MOLECULAR BASIS OF ADRENARCHE--DISSECTION OF P450C17

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We propose to thoroughly define the structural components of human P450c17 (17alpha-hydroxylase/17,20 lyase) that contribute to its two activities and to identify the physiologic factors that regulate the ratio of these two activities. In early childhood, adrenal P450c17 activity is limited almost exclusively to 17-hydroxylation. During adrenarche, adrenal synthesis of the weak androgen DHEA commences, resultant from acquisition of 17,20 lyase activity. The molecular basis for this shift in P450c17 activities, however, remains enigmatic. Based on data from site-directed mutagenesis experiments, we hypothesize that post-translational modification generates the physiologically relevant change in the three- dimensional structure of P450c17 that leads to the rise in adrenal 17,20 lyase activity during adrenarche. Preliminary data from the mentor's laboratory suggest that P450c17 undergoes hormone-responsive phosphorylation. We propose to characterize post-translational modifications of P450c17, regulation of this modification, and its effect on P450c17 activities. We will study how structure relates to function by building a computer model of P450c17 based on X-ray crystal structures of the 3 soluble P450's. We will substitute core structural units (alpha- helicies and beta-sheets) of homologous regions in P450c17 for those in P450's -cam, -terp, and -BM3; energy minimize; and add connecting loops. We will use the model to identify residues surrounding the substrate binding pocket likely to participate in substrate binding, reduction by P450-oxidoreductase, and catalysis for each of the 2 separate activities. Finally, we will test the model using site-directed mutagenesis to introduce structural changes predicted to alter one of the two activities. We will thoroughly characterize the activities of each mutant and post- translationally modified protein, including inhibition by alternate substrates and oxygenation at adjacent carbon atoms. We will incorporate the data from mutagenesis and modification experiments back into the model to refine our structure of P450c17. This proposal teams a PI with graduate training in steroid biochemistry, enzymology, and spectroscopic methods, a mentor who is a leader in the molecular biology of human steroidogenesis, and a collaborator with expertise in crystallography and computer modeling P450 enzymes. This project will complete the PI's training in molecular biology and preparation for independent investigation; improve the technology of building structural protein models based on known structures of related proteins; and advance our understanding of and ability to modulate androgen (and consequently estrogen) biosynthesis.