Relationship between P-box amino acid sequence and DNA binding specificity of the thyroid hormone receptor. The effects of sequences flanking half-sites in thyroid hormone response elements.

The three P-box amino acids in the DNA recognition alpha-helix of steroid/thyroid hormone receptors participate in the discrimination of the central base pairs of the hexameric half-sites of receptor response elements in DNA. Using a series of variant receptors incorporating all 19 possible substitutions for each individual P-box amino acid of the human thyroid hormone receptor (hT3R beta), we demonstrated that the first P-box position must have a glutamate, and the second P-box position must have either an alanine or a glycine for high affinity binding to everted repeat elements with half-site sequences of AGGNCA. In the present study, the influence of half-site flanking sequence on the compatibility of P-box amino acids in hT3R beta with DNA binding was investigated. When a 5' sequence of CTG flanked AGGNCA half-sites in an everted repeat, several additional P-box variant receptors were able to bind to the DNA that were not able to bind when the half-sites were flanked with the 5' sequence CAG. Flanking sequence had the most dramatic effects on amino acid substitutions at the first P-box position, with smaller effects observed at the second P-box position and only subtle effects observed at the third P-box position. Expansion of the number of P-box sequences compatible with binding of hT3R beta to thyroid hormone response elements required the thymidine in the CTG flanking sequence, an everted repeat of the AGGNCA half-sites, and an intermolecular interaction in the C terminus of the receptor.