The dynamic process of GPCR activation: Insights from the human β 2 AR

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1 The structural basis of G protein coupled receptor signaling The dynamic process of GPCR activation: Insights from the human β 2 AR Brian Kobilka Department of Molecular and Cellular Physiology Brian Kobilka Molecular Stanford and Cellular Physiology Stanford University

2 The β 2 AR modulates the activity of multiple signaling pathways Ca 2+ Channel Adenylyl Cyclase Efficacy Basal

3 Agonist binding G protein coupling and nucleotide exchange Activated G protein subunits regulate effector proteins GTP hydrolysis and inactivation of Gα protein GPCR-G Protein Cycle

4 Outline Overview of approaches to characterize GPCR structure GPCR crystallography Mechanistic insights into GPCR-G protein activation Agonist binding G protein coupling and nucleotide exchange Activated G protein subunits regulate effector proteins GTP hydrolysis and inactivation of Gα protein GPCR-G Protein Cycle

5 Approaches to characterizing β 2 AR structure: Cloned DNA Sequence (1986) GAATTCATGCCGCGTTTCTGTGTTGGACAGGGGTGACTTTGTGCC GGATGGCTTCTGTGTGAGAGCGCGCGCGAGTGTGCATGTCGGTGA GCTGGGAGGGTGTGTCTCAGTGTCTATGGCTGTGGTTCGGTATAAG TCTAAGCATGTCTGCCAGGGTGTATTTGTGCCTGTATGTGCGTGCCT CGGTGGGCACTCTCGTTTCCTTCCGAATGTGGGGCAGTGCCGGTG TGCTGCCCTCTGCCTTGAGACCTCAAGCCGCGCAGGCGCCCAGGG CAGGCAGGTAGCGGCCACAGAAGAGCCAAAAGCTCCCGGGTTGG CTGGTAAGCACACCACCTCCAGCTTTAGCCCTCTGGGGCCAGCCA GGGTAGCCGGGAAGCAGTGGTGGCCCGCCCTCCAGGGAGCAGTT GGGCCCCGCCCGGGCCAGCCTCAGGAGAAGGAGGGCGAGGGGA GGGGAGGGAAAGGGGAGGAGTGCCTCGCCCCTTCGCGGCTGCC GGCGTGCCATTGGCCGAAAGTTCCCGTACGTCACGGCGAGGGCA GTTCCCCTAAAGTCCTGTGCACATAACGGGCAGAACGCACTGCGA AGCGGCTTCTTCAGAGCACGGGCTGGAACTGGCAGGCACCGCGA GCCCCTAGCACCCGACAAGCTGAGTGTGCAGGACGAGTCCCCACC ACACCCACACCACAGCCGCTGAATGAGGCTTCCAGGCGTCCGCTC GCGGCCCGCAGAGCCCCGCCGTGGGTCCGCCTGCTGAGGCGCCC CCAGCCAGTGCGCTTACCTGCCAGACTGCGCGCCATGGGGCAACC CGGGAACGGCAGCGCCTTCTTGCTGGCACCCAATAGAAGCCATGC GCCGGACCACGACGTCACGCAGCAAAGGGACGAGGTGTGGGTG GTGGGCATGGGCATCGTCATGTCTCTCATCGTCCTGGCCATCGTGTT TGGCAATGTGCTGGTCATCACAGCCATTGCCAAGTTCGAGCGTCTG CAGACGGTCACCAAC Sequence analysis secondary structure (transmembrane domains) Amino Acid Sequence MGQPGNGSAFLLAPNRSHAPDHDVT QQRDEVWVVGMGIVMSLIVLAIVFGN VLVITAIAKFERLQTVTNYFITSLACADLV MGLAVVPFGAHILMKMWTFGNFWCE FWTSIDVLCVTASIETLCVIAVDRYFAITS PFKYQSLLTKNKARVIILMVWIVSGLTSF LPIQMHWYRATHQEAINCYANETCCDF FTNQAYAIASSIVSFYVPLVIMVFVYSRV FQEAKRQLQKIDKSEGRFHVQNLSQVE QDGRTGHGLRRSSKFCLKEHKALKTLGII MGTFTLCWLPFFIVNIVHVIQDNLIRKE VYILLNWIGYVNSGFNPLIYCRSPDFRIA FQELLCLRRSSLKAYGNGYSSNGNTGEQ SGYHVEQEKENKLLCEDLPGTEDFVGH QGTVPSDNIDSQGRNCSTNDSLL

6 Approaches to characterizing β 2 AR structure: Cloned DNA Sequence (1986) GAATTCATGCCGCGTTTCTGTGTTGGACAGGGGTGACTTTGTGCC GGATGGCTTCTGTGTGAGAGCGCGCGCGAGTGTGCATGTCGGTGA GCTGGGAGGGTGTGTCTCAGTGTCTATGGCTGTGGTTCGGTATAAG TCTAAGCATGTCTGCCAGGGTGTATTTGTGCCTGTATGTGCGTGCCT CGGTGGGCACTCTCGTTTCCTTCCGAATGTGGGGCAGTGCCGGTG TGCTGCCCTCTGCCTTGAGACCTCAAGCCGCGCAGGCGCCCAGGG CAGGCAGGTAGCGGCCACAGAAGAGCCAAAAGCTCCCGGGTTGG CTGGTAAGCACACCACCTCCAGCTTTAGCCCTCTGGGGCCAGCCA GGGTAGCCGGGAAGCAGTGGTGGCCCGCCCTCCAGGGAGCAGTT GGGCCCCGCCCGGGCCAGCCTCAGGAGAAGGAGGGCGAGGGGA GGGGAGGGAAAGGGGAGGAGTGCCTCGCCCCTTCGCGGCTGCC GGCGTGCCATTGGCCGAAAGTTCCCGTACGTCACGGCGAGGGCA GTTCCCCTAAAGTCCTGTGCACATAACGGGCAGAACGCACTGCGA AGCGGCTTCTTCAGAGCACGGGCTGGAACTGGCAGGCACCGCGA GCCCCTAGCACCCGACAAGCTGAGTGTGCAGGACGAGTCCCCACC ACACCCACACCACAGCCGCTGAATGAGGCTTCCAGGCGTCCGCTC GCGGCCCGCAGAGCCCCGCCGTGGGTCCGCCTGCTGAGGCGCCC CCAGCCAGTGCGCTTACCTGCCAGACTGCGCGCCATGGGGCAACC CGGGAACGGCAGCGCCTTCTTGCTGGCACCCAATAGAAGCCATGC GCCGGACCACGACGTCACGCAGCAAAGGGACGAGGTGTGGGTG GTGGGCATGGGCATCGTCATGTCTCTCATCGTCCTGGCCATCGTGTT TGGCAATGTGCTGGTCATCACAGCCATTGCCAAGTTCGAGCGTCTG CAGACGGTCACCAAC Sequence analysis secondary structure (transmembrane domains) Amino Acid Sequence MGQPGNGSAFLLAPNRSHAPDHDVT QQRDEVWVVGMGIVMSLIVLAIVFGN VLVITAIAKFERLQTVTNYFITSLACADLV MGLAVVPFGAHILMKMWTFGNFWCE FWTSIDVLCVTASIETLCVIAVDRYFAITS PFKYQSLLTKNKARVIILMVWIVSGLTSF LPIQMHWYRATHQEAINCYANETCCDF FTNQAYAIASSIVSFYVPLVIMVFVYSRV FQEAKRQLQKIDKSEGRFHVQNLSQVE QDGRTGHGLRRSSKFCLKEHKALKTLGII MGTFTLCWLPFFIVNIVHVIQDNLIRKE VYILLNWIGYVNSGFNPLIYCRSPDFRIA FQELLCLRRSSLKAYGNGYSSNGNTGEQ SGYHVEQEKENKLLCEDLPGTEDFVGH QGTVPSDNIDSQGRNCSTNDSLL

7 Approaches to characterizing β 2 AR structure: Sequence analysis secondary structure (transmembrane domains) post-translational modifications glycosylation palmitoylation phosphorylation

8 Approaches to characterizing β 2 AR structure: Sequence analysis secondary structure (transmembrane domains) post-translational modifications Chimeric Receptors and site-directed mutagenesis ligand binding and G protein coupling Ligand binding G protein coupling specificity

9 Approaches to characterizing β 2 AR structure: FLAG Signal Sequence Sequence analysis secondary structure (transmembrane domains) post-translational modifications Chimeric Receptors and Site-directed mutagenesis ligand binding and G protein coupling enhance expression and purification HHHHHH

10 Biochemistry Spectroscopy Crystallography

11 Approaches to characterizing β 2 AR structure: FLAG Signal Sequence Sequence analysis secondary structure (transmembrane domains) post-translational modifications Chimeric Receptors and site-directed mutagenesis ligand binding and G protein coupling enhance expression and purification HHHHHH

12 Approaches to characterizing β 2 AR structure: FLAG Signal Sequence Sequence analysis secondary structure (transmembrane domains) post-translational modifications Chimeric Receptors and site-directed mutagenesis ligand binding and G protein coupling enhance expression and purification Biochemistry unstructured, flexible sequence HHHHHH

13 Approaches to characterizing β 2 AR structure: TM6 Cysteine 265 FLAG Signal Sequence Sequence analysis secondary structure (transmembrane domains) post-translational modifications Chimeric Receptors and site-directed mutagenesis ligand binding and G protein coupling enhance expression and purification Biochemistry unstructured, flexible sequence Spectroscopy (Fluorescence, EPR, NMR) ligand-specific conformational states dynamic, flexible character useful tool for monitoring receptor activity HHHHHH

14 Conformational changes in TM6 in response to norepinepherine: % Change in Intensity Rhodamine Time (sec) Gayathri Swaminath, 2004

15 Conformational changes in TM6 in response to norepinepherine: % Change in Intensity Biphasic changes in intensity over time Rapid t1/2 < 5 sec Slow t1/2 = 70 sec Rhodamine Time (sec) Gayathri Swaminath, 2004

16 Conformational changes in TM6 in response to norepinepherine: Not consistent with single active state A + R AR* % Change in Intensity Biphasic changes in intensity over time Rapid t1/2 < 5 sec Slow t1/2 = 70 sec Rhodamine Time (sec) Gayathri Swaminath, 2004

17 Conformational changes in TM6 in response to norepinepherine: Sequential conformational changes A + R AR + AR* % Change in Intensity Biphasic changes in intensity over time Rapid t1/2 < 5 sec Slow t1/2 = 70 sec Rhodamine Time (sec) Gayathri Swaminath, 2004

18 Fluorescence lifetime experiments Multiple agonist states Ligand-specific conformational states Fluorescein A + R AR + AR* P + R PR + PR # Efficacy AR + Basal AR* AR* PR # Pejman Ghanouni, 2001

19 Insights from spectroscopy studies -fluorescence, NMR, EPR- TM6 Ansgar Philippsen & Ron Dror (D.E. Shaw Research) The β 2 AR is flexible and dynamic TM6 undergoes the largest changes in response to agonists Agonist binding and activation occur through a series of conformational intermediates Agonists and partial agonists stabilize distinct conformational states Agonists alone do not stabilize a single active conformation. Fluorescence spectroscopy aided in identifying optimal conditions for crystallography

20 Outline Overview of approaches to characterize GPCR structure GPCR crystallography Mechanistic insights into GPCR-G protein activation Agonist binding G protein coupling and nucleotide exchange Activated G protein subunits regulate effector proteins GTP hydrolysis and inactivation of Gα protein GPCR-G Protein Cycle

21 Crystals of wild-type β 2 AR Nov 2004 ESRF microfocus beamline ID13, July µm 20Å

22 TM5 Purified protein TM6 Conformationally uniform GPCR High-quality crystal

23 TM5 Purified protein TM6 Conformationally heterogeneous GPCR Poor quality crystal

24 Challenges for crystallography Protein dynamics TM5 TM6

25 Challenges for crystallography Protein dynamics Little polar surface TM5 TM6

26 Challenges for crystallography Protein dynamics Little polar surface Stabilize and increase polar surface Antibodies Protein Engineering

27 Approaches for GPCR crystallogenesis: Antibodies and protein engineering 2007 Søren Rasmussen Dan Rosenbaum TM5 TM6 TM5 TM6 T4L Fab

28 Approaches for GPCR crystallogenesis: Antibodies and protein engineering Lipid-based media: bicelles and lipidic cubic phase 2007 Søren Rasmussen Dan Rosenbaum TM5 TM6 TM5 TM6 T4L Fab

29 Approaches for GPCR crystallogenesis: Antibodies and protein engineering Lipid-based media: bicelles and lipidic cubic phase 2007 Søren Rasmussen Dan Rosenbaum T4L TM5 TM6 TM5 TM6 TM5 TM6 T4L Fab

30 Inactive-state GPCR structures Antibodies and Protein Engineering T4L Other Approaches Thermostabilization through alanine scanning mutations β 1 AR, Adenosine A2A Tate and Schertler Rhodopsin (native) Schertler (2D crystals) Palczewski and Okada (3D) Ernst and Hofmann (Opsin) Fab T4L Recent GPCR-T4L structures from Kobilka Lab and collaborators Stevens Lab and collaborators M2 Muscarinic M3 Muscarinic δ Opioid µ Opioid PAR1 (Haga and Kobayashi) (Jurgen Wess) (Sebastien Granier) (Shaun Coughlin) Adenosine A2A D3 Dopamine CXCR4 Histamine S1P1 κ-opioid

31 Outline Overview of approaches to characterize GPCR structure GPCR crystallography Mechanistic insights into GPCR-G protein activation Agonist binding G protein coupling and nucleotide exchange Activated G protein subunits regulate effector proteins GTP hydrolysis and inactivation of Gα protein GPCR-G Protein Cycle

32 β 2 AR INACTIVE β 2 AR ACTIVE? Inverse Agonist Agonist (covalent) T4L Fab T4L T4L Dan Rosenbaum Ralph Holl Peter Gmeiner Pipette

33 β 2 AR INACTIVE β 2 AR ACTIVE? Inverse Agonist T4L Agonist (covalent) Agonist alone does not fully stabilize active state β 2 AR β 2 AR + Agonist Fab T4L T4L Pipette

34 β 2 AR INACTIVE β 2 AR ACTIVE? Inverse Agonist T4L Agonist (covalent) Agonist alone does not fully stabilize active state β 2 AR β 2 AR + Agonist Fab T4L T4L Stabilizing protein Pipette

35 β 2 AR INACTIVE β 2 AR ACTIVE? Inverse Agonist T4L Agonist (covalent) Agonist alone does not fully stabilize active state β 2 AR β 2 AR + Agonist Fab T4L T4L Stabilizing protein Nanobody variable domain of a single chain camelid antibody Pipette Jan Steyaert Els Pardon

36 β 2 AR INACTIVE β 2 AR ACTIVE Inverse Agonist Agonist (covalent) Partial Agonist Agonist T4L T4L Fab T4L T4L Nb71 Nb80 Stabilizing protein Nanobody variable domain of a single chain camelid antibody Pipette Jan Steyaert Els Pardon

37 β 2 AR INACTIVE β 2 AR ACTIVE Inverse Agonist Agonist (covalent) Partial Agonist Agonist T4L T4L Fab T4L T4L Nb71 Nb80 β 2 AR-Gs Nanobody variable domain of a single chain camelid antibody Pipette Jan Steyaert Els Pardon

38 Technical contributions to crystallizing the β 2 AR-Gs complex High-affinity agonist BI (1 of ~ 60 screened) Removal of GDP Apyrase Detergent: MNG-3 (long-term storage, aids transition into LCP) New mesophase lipid (7.7 MAG) to accommodate G protein (provided by Martin Caffrey) Nanobody to stabilize G protein complex (Jan Steyaert) Amino Terminal T4 Lysozyme Project guided by data from negative stain single particle EM (Georgios Skiniotis)

39 Microcrystallography GM/CA-CAT at Argonne National Labs β 2 AR-Gs complex Andy Kruse, Brian DeVree, Søren Rasmussen me Roger Sunahara Returning from Argonne with final data set April 2011

40 Gsαβγ β 2 AR

41 Inactive Active β 2 AR-Cz β 2 AR-Gs

42 Active state of β 2 AR Cytoplasmic View 14Å 14Å β 2 AR - Inactive β 2 AR-Gs

43 Extracellular Surface β 2 AR-Cz TM3 Carazolol TM5 S203 D113 S204 S207 N312 W286 TM6 TM7

44 Extracellular Surface β 2 AR-Gs BI TM3 TM5 S203 D113 S204 S207 N312 W286 TM6 TM7

45 Inactive Active

46 Extracellular Surface β 2 AR-Cz β 2 AR-Gs TM3 TM5 S203 D113 S204 S207 N312 2Å TM6 W286 TM7

47 Extracellular TM3 β 2 AR-Inactive TM5 TM7 2Å Pro 211 Ile121 Phe282 Asn 318 Packing of conserved amino acids maintains inactive state. 14Å Cytoplasm TM6

48 Extracellular TM5 TM3 TM3 TM7 TM7 β 2 AR-Inactive β 2 AR-Active 2Å Weak coupling between ligand binding and G protein coupling domains Pro Pro Phe282 Ile121 Ile121 Phe282 Asn 318 Asn 318 Rearrangement of conserved amino acids required to accommodate agonist binding. 14Å Cytoplasm TM6 TM6

49 β 2 AR Gsαβγ Inactive Gα Ras-like domain GDP α-helical domain Interactions between the β 2 AR and Gs promote GDP release.

50 β 2 AR Gsαβγ Inactive Active

51 β 2 AR α helical domain Gsαβγ Active Inactive

52 Mobility of alpha helical domain confirmed by EM G Ras G αh Nb37 Gerwin Westfield and Georgios Skiniotis, Univ. Michigan

53 Ca 2+ Channel Adenylyl Cyclase Future Directions Efficacy Basal

54 β 2 AR-Gs Team GPCR Workshop, Maui, Dec. 2011

55 Many Thanks Tong Sun Kobilka Bob Lefkowitz and colleagues in the Lefkolab Kobilka lab students, postdoctoral fellows and collaborators ( ) Bill Weis and Roger Sunahara Stanford Søren Rasmussen Foon Sun Thian Tong Sun Kobilka Yaozhong Zou Andrew Kruse Ka Young Chung Jesper Mathiesen Bill Weis β 2 AR-Gs Team University of Michigan Brian DeVree Diane Calinski Gerwin Westfield Georgios Skiniotis Roger Sunahara University of Wisconsin Pil Seok Chae Sam Gellman We will miss Virgil Woods (UCSD), Free University of Brussels Els Pardon Jan Steyaert Trinity College Dublin Joseph Lyons Syed Shah Martin Caffrey Financial Support NIH NINDS and NIGMS Gifts from: Mathers Foundation Lundbeck

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