Abstract
The interaction of carbohydrate-binding proteins (CBPs) with their corresponding glycan ligands is challenging to study both experimentally and computationally. This is in part due to their low binding affinity, high flexibility, and the lack of a linear sequence in carbohydrates, as exists in nucleic acids and proteins. We recently described a function-prediction technique called SPOT-Struc that identifies CBPs by global structural alignment and binding-affinity prediction. Here we experimentally determined the carbohydrate specificity and binding affinity of YesU (RCSB PDB ID: 1oq1), an uncharacterized protein from Bacillus subtilis that SPOT-Struc predicted would bind high mannose-type glycans. Glycan array analyses however revealed glycan binding patterns similar to those exhibited by fucose (Fuc)-binding lectins, with SPR analysis revealing high affinity binding to Lewisx and lacto-N-fucopentaose III. Structure based alignment of YesU revealed high similarity to the legume lectins UEA-I and GS-IV, and docking of Lewisx into YesU revealed a complex structure model with predicted binding affinity of −4.3 kcal/mol. Moreover the adherence of B. subtilis to intestinal cells was significantly inhibited by Lex and Ley but by not non-fucosylated glycans, suggesting the interaction of YesU to fucosylated glycans may be involved in the adhesion of B. subtilis to the gastrointestinal tract of mammals.
Highlights
To date many classes of carbohydrate binding proteins (CBPs) have been identified, including lectins such as F-type[1], C-type[2], and Galectins[3], and carbohydrate binding modules (CBMs) associated with glycoside hydrolases or glycosidases[4] that occur ubiquitously in nature
We describe the experimental verification of YesU as a carbohydrate-binding proteins (CBPs), and its functional annotation as a new Lewisx (Lex) binding lectin using a combination of glycan array profiling followed by detailed affinity analysis using surface plasmon resonance (SPR)
YesU displayed no significant binding to any Man structures present on the array, even though VIP36, the lectin matched to YesU using SPOT-Struc, exclusively binds high-Man type glycans[17]
Summary
To date many classes of carbohydrate binding proteins (CBPs) have been identified, including lectins such as F-type[1], C-type[2], and Galectins[3], and carbohydrate binding modules (CBMs) associated with glycoside hydrolases or glycosidases[4] that occur ubiquitously in nature. Employing computational function prediction to guide experimental analysis has the potential to significantly aid in this endeavour. We introduced a template-based method (SPOT-Struc) that predicts CBPs by making structural alignment between a query structure and the template structure of a known CBP. Our SPOT-Struc analysis matched YesU to the integral membrane mammalian protein VIP36 (2e6v)[13], a known leguminous type lectin with a β-sandwich and jellyroll fold[17,18] that recognizes high Man-type glycans[19]. We describe the experimental verification of YesU as a CBP, and its functional annotation as a new Lewisx (Lex) binding lectin using a combination of glycan array profiling followed by detailed affinity analysis using surface plasmon resonance (SPR). Our data demonstrates the complementary role of computational prediction and experimental validation in function annotation
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