Grad Iwona, Kochman Marian, Ozyhar Andrzej
Institute of Organic Chemistry, Biochemistry and Biotechnology, Wroclaw University of Technology, Wrocław, Poland.
Acta Biochim Pol. 2002;49(3):747-56.
Nuclear receptors are ligand-dependent transcription factors responsible for controlling differentiation, growth and development of higher eukaryotes. Three amino acids within the recognition alpha-helix of the DNA-binding domain of the nuclear receptors constitute the so-called "P-box" which determines response element specificity. In the ultraspiracle (Usp) protein, which together with EcR forms the heterodimeric ecdysone receptor, the P-box residues are E19, G20 and G23. Substitution of E19, the most characteristic amino acid for estrogen receptor-like P-boxes, with alanine showed that the mutation did not appreciably alter the affinity of the wild-type Usp DNA-binding domain (UspDBD(WT)) for a probe containing natural ecdysone response element (hsp27(wt)). Since in many cases E19 contacts a G/C base pair in position -4, which is absent in hsp27(wt), we analysed the interaction of UspDBD(WT), E19A and other P-box region mutants with the hsp27(wt) derivative which contains a G/C instead of an T/A base pair in position -4. UspDBD(WT) exhibited higher affinity for this element than for hsp27(wt). Moreover, a different interaction pattern of P-box region mutants was also observed. Thus we conclude that the E19 residue of UspDBD is not involved in any hsp27(wt) sequence-discerning contacts. However, substitution of the hsp27(wt) T/A base pair in position -4 with G/C generates target sequence with distinct functional characteristics and possibly with a new specificity. These results could serve as a basis for understanding the role of the presence of a T/A or G/C base-pair in the position -4 in the two types of ecdysone response elements found in nature.
