The structures of CYP17A1 with steroid inhibitors abiraterone or TOK-001 provide a better understanding of the enzyme’s catalytic capabilities and inhibition, and hence assist in understanding steroidogenic diseases and designing drugs to improve the treatment of prostate and other steroid-responsive cancers. The membrane-bound enzyme cytochrome P450 17A1 (CYP17A1) catalyses the biosynthesis of androgens in humans, and CYP17A1 inhibitors are being investigated as potential therapeutics for castration-resistant prostate cancer. The first X-ray crystal structures of this enzyme are now reported. Structures were obtained in the presence of the steroidal inhibitors abiraterone and TOK-001. The binding mode differs substantially from that predicted by homology models, and is of interest to structural biologists and those involved in developing improved inhibitors as potential therapeutics. Cytochrome P450 17A1 (also known as CYP17A1 and cytochrome P450c17) catalyses the biosynthesis of androgens in humans1. As prostate cancer cells proliferate in response to androgen steroids2,3, CYP17A1 inhibition is a new strategy to prevent androgen synthesis and treat lethal metastatic castration-resistant prostate cancer4, but drug development has been hampered by lack of information regarding the structure of CYP17A1. Here we report X-ray crystal structures of CYP17A1, which were obtained in the presence of either abiraterone, a first-in-class steroidal inhibitor recently approved by the US Food and Drug Administration for late-stage prostate cancer5, or TOK-001, an inhibitor that is currently undergoing clinical trials4,6. Both of these inhibitors bind the haem iron, forming a 60° angle above the haem plane and packing against the central I helix with the 3β-OH interacting with aspargine 202 in the F helix. Notably, this binding mode differs substantially from those that are predicted by homology models and from steroids in other cytochrome P450 enzymes with known structures, and some features of this binding mode are more similar to steroid receptors. Whereas the overall structure of CYP17A1 provides a rationale for understanding many mutations that are found in patients with steroidogenic diseases, the active site reveals multiple steric and hydrogen bonding features that will facilitate a better understanding of the enzyme’s dual hydroxylase and lyase catalytic capabilities and assist in rational drug design. Specifically, structure-based design is expected to aid development of inhibitors that bind only CYP17A1 and solely inhibit its androgen-generating lyase activity to improve treatment of prostate and other hormone-responsive cancers.

The human cytochrome P450 17A1 (CYP17A1) enzyme operates at a key juncture of human steroidogenesis, controlling the levels of mineralocorticoids influencing blood pressure, glucocorticoids involved in immune and stress responses, and androgens and estrogens involved in development and homeostasis of reproductive tissues. Understanding CYP17A1 multifunctional biochemistry is thus integral to treating prostate and breast cancer, subfertility, blood pressure, and other diseases. CYP17A1 structures with all four physiologically relevant steroid substrates suggest answers to four fundamental aspects of CYP17A1 function. First, all substrates bind in a similar overall orientation, rising ∼60° with respect to the heme. Second, both hydroxylase substrates pregnenolone and progesterone hydrogen bond to Asn²⁰² in orientations consistent with production of 17α-hydroxy major metabolites, but functional and structural evidence for an A105L mutation suggests that a minor conformation may yield the minor 16α-hydroxyprogesterone metabolite. Third, substrate specificity of the subsequent 17,20-lyase reaction may be explained by variation in substrate height above the heme. Although 17α-hydroxyprogesterone is only observed farther from the catalytic iron, 17α-hydroxypregnenolone is also observed closer to the heme. In conjunction with spectroscopic evidence, this suggests that only 17α-hydroxypregnenolone approaches and interacts with the proximal oxygen of the catalytic iron-peroxy intermediate, yielding efficient production of dehydroepiandrosterone as the key intermediate in human testosterone and estrogen synthesis. Fourth, differential positioning of 17α-hydroxypregnenolone offers a mechanism whereby allosteric binding of cytochrome b₅ might selectively enhance the lyase reaction. In aggregate, these structures provide a structural basis for understanding multiple key reactions at the heart of human steroidogenesis.

Cytochromes P450 (CYP) from the 2A subfamily are known for their roles in the metabolism of nicotine, the addictive agent in tobacco, and activation of the tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Although both the hepatic CYP2A6 and respiratory CYP2A13 enzymes metabolize these compounds, CYP2A13 does so with much higher catalytic efficiency, but the structural basis for this has been unclear. X-ray structures of nicotine complexes with CYP2A13 (2.5 Å) and CYP2A6 (2.3 Å) yield a structural rationale for the preferential binding of nicotine to CYP2A13. Additional structures of CYP2A13 with NNK reveal either a single NNK molecule in the active site with orientations corresponding to metabolites known to form DNA adducts and initiate lung cancer (2.35 Å) or with two molecules of NNK bound (2.1 Å): one in the active site and one in a more distal staging site. Finally, in contrast to prior CYP2A structures with enclosed active sites, CYP2A13 conformations were solved that adopt both open and intermediate conformations resulting from an ∼2.5 Å movement of the F to G helices. This channel occurs in the same region where the second, distal NNK molecule is bound, suggesting that the channel may be used for ligand entry and/or exit from the active site. Altogether these structures provide multiple new snapshots of CYP2A13 conformations that assist in understanding the binding and activation of an important human carcinogen, as well as critical comparisons in the binding of nicotine, one of the most widely used and highly addictive drugs in human use.

Human xenobiotic‐metabolizing cytochrome P450 (CYP) enzymes can each bind and monooxygenate a diverse set of substrates, including drugs, often producing a variety of metabolites. Additionally, a single ligand can interact with multiple CYP enzymes, but often the protein structural similarities and differences that mediate such overlapping selectivity are not well understood. Even though the CYP superfamily has a highly canonical global protein fold, there are large variations in the active site size, topology, and conformational flexibility. We have determined how a related set of three human CYP enzymes bind and interact with a common inhibitor, the muscarinic receptor agonist drug pilocarpine. Pilocarpine binds and inhibits the hepatic CYP2A6 and respiratory CYP2A13 enzymes much more efficiently than the hepatic CYP2E1 enzyme. To elucidate key residues involved in pilocarpine binding, crystal structures of CYP2A6 (2.4 Å), CYP2A13 (3.0 Å), CYP2E1 (2.35 Å), and the CYP2A6 mutant enzyme, CYP2A6 I208S/I300F/G301A/S369G (2.1 Å) have been determined with pilocarpine in the active site. In all four structures, pilocarpine coordinates to the heme iron, but comparisons reveal how individual residues lining the active sites of these three distinct human enzymes interact differently with the inhibitor pilocarpine.Database Structural data are available in the Protein Data Bank database under the accession numbers 3T3Q (CYP2A6 I208S/I300F/G301A/S369G with pilocarpine), 3T3R (CYP2A6 with pilocarpine), 3T3S (CYP2A13 with pilocarpine), and 3T3Z (CYP2E1 with pilocarpine)

Cytochrome P450s (P450s) metabolize a large number of diverse substrates with specific regio- and stereospecificity. A number of compounds, including nicotine, cotinine, and aflatoxin B₁, are metabolites of the 94% identical CYP2A13 and CYP2A6 enzymes but at different rates. Phenacetin and 4-aminobiphenyl were identified as substrates of human cytochromes P450 1A2 and 2A13 but not of CYP2A6. The purpose of this study was to identify active site amino acids that are responsible for CYP2A substrate specificity using phenacetin as a structural probe. Ten amino acid residues that differ in the CYP2A13 and CYP2A6 active sites were exchanged between the two enzymes. Phenacetin binding revealed that the six substitution, CYP2A13 S208I, A213S, F300I, A301G, M365V, and G369S decreased phenacetin affinity. Although incorporation of individual CYP2A13 residues into CYP2A6 had little effect on this enzyme9s very low levels of phenacetin metabolism, the combination of double, triple, and quadruple substitutions at positions 208, 300, 301, and 369 increasingly endowed CYP2A6 with the ability to metabolize phenacetin. Enzyme kinetics revealed that the CYP2A6 I208S/I300F/G301A/S369G mutant protein O-deethylated phenacetin with a K_m of 10.3 μM and a k_cat of 2.9 min^-1, which compare very favorably with those of CYP2A13 (K_m of 10.7 μM and k_cat of 3.8 min^-1). A 2.15 Å crystal structure of the mutant CYP2A6 I208S/I300F/G301A/S369G protein with phenacetin in the active site provided a structural rationale for the differences in phenacetin metabolism between CYP2A6 and CYP2A13.

Thermal green protein (TGP) is an extremely stable, highly soluble synthetic green fluorescent protein. The quantum yield of TGP is lower than the closest related natural fluorescent protein, monomeric Azami-Green. We improved the thermal recovery of TGP through the introduction of a chromophore mutation, Q66E. Furthermore, we developed a yellow thermal protein (YTP) via mutation of histidine 193 to tyrosine. Incorporation of Q66E into YTP (YTP-E) improved chemostability and pH stability. Both YTP and YTP-E have superior thermostability compared to TGP or TGP-E. These proteins offer a new option for green or yellow fluorescence under harsh chemical or thermal conditions.