The dynamic process of GPCR activation: Insights from the … · 2014. 10. 8. · Therapeutic...
Transcript of The dynamic process of GPCR activation: Insights from the … · 2014. 10. 8. · Therapeutic...
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The dynamic process of GPCR activation: Insights from the
human β2AR
Brian Kobilka Molecular and Cellular
Physiology Stanford University
The structural basis of G protein coupled receptor signaling
Brian Kobilka Department of Molecular and
Cellular Physiology Stanford
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Autonomic nervous system regulates cardiovascular, pulmonary and endocrine function.
Sympathetic (9 Adrenergic receptor subtypes)
Adrenaline and Noradrenaline
Parasympathetic (5 Muscarinic receptor subtypes)
Acetylcholine
Therapeutic targets for intensive care medicine
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Autonomic nervous system regulates cardiovascular, pulmonary and endocrine function.
Sympathetic (9 Adrenergic receptor subtypes)
Adrenaline and Noradrenaline
Parasympathetic (5 Muscarinic receptor subtypes)
Acetylcholine
Therapeutic targets for intensive care medicine
β adrenergic Receptor
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Autonomic nervous system regulates cardiovascular, pulmonary and endocrine function.
Sympathetic (9 Adrenergic receptor subtypes)
Adrenaline and Noradrenaline
Parasympathetic (5 Muscarinic receptor subtypes)
Acetylcholine
Therapeutic targets for intensive care medicine
β adrenergic Receptor
M2 Muscarinic Receptor
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Autonomic nervous system regulates cardiovascular, pulmonary and endocrine function.
Sympathetic (9 Adrenergic receptor subtypes)
Adrenaline and Noradrenaline
Parasympathetic (5 Muscarinic receptor subtypes)
Acetylcholine
Therapeutic targets for intensive care medicine
β adrenergic Receptor
M2 Muscarinic Receptor
µ−Opioid Receptor
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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
G protein
What are G protein coupled receptors? • Integral membrane proteins • Detect hormones, neurotransmitters, ions, light, odors and
flavors • Transmit signals across the cell membrane to change the
behavior of cells • Over 800 members of the GPCR family in the human genome • Largest class (~40%) of pharmaceutical targets
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Largest family of membrane proteins in the human genome
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The Rhodopsin Family GPCRs: 701 Total 241 Non-olfactory
Chemokine Angiotensin Bradykinin
FSH, LH and TSH receptors
Rhodopsin Adrenergic Dopamine Serotonin Histamine Muscarinic
Neuropeptide THRH Oxytocin receptor
Fredriksson et al. Mol Pharm 2003
GPCR Family Tree
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Rhodopsin Family Tree
The Rhodopsin Family (241 Nonolfactory, Total of 701) Adrenergic
Serotonin Dopamine
Muscarinic
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• Many targets that share sequence and structural homology
• Complex signaling behavior o Multiple cell-specific signaling pathways o Different types of ligands (Efficacy) o Receptor oligomers (homo- and heteromers)
G Protein Coupled Receptors –Challenges Drug Discovery
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GPCR Signaling Adenylyl Cyclase Ca2+ Channel
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GPCR Signaling
Basal
Adenylyl Cyclase Ca2+ Channel
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Basal
Adenylyl Cyclase Ca2+ Channel
GPCR Signaling
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Basal
Adenylyl Cyclase Ca2+ Channel
GPCR Signaling
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Basal
Adenylyl Cyclase Ca2+ Channel
GPCR Signaling
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Basal
Adenylyl Cyclase Ca2+ Channel
GPCR Signaling
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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
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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
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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
Outline •Challenges in obtaining GPCR crystals •Inactive state structures •Active state structures
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Biochemistry Spectroscopy
Crystallography
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Crystals grown from wild-type β2AR
50µm
247d3
249d4
245c5
June 05 Nov 05
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50 µm
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Conventional beam (100 micron)
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Microfocus beam (5 micron)
ESRF microfocus beamline ID13, July 2005
Gebhard Schertler
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Microfocus beam
20Å
ESRF microfocus beamline ID13, July 2005
Gebhard Schertler
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Insights from fluorescence, EPR and NMR spectroscopy studies
•TM6 undergoes largest structural changes following agonist binding
•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
TM6
Ansgar Philippsen & Ron Dror (D.E. Shaw Research)
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Gs
β2AR No ligand
β2AR + agonist
β2AR + agonist + Gs
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Gs
β2AR No ligand
β2AR + agonist
β2AR + agonist + Gs
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Gs
β2AR No ligand
β2AR + agonist
β2AR + agonist + Gs
Gs
Structural insights from fluorescence, EPR and NMR spectroscopy
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TM5
TM6
Purified protein
High-quality crystal
Conformationally uniform GPCR
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TM5
TM6
Poor quality crystal
Conformationally heterogeneous GPCR
Purified protein
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TM5 TM6
Challenges for crystallography •Protein dynamics
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TM5 TM6
Challenges for crystallography •Protein dynamics •Little polar surface
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Challenges for crystallography •Protein dynamics •Little polar surface
Stabilize and increase polar surface •Antibodies •Protein Engineering
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3.5 Å
50µm
A B
Primitive monoclinic a = 114.5 Å b = 65.5 Å c = 127.6 Å β = 95.8°
β2AR-T4L
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3.0Å
β2AR-Fab5 complex
50µm
Space group C2 Unit cell parameters a=336.9 Å b=48.6 Å c=89.0 Å β=104.6º one β2AR-Fab complex in the asymmetric unit.
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Rock II Crystals
β2AR-T4L
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Fab
T4L
TM5 TM6
TM5 TM6
Approaches for GPCR crystallogenesis: •Antibodies and protein engineering •Lipid-based media: bicelles and lipidic cubic phase
2007 Søren Rasmussen Dan Rosenbaum
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Fab
T4L
TM5 TM6
TM5 TM6
Approaches for GPCR crystallogenesis: •Antibodies and protein engineering •Lipid-based media: bicelles and lipidic cubic phase
2007 Søren Rasmussen Dan Rosenbaum
T4L
TM5 TM6
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Fab
β2AR INACTIVE
Inverse Agonist
T4L
T4L
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Fab
β2AR INACTIVE
Inverse Agonist
T4L
Pipette Pipette
Other inactive state structures
PAR1 µ Opioid M3 Muscarinic δ Opioid M2 Muscarinic
(Haga and Kobayashi) (Jurgen Wess) (Sebastien Granier) (Shaun Coughlin)
T4L
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Fab
β2AR INACTIVE
Inverse Agonist
T4L
Pipette Pipette
Other inactive state structures
PAR1 µ Opioid M3 Muscarinic δ Opioid M2 Muscarinic
(Haga and Kobayashi) (Jurgen Wess) (Sebastien Granier) (Shaun Coughlin)
T4L
Thermostabilization through alanine scanning mutations – β1AR, Adenosine A2A – Tate and Schertler
Other Approaches
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Fab
β2AR INACTIVE
Inverse Agonist
T4L
Pipette
β2AR ACTIVE
Pipette
Other inactive state structures
PAR1 µ Opioid M3 Muscarinic δ Opioid M2 Muscarinic
(Haga and Kobayashi) (Jurgen Wess) (Sebastien Granier) (Shaun Coughlin)
T4L
Thermostabilization through alanine scanning mutations – β1AR, Adenosine A2A – Tate and Schertler
Rhodopsin (native) •Schertler (2D crystals) - 1997 •Palczewski and Okada (3D) - 2000 •Ernst and Hofmann (Opsin) - 2008
Other Approaches
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Fab
β2AR INACTIVE
Inverse Agonist
T4L
Pipette
β2AR ACTIVE
Pipette
Other inactive state structures
PAR1 µ Opioid M3 Muscarinic δ Opioid M2 Muscarinic
(Haga and Kobayashi) (Jurgen Wess) (Sebastien Granier) (Shaun Coughlin)
T4L
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Fab
β2AR INACTIVE
Inverse Agonist
T4L
Pipette
β2AR ACTIVE
Pipette
Other inactive state structures
PAR1 µ Opioid M3 Muscarinic δ Opioid M2 Muscarinic
(Haga and Kobayashi) (Jurgen Wess) (Sebastien Granier) (Shaun Coughlin)
T4L
Unable to grow crystals of agonist bound receptor
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Fab
β2AR INACTIVE
T4L
Agonist (covalent)
Inverse Agonist
T4L
Pipette
β2AR ACTIVE
Pipette
Other inactive state structures
PAR1 µ Opioid M3 Muscarinic δ Opioid M2 Muscarinic
(Haga and Kobayashi) (Jurgen Wess) (Sebastien Granier) (Shaun Coughlin)
T4L
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Fab
β2AR INACTIVE
T4L
Agonist (covalent)
Inverse Agonist
T4L
Pipette
β2AR ACTIVE
Pipette
Other inactive state structures
PAR1 µ Opioid M3 Muscarinic δ Opioid M2 Muscarinic
(Haga and Kobayashi) (Jurgen Wess) (Sebastien Granier) (Shaun Coughlin)
T4L
β2AR ACTIVE
β2AR β2AR + Agonist
Stabilizing protein
Agonist alone does not fully stabilize active state
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Conventional vs. Camelid IgG
Jan Steyaert Els Pardon
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Conventional vs. Camelid IgG
Jan Steyaert Els Pardon
Fab fragment
Nanobody
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Generation of conformationally sensitive antibodies to the β2AR
•Purification of functional β2AR antigen •Reconstitution into proteoliposomes at a high protein/lipid ratio •Immunize llamas > prepare nanobody-display library > select binders
(Immunogenic)
Immunization Phage display
(Collaboration with Jan Steyaert and Els Pardon, Free University Brussels)
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Nanobody vs. Fab
7 of 16 Nb clones exhibited: 1. Agonist dependent binding to β2AR 2. Stabilize an active conformation 3. Stabilize agonist high-affinity state
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Fab
β2AR INACTIVE
T4L
Agonist (covalent)
Inverse Agonist
T4L
Pipette
β2AR ACTIVE
Pipette
Other inactive state structures
PAR1 µ Opioid M3 Muscarinic δ Opioid M2 Muscarinic
(Haga and Kobayashi) (Jurgen Wess) (Sebastien Granier) (Shaun Coughlin)
T4L
β2AR ACTIVE
Nb80
Stabilizing protein
Agonist
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Fab
β2AR INACTIVE
T4L
Agonist (covalent)
Inverse Agonist
T4L
Pipette
β2AR ACTIVE
Pipette
Other inactive state structures
PAR1 µ Opioid M3 Muscarinic δ Opioid M2 Muscarinic
(Haga and Kobayashi) (Jurgen Wess) (Sebastien Granier) (Shaun Coughlin)
T4L
Agonist Partial Agonist
β2AR ACTIVE
T4L
Nb71 Nb80
Stabilizing protein
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Fab
β2AR INACTIVE
T4L
Agonist (covalent)
Inverse Agonist
T4L
Pipette
β2AR ACTIVE
Pipette
Other inactive state structures
PAR1 µ Opioid M3 Muscarinic δ Opioid M2 Muscarinic
(Haga and Kobayashi) (Jurgen Wess) (Sebastien Granier) (Shaun Coughlin)
T4L
Agonist Partial Agonist
β2AR ACTIVE
T4L
Nb71 Nb80
Active M2 Muscarinic
Nb9-8
Stabilizing protein
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Fab
β2AR INACTIVE
T4L
Agonist (covalent)
Inverse Agonist
T4L
Pipette
β2AR ACTIVE
Pipette
Other inactive state structures
PAR1 µ Opioid M3 Muscarinic δ Opioid M2 Muscarinic
(Haga and Kobayashi) (Jurgen Wess) (Sebastien Granier) (Shaun Coughlin)
T4L
Agonist Partial Agonist
β2AR ACTIVE
T4L
Nb71 Nb80
Active M2 Muscarinic
Nb9-8
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Technical contributions to crystallizing the β2AR-Gs complex
• High-affinity agonist BI-167107 (1 of ~ 60 screened) • Detergent: MNG-3 (long-term storage, aids transition into LCP) • Lipidic cubic phase crystallography: New mesophase lipid (7.7 MAG) to
accommodate G protein (provided by Martin Caffrey) • Nanobody to stabilize G protein complex (Jan Steyaert) • Protein engineering: amino terminal T4 Lysozyme • Project guided by data from negative stain single particle EM
(Georgios Skiniotis) • Mini-beam X-ray technology (GM/CA, Argonne National Labs)
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Microcrystallography
GM/CA-CAT at Argonne National Labs
β2AR-Gs complex Andy Kruse, Brian DeVree, Søren Rasmussen me Roger Sunahara
Returning from Argonne with final data set April 2011
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β2AR
Gsαβγ
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β2AR - Inactive β2AR-Gs
Cytoplasmic View
14Å
Active state of β2AR
TM6 TM5 2Å
14Å TM6
TM5
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β2AR
Gα
α-helical domain
Ras-like domain
GDP
Inactive
Gsαβγ
Interactions between the β2AR and Gs promote GDP release….
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β2AR
Inactive
Gsαβγ
β2AR
Gsαβγ
Active
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β2AR
Inactive Active Gsαβγ
α−helical domain
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Computational approaches to drug discovery
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Future Directions
Basal
Adenylyl Cyclase Ca2+ Channel
Efficacy
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GPCR Workshop, Maui, Dec. 2011
β2AR-Gs Team
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•Tong Sun Kobilka •Kobilka lab students, postdoctoral fellows and collaborators (1989-2012) •Bill Weis and Roger Sunahara
Many Thanks
Stanford Søren Rasmussen Foon Sun Thian Tong Sun Kobilka Yaozhong Zou Andrew Kruse Ka Young Chung Jesper Mathiesen Bill Weis
University of Michigan Brian DeVree Diane Calinski Gerwin Westfield Georgios Skiniotis Roger Sunahara
Free University of Brussels Els Pardon Jan Steyaert
β2AR-Gs Team
Trinity College Dublin Joseph Lyons Syed Shah Martin Caffrey
University of Wisconsin Pil Seok Chae Sam Gellman
Financial Support NIH – NINDS and NIGMS Gifts from:
Mathers Foundation Lundbeck We will miss Virgil Woods (UCSD) , 1948-2012
GM/CA staff at Argonne