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PDBsum entry 2v8s
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Protein transport
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PDB id
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2v8s
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References listed in PDB file
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Key reference
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Title
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A snare-Adaptor interaction is a new mode of cargo recognition in clathrin-Coated vesicles.
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Authors
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S.E.Miller,
B.M.Collins,
A.J.Mccoy,
M.S.Robinson,
D.J.Owen.
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Ref.
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Nature, 2007,
450,
570-574.
[DOI no: ]
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PubMed id
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Abstract
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Soluble NSF attachment protein receptors (SNAREs) are type II transmembrane
proteins that have critical roles in providing the specificity and energy for
transport-vesicle fusion and must therefore be correctly partitioned between
vesicle and organelle membranes. Like all other cargo, SNAREs need to be sorted
into the forming vesicles by direct interaction with components of the vesicles'
coats. Here we characterize the molecular details governing the sorting of a
SNARE into clathrin-coated vesicles, namely the direct recognition of the
three-helical bundle H(abc) domain of the mouse SNARE Vti1b by the human
clathrin adaptor epsinR (EPNR, also known as CLINT1). Structures of each domain
and of their complex show that this interaction (dissociation constant 22 muM)
is mediated by surface patches composed of approximately 15 residues each, the
topographies of which are dependent on each domain's overall fold. Disruption of
the interface with point mutations abolishes the interaction in vitro and causes
Vti1b to become relocalized to late endosomes and lysosomes. This new class of
highly specific, surface-surface interaction between the clathrin coat component
and the cargo is distinct from the widely observed binding of short, linear
cargo motifs by the assembly polypeptide (AP) complex and GGA adaptors and is
therefore not vulnerable to competition from standard motif-containing cargoes
for incorporation into clathrin-coated vesicles. We propose that conceptually
similar but mechanistically different interactions will direct the post-Golgi
trafficking of many SNAREs.
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Figure 1.
Figure 1: Mapping the binding sites on the Vti1b H[abc] domain
and the EPNR ENTH Delta- alpha- 0
domain on their isolated structures. a, b, Ribbon diagram
showing the three-helix bundle (H[abc] domain) of uncomplexed
Vti1b (a, light green) and of uncomplexed EPNR ENTH   0
(b, pale pink). Surface views are shown in the same
orientations. Mutated residues on both representations are
coloured green if they affected binding to EPNR, pink if they
affected binding to Vti1b H[abc] and grey-blue if there was no
effect (surface views only). c–f, Pull-down experiments
detecting the binding of EPNR ENTH–Myc constructs to
GST–Vti1b by western blotting for the Myc tag. c, The effect
of mutations in GST–Vti1b on their binding of wild-type EPNR
ENTH–Myc. d, e, The effect of point (d) and helix-deletion (e)
mutations in EPNR ENTH–Myc on their binding to wild-type
GST–Vti1b. f, The effect of the charge-swap mutations
EPNR(R146E) and Vti1b(E23R) introduced on the basis of the
complex structure.
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Figure 2.
Figure 2: The EPNR ENTH Delta-alpha-0–Vti1b
H[abc] domain complex. a, The complex is shown, with Vti1b
coloured dark green to pale green (N to C) and EPNR coloured
pale pink to dark pink (N to C). Enlarged views are shown of key
residues in the binding interface. The putative lipid binding
helix 0
of EPNR is shown in grey. The proposed orientation of the
remaining portions of Vti1b and EPNR are indicated by dotted
lines. Charge-swap mutations are boxed. b, Surface
representations of the complex, with each rotated by 90 degrees
to show the 'footprint' of interaction coloured green on Vti1b
H[abc] and pink on EPNR ENTH. C ribbons
and side chains participating in the interaction are shown
through the different surfaces. Mutated residues that have been
demonstrated to affect binding are boxed. c, Structure-based
sequence alignment of the H[abc] domains of mouse Vti1b, mouse
Vti1a and yeast Vti1 (with conserved residues boxed in green),
and of human EPNR ENTH domain with yeast Ent3 (with conserved
residues shown in pink). Residues in which mutation abolishes
binding between Vti1b and EPNR are marked with an asterisk.
Residues that have significant roles in the binding interface
are indicated by a triangle.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nature
(2007,
450,
570-574)
copyright 2007.
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