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International Journal of Molecular Medical Science
4
N terminus that contribute to sequence-specificDNA
binding (Georgopoulos, 2002; Thompson et al., 2007).
Our study indicates that these motifs, especially ZF
(2-4), are also important for its interaction with
Ku
. To
bind all three ZF at once, it would require a protein to
have a special helical conformation to fit the extended
contours in which three ZFs bind to DNA along its
major groove. Ku protein that binds DNA along its
minor groove with its Ku80 component fitting the
contours of IK1 ZF (2-4) is uniquely suited to bind
the ZF of IK1, as confirmed by our modeling
studies.
Figure 1 Ikaros-Ku molecular complex [A] analysis of Ku-Ikaros binding interactions by surface plasmon resonance (SPR)
Note: Representative BIAcore sensorgrams showing the binding of MBP-IK1 vs. MBP-IK5 to Ku70 (A.1), Ku80 (A.2) and
Ku70/Ku80 (A.3), that were immobilized on an NTA sensor chip. SPR assays were performed and analyzed, as detailed in Materials
and Methods. MBP-IK1 (but not MBP-IK5) exhibited high affinity binding to Ku70, Ku80 as well as Ku70/Ku80. [B] A Model of
the Structural Interface Between IK1 and Ku Proteins. [B1] Space filling model of IK1 and Ku70/Ku80 complex based on the crystal
structure of the Ku heterodimer bound to DNA and a homology model of IK1 ZF fragment (110-256). Ku70/Ku80-light blue;
IK1-white, DNA-atom-type colored (carbon-green, oxygen-red, phosphate-magenta, nitrogen=blue). [B.2&B.3]. Ribbon
representation of the IK1 and Ku70/Ku80 complex model is shown in the following colors: Ku70-green, Ku80-orange, IK1-red,
DNA-white for backbone and blue for groove. The two subunits of the Ku heterodimer share little sequence identity (15%) with each
other. However, the crystal structure of Ku70/Ku80 revealed that both subunits have the same folding, domain structure, and overall
ternary structure except for the terminal regions (shown in different colors). The shared structural features include the N-terminal α/β
domain (34-250 and 6-238 for Ku70 and Ku80 respectively) and the β-barrel domain (116 residues). The α/β domains are positioned
at the periphery of the Ku heterodimer and contribute little to the DNA binding and domain interface. On the other hand, the β-barrel
domains of the heterodimer use seven β-strands to provide a symmetrical circle forming the cradle of the DNA-binding site by
pointing one end of each barrel towards the DNA, as revealed by the crystal structure of Ku/DNA complex. The protein dyad axis of
the dimer coincides with the bound DNA duplex axis. The DNA is positioned centrally through the ring formed by the protein dyad.
All three ZF in IK1 (Ik_Zf2-4) are in close and extensive contact with Ku80. Likewise, a similar interaction is contemplated with
Ku70 with the segment of the βO-to-α12 turn in Ku70 and Ku80 C-terminus (541-545) adopting a different conformation, as
observed on the other end of the ring
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