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Leukemic T-cell Precursors from T-lineage All Patients Characterized by Profound Ku80 Deficiency
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using a 45 Ti rotor. Following centrifugation, the
clarified supernatant was filtered through a 0.22 μm
membrane filter (S100) and dialyzed for 4 h into
buffer A (20 mM sodium phosphate, 10% glycerol, pH
7.2). For purification of Ku70 and Ku80 proteins as
well
as Ku70/Ku80 heterodimer,
dialyzed
supernatants were applied to a Nickel-chelation
column (Pharmacia) that was equilibrated with buffer
B (20 mM sodium phosphate, 500 mM NaCl, 0.5 M
imidazole, 10% glycerol, pH 7.2). Ku70 or Ku80
enriched fractions were dialyzed against buffer C (20
mM Tris, pH 8.0 with 1 M DTT, 10% glycerol)
overnight to remove imidazole and then applied to a
Sepharose Q HP26/10 ion exchange column (column
volume 50 mL; Amersham Pharmacia Biotech)
followed by size exclusion chromatography on a
Superdex 200 HR 10/30 column (Pharmacia) for
further purification (Ozer et al., 2013).
2.2 Monitoring of Binding Interactions Using
Surface Plasmon Resonance (SPR) Technology
A BIAcore X surface plasmon resonance-based
biosensor system (Amersham Pharmacia Biotech
Biosensor AB) was used to measure the kinetic
parameters for the interactions between soluble
recombinant MBP-IK1 vs. MBP-IK5 fusion proteins
(=analytes) and the immobilized 6xHis-tagged
recombinant Ku (Ku70/80 heterodimer), Ku70, Ku80
proteins captured via metal chelation (= ligands), as
previously described (Ozer et al., 2013; Mahajan et al.,
2001; Rajamohan et al., 2001). The nitrilotriacetic
acid (NTA) sensor chip was saturated by injection 20
μl of 500 mM NiCl
2
solution at a flow rate of 20
μL/min in NTA buffer. Ligands (200 nM in NTA
buffer) were immobilized on the sensor chip by
injecting 25 μL of the ligand solution (20 μL/min). A
50-μL sample of either MBP-IK1 or MBP-IK5 fusion
protein (100 nM) was injected at 25
℃
at a flow rate of
20 μL/min onto the sensor chip surface on which a
specific ligand had been immobilized. Between
samples, the binding surface was regenerated by
injection of 40 μL of EDTA-containing regeneration
buffer (10 mM HEPES, 0.15 M NaCl, 0.35 M EDTA,
0.005 Surfactant P20, pH: 8.3) at a flow rate 20
μL/min to remove metal ions. The primary data were
analyzed and association rate constant (kon)/
dissociation rate constant (koff) values were
determined using the BIAevaluation software (Version
3.0) supplied with the instrument (Biacore, Inc.), as
previously described (Mahajan et al., 2001;
Rajamohan et al., 2001). The dissociation constant KD
= koff/kon and association constant KA = kon/koff
were also calculated.
2.3 Molecular Model of Ikaros-Ku Complex
We used the known cDNA sequence of IK1 and the
already published crystal structure of the Ku70/80
heterodimer bound to DNA in order to construct a
molecular model to explore possible modes of
interaction between IK1 and Ku proteins. The IK1
molecule from residue 110 to residue 256 was
modeled based on the homology with two zinc finger
proteins (PDB access codes: 1AAY, 1G2D) using
InsightII (Molecular Simulation Inc. San Diego, CA)
and MOE (Chemical Computing Group Inc., Montreal,
Canada), as previously described (Brady and Stouten,
2000; Vig et al., 1998). The interaction surface
analyses were done with GRASP (Nicholls et al.,
1991). We first explored the interaction of the
Ku70/Ku80 heterodimer bound to DNA (PDB access
code: 1JEY) and the IK1 zinc-finger (ZF) domains
using our IK1 homology model. The initial positions
of both Ku heterodimer and IK1 ZF domains were
placed manually. The DNA major groove contour and
base pair positions were used as a guide to properly
position IK1 ZF domains with respect to Ku. The
initial 55-nucleotide DNA element from the crystal
structure of the Ku heterodimer was extended in a
standard B conformation and fixed in the subsequent
docking calculation. Major steric collisions with Ku
were removed by manually adjusting the torsion
angles of the residues on the interface. We created a
definitive binding set of Ku residues in the binding
pocket to move as a 4.0 Å shell around the manually
docked IK1 ZF domains. This general position of the
Ku, IK1 and DNA were used as a starting point for
subsequent automatic docking trials and energy
minimization. The docking calculation was repeated
several times for several slightly different initial
positions of the complex towards each other to ensure
the stability of the end result and avoid local minimum.
The calculations used a consistent valence force field
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