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PDBsum entry 1emo
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Matrix protein
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PDB id
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1emo
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References listed in PDB file
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Key reference
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Title
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Solution structure of a pair of calcium-Binding epidermal growth factor-Like domains: implications for the marfan syndrome and other genetic disorders.
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Authors
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A.K.Downing,
V.Knott,
J.M.Werner,
C.M.Cardy,
I.D.Campbell,
P.A.Handford.
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Ref.
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Cell, 1996,
85,
597-605.
[DOI no: ]
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PubMed id
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Abstract
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The nuclear magnetic resonance structure of a covalently linked pair of
calcium-binding (cb) epidermal growth factor-like (EGF) domains from human
fibrillin-1, the protein defective in the Marfan syndrome, is described. The two
domains are in a rigid, rod-like arrangement, stabilized by interdomain calcium
binding and hydrophobic interactions. We propose a model for the arrangement of
fibrillin monomers in microfibrils that reconciles structural and antibody
binding data, and we describe a set of disease-causing mutations that provide
the first clues to the specificity of cbEFG interactions. The residues involved
in stabilizing the domain linkage are highly conserved in fibrillin, fibulin,
thrombomodulin, and the low density lipoprotein receptor. We propose that the
relative orientation of tandem cbEGF domains in these proteins is similar, but
that in others, including Notch, pairs adopt a completely different conformation.
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Figure 2.
Figure 2. Schematic Illustration of the Domain–Domain
Packing Interactions of the fib32–33 PairTyr 2157 is
highlighted in yellow, and Ile 2185 and Gly 2186 are in cyan.
β-strands are depicted by green arrows, and calcium atoms are
shown in red. This figure was rendered ([38]) from MOLSCRIPT (
[34]) input.
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Figure 6.
Figure 6. The Domain Structure of Human Fibrillin-1 and a
Model for the Organization of Fibrillin Monomers within
Connective Tissue MicrofibrilsThe domain structure of human
fibrillin-1 ([41]) is shown on top, and the model for the
organization of fibrillin monomers within connective tissue
microfibrils is shown below. The position of the cbEGF domains
(32–36) shown in Figure 6 is highlighted. In the model,
fibrillin monomers are shown as arrows. The monomers are
arranged in a parallel, staggered, head-to-tail arrangement,
with N- and C-termini in the “bead” regions (shaded). The
dimensions of the fib32–33 pair, combined with the results of
antibody labeling data ( [46]), suggest that the extended
fibrillin monomer spans two interbead regions with 50% overlap,
as shown. The inverted Ys denote the position within each
monomer of a putative antibody binding site.
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The above figures are
reprinted
by permission from Cell Press:
Cell
(1996,
85,
597-605)
copyright 1996.
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Secondary reference #1
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Title
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Calcium binding properties of an epidermal growth factor-Like domain pair from human fibrillin-1.
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Authors
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V.Knott,
A.K.Downing,
C.M.Cardy,
P.Handford.
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Ref.
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J Mol Biol, 1996,
255,
22-27.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1. A schematic diagram of the 32nd and 33rd
Ca
2+
binding EGF-like domains from human fibrillin-1.
Ca
2+
ligands, as predicted from the factor IX EGF-like
domain crystal structure, are marked. It should be noted
that the positions of a carboxyamide side-chain and a
carboxylate side-chain are reversed in the Ca
2+
binding
sites of this pair, compared with the factor IX EGF-like
domain (Handford et al., 1995). Residues referred to in the
text are numbered. Pairs of residues for which C
a
H-C
a
H
connectivities were observed in a two-dimensional
NOESY spectrum recorded in 99.996%
2
H2 O are
connected by bold lines.
The domain pair was cloned and expressed as follows.
A DNA fragment encompassing nucleotides 6502 to 6748
of the human fibrillin-1 cDNA and corresponding to
amino acids 2124 to 2205 (numbering according to Pereira
et al., 1993) was amplified by standard PCR techniques
using Pfu polymerase (Stratagene). The cDNA encoded a
Thr2158 : Ile substitution, compared with the published
sequence (Pereira et al., 1993). This observation was
confirmed by genomic analysis of three independent
samples (data not shown). The primers used for the
amplification of DNA encoding the EGF-like domain pair
were 5' TAGTAGGGATCCATAGAAGGACGATCAGCAG-
TTGATATGGACGAATGCAAA (forward primer) and
5' TAGTAGAAGCTTCTATTATTCACATGTCATCATTGG-
ACCG (reverse primer). Each of these primers contained
a restriction site (BamHI-forward primer, HindIII reverse
primer) at the 5'end for cloning purposes, and in addition
the forward primer contained a sequence encoding a
factor Xa cleavage site. After digestion, the amplified
DNA was ligated into BamHI/HindIII cleaved pQE30
(Qiagen). Plasmid pQE30 contains a sequence encoding a
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Figure 4.
Figure 4. The change in chemical shift (r) of the Hd*
resonances of Tyr2149 (Q) and Phe2188 (q) plotted
against [Ca
2+
]free. The concentration of free Ca
2+
concen-
tration was calculated using the equation [Ca
2+
]free =
[Ca
2+
]tot - (DND
-1
No)[protein] - (DCD
-1
Co)[protein], where
DND
-1
No and DCD
-1
Co are fractional changes in Hd* chemical
shift for Tyr2149 and Phe2188, respectively. This equation
assumes independent Ca
2+
binding by the two sites.
Standard regression analysis was used to curve fit the
data for each site using the equation D = Do [Ca
2+
]free /
(Kd + [Ca
2+
]free).
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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