Membrane-associated cytochrome P450s (P450s) work as members of a large enzymatic complex (the P450 monooxygenase system) that catalyze the oxidation of a variety of exogenous and endogenous compounds, particularly the detoxification or bioactivation of thousands of pharmaceutical compounds. Allelic variants of P450s are also implicated in the development of certain cancers and are considered important biomarkers for cancer susceptibility. Catalytic activities of cytP450 are modulated by the microsomal membrane-bound cytochrome b5 (cytb5). The influence of cytb5 on cytP450 activity has been shown to depend on the cytP450 isozyme and the substrate involved. Remarkably, cytb5 enhances some catalytic reactions of cytP450 but does not affect or even inhibits others. The main objective of the proposed research is to obtain an in-depth understanding of the molecular basis of the effects of cytb5 on cytP450 activity by determining the structural interactions between the membrane-bound full-length rabbit cytP4502B4 and cytb5. Structure of the membrane-bound ~70-kDa cytP450-cytb5 complex will provide insights into the mechanism by which cytb5 regulates the enzymatic function of cytP450. We have already determined the first full-length membrane- bound rabbit cytb5 structure. We propose to use a combination of solution and solid-state (both static and magic angle spinning) NMR techniques to obtain structural and dynamic interactions from the rabbit cytP450- cytb5 complex embedded in model membranes such as bicelles, lipid vesicles and nanodiscs. Mutational and functional studies will also be carried out to identify the residues in the interacting interface of the cytP450- cytb5 complex. The effect of selected substrates on the cytP450-cytb5 complex will be investigated using NMR and functional measurements. While our preliminary results demonstrate that NMR measurements on the cytP450-cytb5 complex embedded in bicelles are feasible, we also plan to use nanodisc as it would enable the investigation of the roles of membrane components on the structure and dynamics of the cytP450-cytb5 complex by varying the membrane composition. After completing our investigation on rabbit cytP4502B4, we will use other cytP450s (such as cytP4503A4, cytP4502D6 and cytP45017A1) to compare their structures and membrane orientations and to further understand the role of cytb5 on the function of cytP450.

Public Health Relevance

Cytochromes P450 (cytsP450) are a ubiquitous superfamily of mixed-function oxygenases, which are found in all kingdoms of life but are especially abundant in eukaryotes. Humans possess 57 different membrane-bound cytsP450. They are found in all tissues of the body and are responsible for influencing a dazzling array of biochemical and physiological processes, including embryonic development, blood coagulation, and the metabolism of carcinogens, environmental toxins, over 50% of drugs in use, vitamin D and other exogenous and endogenous compounds. Human cytsP450 are targets for the treatment of prostate and breast cancer, respectively. The outcome of the proposed structural studies on the membrane-bound full-length rabbit cytP450-cytb5 complex will provide insights into molecular mechanism by which cytb5 influences oxidation by cytP450 that metabolizes more than 50% of current-day drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM084018-08
Application #
9452068
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Okita, Richard T
Project Start
2009-01-01
Project End
2019-03-31
Budget Start
2018-04-01
Budget End
2019-03-31
Support Year
8
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biophysics
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Damron, Joshua T; Ma, Jialiu; Kurz, Ricardo et al. (2018) The Influence of Chemical Modification on Linker Rotational Dynamics in Metal-Organic Frameworks. Angew Chem Int Ed Engl 57:8678-8681
Ravula, Thirupathi; Hardin, Nathaniel Z; Bai, Jia et al. (2018) Effect of polymer charge on functional reconstitution of membrane proteins in polymer nanodiscs. Chem Commun (Camb) 54:9615-9618
Ravula, Thirupathi; Hardin, Nathaniel Z; Ramadugu, Sudheer Kumar et al. (2018) Formation of pH-Resistant Monodispersed Polymer-Lipid Nanodiscs. Angew Chem Int Ed Engl 57:1342-1345
Barnaba, Carlo; Sahoo, Bikash Ranjan; Ravula, Thirupathi et al. (2018) Cytochrome-P450-Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs. Angew Chem Int Ed Engl 57:3391-3395
Gentry, Katherine A; Zhang, Meng; Im, Sang-Choul et al. (2018) Substrate mediated redox partner selectivity of cytochrome P450. Chem Commun (Camb) 54:5780-5783
Prade, Elke; Mahajan, Mukesh; Im, Sang-Choul et al. (2018) A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs. Angew Chem Int Ed Engl 57:8458-8462
Barnaba, Carlo; Ravula, Thirupathi; Medina-Meza, Ilce G et al. (2018) Lipid-exchange in nanodiscs discloses membrane boundaries of cytochrome-P450 reductase. Chem Commun (Camb) 54:6336-6339
Naito, Akira; Matsumori, Nobuaki; Ramamoorthy, Ayyalusamy (2018) Dynamic membrane interactions of antibacterial and antifungal biomolecules, and amyloid peptides, revealed by solid-state NMR spectroscopy. Biochim Biophys Acta Gen Subj 1862:307-323
Damron, Joshua T; Kersten, Kortney M; Pandey, Manoj Kumar et al. (2017) Electrostatic Constraints Assessed by 1H MAS NMR Illuminate Differences in Crystalline Polymorphs. J Phys Chem Lett 8:4253-4257
Yamamoto, Kazutoshi; Caporini, Marc A; Im, Sang-Choul et al. (2017) Transmembrane Interactions of Full-length Mammalian Bitopic Cytochrome-P450-Cytochrome-b5 Complex in Lipid Bilayers Revealed by Sensitivity-Enhanced Dynamic Nuclear Polarization Solid-state NMR Spectroscopy. Sci Rep 7:4116

Showing the most recent 10 out of 94 publications