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Insights into the world of GPCRs (Adrenergic Receptors)

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Speaker: Bundit Boonyarit 5814400587
Dept. Biochemistry, Fac. Science, Kasetsart University
2 May, 2016 (11.15 - 12.00 a.m.)

Advanced Protein Biochemistry (01402542)

Published in: Science
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Insights into the world of GPCRs (Adrenergic Receptors)

  1. 1. Insights into the world of GPCRs (Adrenergic Receptors) Speaker: Bundit Boonyarit 5814400587 Dept. Biochemistry, Fac. Science, Kasetsart University 2 May, 2016 (11.15 - 12.00 a.m.) Advanced Protein Biochemistry (01402542)
  2. 2. TOPICS 2 Protein structure Protein function Protein interaction Protein engineering Peptide De novo design
  3. 3. GPCRs Transmembrane protein: largest superfamily of receptors (~50kDa) 3 Drew J. (2000) Biochemical classes of drug targets of current therapies Chemical diversity of ligands e.g. Biogenic amines, Peptides, Glycoproteins, Lipids, Nucleotides, Ions, Odors, Light, Taste, Cannaninoid, Opioid G protein-coupled receptors
  4. 4. 4 HISTORY ( b ( o m c M T c lo ( c th m 2000 Human β -AR 0 10 20 30 40 50 60 70 80 ‘93 ‘94 1995 ‘96 ‘97 ‘98 ‘99 2000 ‘01 ‘02 ‘03 ‘04 2005 ‘06 ‘07 ‘08 ‘09 ‘11 ‘12 First projection map First electon density map Low-resolution structures High-resolution structures First high-resolution structure First active-state structure First receptor–G protein complex structure First NMR structure 2010 Numberofstructures Light-activated Aminergic Nucleoside binding Peptide binding Lipid binding a b RESEARCH REVIEW Venkatakrishnan et al., (2013)
  5. 5. 5 HISTORY Venkatakrishnan et al., (2013) Molecular signatures of the GPC The structure of a GPCR can be divide cellular region, consisting of the N te loops (ECL1–ECL3); (2) the TM regio (TM1–TM7); and (3) the intracellular r cellular loops (ICL1–ICL3), an intracellu the C terminus (Fig. 2a). In a broad modulates ligand access; the TM region ligands and transduces this informat through conformational changes, and th with cytosolic signalling proteins. Extracellular region and ligand-bi Sequence analysis shows that there is a l sequence compositions of the N terminu The class A GPCR structures reveal tw region: those that either occlude the lig ligand-binding pocket water-accessible S1P1 receptor31 have occluded bindin they both bind hydrophobic ligands th the lipid bilayer41 . The N terminus an b-hairpin loops, and together they for pocket. Similarly, the S1P1 receptor co packs against ECL2 and ECL3 (ref. 31). soluble ligands, ECL2 can differ structu structures are likely to be conserved i ECL2 can contain helices (for example, receptors) or sheets (for example, pept 2000 2007 2008 2010 2011 2012 Bovine rhodopsin (1F88) Human β2 -AR (2RH1) Turkey β1 -AR (2VT4) Squid rhodopsin (2Z73) Human Α2Α R (3EML) Human D3R (3PBL) Human CXCR4 (3ODU) Human Α2Α R (3QAK) Bovine rhodopsin (3PQR) Human H1 R (3RZE) Human κ-OR (4DJH) Mouse μ-OR (4DKL) Human N/OFQOR (4EA3) Mouse δ-OR (4EJ4) Human M2R (3UON)N)) Rat M3R (4DAJ)J)J) Rat NTSR1 (4GRV) Active Intermediate-active 0 10 20 ‘93 ‘94 1995 ‘96 ‘97 ‘98 ‘99 2000 ‘01 ‘02 ‘03 ‘04 2005 ‘06 ‘07 ‘08 ‘09 ‘11 ‘12 First projection map First electon density map First high-resolution structure 2010 Nu Light-activated Aminergic Nucleoside binding Peptide binding Lipid binding Human S1P1 R (3V2Y) Human PAR1 (3VW7) Human CXCR1 (2LNL) NMR b Human β2 -AR (3SN6) ** * ** * > >
  6. 6. 6 HISTORY The Nobel Prize in Chemistry 2012 “for studies of G-protein-coupled receptors” Brian K. Kobilka American physiologist Robert J. Lefkowitz American physician
  7. 7. 7 GPCRs http://oldeurope.deviantart.com/art/ GPCR-in-Lipid-Bilayer-focus-129477640 N-terminal segment (Extracellular) 7 Transmembrane domain (TM1-TM7) to form TM core 3 exoloops and 3-4 cytoloops C-terminal segment (Intracellular) EllisClare (2004) Tamas Bart tical industr with what w knowledge- — to cobble have often b than predict Joël Bock ‘correct’, but presence of r structure of Arthur Ch crystal struct has certainly bear in mind similarity to incorporated were of the r necessarily e between this good startin been very su some of the molecules b muscarinic mine H3 and onlyrefersto we are still extracellular Gα N C C Plasma membrane GPCR β γ A N GPCR Gα Gαβγ GDP GTP GDP βγ a b c GPCR B Agonist Agonist T W E N T Y Q U E S T I O N S Tamas Bartfai. Bothinsideandoutsidethepharmaceu- tical industry,the rhodopsin model has been combined with what we know about PHARMACOPHORES — which is a knowledge-rich area for several monoamine receptors — to cobble together models. However, such models have often been of post factum value; explaining rather than predicting results. Joël Bockaert. The predictions have generally been ‘correct’, but the rhodopsin crystal was obtained in the presence of retinal,which is an inverse agonist.So far,the structure of an‘active’rhodopsin molecule is still lacking. Arthur Christopoulos. The determination of the crystal structure of bovine rhodopsin at high resolution59 has certainly been a boon to the GPCR field.One must bear in mind,however,that rhodopsin has low sequence similarity to most other GPCRs, has an inverse agonist incorporated into its structure,and the crystals obtained were of the receptor in its inactive state,so we should not necessarily expect to find high degrees of concordance between this structure and other GPCRs.Nevertheless,a good starting point is better than none, and there have been very successful predictions for the structures of some of the receptors for bioamines and related small molecules based on the rhodopsin model; for example, muscarinic M1 , dopamine D2 , α1 -adrenoceptor, hista- mine H3 and adenosineA1 receptors4,63,64 .Of course,this onlyreferstothetransmembranedomainsof theGPCRs; we are still some way off determining the intra- and extracellularloopstructures. Gα N C C Plasma membrane GPCR β γ A N GPCR Gα Gαβγ GDP GTP GDP βγ a b c GPCR B Agonist Agonist Tam tica with kno — t hav than Joë ‘cor pres stru Art crys has bear sim inco wer nec betw goo bee som mo mu min only we extr Gα N C C Plasma membrane GPCR β γ A N GPCR Gα Gαβγ GDP GTP GDP βγ a b c GPCR B Agonist Agonist
  8. 8. 8 GPCRs T W E N T Y Q U E S T I O N S Tamas Bartfai. Bothinsideandoutsidethepharmaceu tical industry,the rhodopsin model has been combine with what we know about PHARMACOPHORES — which is knowledge-rich area for several monoamine receptor — to cobble together models. However, such model have often been of post factum value; explaining rathe than predicting results. Joël Bockaert. The predictions have generally bee ‘correct’, but the rhodopsin crystal was obtained in th presence of retinal,which is an inverse agonist.So far,th structure of an‘active’rhodopsin molecule is still lacking Arthur Christopoulos. The determination of th crystal structure of bovine rhodopsin at high resolution has certainly been a boon to the GPCR field.One mus bear in mind,however,that rhodopsin has low sequenc similarity to most other GPCRs, has an inverse agonis incorporated into its structure,and the crystals obtaine were of the receptor in its inactive state,so we should no necessarily expect to find high degrees of concordanc between this structure and other GPCRs.Nevertheless, good starting point is better than none, and there hav been very successful predictions for the structures o some of the receptors for bioamines and related sma molecules based on the rhodopsin model; for example muscarinic M1 , dopamine D2 , α1 -adrenoceptor, hista mine H3 and adenosineA1 receptors4,63,64 .Of course,thi Gα N C C Plasma membrane GPCR β γ A N GPCR Gα Gαβγ GDP GTP GDP βγ a b c GPCR B Agonist Agonist Antiparallel alpha helices Venkatakrishnan et al., (2013) TM region EC region IC region β1-AR and β2-AR ICL2 ECL2N terminus S1P1RRhodopsin Rhodopsin β1-AR, β2-AR, M2R, M3R, D3R μ-OR, δ-OR NTSR1 A2A R a μ/κ/δ-OR, NTSR1, NOPR, PAR1, CXCR1/4 A2AR NN 4.504.50 3.503.50 5.505.50 6.506.50 7.507.50 1.501.50 2.502.50 C N 4.50 3.50 5.50 6.50 7.50 1.50 2.50 C Figure 2 | Diversity in the secondary structure elements of GPCRs in the extracellular and intracellular regions. a, TM helices (TM1–TM7) are shown as cartoon (coloured in a spectrum of green) and surface representation. Numbers denote Ballesteros–Weinstein numbering. In this receptor- independent notation, each residue is identified by two numbers that are sequentially, respectiv highly conserved resid and C termini and the extracellular (EC) and type of secondary stru Diversity in the secondary structure elements of GPCRs in the extracellular and intracellular regions TM = Transmembrane, EC = Extracellular, IC = Intracellular, ICL = Intracellular loop (cytoloop) and ECL = Extracellular loop (exoloop)
  9. 9. 9 GPCRs Venkatakrishnan et al., (2013) TM region EC region IC region β1-AR and β2-AR ICL2 ECL2N terminus S1P1RRhodopsin Rhodopsin β1-AR, β2-AR, M2R, μ-OR, δ-OR NTSR1 A2A R a b Occluded ligand-binding pocket Bovine rhodopsin Exposed ligand-binding pocket μ/κ/δ-OR, NTSR1, NOPR, PAR1, CXCR1/4 A2AR NN 4.504.50 3.503.50 5.505.50 6.506.50 7.507.50 1.501.50 2.502.50 C N 4.50 3.50 5.50 6.50 7.50 1.50 2.50 C R Ballesteros–Weinstein numbering First number ranges from 1 to 7 and corresponds to the TM helix The second number indicates position relative to the most conserved residue of the helix, which is assigned the number 50 For example, in bovine rhodopsin, the most conserved residues in each helix are: TM1 Asn55(1.50) (98%) TM2 Asp83(2.50) (92%) TM3 Arg135(3.50) (97%) TM4 Trp161(4.50) (96%) TM5 Pro215(5.50) (77%) TM6 Pro267(6.50) (98%) TM7 Pro303(7.50) (96%)
  10. 10. 10 GPCRs Types of GPCR their radiolabelled counterparts when necessary),which has allowed the determination of receptor distribution, their principal functions and agonist versus antagonist activity. Expression in cells of both human GPCRs and their corresponding rodent proteins is an important step towards a rapid physiopharmacological,and puta- tively clinical, study of the target.At this time, there are s well on. is ave or f 1 ke ch d b Family 2 NH2 45 C C C C C C C C C P W COOH a Family 1 P NH2 C C D P N D R Y C 7 6 5 1 2 3 4 Family A Biogenic amine receptors (adrenergic, serotonin, dopamine, muscarinic, histamine) CCK, endothelia, tachykinin, neuropeptide Y, TRH, neurotensin, bombesin, and growth hormone secretagogues receptors plus vertebrate opsins Invertebrate opsin and bradykinin receptors Adenosine, cannabinoid, melanocortin and olfactory receptors Chemokine, fMLP, C5A, GnRH, eicosanoid, leukotriene, FSH, LH, TSH, nucleotide, opioid, oxytocin, vasopressin Melatonin receptors Disulfide bridge phosphodiesteramine helix broken (Pro) - Small extracellular domain - Ligand binding site is deep within the plane of membrane Family A: Rhodopsin/β2 Adrenergic receptor-like
  11. 11. 11 GPCRs Types of GPCR Calcitonin, CGRP and CRF receptors PTH and PTHrP receptors Glucagon, glucagon-like peptide, GIP, GNRH, PACAP, VIP, and secretin receptors Latrotoxin - Long amino acid at extracellular domain (N- terminal) - No helix broken - Cysteine at N-terminal Family B: Glucagon/VIP/Calcitonin receptor-like . e r b Family 2 NH2 1 2 3 45 6 7 COOH C C C C C C C C C 5 4 c Family 3 NH2 C C P W COOH P D P N D R Y C 7 1 2 3 4 Family B Disulfide bridge
  12. 12. 12 GPCRs Types of GPCR Metabotropic glutamate receptors Matabotropic GABA receptors Calcium receptors Vomeronasal pheromone receptors Taste receptors - Large extracellular domain (N-terminal) - No helix broken Family C: Metabotropic neurotransmitter/ Calcium receptors s) d l, e or y at is n M ce, + -sensing and the GABAB (γ-aminobutyric acid, type B) mino terminus and carboxyl tail. The ligand-binding inus, which has been shown by the crystal structure of the linked dimer103 . It is thought to resemble a Venus fly trap, 1 2 3 45 6 7 COOH P K 5 6 4 2 c Family 3 NH2 1 3 COOH N E A C 7 C Family C Disulfide bridge
  13. 13. T W E N T Y Q U E S T I O N S Tamas Bartfai. Bothinsideandoutsidethepharmaceu- tical industry,the rhodopsin model has been combined with what we know about PHARMACOPHORES — which is a knowledge-rich area for several monoamine receptors — to cobble together models. However, such models have often been of post factum value; explaining rather than predicting results. Joël Bockaert. The predictions have generally been ‘correct’, but the rhodopsin crystal was obtained in the presence of retinal,which is an inverse agonist.So far,the structure of an‘active’rhodopsin molecule is still lacking. Arthur Christopoulos. The determination of the crystal structure of bovine rhodopsin at high resolution59 has certainly been a boon to the GPCR field.One must bear in mind,however,that rhodopsin has low sequence similarity to most other GPCRs, has an inverse agonist incorporated into its structure,and the crystals obtained were of the receptor in its inactive state,so we should not necessarily expect to find high degrees of concordance Gα N C C GPCR β γ N GDP a Agonist T W E N T Y Q U E S T I O N S Tamas Bartfai. Bothinsideandoutsidethepharmaceu- tical industry,the rhodopsin model has been combined with what we know about PHARMACOPHORES — which is a knowledge-rich area for several monoamine receptors — to cobble together models. However, such models have often been of post factum value; explaining rather than predicting results. Joël Bockaert. The predictions have generally been ‘correct’, but the rhodopsin crystal was obtained in the presence of retinal,which is an inverse agonist.So far,the structure of an‘active’rhodopsin molecule is still lacking. Arthur Christopoulos. The determination of the crystal structure of bovine rhodopsin at high resolution59 has certainly been a boon to the GPCR field.One must bear in mind,however,that rhodopsin has low sequence similarity to most other GPCRs, has an inverse agonist incorporated into its structure,and the crystals obtained were of the receptor in its inactive state,so we should not necessarily expect to find high degrees of concordance Gα N C C GPCR β γ N GDP a Agonist Gα N C C Plasma membrane GPCR β γ GPCR Gα Gα Gα Gα Gα βγ βγ GDP βγ GTP GTP GDP GTP GDP Effector Pi βγ a b c d e GPCR B Agonist Agonist GPCRs GPCR activation and inactivation G protein 13EllisClare (2004)
  14. 14. 14 GPCRs G-proteins families Family Some member Action mediated Functions I Gs α Activate adenylyl cyclase, Ca2+ channels Gelf α Activate adenylyl cyclase in olfactory sensory neutron II Gi α Inhibit adenylyl cyclase βγ Activates K+ channel Go βγ Activates K+ channel, Inactivate Ca2+ channels α and βγ Activates phospholipase C-β Gt (tranducin) α Activates cyclic GMP phosphodiesterase in vertebrate photoreceptors III Gq α Activates phospholipase C-β IV G12 α Activates Rho guanine-nucleotide exchange factors (GEFs)
  15. 15. Adrenergic receptors Family A-GPCR
  16. 16. 16 HISTORY 1910 - 1910 • Langley proposes that cells have“receptive substances”
 • Dale refers to“receptive mechanism for adrenalin”
 • Abel isolates epinephrine from the adrenal medulla, the first hormone to be isolated 1941 - 1950 • von Euler demonstrates that norepinephrine is the sympathetic neurotransmitter • Ahlquist defines α− and β-types of adrenergic receptors 1951 - 1960 • Sutherland discovers cyclic AMP, leading to the second messenger concept 1961 - 1970 • Sir James Black develops propranolol, the first clinically useful β-antagonist • Lands defines β1- and β2-subtypes The Adrenergic Receptors in the 21st Century
  17. 17. 17 HISTORY 1971 - 1980 • Langer defines α1 as postsynaptic and α2 as presynaptic
 • Pettinger defines α1- and α2-receptors functionally
 • Snyder and Lefkowitz develop radioligand binding assays for most adrenergic receptors
 • Lefkowitz develops the ternary complex model for G protein-coupled receptors 1981 - 1990 • Khorana clones bacteriorhodopsin, the first of the seven transmembrane receptors
 • Nathans and Hogness clone rhodopsin, the first of the G protein-coupled receptors
 • Arch defines the β3-receptor using pharmacological criteria
 • Bylund defines α1, α2, and β as the three types of adrenergic receptors
 • Dixon, Strader, and Lefkowitz clone the β2-adrenergic receptor
 • Creese proposes α1A- and α1B-subtypes based on radioligand binding
 • Bylund defines α2A-, α2B-, and α2C-subtypes using pharmacological criteria
 • Lefkowitz clones β1-, α2A-, α2B-, α2A-, α1A-, and α1B-receptors
 • Strosberg clones the β3-receptor
 1991 - 2000 • Strader’s laboratory and other laboratories use site-directed mutagenesis to define ligand- binding site and signaling mechanisms
 • Graham and Perez clone α1D
 • Transgenic mice developed by several laboratories
 • Lefkowitz works out desensitization mechanism involving β-adrenergic receptor kinase and β-arrestin
 • Lowell generates β3-knockout mice The Adrenergic Receptors in the 21st Century
  18. 18. 18 HISTORY 1991 - 2000 • Kobilka generates β1-, β2-, α2A-, α2B-, and α2C-knockout mice
 • Cotecchia generates α1B-knockout mice
 • Liggett describes clinically relevant polymorphisms in α2- and β-receptors • Crystal structure of rhodopsin, a G protein-coupled receptor, determined The Adrenergic Receptors in the 21st Century 0 Bylund dioligand-binding studies showing that the two subtypes had differential sen- Fig. 1. A cartoon from 1988 indicating the frustration some investigators felt at the emingly endless proliferation of adrenergic receptor subtypes. (From ref. 40; © 1988, ith permission from Elsevier.) A cartoon from 1988 indicating the frustration some investigators felt at the seemingly endless proliferation of adrenergic receptor subtypes.
  19. 19. 19 Adrenoceptors Types of Adrenoceptors
  20. 20. 20 Adrenoceptors Types of Adrenoceptors
  21. 21. 21 Adrenoceptors Ligands of Adrenoceptors
  22. 22. 22 Adrenoceptors Binding pocket of AdrenoceptorsFinch, Sarramegna, and Graham nergic Receptor Ligand-Binding Sites ding Contacts of the Endogenous Ligands early 1930s, Easson and Stedman proposed that receptor binding of a dpossessingachiralcenterinvolvedinteractionsbetweenthreecontact the receptor and three moieties of the ligand (1). On the basis of ntal data on the activity of the enantiomers of epinephrine, they pro- t epinephrine’s triad consisted of the basic group (the amide), the aro- g with its hydroxyl groups, and the alcoholic chiral, β-carbon hydroxyl (Fig. 1). The importance of these three chemical groups and their n with the adrenergic receptors (ARs) has been borne out by numerous Chemical structure of (–)-epinephrine. Individual moieties, including the para-hydroxyls, catechol ring, protonated amine, alcoholic chiral β-carbon and N-methyl group are indicated. (–)-epinephrine Ligand Binding, Activation, and Agonist Trafficking 27 Table 1 Binding Contacts of Adrenergic Receptors With Endogenous Ligands a,b Moieties of Endogenous Catecholamine Ligands para- meta- Catechol β-Carbon Receptor Amine Hydroxyl Hydroxyl Ring Hydroxyl N-methyl α1A D3.32 S5.46 S5.42 F4.62, F5.41 α1B D3.32 S5.42 S5.42 F6.51 α2A D3.32 S5.46 — F6.52, Y6.55 D3.32, F7.38, S2.61, F7.39 S7.46 β2 D3.32 S5.46 S5.42, S5.43 F6.51, F6.52 N6.55, D3.32, T4.56 a Interaction demonstrated experimentally. b Residues in italics are those that have been proposed to interact based only on molecular modeling studies. Binding Contacts of Adrenergic Receptors With Endogenous Ligands a,b gand Binding, Activation, and Agonist Trafficking 27 Table 1 Binding Contacts of Adrenergic Receptors With Endogenous Ligands a,b Moieties of Endogenous Catecholamine Ligands para- meta- Catechol β-Carbon eceptor Amine Hydroxyl Hydroxyl Ring Hydroxyl N-methyl A D3.32 S5.46 S5.42 F4.62, F5.41 B D3.32 S5.42 S5.42 F6.51 A D3.32 S5.46 — F6.52, Y6.55 D3.32, F7.38, S2.61, F7.39 S7.46 D3.32 S5.46 S5.42, S5.43 F6.51, F6.52 N6.55, D3.32, T4.56 a Interaction demonstrated experimentally. b Residues in italics are those that have been proposed to interact based only on olecular modeling studies. Counterclockwise orientation of 7TM is not yet available for GPCRs. A high-resolution structure of the light-driven proton pump from Halobacterium halobium, bacteriorhodopsin, has been available for several years (30). Since bacteriorhodopsin, similar to the GPCRs, possesses seven-transmembrane ␣-helices and uses retinal as its chro- mophore, it has been considered a bacterial homolog of ver- tebrate rhodopsin. The bacteriorhodopsin structure has ac- cordingly been widely used as a template for tertiary structure models of GPCRs (31–35). However, bacteriorho- dopsin is a proton pump, is not linked to a G protein, and does not even display remote sequence homology with any GPCR. Moreover, the structural information that recently has become available for rhodopsin indicated clear differences between bacteriorhodopsin and rhodopsin (30, 36–39). Over- all, the use of bacteriorhodopsin as a template for molecular models should now be considered obsolete. Using electron cryomicroscopy of two-dimensional crys- tals, Schertler and co-workers (36–39) have succeeded in obtaining low-resolution structures of both bovine and frog rhodopsin. In addition, a low-resolution structure of squid February, 2000 ACTIVATION OF G PROTEIN-COUPLED RECEPTORS 93 The Adrenergic Receptors in the 21st Century Gather U. (2000)
  23. 23. 23 Adrenoceptors Binding pocket of Adrenoceptors seven-transmembrane ␣-helices and uses retinal as its chro- mophore, it has been considered a bacterial homolog of ver- tebrate rhodopsin. The bacteriorhodopsin structure has ac- cordingly been widely used as a template for tertiary structure models of GPCRs (31–35). However, bacteriorho- dopsin is a proton pump, is not linked to a G protein, and does not even display remote sequence homology with any GPCR. Moreover, the structural information that recently has become available for rhodopsin indicated clear differences between bacteriorhodopsin and rhodopsin (30, 36–39). Over- all, the use of bacteriorhodopsin as a template for molecular models should now be considered obsolete. Using electron cryomicroscopy of two-dimensional crys- tals, Schertler and co-workers (36–39) have succeeded in obtaining low-resolution structures of both bovine and frog rhodopsin. In addition, a low-resolution structure of squid rhodopsin has become available (40). The first projection map of bovine rhodopsin at 9 Å resolution provided the first direct insight into how the predicted seven helices are or- ganized relative to one another in the tertiary structure of the receptor (36). Importantly, a very similar arrangement of the transmembrane helices was found in the projection maps of frog and squid rhodopsin at 7 Å and 8 Å resolutions, re- spectively (38, 40). The projection maps are characterized by an arc-shaped feature, which has been interpreted as reflect- ing the presence of three tilted helices (36, 38, 40). Four additional peaks were interpreted as the remaining four transmembrane helices (36, 38, 40). The structural informa- tion achieved from aligning multiple receptor sequences per- mitted assignment of the individual peaks in the projection maps to the individual helices in the receptor (25, 41). As shown in Fig. 2, it is believed that the helices are organized sequentially in a counterclockwise fashion as seen from the extracellular side, with helix 3 being almost in the center of the molecule. Further insight into the packing of the seven- helix bundle and calculation of the tilting angles of the helices Yellow - conserved residues Human β2- Adrenergic receptor FIG. 3. Comparison of ligand-binding domains in a prototype small-molecule family A receptor (the ␤2- February, 2000 ACTIVATION OF G PROTEIN-COUPLED RECEPTORS FIG. 3. Comparison of ligand-binding domains in a prototype small-molecule family A receptor (the ␤2 February, 2000 ACTIVATION OF G PROTEIN-COUPLED RECEPTORS Green - for agonist Red - for antagonist
  24. 24. 24 Adrenoceptors Binding pocket of Adrenoceptors ng binding sites be- . s on receptor nformational changes c receptor have pro- formational changes wever, the data also molecular modes of discussed in Section onal changes in both tor suggest that sim- vation of both recep- fferences underlying th the ␤2-adrenergic s ligand, cis-retinal, is inverse agonist and s to an agonist (trans- FIG. 5. Sequential binding and conformational stabilization model for the molecular mechanisms of ligand action in GPCRs. The hypo- VATION OF G PROTEIN-COUPLED RECEPTORS 105 Gather U. (2000)
  25. 25. 25 Adrenoceptors Binding pocket of Epinephrine S7.46 D3.32 S5.46 S5.42, S5.43 F6.51, F6.52 N6.55, D3.32, T4.56 Interaction demonstrated experimentally. Residues in italics are those that have been proposed to interact based only on ecular modeling studies. Fig. 2. Major interactions between (–)-epinephrine and its β-adrenergic receptor bind- site: (a) ionic interaction between the amino group of epinephrine and the carboxylate Ligand binding (H-bond) Asp113 (TM3) Ser204, Ser207 (TM5) Asn293 (TM6) Receptor activation (Hydrophobic interaction) Phe290 (TM6)
  26. 26. Long-sought image of activated GPCR β-adrenergic receptor 1) just before being activated 2) at the moment of activation
  27. 27. β1 and β2-adrenergic receptor structures interested in researchers Inactive Active 2007 2008 2011 2011 β2AR inactive form β1AR inactive form β1AR active form β2AR active form Rasmussen et al. (2007) Warne et al. (2008) Warne et al. (2011) Rasmussen et al. (2011) Timeline discovery of β1 and β2-adrenergic receptor structures
  28. 28. 28 Adrenoceptorsβ2AR inactive form 2007 tion Facility, Grenoble) or moderately focused and then further colli- mated (23ID-B GM/CA-CAT beamline, Advanced Photon Source) to diameters between 5 and 10mm. The initial images from the best crystals showed diffraction to 3.0 A˚ ; however, resolution was rapidly lost in sequential images from the same crystal volume. Nevertheless, we obtained a complete data set from a single crystal, and determined the structure by molecular replacement using immunoglobulin- domain search models for the Fab. The diffraction is anisotropic, with diffraction extending to 3.4 A˚ in the plane of the membrane and 3.7 A˚ perpendicular to the plane of the membrane. Structure of the b2AR–Fab5 complex Figure 2a shows the packing of the b2AR365–Fab5 complex in the crystals.Thecrystals seem to be formed from stacks of two-dimensional receptor, the electron density is uninterpretable in the extracellular domain (Supplementary Fig. 2), even though this region of two receptor molecules packs together in a head-to-head manner around the crystallographic two-fold axis. The poor packing in this interface probably explains the significant anisotropy and poor overall reso- lution of the crystals. In an effort to improve the packing of the extracellular domains, we further modified b2AR365 by inserting a TEV cleavage site after amino acid 24 (b2AR24/365, Fig. 1). However, crystals of this construct are isomorphous to those made with b2AR365, and the structure (Supplementary Table 1) is virtually identical to that obtained from b2AR365–Fab5. As expected, the overall structure of the b2AR (Fig. 2b) is similar to rhodopsin, with seven transmembrane helices and an eighth helix that runs parallel to the cytoplasmic face of the membrane. Several of P D H D V T A H MGQPN GS GF AL LAPNRS Q Q R D E V W V V G GM I V M S L I V L A I V F G N V L V I T A I L HI A A G F P V V A L G M V L D A C A L S T I A K F E R L Q T V TF Y N M K M W T F G N F W C E WF T S I D V L C V T A S I E T L C V I V D R Y F A I T S P F K Y Q S L L T KNKA Y HW M Q I P L F S T L G S V I W V M L I V RI T A R E Q H N I A C Y A E E T C C D F F T N Q YA A I A S S I V S F V V P L V I M V F Y SV I HV V I N V I F F P L W C L T F T G M I G L E YV I L L N W I G Y V N S G F N L I Y R S Q D N L I R K I C R V F Q E A K R Q L Q K T K L A K H E K L C F K S P D F R I A F Q E L L C L R R S S R R L T G H G I D K Q D G R G E S Q V EH F R NQV L S S L G Y A K Y G N G N S SE G T NG S Q P V H YEQEK E N K L L C E D L P G T E FD V G H QQ GG TS P VD I N DR G Q ST S NL L S D N C 365 24 30 60 100 190 A 140 240 270 300 330 350 390 Extracellular TM1 TM2 TM3 TM4 TM5 TM6 TM7 Intracellular Loop 1 Loop 2 Loop 3 Loop 1 Loop 2 Loop 3 Figure 1 | Schematic diagram of the b2AR. Black circles with white letters indicate disordered residues not included in the model. Grey letters and circles indicate residues not included in the b2AR365 construct used for crystallography. Red letters indicate amino acids for which side-chain electron density was not modelled. Yellow residues indicate amino acids implicated in ligand binding from mutagenesis studies. Orange residues indicate the conserved DRY sequence. Green residues form the Fab5 epitope, and pink residues are packed against the Fab5 constant domain in the lattice. Small blue circles indicate glycosylation sites. Red lines indicate ten-amino-acid increments. 384 Nature©2007 Publishing Group Crystallize the agonist- bound β-adrenergic receptors without significant modification is impossible because the receptors change shapes rapidly Need to reduce wiggly-ness
  29. 29. 29 Adrenoceptorsβ2AR inactive form 2007 Crystallize the agonist- bound β-adrenergic receptors without significant modification is impossible because the receptors change shapes rapidly Need to reduce wiggly-ness Strategy to obtain crystal of carazolol-bound β2AR-Mab5 - Prepare truncated β2365 & β2AR24/365 - Mutation N178E to remove third glycosylation site - Prepare antibody fragment that bind ICL3 of β2AR (Mab5) - Crystallize carazolol-bound β2AR-Mab5 in DMPC bicelles carazolol (antagonist)
  30. 30. 30 Adrenoceptorsβ2AR inactive form 2007 crystals showed diffraction to 3.0 A˚ ; however, resolution was rapidly lost in sequential images from the same crystal volume. Nevertheless, we obtained a complete data set from a single crystal, and determined the structure by molecular replacement using immunoglobulin- domain search models for the Fab. The diffraction is anisotropic, with diffraction extending to 3.4 A˚ in the plane of the membrane and 3.7 A˚ perpendicular to the plane of the membrane. Structure of the b2AR–Fab5 complex Figure 2a shows the packing of the b2AR365–Fab5 complex in the crystals.Thecrystals seem to be formed from stacks of two-dimensional the crystallographic two-fold axis. The poor packing in this interface probably explains the significant anisotropy and poor overall reso- lution of the crystals. In an effort to improve the packing of the extracellular domains, we further modified b2AR365 by inserting a TEV cleavage site after amino acid 24 (b2AR24/365, Fig. 1). However, crystals of this construct are isomorphous to those made with b2AR365, and the structure (Supplementary Table 1) is virtually identical to that obtained from b2AR365–Fab5. As expected, the overall structure of the b2AR (Fig. 2b) is similar to rhodopsin, with seven transmembrane helices and an eighth helix that runs parallel to the cytoplasmic face of the membrane. Several of P D H D V T A H MGQPN GS GF AL LAPNRS Q Q R D E V W V V G GM I V M S L I V L A I V F G N V L V I T A I L HI A A G F P V V A L G M V L D A C A L S T I A K F E R L Q T V TF Y N M K M W T F G N F W C E WF T S I D V L C V T A S I E T L C V I V D R Y F A I T S P F K Y Q S L L T KNKA Y HW M Q I P L F S T L G S V I W V M L I V RI T A R E Q H N I A C Y A E E T C C D F F T N Q YA A I A S S I V S F V V P L V I M V F Y SV I HV V I N V I F F P L W C L T F T G M I G L E YV I L L N W I G Y V N S G F N L I Y R S Q D N L I R K I C R V F Q E A K R Q L Q K T K L A K H E K L C F K S P D F R I A F Q E L L C L R R S S R R L T G H G I D K Q D G R G E S Q V EH F R NQV L S S L G Y A K Y G N G N S SE G T NG S Q P V H YEQEK E N K L L C E D L P G T E FD V G H QQ GG TS P VD I N DR G Q ST S NL L S D N C 365 24 30 60 100 190 A 140 240 270 300 330 350 390 Extracellular TM1 TM2 TM3 TM4 TM5 TM6 TM7 Intracellular Loop 1 Loop 2 Loop 3 Loop 1 Loop 2 Loop 3 Figure 1 | Schematic diagram of the b2AR. Black circles with white letters indicate disordered residues not included in the model. Grey letters and circles indicate residues not included in the b2AR365 construct used for crystallography. Red letters indicate amino acids for which side-chain electron density was not modelled. Yellow residues indicate amino acids implicated in ligand binding from mutagenesis studies. Orange residues indicate the conserved DRY sequence. Green residues form the Fab5 epitope, and pink residues are packed against the Fab5 constant domain in the lattice. Small blue circles indicate glycosylation sites. Red lines indicate ten-amino-acid increments. 384 Black circle: disordered residues not included in the model Grey letter: residues not included in the β2AR365 construct used for crystallography Red letter: amino acids for which side chain electron density was not modelled Yellow circle: amino acids implicated in ligand binding from mutagenesis studies Orange circle: conserved DRY sequence Green circle: residues form the Fab5 epitope Pink circle: residues are packed against the Fab5 constant domain in the lattice N- and C-terminal ends of ICL3 are involved in G-protein activation and selectivity of GPCR-G-protein interaction
  31. 31. 31 Adrenoceptorsβ2AR inactive form 2007 Figure S2. Weak electron density in the extracellular region of the β2AR. The final 2Fo-F map (grey mesh, contoured at 0.7σ) around two receptor molecules packed across the crystallographic twofold (b) axis (horizontal line). The view is the same as the left panel Fig. 2a. Fab provide conformational stability (by binding to ICL3) and increase polar available for crystal contact
  32. 32. 32 Adrenoceptorsβ2AR inactive form 2007 b2AR, which is in agreement with the observation that Fab5 binds to native, but not denatured b2AR protein28 . Additional lattice con- tacts occur between the constant domain of a symmetry-related Fab5 molecule and the second intracellular loop of b2AR (shown in magenta in Fig. 2b). dete F b2A The the t men open ends prov has trast tivel (Sup func b2A (E/D bers E134 bon inte ioni a b 90º
  33. 33. 33 Adrenoceptorsβ2AR inactive form 2007 (Sup funct b2AR (E/D bers) E134 bond intera ionic muta tors l ical s relati How b2AR boun The s simil which dops becau ident of TM ling, b TM5 TM6 TM7 TM1 TM2 TM3 TM5 TM6 TM7 TM1 TM2 TM3 TM4 N OV114 90º 90º c Ligand binding Sites of interaction with Fab5-epitope (with ICL3)
  34. 34. 34 Adrenoceptorsβ2AR inactive form 2007 TM5 TM6 TM5 TM6 TM3 H N OH H O N Carazolol F289 F290 N312 D113 V114 c TM3 TM6 Figure 2 | Structure of the b2AR365–Fab5 complex. a, Packing of the b2AR365–Fab5 complex in crystals formed in DMPC bicelles (b2AR, gold; heavy chain, blue; light chain, red). b, Structure of the b2AR showing sites of the interactions with Fab5. Sites of specific (idiotypic) interactions between
  35. 35. 35 Adrenoceptorsβ2AR inactive form 2007 TM3 TM1 TM4 TM6 TM5 TM2 TM7 TM3 TM4 TM5 TM1 TM6 TM2 TM7 TM5 TM1 Retinal D R Y E R Y β2AR Rhodopsin β2AR Inactive rhodopsin a b Light-activated rhodopsin Figure 3 | Comparis structures. a, The the homologous str is shown in purple a putative ligand-bin mesh. Structures w transmembrane seg represent cross-sect around the horizon extracellular face of of the b2AR with str and light-activated conserved E/DRY s line shows the dista arginine in TM3 an facilitate compariso structures were alig only. ARTICLES NATURE
  36. 36. 36 Adrenoceptorsβ2AR inactive form 2007 disruption of the ionic lock in rhodopsin. It is likely that this muta- tion would produce a more loosely packed, dynamic structure in this region, shifting the equilibrium towards a more active state. It is interesting that pack while the ionic lock inter either E268 or L272 leads both are involved in maint the current structure, we lock and the tight packing even be structurally incom L272 interactions stabilize TM5 TM6 TM7 R E R Y β2AR Inactive rhodopsin b Light-activated rhodopsin TM6 TM3 TM2 E268D130 R131 TM6TM3 TM2 E247 R135 E134 2.9 Å6.2 Å TM6TM3 TM2 E247 R135 E134 4.1 Å Ionic lock relates to inactive conformation Movement of cytoplasmic end of TM3 (E/DRY) relative to TM6 involves in activation of both β2AR and rhodopsin more-open (with carazolol bound)
  37. 37. 37 Adrenoceptorsβ1AR inactive form 2008 Crystallize the agonist- bound β-adrenergic receptors without significant modification is impossible because the receptors change shapes rapidly Need to reduce wiggly-ness Strategy to obtain crystal of cyanopindolol-bound β1AR - Prepare mutated turkey receptor, β1AR-m23 (enhanced thermostability and prefer adopting antagonist state) - Purified in othylthioglucoside and in the presence of cyanopindolol - Crystallize cyanopindolol-bound β1AR-m23 cyanopindolol (antagonist)
  38. 38. 38 Adrenoceptorsβ1AR inactive form 2008 water molecules, 4 Na1 ions and 14 detergent molecules (see Supplementary Information). Unless otherwise stated, all further dis- cussion refers to molecule B, because this molecule has an unkinked H1 and a relatively well-ordered H8. The helix boundaries, disordered regions and overall structural motifs are presented in Fig. 1. The amino acid sequence of turkey b1AR19 is 82% and 67% iden- tical to human b1AR and human b2AR, respectively, over residues Trp401.31 –Asp2425.73 and Glu2856.30 –Cys358H8-Cterm (that is, excluding the N and C termini and most of CL3); it is therefore expected that the structure of the transmembrane regions of b1AR structure of the entrance to the ligand-binding pocket. The large difference in EL2 conformation between the a-helix found in b2AR and the b-hairpin that closes off the retinal-binding site in rhodopsin is confirmed in the structure of b1AR, suggesting that the a-helix may be a common feature in those GPCRs that bind their ligands rapidly and reversibly. Cytoplasmic loop structure In all GPCRs, CL2 and CL3 are believed to have an important role in the binding, selectivity and activation of G proteins, CL2 being EL2 N terminus C terminus CL1 CL2 V P F G A T L V V R G P Q A L K C Y Q D GAEL L W Q Q S E A G M S L L M A LV V L L I V A G N V L V I A A I G S T Q R L Q T L N F L I T S L A C A D L V V G L L T W L W G S F L E L W T S L D V L C V T A S I E T L C V I A I D R Y L A I T S M P F R Y Q S L T R A R AK V II C T VW A IS A L V S F L P I M M H W W R D EP G D F V T N R A Y A I A S S I I S K I D R A S K R K R V M L M R E HK A L KT L GI I M GV F TL C W L P F F L V N I V N V F P C R N R D L V CEGRFYGSQE QPQPPPLPQH QPILGNGR M DGWLPPDCGP HNRSGGGGAT AAPTGSRQVS A DRRLHHHHHH AGGQPAPLPGGFISTLGSPEH SPGGTWSDCNGGTRGGSES SLEERHSKTSRSESKMEREKN ILATTRFYCTFLGNGDKAVFC TVLRIVKLFEDATCTCPHTHK LKMKWRFKQHQA C C C 401.31 681.59 1042.67 762.39 1443.55 1113.22 1784.62 1534.37 2055.36 2355.66 3156.60 2856.30 3227.32 3427.52 3568.57 3478.48 L D F R K A F K R L P A F P R K T D Y L L I M I F V LA V Y R E A K E Q I R PI R M N P I I LW F V A F N W L G Y A N S D A S Y V TS F a b Figure 1 | Schematic representations of the turkey b1AR structure. a, Diagram of the turkey b1AR sequence in relation to secondary structure elements. The residues in white circles indicate regions that are well ordered; the sequences in grey circles were not resolved in the structure. The sequences on an orange background were deleted to make the b1AR construct for expression. Thermostabilizing mutations are in red circles and two other mutations—C116L (increases functional expression) and C358A (eliminates palmitoylation site)—are in blue circles. The Na1 ion is in (blue boxes), with the Ballesteros–Weinstein numbering in superscript. Helices were defined using the Kabsch and Sander algorithm49 , with helix distortions being defined as residues that have main chain torsion angles that differ by more than 40u from standard a-helix values (260u,240u). b, Ribbon representation of the b1AR structure in rainbow colouration (N terminus, blue; C terminus, red), with the Na1 ion in pink, the two near-by disulphide bonds in yellow, and cyanopindolol as a space-filling model. The extracellular loop 2 (EL2) and cytoplasmic loops 1 and 2 (CL1, CL2) are Red circle: Thermostabilizing mutations and C116L (increases functional expression) and C358A (eliminates palmitoylation site) are in blue circles Purple: Na+ ion Grey circle: not resolved in structure Orange boxes: deleted to make the β1AR construct for expression
  39. 39. 39 Adrenoceptorsβ1AR inactive form 2008 0.00 0.25 0.50 0.75 1.00 -9 -8 -7 -6 -5 -4 -3 basal isoprenaline 10μM propranolol 30nM propranolol 300nM propranolol 3000nM adrenaline adrenaline + propranolol 30nM adrenaline + propranolol 300nM adrenaline + propranolol 3000nM log[adrenaline](M) [SPAP](ODunits) 0 1000 2000 3000 4000 5000 6000 -10 -9 -8 -7 -6 -5 -4 basal isoprenaline 10μM 350000 400000 ICI 118551 log[ligand](M) 3 H-cAMPaccumulation (dpm) A B 0.00 0.25 0.50 0.75 1.00 -9 -8 -7 -6 -5 -4 -3 basal isoprenaline 10μM propranolol 30nM propranolol 300nM propranolol 3000nM adrenaline adrenaline + propranolol 30nM adrenaline + propranolol 300nM adrenaline + propranolol 3000nM log[adrenaline](M) [SPAP](ODunits) 0 1000 2000 3000 4000 5000 6000 -10 -9 -8 -7 -6 -5 -4 basal isoprenaline 10μM 350000 400000 ICI 118551 log[ligand](M) 3 H-cAMPaccumulation (dpm) A B (inverse agonist) B A EL2 H3 H2 CYP H6W303 D121 water Na water water H2 Na+ stabilizes helical conformation of EL2 and structure of the entrance to the ligand-binding pocket
  40. 40. 40 Adrenoceptorsβ1AR inactive form 2008 ose GPCRs that bind their ligands rapidly re 3 are believed to have an important role in nd activation of G proteins, CL2 being EL2 N terminus C terminus CL1 CL2 A DRRL GFISTLGSPEH 7.32 3427.52 568.57 LL A F P R K b Roles of CL2 and CL3 in GPCRs - binding of G-protein - selectivity of G-protein - activation of G-protein Role of CL2 involve in strength of interaction Role of CL3 involve in specificity of interaction
  41. 41. 41 Adrenoceptorsβ1AR inactive form 2008 H3 H4 CL2 Y149 α-helix of CL2 interacts with D/ERY in H3 through H-bonding with Y149
  42. 42. 42 Adrenoceptorsβ1AR inactive form 2008 Amino acid residues that interact with the ligand cyanopindolol (yellow) by polar interactions (aquamarine) or non-polar interactions (grey) H7 H2 EL2 H5 H3 T203 S211 S212 S215 Cyanopindolol Adrenaline D 5.0 5.9 4.5 2.8 2.5 b Figure 4 | Comparisons between b receptor ligand-binding p binding of different ligands. a, Superposition of b1AR molecu (PDB code 2RH1, ref. 10) in the region surrounding the ligan Shown are side chains that have different rotamer conforma and S2115.42 ) along with two residues that are conserved yet different between b1 and b2 receptors (F325/Y3087.35 and V1 Cyanopindolol (CYP) is in the ligand-binding pocket of the b carazolol (CAR) is in the b2 receptor. The biggest backbone d at the V172/T1644.56 position. b, Superposition of a model o adrenaline (yellow), with the structure of the antagonist, cya (pink), as it binds to b1AR, showing the distances (in A˚ , red) side chains known to interact with the hydoxyl groups on the the agonist. It is clear that a 2–3 A˚ tightening of the pocket aro b EL2 T203 T118 F201 W117 H3 D121 N329 Y333 W303 F306 F307 N310 S215 S211 A208 V122 H5 H7 H6 F307 W303 H7 F299 Figure 3 | Structure of the ligand-binding pocket. a, 2Fo–Fc map before inclusion of cyanopindolol (CYP) in the model, showing the interaction of CYP with Thr 203 and Phe 201 in EL2. b, Amino acid residues that interact with the ligand cyanopindolol (yellow) by polar interactions (aquamarine)
  43. 43. 43 Adrenoceptorsβ1AR inactive form 2008 Yellow - β1AR inactive form Grey - β2AR inactive form or her or, ds est expected to hydrogen bond with the meta- and para-hydroxyl groups on the catechol ring36–38 . As noticed previously39 , the catechol hydroxyl groups are well spaced and well oriented to interact with the side chain hydroxyl groups of Ser 2115.42 , Ser 2125.43 and Ser 2155.46 on H5, but cannot reach far enough to make good hydro- gen bonds if the amine occupies the same position as it does adjacent to Asp 1213.32 in the cyanopindolol complex, without a substantial structural change in the receptor. It seems very reasonable that the H3 H4H5 H6 H7 H2 β1 V172 β2 T164 β2 Y308 β1 F325 β2 N293 β1 N310 β1 CYP β2 CAR β1 S211 β2 S203 EL2 a b
  44. 44. 44 Adrenoceptorsβ1AR inactive form 2008 Yellow - Adrenaline (agonist) Pink - Cyanopindolol (agonist) H7 H2 β2 Y308 β1 F325 EL2 H5 H3 T203 S211 S212 S215 Cyanopindolol Adrenaline D121 5.0 5.9 4.5 2.8 2.5 b Figure 4 | Comparisons between b receptor ligand-binding pockets and the binding of different ligands. a, Superposition of b1AR molecule B with b2AR
  45. 45. Strategies to stabilise the agonist-bound receptors R R* Agonist Inverse agonist 1. Genetically mutated the receptor at six points, to make it less sensitive to heat 2.“handcuffed”the modified agonist to the binding pocket by chemical bond 3. Stabilised the agonist-bound receptor in an active state by binding to llama antibody that act like as G-protein
  46. 46. 46 Adrenoceptors β1AR active form 2011 cussion refers to molecule B, because this molecule has an unkinked H1 and a relatively well-ordered H8. The helix boundaries, disordered regions and overall structural motifs are presented in Fig. 1. The amino acid sequence of turkey b1AR19 is 82% and 67% iden- tical to human b1AR and human b2AR, respectively, over residues Trp401.31 –Asp2425.73 and Glu2856.30 –Cys358H8-Cterm (that is, excluding the N and C termini and most of CL3); it is therefore expected that the structure of the transmembrane regions of b1AR and the b-hairpin that closes off the retinal-bind is confirmed in the structure of b1AR, suggesting be a common feature in those GPCRs that bind and reversibly. Cytoplasmic loop structure In all GPCRs, CL2 and CL3 are believed to have the binding, selectivity and activation of G p N terminus C terminus V P F G A T L V V R G P Q A L K C Y Q D GAEL L W Q Q S E A G M S L L M A LV V L L I V A G N V L V I A A I G S T Q R L Q T L N F L I T S L A C A D L V V G L L T W L W G S F L E L W T S L D V L C V T A S I E T L C V I A I D R Y L A I T S M P F R Y Q S L T R A R AK V II C T VW A IS A L V S F L P I M M H W W R D EP G D F V T N R A Y A I A S S I I S K I D R A S K R K R V M L M R E HK A L KT L GI I M GV F TL C W L P F F L V N I V N V F P C R N R D L V CEGRFYGSQE QPQPPPLPQH QPILGNGR M DGWLPPDCGP HNRSGGGGAT AAPTGSRQVS A DRRLHHHHHH AGGQPAPLPGGFISTLGSPEH SPGGTWSDCNGGTRGGSES SLEERHSKTSRSESKMEREKN ILATTRFYCTFLGNGDKAVFC TVLRIVKLFEDATCTCPHTHK LKMKWRFKQHQA C C C 401.31 681.59 1042.67 762.39 1443.55 1113.22 1784.62 1534.37 2055.36 2355.66 3156.60 2856.30 3227.32 3427.52 3568.57 3478.48 L D F R K A F K R L P A F P R K T D Y L L I M I F V LA V Y R E A K E Q I R PI R M N P I I LW F V A F N W L G Y A N S D A S Y V TS F a b Figure 1 | Schematic representations of the turkey b1AR structure. a, Diagram of the turkey b1AR sequence in relation to secondary structure elements. The residues in white circles indicate regions that are well ordered; the sequences in grey circles were not resolved in the structure. The sequences on an orange background were deleted to make the b1AR (blue boxes), with the Ballesteros–Weinstein numbe Helices were defined using the Kabsch and Sander a distortions being defined as residues that have main that differ by more than 40u from standard a-helix v b, Ribbon representation of the b1AR structure in ra - Prepare mutated turkey receptor, β1AR-m23 - Co-crystallized β1AR-m23 in the presence of full agonist (carmoterol, isoprenaline) and partial agonist (salbutanol, dobutamine)
  47. 47. 47 Adrenoceptors β1AR active form 2011 Structure of the β1AR bound to agonists (with carmoterol bound in the active state) Cyanopindolol N C H1 H4 EL2 H3 H2 H5EL2 T203 Salbutamol a b d Dobutaminec N329 H7 H5H6 H3 S211 N310 D121 EL2 W330 W H7 H5H6 EL2
  48. 48. 48 Adrenoceptors β1AR active form 2011 inverse agonist Carmoterol N329 H7 H5H6 H3 S211 S215 N310 D121 EL2 N329 H7 H5H6 H3 S211 S215 N310 D121 EL2 Cyanopindolol C H1 H4 EL2 H3 N329 H7 H5 H6 H3 S211 D121 EL2 T203 Salbutamolb d f N329 H7 H5H6 H3 S211 N310 D121 EL2 W330 W N329 H7 H5H6 H3 S211 D121 EL2 of the b1-adrenergic receptor bound to agonists. shown in cartoon representation with the intracellular side figure. The ligand carmoterol is shown as a space filling red; N, blue). The amino terminus (N), carboxy terminus p 2 (EL2), and transmembrane helices 1–4 (H1–H4) are me orientation of receptor is shown in panels b, the dolol; c, d, the partial agonists dobutamine and salbutamol; isoprenaline and carmoterol. The colour scheme of the ligand and labelling of the receptor is identical in all panel side chains that make hydrogen bonds to the ligands depicte N, blue). For clarity, residues 171–196 and 94–119 have be b–f, which correspond to the C-terminal region of H4 and the C-terminal region of H2 and N-terminal region of H3 structures shown are of monomer B (Supplementary Fig. 2) using Pymol (DeLano Scientific). For a comparison of the ligands when bound to the receptor, see Supplementary Fi Isoprenaline Carmoterol N329 H7 H5H6 H3 S211 S215 N310 D121 EL2 N329 H7 H5H6 H3 S211 S215 N310 D121 EL2 Cyanopindolol N C H1 H4 EL2 H3 H2 N329 H7 H5 H6 H3 S211 D121 EL2 T203 Salbutamol a b d e f Dobutaminec N329 H7 H5H6 H3 S211 N310 D121 EL2 W330 W N329 H7 H5H6 H3 S211 D121 EL2 f the b1-adrenergic receptor bound to agonists. own in cartoon representation with the intracellular side ligand and labelling of the receptor is identical in all pane side chains that make hydrogen bonds to the ligands depic TER partial agonist full agonist Isoprenaline Carmoterol N329 H7 H5H6 H3 S211 S215 N310 D121 EL2 N329 H7 H5H6 H3 S211 S215 N310 D121 EL2 Cyanopindolol N C H1 H4 EL2 H3 H2 N329 H7 H5 H6 H3 S211 D121 EL2 T203 Salbutamol a b d e f Dobutaminec N329 H7 H5H6 H3 S211 N310 D121 EL2 W330 W N329 H7 H5H6 H3 S211 D121 EL2 the b1-adrenergic receptor bound to agonists. own in cartoon representation with the intracellular side ligand and labelling of the receptor is identical in all panels, side chains that make hydrogen bonds to the ligands depicted ER
  49. 49. 49 Adrenoceptors β1AR active form 2011 partial agonist H3 S215 H3 D121 H3 Figure 1 | Structure of the b1-adrenergic receptor bound to agonists. a, Structure of b1AR shown in cartoon representation with the intracellular side at the bottom of the figure. The ligand carmoterol is shown as a space filling model (C, yellow; O, red; N, blue). The amino terminus (N), carboxy terminus (C), extracellular loop 2 (EL2), and transmembrane helices 1–4 (H1–H4) are labelled. b–f, The same orientation of receptor is shown in panels b, the antagonist cyanopindolol; c, d, the partial agonists dobutamine and salbutamol; e, f, the full agonists isoprenaline and carmoterol. The colour scheme of the ligand and labelling of the receptor is identical in all panels, with amino acid side chains that make hydrogen bonds to the ligands depicted (C, green; O, red; N, blue). For clarity, residues 171–196 and 94–119 have been removed in b–f, which correspond to the C-terminal region of H4 and EL2, and EL1 with the C-terminal region of H2 and N-terminal region of H3, respectively. All structures shown are of monomer B (Supplementary Fig. 2) and were generated using Pymol (DeLano Scientific). For a comparison of the positions of the ligands when bound to the receptor, see Supplementary Fig. 5. Carmoterol Isoprenaline Phe 306 Phe 307 Val 122 Asp 121 Asn 310 Ser 211 Ser 215 Asn 329 Trp 117 Tyr 333 Val 125 OHH3 C CH3 OH H OH N B* B* B* a* Salbutamol Val 122 HO OH OH H N CH3 H3 C Val 125 Asp 121 Asn 329 Asn 310 Ser 211 Ser 215 Phe 306 Tyr 333 Thr 118 Trp 117 Phe 201 Phe 307 A* H3 C Tyr 207 B* B* b* Dobutamine Val 122 OH HO OH CH3 H N Val 125 Phe 307 Ser 211 Ser 215 W Asn 310 Phe 306 Phe 201 Asp 121 Asn 329 Leu 101 Trp 117 Tyr 333 Val 102 Gly 98 Thr 118 β3: Ala 94 β3: Ala 98 β2: Ile 94 β2: His 93 β1: Ile 118 <B*> Trp 330 B* [B*] B* Ser 212 <B*> Trp 330 A*a b c d Phe 307 Val 122Val 125 O O OH CH3 H OH H N N Phe 201 Cys 199 Tyr 333 Ala 208 Trp 117 Asn 310 Ser 215 Phe 306 Asp 121 Asn 329 Asp 200 β3: Ala 197 β3: Val 205 Ser 211 H3C Thr 203 Thr 118 b* W A* A* B* A* B* b* a* b* Figure 2 | Polar and non-polar interactions involved in agonist binding to b1-adrenergic receptor. a–d, Amino acid residues within 3.9A˚ of the ligands are depicted, with residues highlighted in blue making van der Waals contacts (blue rays) and residues highlighted in red making potential hydrogen bonds found only in monomer B of dob92, whereas another contact, labelled [B*], is found only in monomer B of dob102 (Supplementary Fig. 6 and also see Supplementary Table 6 for further details and for the Ballesteros–Weinstein numbering). If specific van der Waals interactions or polar interactions are full agonist Figure 1 | Structure of the b1-adrenergic receptor bound to agonists. a, Structure of b1AR shown in cartoon representation with the intracellular side at the bottom of the figure. The ligand carmoterol is shown as a space filling model (C, yellow; O, red; N, blue). The amino terminus (N), carboxy terminus (C), extracellular loop 2 (EL2), and transmembrane helices 1–4 (H1–H4) are labelled. b–f, The same orientation of receptor is shown in panels b, the antagonist cyanopindolol; c, d, the partial agonists dobutamine and salbutamol; e, f, the full agonists isoprenaline and carmoterol. The colour scheme of the ligand and labelling of the receptor is identical in all panels, with amino acid side chains that make hydrogen bonds to the ligands depicted (C, green; O, red; N, blue). For clarity, residues 171–196 and 94–119 have been removed in b–f, which correspond to the C-terminal region of H4 and EL2, and EL1 with the C-terminal region of H2 and N-terminal region of H3, respectively. All structures shown are of monomer B (Supplementary Fig. 2) and were generated using Pymol (DeLano Scientific). For a comparison of the positions of the ligands when bound to the receptor, see Supplementary Fig. 5. Carmoterol Isoprenaline Phe 306 Phe 307 Val 122 Asp 121 Asn 310 Ser 211 Ser 215 Asn 329 Trp 117 Tyr 333 Val 125 OHH3 C CH3 OH H OH N B* B* B* a* Salbutamol Val 122 HO OH OH H N CH3 H3 C Val 125 Asp 121 Asn 329 Asn 310 Ser 211 Ser 215 Phe 306 Tyr 333 Thr 118 Trp 117 Phe 201 Phe 307 A* H3 C Tyr 207 B* B* b* Dobutamine Val 122 OH HO OH CH3 H N Val 125 Phe 307 Ser 211 Ser 215 W Asn 310 Phe 306 Phe 201 Asp 121 Asn 329 Leu 101 Trp 117 Tyr 333 Val 102 Gly 98 Thr 118 β3: Ala 94 β3: Ala 98 β2: Ile 94 β2: His 93 β1: Ile 118 <B*> Trp 330 B* [B*] B* Ser 212 <B*> Trp 330 A*a b c d Phe 307 Val 122Val 125 O O OH CH3 H OH H N N Phe 201 Cys 199 Tyr 333 Ala 208 Trp 117 Asn 310 Ser 215 Phe 306 Asp 121 Asn 329 Asp 200 β3: Ala 197 β3: Val 205 Ser 211 H3C Thr 203 Thr 118 b* W A* A* B* A* B* b* a* b* Figure 2 | Polar and non-polar interactions involved in agonist binding to b1-adrenergic receptor. a–d, Amino acid residues within 3.9A˚ of the ligands are depicted, with residues highlighted in blue making van der Waals contacts (blue rays) and residues highlighted in red making potential hydrogen bonds found only in monomer B of dob92, whereas another contact, labelled [B*], is found only in monomer B of dob102 (Supplementary Fig. 6 and also see Supplementary Table 6 for further details and for the Ballesteros–Weinstein numbering). If specific van der Waals interactions or polar interactions are Catecholamine meta- hydroxy form a hydrogen bond with Asn310 (H6) All agonist forms hydrogen bond with Ser211 (H5) Only full agonist forms hydrogen bond with Ser212 (H5)
  50. 50. 50 Adrenoceptors β1AR active form 2011 Change in rotamer conformation o side chain Ser212 and Ser215 in H5 combined effects of strengthening the H5–H6 interface and weakening the H4–H5 interface could facilitate the subsequent movements of H5 and H6, as observed in the activation of rhodopsin. Stabilization of the contracted catecholamine binding pocket is prob- ably the most important role of bound agonists in the activation process (Fig. 4). This probably requires strong hydrogen bonding interactions between the catechol (or equivalent) moiety and both H5 and H6, and strong interactions between the secondary amine and b-hydroxyl groups in the agonist and the amino acid side chains in helices H3 andH7.Reductioninthestrengthoftheseinteractionsislikelytoreduce the efficacy of a ligand29 . Both salbutamol and dobutamine are partial agonists of b1AR-m23 (Supplementary Table 3) and human b1AR. In the case of salbutamol, there are only two predicted hydrogen bonds between the headgroup and H5/H6, compared to three–four potential hydrogen bonds for isoprenaline and carmoterol. Dobutamine lacks the b-hydroxyl group, which similarly reduces the number of potential hydrogen bonds to H3/H7 from three–four seen in the other agonists toonlytwo.Weproposethatthisweakeningofagonistinteractionswith H5/H6 for salbutamol and H3/H7 for dobutamine is a major contri- buting factor in making these ligands partial agonists rather than full agonists. Theagonist-bound structuresofb1ARindicatetherearethree major determinants that dictate the efficacy of any ligand: ligand-induced rotamer conformational changes of (1) Ser 2125.43 and (2) Ser 2155.46 and (3) stabilization of the contracted ligand-binding pocket. The full agonistsstudiedhere achieve allthree.Thepartial agonistsstudied here S212 N310 D121 N329 Y333 S211 S215 F325 H7 H6 H5 H3 c S212 S215 N310 D121 N329 Y333 S211 F325 H7H6 H5 H3 b Y308 N293 N312 D113 S204 Y316 H7 H6 H5 H3 Figure 3 | Comparison of the ligand-binding pockets of the b1 and b2 adrenergic receptors. The ligand-binding pockets are shown as viewed from the extracellular surface with EL2removed for clarity (same colourscheme as in Fig. 1). a, b AR with the antagonist carazolol bound (PDB code 2RH1); b, b AR H7 H5 S211 S215 N329 EL2 Figure 4 | Differencesin theligand-bindingpocketbetween antagonist-and agonist-bound b1-adrenergic receptor. An alignment was performed (see Methods) between the structures of b1AR-m23 bound to either cyanopindolol (grey) or isoprenaline (orange) and the relative positions of the ligands and the transmembrane helices H5 and H7 are depicted. The 1 A˚ contraction of the ligand-binding pocket between H5 and H7 is clear. combined effects of strengthening the H5–H6 interface and weakening the H4–H5 interface could facilitate the subsequent movements of H5 and H6, as observed in the activation of rhodopsin. Stabilization of the contracted catecholamine binding pocket is prob- ably the most important role of bound agonists in the activation process (Fig. 4). This probably requires strong hydrogen bonding interactions between the catechol (or equivalent) moiety and both H5 and H6, and strong interactions between the secondary amine and b-hydroxyl groups in the agonist and the amino acid side chains in helices H3 andH7.Reductioninthestrengthoftheseinteractionsislikelytoreduce the efficacy of a ligand29 . Both salbutamol and dobutamine are partial agonists of b1AR-m23 (Supplementary Table 3) and human b1AR. In the case of salbutamol, there are only two predicted hydrogen bonds between the headgroup and H5/H6, compared to three–four potential hydrogen bonds for isoprenaline and carmoterol. Dobutamine lacks the b-hydroxyl group, which similarly reduces the number of potential hydrogen bonds to H3/H7 from three–four seen in the other agonists toonlytwo.Weproposethatthisweakeningofagonistinteractionswith H5/H6 for salbutamol and H3/H7 for dobutamine is a major contri- buting factor in making these ligands partial agonists rather than full agonists. Theagonist-bound structuresofb1ARindicatetherearethree major determinants that dictate the efficacy of any ligand: ligand-induced rotamer conformational changes of (1) Ser 2125.43 and (2) Ser 2155.46 S212 N310 D121 N329 Y333 S211 S215 F325 H7 H6 H5 H3 c S212 S215 N310 D121 N329 Y333 S211 F325 H7H6 H5 H3 b Y308 N293 N312 D113 S204 Y316 H7 H6 H5 H3 Figure 3 | Comparison of the ligand-binding pockets of the b1 and b2 adrenergic receptors. The ligand-binding pockets are shown as viewed from H7 H5 S211 S215 N329 EL2 Figure 4 | Differencesin theligand-bindingpocketbetween antagonist-and agonist-bound b1-adrenergic receptor. An alignment was performed (see Methods) between the structures of b1AR-m23 bound to either cyanopindolol (grey) or isoprenaline (orange) and the relative positions of the ligands and the transmembrane helices H5 and H7 are depicted. The 1 A˚ contraction of the ligand-binding pocket between H5 and H7 is clear. cyanopindolol (inverse agonist) isoprenaline (full agonist) S215 D121 Y333 S211 H3 b Y308 N293 N312 D113 S204 Y316 S207 S203 H7 H6 H5 H3 a H7 H5 S211 S215 N329 EL2 Figure 4 | Differencesin theligand-bindingpocketbetween agonist-bound b1-adrenergic receptor. An alignment was Methods) between the structures of b1AR-m23 bound to eith (grey) or isoprenaline (orange) and the relative positions of th transmembrane helices H5 and H7 are depicted. The 1 A˚ co ligand-binding pocket between H5 and H7 is clear. LETTER Contraction of the catecholamine binding pocket by ~1Å between Cα atom of Asn329 (H7) and Ser211 (H5) Gold - Full agonist Gray - Antagonist
  51. 51. CHALLENGE Agonist are much less efficient at stabilizing the active state of β2AR, making its difficult to capture this state in a crystal structure. BI-167107 (agonist) Receptor (β2 adrenergic receptor) Stimulatory G protein (GS) GTP Instability β2AR active form Adrenoceptors 2011 51
  52. 52. 52 BI-167107 (agonist) Receptor (β2 adrenergic receptor) T4 lysozyme (T4L) Nanobody (Nb80) STRATEGY Develop a binding protein that preferentially binds to and stabilises an active conformation, acting as a surrogate for Gs (Nanobody, Nb80). Adrenoceptors β2AR active form 2011 52
  53. 53. 53 Adrenoceptors β2AR active form 2011 Nanobody: Antibody fragment consisting of a single monomeric variable antibody domain that secreted by a type of white blood cell called a plasma cell 150 kDa Camelidae Antibody 14 kDaFab region Fc region Heavy chain Light chain 53
  54. 54. 54 Adrenoceptors β2AR active form 2011 BI-167107 (agonist) Receptor (β2 adrenergic receptor) from Homo sapiens T4 lysozyme (T4L) from Enterobacteria phage T4 Nanobody (Nb80) from Lama glama High-density lipoprotein (HDL) 54 Adrenoceptors the b2AR-T4L, there are subtle differences in the baseline spectrum of the bimane-labeled fusion protein, as might be expected if the environment around Cys2656.27 is altered by T4L. However, the full agonist isoproterenol induces a qualita- tively similar decrease in intensity and rightward shift in lmax. Thus, the presence of the fused T4L does not prevent agonist-induced confor- mational changes. The partial agonist salbuta- mol induced larger responses in b2AR-T4L than were observed in WT b2AR, and there was a small increase in fluorescence in response to the inverse agonist ICI-118,551. These properties are observed in CAMs (15, 22) and are consistent with the higher affinities for agonists and partial b2AR crystallization, in that both strategies rely on attachment (covalent or noncovalent, respec- tively) of a soluble protein partner between helices V and VI. A major difference between the two structures is that the extracellular loops and the carazolol ligand could not be modeled in the b2AR-Fab complex, whereas these regions are resolved in the structure of b2AR-T4L. None- theless, it is clear that the T4L insertion does not substantially alter the receptor. Superposition of the two structures (fig. S4) illustrates that the trans- membrane helices of the receptor components are very similar (root mean square deviation = 0.8 Å for 154 common modeled transmembrane Ca positions versus 2.3 Å between b2AR-T4L b2AR-T4L could contribute to the higher agonist binding affinity characteristic of a CAM. An unexpected difference between the struc- ture of rhodopsin and the b2AR-T4L involves the sequence E/DRY (24) found at the cytoplasmic end of helix III in 71% of class A GPCRs. In rhodopsin, Glu1343.49 and Arg1353.50 form a network of hydrogen bond and ionic interac- tions with Glu2476.30 at the cytoplasmic end of helix VI. These interactions have been referred to as an “ionic lock” that stabilizes the inactive state of rhodopsin and other class A members (25). However, the arrangement of the homolo- gous residues is considerably different in b2AR- T4L: Arg1313.50 interacts primarily with Asp1303.49 C D F F T N Q YA A I A S S I V S F V V P L V I M V F D Q Y SV R V F Q E A K R Q L Q K I Y G N GN S S EGTN G SQ V H Y EQQEKKENKK L L C E D L P G T E FD V G H QQ GG TS P VD I N D R GG QQ S TS N LLSDDN C 365 190 230 400 Extracellular Helix V Intracellular I HV V I N V I F F P L W C L T F T G M I G L E YV I L L N W I G Y V N S G F N L I Y R S Q D N L I R K I C T K L A K H E K L C F K S P D R L T G H GG D G R F R I A F Q E L L C L R R S S R S L G Y A K P 260 300 330 350 Helix VI Helix VII ECL3 ILC3 N A D W T L N K Y A B C D E 1 2 3 4 5 T4-Lysozyme C ECL2 BA Positive Control: FLAG-β1AR D3 C3 D5 D1 Negative Control: pCDNA3 β2AR-T4L M1 M1+DAPI Fig. 1. Design and optimization of the b2AR-T4L fusion protein. (A) The sequence of the region of the b2AR targeted for insertion of a crystallizable domain is shown, and the positions of the junctions between the receptor and T4L (red) for various constructs are indicated. The sequences that were initially replaced or removed are faded. Red lines are shown after every tenth residue. ECL, extracellular loop. (B) Immunofluorescence images of HEK293 cells expressing selected fusion constructs. (Left) M1 anti-FLAG signal corre- sponding to antibody bound to the N terminus of the receptor. (Right) Same signal merged with blue emission from 4´,6´-diamidino-2-phenylindole (nuclear staining for all cells). Plasma membrane staining is observed in the positive control, D3, and D1, whereas C3 and D5 are retained in the endoplasmic reticulum. Rosenbaum, D.M. et al.(2004)
  55. 55. 55 Adrenoceptors β2AR active form 2011 Bimane fluorescence spectroscopy This effect was not observed in b2AR bound to the inverse CI-118,551. The effect of Nb80 was increased in the presence isoproterenol. These results show that Nb80 does not recog- nactive conformation of the b2AR, but binds efficiently to stab exp doe ma 425 450 4 0.4 0.6 0.8 1.0 Wavelengt Fluorescenceintensity (normalizedtounliganded) U mBB-β2 AR/H with Gs ba Activation TM6 TM5 TM3 TM5 TM3 TM6Monobromobimane (mBBr) change conformation to activation Cys265 on TM6 of β2AR
  56. 56. 56 Adrenoceptors β2AR active form 2011 is similar to that of Gs AR bound to the inverse increased in the presence hat Nb80 does not recog- , but binds efficiently to b2AR and b2AR–T4L in the presence of Nb80 in stabilizes a similar conformation in these two protein explanation for the ability of Nb80 to bind to b2AR does not is the difference in size of these two proteins mately 14kDa whereas the Gs heterotrimer is appro 425 450 475 500 0.4 0.6 0.8 1.0 425 450 475 500 0.4 0.6 0.8 1.0 Wavelength (nm) Fluorescenceintensity (normalizedtounliganded) Gs + ISO ISO Gs Unliganded Nb80 + ISO Nb80 Nb80 + ICI ICI mBB-β2 AR/HDL with Gs mBB-β2 AR/HDL with Nb80 DL β2 AR–T4L/HDLβ2 AR/HDL 100 100 b c e f TM5 TM6 (mBBr) Wavelength (nm) Isoproterenol (ISO) ICI-188,551 (ICI) (agonist) s similar to that of Gs R bound to the inverse creased in the presence at Nb80 does not recog- but binds efficiently to i b2AR and b2AR–T4L in the presence of Nb80 indicat stabilizes a similar conformation in these two proteins. Th explanation for the ability of Nb80 to bind to b2AR–T4L does not is the difference in size of these two proteins. Nb8 mately 14kDa whereas the Gs heterotrimer is approxima 425 450 475 500 0.4 0.6 0.8 1.0 425 450 475 500 0.4 0.6 0.8 1.0 Wavelength (nm) Fluorescenceintensity (normalizedtounliganded) Gs + ISO ISO Gs Unliganded Nb80 + ISO Nb80 Nb80 + ICI ICI mBB-β2 AR/HDL with Gs mBB-β2 AR/HDL with Nb80 L β2 AR–T4L/HDLβ2 AR/HDL 100 100 b c e f TM5 TM6 mBBr) Wavelength (nm) (inverse agonist)
  57. 57. 57 Adrenoceptors β2AR active form BI-167107 (agonist) β2AR Nanobody 2011 carazolol using two different approaches. The first crystals were obtained from b2AR bound to a Fab fragment that recognized an epitope composed of the amino and carboxyl terminal ends of the third intracellular loop connecting TMs 5 and 6 (ref. 8). In the second approach, the third intracellular loop was replaced by T4 lysozyme (b2AR–T4L)7 . Efforts to crystallize b2AR–Fab complex and b2AR– T4L bound to BI-167107 and other agonists failed to produce crystals of sufficient quality for structure determination. We therefore attempted to crystallize BI-167107 bound to b2AR and b2AR–T4L tor (Fig. 2a, and Sup Agonist-stabilize Figure 2 b–d compar lol bound b2AR–T4L ponent of the b2AR– found at the cytoplas ment of TM5 and TM the b2AR–T4L–Nb80 a b c TM5 TM6 C terminus N terminus 90º β2 ANb80 BI-167107 (agonist) β2AR-Nb80 (active) carazolol (inverse agonist) β2AR-Cz (inactive)
  58. 58. 58 Adrenoceptors β2AR active form 2011 d TM5 TM6 C terminus TM7 90º e TM3 (DRY) TM5 TM6 TM7 (NPxxY) TM1TM2 TM4 11.4 Å β2 AR–Nb80 β2 AR–Nb80 D/E3.49 R3.50 Y7.53Y5.58 E6.30 Y3.51 β2 AR–Cz Opsin N terminus β2 AR–Nb80β2 AR–CzNb80 Comparison of the agonist-Nb80 stabilized crystal structures of with inverse agonist bound b2AR and opsin. The structure of onist carazolol-bound b2AR–T4L (b2AR–Cz) is shown in blue with ol in yellow. The structure of BI-167107 agonist-bound and Nb80- binding domains showing modest structural changes. d, showing the ionic lock interaction between Asp 3.49 and motif in TM3 is broken in the b2AR–Nb80 structure. Th TM6 is moved outward and away from the core of the r β2AR-Nb80 (active) β2AR-Cz (inactive) 11.4Å 6Å 2.5Å 4Å Asn 2936.55 has a stronger influence on the preference f of the b-OH of catecholamine agonists, compared wit agonists and antagonists20 . Trp 6.48 is highly conserved in Family A GPCRs, a proposedthatits rotameric state has aroleinGPCRactiv β2 AR–Nb80 Pro 211 Asn 318Phe 282 Ile 121 Ser 207 Ser 203 2.1 Å Trp 286 a β2 AR–Cz Figure 4 | Rearrangement of transmembrane segment pack upon agonist binding a, The BI-167107- and carazolol-bou superimposed to show structural differences propagating fro binding pocket. BI-167107 and carazolol are shown in green inward bulge of TM5 centred around Ser207 2.1Å
  59. 59. 59 Adrenoceptors β2AR active form 2011 Agonist interaction with Ser203 and Ser207 stabilize a receptor conformation that includes a 2.1Å inward movement in TM5 S204 Y308 N293 N312 Y316 TM6 TM7 TM6 TM7 β2 AR–Czβ2 AR–Nb80 CarazololBI-167107 F290 V117 I309 S204 Y308N293 N312 Y316 F290 V117 I309 OH N O HN HO S2045.43 S2075.46 Y3087.35 S2035.42 N2936.55 V1173.36 F2906.52 A2005.39 OH F1935.32 F2896.51 S2045.43 S2075.46 Y3087.35 Hydrophobic contacts Polar interactions V1143.33 T1183.37 Mutation disrupts antagonist and agonist binding Mutation disrupts agonist binding 19 17 N2936.55 V1173.36 W2866.48 F2906.52 A2005.39 Y1995.38 F1935.32 W1093.28 F2896.51 OH BI-167107 Carazolol O HN I3097.36 W1093.28 O c d OH O H2N O H2N O O HO S2035.42 OH O NH2 O O HO D1133.32 N3127.39 Y3167.43 D1133.32 N3127.39 Y3167.43 H2 NH2 e 3 | Ligand binding pocket of BI-167107 and carazolol-bound b2AR ures. a, b, Extracellular views of the agonist BI-167107-bound (a) and lol-bound (b) structures, respectively. Residues within 4 A˚ of one or both s are shown as sticks. In all panels, red and blue represent oxygen and en, respectively. c, d, Schematic representation of the interactions en the b2AR and theligands BI-167107(c) andcarazolol (d). The residues shown here have at least one atom within 4 A˚ of the ligand in the crystal structures. Mutations of amino acids in orange boxes have been shown t disrupt both antagonist and agonist binding. Mutations of amino acids in boxes have been shown to disrupt agonist binding. Green lines indicate potential hydrophobic interactions and orange lines indicate potential po interactions. N A T U R E | V O L 4 6 9 | 1 3 J A N U A R Y 2 0 1 1 Y3087.35 N2936.55 S2075.46 S2035.42 S2045.43 N3127.39 D1133.32 N3127.39 D1133.32 V1143.33 S2075.46 S2035.42 S2045.43 Y3087.35 N2936.55
  60. 60. 60 Adrenoceptors β2AR active form 2011 proposed to hold together the cytoplasmic ends of TM3 and TM6 in the resting state of different amine receptors (Ballesteros et al., 2001a; Greasley et al., 2002; Shapiro et al., 2002). This interaction is also observed in the crystal structures of inactive rhodopsin (Li et al., 2004; Okada, 2004; Okada et al., 2002; Palczewski et al., 2000; Teller et al., 2001), and disruption of this interaction during activation is suggested by various biophysical (Farrens et al., 1996; Gether et al., 1997b), biochemical (Arnis et al., 1994; Ghanouni et al., 2000; Sheikh et al., 1996, 1999), and mutagenesis (Alewijnse et al., 2000; Fig. 7. The ionic lock stabilizes interactions between the cytoplasmic ends of TM3 and TM6 in the inactive state. Agonist binding disrupts these interactions. ionic lock interaction between Asp130 and Arg131 of the DRY motif in TM3 is broken in the β2AR-Nb80 structure
  61. 61. 61 Adrenoceptors β2AR active form 2011 d TM5 TM6 C terminus TM7 e TM3 (DRY) TM5 TM6 TM7 (NPxxY) TM1TM2 TM4 11.4 Å β2 AR–Nb80 β2 AR–N D/E3.49 R3.50 Y7.53Y5.58 E6.30 Y3.51 β2 AR–Cz Opsin N ter β2 AR–Nbβ2 AR–CzNb80 Comparison of the agonist-Nb80 stabilized crystal structures of with inverse agonist bound b2AR and opsin. The structure of onist carazolol-bound b2AR–T4L (b2AR–Cz) is shown in blue with binding domains showing modest structu showing the ionic lock interaction betwee motif in TM3 is broken in the b2AR–Nb8 β2AR-Nb80 (active) β2AR-Cz (inactive) Arg131 Asp130 BROKEN ionic lock interaction between Asp130 and Arg131 of the DRY motif in TM3 is broken in the β2AR-Nb80 structure 4Å
  62. 62. 62 Adrenoceptors β2AR active form 2011 C terminus TM7 e 1 Nb80 β2 AR–Nb80 D/E3.49 R3.50 Y7.53Y5.58 E6.30 Y3.51 Cz Opsin N terminus β2 AR–Nb80β2 AR–Cz ctures of ure of blue with nd Nb80- binding domains showing modest structural changes. d, Cytoplasmic view showing the ionic lock interaction between Asp 3.49 and Arg 3.50 of the DRY motif in TM3 is broken in the b2AR–Nb80 structure. The intracellular end of TM6 is moved outward and away from the core of the receptor. The arrow ˚ the ionic lock between highly conserved Asp130 and Arg/Glu131 in TM3 is brokenβ2AR-Nb80 (active) Opsin (active) BROKEN
  63. 63. Peptide binding in GPCRs
  64. 64. 64 Peptide binding TM1 TM2 TM3 TM4 TM5 TM7 TM6 C1 C2 C3 CO2 G-proteins neral structure of GPCR; E = exoloop; C = Cytoloop. (b) Schematic pre- hormone-receptor interaction for peptides of ≤40 amino acids. en refined at resolutions as high as 2.2 Å [19–21]. The structure of confirms the anticlockwise bundle of 7 TM α-helices, connected by s of varying lengths [18]. Also, the amino terminal ligand binding llicle-stimulating hormone (FSH) receptor was crystallized in com- d to 2.9 Å, which shed light on the receptor–ligand interactions and n [22]. Peptide binding - Peptides ≤40 amino acids have been reported to bind to both the GPCR core and exoloops - Polypeptides ≤90 amino acids bind to exoloops and N-terminal segment - For the majority of family A peptide receptors, ligands have been postulated to interact with the receptor at the amino terminus and extracellular loop regions. - This includes the receptors for angiotensin, neuropeptide Y, chemokines (interleukin-8, IL-8), vasopressin/oxytocin, Gonadotrophin-releasing hormone (GnRH), formyl-Leu-Met-Phe, somato- statin, bradykinins, and cholecystokinin (CCK)/gastrin - Tripeptide N-formyl-Met-Leu-Phe binds in the TM core around TMs 2 and 3, whereas the C-terminal region of the ligands associates with the N-terminal segment and exoloops 1 and 2 Peptide chemistry and drug design
  65. 65. 65 Peptide binding Peptide ligand design100 PEPTIDE DESIGN STRATEGIES FOR G-PROTEIN COUPLED RECEPTORS (GPCRs) Biologically active peptide Biophysical studies • NMR • X-ray • Computer assisted modeling Structure–activity studies • Truncation and deletion (define active core) • Alanine and D-amino acid scans (critical side chain residue) • Single and multiple substitutions Indentification of: 1) message sequence 2) address sequence 3) stereochemistry feature of each amino acid residue Receptor mutagenesis • Functional assay • Binding assay Structure–activity studies Conformational constrained peptide analogs Cyclization, amide bond modification, turn mimetics Chimeric and Unusual amino acids Prediction of: 1) Local conforamtion parameters 2) Global conformation parameters Bioactive conformation and 3D pharmacophore model 3D receptor modeling and pharmacophore docking Figure 3.4 Strategy to design receptor-selective peptide ligands. Strategy to design receptor-selective peptide ligands Peptide chemistry and drug design
  66. 66. 66 Peptide binding Peptide ligand design General SAR Studies to Design Receptor Selective and Potent Ligands 102 PEPTIDE DESIGN STRATEGIES FOR G-PROTEIN COUPLED RECEPTORS (GPCRs) TABLE 3.3 General SAR Studies to Design Receptor Selective and Potent Ligands. Study Feature 1 Substitution by d-amino acids Stereochemical requirement; secondary structures (β-turns, a-helix, etc.) 2 Substitution of side chain moieties by a methyl group Stereoelectronic properties of the side chain and its importance in interaction 3 Substitution of peptide bonds Importance of specific amide bonds for ligand–receptor interactions 4 Cyclization approaches Define topography of the amino acid residues; secondary structure 5 Reduction or increase in ring size The optimum ring size for biological activity 6 Backbone N𝛼 -alkylation Conformational constraint; less prone to enzymatic hydrolysis 7 Backbone C𝛼 -alkylation Conformational constraint, generally to α-helix Peptide chemistry and drug design
  67. 67. 67 Peptide binding Peptide ligand design Amide Bond Replacements and Their Applications for Peptide Ligands DESIGN APPROACHES FOR GPCR SELECTIVE PEPTIDE LIGANDS 103 TABLE 3.4 Amide Bond Replacements and Their Applications for Peptide Ligands [98]. Amide Bond Replacement Application References Ψ[CH2NH] Neurokinin antagonist [99] Ψ[CH2O] Gastrin releasing peptide antagonist [100] Ψ[CH2S] and Ψ[CH2SO] Reverse turn stabilizers [101] Ψ[COCH2] Neurotensin analog [102] Ψ[(E)-CH=CH] Determination of bioactive conformation of cholecystokinin terminal hexapeptide [103] Ψ[(E)-CF=CH] Opioid agonist [104] Ψ[CN4] Somatostatin and bradykinin analogs [105] Ψ[CH(CN)NH] Neurotensin analog [102] and/or biologically active conformations are generated, a common approach is then Peptide chemistry and drug design
  68. 68. 68 Peptide binding Peptide ligand design Krumm B. E. and Grisshammer R. (2015) nd Grisshammer Pept E 1 | Crystal structures of peptide receptors. Receptors were in PyMol. Ligands are shown as yellow sticks, receptors are as cartoons. CXCR4 with the cyclic peptide antagonist CVX15 code 3OE0), DOR with the morphinan antagonist naltrindole (PDB 4EJ4), NTSR1 with the peptide agonist NTS8−13 (PDB code 4GRV), AR1 with the antagonist vorapaxar (PDB code 3VW7). For comparison, the α group member β2-adrenergic receptor with the inverse agonist carazolol (PDB code 2RH1) is shown. Red lines i the putative depth of peptide ligand binding as discussed in the black lines indicate the depth of ligand binding as seen in the re structures. Residues of PAR1, implicated in tethered ligand bindin shown as purple sticks. the binding site, whereas the negatively charged car- e of Leu13 resides in an electropositive environment. are also extensive van der Waals interactions between 13 and the receptor; key NTSR1 residues are in con- similar positions as agonists and antagonists in the β-ad receptor, forming ionic interactions with an aspartate (Asp3.32) conserved in all opioid receptors, suggesting a tial role of Asp3.32 in anchoring positively charged peptide agonist NTS8−13 (PDB code 4GRV) (GLN,DPR,TYR,LYS,ARG, CYS,PRO,GLY,CIR,ALN) cyclic peptide antagonist CVX15 (PDB code 3OE0) PAR1 with the antagonist vorapaxar (PDB code 3VW7) morphinan antagonist naltrindole (PDB code 4EJ4)
  69. 69. 69 Peptide binding Peptide ligand design Krumm B. E. and Grisshammer R. (2015) Grisshammer Peptide GPCRs 2 | Electrostatic surface properties contribute to discrimination peptide ligands. View from the extracellular side. The receptor (PDB code 4KDL); CXCR4 (PDB code 3OE0); CCR5 (PDB code 4MBS); NTSR1 (PDB code 4GRV); PAR1 (PDB code 3VW7). For orientation, the position of View from the extracellular side red, negative blue, positive position of transmembrane helix 1 (TM1) and ECL2
  70. 70. 70 Peptide binding Peptide ligand design Krumm B. E. and Grisshammer R. (2015) Possible Binding Modes of Peptides 1. Peptides may reach deeply into the receptor core (opioid peptides); bind closer to the receptor surface NTS; or are in contact with superficial receptor areas (tethered PAR1 ligand) 2. Matching electrostatic properties between peptide ligand and binding pocket (or their absence) allows discrimination between ligands 3. Subtype specificity and ligand affinity are given by the complementary shape and property of the binding site 4. Most peptide receptor structures show inactive, signaling incompetent conformations
  71. 71. Insights into the world of GPCRs (Adrenergic Receptors) Speaker: Bundit Boonyarit 5814400587 Dept. Biochemistry, Fac. Science, Kasetsart University 2 May, 2016 (11.15 - 12.00 a.m.) Advanced Protein Biochemistry (01402542)

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