Kinetic and thermodynamic evidence for flipping of a methyl-CpG binding domain on methylated DNA.
The methyl-CpG binding domain (MBD) is a conserved domain in transcriptional factors that binds to methylated CpG dinucleotide DNA sequences in vertebrates. The complex is comprised of an asymmetric MBD monomer and a symmetric DNA duplex. Therefore, in the complex, each strand of the duplex DNA is in contact with the protein at a distinct surface and thus exhibits a different chemical shift in NMR spectra. Two-dimensional chemical exchange spectroscopy revealed the presence of a stochastic exchange of the two strands of the duplex DNA in the complex at a rate of 4 s (-1) at 25 degrees C, which indicates the existence of a motion of the MBD such that the orientation of the MBD becomes reversed with respect to the DNA duplex. Kinetic and thermodynamic analyses using surface plasmon resonance, quartz crystal microbalance, and isothermal titration calorimetry suggest that the reversal of MBD with respect to the DNA duplex takes place without its complete dissociation from DNA, indicating the presence of an intermediate protein-DNA binding state that allows the protein to undergo a flip motion upon DNA.
Inomata
Kosuke
K
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan.
Ohki
Izuru
I
Tochio
Hidehito
H
Fujiwara
Kenichiro
K
Hiroaki
Hidekazu
H
Shirakawa
Masahiro
M
D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D018899:CpG Islands; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
D004247:DNA; D004268:DNA-Binding Proteins; D015226:Dinucleoside Phosphates; C015772:CpG dinucleotide