Structural basis for mannose recognition by alectin from opportunistic bacteria Burkholderia cenocepacia
Résumé
Chronic colonization of the lungs by opportunist bacteria such as Pseudomonas aeruginosa and members of the Burkholderia cepacia complex (Bcc) is the major cause of morbidity and mortality among cystic fibrosis (CF) patients. PA-IIL (lecB gene), a soluble lectin from P. aeruginosa, has been the subject of much interest because of its very strong affinity for fucose. Orthologs have been identified in the opportunist bacteria Ralstonia solanacearum, Chromobacterium violaceum and Burkholderia of Bcc. The genome of the J2315 strain of B. cenocepacia, responsible for epidemia in CF centers, contains three genes that code for proteins with PA-IIL domains. The shortest gene was cloned in Escherichia coli and pure recombinant protein, BclA, obtained. The presence of native BclA in B. cenocepacia extracts was checked by proteomics approach. The specificity of recombinant BclA was characterized using surface plasmon resonance showing a preference for mannosides and supported with glycan array experiments demonstrating a strict specificity for oligomannose-type N-glycan structures. The interaction thermodynamics of BclA with α-methyl-mannoside (αMeMan) demonstrates a dissociation constant (Kd) of 2.75 x 10-6 M. The X-ray crystal structure of the complex with αMeMan was determined at 1.7Å resolution. The lectin forms homodimers with one binding site per monomer, acting cooperatively with the second dimer site. Each monomer contains two calcium ions and one sugar ligand. Despite strong sequence similarity, the differences between BclA and PA-IIL in their specificity, binding site, and oligomerisation mode indicate that the proteins should have different roles in the bacteria.
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