Right: Alphafold2-predicted structures (I-III). CBM102_III is missing one of the groove-forming loops.

Functionalities

The CBM102-containing SGBP is tethered to the outer membrane of C. forsetii and is abundantly produced in laminarin-grown cells [3]. With four CBM102s, the SGBP provides multiple laminarin docking sites [1]. The protein further contains two N-terminal Ig-like domains that precede the CBM102s, which presumably act as spacers to ensure distance to the membrane and exposure of binding sites as suggested before for β-glucan-binding CBM-containing SGBPs [4]. The four repeating CBM102s in the C. forsetii protein were suggested to render the SGBP an optimal 'sugar-trapping antenna' on the bacterial surface; this is supported by an elongated protein shape (R_g 52 Å and D_max 178 Å) as determined by small angle X-ray scattering [1]. Unexpectedly, isothermal titration calorimetry showed that maximum two laminarin molecules were bound by the four CBM102s and that affinity was not increased by multiple CBM102s. In bacterial metagenome-assembled genomes from phytoplankton blooms in the North Sea, CBM102 was detected together with appended catalytic modules, indicating other putative functions (for example enhancing enzyme concentration on substrate to improve catalysis) [1]. The majority of associated catalytic modules were GH16_3s, but also rare examples of GH2, GH5_34, GH81, and GH162. Within this dataset, CBM102 also co-occurred with CBM4, CBM6 and CBM103 in multimodular proteins. CBM102-only-proteins contained up to eight repeats of CBM102. The production of CBM102-containing proteins correlated with the course of the phytoplankton bloom, which underlines the relevance of CBM102 in marine β-glucan use.

Family Firsts

First Identified

CBM102 was first identified in an SGBP encoded in a laminarin utilization locus of Christiangramia forsetii KT0803^T [1], a marine flavobacterium isolated from surface waters in the North Sea [5]. More than 350 CBM102-containing sequences were detected in bacterial metagenome-assembled genomes (555 representative MAGs of which 201 belonged to Bacteroidota) retrieved from bacterial biomass from phytoplankton blooms of three respective years (2016, 2018 and 2020) in the North Sea. Sequences mostly belonged to Bacteroidota, but also to Proteobacteria, Myxococcota and Actinobacteriota [1]. The association with GHs (mostly GH16_3) within this dataset led to the creation of the CBM family 102.

First Structural Characterization

The C-terminal CBM102_IV of the laminarin PUL-encoded SGBP of C. forsetii KT0803^T is the first structurally characterized member of the family (PDB ID 8QX6). The 3D crystal structure was obtained in complex with laminaritriose.

References

1. Zühlke MK, Ficko-Blean E, Bartosik D, Terrapon N, Jeudy A, Jam M, Wang F, Welsch N, Dürwald A, Martin LT, Larocque R, Jouanneau D, Eisenack T, Thomas F, Trautwein-Schult A, Teeling H, Becher D, Schweder T, and Czjzek M. (2024). Unveiling the role of novel carbohydrate-binding modules in laminarin interaction of multimodular proteins from marine Bacteroidota during phytoplankton blooms. Environ Microbiol. 2024;26(5):e16624. DOI:10.1111/1462-2920.16624 |
2. Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, and Steinegger M. (2022). ColabFold: making protein folding accessible to all. Nat Methods. 2022;19(6):679-682. DOI:10.1038/s41592-022-01488-1 |
3. Kabisch A, Otto A, König S, Becher D, Albrecht D, Schüler M, Teeling H, Amann RI, and Schweder T. (2014). Functional characterization of polysaccharide utilization loci in the marine Bacteroidetes 'Gramella forsetii' KT0803. ISME J. 2014;8(7):1492-502. DOI:10.1038/ismej.2014.4 |
4. Tamura K, Dejean G, Van Petegem F, and Brumer H. (2021). Distinct protein architectures mediate species-specific beta-glucan binding and metabolism in the human gut microbiota. J Biol Chem. 2021;296:100415. DOI:10.1016/j.jbc.2021.100415 |
5. Eilers H, Pernthaler J, Peplies J, Glöckner FO, Gerdts G, and Amann R. (2001). Isolation of novel pelagic bacteria from the German bight and their seasonal contributions to surface picoplankton. Appl Environ Microbiol. 2001;67(11):5134-42. DOI:10.1128/AEM.67.11.5134-5142.2001 |