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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
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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.
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We thank Prof. Carlos Fontes and Dr Joana Bras (NZYTech, Portugal) for their assistance in obtaining the initial BT0996-C clone. We are grateful to Prof Ten Feizi, Dr Yan Liu and Dr Lisete Silva from the Glycosciences Laboratory (Imperial College London, UK) for their support and assistance on robotic microarray printing. This work was supported by the FCT - Fundação para a Ciência e a Tecnologia, I.P., through the DL-57/2016 Program Contract (BP). This work is financed by national funds from FCT - Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB. The authors acknowledge the European Synchrotron Radiation Facility (Grenoble, France) and ALBA (Barcelona, Spain) for access to beamlines ID30B and BL-13 XALOC, respectively.
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© 2022
Palavras-chave
Bacteroides thetaiotaomicron Carbohydrate binding module Carbohydrates Human gut microbiota Rhamnogalacturonan II Structural Biology