Structural relationship of lithocholic acid derivatives binding to the N-terminal 8-kDa domain of DNA polymerase beta.
We reported previously that lithocholic acid (LCA, 3-alpha-hydroxy-5-beta-cholan-24-oic acid), one of the major compounds in the secondary bile acids, selectively inhibited the activity of mammalian DNA polymerase beta (pol beta) [Mizushina, Y., Ohkubo, T., Sugawara, F., and Sakaguchi, K. (2000) Biochemistry 39, 12606-12613]. The purpose of this study was to investigate the molecular structural relationship of LCA and its 10 chemically synthesized derivatives. The inhibitory activities of pol beta by some derivative compounds were stronger than that by LCA, and these compounds bound tightly to the 8-kDa domain fragment but not to the 31-kDa domain fragment of pol beta. Biacore analysis demonstrated that the 8-kDa domain bound selectively to compound 9 (3-alpha-O-lauroyl-5-beta-cholan-24-oic acid), which was the strongest pol beta inhibitor tested, as a 1:1 complex with a dissociation constant (K(d)) of 1.73 nM. From computer modeling analysis (i.e., molecular dynamics analysis), the 8-kDa domain had two inhibitor binding areas. Three amino acid residues (Lys60, Leu77, and Thr79) of the 8-kDa domain bound to LCA and compound 2 (3-alpha-methoxy-5-beta-cholan-24-oic acid), and four amino acid residues (Leu11, Lys35, His51, and Thr79) of the 8-kDa domain bound to compound 9. From these results, the structure-function relationship among pol beta and its selective inhibitors was discussed.
Mizushina
Yoshiyuki
Y
Laboratory of Food and Nutritional Sciences, Department of Nutritional Science, and High Technology Research Center, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan. mizushin@nutr.kobegakuin.ac.jp
Kasai
Nobuyuki
N
Miura
Katsumi
K
Hanashima
Shinya
S
Takemura
Masaharu
M
Yoshida
Hiromi
H
Sugawara
Fumio
F
Sakaguchi
Kengo
K
D000818:Animals; D001665:Binding Sites; D003198:Computer Simulation; D019951:DNA Polymerase beta / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry; D008095:Lithocholic Acid / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats
D004791:Enzyme Inhibitors; D008095:Lithocholic Acid; D019951:DNA Polymerase beta