2w68

ENHANCING THE RECEPTOR AFFINITY OF THE SIALIC ACID-BINDING DOMAIN OF VIBRIO CHOLERAE SIALIDASE THROUGH MULTIVALENCY

spinqualitylabelsall models 2w68, resolution 2.50Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 2w68 is a 3 chain structure with sequence from Vibrio cholerae. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2.5Å Ligands: BGC, CA, GAL, SIA Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function NANH_VIBCH Cleaves the terminal sialic acid (N-acetyl neuraminic acid) from carbohydrate chains in glycoproteins providing free sialic acid which can be used as carbon and energy sources. Sialidases have been suggested to be pathogenic factors in microbial infections. Facilitates cholera toxin binding to host intestinal epithelial cells by converting cell surface polysialogangliosides to GM1 monogangliosides. Publication Abstract from PubMed Many glycoside hydrolases possess carbohydrate-binding modules (CBMs) that help target these enzymes to appropriate substrates and increase their catalytic efficiency. The Vibrio cholerae sialidase contains two CBMs, one of which is designated as a family CBM40 module and has been shown through structural and calorimetry studies to recognize the alpha-anomer of sialic acid with a KD of approximately 30 microM at 37 degrees C. The affinity of this V. cholerae CBM40 module for sialic acid is one of the highest reported for recognition of a monosaccharide by a CBM. As Nature often increases a weak substrate affinity through multivalency, we have explored the potential of developing reagents with an increased affinity for sialic acid receptors through linking CBM40 modules together. The V. cholerae CBM40 was subcloned and crystallized in the presence of sialyllactose confirming its ability to recognize sialic acid. Calorimetry revealed that this CBM40 demonstrated specificity to alpha(2,3)-, alpha(2,6)-, and alpha(2,8)-linked sialosides. Polypeptides containing up to four CBM40 modules in tandem were created to determine if an increase in affinity to sialic acid could be achieved through an avidity effect. Using SPR and a multivalent alpha(2,3)-sialyllactose ligand, we show that increasing the number of linked modules does increase the affinity for sialic acid. The four-CBM40 module protein has a 700- to 1500-fold increase in affinity compared with the single-CBM40 module. Varying the linker length of amino acids between each CBM40 module had little effect on the binding of these polypeptides. Finally, fluorescence-activated cell sorting analysis demonstrated that a green fluorescent protein fused to three CBM40 modules bound to subpopulations of human leukocytes. These studies lay the foundation for creating high affinity, multivalent CBMs that could have broad application in glycobiology. Enhancing the receptor affinity of the sialic acid-binding domain of Vibrio cholerae sialidase through multivalency.,Connaris H, Crocker PR, Taylor GL J Biol Chem. 2009 Mar 13;284(11):7339-51. Epub 2009 Jan 5. PMID:19124471 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Neuraminidase 3D structures

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