Analysis of estrogen response element binding by genetically selected steroid receptor DNA binding domain mutants exhibiting altered specificity and enhanced affinity

To analyze the role of amino acids in the steroid receptor DNA binding doma in (DBD) recognition helix in binding of the receptor to the estrogen respo nse element (ERE), we adapted the powerful P22 challenge phage selection sy stem for use with a vertebrate protein. We used the progesterone receptor D NA binding domain and selected for mutants that gained the ability to bind to the ERE. We used a mutagenesis protocol based on degenerate oligonucleot ides to create a large and diverse pool of mutants in which 10 nonconsensus amino acids in the DNA recognition helix of the progesterone receptor DNA binding domain were randomly mutated. After a single cycle of modified P22 challenge phage selection, 37 mutant proteins were identified, all of which lost the ability to bind to the progesterone response element. In gel mobi lity shift assays, approximately 70% of the genetically selected mutants bo und to the consensus ERE with a >4-fold higher affinity than the naturally occurring estrogen receptor DBD, In the P-box region of the DNA recognition helix, the selected mutants contained the amino acids found in the wildtyp e estrogen receptor DBD, as well as other amino acid combinations seen in n aturally occurring steroid/nuclear receptors that bind the aGGTCA half-site . We also obtained high affinity DBDs with Trp(585) as the first amino acid of the P-box, although this is not found in the known steroid/nuclear rece ptors, In the linker region between the two zinc fingers, G597R was by far the most common mutation. In transient transfections in mammalian cells usi ng promoter interference assays, the mutants displayed enhanced affinity fo r the ERE, When linked to an activation domain, the transfected mutants act ivated transcription from ERE containing reporter genes. We conclude that the P-box amino acids can display considerable variation a nd that the little studied linker amino acids play an important role in det ermining affinity for the ERE, This work also demonstrates that the P22 cha llenge phage genetic selection system, modified for use with a mammalian pr otein, provides a novel, single cycle selection for steroid/nuclear recepto r DBDs with altered specificity and greatly enhanced affinity for their res ponse elements.