PDBsum entry 2w68

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Hydrolase PDB id
Protein chains
195 a.a.
Waters ×133
PDB id:
Name: Hydrolase
Title: Enhancing the receptor affinity of the sialic acid-binding domain of vibrio cholerae sialidase through multivalency
Structure: Sialidase. Chain: a, b, c. Fragment: cbm40 domain, residues 25-216. Synonym: sialic acid binding domain from v. Choleraesialidase, nanase, neuraminidase. Engineered: yes
Source: Vibrio cholerae. Organism_taxid: 666. Expressed in: escherichia coli. Expression_system_taxid: 562
2.50Å     R-factor:   0.217     R-free:   0.256
Authors: H.Connaris,P.R.Crocker,G.L.Taylor
Key ref:
H.Connaris et al. (2009). Enhancing the receptor affinity of the sialic acid-binding domain of Vibrio cholerae sialidase through multivalency. J Biol Chem, 284, 7339-7351. PubMed id: 19124471 DOI: 10.1074/jbc.M807398200
17-Dec-08     Release date:   30-Dec-08    
Go to PROCHECK summary

Protein chains
Pfam   ArchSchema ?
P0C6E9  (NANH_VIBCH) -  Sialidase
781 a.a.
195 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 3 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.  - Exo-alpha-sialidase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Hydrolysis of alpha-(2->3)-, alpha-(2->6)-, alpha-(2->8)-glycosidic linkages of terminal sialic residues in oligosaccharides, glycoproteins, glycolipids, colominic acid and synthetic substrates.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biochemical function     sialic acid binding     1 term  


DOI no: 10.1074/jbc.M807398200 J Biol Chem 284:7339-7351 (2009)
PubMed id: 19124471  
Enhancing the receptor affinity of the sialic acid-binding domain of Vibrio cholerae sialidase through multivalency.
H.Connaris, P.R.Crocker, G.L.Taylor.
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.
  Selected figure(s)  
Figure 1.
a, schematic drawing of the V. cholerae sialidase showing the central catalytic domain flanked by the lectin-like CBMs. The sialic acid recognizing CBM, CBM40, is on the right. b, schematic view of the constructs made in this study.
Figure 2.
Sialosides used in the ITC study. a, 3′SL; b, 6′SL; c, DSL; and d, DSLNT.
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 7339-7351) copyright 2009.  
  Figures were selected by an automated process.