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PDBsum entry 2c08
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References listed in PDB file
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Key reference
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Title
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Mechanism of endophilin n-Bar domain-Mediated membrane curvature.
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Authors
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J.L.Gallop,
C.C.Jao,
H.M.Kent,
P.J.Butler,
P.R.Evans,
R.Langen,
H.T.Mcmahon.
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Ref.
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EMBO J, 2006,
25,
2898-2910.
[DOI no: ]
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PubMed id
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Abstract
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Endophilin-A1 is a BAR domain-containing protein enriched at synapses and is
implicated in synaptic vesicle endocytosis. It binds to dynamin and synaptojanin
via a C-terminal SH3 domain. We examine the mechanism by which the BAR domain
and an N-terminal amphipathic helix, which folds upon membrane binding, work as
a functional unit (the N-BAR domain) to promote dimerisation and membrane
curvature generation. By electron paramagnetic resonance spectroscopy, we show
that this amphipathic helix is peripherally bound in the plane of the membrane,
with the midpoint of insertion aligned with the phosphate level of headgroups.
This places the helix in an optimal position to effect membrane curvature
generation. We solved the crystal structure of rat endophilin-A1 BAR domain and
examined a distinctive insert protruding from the membrane interaction face.
This insert is predicted to form an additional amphipathic helix and is
important for curvature generation. Its presence defines an endophilin/nadrin
subclass of BAR domains. We propose that N-BAR domains function as low-affinity
dimers regulating binding partner recruitment to areas of high membrane
curvature.
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Figure 2.
Figure 2 Membrane insertion and orientation of endophilin
N-terminal amphipathic helix. (A) Oxygen (red circles) and
NiEDDA (green squares) accessibilities (  )
of membrane-bound N-BAR domain as a function of label position.
The graph below shows a ln( ratio)
plot (  )
showing the differential access of colliders to the spin label
and the penetration of hydrophobic residues into the membrane.
The periodic oscillation is indicative of a helical structure.
Equivalent maxima indicate that the helix lies planar to the
membrane. (B) Helical wheel representation showing hydrophobic
and charged faces. (C) Model of the amphipathic helix, residues
1–16 with hydrophobic residues coloured green and surface
charge potential also shown. (D) Model of the N-BAR amphipathic
helix to scale with PtdIns(4,5)P[2] and PtdSer lipids showing
the depth of penetration of the helix as calculated from data in
(A) and penetration measurements, described in Materials and
methods.
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Figure 7.
Figure 7 High membrane curvature promotes membrane fusion. (A)
Example of endophilin N-BAR tubules made from liposomes extruded
to a size cutoff of 400 nm. (B) Emission spectra from mixed
liposomes in the absence and presence of calcium (which promotes
fusion) and Triton (to obtain total donor fluorescence). See
Materials and methods for details of the assay. (C) FRET assay
of membrane fusion, showing dilution of the FRET pair into
unlabelled liposome in the presence of the N-BAR (see Results).
The N-BAR control has no unlabelled liposomes and thus there can
be no dilution of the FRET pair. The BAR domain does not lead to
membrane fusion. As highly curved membranes are more fusogenic,
we believe that the fusion seen with the N-BAR domain is a
readout of the efficiency of curvature generation. (D)
Comparison of mutants and WT N-BAR domains at 55  M
in the fusion assay. Values displayed in the bar chart  s.e.m.
are the difference in the ratios of emission maxima between
experiment (mixed liposomes (530/585 nm)) and controls
(uniformly labelled liposomes (530/585 nm)) (see Results).
Studentʼs t-test ^*P<0.001, ^**P<0.2.
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The above figures are
reprinted
from an Open Access publication published by Macmillan Publishers Ltd:
EMBO J
(2006,
25,
2898-2910)
copyright 2006.
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