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Structural Features of Β2 Adrenergic Receptor

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Structural Features of Β2 Adrenergic Receptor Mol. Cells 2015; 38(2): 105-111 http://dx.doi.org/10.14348/molcells.2015.2301 Molecules and Cells http://molcells.org Established in 1990 Structural Features of 2 Adrenergic Receptor: Crystal Structures and Beyond Injin Bang, and Hee-Jung Choi* The beta2-adrenergic receptor (2AR) belongs to the G class A, also referred to as rhodopsin type GPCRs. In 2000, the protein coupled receptor (GPCR) family, which is the larg- first GPCR structure was visualized by using bovine rhodopsin est family of cell surface receptors in humans. Extra atten- complexed with 11-cis-retinal and this structure has been used tion has been focused on the human GPCRs because they as an important template for GPCR modeling (Palczewski et al., have been studied as important protein targets for phar- 2000). The overall rhodopsin structure consists of seven trans- maceutical drug development. In fact, approximately 40% membrane (TM) helices and three loop regions at the extracel- of marketed drugs directly work on GPCRs. GPCRs re- lular and the cytoplasmic sides. The ligand binding pocket of spond to various extracellular stimuli, such as sensory rhodopsin is formed by hydrophobic residues from TM5 and signals, neurotransmitters, chemokines, and hormones, to TM6 to stabilize the hydrocarbone backbone of retinal, which is induce structural changes at the cytoplasmic surface, ac- covalently bound to Lys296 of TM7. tivating downstream signaling pathways, primarily through One of the common sequence motifs in rhodopsin type interactions with heterotrimeric G proteins or through G- GPCRs is the D[E]RY motif on TM3, which forms an ionic lock protein independent pathways, such as arrestin. Most by making a salt bridge between Arg3.50 of the D[E]RY motif GPCRs, except for rhodhopsin, which contains covalently and Asp/Glu6.30 of TM6. The ionic lock was suggested as a linked 11 cis-retinal, bind to diffusible ligands, having vari- characteristic of inactive conformation of GPCR, to block the G ous conformational states between inactive and active protein binding at the cytoplasmic region. The other common structures. The first human GPCR structure was deter- motif is the NPXXY motif on TM7. In contrast to the ionic lock, mined using an inverse agonist bound 2AR in 2007 and which stabilizes inactive conformation, it has been suggested to since then, more than 20 distinct GPCR structures have play an important role in GPCR activation. Although rhodopsin been solved. However, most GPCR structures were solved structure provided the first structural aspects of GPCR, it was as inactive forms, and an agonist bound fully active struc- suggested that most other GPCRs, which interact with diffusible ture is still hard to obtain. In a structural point of view, ligands with different efficacy, would have different structural 2AR is relatively well studied since its fully active struc- features from rhodopsin since rhodopsin has a covalently ture as a complex with G protein as well as several inactive bound ligand. structures are available. The structural comparison of in- Human 2AR was first identified in the 1990s but its struc- active and active states gives an important clue in under- tural study hadn’t begun until 2007. Unlike rhodopsin, 2AR standing the activation mechanism of 2AR. In this review, shows conformational instability, suggested by its agonist inde- structural features of inactive and active states of 2AR, pendent basal activity. Also, 2AR has a much longer flexible the interaction of 2AR with heterotrimeric G protein, and intracellular loop 3 (IL3), which could be an obstacle for crystal- the comparison with 1AR will be discussed. lization. A high affinity inverse agonist, carazolol, was used to stabilize the inactive conformation of 2AR and the flexibility of IL3 was reduced by making a complex with IL3-specific Fab OVERALL STRUCTURE OF BETA2-ADRENERGIC RECEPTOR fragment or by replacing it with T4 lysozyme (T4L) (Fig. 1 1A) (Cherezov et al., 2007; Rosenbaum et al., 2007; Rasmussen Based on sequence similarity, GPCRs can be divided into four et al., 2007). Although the 2AR-Fab complex structure didn’t classes: class A, B, C, and F. The majority of GPCRs belong to resolve the carazolol binding site, the native conformation around IL3 showed that the ionic lock was broken in its inactive structure, explaining why 2AR shows basal activity even in the School of Biological Sciences, College of Natural Sciences, Seoul Na- presence of an inverse agonist. High resolution structure of tional University, Seoul 151-747, Korea 2AR with T4L fusion was obtained from the crystals in lipid *Correspondence: [email protected] cubic phase (LCP). The T4L region greatly facilitates crystalli- Received 10 November, 2014; accepted 13 November, 2014; published zation by making favorable crystal packing interaction of T4L online 24 December, 2014 with the extracellular loop regions of neighboring 2AR. LCP crystallization method and T4L fusion strategy are now com- Keywords: beta2-adrenergic receptor (2AR), conformational change, monly used for GPCR structure determination. High resolution crystal structure, G-protein coupled receptor (GPCR), heterotrimeric G structure of 2AR provides invaluable information on the ligand protein binding site of 2AR. Carazolol forms hydrogen bonding inter- eISSN: 0219-1032 The Korean Society for Molecular and Cellular Biology. All rights reserved. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/. Structural Features of 2 Adrenergic Receptor: Crystal Structures and Beyond Injin Bang & Hee-Jung Choi A B Fig. 1. Carazolol bound inactive structures of 2AR. (A) Crystal structures of carazolol bound 2AR with T4L fusion (left) and that complexed with Fab (right) are shown in green and cyan, respectively (pdb ID: 2RH1, 2R4R). T4L and Fab are colored in grey and carazolol is shown as yellow spheres. (B) Close view of the carazolol binding site in 2AR-T4L. Carazolol is shown as yellow sticks and 2AR amino acids making polar and hydrophobic interactions are labeled in black and blue, respectively. actions with Asp1133.32, Asn3127.39, and Tyr3167.43 and has AGONIST INDUCED CONFORMATIONAL CHANGE OF hydrophobic interactions with Val1143.33, Phe2906.52, and Phe1935.32 BETA2-ADRENERGIC RECEPTOR of 2AR (Fig. 1B). Its binding site is partially overlapped with that of retinal in rhodopsin. The -ionone ring of retinal probes Since the inactive structure of 2AR was first reported in 2007, deep inside rhodopsin to interact with Trp2866.48, which is lots of effort had been made to determine the agonist bound known as the “toggle switch” for receptor activation. In contrast, active conformation of 2AR. One of the approaches was to carazolol cannot reach deep enough to interact with the toggle design the covalently bound agonist to stabilize the agonist switch. The ligand binding site of 2AR is relatively open to the bound active form of 2AR. For this purpose, Cys was incorpo- solvent which enables a ligand to diffuse in and out easily. In rated into residue 93 of 2AR instead of His, to make a disulfide the case of retinal-bound rhodopsin, direct access to the ligand bond with an agonist, FAUC50 (Rosenbaum et al., 2011). binding site is restricted by extracellular loop 2 (ECL2), which Structural study of 2AR with the covalently linked agonist dis- forms a sheet above the retinal binding site by interacting with covered an interesting result that the agonist alone was not the N-terminus. ECL2 in 2AR, which doesn’t make any direct sufficient to stabilize the active conformation of 2AR, which contact with the N-terminus, contains an alpha helix and a dis- was unexpected since the structure of metarhodopsin II ulfide bond between Cys1844.76 and Cys1905.29. Another disul- showed the active state conformation, like outward movement fide bond between Cys1915.30 and Cys1063.25 from TM3 contri- of the cytopalsmic end of TM6, in the absence of a cytoplasmic butes to the stabilization of ECL2. A 2.4Å resolution structure of binding partner. In 2011, two active structures of 2AR bound T4L fusion of 2AR clearly showed water mediated hydrogen to a high affinity agonist (BI-167107) were determined using bonding network between TM residues. These water-filled, either Nb80 (nanobody 80) or Gs protein bound to the cytop- loosely packed regions may allow for conformational changes lasmic side of 2AR (Figs. 2A and 2B) (Rasmussen et al., upon activation. 2011a; 2011b). RMSD evaluation found that the structural dif- Other inactive 2AR structures in a complex with the partial ference between Nb80 bound and Gs bound 2AR was minim- inverse agonist, timolol or antagonist, alprenolol, have been al (Fig. 2C). Detailed structural analysis of G protein bound reported and their overall structural folds are maintained with 2AR will be discussed later. minor structural rearrangements of the ligand binding site to The comparison between a carazolol bound inactive struc- accommodate different chemical properties of ligands (Hanson ture and an agonist bound active structure shows that only little et al., 2008; Wacker et al., 2010). While the hydrogen bond changes occur on the extracellular side of the receptor. In fact, network with Asp1133.32, Asn3127.39, and Tyr3167.43 of 2AR is the interaction pattern in the agonist-binding pocket differs only conserved in the interactions of carazolol, timolol and alprenolol, slightly between carazolol and BI-167107. The key change additional hydrogen bonding interactions and hydrophobic inte- appears to be in the interaction with Ser2045.43 and Ser2075.46 ractions are varied for each ligand, which could be related to on TM5. The hydrogen bonding between BI-167107 and the the strength of inverse agonism or antagonism. polar pocket residues, including the two serines, causes a 2Å inward movement of TM5 at position Ser2075.46, resulting in the 106 Mol.
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