CRYSTAL STRUCTURE OF PHO4 bHLH DOMAIN-DNA COMPLEX
T. Shimizu1, A. Toumoto1, K. Ihara1, M. Shimizu2, Y. Kyogoku3, N. Ogawa4, Y. Oshima4 and T. Hakoshima1
1Department of Bioscience, Nara
Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara
630-0101, Japan
2Biomolecular Engineering Research
Institute, Furuedai, Suita, Osaka 565
3Institute for Protein Research,
Osaka University, Yamadaoka, Suita, Osaka 565
4Faculty of Engineering, Osaka
University, Yamadaoka, Suita, Osaka 565, Japan
Keywords: crystal structure, PHO4, basic-helix-loop-helix, E-box, flanking base recognition
The transcription of the genes relevant to phosphatase(PHO) system in Saccharomyces cerevisiae are regulated by intracelluar levels of phosphate. PHO4 is a positive regulatory factor in this regulon and is to be indispensable for transcription of several genes. PHO4 consists of 312 amino acid residues and the C terminal region is a basic-helix-loop-helix(bHLH) DNA binding domain. The consensus sequence of the recognition is CACGT(G/T) known as E-box. Several studies(1,2) showed that bases outside the E-box were involved in determining the binding specificity in PHO4. Here, we have determined the 3D structure of a bHLH domain of PHO4, which is composed of 63 residues, complexed with DNA by X ray crystallography(3). The crystal structure at 2.8 A (revealed that the domain folds into a bHLH motif with a long but compact loop that contains a short -helical segment. This helical structure positions a Trp into an aromatic cluster so as to make the loop compact. PHO4 binds to DNA as a homodimer with direct reading of the core E box sequence but also of its 3'-flanking bases. The 3'-flanking bases GG are recognized by Arg2 and His5. The residues involved in the E-Box recognition are His5, Glu9, and Arg13, as already reported for bHLH/Zip proteins MAX and USF, and are different from those by bHLH proteins MyoD and E47, though PHO4 is one of the bHLH proteins.