Over the past few years, we have determined the structures of the cytochrome bc1 complex from the photosynthetic bacterium R. sphaeroides (Rsbc1) in various forms, proposed an hypothesis for the mechanism of the surface-affinity modulated iron-sulfur protein (ISP) conformation switch to account for the bifurcated electron transfer (ET) at the quinol oxidation (QP) site, provided experimental evidence to support this hypothesis, and identified substrate ubiquinol (QH2) in the QP site for the first time. All these achievements were rooted in our relentless pursuit of better diffracting crystals. The structure solution of Rsbc1 accomplishes one of our goals in establishing a model system to systematically study the bc1 complex by combining structural, genetic, and biochemical techniques. Our structural studies of bovine bc1 suggested that the key to the bifurcated ET at the QP site is the control of the ISP-ED movement, which regulates the distance between the 2Fe2S cluster and c1 heme. The distance is too long to permit ET between the two sites when ISP-ED is in the fixed conformation;ET is only possible when ISP-ED is in the mobile conformation. We hypothesized that by modulating the shape of the binding surface, the cyt b subunit effectively controls its affinity for the ISP-ED, the movement of the ISP, and thereby the directions of the two electrons from the substrate ubiquinol. Data from reports in the literature and new experiments can be used to verify this hypothesis;and this is an ongoing process. (i) We were able to crystallize Rsbc1 only in the presence of Pf inhibitors such as stigmatellin, JG144 and famoxadone. (ii) The significance of the extraordinarily high sequence conservation of the cd1 helix and the PEWY motif in the ef loop of the cyt b subunit can now be appreciated, as both motifs contribute to the ISP binding surface. (iii) Based on our hypothesis, the b hemes should be reduced before the c1 heme, which were experimentally observed both from reports in the literature and in our measurement under pre-steady state conditions. (iv) In contrast to the previous hypothesis that the conformational fixation of ISP in the presence of stigmatellin was due to a H-bond between the bound inhibitor and ISP, we found that the elimination of this H-bond still promotes immobility of ISP in the cases of famoxadone or JG144 binding. These findings strongly support the idea that the switch of the ISP conformation is an intrinsic property of the bc1 complex. If this H-bond were indeed important for the conformational fixation of ISP, the QH2/Q would have served as an inhibitor rather than a substrate, as the same H-bond exists between bound Q/QH2 and ISP. (v) Mutagenesis of residues contributing to the ISP binding surface on cyt b invariably changes ET kinetics between ISP and c1 but does not affect QH2 binding to the QP site. (vi) For Rsbc1, the I292M and I292A mutants that have significantly slowed down ET between ISP and c1 can be crystallized in the absence of Pf inhibitors. Our current studies focus on investigating the conformational transition of ISP in the absence of respiratory inhibitors. We have discovered that ISP conformation switch induced by redox potentials changes. Since its approval by the FDA in the 1970s, cisplatin chemotherapy has become the cornerstone of a broad spectrum of cancer treatments and it is one of the most commonly used chemotherapy drugs in cancer medicine today. Like many other chemotherapeutic agents, cisplatin is facing a growing problem of resistance by cancers in its clinical application. A number of mechanisms have been proposed for the development of cisplatin resistance, including changes in cellular uptake and efflux of the drug, increased detoxification of the drug, inhibition of apoptosis, and increased DNA repair. Despite extensive research in the field, molecular mechanisms of cisplatin resistance remain elusive. The hypothetical protein TMEM205, formerly known as MBC3205, was speculated to be a secreted integral membrane protein by the Secreted Protein Discovery Initiative. This protein consists of 189 amino acid residues with four predicted trans-membrane helices (TMHs). Little was known about this protein and its cellular function until recently when we reported its role in cisplatin resistance in cancer cell lines. Using a fluorescence-labeled cisplatin, it was shown that overexpression of TMEM205 reduces accumulation of cisplatin in cancer cell lines;this reduction correlates with the cisplatin resistance of the cells. TMEM205 is shown to be a membrane protein localized to the cell surface and is highly expressed in both human cisplatin-resistant cervical carcinoma and hepatoma cells internally near the trans-golgi network. High expression levels of this protein are found in certain secretory tissues, such as those of the liver, pancreas, and adrenal glands, consistent with the postulated role of TMEM205 in cisplatin resistance. To achieve structural solution of TMEM205, we overexpressed this integral membrane protein, determined its oligomeric state, and crystallized it. The TMEM205 crystals diffracted X-rays to 2 A resolution. Multidrug resistance (MDR) is a long-standing clinic challenge in cancer therapies. MDR is associated with over expression of efflux ABC transporters such as P-glycoproteins (P-gp) on cell surface. Efforts to stop P-gp during cancer treatment have not been successful. My lab has been working on elucidating the structure of P-gp for a long time in our attempts to uncover the mechanism of P-gp function from a structural perspective. Recently, we have successfully expressed both human and mouse P-gp in yeast expression systems. More importantly, we were able to crystallize mouse P-gp and crystals of mouse P-gp diffracted to 3.5 A resolution. This success provides us an opportunity to investigate the differences in solution behavior of human and mouse P-gp and potentially to provide a better structure of mouse P-gp. My lab also engages in molecular modeling studies of ABC transporters, which has become an important tool to gain structural and functional insights into proteins whose atomic structures are unknown. Over the years, we have constructed structural models for a number of ABC transporters such as ABCB1, ABCG2, Pdr5p, etc. These models are useful as guidance for further characterizations of these proteins.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010319-13
Application #
8348974
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
2011
Total Cost
$790,311
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Esser, Lothar; Zhou, Fei; Pluchino, Kristen M et al. (2017) Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity. J Biol Chem 292:446-461
Esser, Lothar; Shukla, Suneet; Zhou, Fei et al. (2016) Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein. Acta Crystallogr F Struct Biol Commun 72:636-41
Pluchino, Kristen M; Hall, Matthew D; Moen, Janna K et al. (2016) Human-Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P-Glycoprotein (ABCB1). Biochemistry 55:1010-23
Esser, Lothar; Zhou, Fei; Zhou, Yihui et al. (2016) Hydrogen Bonding to the Substrate Is Not Required for Rieske Iron-Sulfur Protein Docking to the Quinol Oxidation Site of Complex III. J Biol Chem 291:25019-25031
Frank, Gabriel A; Shukla, Suneet; Rao, Prashant et al. (2016) Cryo-EM Analysis of the Conformational Landscape of Human P-glycoprotein (ABCB1) During its Catalytic Cycle. Mol Pharmacol 90:35-41
Jian, Xiaoying; Tang, Wai-Kwan; Zhai, Peng et al. (2015) Molecular Basis for Cooperative Binding of Anionic Phospholipids to the PH Domain of the Arf GAP ASAP1. Structure 23:1977-88
Esser, Lothar; Yu, Chang-An; Xia, Di (2014) Structural basis of resistance to anti-cytochrome bc? complex inhibitors: implication for drug improvement. Curr Pharm Des 20:704-24
Xiao, Yu-Mei; Esser, Lothar; Zhou, Fei et al. (2014) Studies on inhibition of respiratory cytochrome bc1 complex by the fungicide pyrimorph suggest a novel inhibitory mechanism. PLoS One 9:e93765
Xia, Di; Esser, Lothar; Tang, Wai-Kwan et al. (2013) Structural analysis of cytochrome bc1 complexes: implications to the mechanism of function. Biochim Biophys Acta 1827:1278-94
Ma, Jichun; Tang, Wai Kwan; Esser, Lothar et al. (2012) Recognition of mesothelin by the therapeutic antibody MORAb-009: structural and mechanistic insights. J Biol Chem 287:33123-31

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