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PDBsum entry 1v0e
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References listed in PDB file
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Key reference
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Title
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Crystal structure of the polysialic acid-Degrading endosialidase of bacteriophage k1f.
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Authors
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K.Stummeyer,
A.Dickmanns,
M.Mühlenhoff,
R.Gerardy-Schahn,
R.Ficner.
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Ref.
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Nat Struct Mol Biol, 2005,
12,
90-96.
[DOI no: ]
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PubMed id
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Abstract
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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.
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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.
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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).
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Struct Mol Biol
(2005,
12,
90-96)
copyright 2005.
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Secondary reference #1
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Title
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Proteolytic processing and oligomerization of bacteriophage-Derived endosialidases.
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Authors
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M.Mühlenhoff,
K.Stummeyer,
M.Grove,
M.Sauerborn,
R.Gerardy-Schahn.
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Ref.
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J Biol Chem, 2003,
278,
12634-12644.
[DOI no: ]
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PubMed id
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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.
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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.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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