The C-terminal domains of two homologous Oleaceae β-1,3-glucanases recognise carbohydrates differently: Laminarin binding by NMR

• Published in: Archives of biochemistry and biophysics 2015-08, Vol.580, p.93-101
• Main Authors: Zamora-Carreras, Héctor, Torres, María, Bustamante, Noemí, Macedo, Anjos L., Rodríguez, Rosalía, Villalba, Mayte, Bruix, Marta
• Format: Article
• Language: English
• Subjects: Allergens - chemistry; Allergens - genetics; Allergens - immunology; Allergy; Amino Acid Sequence; Antigens, Plant - chemistry; Antigens, Plant - genetics; Antigens, Plant - immunology; Ash pollen; beta-Glucosidase - chemistry; beta-Glucosidase - genetics; beta-Glucosidase - immunology; Binding Sites; Carbohydrate-binding protein; Fra e 9; Fraxinus - chemistry; Fraxinus - enzymology; Gene Expression; Glucan 1,3-beta-Glucosidase - chemistry; Glucan 1,3-beta-Glucosidase - genetics; Glucan 1,3-beta-Glucosidase - immunology; Glucans - chemistry; Humans; Models, Molecular; Molecular Sequence Data; NMR; Nuclear Magnetic Resonance, Biomolecular; Ole e 9; Olea - chemistry; Olea - enzymology; Pichia - genetics; Pichia - metabolism; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - immunology; Pollen - chemistry; Pollen - immunology; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Sequence Homology, Amino Acid; β-1,3-Glucanase
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2015.07.004

[Display omitted] •Homologous CtD-Fra e 9 and CtD-Ole e 9 differ in their carbohydrate-binding ability.•CtD-Ole e 9 shows a higher affinity for laminarin than CtD-Fra e 9.•Results point to the formation of 1:1 stoichiometry protein–sugar complexes.•Protein/laminarin interaction occurs in the aromatic-rich inter-helical region.•rCtD-Ole e 9 higher affinity for laminarin can arise from its larger binding surface. Ole e 9 and Fra e 9 are two allergenic β-1,3-glucanases from olive and ash tree pollens, respectively. Both proteins present a modular structure with a catalytic N-terminal domain and a carbohydrate-binding module (CBM) at the C-terminus. Despite their significant sequence resemblance, they differ in some functional properties, such as their catalytic activity and the carbohydrate-binding ability. Here, we have studied the different capability of the recombinant C-terminal domain of both allergens to bind laminarin by NMR titrations, binding assays and ultracentrifugation. We show that rCtD-Ole e 9 has a higher affinity for laminarin than rCtD-Fra e 9. The complexes have different exchange regimes on the NMR time scale in agreement with the different affinity for laminarin observed in the biochemical experiments. Utilising NMR chemical shift perturbation data, we show that only one side of the protein surface is affected by the interaction and that the binding site is located in the inter-helical region between α1 and α2, which is buttressed by aromatic side chains. The binding surface is larger in rCtD-Ole e 9 which may account for its higher affinity for laminarin relative to rCtD-Fra e 9.