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PDBsum entry 2zs6
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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 ha3 subcomponent of clostridium botulinum type c progenitor toxin.
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Authors
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T.Nakamura,
M.Kotani,
T.Tonozuka,
A.Ide,
K.Oguma,
A.Nishikawa.
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Ref.
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J Mol Biol, 2009,
385,
1193-1206.
[DOI no: ]
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PubMed id
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Abstract
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The Clostridium botulinum type C 16S progenitor toxin contains a neurotoxin and
several nontoxic components, designated nontoxic nonhemagglutinin (HA), HA1
(HA-33), HA2 (HA-17), HA3a (HA-22-23), and HA3b (HA-53). The HA3b subcomponent
seems to play an important role cooperatively with HA1 in the internalization of
the toxin by gastrointestinal epithelial cells via binding of these
subcomponents to specific oligosaccharides. In this study, we investigated the
sugar-binding specificity of the HA3b subcomponent using recombinant protein
fused to glutathione S-transferase and determined the three-dimensional
structure of the HA3a-HA3b complex based on X-ray crystallography. The crystal
structure was determined at a resolution of 2.6 A. HA3b contains three domains,
domains I to III, and the structure of domain I resembles HA3a. In crystal
packing, three HA3a-HA3b molecules are assembled to form a three-leaved
propeller-like structure. The three HA3b domain I and three HA3a alternate,
forming a trimer of dimers. In a database search, no proteins with high
structural homology to any of the domains (Z score >10) were found.
Especially, HA3a and HA3b domain I, mainly composed of beta-sheets, reveal a
unique fold. In binding assays, HA3b bound sialic acid with high affinity, but
did not bind galactose, N-acetylgalactosamine, or N-acetylglucosamine. The
electron density of liganded N-acetylneuraminic acid was determined by crystal
soaking. In the sugar-complex structure, the N-acetylneuraminic acid-binding
site was located in the cleft formed between domains II and III of HA3b. This
report provides the first determination of the three-dimensional structure of
the HA3a-HA3b complex and its sialic acid binding site. Our results will provide
useful information for elucidating the mechanism of assembly of the C16S toxin
and for understanding the interactions with oligosaccharides on epithelial cells
and internalization of the botulinum toxin complex.
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Figure 1.
Fig. 1. Three-dimensional structure of HA3. (a) The trimer
structure of HA3. In Mol-A, HA3a and the domains of HA3b are
colored green, purple, yellow, and magenta. Two other HA3a and
HA3b molecules, Mol-B and Mol-C, are shown in light blue and
olive, respectively. (b) Wall-eye stereo view of the overall
structure of HA3 as a complex of HA3a and HA3b. There is one
molecule in an asymmetric unit. The structure of HA3 consists of
two components, HA3a (green) and HA3b, and HA3b is composed of
three domains, domain I (purple), domain II (yellow), and domain
III (magenta). The C-terminal residue of HA3a and the N-terminal
residue of HA3b domain I are indicated by red triangles. The
sugar-binding position located on HA3b domain III is shown as a
purple circle.
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Figure 2.
Fig. 2. Schematic representation of HA3a and each domain of
HA3b. β-Strands of each domain are indicated with arrowheads
and numbered. The helices are illustrated as black columns and
labeled alphabetically. Domain I of HA3a and HA3b are
structurally similar to each other. However, HA3b domain I
contains the Pro-loop and the short β-strands, βI and βII,
which run between the β6–β6′ and β7–β7′ strands, and
these structural features differ from HA3a. HA3b domain II and
domain III form similar structural compositions, jelly-roll-like
folds.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2009,
385,
1193-1206)
copyright 2009.
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Secondary reference #1
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Title
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Sugar-Binding sites of the ha1 subcomponent of clostridium botulinum type c progenitor toxin.
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Authors
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T.Nakamura,
T.Tonozuka,
A.Ide,
T.Yuzawa,
K.Oguma,
A.Nishikawa.
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Ref.
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J Mol Biol, 2008,
376,
854-867.
[DOI no: ]
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PubMed id
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Figure 2.
Fig. 2. Three-dimensional structures of HA1–sugar complex
and F[o] − F[c] omit maps for the ligands with 2.0 σ
contoured level. (a) The overall structure of the HA1–Neu5Ac
complex. There are two molecules (Mol-A and Mol-B) in an
asymmetric unit; Mol-A and Mol-B are structurally almost
identical. HA1 is composed of two β-trefoil domains, N- and
C-terminal domains, linked by an α-helix (gray). The three
repeats of each domain are displayed in lime green (α-repeat),
blue (β-repeat), and magenta (γ-repeat). Two Neu5Ac molecules
are indicated by the stick models. The ligands are found at
binding sites I and III. At the site I position, Neu5Ac
molecules are found not only in Mol-A but also in Mol-B (site
I[B]). The binding sites I[A] and I[B] illustrated in the
figures are in the equivalent position for each molecule. At the
position indicated as binding site III, Neu5Ac is only seen in
Mol-A. (b) The HA1–Gal complex structure. Gal molecules are
seen at site I[B] located at the 2α-repeat and at site II
located at the 2γ-repeat of Mol-B; no electron density for the
ligands was observed for Mol-A. The β-strands of 2α- and
2γ-repeat (site I and II) were numbered. (c) The density of
Neu5Ac located at binding sites I and III. At binding site I[B],
the electron density of the sugar is clearly seen, almost to the
same degree as that at binding site I (data not shown). The
electron density of Neu5Ac observed at site II is relatively
weak. (d) The electron density of GalNAc located at binding site
I[B]. (e) The density of Gal located at binding sites I[B] and
II[B].
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Figure 4.
Fig. 4. Superimposed illustrations of the area near the
sugar-binding sites of type C and type A HA1. (a) Close-up view
of the 2α-repeat region of type C HA1 superimposed on the same
region of type A HA1 with a Gal molecule model. The putative
sugar-binding residues as observed in type C HA1 (pink) and
their counterparts in type A HA1 (blue). Residue labels are
indicated for type C HA1; those from the type A HA1 structure
are in parentheses. (b) Overlay of the two types of HA1 on the
2γ-repeat region with a stick-modeled Gal.
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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Secondary reference #2
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Title
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Binding properties of clostridium botulinum type c progenitor toxin to mucins.
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Authors
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T.Nakamura,
N.Takada,
T.Tonozuka,
Y.Sakano,
K.Oguma,
A.Nishikawa.
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Ref.
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Biochim Biophys Acta, 2007,
1770,
551-555.
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PubMed id
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