Microsomal cytochromes P450 (CYPs) require electrons transferred from NADPH by cytochrome P450 reductase (CPR) to support O2 reduction and substrate oxidation. The CPR catalytic domain is tethered by a linker to an N-terminal transmembrane domain, both of which are required to support CYP catalysis. However, details of the interactions of the tether and catalytic domains with the membrane surface are poorly understood. Herein, we describe the assembly, characterization, and interrogation of the structures of an ancestral CPR embedded in lipoprotein nanodiscs (NDs) composed of defined and microsomal lipids. No significant differences in the rates of NADPH oxidation between the NDs and detergent-solubilized CPR were observed; however, there were differences in the rates of cytochrome c reduction. Using multicontrast small angle neutron scattering (SANS) and a grid-based molecular dynamics strategy to refine structures to density maps, it was determined that CPR-NDs assume a more compact solution structure than predicted by unrestrained molecular dynamics simulations. The tether domain lines the membrane surface, and the catalytic domain is positioned at the ND edge. To the extent that the SANS-based structure is representative of CPR-NDs with varying lipid composition, the interactions of the catalytic domain and scaffold protein likely interfere with cytochrome c interactions, thereby resulting in varying rates of reduction. These studies report the first experimentally grounded structure of membrane embedded CPR and demonstrate that interactions between embedded and ND scaffold proteins cannot be neglected. This strategy is expected to contribute to the repertoire of methods to probe membrane protein-lipid interactions.
