Mapping Protein-Protein Interactions in Self-Sufficient Cytochrome P450 Enzymes
Felker, Dana
2021
Abstract
Cytochrome P450 (P450) proteins are heme-containing mixed function oxidase enzymes that play a critical role in the metabolism of drugs and toxicants. For P450 enzyme catalysis, a flavin-containing reductase must shuttle electrons from NADPH to the heme prosthetic group of P450. Although in most cases the reductase enzyme is a separate protein partner, there are self- sufficient P450 enzymes containing the reductase enzyme fused with the P450 enzyme in a single polypeptide chain. These self-sufficient P450 enzymes are excellent model systems to study the mechanism of P450 catalysis and electron transfer reactions. There are no crystal structures of the full-length self-sufficient P450 enzymes, although recently low-resolution cryo-EM derived structures of two self-sufficient P450 enzymes, CYP102A1 and neuronal nitric oxide synthase (nNOS), have been reported by our lab. A covalent crosslinking and mass spectrometry (CXL-MS) workflow was developed in this thesis to study interdomain and interprotein interactions within these two self-sufficient P450 enzymes, in order to learn how P450 and reductase domains interact in detail. In these studies, I refined the workflow to analyze and interpret MS data of crosslinks specifically from homodimeric proteins by utilizing existing structural data in combination with an established subtractive method to identify intra- and inter-protein crosslinks. CXL-MS of the CYP102A1 homodimer yielded 31 unique crosslinks, 26 of which mapped in accordance with the cryo-EM based structural models of the protein. Furthermore, I identified a novel conformational state of the CYP102A1 homodimer based on modeling studies of the remaining five crosslinks that likely represent a structure favorable for electron transport to the active site heme. I then applied CXL-MS to a homodimer of nNOS bound to its regulatory protein calmodulin (CaM), which activates the enzyme for catalysis. The resulting 74 crosslinks, similar to those found for CYP102A1, are consistent with a conformation where the oxygenase and reductase domains are in closer proximity than seen in previous structural studies. My studies identify novel heme and reductase interdomain interactions for both of the analyzed self-sufficient P450 enzymes and provide insight on domain organization during catalysis. The CXL-MS workflow developed here will be applicable in investigating other homomeric multiprotein machineries. Most importantly, I have identified new interdomain and interprotein interfaces that can serve as regulatory targets in modulating these important metabolizing systems. This work provides new insights on the catalytic mechanism of these powerful mixed function oxidase enzymes, which are important protective enzymes from environmental toxicants as well as potential biocatalysts for use in environmental waste remediation.Deep Blue DOI
Subjects
Cytochrome P450 Crosslinking CXL-MS Protein-Protein Interactions CYP102A1 Neuronal Nitric Oxide Synthase
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