Photon Factory Activity Report 2014 #32 (2015) B

BL-5A, BL-17A/2013G728

X-ray crystallographic analysis of the (Runx1)2-DNA complex assembled on theenhancer of T cell receptor α chain gene; a possible role of the intrinsically disorderedregion of Runx1
Masaaki SHIINA, Keisuke HAMADA, Taiko BUNGO, Mariko SHIMAMURA, Shiho BABA,
Kazuhiro OGATA*
Dept. of Biochemistry, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku,
Yokohama 236-0004, Japan1 Introduction
Gene-specific transcription is initially regulated
through assembly of transcription factors (TFs)-DNAcomplex formed on an enhancer.
To reveal the mechanism of the TF-DNA assembly, we
have been studying the T cell-specific assembly formedon the T cell antigen receptor (TCR) α chain enhancer.
This complex is comprised of multiple TFs including two
Runx1-CBFβ heterodimers, LEF1, Ets1, and CREB,
which bind cooperatively to the TCRα enhancer. In thisstudy, we solved the crystal structure of the (Runx1)2DNA complex formed on the TCRα enhancer, using theRunx1 fragment containing its N-terminal intrinsicallydisordered region (IDR). We found that the IDR of Runx1
regulates cooperative DNA binding between two Runx1
molecules.
2 ExperimentBacterially expressed Runx1 fragment containing theN-terminal IDR was purified and mixed with doublestranded DNA having TCRα enhancer sequence, which
includes two binding sites of Runx1, in the two to one
molar ratio of protein and DNA. The complex formation
was checked by electrophoretic mobility shift assay(EMSA) and SDS-polyacrylamide gel electrophoresis.
Then the complex solution was subjected to initial
crystallization screening using Natrix, a crystallization
reagent kit for nucleic acids (Hampton Research). By
modifying crystallization conditions and procedures, weobtained crystals of the (Runx1)2-DNA complex. Thenthe obtained crystals were soaked in cryoprotectant
solutions, followed by flush-cooling in a stream of cold
nitrogen gas at 100 K. Diffraction images were collectedwith synchrotron radiation at BL-5A and BL-17A in
Photon Factory and were processed using HKL2000.
3 Results and Discussion
The crystals belong to the space group P222, with unit
cell dimensions of a = 87.0 Å, b = 88.0 Å, c = 97.6 Å.
The crystals were relatively resistant to damage by X-rays
and diffracted to 2.2 Å (Table 1). The structure wassolved by molecular replacement with a published Runx1structure. The obtained structure showed that the two
Runx1 molecules bind to the TCRα enhancer DNA in thehead-to-tail orientation. The electron densities

corresponding to the N-terminal IDRs of the boundRunx1 molecule were clearly observed in one of the two
Runx1 molecules. This suggests that the N-terminal IDRof one of the two Runx1 molecules becomes folded bycooperative binding of them to the TCRα enhancer. We
are now exploring biological implication of the induced
folding of the N-terminal IDR of Runx1 using sitedirected mutagenesis based on the crystal structure.
Table 1: Data-collection statistics of(Runx1)2-DNA complex
Beam-line
BL-5A
Wavelength
1.00
Resolution (Å)
50.0-2.2
Space group
P222
Unit-cell parameters (Å)
a87.0
b88.0
c
97.6
90°, 90°, 90°α, β, γNo. of reflections
Observed
415812
Unique38543
Completeness (%)
99 (100)#I/σ (I)56.6 (8.0)#
Rmerge (%)
6.9 (49.1)#Numbers in parentheses refer to data for highresolution outer shell 2.24-2.28 Å* ogata@med.yokohama-cu.ac.jp