The Structural Basis for the Ligand Specificity of Family 2 Carbohydrate-binding Modules

Abstract The conserved N-glycan on Asn297 of immunoglobulin G (IgG) has significant impacts on antibody effector functions, and is a frequent target for antibody engineering. Chemoenzymatic synthesis has emerged as a strategy for producing antibodies with homogenous glycosylation and improved effector functions. Central to this strategy is the use of enzymes with activity on the Asn297 glycan. EndoS and EndoS2, produced by Streptococcus pyogenes, are endoglycosidases with remarkable specificity for Asn297 glycosylation, making them ideal tools for chemoenzymatic synthesis. Although both enzymes are specific for IgG, EndoS2 recognizes a wider range of glycans than EndoS. Recent progress has been made in understanding the structural basis for their activities on antibodies. In this review, we examine the molecular mechanism of glycosidic bond cleavage by these enzymes and how specific point mutations convert them into glycosynthases. We also discuss the structural basis for differences in the glycan repertoire that IgG-active endoglycosidases recognize, which focuses on the structure of the loops within the glycoside hydrolase (GH) domain. Finally, we discuss the important contributions of carbohydrate binding modules (CBMs) to endoglycosidase activity, and how CBMs work in concert with GH domains to produce optimal activity on IgG.

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Carbohydrate-binding modules: fine-tuning polysaccharide recognition

Biochemical Journal ◽

10.1042/bj20040892 ◽

2004 ◽

Vol 382 (3) ◽

pp. 769-781 ◽

Cited By ~ 1241

Author(s):

Alisdair B. BORASTON ◽

David N. BOLAM ◽

Harry J. GILBERT ◽

Gideon J. DAVIES

Keyword(s):

Glycoside Hydrolases ◽

Fine Tuning ◽

Carbohydrate Binding ◽

Ligand Specificity ◽

Glycosidic Bonds ◽

Carbohydrate Binding Modules ◽

Bioinformatic Approaches ◽

The Impact ◽

And Storage ◽

Hydrolysis Of

The enzymic degradation of insoluble polysaccharides is one of the most important reactions on earth. Despite this, glycoside hydrolases attack such polysaccharides relatively inefficiently as their target glycosidic bonds are often inaccessible to the active site of the appropriate enzymes. In order to overcome these problems, many of the glycoside hydrolases that utilize insoluble substrates are modular, comprising catalytic modules appended to one or more non-catalytic CBMs (carbohydrate-binding modules). CBMs promote the association of the enzyme with the substrate. In view of the central role that CBMs play in the enzymic hydrolysis of plant structural and storage polysaccharides, the ligand specificity displayed by these protein modules and the mechanism by which they recognize their target carbohydrates have received considerable attention since their discovery almost 20 years ago. In the last few years, CBM research has harnessed structural, functional and bioinformatic approaches to elucidate the molecular determinants that drive CBM–carbohydrate recognition. The present review summarizes the impact structural biology has had on our understanding of the mechanisms by which CBMs bind to their target ligands.

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Molecular determinants of ligand specificity in family 11 carbohydrate binding modules - an NMR, X-ray crystallography and computational chemistry approach

FEBS Journal ◽

10.1111/j.1742-4658.2008.06401.x ◽

2008 ◽

Vol 275 (10) ◽

pp. 2524-2535 ◽

Cited By ~ 24

Author(s):

Aldino Viegas ◽

Natércia F. Brás ◽

Nuno M. F. S. A. Cerqueira ◽

Pedro Alexandrino Fernandes ◽

José A. M. Prates ◽

...

Keyword(s):

Computational Chemistry ◽

Carbohydrate Binding ◽

Ligand Specificity ◽

X Ray ◽

X Ray Crystallography ◽

Carbohydrate Binding Modules ◽

Molecular Determinants

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Structural basis for binding uronic acids by family 32 carbohydrate-binding modules

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2020.09.064 ◽

2020 ◽

Vol 533 (3) ◽

pp. 257-261

Author(s):

Aik-Hong Teh ◽

Pei-Fang Sim ◽

Tamao Hisano

Keyword(s):

Structural Basis ◽

Carbohydrate Binding ◽

Uronic Acids ◽

Carbohydrate Binding Modules

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Structural basis for carbohydrate-binding specificity—A comparative assessment of two engineered carbohydrate-binding modules

Glycobiology ◽

10.1093/glycob/cws063 ◽

2012 ◽

Vol 22 (7) ◽

pp. 948-961 ◽

Cited By ~ 19

Author(s):

Laura von Schantz ◽

Maria Håkansson ◽

Derek T Logan ◽

Björn Walse ◽

Jacob Österlin ◽

...

Keyword(s):

Binding Specificity ◽

Comparative Assessment ◽

Structural Basis ◽

Carbohydrate Binding ◽

Carbohydrate Binding Modules

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Atypical Cohesin-Dockerin Complex Responsible for Cell Surface Attachment of Cellulosomal Components

Journal of Biological Chemistry ◽

10.1074/jbc.m113.466672 ◽

2013 ◽

Vol 288 (23) ◽

pp. 16827-16838 ◽

Cited By ~ 28

Author(s):

Orly Salama-Alber ◽

Maroor K. Jobby ◽

Seth Chitayat ◽

Steven P. Smith ◽

Bryan A. White ◽

...

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Calcium Binding ◽

Plant Cell Wall ◽

Structural Basis ◽

Single Type ◽

Rumen Bacterium ◽

Carbohydrate Binding ◽

Surface Attachment ◽

Carbohydrate Binding Modules

The rumen bacterium Ruminococcus flavefaciens produces a highly organized multienzyme cellulosome complex that plays a key role in the degradation of plant cell wall polysaccharides, notably cellulose. The R. flavefaciens cellulosomal system is anchored to the bacterial cell wall through a relatively small ScaE scaffoldin subunit, which bears a single type IIIe cohesin responsible for the attachment of two major dockerin-containing scaffoldin proteins, ScaB and the cellulose-binding protein CttA. Although ScaB recruits the catalytic machinery onto the complex, CttA mediates attachment of the bacterial substrate via its two putative carbohydrate-binding modules. In an effort to understand the structural basis for assembly and cell surface attachment of the cellulosome in R. flavefaciens, we determined the crystal structure of the high affinity complex (Kd = 20.83 nm) between the cohesin module of ScaE (CohE) and its cognate X-dockerin (XDoc) modular dyad from CttA at 1.97-Å resolution. The structure reveals an atypical calcium-binding loop containing a 13-residue insert. The results further pinpoint two charged specificity-related residues on the surface of the cohesin module that are responsible for specific versus promiscuous cross-strain binding of the dockerin module. In addition, a combined functional role for the three enigmatic dockerin inserts was established whereby these extraneous segments serve as structural buttresses that reinforce the stalklike conformation of the X-module, thus segregating its tethered complement of cellulosomal components from the cell surface. The novel structure of the RfCohE-XDoc complex sheds light on divergent dockerin structure and function and provides insight into the specificity features of the type IIIe cohesin-dockerin interaction.

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Functional diversity of three tandem C-terminal carbohydrate-binding modules of a β-mannanase

Journal of Biological Chemistry ◽

10.1016/j.jbc.2021.100638 ◽

2021 ◽

pp. 100638

Author(s):

Marie Sofie Møller ◽

Souad El Bouaballati ◽

Bernard Henrissat ◽

Birte Svensson

Keyword(s):

Functional Diversity ◽

Carbohydrate Binding ◽

Carbohydrate Binding Modules

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Functionalization of Cellulose-Based Hydrogels with Bi-Functional Fusion Proteins Containing Carbohydrate-Binding Modules

Materials ◽

10.3390/ma14123175 ◽

2021 ◽

Vol 14 (12) ◽

pp. 3175

Author(s):

Mariana Barbosa ◽

Hélvio Simões ◽

Duarte Miguel F. Prazeres

Keyword(s):

Fusion Proteins ◽

Fluorescent Protein ◽

Protein A ◽

Chemical Properties ◽

Main Idea ◽

Bioactive Molecules ◽

Scaffolding Protein ◽

Carbohydrate Binding ◽

Biological Interactions ◽

Carbohydrate Binding Modules

Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. This functionalization can deliver tailored cellulose-based materials with enhanced physical and chemical properties and control of biological interactions that match specific applications. One of the foundations for the success of such biomaterials is to efficiently control the capacity to combine relevant biomolecules into cellulose materials in such a way that the desired functionality is attained. In this context, our main goal was to develop bi-functional biomolecular constructs for the precise modification of cellulose hydrogels with bioactive molecules of interest. The main idea was to use biomolecular engineering techniques to generate and purify different recombinant fusions of carbohydrate binding modules (CBMs) with significant biological entities. Specifically, CBM-based fusions were designed to enable the bridging of proteins or oligonucleotides with cellulose hydrogels. The work focused on constructs that combine a family 3 CBM derived from the cellulosomal-scaffolding protein A from Clostridium thermocellum (CBM3) with the following: (i) an N-terminal green fluorescent protein (GFP) domain (GFP-CBM3); (ii) a double Z domain that recognizes IgG antibodies; and (iii) a C-terminal cysteine (CBM3C). The ability of the CBM fusions to bind and/or anchor their counterparts onto the surface of cellulose hydrogels was evaluated with pull-down assays. Capture of GFP-CBM3 by cellulose was first demonstrated qualitatively by fluorescence microscopy. The binding of the fusion proteins, the capture of antibodies (by ZZ-CBM3), and the grafting of an oligonucleotide (to CBM3C) were successfully demonstrated. The bioactive cellulose platform described here enables the precise anchoring of different biomolecules onto cellulose hydrogels and could contribute significatively to the development of advanced medical diagnostic sensors or specialized biomaterials, among others.

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Integrated Counts of Carbohydrate-Active Protein Domains as Metabolic Readouts to Distinguish Probiotic Biology and Human Fecal Metagenomes

Scientific Reports ◽

10.1038/s41598-019-53173-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Hong-Hsing Liu ◽

Yu-Chen Lin ◽

Chen-Shuan Chung ◽

Kevin Liu ◽

Ya-Hui Chang ◽

...

Keyword(s):

Markov Models ◽

Protein Domains ◽

Degradation Pathway ◽

The Other ◽

Carbohydrate Binding ◽

Gram Stain ◽

Active Protein ◽

Metabolic Potential ◽

Independent Validation ◽

Carbohydrate Binding Modules

AbstractBowel microbiota is a “metaorgan” of metabolisms on which quantitative readouts must be performed before interventions can be introduced and evaluated. The study of the effects of probiotic Clostridium butyricum MIYAIRI 588 (CBM588) on intestine transplantees indicated an increased percentage of the “other glycan degradation” pathway in 16S-rRNA-inferred metagenomes. To verify the prediction, a scoring system of carbohydrate metabolisms derived from shotgun metagenomes was developed using hidden Markov models. A significant correlation (R = 0.9, p < 0.015) between both modalities was demonstrated. An independent validation revealed a strong complementarity (R = −0.97, p < 0.002) between the scores and the abundance of “glycogen degradation” in bacteria communities. On applying the system to bacteria genomes, CBM588 had only 1 match and ranked higher than the other 8 bacteria evaluated. The gram-stain properties were significantly correlated to the scores (p < 5 × 10−4). The distributions of the scored protein domains indicated that CBM588 had a considerably higher (p < 10−5) proportion of carbohydrate-binding modules than other bacteria, which suggested the superior ability of CBM588 to access carbohydrates as a metabolic driver to the bowel microbiome. These results demonstrated the use of integrated counts of protein domains as a feasible readout for metabolic potential within bacteria genomes and human metagenomes.

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