Effect of Mutations Within the DNA Binding Domain on Ancestral Steroid Hormone Receptors’ Specificity to DNA Response Elements and Protein Stability

Presenter: Greg Hanak, Biology

The steroid hormone receptor (SR) family is a group of proteins that activate transcription via the binding of DNA response elements. We believe that the present day SRs found in animals, which include the estrogen, androgen, progestagen, mineralocorticoid and glucocorticoid receptor, all evolved from one ancestral receptor protein (ancSR1). The ancSR1, which had the ability to activate transcription via the binding of DNA estrogen response elements (EREs) in response to estrogens, gave rise to another ancestral receptor (ancSR2) that carried the ability to bind the DNA response elements associated with all the SRs except the EREs that were recognized by ancSR1. Examination of the ancSR1 and ancSR2 on a molecular level showed a number of differences in the amino acid sequence between the two receptors, some being within the P-box of the DNA binding domain (DBD). The question then becomes how are these P-box mutations contributing to the specificity of the ancSR2 protein. We predict that the derived P-box in ancSR2 is eliminating positive contact with EREs. To prove this, biophysical and biochemical experiments such as thermal melts, guanidine melts and DNA binding assays will be run on the DBDs of these two ancestral proteins and two different supposed intermediates of ancSR1 and ancSR2. The data gained from these experiments and the inferences made will help us better understand the series of events leading up to the formation of the current, distinct SRs and, more importantly, how and why they function as they do.