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Stabilization of Functional Recombinant Cannabinoid Receptor CB2 in Detergent Micelles and Lipid Bilayers

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Stabilization of Functional Recombinant Cannabinoid Receptor CB2 in Detergent Micelles and Lipid Bilayers Stabilization of Functional Recombinant Cannabinoid Receptor CB2 in Detergent Micelles and Lipid Bilayers Krishna Vukoti¤, Tomohiro Kimura, Laura Macke, Klaus Gawrisch, Alexei Yeliseev* National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland, United States of America Abstract Elucidation of the molecular mechanisms of activation of G protein-coupled receptors (GPCRs) is among the most challenging tasks for modern membrane biology. For studies by high resolution analytical methods, these integral membrane receptors have to be expressed in large quantities, solubilized from cell membranes and purified in detergent micelles, which may result in a severe destabilization and a loss of function. Here, we report insights into differential effects of detergents, lipids and cannabinoid ligands on stability of the recombinant cannabinoid receptor CB2, and provide guidelines for preparation and handling of the fully functional receptor suitable for a wide array of downstream applications. While we previously described the expression in Escherichia coli, purification and liposome-reconstitution of multi-milligram quantities of CB2, here we report an efficient stabilization of the recombinant receptor in micelles - crucial for functional and structural characterization. The effects of detergents, lipids and specific ligands on structural stability of CB2 were assessed by studying activation of G proteins by the purified receptor reconstituted into liposomes. Functional structure of the ligand binding pocket of the receptor was confirmed by binding of 2H-labeled ligand measured by solid- state NMR. We demonstrate that a concerted action of an anionic cholesterol derivative, cholesteryl hemisuccinate (CHS) and high affinity cannabinoid ligands CP-55,940 or SR-144,528 are required for efficient stabilization of the functional fold of CB2 in dodecyl maltoside (DDM)/CHAPS detergent solutions. Similar to CHS, the negatively charged phospholipids with the serine headgroup (PS) exerted significant stabilizing effects in micelles while uncharged phospholipids were not effective. The purified CB2 reconstituted into lipid bilayers retained functionality for up to several weeks enabling high resolution structural studies of this GPCR at physiologically relevant conditions. Citation: Vukoti K, Kimura T, Macke L, Gawrisch K, Yeliseev A (2012) Stabilization of Functional Recombinant Cannabinoid Receptor CB2 in Detergent Micelles and Lipid Bilayers. PLoS ONE 7(10): e46290. doi:10.1371/journal.pone.0046290 Editor: Hendrik W. van Veen, University of Cambridge, United Kingdom Received May 31, 2012; Accepted August 28, 2012; Published October 3, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This study was supported by the intramural research program of the National Institute on Alcohol Abuse and Alcoholism (NIAAA), National Institutes of Health (NIH), to KG. KG participated in study design, data collection and analysis, decision to publish, and preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] ¤ Current address: Case Center for Proteomics and Bioformatics, Case Western Reserve University, Cleveland, Ohio, United States of America Introduction stable over extended periods of time. While solubilization in detergents is needed for extraction of GPCRs from cell Heptahelical G protein-coupled receptors (GPCRs) are integral membranes and chromatographic purification, the preservation membrane proteins involved in a wide range of physiological of the structural integrity of receptors in micelles is a notoriously processes including sensory transduction and cell-to-cell commu- difficult task. Unlike dark-adapted bovine rhodopsin which nication [1]. The cannabinoid receptor CB2 is an attractive target exhibits significant stability in detergent solutions, many GPCRs, for the development of drugs for management of pain, inflamma- when removed from membranes and exposed to detergents, lose tion, osteoporosis, inhibition of growth of malignant gliomas and their functional fold in a matter of minutes and, therefore, require tumors of immune origin, and treatment of immunological additional efficient stabilization [7,8]. The relatively few successful disorders such as multiple sclerosis [2,3,4]. High resolution attempts to preserve the functional structure of purified GPCRs structural studies will provide critical insights into the molecular relied on a careful selection of mild solubilizing detergents as well mechanisms of ligand binding and activation of CB , and may 2 as on supplementation of buffers used for purification with guide the rational design of novel, highly specific pharmaceuticals. stabilizers such as lipids and ligands, adjustment of ionic strength Although GPCRs represent as much as 50% of pharmaceutical and glycerol content. Yet, no general methodology for an efficient drug targets currently under development, the progress with stabilization of GPCRs has been developed yet, and their low structural studies has been relatively slow, in part due to the stability remains a major bottleneck for structural biology [9]. difficulties in obtaining large quantities of sufficiently pure, A recently introduced approach to stabilization by site-directed homogenous and functional protein. With the notable exception mutagenesis [10,11] as well as by replacement of the large of rhodopsin, GPCRs are naturally expressed at low levels, and intracellular loop 3 and truncation of flexible N- and C-terminal heterologous production is currently the only technically feasible domains succeeded in obtaining well-diffracting crystals of several way to prepare these proteins [5,6]. class A GPCRs [12,13,14]. However, such modifications alter the In addition to the availability of large quantities of purified wild type structure and are known to affect the functional receptor, structural methods require that the protein is sufficiently PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e46290 Stabilization of Cannabinoid Receptor CB2 properties of receptors significantly [10,11,15,16,17,18]. There- following published procedures [29,30], and their correct fore, rather than performing an extensive modification of the posttranslational modifications, adequate purity and functional structure of CB2 with poorly predictable functional consequences, activity ensured. The reaction conditions have been optimized in this study we explored the stabilization potential of carefully such that the individual subunits of G proteins were provided at selected detergents, lipids and high affinity ligands. The minimal concentrations significantly higher than those of the receptor to 35 alteration to the native amino acid sequence of CB2 used in this maintain linear rates of accumulation of Gai1 bound to c- S- work included addition of the small affinity tags at the N- and C- GTP, a non-hydrolizable analog of GTP [20] and to ensure that terminus of the protein [19] that, as we demonstrated, do not they accurately represent the fraction of the ligand-binding affect function of the receptor as determined by G protein competent and functional protein. Furthermore, these proteolipo- activation and ligand-binding tests [20]. somes model a physiologically relevant environment enabling We focus on developing applications of spectroscopic tech- studies of structure and function of CB2 as well as its interaction niques, in particular nuclear magnetic resonance (NMR) to studies with the lipid matrix. of the full length, structurally unperturbed CB2 in lipid bilayers [5,6,19,21,22]. Emphasis is on studies by the solid-state NMR on Experimental Strategy purified CB2 reconstituted at a high protein-to-lipid ratio into lipid The main objective of this study has been development of bilayers of a defined composition. While the high-density procedures for efficient stabilization of the recombinant CB2 in homogenous reconstitution of CB2 into liposomes was discussed detergent micelles and improvement of yield of purified, functional in a recent publication [19], the present work addresses protein. The general outline of the experiments is presented in stabilization of CB2 during its expression, solubilization in Fig. 1, A. The influence of detergents, cholesteryl hemisuccinate detergent micelles, and chromatographic purification, with a goal (CHS) and ligands is tested at various stages of protein of maximizing yield of fully functional receptor, in micelles as well preparation. The effects of stabilizers are assessed after reconsti- as in lipid bilayers. tution of the receptor into liposomes, by studying G protein Several earlier attempts to express the recombinant CB2 for activation. Whenever possible, this was done by reconstitution of structural investigations failed to produce pure and fully the receptor into lipid bilayers of equal composition to avoid an functional receptor [23,24,25,26,27]. Reasons for the poor influence from lipid composition on G protein activation rates as recovery of functional receptor vary and depend on the reported earlier [19]. approach used to expression and purification of this protein. To assess the effect of lipids on stability of purified CB2 in As we demonstrated earlier, an expression
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