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PDBsum entry 1nub
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Extracellular module
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PDB id
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1nub
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Contents |
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* Residue conservation analysis
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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 and mapping by site-Directed mutagenesis of the collagen-Binding epitope of an activated form of bm-40/sparc/osteonectin.
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Authors
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T.Sasaki,
E.Hohenester,
W.Göhring,
R.Timpl.
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Ref.
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Embo J, 1998,
17,
1625-1634.
[DOI no: ]
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PubMed id
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Abstract
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The extracellular calcium-binding domain (positions 138-286) of the matrix
protein BM-40 possesses a binding epitope of moderate affinity for several
collagen types. This epitope was predicted to reside in helix alphaA and to be
partially masked by helix alphaC. Here we show that deletion of helix alphaC
produces a 10-fold increase in collagen affinity similar to that seen after
proteolytic cleavage of this helix. The predicted removal of the steric
constraint was clearly demonstrated by the crystal structure of the mutant at
2.8 A resolution. This constitutively activated mutant was used to map the
collagen-binding site following alanine mutagenesis at 13 positions. Five
residues were crucial for binding, R149 and N156 in helix alphaA, and L242, M245
and E246 in a loop region connecting the two EF hands of BM-40. These residues
are spatially close and form a flat ring of 15 A diameter which matches the
diameter of a triple-helical collagen domain. The mutations showed similar
effects on binding to collagens I and IV, indicating nearly identical binding
sites on both collagens. Selected mutations in the non-activated mutant DeltaI
also reduced collagen binding, consistent with the same location of the epitope
but in a more cryptic form in intact BM-40.
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Figure 2.
Figure 2 Structure of BM-40  I,
 C.
(A) Two orthogonal views (Kraulis, 1991) related by a rotation
of 90° about the vertical axis. The FS domain is in green and
the EC domain is in red. The calcium ions bound to the EF hand
pair in the EC domain (Ca1 and Ca2) are in yellow; the calcium
ion bound at the tip of the E
- F
loop (Ca3) is in blue. Helices in the EC domain are labelled.
Note that five residues around the deletion site (marked by a
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Figure 3.
Figure 3 Stereo view (Kraulis, 1991) of a C[  ]trace
of the BM-40 I,
C
structure showing the residues that were mutated in this study
(see text). Disulfide bridges are shown with thick bonds.
Mutated residues are identified by position numbers and their
side chains. The three calcium ions are shown as black spheres.
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The above figures are
reprinted
from an Open Access publication published by Macmillan Publishers Ltd:
Embo J
(1998,
17,
1625-1634)
copyright 1998.
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Secondary reference #1
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Title
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Crystal structure of a pair of follistatin-Like and ef-Hand calcium-Binding domains in bm-40.
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Authors
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E.Hohenester,
P.Maurer,
R.Timpl.
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Ref.
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Embo J, 1997,
16,
3778-3786.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1 Stereo view (Kraulis, 1991) of the BM-40 FS -EC
structure and its secondary structure elements. The FS domain
(residues 54 -137) is shown in green and consists of an
N-terminal  -hairpin
and a small hydrophobic core of /
structure.
The EC domain (residues 138 -286) is in red and consists of a
pair of EF-hand calcium-binding sites and helices A, B and C.
The calcium ions bound to the EF-hands are shown as yellow
spheres. The FS and EC domains interact through a small
interface involving mainly 5
and the preceding loop of the FS domain and E
of the EC domain. The FS domain is glycosylated at Asn99; the
first two N-acetylglucosamine (NAG) sugar moieties are included
in the crystallographic model and are shown in atomic detail.
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Figure 6.
Figure 6 Model of a tandem of FS domains in follistatin and
agrin. The N-terminal domain (in green) and the C-terminal
domain (in red) are linked by an extended segment of three
residues (in blue); the relative rotation between the two
domains is  0°.
The tip of the N-terminal -hairpin
of the C-terminal domain contacts the N-terminal domain at the
small three-stranded -sheet
and stabilizes the linear arrangement of domains. In this model,
helix 2,
which is unique to BM-40, has been replaced with the
corresponding segment of ovomucoid (compare Figures 3 and 4A).
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The above figures are
reproduced from the cited reference
which is an Open Access publication published by Macmillan Publishers Ltd
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Secondary reference #2
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Title
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The biology of sparc, A protein that modulates cell-Matrix interactions.
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Authors
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T.F.Lane,
E.H.Sage.
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Ref.
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Faseb J, 1994,
8,
163-173.
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PubMed id
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