Our major research goal is elucidating the mechanisms of molecular recognition in metalloprotein systems, including the recognition of proteins with other proteins, with small molecule substrates/inhibitors and with the lipid membrane components that establish and control the assembly of supra-molecular membrane complexes that affect all aspects of recognition and catalysis. Efforts focus on the human hepatic cytochromes P450 that are central players in drug metabolism. Due to their important role in human health and disease, these enzyme systems have occupied investigators for many decades. Despite an intense research effort, a complete molecular understanding of the biophysical and biochemical mechanisms of P450 catalysis remain important goals. One obstacle has been that the human cytochrome P450s are integral membrane proteins. While much has been learned by investigating their function in native membrane preparations or by detergent solubilization, critical knowledge on the role of the membrane in facilitating function and the basis for substrate recognition is lacking. In a novel approach, we surmount this challenge by making make use of the Nanodisc system (developed under support from this grant) that self-assembles the membrane protein target into a discoidal nanometer size lipid bilayer that is soluble in aqueous solution. We use a variety of biophysical approaches in order to define the mechanism of protein self-assembly into these robust bilayer structures and to reveal the topology of P450 and its redox partner, cytochrome P450 reductase (CPR). In seeking to understand the details of how lipids interact with proteins and multi-protein complexes, we define the mechanisms of complex formation between human hepatic P450 and CPR as well as explore the potential role of P450 oligomerization in drug metabolism. The human P450 detoxifying enzymes require a highly plastic active site in order to functionally accommodate a wide spectrum of substrate structures. The ability to bind and metabolize multiple substrates in the P450 active site is the origin of "drug-drug" interactions, which in many cases, is responsible for deleterious side effects and limitations in the therapeutic window between efficacy and toxicity. Hence, understanding this aspect of molecular recognition in the precise mechanisms and magnitude of cooperative substrate binding is also an important goal of our future work. These efforts to reveal the principles of molecular recognition in metalloprotein mechanisms are linked through application of a breadth of biophysical, biochemical and structural techniques to reveal the molecular details of self-assembly, the role of the membrane environment and protein conformational determinants of cytochrome P450 catalysis.

Public Health Relevance

This research program seeks to understand the mechanisms of molecular recognition in the cytochrome P450 oxygenases involved in human drug metabolism using a combination of biophysical and biochemical techniques in concert with a novel self-assembly system (Nanodiscs) for elucidating the structure and function of integral membrane proteins. !

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033775-29
Application #
8477045
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
1984-12-01
Project End
2015-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
29
Fiscal Year
2013
Total Cost
$352,532
Indirect Cost
$125,757
Name
University of Illinois Urbana-Champaign
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
Reichart, Timothy M; Baksh, Michael M; Rhee, Jin-Kyu et al. (2016) Trimerization of the HIV Transmembrane Domain in Lipid Bilayers Modulates Broadly Neutralizing Antibody Binding. Angew Chem Int Ed Engl 55:2688-92
Denisov, Ilia G; Mak, Piotr J; Grinkova, Yelena V et al. (2016) The use of isomeric testosterone dimers to explore allosteric effects in substrate binding to cytochrome P450 CYP3A4. J Inorg Biochem 158:77-85
Wilcox, Kyle C; Marunde, Matthew R; Das, Aditi et al. (2015) Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer's-Associated Aβ Oligomers. PLoS One 10:e0125263
Carney, Christiane E; Lenov, Ivan L; Baker, Catherine J et al. (2015) Nanodiscs as a Modular Platform for Multimodal MR-Optical Imaging. Bioconjug Chem 26:899-905
Denisov, Ilia G; Grinkova, Yelena V; Baylon, Javier L et al. (2015) Mechanism of drug-drug interactions mediated by human cytochrome P450 CYP3A4 monomer. Biochemistry 54:2227-39
Mak, Piotr J; Gregory, Michael C; Denisov, Ilia G et al. (2015) Unveiling the crucial intermediates in androgen production. Proc Natl Acad Sci U S A 112:15856-61
Skar-Gislinge, Nicholas; Kynde, Søren A R; Denisov, Ilia G et al. (2015) Small-angle scattering determination of the shape and localization of human cytochrome P450 embedded in a phospholipid nanodisc environment. Acta Crystallogr D Biol Crystallogr 71:2412-21
Mak, Piotr J; Gregory, Michael C; Sligar, Stephen G et al. (2014) Resonance Raman spectroscopy reveals that substrate structure selectively impacts the heme-bound diatomic ligands of CYP17. Biochemistry 53:90-100
Khatri, Yogan; Gregory, Michael C; Grinkova, Yelena V et al. (2014) Active site proton delivery and the lyase activity of human CYP17A1. Biochem Biophys Res Commun 443:179-84
Mak, Piotr J; Luthra, Abhinav; Sligar, Stephen G et al. (2014) Resonance Raman spectroscopy of the oxygenated intermediates of human CYP19A1 implicates a compound i intermediate in the final lyase step. J Am Chem Soc 136:4825-8

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