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Projeto de investigação
Translating the human-microbiome molecular cross-talk using glycan microarray and structural biology strategies
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The structure of a Bacteroides thetaiotaomicron carbohydrate-binding module provides new insight into the recognition of complex pectic polysaccharides by the human microbiome
Publication . Trovão, Filipa; Correia, Viviana G.; Lourenço, Frederico M.; Ribeiro, Diana O.; Carvalho, Ana Luísa; Palma, Angelina S.; Pinheiro, Benedita A.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Academic Press Inc.
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 β-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.
A molecular view on how a commensal bacterium thrives in the human gut
Publication . Candeias, Alícia Isabel Martins; Carvalho, Ana; Palma, Maria Angelina
The human gut contains a community of commensal bacteria, named gut microbiota, which are essential to breakdown the dietary polysaccharides indigestible by humans, by producing carbohydrate-active enzymes (CAZymes). The Bacteroidetes phylum possess polysaccharide utilization loci (PUL) that code for CAZymes, along with other proteins responsible for transport and uptake of dietary polysaccharides and host-derived glycans. The CAZymes are usually associated with non-catalytic ancillary modules, nominated carbohydrate-binding modules (CBMs), important for targeting carbohydrates. Multiple CAZymes and CBMs are still not well characterized and require further structural and functional studies.
The main goal of the work reported in this thesis was to structurally characterize three putative family 32 CBMs identified in the genome of Bacteroides caccae, a member of the gut microbiota. These putative CBM32s are the non-catalytic ancillary modules found in glycoside hydrolases family 31 (BC03580) and in peptidases from the M60-like family (BC16100-C and BC07535-C). A preliminary three-dimensional (3D) structure of BC03580 (PUL 22) was solved by X-ray crystallography at a resolution of 2.97Å, revealing multiple regions of disordered electron density. In addition, the 3D structures of BC16100-C from PUL 53 and BC16100-C bound to GalNAc were obtained with a resolution of 1.67Å and 1.6Å, respectively. A prediction of the 3D structure of BC07535-C (PUL 35) was also modelled using the machine-learning approach of AlphaFold2. The comparison with a characterized CBM32, BT3015-C, revealed the conservation of a -sandwich fold in the three putative CBM32s and a lack of conservation of some amino acids belonging to the putative binding site in BC16100-C and BC07535-C. This could result in different modes of binding to their respective carbohydrates ligands, Tn and core 1 and core 2 O-glycans. These results pave the way to an increased knowledge on the function of these important modules and their influence in the action of the enzyme partners.
Mapping Molecular Recognition of β1,3-1,4-Glucans by a Surface Glycan-Binding Protein from the Human Gut Symbiont Bacteroides ovatus
Publication . Correia, Viviana G.; Trovão, Filipa; Pinheiro, Benedita A.; Brás, Joana L. A.; Silva, Lisete M.; Nunes, Cláudia; Coimbra, Manuel A.; Liu, Yan; Feizi, Ten; Fontes, Carlos M. G. A.; Mulloy, Barbara; Chai, Wengang; Carvalho, Ana Luísa; Palma, Angelina S.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; American Society for Microbiology
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 β1,3-1,4-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 β1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward β1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of β1,3 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 β1,3-1,4-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 β-glucan backbone imposed by the β1,3 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 β1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows Bacteroidetes to outcompete bacteria that lack this PUL for utilization of β1,3-1,4-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 β1,3-1,4-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.
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Fundação para a Ciência e a Tecnologia
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OE
Número da atribuição
SFRH/BD/143494/2019