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Cholesterol interaction motifs in G protein‐coupled receptors: Slippery hot spots?

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Abstract G protein‐coupled receptors (GPCRs) are cell membrane associated signaling hubs that orchestrate a multitude of cellular functions upon binding to a diverse variety of extracellular ligands. Since GPCRs are integral membrane proteins with seven‐transmembrane domain architecture, their function, organization and dynamics are intimately regulated by membrane lipids, such as cholesterol. Cholesterol is an extensively studied lipids in terms of its effects on GPCR structure and function. One of the possible mechanisms underlying modulation of GPCR function by cholesterol is via specific interaction of GPCRs with membrane cholesterol. These interactions of GPCRs with membrane cholesterol are often attributed to structural features of GPCRs that could facilitate their preferential association with cholesterol. In this backdrop, cholesterol interaction motifs represent putative interaction sites on GPCRs that could facilitate cholesterol‐sensitive function of these receptors. In this review, we provide an overview of cholesterol interaction motifs found in GPCRs, which have been identified through a combination of crystallography, bioinformatics analysis, and functional studies. In addition, we will highlight, using specific examples, why mere presence of a cholesterol interaction motif at a given site may not directly implicate its role in interaction with membrane cholesterol. We therefore believe that experimental approaches, followed by functional analysis of cholesterol sensitivity of GPCRs, would provide a better understanding of the role played by these motifs in cholesterol‐sensitive function. We envision that a comprehensive knowledge of cholesterol interaction sites in GPCRs would allow us to develop a better understanding of GPCR structure‐function paradigm, and could be useful in future therapeutics. This article is categorized under: Models of Systems Properties and Processes > Mechanistic Models Analytical and Computational Methods > Computational Methods Laboratory Methods and Technologies > Macromolecular Interactions, Methods
A schematic representation of the membrane‐embedded human serotonin1A receptor highlighting overlapping lipid binding/interaction motifs. The amino acids in the receptor sequence are shown as circles. The sphingolipid binding domain (SBD) and sphingolipid binding motif (SBM) in TM II and TM V, respectively, are highlighted in cyan. Enlarged representations of TM II and TM V of the human serotonin1A receptor showing the overlap of SBD and SBM (highlighted in cyan) with CRAC motif I and II (highlighted in yellow), respectively. Residues common to both SBM/SBD and CRAC motifs are shown in a combination of cyan and yellow. (Reprinted with permission from Shrivastava et al. (). Copyright 2018 Springer Nature Singapore Pte Ltd.)
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Cholesterol interaction hot spots in the serotonin1A receptor. (a) Residue‐wise maximum occupancy of cholesterol bound to the serotonin1A receptor, obtained by coarse‐grain molecular dynamics simulations. Maximum occupancy time (defined as the maximum time a given cholesterol molecule is found at a particular residue during the course of the simulation; see text for details) of cholesterol at each amino acid of the serotonin1A receptor was averaged and normalized over simulations carried out at varying concentrations of cholesterol. The transmembrane helices are represented as gray bands, and CRAC motifs are highlighted with the same color coding as in Figure a. The high cholesterol occupancy observed at the CRAC motif on transmembrane helix V (CRAC II) is noteworthy (Reprinted with permission from Sengupta and Chattopadhyay (). Copyright 2012 American Chemical Society). (b) A schematic energy landscape corresponding to cholesterol interaction sites in GPCRs. The interaction of cholesterol with GPCRs is weak, yet dynamic with varying occupancy times ranging from ns to μs time scale. This aspect of the interaction of cholesterol with GPCRs is reflected in the energy landscape of cholesterol interaction which is represented as a series of shallow minima interconnected by low energy barriers. The abscissa can be thought to correspond to individual occupancy sites represented by single residues or by a sub‐space at the receptor surface (such as cholesterol consensus motif [CCM] or CRAC sites). The occupancy sites are most likely to be accessed via an exchange with the annular lipids and less often by direct site hopping of cholesterol. Note that the energy barriers and the minima could be modulated by other membrane lipids such as sphingolipids (Reprinted with permission from Sengupta and Chattopadhyay (). Copyright 2015 Elsevier B.V.)
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Cholesterol recognition/interaction amino acid consensus (CRAC) motifs in G protein‐coupled receptors. (a) CRAC motifs in representative GPCRs. The numbers corresponding to the starting amino acid position in the respective sequences are mentioned in parentheses before the sequences. The putative positions of the CRAC motifs mapped to individual helices in these GPCRs are indicated at the top. The central aromatic amino acid residues (tyrosine) of the CRAC motifs are highlighted in maroon. The basic amino acid residues (arginine or lysine) at the C‐termini are highlighted in blue and the nonpolar branched aliphatic amino acid residues (leucine or valine) at the N‐termini are highlighted in green. (b) A schematic representation depicting the topological features and amino acid sequence of the human serotonin1A receptor embedded in a membrane bilayer consisting of phospholipids and cholesterol. The putative positions of the transmembrane helices of the human serotonin1A receptor was predicted using the crystal structure of the human serotonin1B receptor (PDB ID: 6G79) and the amino acids in the receptor sequence are shown as circles. The receptor has seven transmembrane stretches, each composed of ~22 amino acids, that are depicted as putative α‐helices and are marked as I–VII. Since there are no crystal structures available for the serotonin1A receptor, the exact boundary between the membrane and the aqueous phase is not known and therefore location of the amino acid residues relative to the membrane bilayer is putative. The serotonin1A receptor consists of three CRAC motifs (highlighted in yellow) in TM II (CRAC I, boxed in red), TM V (CRAC II, boxed in green), and TM VII (CRAC III, boxed in blue). (Reprinted with permission from Jafurulla et al. (). Copyright 2010 Elsevier Inc.)
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Identification of cholesterol recognition/interaction amino acid consensus (CRAC) motifs in proteins. (a) The key elements of the CRAC motif. CRAC is a short linear motif that fulfills the following sequence algorithm from the N‐terminus to C‐terminus: a branched apolar leucine or valine residue, followed by a segment containing 1–5 of any amino acid residues, an aromatic residue that is specifically tyrosine, followed by a stretch of 1–5 of any amino acid residues, and finally a basic lysine or arginine residue at the C‐terminus. (b) CRAC motifs in representative proteins that have been shown to interact with cholesterol. The numbers corresponding to the starting amino acid position in the respective sequences are mentioned before the CRAC motif for each protein. The positions of the central aromatic amino acid residues (tyrosine) are highlighted in maroon. The basic amino acid residues (arginine or lysine) at the C‐termini are highlighted in blue and the nonpolar branched aliphatic amino acid residues (leucine or valine) at the N‐termini are highlighted in green. The sequences containing the CRAC motifs are taken from Li and Papadopoulos ()
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Closely associated cholesterol molecules in G protein‐coupled receptor crystal structures. (a) Crystal structure of the human β2‐adrenergic receptor (Cherezov et al., ; Rosenbaum et al., ; PDB ID: 2RH1). The receptor monomers (shown in blue) pack in a parallel orientation with three cholesterol molecules (shown in orange) bound to each monomer and a palmitic acid alkyl chain is located between cholesterol 2 and 3. The receptor construct was modified for enhanced crystallizability by incorporation of T4‐lysozyme between helices V and VI (shown in green) (Reprinted with permission from Hanson et al. (). Copyright 2008 Elsevier Ltd.). (b) The key amino acid residues in the strict cholesterol consensus motif (CCM) from the crystal structure of the human β2‐adrenergic receptor (Hanson et al., ; PDB ID: 3D4S). Two bound cholesterol molecules are shown in yellow and the side chain positions of the crucial amino acids in the CCM are highlighted. Site 1 (blue) at the cytoplasmic end of transmembrane helix IV (TM IV) spanning positions 4.39–4.43 fulfills the CCM requirement, if one or more of these positions contain a basic amino acid residue (arginine or lysine). Site 2 (cyan) at position 4.50 on TM IV contributes to CH‐π interactions (represented as space‐filling cyan side‐chain atoms) and is the most conserved site with tryptophan occupying the position in ~94% of class A GPCRs. The other allowed amino acid in this position is tyrosine. Site 3 (represented as space‐filling side‐chain atoms in green) at position 4.46 on TM IV contributes via van der Waals interaction to cholesterol binding and fulfills the CCM requirement, if isoleucine, leucine, or valine is present in this position. Site 4 (maroon) on TM II at position 2.41 can be either tryptophan or phenylalanine or tyrosine. Sites 1–3 together defines CCM, whereas the presence of site 4 along with other three sites defines the four component strict CCM (Reprinted with permission from Hanson et al. (). Copyright 2008 Elsevier Ltd.). (c) A representative list of GPCRs with their CCM motifs. The positions of the aromatic amino acid residues (tryptophan or tyrosine or phenylalanine) in TM II and TM IV are highlighted in maroon and cyan, respectively. The basic amino acid residues (arginine or lysine) at the cytoplasmic end of TM IV are highlighted in blue and the central aliphatic amino acid residues (isoleucine, leucine, or valine) in TM IV are highlighted in green. The numbers above the amino acid sequence represent the Ballesteros–Weinstein numbering scheme for GPCRs. The corresponding protein accession numbers are indicated in parentheses
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Chemical structure of cholesterol. Chemical structure of cholesterol with its three structurally distinct regions (shown as shaded boxes): the polar 3β‐hydroxyl group, the rigid tetracyclic fused ring (shown as A–D), and the flexible isooctyl side chain
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Diversification of cholesterol interaction motifs. (a) The CARC motif is similar to the CRAC sequence, but exhibits the opposite orientation (“inverted CRAC”) along the polypeptide chain. For CARC motif, the central residue is still aromatic, but unlike CRAC which by definition has a specific requirement for tyrosine, the CARC motif could have tyrosine, phenylalanine or tryptophan as a central aromatic residue. In case of CRAC motif, the aromatic ring of phenylalanine could sustain the interaction with cholesterol when tyrosine is not available. This constitutes the CRAC‐like motif, where the central aromatic residue is phenylalanine. (b) For GPCRs (where the N‐terminus is extracellular), in TM I, III, V, and VII, the CARC motif is located in the outer leaflet and the CRAC domain is in the inner leaflet. In case of TM II, IV, and VI, the arrangement still holds but in this case CARC is located in the inner leaflet and CRAC in the outer leaflet. (c) The simultaneous presence of CRAC and CARC motifs within the same transmembrane helix constitutes a “mirror code” that could accommodate two cholesterol molecules (shown in green) in a typical tail‐to‐tail orientation, one bound to CRAC and the other to CARC. (Reprinted with permission from Fantini et al., (). Copyright 2016 Springer Nature Ltd.)
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An evolutionarily conserved collage of four categories of cholesterol interaction motifs associated with TM V and the adjacent intracellular loop 3 fragment of the vertebrate serotonin1A receptor. (a) A schematic representation of phylogenetic clades: Teleost, Aves, and Mammalia (includes Primate and Rodentia subclades), used for this analysis. Aves and Mammalia represent endotherms, and Teleostei represent ectotherms. The dashed line in the Mammalia clade represents the species that are not categorized into subclades. (b) A collage of putative cholesterol interaction motifs overlaid on the sequence logo of serotonin1A receptor TM V and intracellular loop 3 (ICL3) fragment. Multiple sequence alignment (MSA) positions in TM V are represented by Ballesteros–Weinstein (BW) indices from 5.34 to 5.50. Position 5.50 represents the position of the evolutionarily conserved proline. In addition to TM V and ICL3, the juxtamembrane regions from extracellular loop 2 (ECL2) is shown. Colored boxes represent various cholesterol‐sensitive motifs (CRAC, CRAC‐like, CARC, and cholesterol consensus motif [CCM] motifs), and numbers in parentheses represent the total number of configurations possible for each motif. The boxes labeled as CCM* may complement an existing CCM in its spatial proximity to constitute a strict CCM (Reprinted with permission from Fatakia et al. (). Copyright 2019 Elsevier B.V.)
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