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PDBsum entry 2odc
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Membrane protein
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PDB id
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2odc
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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 nmr structure of the barrier-To-Autointegration factor-Emerin complex.
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Authors
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M.Cai,
Y.Huang,
J.Y.Suh,
J.M.Louis,
R.Ghirlando,
R.Craigie,
G.M.Clore.
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Ref.
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J Biol Chem, 2007,
282,
14525-14535.
[DOI no: ]
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PubMed id
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Abstract
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The barrier-to-autointegration factor BAF binds to the LEM domain (Em(LEM)) of
the nuclear envelope protein emerin and plays an essential role in the nuclear
architecture of metazoan cells. In addition, the BAF(2) dimer bridges and
compacts double-stranded DNA nonspecifically via two symmetry-related DNA
binding sites. In this article we present biophysical and structural studies on
a complex of BAF(2) and Em(LEM). Light scattering, analytical
ultracentrifugation, and NMR indicate a stoichiometry of one molecule of Em(LEM)
bound per BAF(2) dimer. The equilibrium dissociation constant (K(d)) for the
interaction of the BAF(2) dimer and Em(LEM), determined by isothermal titration
calorimetry, is 0.59 +/- 0.03 microm. Z-exchange spectroscopy between
corresponding cross-peaks of the magnetically non-equivalent subunits of the
BAF(2) dimer in the complex yields a dissociation rate constant of 78 +/-
2s(-1). The solution NMR structure of the BAF(2)-Em(LEM) complex reveals that
the LEM and DNA binding sites on BAF(2) are non-overlapping and that both
subunits of the BAF(2) dimer contribute approximately equally to the Em(LEM)
binding site. The relevance of the implications of the structural and
biophysical data on the complex in the context of the interaction between the
BAF(2) dimer and Em(LEM) at the nuclear envelope is discussed.
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Figure 5.
FIGURE 5. Thermodynamics and kinetics of the BAF[2]-Em^LEM
complex. A, ITC titration of BAF[2] with Em^LEM. The titration
(3 µl per injection of 854 µM Em^LEM) was performed
at 30 °C in a calorimetric cell (  1.8 ml) containing 31
µM BAF[2] dimer in 25 mM Tris-HCl, pH 6.5, and 0.2 M NaCl.
The experimental data are shown as solid circles. The best-fit
curve to a one site binding equilibrium is shown as a solid line
and yields a value of K[d] = 0.59 ± 0.03 µM. B,
because one molecule of Em^LEM binds to the BAF[2] dimer, the
chemical environments of equivalent residues from the two
subunits of BAF are no longer identical and display different
chemical shifts, as illustrated for Gly^47. Z-exchange
spectroscopy reveals the presence of exchange cross-peaks
(indicated by ex) between equivalent residues in addition to the
auto-peaks (labeled as G47 and g47'). This arises from the fact
that Em^LEM can bind to the BAF[2] dimer in two chemically
equivalent ways related by a 180° rotation (see Fig. 6). C,
kinetic scheme describing the magnetization transfer involving
dissociation and reassociation of Em^LEM to BAF[2] in two
chemically equivalent orientations. Cross-peaks corresponding to
the two magnetically inequivalent subunits of BAF[2] in the
complex are simply interchanged in the two bound states. M[F] is
the magnetization of free BAF[2]; M[B] and M[B]['] are the
magnetizations of the two bound states of BAF[2] related by the
180° rotation of Em^LEM; k[on] and k[off] are the
association and dissociation rate constants, and [Em^LEM][F] is
the concentration of free Em^LEM;  [F] and [B] are
the spin-lattice relaxation rates for free and bound BAF[2] and
for simplicity are considered equal because [F] cannot be determined
from the present data. D, time course of the normalized
auto-(open circles) and exchange-(closed circles) peaks of
Gly^47 together with the best-fit curves (red and blue lines,
respectively) obtained for the kinetic model shown in C. The
experimental data are shown at three different concentrations of
free Em^LEM (0.39, 0.68, and 0.89 mM) with total concentrations
of U-^15N/^13C/^2H/[methyl-^1H]Val/Leu/Ile-labeled of 0.42,
0.35, and 0.30 mM, respectively, and total concentration of
unlabeled Em^LEM of 0.81, 1.03, and 1.19 mM, respectively. E,
selected strips from a three-dimensional
^12C-filtered/^13C-separated NOE spectrum illustrating
intermolecular NOEs from ^12C-attached protons of the BAF[2]
dimer (F[1]dimension) to ^13C-attached protons of Em^LEM (F[3]
dimension). The spectrum was recorded in 95% H[2]O, 5% D[2]O.
The cross-peaks involving equivalent residues in the two
subunits of BAF[2] are indicated by upper and lowercase
one-letter codes.
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Figure 8.
FIGURE 8. The BAF[2]-Em^LEM interface. A, ribbon diagram of
the BAF[2]-Em^LEM complex (color coded as in Fig. 7A) also
illustrating the position of the two DNA duplexes observed in
the crystal structure of the BAF[2]-DNA[2] complex (5). B,
surface representations illustrating the binding surfaces
involved in the BAF[2]-Em^LEM complex. The binding surface on
BAF[2] is shown on the left panel and on Em^LEM on the right
panel. The binding surfaces are color coded with hydrophobic
residues in green, polar residues in light blue, positively
charged residues in dark blue, and negatively charged residues
in red. The relevant portions of the interacting partner are
shown as gold tubes. The surface of the non-interacting residues
of the BAF[2] dimer is shown in dark gray for the red subunit
and light gray for the blue subunit (as depicted in A). Residues
of the blue subunit of BAF[2] are labeled in lowercase, and
residues of Em^LEM in italics. The view in the right-hand panel
is related to that in the left-hand panel by a 180° rotation
about an axis parallel to the printed lines on the page.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
14525-14535)
copyright 2007.
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