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Cholesterol Increases Kinetic, Energetic, and Mechanical Stability Cholesterol increases kinetic, energetic, and PNAS PLUS mechanical stability of the human β2-adrenergic receptor Michael Zochera, Cheng Zhangb, Søren G. F. Rasmussenb, Brian K. Kobilkab,1, and Daniel J. Müllera,1 aDepartment of Biosystems Science and Engineering, Eidgenössiche Technische Hochschule (ETH) Zurich, 4058 Basel, Switzerland; and bDepartment of Molecular and Cellular Physiology, Stanford University School of Medicine, Palo Alto, CA 94305 Edited by Wolfgang Baumeister, Max Planck Institute of Biochemistry, Martinsried, Germany, and approved October 10, 2012 (received for review July 18, 2012) The steroid cholesterol is an essential component of eukaryotic Similar to other GPCRs, cholesterol modulates the physio- membranes, and it functionally modulates membrane proteins, logical function of β2AR (26, 28). Furthermore, cholesterol and including G protein-coupled receptors. To reveal insight into how the more water-soluble cholesterol analog cholesteryl hemi- cholesterol modulates G protein-coupled receptors, we have used succinate (CHS) enhance the thermal stability of β2AR (26, 29). dynamic single-molecule force spectroscopy to quantify the me- Additionally, cholesterol facilitates interactions between GPCRs fl chanical strength and exibility, conformational variability, and and seems to be helpful in crystallizing β2AR (9). A recently kinetic and energetic stability of structural segments stabilizing published X-ray crystallography model of human β2AR showed β β the human 2-adrenergic receptor ( 2AR) in the absence and pres- cholesterol to fit into a shallow surface groove formed by ence of the cholesterol analog cholesteryl hemisuccinate (CHS). transmembrane α-helices H1, H2, H3, and H4 (30). This struc- CHS considerably increased the kinetic, energetic, and mechanical tural model unraveled possible interactions between cholesterol fi stability of almost every structural segment at suf cient magni- and β2AR. However, understanding the extent to which the β tude to alter the structure and functional relationship of 2AR. One interactions established by cholesterol change kinetic, energetic, β exception was the structural core segment of 2AR, which estab- and mechanical properties of structural regions in the receptor BIOPHYSICS AND lishes multiple ligand binding sites, and its properties were not requires additional insight. significantly influenced by CHS. Intra- and intermolecular interactions stabilizing membrane COMPUTATIONAL BIOLOGY proteins can be quantified and localized using atomic force mi- atomic force microscopy | energy landscape | intermolecular and croscopy (AFM) -based single-molecule force spectroscopy (SMFS) intramolecular interactions | proteoliposomes (31, 32). SMFS enables the assignment of these interactions to in- dividual secondary structure elements, such as transmembrane he human β2-adrenergic receptor (β2AR) is one of the best- α-helices, β-strands, or polypeptide loops of membrane proteins β Tcharacterized G protein-coupled receptors (GPCRs). 2AR embedded in lipid membranes. Measuring the kinetic response of belongs to the class A GPCRs and is expressed in pulmonary and the individual structural elements by dynamic SMFS (DFS) allows cardiac myocyte tissue (1, 2). Together with its close relative quantification of their unfolding free energy, kinetic stability, con- β1AR, β2AR senses adrenalin in bronchial vasculature and nor- formational variability, and mechanical flexibility (33). Both SMFS adrenalin in cardiac muscle. The implication in a broad spectrum and DFS have been applied to characterize these parameters of of diseases, like asthma or heart failure, makes β2AR a potential various membrane proteins and follow how they change on chang- therapeutic target (3–7). Several crystal structures of β2AR have ing their functional state or physiological environment (34–39). – been determined during the last years (8 12), providing unique In this work, we have reconstituted human β2AR into phos- insights into the structure–function relationship of GPCRs. pholipid bilayers in the presence and absence of the cholesterol Cellular membranes functionally modulate a large number analog CHS and used SMFS and DFS to quantify and localize – of membrane proteins (13 18). Such functional modulation is the inter- and intramolecular interactions established in β2AR. facilitated by chemical and physical interactions between mem- CHS is a more water-soluble cholesterol ester, and it is much brane proteins and phospholipids, sphingolipids, cholesterol, and easier to handle for in vitro studies than cholesterol, which is one other components of the cell membrane. Similar to many other main reason that CHS is widely used in structural biology and membrane proteins, GPCRs are regulated by their hetero- and biophysical studies as the cholesterol analog. Because CHS and homooligomeric assembly and the membrane composition. Be- cholesterol are structurally quite similar, they are supposed to cause the heterogeneous composition of cellular membranes show similar properties in interacting and functionally modulating changes dynamically, the functional state of GPCRs depends on membrane proteins, such as GPCRs (29, 40, 41). Accordingly, the location in the cell membrane and the state of the cell (19, GPCRs are frequently characterized in the presence of CHS, and 20). The steroid cholesterol modulates chemical and physical the structural and functional results are interpreted as being properties of cellular membranes and plays a role in the dynamic formation of sphingolipid-enriched assemblies of lipids and mem- brane proteins. These metastable assemblies, or lipid rafts, can functionally regulate membrane proteins by different mecha- Author contributions: M.Z., B.K.K., and D.J.M. designed research; M.Z. performed re- nisms (19). Indirect regulation of membrane proteins, including search; C.Z., S.G.F.R., B.K.K., and D.J.M. contributed new reagents/analytic tools; M.Z., GPCRs, can be observed through the ability of cholesterol to S.G.F.R., and D.J.M. analyzed data; and M.Z., B.K.K., and D.J.M. wrote the paper. modulate biophysical properties of a lipid bilayer (21), whereas The authors declare no conflict of interest. the direct regulation of membrane proteins can occur through This article is a PNAS Direct Submission. fi – speci c interactions (22 25). Although it is not completely un- Freely available online through the PNAS open access option. derstood how cells control the distribution of cholesterol and by 1To whom correspondence may be addressed. E-mail: [email protected] or daniel. which mechanisms cholesterol functionally regulates GPCRs, [email protected]. insights into these processes are of cell biological and pharma- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cological importance (26, 27). 1073/pnas.1210373109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1210373109 PNAS Early Edition | 1of10 Downloaded by guest on September 28, 2021 obtained in the presence of cholesterol (40–44). To correlate our density plots (Fig. 1 B and C, Lower). The superimpositions of SMFS measurements with complementary structural and func- F-D curves recorded of β2AR reconstituted in DOPC (Fig. 1B) tional insights revealed of β2AR reconstituted into liposomes and DOPC/CHS (Fig. 1C) showed a characteristic pattern of containing CHS (29, 30, 40), we characterized β2AR reconstituted eight force peaks. The presence of the cholesterol analog CHS into lipid membranes in the presence and absence of the choles- did not change the positions of the force peaks. However, the terol analog CHS. This approach allows a precise correlation of magnitude of the force peaks increased in presence of CHS (Fig. the CHS-induced interactions quantified by SMFS and provides 1 and Fig. S2). This difference implies that the cholesterol analog unique insight into how cholesterol modulates kinetic, energetic, increases the strength of interactions stabilizing β2AR. and mechanical properties of structural regions of β2AR. Mapping Interactions That Stabilize Structural Segments of β2AR. Results After having identified that the superimposed F-D spectra B C Lower β SMFS of Human β2AR in the Presence and Absence of Cholesterol. To (Fig. 1 and , ) correspond to the unfolding of 2AR characterize the influence of cholesterol on the interactions from the N-terminal end (SI Text and Figs. S3 and S4), we β stabilizing human β2AR, we reconstituted the receptor into lip- mapped the interactions to the 2AR structure. When exerting osomes containing phospholipids [1,2-dioleoyl-sn-glycero-3- sufficient force to the N-terminal end, β2AR unfolds in a se- phosphocholine (DOPC)] or phospholipids and CHS (DOPC/ quence of steps. Every unfolding step is reported by a force peak CHS) (Materials and Methods). For SMFS, the proteoliposomes of the F-D curve (Fig. 1B). An unfolding step, in which a stable were densely adsorbed onto freshly cleaved mica (Fig. S1). The structural segment unfolds, describes the transfer of one AFM stylus was approached and pushed onto the proteolipo- unfolding intermediate to the next intermediate (31). To assign somes with a force of ∼700 pN for 0.5 s (Fig. 1A). Subsequently, the unfolding steps and stable structural segments, every force the cantilever was retracted, and its deflection was recorded as a peak of an F-D curve was fitted using the worm-like chain force-distance (F-D) curve. In ∼0.5% of all approach-and-retract (WLC) model (Materials and Methods). Each WLC fit revealed cycles (n = 300,000),
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