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PDBsum entry 1v0f

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Hydrolase PDB id
1v0f
Jmol
Contents
Protein chains
(+ 0 more) 666 a.a.
Ligands
SLB ×6
PO4 ×6
SIA ×4
SIA-SIA
Waters ×921

References listed in PDB file
Key reference
Title Crystal structure of the polysialic acid-Degrading endosialidase of bacteriophage k1f.
Authors K.Stummeyer, A.Dickmanns, M.Mühlenhoff, R.Gerardy-Schahn, R.Ficner.
Ref. Nat Struct Mol Biol, 2005, 12, 90-96. [DOI no: 10.1038/nsmb874]
PubMed id 15608653
Abstract
Phages infecting the polysialic acid (polySia)-encapsulated human pathogen Escherichia coli K1 are equipped with capsule-degrading tailspikes known as endosialidases, which are the only identified enzymes that specifically degrade polySia. As polySia also promotes cellular plasticity and tumor metastasis in vertebrates, endosialidases are widely applied in polySia-related neurosciences and cancer research. Here we report the crystal structures of endosialidase NF and its complex with oligomeric sialic acid. The structure NF, which reveals three distinct domains, indicates that the unique polySia specificity evolved from a combination of structural elements characteristic of exosialidases and bacteriophage tailspike proteins. The endosialidase assembles into a catalytic trimer stabilized by a triple beta-helix. Its active site differs markedly from that of exosialidases, indicating an endosialidase-specific substrate-binding mode and catalytic mechanism. Residues essential for endosialidase activity were identified by structure-based mutational analysis.
Figure 1.
Figure 1. Structure of endoNF (residues 245 -911). (a) Ribbon diagram of one of the endoNF homotrimers present in the asymmetric unit. The three monomers are red, blue and yellow. (b) Structure of the endoNF monomer with its three domains. The chain is colored from blue at the N terminus to red at the C terminus. (c) Bottom view of the homotrimer with cross-section through its triangular tail domain. An 2,8-linked sialic acid dimer bound to the yellow -barrel domain is in stick representation.
Figure 5.
Figure 5. Surface representation of the endoNF homotrimer in complex with sialic acid. The molecular surface of the left trimer is colored by electrostatic potential (blue, positive; red, negative),whereas the transparent surface of the right trimer shows the underlying ribbon model. Sialic acid residues bound to the -barrel as 2,8-linked dimer and as monomer to the -prism domain of the spike are green spheres. Polymeric sialic acid could simultaneously interact with both binding sites, on the spike (yellow monomer) and the -barrel (red monomer) and be cleaved by the active site of the third subunit (blue monomer).
The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Mol Biol (2005, 12, 90-96) copyright 2005.
Secondary reference #1
Title Proteolytic processing and oligomerization of bacteriophage-Derived endosialidases.
Authors M.Mühlenhoff, K.Stummeyer, M.Grove, M.Sauerborn, R.Gerardy-Schahn.
Ref. J Biol Chem, 2003, 278, 12634-12644. [DOI no: 10.1074/jbc.M212048200]
PubMed id 12556457
Full text Abstract
Figure 3.
Fig. 3. C-terminal truncations of endosialidases. Schematic representations of wild type (wt) and C-terminal truncated forms are shown in A and B for endoNE and endoNF, respectively. The positions of the identified cleavage site are indicated by arrowheads. The C-terminal amino acid and the number of deleted ( ) amino acids are shown. In the case of the C-terminal fragment of endoNE the numbers of the first and last amino acid are shown. C, soluble fractions of E. coli BL21(DE3) expressing C-terminally His[6]-tagged wild type or C-terminally truncated constructs of endoNE were analyzed by 12% ProSieve SDS-PAGE and immunoblotting. A polyclonal anti-endoNE guinea pig serum was used for detection. A faint 75 kDa band that is also visible in the first lane showing lysate of mock transformed bacteria is visualized by cross-reactivity of the polyclonal serum. Bands corresponding to full-length and cleaved wild type endoNE are indicated with arrows. D, wild type and C-terminally truncated endoNF containing an N-terminal T7 tag were analyzed by 10% SDS-PAGE and immunoblotting using an anti-T7 antibody.
Figure 6.
Fig. 6. Trimer formation of endoNF. A, wild type and mutant endoNF were expressed in E. coli BL21(DE3), and soluble fractions of the bacterial lysates were analyzed by 6% SDS-PAGE and Western blot. To visualize SDS-resistant trimers, one aliquot of each sample was analyzed omitting the boiling step before electrophoresis. The upper blot was developed with a combination of anti-T7 and anti-His[6] antibody and the lower blot with anti-His[6] antibody, exclusively. EndoNF mutants with the indicated amino acid exchanges are shown in lanes 3-10 and C-terminal truncated forms in lanes 11-14. Bands corresponding to trimers, N-terminal fragments, and full-length endoNF are indicated with arrows. B, wild type endoNF, endoNE, and the chimera endoNF-E and endoNE-F were monitored for trimer formation as described above. Bands corresponding to an SDS-resistant complex and the N-terminal catalytic domains of endoNF and endoNE are marked with arrows.
The above figures are reproduced from the cited reference with permission from the ASBMB
PROCHECK
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