Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

15A/2003G322

DNA-binding properties of human NF-κB p50 subunit and its mutant form R59E
studied by synchrotron small-angle x-ray scattering
Alexander Timchenko1, Maria Timchenko2, Viktoria Shyp1, Kazumoto Kimura3, Hiroshi Kihara4*
1
Institute of Protein Research, Pushchino, Russia, 142290;
2
Institute of Theoretical and Experimental Biophysics, Pushchino, Russia, 142290;
3
Department of Biochemistry Dokkyo University, Mibu, Tochigi 321-02, Japan4
Department of Physics, Kansai Medical University Uyamahigashi, Hirakata Osaka 573, Japan
Introduction
The human transcription factor NF-κB plays animportant role in tumorigenesis participating in theexpression of oncogenes. NF-κB is presented in a tumor
cell mainly as heterodimer p50-p65, where p50 subunit is
responsible for DNA binding and p65 - for transcriptional
activity [1]. For prevention of tumor progression it'snecessary to inhibit NF-κB activity. Therefore theproblem of NF-κB inhibition is intensively studied now[2]. One of the possible way to inhibit the NF-κB is theusage of mutant forms of NF-κB incapable for DNAbinding but keeping the stability and dimerization
properties close to those of wild type of protein. Here wepresent the DNA-binding properties of wild type p50
subunit of NF-κB and its mutant form R59E withconsiderably reduced DNA-binding activity.
Experimental
Wild type (WT) and mutant form R59E of human p50
subunit of NF-κB (replacement of Arg59 for Glu) wereisolated from the E.coli BL21(DE3) carried the plasmids
encoding corresponding gene. For the construction ofgene encoding mutated p50 molecules a plasmid pEt-14b
encoding wild type of p50 protein (kindly provided byA.Israel) was used as template. The buffer conditions are:
7.5mM HEPES (pH8.0), 34mM NaCl, 1mM MgCl2,0.5mM DTT, 0.05mM EDTA. Protein concentrations
were in the range 1.5-2.0 mg/ml. 50mkM concentrationsof synthetic specific DNA duplexes were used.
Synchrotron X-ray measurements were done on thesmall-angle camera BL-15A (Photon Factory, Tsukuba)using CCD-detector. The range of scattering vectors Q=-1
0.008-0.15 nm .
Results
One can see in Fig. 1 and Fig. 2 that Kratky plots (I*Q2versus Q) exhibit bell shape for the studied protein and its
complexes with DNA-duplexes, indicating a compact
structure of particles. The position of maximum
corresponds to a dimer of protein. Guinier plots (log I2versus Q ) have nonlinear behavior (data not shown)pointing out some association of protein. The evaluationof radius of gyration and molecular mass from these plots
gives values about 4 nm and 90 kD, respectively. These
meanings correspond to the dimer of protein. Addition ofDNA-duplexes shifts the position of maximum on Kratky

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plot toward higher Q values indicating somecompactization of protein-DNA complexes. At the sametime some shift to smaller Q-values is observed for R59E
complexes only at the excess of DNA over protein thatlikely demonstrates the unspecific binding of DNA toprotein. This fact correlates with high dissociation
constant for R59E found by us in gel-shift experiments. It
permits to consider the R59E mutant as perspective one
for NF-κB inhibition in a cell.

Fig. 1 Kratky plot for WT-p50 subunit (1) and its
complex with DNA at the indicating p50:DNA ratio.

Fig. 2 Kratky plot for R59E (1) and its complex with
DNA at the indicating p50:DNA ratio.
References[1] Perkins, TIBS, 25, 434, 2000
[2] Huang et al. Oncogene, 20, 4188, 2001
kihara@makino.kmu.ac.jp