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G PROTEIN COUPLED RECEPTORS

G PROTEIN COUPLED RECEPTORS. GPCR FAMILY CLASS A STRUCTURAL ANALYSIS TASTE RECEPTORS CONCLUSIONS & QUESTIONS. GPCRS. OVERVIEW . Also known as 7TM receptors Largest family of proteins in the human genome (Nearly 1000 such receptors are though to be present )

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G PROTEIN COUPLED RECEPTORS

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  1. G PROTEIN COUPLED RECEPTORS

  2. GPCR FAMILY • CLASS A STRUCTURAL ANALYSIS • TASTE RECEPTORS • CONCLUSIONS & QUESTIONS

  3. GPCRS. OVERVIEW • Also known as 7TM receptors • Largest family of proteins in the human genome (Nearly 1000 such receptors are though to be present ) • Mediate signal transduction by recognizing different stimuli such as photons of light, biogenic amines, peptides…. • Mediates responses to visual, olfactory, hormonal, neurotransmitter and others… • Involved in many different diseases so half of the drug targets in the pharmaceutical industry are GPCRs

  4. Membrane proteins with seven transmembrane domains • Upon activation, signal gets transmitted to the cytoplasmatic face and amplifies through heterotrimeric G protein complex

  5. GPCRS. OVERVIEW (II) • Very hard-to-crystalize proteins • First high resolution cristal was Rhodopsin • Currently just four groups of proteins have an available PDB structure • Three differentiated regions: extracellular, transmembrane and intracelullar

  6. GPCRS. STRUCTURAL OVERVIEW (III) • There is a large gap in experimental GPCR structural space • Currently just 5 groups of GPCRs structurally solved • ADENOSINE-2A RECEPTOR • β-1 ADRENERGIC RECEPTOR • β-2 ADRENERGIC RECEPTOR • RHODOPSIN • RHODOPSIN (ALL OF THEM BELONGING TO CLASS A GPCRs)

  7. GPCRs CLASS A - STRUCTURAL ANALYSIS

  8. CLASS A FAMILY OVERVIEW • SEQUENCE SIMILARITIES. CONSERVED MOTIFS • STRUCTURAL ANALYSIS • EXTRACELLULAR REGION • LIGAND BINDING POCKET (TRANSMEMBRANE) • INTRACELLULAR REGION • CONCLUSIONS & QUESTIONS

  9. CLASS A - STRUCTURAL ANALYSIS • Main common regions: • N-terminus • Extracellular loops (ECL1, 2, 3) • Transmembrane Helices (TMH1, 2, 3, 4, 5, 6, 7,8) • Intracellular loops (ICL1, 2, 3) • C-terminus • Some structural features are shared by all • Pro distortions in TMHs 4,5,6 and 7 • Disulphide bridge between TMH3 and ECL2 • Some other features are either unique to a particular receptor or shared by a subset (i.e specific loop conformation) • The most distinct features are observed in the extracellular and intracellular loops

  10. GPCRS. STRUCTURAL OVERVIEW GRAFS systemconsidersfivemainfamilies: • GLUTAMATE (G) (CLASS C*) • RHODOPSIN (R) (CLASS A*) • ADHESION (A) (CLASS B*) • FRIZZLED/TASTE2 (F) (FRIZZLED CLASS*) • SECRETIN (S) (CLASS B*) * NC-IUPHAR NOMENCLATURE SYSTEM

  11. CLASS A - STRUCTURAL ANALYSIS • PDBs used as representative structures in the structural analysis: • ADENOSINE-2A RECEPTOR (Human): 3EML • β-1 ADRENERGIC RECEPTOR (Turkey): 2VT4 • β-2 ADRENERGIC RECEPTOR (Human): 2RH1 • RHODOPSIN (Squid): 2Z73 • RHODOPSIN (Bovine): 1U19

  12. CLASS A - STRUCTURAL ANALYSIS • Comparison of amino acid sequences of these receptors reveal modest conservation ranging from 22% to 64% sequence identity

  13. CLASS A - STRUCTURAL ANALYSIS • Percentage of sequence identity within receptors

  14. CLASS A - STRUCTURAL ANALYSIS • Comparison of amino acid sequences of these receptors reveal modest conservation ranging from 22% to 64% sequence identity • When restricting the comparison to individual helices, differences in sequence similarity between each receptor are higher (although still small…) MSA of the firs Transmembrane Helix I (TMH1) of all 5 receptors

  15. CLASS A - STRUCTURAL ANALYSIS • MSA of the five receptors structurally solved identified 25 conserved residues:

  16. CLASS A - STRUCTURAL ANALYSIS • Conserved segments are localized in the transmembrane domains, among them the most highly conserved are: • E/DRY motif in TMH3 MSA of Transmembrane Helix III (TMH3) of all 5 receptors

  17. CLASS A - STRUCTURAL ANALYSIS • WXPF/Y motif in TMH6 MSA of Transmembrane Helix VI (TMH6) of all 5 receptors

  18. CLASS A - STRUCTURAL ANALYSIS • NPXIY motif in TMH7 MSA of Helix VII (TMH7) of all 5 receptors

  19. CLASS A - STRUCTURAL ANALYSIS ADENOSINE-2A RECEPTOR) β-2 ADRENERGIC RECEPTOR RHODOPSIN (Bovine) β-1 ADRENERGIC RECEPTOR RHODOPSIN (Squid)

  20. CLASS A - STRUCTURAL ANALYSIS • Structural superpositioning of the 5 receptors demonstrating a high level of overall structure similarity • RMSDs of superimposition ranging from 0.63Å to 4.03Å • Slightly more variation at the extracellular side of the membrane surface

  21. CLASS A - STRUCTURAL ANALYSIS • EXTRACELLULAR REGION • RHODOPSIN • Extensive secondary and tertiary structure to completely occlude the binding site from solvent access (“retinal plug”) • N-terminus along with ECL2 form a four-stranded β-sheet with additional interactions ECL3-ECL1 • Access to retinal binding pocket severely restricted

  22. CLASS A - STRUCTURAL ANALYSIS N-TERMINUS ECL-1 ECL-2 ECL-3

  23. CLASS A - STRUCTURAL ANALYSIS • EXTRACELLULAR REGION • RHODOPSIN • Extensive secondary and tertiary structure to completely occlude the binding site from solvent access (“retinal plug”) • N-terminus along with ECL2 form a four-stranded β-sheet with additional interactions ECL3-ECL1 • Access to retinal binding pocket severely restricted • One disulfide bridge (it has been shown to be essential for the normal function of Rhodopsin)

  24. CLASS A - STRUCTURAL ANALYSIS CYS 187 (ECL2) CYS 110 (TMH3)

  25. CLASS A - STRUCTURAL ANALYSIS

  26. CLASS A - STRUCTURAL ANALYSIS • Β-ADRENERGIC RECEPTORS • Extracellular region much more open • Short helical segment within ECL2: • Limited interactions with ECL1 • 2 disulfide bridges: one with a coil segment of ECL2 and the other fixing the entire loop to the top of TMH3 • The random coil section of ECL2 forms the top of the ligand binding pocket (only partially occluded) • ECL3 forms no interaction with ECL1 or ECL2

  27. CLASS A - STRUCTURAL ANALYSIS CYS 184 (ECL2) CYS 106 (TMH3) CYS 191 (ECL2) CYS 190 (ECL2)

  28. CLASS A - STRUCTURAL ANALYSIS • Β-ADRENERGIC • Extracellular region much more open • Short helical segment within ECL2: • Limited interactions with ECL1 • 2 disulfide bridges: one with a coil segment of ECL2 and the other fixing the entire loop to the top of TMH3 • The random coil section of ECL2 forms the top of the ligand binding pocket (only partially occluded) • ECL3 forms no interaction with ECL1 or ECL2 • Entire 28-resiude N -terminus completely disordered in the four structures solved to date Does the extracellular region of the β-Adrenergic family has evolved to allow access to the ligand binding site?

  29. CLASS A - STRUCTURAL ANALYSIS ? RHODOPSIN Β-ADRENERGIC RECEPTOR

  30. CLASS A - STRUCTURAL ANALYSIS • ADENOSIN RECEPTORS • Highly constrained by four disulfide bridges and multiple ligand binding interactions • Three out of the four disulfide bridges constrain the position of ECL2 anchoring this loop to ECL1 and the top of TMH3

  31. CYS 259 (ECL3) CLASS A - STRUCTURAL ANALYSIS CYS 262 (TMH6) CYS 71(ECL1) CYS 166 (ECL2) CYS 77 (TMH3) CYS 159 (ECL2) CYS 146 (N-TERMINUS) CYS 74 (TMH3)

  32. CLASS A - STRUCTURAL ANALYSIS • ADENOSIN RECEPTORS • Highly constrained by four disulfide bridges and multiple ligand binding interactions • Three out of the four disulfide bridges constrain the position of ECL2 anchoring this loop to ECL1 and the top of TMH3 • The former three disulfide bridges probably stabilize a short helical segment N terminal of TMH5 containing Phe168 and Glu169 . This segment is considered to be an important region for ligand binding

  33. CLASS A - STRUCTURAL ANALYSIS PHE 168 DISULFIDE BRIDGES GLU 169 RANDOM COIL (ECL2)

  34. RANDOM COIL (ECL2) CLASS A - STRUCTURAL ANALYSIS DISULFIDE BRIDGE GLU 169 PHE 168

  35. CLASS A - STRUCTURAL ANALYSIS • ADENOSIN RECEPTORS • Highly constrained by four disulfide bridges and multiple ligand binding interactions • Three out of the four disulfide bridges constrain the position of ECL2 anchoring this loop to ECL1 and the top of TMH3 • The former three disulfide bridges probably stabilize a short helical segment N terminal of TMH5 containing Phe168 and Glu169 . This segment is considered to be an important region for ligand binding • ECL3 contains another disulfide bridge that might constrain His264 position, which in turn forms a polar interaction with Glu169

  36. CLASS A - STRUCTURAL ANALYSIS • LIGAND BINDING POCKET • RHODOPSIN (I) • 11-cis-retinal is covalently bound to Lys296 in TMH7 by a protonatedShiff base • This ligand stabilizes the inactive state of rhodopsin until photon absorption occurs.

  37. CLASS A - STRUCTURAL ANALYSIS • LIGAND BINDING POCKET • RHODOPSIN (I) • 11-cis-retinal covalently bound to Lys296 in TMH7 by a protonatedShiff base. This ligand stabilizes the inactive state of rhodopsin until photon absorption • The molecular switchinvolved in theactivation of the receptor is a is a rotamertoogleswitch • The indole chain of the highly conserved W265 is in van der Waals contact with the β-ionone ring of retinal

  38. W265 (Toggleswitch) 11-CIS-RETINAL

  39. CLASS A - STRUCTURAL ANALYSIS 11-CIS-RETINAL

  40. CLASS A - STRUCTURAL ANALYSIS

  41. CLASS A - STRUCTURAL ANALYSIS TYR191 MET207 GLU 181 GLU 113 PHE 212 LYS 296 TRP265 PHE 261

  42. CLASS A - STRUCTURAL ANALYSIS • LIGAND BINDING POCKET • RHODOPSIN (II) • Binding pocket comprises a cluster of the following residues: Glu113, Glu181, Tyr191, Met207, Phe212, Phe261, Phe293, Lys296 and Trp265 • The position of this binding pocket does not vary too much between different subspecies • Prior to activation, a chained series of conformational changes occur. Among this changes, it’s worth highlighting that Lys296 releases from ligand

  43. CLASS A - STRUCTURAL ANALYSIS 11-CIS-RETINAL TRP265 LYS 296

  44. CLASS A - STRUCTURAL ANALYSIS • LIGAND BINDING POCKET • RHODOPSIN (III) • Binding pocket comprises a cluster of the following residues: Glu113, Glu181, Tyr191, Met207, Phe212, Phe261, Phe293, Lys296 and Trp265 • The position of this binding pocket does not vary too much between different subspecies • An extended hydrogen-bonded network (ionic lock) between TMH3 and TMH6 is present. Breakage of this ionic lock needs to happen for receptor’s activation

  45. CLASS A - STRUCTURAL ANALYSIS BINDING POCKET TMH6 ARG135 THR251 TMH3 GLU134 IONIC LOCK GLU 247

  46. CLASS A - STRUCTURAL ANALYSIS • β-ADRENERGIC RECEPTORS • Similar binding pocket to the Rhodopsin’s one, position does not vary considerably with alternate ligands or between different species (Hanson et al.2008; Warne et al.2008) As a representative ligand, carazolol follows a similar path as that of rhodopsin

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