Abstract
Crystallographic studies of different membrane cytochrome P450 enzymes have provided examples of distinct structural conformations, suggesting protein flexibility. It has been speculated that conformational selection is an integral component of substrate recognition and access, but direct evidence of such substate interconversion has thus far remained elusive. In the current study, solution NMR revealed multiple and exchanging backbone conformations for certain structural features of the human steroidogenic cytochrome P450 17A1 (CYP17A1). This bifunctional enzyme is responsible for pregnenolone C17 hydroxylation, followed by a 17,20-lyase reaction to produce dehydroepiandrosterone, the key intermediate in human synthesis of androgen and estrogen sex steroids. The distribution of CYP17A1 conformational states was influenced by temperature, binding of these two substrates, and binding of the soluble domain of cytochrome b5 (b5). Notably, titration of b5 to CYP17A1·pregnenolone induced a set of conformational states closely resembling those of CYP17A1·17α-hydroxypregnenolone without b5, providing structural evidence consistent with the reported ability of b5 to selectively enhance 17,20-lyase activity. Solution NMR thus revealed a set of conformations likely to modulate human steroidogenesis by CYP17A1, demonstrating that this approach has the potential to make similar contributions to understanding the functions of other membrane P450 enzymes involved in drug metabolism and disease states.
Highlights
Crystallography provides a static structure of cytochrome P450 17A1 (CYP17A1)
Solution NMR revealed a set of conformations likely to modulate human steroidogenesis by CYP17A1, demonstrating that this approach has the potential to make similar contributions to understanding the functions of other membrane P450 enzymes involved in drug metabolism and disease states
Generation of CYP17A1 NMR Spectra and Spectral Assignments—The major technical difficulties involved in the application of solution protein NMR to membrane cytochrome P450 enzymes have been 1) the ability to generate the required quantities of appropriately labeled protein in minimal medium and 2) the moderate stability and solubility of these enzymes in conditions suitable for the NMR experiment
Summary
Crystallography provides a static structure of cytochrome P450 17A1 (CYP17A1). Results: Solution NMR reveals an ensemble of CYP17A1 conformational substates. Its inhibition by the active drug abiraterone for metastatic castration-resistant prostate cancer [24] recently provided an exciting new option for treating the second leading cause of cancer death in United States men [25] This enzyme is intriguing from a biochemical perspective because it binds and turns over a set of substrates and performs both hydroxylation and lyase reactions via distinct catalytic intermediates, with only the lyase reaction altered by the presence of cytochrome b5. The realization of solution NMR as a viable tool to probe ensembles of membrane CYP17A1 substates provides the potential to examine numerous human P450 enzymes with key roles in human drug metabolism and other endogenous processes
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