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

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

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

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

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

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

Largest family of membrane proteins in the human genome

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

Rhodopsin Family Tree

The Rhodopsin Family (241 Nonolfactory, Total of 701) Adrenergic

Serotonin Dopamine

Muscarinic

• 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

GPCR Signaling Adenylyl Cyclase Ca2+ Channel

GPCR Signaling

Basal

Adenylyl Cyclase Ca2+ Channel

Basal

Adenylyl Cyclase Ca2+ Channel

GPCR Signaling

Basal

Adenylyl Cyclase Ca2+ Channel

GPCR Signaling

Basal

Adenylyl Cyclase Ca2+ Channel

GPCR Signaling

Basal

Adenylyl Cyclase Ca2+ Channel

GPCR Signaling

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

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

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

Biochemistry Spectroscopy

Crystallography

Crystals grown from wild-type β2AR

50µm

247d3

249d4

245c5

June 05 Nov 05

50 µm

Conventional beam (100 micron)

Microfocus beam (5 micron)

ESRF microfocus beamline ID13, July 2005

Gebhard Schertler

Microfocus beam

20Å

ESRF microfocus beamline ID13, July 2005

Gebhard Schertler

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)

Gs

β2AR No ligand

β2AR + agonist

β2AR + agonist + Gs

Gs

β2AR No ligand

β2AR + agonist

β2AR + agonist + Gs

Gs

β2AR No ligand

β2AR + agonist

β2AR + agonist + Gs

Gs

Structural insights from fluorescence, EPR and NMR spectroscopy

TM5

TM6

Purified protein

High-quality crystal

Conformationally uniform GPCR

TM5

TM6

Poor quality crystal

Conformationally heterogeneous GPCR

Purified protein

TM5 TM6

Challenges for crystallography •Protein dynamics

TM5 TM6

Challenges for crystallography •Protein dynamics •Little polar surface

Challenges for crystallography •Protein dynamics •Little polar surface

Stabilize and increase polar surface •Antibodies •Protein Engineering

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

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.

Rock II Crystals

β2AR-T4L

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

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

Fab

β2AR INACTIVE

Inverse Agonist

T4L

T4L

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

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

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

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

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

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

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

Conventional vs. Camelid IgG

Jan Steyaert Els Pardon

Conventional vs. Camelid IgG

Jan Steyaert Els Pardon

Fab fragment

Nanobody

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)

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

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

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

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

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

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)

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

β2AR

Gsαβγ

β2AR - Inactive β2AR-Gs

Cytoplasmic View

14Å

Active state of β2AR

TM6 TM5 2Å

14Å TM6

TM5

β2AR

Gα

α-helical domain

Ras-like domain

GDP

Inactive

Gsαβγ

Interactions between the β2AR and Gs promote GDP release….

β2AR

Inactive

Gsαβγ

β2AR

Gsαβγ

Active

β2AR

Inactive Active Gsαβγ

α−helical domain

Computational approaches to drug discovery

Future Directions

Basal

Adenylyl Cyclase Ca2+ Channel

Efficacy

GPCR Workshop, Maui, Dec. 2011

β2AR-Gs Team

•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