Functionality versus strength - has functional selection taken place in the case of the ecdysteroid receptor response element?

Nuclear receptors are ligand-dependent transcription factors responsible for controlling differentiation, growth and development of higher eukaryotes. Three amino acids within the recognition -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 (UspDBDWT) for a probe containing natural ecdysone response element (hsp27wt). Since in many cases E19 contacts a G/C base pair in position –4, which is absent in hsp27wt, we analysed the interaction of UspDBDWT, E19A and other P-box region mutants with the hsp27wt derivative which contains a G/C instead of an T/A base pair in position –4. UspDBDWT exhibited higher affinity for this element than for hsp27wt. Moreover, a different interaction pattern of P-box region mu