title: The C-terminal domains of two homologous Oleaceae β-1,3-glucanases recognise carbohydrates differently: Laminarin binding by NMR.
abstract: 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.
authors.affiliation: Departamento de Química Física Biológica, Instituto de Química Física "Rocasolano", CSIC, Serrano 119, 28006 Madrid, Spain.
authors.forename: Héctor
authors.initials: H
authors.lastname: Zamora-Carreras
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Facultad de Química, Universidad Complutense, 28040 Madrid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Torres
authors.affiliation: Departamento de Química Física Biológica, Instituto de Química Física "Rocasolano", CSIC, Serrano 119, 28006 Madrid, Spain.
authors.forename: Noemí
authors.initials: N
authors.lastname: Bustamante
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors.forename: Anjos L
authors.initials: AL
authors.lastname: Macedo
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Facultad de Química, Universidad Complutense, 28040 Madrid, Spain.
authors.forename: Rosalía
authors.initials: R
authors.lastname: Rodríguez
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Facultad de Química, Universidad Complutense, 28040 Madrid, Spain.
authors.forename: Mayte
authors.initials: M
authors.lastname: Villalba
authors.affiliation: Departamento de Química Física Biológica, Instituto de Química Física "Rocasolano", CSIC, Serrano 119, 28006 Madrid, Spain. Electronic address: mbruix@iqfr.csic.es.
authors.forename: Marta
authors.initials: M
authors.lastname: Bruix
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D001665:Binding Sites; D031661:Fraxinus / Q000737:chemistry / Q000201:enzymology; D015870:Gene Expression; D043326:Glucan 1,3-beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D005936:Glucans / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D031658:Olea / Q000737:chemistry / Q000201:enzymology; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011058:Pollen / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D005936:Glucans; D010940:Plant Proteins; D011994:Recombinant Proteins; C008247:laminaran; C433244:OLE9 protein, Olea europaea; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase; C015968:lichenin
keywords: Allergy; Ash pollen; Carbohydrate-binding protein; Fra e 9; NMR; Ole e 9; β-1,3-Glucanase