![Page 1: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/1.jpg)
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
![Page 2: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/2.jpg)
The β2AR modulates the activity of multiple signaling pathways
Basal
Adenylyl Cyclase Ca2+ Channel
Efficacy
![Page 3: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/3.jpg)
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
![Page 4: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/4.jpg)
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
![Page 5: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/5.jpg)
MGQPGNGSAFLLAPNRSHAPDHDVTQQRDEVWVVGMGIVMSLIVLAIVFGNVLVITAIAKFERLQTVTNYFITSLACADLVMGLAVVPFGAHILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQSLLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYANETCCDFFTNQAYAIASSIVSFYVPLVIMVFVYSRVFQEAKRQLQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQDNLIRKEVYILLNWIGYVNSGFNPLIYCRSPDFRIAFQELLCLRRSSLKAYGNGYSSNGNTGEQSGYHVEQEKENKLLCEDLPGTEDFVGHQGTVPSDNIDSQGRNCSTNDSLL
Cloned DNA Sequence (1986) Amino Acid Sequence GAATTCATGCCGCGTTTCTGTGTTGGACAGGGGTGACTTTGTGCCGGATGGCTTCTGTGTGAGAGCGCGCGCGAGTGTGCATGTCGGTGAGCTGGGAGGGTGTGTCTCAGTGTCTATGGCTGTGGTTCGGTATAAGTCTAAGCATGTCTGCCAGGGTGTATTTGTGCCTGTATGTGCGTGCCTCGGTGGGCACTCTCGTTTCCTTCCGAATGTGGGGCAGTGCCGGTGTGCTGCCCTCTGCCTTGAGACCTCAAGCCGCGCAGGCGCCCAGGGCAGGCAGGTAGCGGCCACAGAAGAGCCAAAAGCTCCCGGGTTGGCTGGTAAGCACACCACCTCCAGCTTTAGCCCTCTGGGGCCAGCCAGGGTAGCCGGGAAGCAGTGGTGGCCCGCCCTCCAGGGAGCAGTTGGGCCCCGCCCGGGCCAGCCTCAGGAGAAGGAGGGCGAGGGGAGGGGAGGGAAAGGGGAGGAGTGCCTCGCCCCTTCGCGGCTGCCGGCGTGCCATTGGCCGAAAGTTCCCGTACGTCACGGCGAGGGCAGTTCCCCTAAAGTCCTGTGCACATAACGGGCAGAACGCACTGCGAAGCGGCTTCTTCAGAGCACGGGCTGGAACTGGCAGGCACCGCGAGCCCCTAGCACCCGACAAGCTGAGTGTGCAGGACGAGTCCCCACCACACCCACACCACAGCCGCTGAATGAGGCTTCCAGGCGTCCGCTCGCGGCCCGCAGAGCCCCGCCGTGGGTCCGCCTGCTGAGGCGCCCCCAGCCAGTGCGCTTACCTGCCAGACTGCGCGCCATGGGGCAACCCGGGAACGGCAGCGCCTTCTTGCTGGCACCCAATAGAAGCCATGCGCCGGACCACGACGTCACGCAGCAAAGGGACGAGGTGTGGGTGGTGGGCATGGGCATCGTCATGTCTCTCATCGTCCTGGCCATCGTGTTTGGCAATGTGCTGGTCATCACAGCCATTGCCAAGTTCGAGCGTCTGCAGACGGTCACCAAC
Approaches to characterizing β2AR
structure:
•Sequence analysis • secondary structure (transmembrane domains)
![Page 6: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/6.jpg)
MGQPGNGSAFLLAPNRSHAPDHDVTQQRDEVWVVGMGIVMSLIVLAIVFGNVLVITAIAKFERLQTVTNYFITSLACADLVMGLAVVPFGAHILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQSLLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYANETCCDFFTNQAYAIASSIVSFYVPLVIMVFVYSRVFQEAKRQLQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQDNLIRKEVYILLNWIGYVNSGFNPLIYCRSPDFRIAFQELLCLRRSSLKAYGNGYSSNGNTGEQSGYHVEQEKENKLLCEDLPGTEDFVGHQGTVPSDNIDSQGRNCSTNDSLL
GAATTCATGCCGCGTTTCTGTGTTGGACAGGGGTGACTTTGTGCCGGATGGCTTCTGTGTGAGAGCGCGCGCGAGTGTGCATGTCGGTGAGCTGGGAGGGTGTGTCTCAGTGTCTATGGCTGTGGTTCGGTATAAGTCTAAGCATGTCTGCCAGGGTGTATTTGTGCCTGTATGTGCGTGCCTCGGTGGGCACTCTCGTTTCCTTCCGAATGTGGGGCAGTGCCGGTGTGCTGCCCTCTGCCTTGAGACCTCAAGCCGCGCAGGCGCCCAGGGCAGGCAGGTAGCGGCCACAGAAGAGCCAAAAGCTCCCGGGTTGGCTGGTAAGCACACCACCTCCAGCTTTAGCCCTCTGGGGCCAGCCAGGGTAGCCGGGAAGCAGTGGTGGCCCGCCCTCCAGGGAGCAGTTGGGCCCCGCCCGGGCCAGCCTCAGGAGAAGGAGGGCGAGGGGAGGGGAGGGAAAGGGGAGGAGTGCCTCGCCCCTTCGCGGCTGCCGGCGTGCCATTGGCCGAAAGTTCCCGTACGTCACGGCGAGGGCAGTTCCCCTAAAGTCCTGTGCACATAACGGGCAGAACGCACTGCGAAGCGGCTTCTTCAGAGCACGGGCTGGAACTGGCAGGCACCGCGAGCCCCTAGCACCCGACAAGCTGAGTGTGCAGGACGAGTCCCCACCACACCCACACCACAGCCGCTGAATGAGGCTTCCAGGCGTCCGCTCGCGGCCCGCAGAGCCCCGCCGTGGGTCCGCCTGCTGAGGCGCCCCCAGCCAGTGCGCTTACCTGCCAGACTGCGCGCCATGGGGCAACCCGGGAACGGCAGCGCCTTCTTGCTGGCACCCAATAGAAGCCATGCGCCGGACCACGACGTCACGCAGCAAAGGGACGAGGTGTGGGTGGTGGGCATGGGCATCGTCATGTCTCTCATCGTCCTGGCCATCGTGTTTGGCAATGTGCTGGTCATCACAGCCATTGCCAAGTTCGAGCGTCTGCAGACGGTCACCAAC
Approaches to characterizing β2AR
structure:
•Sequence analysis • secondary structure (transmembrane domains)
Cloned DNA Sequence (1986) Amino Acid Sequence
![Page 7: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/7.jpg)
Approaches to characterizing β2AR
structure:
glycosylation
palmitoylation
•Sequence analysis • secondary structure (transmembrane domains) • post-translational modifications
phosphorylation
![Page 8: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/8.jpg)
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
![Page 9: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/9.jpg)
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
![Page 10: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/10.jpg)
Biochemistry Spectroscopy
Crystallography
![Page 11: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/11.jpg)
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
![Page 12: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/12.jpg)
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
![Page 13: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/13.jpg)
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
![Page 14: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/14.jpg)
% C
hang
e in
Inte
nsity
Conformational changes in TM6 in
response to norepinepherine:
Time (sec) Gayathri Swaminath, 2004
Rhodamine
![Page 15: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/15.jpg)
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
![Page 16: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/16.jpg)
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
![Page 17: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/17.jpg)
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
![Page 18: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/18.jpg)
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
![Page 19: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/19.jpg)
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)
![Page 20: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/20.jpg)
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
![Page 21: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/21.jpg)
Crystals of wild-type β2AR
Nov 2004
50 µm
ESRF microfocus beamline ID13, July 2005
20Å
![Page 22: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/22.jpg)
TM5
TM6
Purified protein
High-quality crystal
Conformationally uniform GPCR
![Page 23: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/23.jpg)
TM5
TM6
Poor quality crystal
Conformationally heterogeneous GPCR
Purified protein
![Page 24: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/24.jpg)
TM5 TM6
Challenges for crystallography •Protein dynamics
![Page 25: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/25.jpg)
TM5 TM6
Challenges for crystallography •Protein dynamics •Little polar surface
![Page 26: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/26.jpg)
Challenges for crystallography •Protein dynamics •Little polar surface
Stabilize and increase polar surface •Antibodies •Protein Engineering
![Page 27: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/27.jpg)
Fab
T4L
TM5 TM6
TM5 TM6
Approaches for GPCR crystallogenesis: •Antibodies and protein engineering
2007 Søren Rasmussen Dan Rosenbaum
![Page 28: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/28.jpg)
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
![Page 29: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/29.jpg)
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
![Page 30: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/30.jpg)
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
![Page 31: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/31.jpg)
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
![Page 32: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/32.jpg)
Fab T4L
β2AR INACTIVE
T4L
Agonist (covalent)
Inverse Agonist
T4L
Pipette
β2AR ACTIVE ?
Dan Rosenbaum Ralph Holl Peter Gmeiner
![Page 33: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/33.jpg)
Fab T4L
β2AR INACTIVE
T4L
Agonist (covalent)
Inverse Agonist
T4L
Pipette
β2AR ACTIVE ?
β2AR β2AR + Agonist
Agonist alone does not fully stabilize active state
![Page 34: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/34.jpg)
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
![Page 35: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/35.jpg)
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
![Page 36: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/36.jpg)
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
![Page 37: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/37.jpg)
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
![Page 38: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/38.jpg)
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)
![Page 39: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/39.jpg)
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
![Page 40: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/40.jpg)
β2AR
Gsαβγ
![Page 41: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/41.jpg)
β2AR-Gs β2AR-Cz
Active Inactive
![Page 42: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/42.jpg)
β2AR - Inactive β2AR-Gs
Cytoplasmic View
14Å
Active state of β2AR
14Å
![Page 43: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/43.jpg)
β2AR-Cz
TM5
TM7 TM6
TM3
Extracellular Surface
Carazolol
D113
N312
S203
W286
S204
S207
![Page 44: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/44.jpg)
β2AR-Gs
TM5
TM7 TM6
TM3
Extracellular Surface
D113
N312
S203
W286
S204
S207
BI-167107
![Page 45: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/45.jpg)
Inactive Active
![Page 46: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/46.jpg)
β2AR-Gs
TM5
TM7 TM6
TM3
Extracellular Surface β2AR-Cz
D113
N312
S203
W286
S204
S207
2Å
![Page 47: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/47.jpg)
Pro 211 Ile121
Phe282
Asn 318
TM5
TM3
TM6
TM7
Extracellular
Cytoplasm
β2AR-Inactive
Packing of conserved amino acids maintains inactive state.
2Å
14Å
![Page 48: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/48.jpg)
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
![Page 49: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/49.jpg)
β2AR
Gα
α-helical domain
Ras-like domain
GDP
Inactive
Gsαβγ
Interactions between the β2AR and Gs promote GDP release….
![Page 50: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/50.jpg)
β2AR
Inactive
Gsαβγ
β2AR
Gsαβγ
Active
![Page 51: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/51.jpg)
β2AR
Inactive Active Gsαβγ
α−helical domain
![Page 52: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/52.jpg)
Mobility of alpha helical domain confirmed by EM
GRas
GαH
Nb37
Gerwin Westfield and Georgios Skiniotis, Univ. Michigan
![Page 53: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/53.jpg)
Future Directions
Basal
Adenylyl Cyclase Ca2+ Channel
Efficacy
![Page 54: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/54.jpg)
GPCR Workshop, Maui, Dec. 2011
β2AR-Gs Team
![Page 55: The dynamic process of GPCR activation: Insights from the ...β. 2. AR-Inactive Cytoplasm β. 2. AR-Active . Pro 211 Ile121 . Phe282 . Asn 318 . TM5 TM3 TM6 TM7 . Rearrangement of](https://reader034.vdocuments.mx/reader034/viewer/2022050602/5fa966e7cc0fd720df15225f/html5/thumbnails/55.jpg)
•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