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

The β2AR modulates the activity of multiple signaling pathways

Basal

Adenylyl Cyclase Ca2+ Channel

Efficacy

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 •Overview of approaches to characterize GPCR structure •GPCR crystallography •Mechanistic insights into GPCR-G protein activation

MGQPGNGSAFLLAPNRSHAPDHDVTQQRDEVWVVGMGIVMSLIVLAIVFGNVLVITAIAKFERLQTVTNYFITSLACADLVMGLAVVPFGAHILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQSLLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYANETCCDFFTNQAYAIASSIVSFYVPLVIMVFVYSRVFQEAKRQLQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQDNLIRKEVYILLNWIGYVNSGFNPLIYCRSPDFRIAFQELLCLRRSSLKAYGNGYSSNGNTGEQSGYHVEQEKENKLLCEDLPGTEDFVGHQGTVPSDNIDSQGRNCSTNDSLL

Cloned DNA Sequence (1986) Amino Acid Sequence GAATTCATGCCGCGTTTCTGTGTTGGACAGGGGTGACTTTGTGCCGGATGGCTTCTGTGTGAGAGCGCGCGCGAGTGTGCATGTCGGTGAGCTGGGAGGGTGTGTCTCAGTGTCTATGGCTGTGGTTCGGTATAAGTCTAAGCATGTCTGCCAGGGTGTATTTGTGCCTGTATGTGCGTGCCTCGGTGGGCACTCTCGTTTCCTTCCGAATGTGGGGCAGTGCCGGTGTGCTGCCCTCTGCCTTGAGACCTCAAGCCGCGCAGGCGCCCAGGGCAGGCAGGTAGCGGCCACAGAAGAGCCAAAAGCTCCCGGGTTGGCTGGTAAGCACACCACCTCCAGCTTTAGCCCTCTGGGGCCAGCCAGGGTAGCCGGGAAGCAGTGGTGGCCCGCCCTCCAGGGAGCAGTTGGGCCCCGCCCGGGCCAGCCTCAGGAGAAGGAGGGCGAGGGGAGGGGAGGGAAAGGGGAGGAGTGCCTCGCCCCTTCGCGGCTGCCGGCGTGCCATTGGCCGAAAGTTCCCGTACGTCACGGCGAGGGCAGTTCCCCTAAAGTCCTGTGCACATAACGGGCAGAACGCACTGCGAAGCGGCTTCTTCAGAGCACGGGCTGGAACTGGCAGGCACCGCGAGCCCCTAGCACCCGACAAGCTGAGTGTGCAGGACGAGTCCCCACCACACCCACACCACAGCCGCTGAATGAGGCTTCCAGGCGTCCGCTCGCGGCCCGCAGAGCCCCGCCGTGGGTCCGCCTGCTGAGGCGCCCCCAGCCAGTGCGCTTACCTGCCAGACTGCGCGCCATGGGGCAACCCGGGAACGGCAGCGCCTTCTTGCTGGCACCCAATAGAAGCCATGCGCCGGACCACGACGTCACGCAGCAAAGGGACGAGGTGTGGGTGGTGGGCATGGGCATCGTCATGTCTCTCATCGTCCTGGCCATCGTGTTTGGCAATGTGCTGGTCATCACAGCCATTGCCAAGTTCGAGCGTCTGCAGACGGTCACCAAC

Approaches to characterizing β2AR

structure:

•Sequence analysis • secondary structure (transmembrane domains)

MGQPGNGSAFLLAPNRSHAPDHDVTQQRDEVWVVGMGIVMSLIVLAIVFGNVLVITAIAKFERLQTVTNYFITSLACADLVMGLAVVPFGAHILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQSLLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYANETCCDFFTNQAYAIASSIVSFYVPLVIMVFVYSRVFQEAKRQLQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQDNLIRKEVYILLNWIGYVNSGFNPLIYCRSPDFRIAFQELLCLRRSSLKAYGNGYSSNGNTGEQSGYHVEQEKENKLLCEDLPGTEDFVGHQGTVPSDNIDSQGRNCSTNDSLL

GAATTCATGCCGCGTTTCTGTGTTGGACAGGGGTGACTTTGTGCCGGATGGCTTCTGTGTGAGAGCGCGCGCGAGTGTGCATGTCGGTGAGCTGGGAGGGTGTGTCTCAGTGTCTATGGCTGTGGTTCGGTATAAGTCTAAGCATGTCTGCCAGGGTGTATTTGTGCCTGTATGTGCGTGCCTCGGTGGGCACTCTCGTTTCCTTCCGAATGTGGGGCAGTGCCGGTGTGCTGCCCTCTGCCTTGAGACCTCAAGCCGCGCAGGCGCCCAGGGCAGGCAGGTAGCGGCCACAGAAGAGCCAAAAGCTCCCGGGTTGGCTGGTAAGCACACCACCTCCAGCTTTAGCCCTCTGGGGCCAGCCAGGGTAGCCGGGAAGCAGTGGTGGCCCGCCCTCCAGGGAGCAGTTGGGCCCCGCCCGGGCCAGCCTCAGGAGAAGGAGGGCGAGGGGAGGGGAGGGAAAGGGGAGGAGTGCCTCGCCCCTTCGCGGCTGCCGGCGTGCCATTGGCCGAAAGTTCCCGTACGTCACGGCGAGGGCAGTTCCCCTAAAGTCCTGTGCACATAACGGGCAGAACGCACTGCGAAGCGGCTTCTTCAGAGCACGGGCTGGAACTGGCAGGCACCGCGAGCCCCTAGCACCCGACAAGCTGAGTGTGCAGGACGAGTCCCCACCACACCCACACCACAGCCGCTGAATGAGGCTTCCAGGCGTCCGCTCGCGGCCCGCAGAGCCCCGCCGTGGGTCCGCCTGCTGAGGCGCCCCCAGCCAGTGCGCTTACCTGCCAGACTGCGCGCCATGGGGCAACCCGGGAACGGCAGCGCCTTCTTGCTGGCACCCAATAGAAGCCATGCGCCGGACCACGACGTCACGCAGCAAAGGGACGAGGTGTGGGTGGTGGGCATGGGCATCGTCATGTCTCTCATCGTCCTGGCCATCGTGTTTGGCAATGTGCTGGTCATCACAGCCATTGCCAAGTTCGAGCGTCTGCAGACGGTCACCAAC

Approaches to characterizing β2AR

structure:

•Sequence analysis • secondary structure (transmembrane domains)

Cloned DNA Sequence (1986) Amino Acid Sequence

Approaches to characterizing β2AR

structure:

glycosylation

palmitoylation

•Sequence analysis • secondary structure (transmembrane domains) • post-translational modifications

phosphorylation

Approaches to characterizing β2AR

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

Approaches to characterizing β2AR

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

Biochemistry Spectroscopy

Crystallography

Approaches to characterizing β2AR

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

Approaches to characterizing β2AR

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

Approaches to characterizing β2AR

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

•Spectroscopy (Fluorescence, EPR, NMR) •ligand-specific conformational states •dynamic, flexible character •useful tool for monitoring receptor activity

TM6 Cysteine 265

HHHHHH

% C

hang

e in

Inte

nsity

Conformational changes in TM6 in

response to norepinepherine:

Time (sec) Gayathri Swaminath, 2004

Rhodamine

Rapid t1/2 < 5 sec

Slow t1/2 = 70 sec

Conformational changes in TM6 in response to norepinepherine:

% C

hang

e in

Inte

nsity

Time (sec) Gayathri Swaminath, 2004

Biphasic changes in intensity over time

Rhodamine

Rapid t1/2 < 5 sec

Slow t1/2 = 70 sec

Conformational changes in TM6 in response to norepinepherine:

Not consistent with single active state

% C

hang

e in

Inte

nsity

Time (sec)

A + R AR*

Gayathri Swaminath, 2004

Biphasic changes in intensity over time

Rhodamine

Rapid t1/2 < 5 sec

Slow t1/2 = 70 sec

Conformational changes in TM6 in response to norepinepherine:

Sequential conformational changes

% C

hang

e in

Inte

nsity

Time (sec)

A + R AR+ AR*

Gayathri Swaminath, 2004

Biphasic changes in intensity over time

Rhodamine

Fluorescence lifetime experiments •Multiple agonist states •Ligand-specific conformational states

Basal

Efficacy

A + R AR+ AR* P + R PR+ PR#

AR* PR# AR*

AR+

Pejman Ghanouni, 2001

Fluorescein

Insights from spectroscopy studies -fluorescence, NMR, EPR-

•The β2AR 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

TM6 Ansgar Philippsen & Ron Dror (D.E. Shaw Research)

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 •Overview of approaches to characterize GPCR structure •GPCR crystallography •Mechanistic insights into GPCR-G protein activation

Crystals of wild-type β2AR

Nov 2004

50 µm

ESRF microfocus beamline ID13, July 2005

20Å

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

Fab

T4L

TM5 TM6

TM5 TM6

Approaches for GPCR crystallogenesis: •Antibodies and protein engineering

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

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 T4L

T4L

Recent GPCR-T4L structures from Kobilka Lab and collaborators

PAR1

Antibodies and Protein Engineering

Thermostabilization through alanine scanning mutations – β1AR, Adenosine A2A – Tate and Schertler

Inactive-state GPCR structures

Adenosine A2A D3 Dopamine CXCR4 Histamine S1P1 κ-opioid

Rhodopsin (native) •Schertler (2D crystals) - 1997 •Palczewski and Okada (3D) - 2000 •Ernst and Hofmann (Opsin) - 2008

Other Approaches

µ Opioid M3 Muscarinic δ Opioid M2 Muscarinic

(Haga and Kobayashi) (Jurgen Wess) (Sebastien Granier) (Shaun Coughlin)

Stevens Lab and collaborators

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 •Overview of approaches to characterize GPCR structure •GPCR crystallography •Mechanistic insights into GPCR-G protein activation

Fab T4L

β2AR INACTIVE

T4L

Agonist (covalent)

Inverse Agonist

T4L

Pipette

β2AR ACTIVE ?

Dan Rosenbaum Ralph Holl Peter Gmeiner

Fab T4L

β2AR INACTIVE

T4L

Agonist (covalent)

Inverse Agonist

T4L

Pipette

β2AR ACTIVE ?

β2AR β2AR + Agonist

Agonist alone does not fully stabilize active state

Fab T4L

β2AR INACTIVE

T4L

Agonist (covalent)

Inverse Agonist

T4L

Pipette

β2AR ACTIVE ?

β2AR β2AR + Agonist

Stabilizing protein

Agonist alone does not fully stabilize active state

Fab T4L

β2AR INACTIVE

T4L

Agonist (covalent)

Inverse Agonist

T4L

Jan Steyaert Els Pardon

Nanobody variable domain of a

single chain camelid antibody

β2AR ACTIVE ?

β2AR β2AR + Agonist

Stabilizing protein

Agonist alone does not fully stabilize active state

Pipette

Fab T4L

β2AR INACTIVE

T4L

Agonist (covalent)

Inverse Agonist Agonist

β2AR ACTIVE

Nb80

T4L

Partial Agonist

Nb71

T4L

Jan Steyaert Els Pardon

Nanobody variable domain of a

single chain camelid antibody

Stabilizing protein

Pipette

Fab T4L

β2AR INACTIVE

T4L

Agonist (covalent)

Inverse Agonist Agonist

β2AR ACTIVE

Nb80

T4L

Partial Agonist

Nb71

T4L

Jan Steyaert Els Pardon

Nanobody variable domain of a

single chain camelid antibody

β2AR-Gs

Pipette

Technical contributions to crystallizing the β2AR-Gs complex

• High-affinity agonist BI-167107 (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)

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-Gs β2AR-Cz

Active Inactive

β2AR - Inactive β2AR-Gs

Cytoplasmic View

14Å

Active state of β2AR

14Å

β2AR-Cz

TM5

TM7 TM6

TM3

Extracellular Surface

Carazolol

D113

N312

S203

W286

S204

S207

β2AR-Gs

TM5

TM7 TM6

TM3

Extracellular Surface

D113

N312

S203

W286

S204

S207

BI-167107

Inactive Active

β2AR-Gs

TM5

TM7 TM6

TM3

Extracellular Surface β2AR-Cz

D113

N312

S203

W286

S204

S207

2Å

Pro 211 Ile121

Phe282

Asn 318

TM5

TM3

TM6

TM7

Extracellular

Cytoplasm

β2AR-Inactive

Packing of conserved amino acids maintains inactive state.

2Å

14Å

Pro 211 Ile121

Phe282

Asn 318

TM5

TM3

TM6

TM7

β2AR-Inactive Extracellular

Cytoplasm

β2AR-Active

Pro 211

Ile121

Phe282

Asn 318

TM5 TM3

TM6

TM7

Rearrangement of conserved amino acids required to

accommodate agonist binding.

2Å

14Å

Weak coupling between ligand binding and G protein coupling domains

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

Mobility of alpha helical domain confirmed by EM

GRas

GαH

Nb37

Gerwin Westfield and Georgios Skiniotis, Univ. Michigan

Future Directions

Basal

Adenylyl Cyclase Ca2+ Channel

Efficacy

GPCR Workshop, Maui, Dec. 2011

β2AR-Gs Team

•Tong Sun Kobilka •Bob Lefkowitz and colleagues in the Lefkolab •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