Pyridoxal 5'-phosphate inhibits DNA binding of HNF1.
An efficient Escherichia coli expression system was constructed for the production of a variant form of HNF1 protein having the additional five amino acid residues (Asp-Arg-Trp-Gly-Ser) at the NH(2)-terminal. The cDNA encoding HNF1 was ligated to 6 x His tag and inserted into an inducible bacterial expression vector pRSET A. After expression in E. coli, the recombinant product was purified by Ni-NTA affinity column chromatography. The purified product showed expected NH(2)-terminal sequence and specific binding to the HNF1 site. The effect of pyridoxal 5'-phosphate and its analogues on the binding activity of recombinant HNF1 was examined and found that only pyridoxal 5'-phosphate effectively inhibited the DNA binding. The concentration of pyridoxal 5'-phosphate that inhibited 50% of DNA binding was around 100 microM. Furthermore, we identified Lys197 of HNF1 molecule as the essential residue of DNA binding. These observations suggest that pyridoxal 5'-phosphate directly interacts with tissue-specific transcription factor HNF1 and modulates the binding to DNA.
Oka
T
T
Department of Veterinary Physiology, Faculty of Agriculture, Kagoshima University, Japan. oka@vet.agri.kagoshima-u.ac.jp
Sugitatsu
H
H
Nordin
H
H
Thakur
M K
MK
Aoyama
M
M
Sasagawa
T
T
Suzuki
I
I
Tsuji
H
H
D000595:Amino Acid Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D007544:Isopropyl Thiogalactoside; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010957:Plasmids; D011485:Protein Binding / Q000187:drug effects; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D007544:Isopropyl Thiogalactoside; D011732:Pyridoxal Phosphate