A subset of cytochrome P450 enzymes perform the first and rate-limiting step in the clearance of foreign small molecule drugs and toxins from the human body, while others play key roles in endogenous pathways. Of necessity the former evolved the flexibility to bind and oxidize a broad range of small molecule chemical scaffolds, while the latter appear to be less flexible and have more substrate specificity. What we know about the structures of all of these membrane proteins has been determined solely by X-ray crystallography. This approach provides detailed information about atomic-level protein/ligand interactions, but has not been applied across the human spectrum of P450 enzymes and does not capture the range of conformations these enzymes are capable of adopting or their interactions with other proteins. Thus application of a cross-section of structural techniques is essential to provide the information needed to understand which P450 enzymes bind which small molecules, how they are bound, and what the products will be. This information is critical for understanding drug/toxin metabolism to forms that may be either active or inactive, adverse interactions of two drugs at the same P450 active site, and endogenous pathways related to diverse diseases. The applicant's long-term research goal is to promote understanding of the structure/function principles that control substrate and inhibitor interactions with P450 enzymes, in order that this information can be exploited to more effectively prevent and treat human disease. The objective of this proposal is to generate structures of new human cytochrome P450 enzymes with the critical components of the catalytic system: their ligands·, redox partner proteins, and eventually the membrane. A number of human cytochrome P450 enzymes do not have structures available and none have structures with their catalytic partner proteins. These are gaps we propose to bridge using the following approaches, building on our previous structural expertise with more than 20 human cytochrome P450 enzymes and a collaboration with cryo-electron microscopist Dr. Melanie Ohi. Specific aim 1: Determine structures of human P450 enzymes. A structure exists for only about half of the 57 human cytochrome P450 enzymes. Many of those without structures are involved in key homeostatic pathways involving bile acids, fatty acids, eicosanoids, and vitamins, impeding our understanding of a number of corresponding diseases. We take advantage of this R37 extension opportunity to propose a small-scale structural genomics project to "close the gap" by determining structures of as many of these P450 enzymes as possible. As we have done successfully for many other human P450 enzymes, we will 1) engineer synthetic genes in ways that usually produce P450 holoproteins, 2) undertake expression and purification trials, and 3) subject those yielding enough active P450 protein to crystallization for X-ray structure determination. We will initially ...