Differences in DNA recognition and conformational change activity between boxes A and B in HMG2 protein

High mobility group (HMG) 2 is a sequence-nonspecific DNA-binding protein consisting of a repeat of DNA-binding domains called HMG1/2 boxes A and B and an acidic C-terminal. To understand the mode of HMG2 interaction with DNA, we expressed various HMG2 peptides containing HMG1/2 box(es) in Escherichia coli cells and purified them. Gel retardation and DNA supercoiling assay indicated that the region essential for the preferential binding of HMG2 with negatively supercoiled DNA and DNA unwinding activity is located in box B, but not sufficient alone. The flanking C-terminal basic region or box A linked by a linker region is necessary to express activities. The SPR measurements certified that the intrinsic DNA binding affinity of box B is weaker (Kd = 170 microM), and these adjoining regions largely strengthen the affinity (Kd