GPCR stabilization using the bicelle-like architecture of mixed sterol-detergent micelles☆
Introduction
Recent attempts at characterizing the human membrane proteome identified approximately 6000 human membrane proteins constituting approximately 30% of the human genome [1], [2]. The characterization of membrane proteins typically requires their extraction from the lipid membrane bilayer, an environment that bestows considerable stabilization on these embedded proteins. Protein extraction from the membrane and its subsequent purification therefore represents a major obstacle that requires the use of detergents/surfactants, which assemble into globular micelles that adopt different geometrical shapes in solution [3], [4], and shield the hydrophobic core of membrane proteins from exposure to aqueous medium through formation of a protein–detergent complex (PDC) [5]. Finding suitable detergent(s) that will stabilize the protein’s structure and maintain its activity is therefore pivotal.
G protein-coupled receptors (GPCRs) represent the largest class of human membrane proteins involved in signal transduction (80%) [6], and remain a largely untapped reservoir of pharmaceutical targets as ∼40% of all marketed drugs target only a small fraction of the 800+ members in this protein family [7], [8]. GPCRs share a common fold consisting of a seven transmembrane (TM) alpha helical bundle flanked by an N-terminus or a globular extracellular domain that plays a role in ligand recognition and/or binding in some receptors, and an amphipathic helix (helix VIII) followed by an intracellular domain at the C-terminus [9]. Common among the rhodopsin family of GPCRs is a palmitoylation site immediately following helix VIII, which anchors this region to the membrane bilayer and may target these proteins for partitioning into cholesterol rich rafts and caveolae [10], [11]. Analysis of the crystal structures of the β1 and β2 adrenergic receptors (β1AR and β2AR) has led to the identification and characterization of a specific cholesterol binding site at a cleft created by helices II, III, and IV [12], [13], [14], [15], [16]. Many residues within this specific binding site have been found to be conserved among the GPCR families, and thus this region has been termed the cholesterol consensus motif (CCM) [13].
The nonionic alkyl glucoside detergents, specifically n-dodecyl-β-d-maltoside (DDM), are the most widely used detergents for the isolation and purification of GPCRs. Above the critical micelle concentration (CMC), DDM micelles form oblate ellipsoids where the polar axis is shorter than the equatorial axes [3], [4], [17]. DDM mixtures with various additive detergents containing various acyl chain lengths or different head groups can be used to adjust the micelle’s physiochemical properties and dimensions for optimal membrane protein stability. Additionally, the use of detergent soluble cholesterol analogs is becoming increasingly popular, with numerous reports showing that these compounds may help protect GPCRs from thermal inactivation [13], [18], [19], [20]. Several successful GPCR structure determination efforts have shown that the addition of cholesterol analogs is often critical for maintaining receptor stability [13], [14], [15], [21], [22], [23], [24], [25]. However, it is unclear if the protective function of these sterol analogs is achieved through modulation of detergent micelle morphology and physiochemical properties, or through a specific sterol-protein interaction.
This study is focused on understanding the effect of cholesterol analogs on DDM/GPCR PDCs. In a case study, we show that the type of cholesterol analog supplemented to the PDC substantially influenced the thermostability of ORL-1 (opioid receptor like -1; also known as NOP (nociceptin opioid peptide) receptor), a member of the opioid receptor (OR) GPCR family. All four opioid receptors have been shown to partition into lipid rafts, and methyl-β-cyclodextrin (MβCD) mediated depletion of cholesterol has been shown to modulate their downstream signaling [26], [27], [28], [29], [30], [31], [32], [33]. Importantly, the ORs contain a tryptophan residue, Trp4.50 (superscript corresponds to the Ballesteros-Weinstein numbering [34]), located at the heart of a putative CCM, which is critical for π-stacking interactions with cholesterol’s sterol core [13]. The importance of ORL-1’s Trp4.50 residue has been underscored through mutagenesis studies. In an effort to understand the differences in effects attributed to the different cholesterol analogs, biophysical studies of various sterol/DDM micelle mixtures (free of protein) were conducted to measure differences in the mixed micelle morphologies. Using dynamic light scattering and small-angle X-ray scattering, we have determined that the most thermal stabilizing sterol, cholesteryl hemisuccinate, induces the formation of a bicelle-like architecture when mixed with DDM detergent.
Section snippets
ORL-1 cloning, expression and purification
The wild type (WT) human ORL-1 gene (UniProt accession code P41146) was synthesized by DNA2.0 and then cloned into a modified pFastBac1 vector (Invitrogen) containing an expression cassette with an HA signal sequence followed by a FLAG tag at the N-terminus, and a PreScission protease site followed by a 10×His tag at the C-terminus. Thirty amino acids were deleted from the C-terminus, which helped to increase receptor yield (data not shown). Recombinant baculoviruses were generated by using the
Cholesterol derivatives and ORL-1 thermostability
ORL-1 was expressed in Sf9 insect cells, and a thorough search for optimal detergents identified DDM as the best detergent for extraction and purification of this receptor (data not shown). To date, little is known about the thermostabilizing effect of sterol addition to OR PDCs. Because cholesterol is insoluble in the absence of lipids (unpublished observations), we tested the effect of cholesterol analogs mixed with DDM on their ability to promote the thermal stabilization of ORL-1 using a
Discussion
Having evolved under pressure of natural selection, cholesterol is the primary sterol found in higher eukaryotes [43]. In the case of plants and fungi, cholesterol is replaced by phytosterols and ergosterol, respectively. The unique rigid planar structure of sterols imparts special properties on membranes by modulating its fluidity [44], thickness [45], curvature [46], and permeability [47]. Many of these “effects” are due to the increase in ordering of lipid acyl tails when packed against the
Conclusion
A number of different sterol/DDM mixtures were tested for their ability to stabilize the opioid receptor ORL-1 for structural studies. Out of nine sterol-like detergents studied, only CHS and CHOBIMALT showed substantial thermostabilization of ORL-1. These two cholesterol analogs are chemically similar, each having a cholesterol-like tetracyclic ring system and non-polar tails, thereby permitting their penetration in the micelle core. The other seven sterols tested contained polar groups
Acknowledgments
The authors acknowledge the assistance of Tsutomu Matsui for support at SSRL BL 4.2; Jan Lipfert for access to the two-shell ellipsoid fitting script for MatLab; Girolamo Calo, Remo Guerrini and Stephano Molinari for the synthesis of Compound-24; and Angela Walker, Enrique Abola and Jeremiah S. Joseph for critical reading of the manuscript.
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This work was supported by the NIH Common Fund grant P50 GM073197 (R.C.S. and V.C.) and NIH grant R01 GM089857 (V.C.). D.W. is supported by a Boehringer Ingelheim Fonds PhD Fellowship.