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 </= 1.2 microM). In contrast, box A, even in the presence of the adjoining basic linker region, showed no such activities, indicating that boxes A and B are different in their DNA recognition mode. The computer modeling suggested that the side chain of Phe-102 in box B is inserted into the base stack to cause DNA conformational changes, while the side chain of Ala-16 in box A is too small to intercalate. These represent that boxes A and B have similar tertiary structures but their activities for DNA conformational changes obviously differ. Box B is the main region for DNA recognition and conformational changes, and box A must play an assistant to increase its DNA recognition.
Yoshioka
K i
Ki
Department of Biological Science and Technology, Science University of Tokyo, Chiba, Japan.
Saito
K
K
Tanabe
T
T
Yamamoto
A
A
Ando
Y
Y
Nakamura
Y
Y
Shirakawa
H
H
Yoshida
M
M
D000818:Animals; D003198:Computer Simulation; D004278:DNA, Superhelical / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006609:High Mobility Group Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D020349:Surface Plasmon Resonance; D013552:Swine
D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D010455:Peptides; D011994:Recombinant Proteins