The structure of a Bacteroides thetaiotaomicron carbohydrate-binding module provides new insight into the recognition of complex pectic polysaccharides by the human microbiome.

The Bacteroides thetaiotaomicron has developed a consortium of enzymes capable of overcoming steric constraints and degrading, in a sequential manner, the complex rhamnogalacturonan II (RG-II) polysaccharide. BT0996 protein acts in the initial stages of the RG-II depolymerisation, where its two catalytic modules remove the terminal monosaccharides from RG-II side chains A and B. BT0996 is modular and has three putative carbohydrate-binding modules (CBMs) for which the roles in the RG-II degradation are unknown. Here, we present the characterisation of the module at the C-terminal domain, which we designated BT0996-C. The high-resolution structure obtained by X-ray crystallography reveals that the protein displays a typical ß-sandwich fold with structural similarity to CBMs assigned to families 6 and 35. The distinctive features are: 1) the presence of several charged residues at the BT0996-C surface creating a large, broad positive lysine-rich patch that encompasses the putative binding site; and 2) the absence of the highly conserved binding-site signatures observed in CBMs from families 6 and 35, such as region A tryptophan and region C asparagine. These findings hint at a binding mode of BT0996-C not yet observed in its homologues. In line with this, carbohydrate microarrays and microscale thermophoresis show the ability of BT0996-C to bind α1-4-linked polygalacturonic acid, and that electrostatic interactions are essential for the recognition of the anionic polysaccharide. The results support the hypothesis that BT0996-C may have evolved to potentiate the action of BT0996 catalytic modules on the complex structure of RG-II by binding to the polygalacturonic acid backbone sequence.

Mapping Molecular Recognition of ß1,3-1,4-Glucans by a Surface Glycan-Binding Protein from the Human Gut Symbiont Bacteroides ovatus.

A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent members of Bacteroidetes in the human gut and characterized in Bacteroides ovatus. This PUL-encoded system specifically targets mixed-linkage ß1,3-1,4-glucans, a group of diet-derived carbohydrates that promote a healthy microbiota and have potential as prebiotics. The BoSGBPMLG-A protein encoded by the BACOVA_2743 gene is a SusD-like protein that plays a key role in the PUL's specificity and functionality. Here, we perform a detailed analysis of the molecular determinants underlying carbohydrate binding by BoSGBPMLG-A, combining carbohydrate microarray technology with quantitative affinity studies and a high-resolution X-ray crystallography structure of the complex of BoSGBPMLG-A with a ß1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward ß1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of ß1,3 linkages. The interaction is defined by a 41-Å-long extended binding site that accommodates the oligosaccharide in a mode distinct from that of previously described bacterial ß1,3-1,4-glucan-binding proteins. In addition to the shape complementarity mediated by CH-π interactions, a complex hydrogen bonding network complemented by a high number of key ordered water molecules establishes additional specific interactions with the oligosaccharide. These support the twisted conformation of the ß-glucan backbone imposed by the ß1,3 linkages and explain the dependency on the oligosaccharide chain length. We propose that the specificity of the PUL conferred by BoSGBPMLG-A to import long ß1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows Bacteroidetes to outcompete bacteria that lack this PUL for utilization of ß1,3-1,4-glucans. IMPORTANCE With the knowledge of bacterial gene systems encoding proteins that target dietary carbohydrates as a source of nutrients and their importance for human health, major efforts are being made to understand carbohydrate recognition by various commensal bacteria. Here, we describe an integrative strategy that combines carbohydrate microarray technology with structural studies to further elucidate the molecular determinants of carbohydrate recognition by BoSGBPMLG-A, a key protein expressed at the surface of Bacteroides ovatus for utilization of mixed-linkage ß1,3-1,4-glucans. We have mapped at high resolution interactions that occur at the binding site of BoSGBPMLG-A and provide evidence for the role of key water-mediated interactions for fine specificity and affinity. Understanding at the molecular level how commensal bacteria, such as prominent members of Bacteroidetes, can differentially utilize dietary carbohydrates with potential prebiotic activities will shed light on possible ways to modulate the microbiome to promote human health.

Noncovalent microarrays from synthetic amino-terminating glycans: Implications in expanding glycan microarray diversity and platform comparison.

Glycan microarrays have played important roles in detection and specificity assignment of glycan recognition by proteins. However, the size and diversity of glycan libraries in current microarray systems are small compared to estimated glycomes, and these may lead to missed detection or incomplete assignment. For microarray construction, covalent and noncovalent immobilization are the two types of methods used, but a direct comparison of results from the two platforms is required. Here we develop a chemical strategy to prepare lipid-linked probes from both naturally derived aldehyde-terminating and synthetic amino-terminating glycans that addresses the two aspects: expansion of sequence-defined glycan libraries and comparison of the two platforms. We demonstrate the specific recognition by plant and mammalian lectins, carbohydrate-binding modules and antibodies and the overall similarities from the two platforms. Our results provide new knowledge on unique glycan-binding specificities for the immune receptor Dectin-1 toward ß-glucans and the interaction of rotavirus P[19] adhesive protein with mucin O-glycan cores.

Structural Insights into the Molecular Recognition Mechanism of the Cancer and Pathogenic Epitope, LacdiNAc by Immune-Related Lectins.

Interactions of glycan-specific epitopes to human lectin receptors represent novel immune checkpoints for investigating cancer and infection diseases. By employing a multidisciplinary approach that combines isothermal titration calorimetry, NMR spectroscopy, molecular dynamics simulations, and X-ray crystallography, we investigated the molecular determinants that govern the recognition of the tumour and pathogenic glycobiomarker LacdiNAc (GalNAcß1-4GlcNAc, LDN), including their comparison with the ubiquitous LacNAc epitope (Galß1-4GlcNAc, LN), by two human immune-related lectins, galectin-3 (hGal-3) and the macrophage galactose C-type lectin (hMGL). A different mechanism of binding and interactions was observed for the hGal-3/LDN and hMGL/LDN complexes, which explains the remarkable difference in the binding specificity of LDN and LN by these two lectins. The new structural clues reported herein are fundamental for the chemical design of mimetics targeting hGal-3/hMGL recognition process.

Molecular Recognition of a Thomsen-Friedenreich Antigen Mimetic Targeting Human Galectin-3.

Overexpression of the Thomsen-Friedenreich (TF) antigen in cell membrane proteins occurs in 90 % of adenocarcinomas. Additionally, the binding of the TF antigen to human galectin-3 (Gal-3), also frequently overexpressed in malignancy, promotes cancer progression and metastasis. In this context, structures that interfere with this specific interaction have the potential to prevent cancer metastasis. A multidisciplinary approach combining the optimized synthesis of a TF antigen mimetic with NMR, X-ray crystallography methods, and isothermal titration calorimetry assays was used to unravel the molecular structural details that govern the Gal-3/TF mimetic interaction. The TF mimetic has a binding affinity for Gal-3 similar to that of the TF natural antigen and retains the binding epitope and bioactive conformation observed for the native antigen. Furthermore, from a thermodynamic perspective, a decrease in the enthalpic contribution was observed for the Gal-3/TF mimetic complex; however, this behavior is compensated by a favorable gain in entropy. From a structural perspective, these results establish our TF mimetic as a scaffold to design multivalent solutions to potentially interfere with Gal-3 aberrant interactions and for likely use in hampering Gal-3-mediated cancer cell adhesion and metastasis.

The Crystal Structure of the R280K Mutant of Human p53 Explains the Loss of DNA Binding.

The p53 tumor suppressor is widely found to be mutated in human cancer. This protein is regarded as a molecular hub regulating different cell responses, namely cell death. Compelling data have demonstrated that the impairment of p53 activity correlates with tumor development and maintenance. For these reasons, the reactivation of p53 function is regarded as a promising strategy to halt cancer. In the present work, the recombinant mutant p53R280K DNA binding domain (DBD) was produced for the first time, and its crystal structure was determined in the absence of DNA to a resolution of 2.0 Å. The solved structure contains four molecules in the asymmetric unit, four zinc(II) ions, and 336 water molecules. The structure was compared with the wild-type p53 DBD structure, isolated and in complex with DNA. These comparisons contributed to a deeper understanding of the mutant p53R280K structure, as well as the loss of DNA binding related to halted transcriptional activity. The structural information derived may also contribute to the rational design of mutant p53 reactivating molecules with potential application in cancer treatment.