![Figure 6 | Possible sequence of b2AR–Gs complex formation. a, b, Comparison of the b2AR–Gs structure (green and orange) with metarhodopsin II32 (PDB ID: 3PQR) (purple) bound with thecarboxy-terminal peptide of transducin (Gt) (blue). TM7 has been omitted in panel a to better visualize theGproteins. c, Cartoonof theb2AR–Gas peptide fusion construct used in the binding experiments (d). d, Competition binding experiments between [3H]dihydroalprenolol ([3H]DHA) and full agonist isoproterenol. Top panel shows binding data (reproduced from ref. 12) on b2AR reconstituted in HDL particles with and withoutGsheterotrimer. The fractionofb2AR in the Ki High state for the b2AR with Gs is 0.55. Bottom panel shows binding to b2AR and a b2AR–Gas peptide fusion expressed in Sf9 cell membranes. The fraction of b2AR in the Ki High state for the b2AR– Gas peptide fusion is 0.68. e, The initial interaction of agonist-bound b2AR and GasRasmay involve an orientation of the carboxy terminus of GasRas similar to that of the carboxy-terminal peptide of transducin in the structure of metarhodopsin II. f, The final position of GasRas on the b2AR as observed in the b2AR–Gs complex.](/figures/figure-6-possible-sequence-of-b2ar-gs-complex-formation-a-b-niqj4j6j.webp)
Vrije Universiteit Brussel
Crystal structure of the β2 adrenergic receptor–Gs protein complex
Rasmussen, Soren G. F.; Devree, Brian T.; Zou, Yaozhong; Kruse, A.; Chung, Ka Young;
Kobilka, Tong Sun; Thian, Foon Sun; Chae, Pil Seok; Pardon, Els; Calinski, Diane;
Mathiesen, Jesper M.; Shah, Syed T. A.; Lyons, Joseph A.; Martin, Caffrey; Steyaert, Jan;
Skiniotis, Georgios; Weis, William I.; Sunahara, Roger K.; Kobilka, Brian K
Published in:
Nature
DOI:
10.1038/nature10361
Publication date:
2011
Document Version:
Final published version
Link to publication
Citation for published version (APA):
Rasmussen, S. G. F., Devree, B. T., Zou, Y., Kruse, A., Chung, K. Y., Kobilka, T. S., ... Kobilka, B. K. (2011).
Crystal structure of the 2 adrenergic receptor–Gs protein complex. Nature, 477, 549-555.
https://doi.org/10.1038/nature10361
Copyright
No part of this publication may be reproduced or transmitted in any form, without the prior written permission of the author(s) or other rights
holders to whom publication rights have been transferred, unless permitted by a license attached to the publication (a Creative Commons
license or other), or unless exceptions to copyright law apply.
Take down policy
If you believe that this document infringes your copyright or other rights, please contact openaccess@vub.be, with details of the nature of the
infringement. We will investigate the claim and if justified, we will take the appropriate steps.
ARTICLE
doi:10.1038/nature10361
Crystal structure of the b
2
adrenergic
receptor–Gs protein complex
Søren G. F. Rasmussen
1,2
*, Brian T. DeVree
3
*,YaozhongZou
1
,AndrewC.Kruse
1
, Ka Young Chung
1
, Tong Sun Kobilka
1
,
Foon Sun Thian
1
, Pil Seok Chae
4
,ElsPardon
5,6
, Diane Calinski
3
,JesperM.Mathiesen
1
, Syed T. A. Shah
7
,JosephA.Lyons
7
,
Martin Caffrey
7
, Samuel H. Gellman
4
, Jan Steyaert
5,6
, Georgios Skiniotis
8
, William I. Weis
1,9
,RogerK.Sunahara
3
& Brian K. Kobilka
1
G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and
neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation
of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The b
2
adrenergic receptor (b
2
AR)
activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here
we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric b
2
AR and
nucleotide-free Gs heterotrimer. The principal interactions between the b
2
AR and Gs involve the amino- and
carboxy-terminal a-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The
largest conformational changes in the b
2
AR include a 14 A
˚
outward movement at the cytoplasmic end of
transmembrane segment 6 (TM6) and an a-helical extension of the cytoplasmic end of TM5. The most surprising
observation is a major displacement of the a-helical domain of Gas relative to the Ras-like GTPase domain. This
crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.
Introduction
The b
2
adrenergic receptor (b
2
AR) has been a model system for the
large and diverse family of G protein-coupled receptors (GPCRs) for
over 40 years. It was one of the first GPCRs to be characterized by
radioligand binding, and it was the first neurotransmitter receptor to
be cloned
1
and structurally determined by crystallography
2,3
. The
b
2
AR was initially identified based on its physiological and phar-
macological properties, but it was not known if receptors and G
proteins were separate entities, or parts of the same protein
4
.
Subsequent biochemical studies led to the isolation and purification
of functional b
2
AR and Gs, the stimulatory G protein that activates
adenylyl cyclase, and the reconstitution of this signalling complex in
phospholipid vesicles
5,6
. The cooperative interactions of b
2
AR and Gs
observed in ligand binding assays formed the foundation of the ternary
complex model of GPCR activation
7,8
. In the ternary complex consist-
ing of agonist, receptor and G protein, the affinity of the receptor for
agonist is enhanced and the specificity of the G protein for guanine
nucleotides changes in favour of GTP over GDP. The GPCR field has
evolved markedly since these initial studies. Isolation of the genes and
cDNAs for the b
2
AR and other GPCRs using protein sequencing and
expression cloning led to the expansion of the family by homology
cloning. More recently, sequencing of the human genome led to the
identification of over 800 GPCR genes
9
. Experimental tools for iden-
tifying protein–protein interactions and for expression and silencing
of genes have revealed a complex network of cellular signalling and
regulatory pathways including G protein-independent activation of
cytosolic kinases
10,11
. Nevertheless, the b
2
AR continues to be a relevant
model for most aspects of GPCR pharmacology, signalling and
regulation.
Notwithstanding the remarkable advances in this field, we still
know relatively little about the structural basis for transmembrane
signalling by GPCRs. Figure 1 shows the G protein cycle for the
b
2
AR–Gs complex. Agonist binding to the b
2
AR promotes interac-
tions with GDP-bound Gsabc heterotrimer, leading to the exchange
of GDP for GTP, and the functional dissociation of Gs into G a-GTP
and Gbc subunits. The separate Ga-GTP and Gbc subunits can
modulate the activity of different cellular effectors (channels, kinases
or other enzymes). The intrinsic GTPase activity of Gas leads to
hydrolysis of GTP to GDP and the reassociation of Ga-GDP and
Gbc subunits, and the termination of signalling. The active state of
a GPCR can be defined as that conformation that couples to and
stabilizes a nucleotide-free G protein. In this agonist-b
2
AR–Gs ternary
complex, Gs has a higher affinity for GTP than GDP, and the b
2
AR has
an approximately 100-fold higher affinity for agonists than does b
2
AR
alone. In an effort to understand the structural basis for GPCR signal-
ling, we crystallized the b
2
AR–Gs complex.
Crystallization of the b
2
AR–Gs complex
The first challenge for crystallogenesis was to prepare a stable b
2
AR–Gs
complex in detergent solution. The b
2
AR and Gs couple efficiently in
lipid bilayers, but not in detergents used to solubilize and purify these
proteins. We found that a relatively stable b
2
AR–Gs complex could be
prepared by mixing purified GDP-Gs (approximately 100 mM final
concentration) with a molar excess of purified b
2
AR bound to a high
affinity agonist (BI-167107, Boehringer Ingelheim)
12
in dodecylmalto-
side solution. Apyrase, a non-selective purine pyrophosphatase, was
added to hydrolyse GDP released from Gs on forming a complex with
the b
2
AR. Removal of GDP was essential because both GDP and GTP
*These authors contributed equally to this work.
1
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.
2
Department of Neuroscience and Pharmacology, The Panum Institute,
University of Copenhagen, 2200 Copenhagen N, Denmark.
3
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
4
Department of Chemistry, University of
Wisconsin, Madison, Wisconsin 53706, USA.
5
Department of Molecular and Cellular Interactions, Vlaams Instituut voor Biotechnologie (VIB), Vrije Universiteit Brussel, B-1050 Brussel, Belgium.
6
Structural
Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium.
7
Membrane Structural and Functional Biology Group, Schools of Medicine and Biochemistry & Immunology, Trinity College, Dublin 2,
Ireland.
8
Life Sciences Institute and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
9
Department of Structural Biology, Stanford University School of
Medicine, Stanford, California 94305, USA.
29 SEPTEMBER 2011 | VOL 477 | NATURE | 549
Macmillan Publishers Limited. All rights reserved
©2011
can disrupt the high-affinity interaction between b
2
AR and Gs
(Supplementary Fig. 1a). The complex was subsequently purified by
sequential antibody affinity chromatography and size-exclusion chro-
matography. The stability of the complex was enhanced by exchanging
it into a recently developed maltose neopentyl glycol detergent MNG-3
(NG310, Anatrace)
13
. The complex could be incubated at room tem-
perature for 24 h without any noticeable degradation; however, initial
efforts to crystallize the complex using sparse matrix screens in deter-
gent micelles, bicelles and lipidic cubic phase (LCP) failed.
To further assess the quality of the complex, we analysed the protein
by single particle electron microscopy
34
. The results confirmed that the
complex was monodisperse, but revealed two potential problems for
obtaining diffraction of quality crystals. First, the detergent used to sta-
bilize the complex formed a la rge micelle, leaving little polar surface on
the extracellular side of the b
2
AR–Gs complex for the formation of
crystal lattice contacts. Our initial approach to this problem, which
was to generate antibodies to the extracellular surface, was not successful.
As an alternative approach, we replaced the unstructured amino ter-
minus of the b
2
AR with T4 lysozyme (T4L). We pr eviously used T4L
to facilitate crystallogenesis of the inactive b
2
AR by inserting T4L
between the cytoplasmic ends of TM5 and TM6 (ref. 3). Several different
amino-terminal fusion proteins were prepared and single particle elec-
tron microscopy was used to identify a fusion with a relatively fixed
orientation of T4L in relation to the b
2
AR.
The second problem revealed by single particle electron micro-
scopy analysis was increased variability in the positioning of the
a-helical component of the Gas subunit. Gas consists of two domains,
the Ras-like GTPase domain (GasRas), which interacts with the b
2
AR
and the Gb subunit, and the a-helical domain (GasAH)
14
. The inter-
face of the two Gas subdomains forms the nucleotide-binding pocket
(Fig. 1), and electron microscopy two-dimensional (2D) averages and
three-dimensional (3D) reconstructions show that in the absence of
guanine nucleotide, GasAH has a variable position relative to the
complex of T4L–b
2
AR–GasRas–Gbc (Fig. 1b)
34
.
We attributed the variable position of GasAH to the empty
nucleotide-binding pocket. However, as noted above both GDP and
non-hydrolysable GTP analogues disrupt the b
2
AR–Gs complex (Sup-
plementary Fig. 1). The addition of the pyrophosphate analogue phos-
phonoformate (foscarnet) led to a significant increase in stabiliza-
tion of GasAH as determined by electron microscopy analysis of the
detergent-solubilized complex
34
. Crystallization trials were carried out
in LCP using a modified monolein (7.7 MAG, see Methods) designed
to accommodate the large hydrophilic component of the T4L–b
2
AR–
Gs complex
15
. Although we were able to obtain small crystals that
diffracted to 7 A
˚
, we were unable to improve their quality through
the use of additives and other modifications.
In an effort to generate an antibody that would further stabilize the
complex and facilitate crystallogenesis, we crosslinked b
2
AR and the
Gs heterotrimer with a small, homobifunctional amine-reactive cross-
linker and used this stabilized complex to immunize llamas. Llamas
and other camelids produce antibodies devoid of light chains. The
single domain antigen binding fragments of these heavy-chain-only
antibodies, known as nanobodies, are small (15 kDa), rigid, and are
easily cloned and expressed in Escherichia coli (Methods)
16
.We
obtained a nanobody (Nb35) that binds to the complex and prevents
dissociation of the complex by GTPcS (Supplementary Fig. 1). The
T4L–b
2
AR–Gs–Nb35 complex was used to obtain crystals that grew
to 250 mm (Supplementary Fig. 2) in LCP (7.7 MAG) and diffracted to
2.9 A
˚
. A 3.2 A
˚
data set was obtained from 20 crystals and the structure
was determined by molecular replacement (Methods).
The b
2
AR–Gs complex crystallized in primitive monoclinic space
group P2
1
, with a single complex in each asymmetric unit. Figure 2a
shows the crystallographic packing interactions. Complexes are arrayed
in alternating aqueous and lipidic layers with lattice contacts formed
almost exclusively between soluble components of the complex, leaving
receptor molecules suspended between G protein layers and widely
separated from one another in the plane of the membrane. Extensive
lattice contacts are formed among all the soluble proteins, probably
accounting for the strong overall diffraction and remarkably clear elec-
tron density for the G protein. Nb35 and T4L facilitated crystal forma-
tion. Nb35 packs at the interface of the Gb and Ga subunits, with the
complementarity determining region (CDR) 1 interacting primarily
with Gb and a long CDR3 loop interacting with both Gb and Ga
subunits. The framework regions of Nb35 from one complex also inter-
act with Ga subunits from two adjacent complexes. T4L is linked to the
b
2
AR only through amino-terminal fusion, but packs against the amino
terminus of the Gb subunit of one complex, the carboxy terminus of the
Gc subunit of another complex, and the Ga subunit of yet another
complex.Figure 2bshows the structure of thecomplete complex includ-
ing T4L and Nb35, and Fig. 2c shows the b
2
AR–Gs complex alone.
Structure of the active-state b
2
AR
The b
2
AR–Gs structure provides the first high-resolution insight into
the mechanism of signal transduction across the plasma membrane
by a GPCR, and the structural basis for the functional properties of
the ternary complex. Figure 3a compares the structures of the
agonist-bound receptor in the b
2
AR–Gs complex and the inactive
RR*
Agonist
binding
G protein coupling
and nucleotide exchange
Activated G protein subunits
regulate effector proteins
GTP hydrolysis and
inactivation of Gα protein
Reassembly of heterotrimeric G protein
GTP
GDP
ααα
αα
Ras
α
AH
β
γ
β
β
β
γ
γ
γ
AC
ATP
cAMP
Ca
2+
P
i
β
2
AR
a b
Figure 1
|
G protein cycle for the b
2
AR–Gs complex. a, Extracellular agonist
binding to the b
2
AR leads to conformational rearrangements of the
cytoplasmic ends of transmembrane segments that enable the Gs heterotrimer
(a, b, and c) to bind the receptor. GDP is released from the a subunit upon
formation of b
2
AR–Gs complex. The GTP binds to the nucleotide-free a
subunit resulting in dissociation of the a and bc subunits from the receptor.
The subunits regulate their respective effector proteins adenylyl cyclase (AC)
and Ca
21
channels. The Gs heterotrimer reassembles from a and bc subunits
following hydrolysis of GTP to GDP in the a subunit. b, The purified
nucleotide-free b
2
AR–Gs protein complex maintained in detergent micelles.
The Gas subunit consists of two domains, the Ras domain (aRas) and the
a-helical domain (aAH). Both are involved in nucleotide binding. In the
nucleotide-free state, the aAH domain has a variable position relative the aRas
domain.
RESEARCH ARTICLE
550 | NATURE | VOL 477 | 29 SEPTEMBER 2011
Macmillan Publishers Limited. All rights reserved
©2011
carazolol-bound b
2
AR. The largest difference between the inactive
and active structures is a 14 A
˚
outward movement of TM6 when
measured at the Ca carbon of E268. There is a smaller outward move-
ment and extension of the cytoplasmic end of the TM5 helix by 7
residues. A stretch of 26 amino acids in the third intracellular loop
(ICL3) is disordered. Another notable difference between inactive and
active structures is the second intracellular loop (ICL2), which forms
an extended loop in the inactive b
2
AR structure and an a-helix in the
b
2
AR–Gs complex. This helix is also observed in the b
2
AR–Nb80
structure (Fig. 3b); however, it may not be a feature that is unique
to the active state, because it is also observed in the inactive structure
of the highly homologous avian b
1
AR (ref. 17).
The quality of the electron density maps for the b
2
AR is highest at
the b
2
AR–GasRas interface, and much weaker for the extracellular
half. The extracellular half of the receptor is not stabilized by any
packing interactions either laterally with adjacent receptors in the
membrane or through the extracellular surface. Instead, the extracel-
lular region is indirectly tethered to the well-packed soluble com-
ponents by the amino-terminal fusion to T4 lysozyme (Fig. 2a).
Given the flexible and dynamic nature of GPCRs, the absence of
stabilizing packing interactions may lead to structural heterogeneity
in the extracellular half of the receptor and, consequently, to the limited
quality of the electron density maps. However, the overall structure of
the b
2
AR in the T4L–b
2
AR–Gs complex is very similar to our recent
active-state structure of b
2
AR stabilized by a G protein mimetic nano-
body (Nb80)
12
. In the b
2
AR–Nb80 crystal, each receptor molecule has
extensive packing interactions with adjacent receptors and the quality
of the electron density maps for the agonist-bound b
2
AR in this
complex is remarkably good for a 3.5 A
˚
structure. Therefore, the
b
2
AR–Nb80 structure allows us to confidently model BI-167107 here,
and provide a more reliable view of the conformational rearrange-
ments of amino acids around the ligand-binding pocket and between
the ligand-binding pocket and the Gs-coupling interface
12
.
The overall root mean square deviation between the b
2
AR compo-
nents in the b
2
AR–Gs and b
2
AR–Nb80 structures is approximately
0.6 A
˚
, and they differ most at the cytoplasmic ends of transmembrane
helices 5 and 6 where they interact with the different proteins (Fig. 3b–d).
The largest divergence is a 3 A
˚
outward movement at the end of helix 6
in the b
2
AR–Gs complex. However, the differences between these two
structures are very small at the level of the most highly conserved
amino acids (E/DRY and NPxxY), which are located at the cytoplasmic
ends of the transmembrane segments (Fig. 3c, d). These conserved
sequences have been proposed to be important for activation or for
maintaining the receptor in the inactive state
18
. Of these residues, only
Arg 131 differs significantly between these two structures. In b
2
AR–
Nb80 Arg 131 interacts with Nb80, whereas in the b
2
AR–Gs structure
Arg 131 packs against Tyr 391 of Gas (Supplementary Fig. 3). The high
structural similarity is in agreement with the functional similarity of
these two proteins. The b
2
AR–Nb80 complex shows the same high
affinity for the agonist isoproterenol as does the b
2
AR–Gs complex
12
,
consistent with high structural homology around the ligand binding
pocket.
The active state of the b
2
AR is stabilized by extensive interactions
with GasRas (Fig. 4). There are no direct interactions with G b or Gc
subunits. The total buried surface of the b
2
AR–GasRas interface is
2,576 A
˚
2
(1,300 A
˚
2
for GasRas and 1,276 A
˚
2
for the b
2
AR). This inter-
face is formed by ICL2, TM5 and TM6 of the b
2
AR, and by a5-helix,
the aN–b1 junction, the top of the b3-strand, and the a4-helix of
GasRas (see Supplementary Table 1 for specific interactions). Some
of the b
2
AR sequences involved in this interaction have been shown to
have a role in G protein coupling; however, there is no clear consensus
sequence for Gs-coupling specificity when these segments are aligned
with other GPCRs. Perhaps this is not surprising considering that the
b
2
AR also couples to Gi and that many GPCRs couple to more than
one G protein isoform. Of the 21 amino acids of Gs that are within 4 A
˚
of the b
2
AR, only five are identical between Gs and Gi, and all of these
T4L
β
2
AR
Nb35
Gβ
G
γ
G
αs
BI-167107
BI-167107
90°
ab
c
TM5
αN
Extracellular
α
Ras
α5
α
AH
TM5
TM4
90°
90º
Gβ
Gγ
Cytoplasmic view
α
Ras
α
AH
α
Ras
αN
α5
Figure 2
|
Overall structure of the b
2
AR–Gs
complex. a, Lattice packing of the complex shows
alternating layers of receptor and G protein within
the crystal. Abundant contacts are formed among
proteins within the aqueous layers. b, The overall
structure of the asymmetric unit contents shows
the b
2
AR (green) bound to an agonist (yellow
spheres) and engaged in extensive interactions with
Gas (orange). Gas together with Gb (cyan) and Gc
(purple) constitute the heterotrimeric G protein
Gs. A Gs-binding nanobody (red) binds the G
protein between the a and b subunits. The
nanobody (Nb35) facilitates crystallization, as does
T4 lysozyme (magenta) fused to the amino
terminus of the b
2
AR. c, The biological complex
omitting crystallization aids, showing its location
and orientation within a cell membrane.
ARTICLE RESEARCH
29 SEPTEMBER 2011 | VOL 477 | NATURE | 551
Macmillan Publishers Limited. All rights reserved
©2011
are in the carboxy-terminal a helix. The structural basis for G protein
coupling specificity must therefore involve more subtle features of the
secondary and tertiary structure. Nevertheless, a noteworthy inter-
action involves Phe 139, which is located at the beginning of the ICL2
helix and sits in a hydrophobic pocket formed by Gas His 41 at the
beginning of the b1-strand, Val 217 at the start of the b3-strand and
Phe 376, Cys 379, Arg 380 and Ile 383 in the a5-helix (Fig. 4c). The
b
2
AR mutant F139A has severely impaired coupling to Gs
19
. The
residue corresponding to Phe 139 is a Phe or Leu on almost all Gs
coupled receptors, but is more variable in GPCRs known to couple to
other G proteins. Of interest, the ICL2 helix is stabilized by an inter-
action between Asp 130 of the conserved DRY sequence and Tyr 141
in the middle of the ICL2 helix (Fig. 4c). Tyr 141 has been shown to be
a substrate for the insulin receptor tyrosine kinase
20
; however, the
functional significance of this phosphorylation is currently unknown.
The lack of direct interactions between the b
2
AR and Gbc is some-
what unexpected given that a heterotrimer is required for efficient
coupling to a GPCR. Whereas Gb does not interact directly with the
b
2
AR, it has an indirect but important role in coupling by stabilizing
the amino-terminal a helix of Gas (Fig. 2c). Several models involving
GPCR dimers propose that one of the protomers interacts predomi-
nantly with Ga while the other interacts with Gbc
21–23
. Consistent
with these models, biochemical and biophysical evidence suggests
that Gai2 forms a stable complex with a LTB4 receptor dimer
24
.
Extracellular
TM5
TM6
TM1
β
2
AR–Cz (inactive)
TM5
TM6
ab
c
14 Å
Cytoplasmic view
TM6
TM5
ICL2
TM7
TM3
TM4
H8
TM1
d
β
2
AR–Gs β
2
AR–Gs β
2
AR–Nb80
Y132
Y219
Y326
R131
D130
N322
P323
TM6
E268
TM5
TM7
TM3
β
2
AR–Nb80β
2
AR–Gs
TM5
TM6
E286
Y132
D130
Y326
N322
ICL2
R131
P323
R131
Y219
Cytoplasmic view
TM7
3 Å
Figure 3
|
Comparison of active and inactive
b
2
AR structures. a, Side and cytoplasmic views of
the b
2
AR–Gs structure (green) compared to the
inactive carazolol-bound b
2
AR structure
3
(blue).
Significant structural changes are seen for the
intracellular domains of TM5 and TM6. TM5 is
extended by two helical turns whereas TM6 is
moved outward by 14 A
˚
as measured at the
a-carbons of Glu 268 (yellow arrow) in the two
structures. b, b
2
AR–Gs compared with the
nanobody-stabilized active state b
2
AR–Nb80
structure
12
(orange). c, The positions of residues in
the E/DRY and NPxxY motifs and other key
residues of the b
2
AR–Gs and b
2
AR–Nb80
structures. All residues occupy very similar
positions except Arg 131 which in the b
2
AR–Nb80
structure interacts with the nanobody. d, View
from the cytoplasmic side of residues shown in c.
b
E225
TM5
α5
TM3
T136
R380
Q384
K232
D381
Q229
R228
a
Y326
R131
Y391
L230
L394
A226
V222
I135
A134
N142
H387
L393
L388
I383
TM5
TM3
TM7
α5
c
αN
TM5
TM3
IL2
α5
β1
β3
V217
F376
R380
I383
H41
F139
F139
D130
Y141
T68
TM2
Figure 4
|
Receptor-G protein interactions. a, b,Thea5-helix of Gas docks
into a cavity formed on the intracellular side of the receptor by the opening of
transmembrane helices 5 and 6. a, Within the transmembrane core, the
interactions are primarily non-polar. An exception involves packing of Tyr 391
of the a5-helix against Arg 131 of the conserved DRY sequence in TM3 (see also
Supplementary Fig. 3). Arg 131 also packs against Tyr 326 of the conserved
NPxxY sequence in TM7. b,Asa5-helix exits the receptor it forms a network of
polar interactions with TM5 and TM3. c, Receptor residues Thr 68 and Asp 130
interact with the ICL2 helix of the b
2
AR via Tyr 141, positioning the helix so
that Phe 139 of the receptor docks into a hydrophobic pocket on the G protein
surface, thereby structurally linking receptor–G protein interactions with the
highly conserved DRY motif of the b
2
AR.
RESEARCH ARTICLE
552 | NATURE | VOL 477 | 29 SEPTEMBER 2011
Macmillan Publishers Limited. All rights reserved
©2011