Transmembrane Interactions of Full-length Mammalian Bitopic Cytochrome-P450-Cytochrome-b(5) Complex in Lipid Bilayers Revealed by Sensitivity-Enhanced Dynamic Nuclear Polarization Solid-state NMR Spectroscopy

The dynamic protein-protein and protein-ligand interactions of integral bitopic membrane proteins with a single membrane-spanning helix play a plethora of vital roles in the cellular processes associated with human health and diseases, including signaling and enzymatic catalysis. While an increasing number of high-resolution structural studies of membrane proteins have successfully manifested an in-depth understanding of their biological functions, intact membrane-bound bitopic protein-protein complexes pose tremendous challenges for structural studies by crystallography or solution NMR spectroscopy. Therefore, there is a growing interest in developing approaches to investigate the functional interactions of bitopic membrane proteins embedded in lipid bilayers at atomic-level. Here we demonstrate the feasibility of dynamic nuclear polarization (DNP) magic-angle-spinning NMR techniques, along with a judiciously designed stable isotope labeling scheme, to measure atomistic-resolution transmembrane-transmembrane interactions of full-length mammalian ~72-kDa cytochrome P450-cytochrome b(5) complex in lipid bilayers. Additionally, the DNP sensitivity-enhanced two-dimensional (13)C/(13)C chemical shift correlations via proton driven spin diffusion provided distance constraints to characterize protein-lipid interactions and revealed the transmembrane topology of cytochrome b(5). The results reported in this study would pave ways for high-resolution structural and topological investigations of membrane-bound full-length bitopic protein complexes under physiological conditions.