 |
PDBsum entry 3fex
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Translation, protein transport
|
PDB id
|
|
|
|
3fex
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
Contents |
 |
|
|
|
|
|
|
|
|
|
|
|
756 a.a.
|
|
|
|
|
|
|
|
|
|
|
80 a.a.
|
|
|
|
|
|
|
|
|
|
|
423 a.a.
|
|
|
|
|
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
The molecular basis for the regulation of the cap-Binding complex by the importins.
|
|
|
Authors
|
|
S.M.Dias,
K.F.Wilson,
K.S.Rojas,
A.L.Ambrosio,
R.A.Cerione.
|
|
|
Ref.
|
|
Nat Struct Biol, 2009,
16,
930-937.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
The binding of capped RNAs to the cap-binding complex (CBC) in the nucleus, and
their dissociation from the CBC in the cytosol, represent essential steps in RNA
processing. Here we show how the nucleocytoplasmic transport proteins
importin-alpha and importin-beta have key roles in regulating these events. As a
first step toward understanding the molecular basis for this regulation, we
determined a 2.2-A resolution X-ray structure for a CBC-importin-alpha complex
that provides a detailed picture for how importin-alpha binds to the CBP80
subunit of the CBC. Through a combination of biochemical studies, X-ray
crystallographic information and small-angle scattering experiments, we then
determined how importin-beta binds to the CBC through its CBP20 subunit.
Together, these studies enable us to propose a model describing how
importin-beta stimulates the dissociation of capped RNA from the CBC in the
cytosol following its nuclear export.
|
|
|
|
|
|
|
|
Figure 1.
X-ray crystal structure of the CBC–importin-   IBB
complex. CBP80 is shown as a surface (gray), whereas CBP20 and
importin- are
depicted by ribbon diagrams (orange and green, respectively).
Shown above in stereoview is the N-terminal region of CBP80,
residues 2–22 (represented by sticks), which contains the NLS
segment of this protein, along with its 2F[o] – F[c] map
contoured at 1  .
|
|
Figure 3.
(a) Surface (CBP80) and ribbon (CBP20) representations of the
CBC (PDB 1N52 (ref. 27)) illustrate the stabilization of the
N-terminal hinge of CBP20 by residues in its binding groove
within CBP80. The N-terminal region of CBP20 (which includes
residues Ser11, Asp12 and Ser13, shown in dark blue) enters a
groove formed by residues Lys327 and Glu328 of CBP80 (green),
making contacts that stabilize residues of CBP20 in loop 2-
3
(magenta) in the cap-bound conformation. The inset shows these
interactions in detail. Residues 26–36 of the N-terminal
region of CBP80 are in black. (b) HeLa cells were transiently
transfected with constructs encoding V5-CBP80 or V5-CBP80-K327A
E328A and HA-CBP20, as indicated, for 24 h. The expressed CBP80
proteins were immunoprecipitated with anti-V5 and western
blotted with anti-HA to show that CBP20 co-imunoprecipitated
equally well with wild-type and mutated CBP80 (as quantified by
densitometry). Immunoprecipitated CBC was assayed for UV
cross-linking incorporation of [ ^32P]GTP
(c) and ^32P-labeled capped mRNA (d), with and without an excess
of cap.
|
|
|
|
|
|
The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Struct Biol
(2009,
16,
930-937)
copyright 2009.
|
|
|
|
|
|
|
