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
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