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PDBsum entry 1w16
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Metal binding protein
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PDB id
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1w16
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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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Structural basis for the evolutionary inactivation of ca2+ binding to synaptotagmin 4.
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Authors
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H.Dai,
O.H.Shin,
M.Machius,
D.R.Tomchick,
T.C.Südhof,
J.Rizo.
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Ref.
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Nat Struct Mol Biol, 2004,
11,
844-849.
[DOI no: ]
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PubMed id
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Abstract
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The neuronal protein synaptotagmin 1 functions as a Ca(2+) sensor in exocytosis
via two Ca(2+)-binding C(2) domains. The very similar synaptotagmin 4, which
includes all the predicted Ca(2+)-binding residues in the C(2)B domain but not
in the C(2)A domain, is also thought to function as a neuronal Ca(2+) sensor.
Here we show that, unexpectedly, both C(2) domains of fly synaptotagmin 4
exhibit Ca(2+)-dependent phospholipid binding, whereas neither C(2) domain of
rat synaptotagmin 4 binds Ca(2+) or phospholipids efficiently. Crystallography
reveals that changes in the orientations of critical Ca(2+) ligands, and perhaps
their flexibility, render the rat synaptotagmin 4 C(2)B domain unable to form
full Ca(2+)-binding sites. These results indicate that synaptotagmin 4 is a
Ca(2+) sensor in the fly but not in the rat, that the Ca(2+)-binding properties
of C(2) domains cannot be reliably predicted from sequence analyses, and that
proteins clearly identified as orthologs may nevertheless have markedly
different functional properties.
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Figure 2.
Figure 2. Intrinsic Ca^2+-binding properties of the
synaptotagmin 4 and 11 C[2] domains. (a -f) 1H-15N HSQC
spectra obtained at different Ca^2+ concentrations for the rat
synaptotagmin 4 C[2]A (a) and C[2]B (b) domains, the D.
melanogaster synaptotagmin 4 C[2]A (c) and C[2]B (d) domains,
and the rat synaptotagmin 11 C[2]A (e) and C[2]B (f) domains.
The protein concentrations were 120  M
and the total Ca^2+ concentrations used (mM) were: a, 0, 0.2, 1,
5, 10, 20, 40, 80; b, 0, 20; c, 0, 5, 10, 20, 40, 80; d, 0, 0.2,
1, 3, 10, 20, 40; e, 0, 0.2, 1, 3, 5, 10, 20, 40, 80; f, 0, 20.
The 1H-15N HSQC spectra acquired in the absence of Ca^2+ and the
highest Ca^2+ concentrations are shown with multiple red
contours and multiple black contours, respectively, whereas
spectra obtained at intermediate Ca^2+ concentrations are shown
with single black contours.
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Figure 4.
Figure 4. Crystal structure of the rat synaptotagmin 4 C[2]B
domain. (a) Ribbon diagram of the rat synaptotagmin 4 C[2]B
domain. Strands (cyan) are labeled 1 -8 and the single helix
(orange) is labeled HA. (b) Electron density map of the
Ca^2+-binding site of the rat synaptotagmin 4 C[2]B domain.
Contours are drawn at 1.0  the
r.m.s. level of the map. Oxygen atoms are red, nitrogen atoms
are blue and carbon atoms are yellow. The Ca^2+ ion is labeled
Ca, the protein Ca^2+ ligands are labeled D2, D3 and E4, and a
coordinating water molecule is labeled W. (c) Backbone
superposition of the structures of the rat synaptotagmin 4 C[2]B
domain (orange) and the rat synaptotagmin 1 C[2]B domain8
(cyan). (d) Superposition of the Ca^2+-binding loops of the rat
synaptotagmin 4 C[2]B domain (orange) and the rat synaptotagmin
1 C[2]B domain (cyan). (e) Superposition of the Ca^2+-binding
loops of the rat synaptotagmin 4 C[2]B domain (orange) and the
rat synaptotagmin 1 C[2]A domain (dark blue). In d and e, the
Ca^2+ ions are in the same color as the protein. The figure was
generated with InsightII (MSI) and MolScript35.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Struct Mol Biol
(2004,
11,
844-849)
copyright 2004.
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