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Two distinct conformations of helix 6 observed
in antagonist-bound structures of a
β1-adrenergic receptor
Rouslan Moukhametzianov, Tony Warne, Patricia C. Edwards, Maria J. Serrano-Vega, Andrew G. W. Leslie,
Christopher G. Tate1, and Gebhard F. X. Schertler,1,2
Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, United Kingdom
Edited by Martin J. Lohse, Universitat Wurzburg Germany, Wurzburg, Germany, and accepted by the Editorial Board March 25, 2011 (received for review
January 5, 2011)
The β1 -adrenergic receptor (β1 AR) is a G-protein-coupled receptor
whose inactive state structure was determined using a thermostabilized mutant (β1 AR–M23). However, it was not thought to be in a
fully inactivated state because there was no salt bridge between
Arg139 and Glu285 linking the cytoplasmic ends of transmembrane
helices 3 and 6 (the R3.50 − D∕E6.30 “ionic lock”). Here we compare
eight new structures of β1 AR–M23, determined from crystallographically independent molecules in four different crystals with three
different antagonists bound. These structures are all in the inactive
R state and show clear electron density for cytoplasmic loop 3
linking transmembrane helices 5 and 6 that had not been seen previously. Despite significantly different crystal packing interactions,
there are only two distinct conformations of the cytoplasmic end
of helix 6, bent and straight. In the bent conformation, the Arg139Glu285 salt bridge is present, as in the crystal structure of darkstate rhodopsin. The straight conformation, observed in previously
solved structures of β-receptors, results in the ends of helices 3 and
6 being too far apart for the ionic lock to form. In the bent conformation, the R3.50 − E6.30 distance is significantly longer than in rhodopsin, suggesting that the interaction is also weaker, which could
explain the high basal activity in β1 AR compared to rhodopsin.
Many mutations that increase the constitutive activity of G-protein-coupled receptors are found in the bent region at the cytoplasmic end of helix 6, supporting the idea that this region plays an
important role in receptor activation.
membrane protein ∣ seven-transmembrane receptor ∣ conformational
thermostabilization
T
he G-protein-coupled receptor (GPCR) superfamily is responsible for the transduction of a wide variety of extracellular signals, and they are found in almost all eukaryotes, with over
800 different GPCRs in humans (1, 2). Their common seventransmembrane-domain architecture has been preserved and
evolved to sense agonists with remarkable structural diversity and
to couple to a variety of specific signaling pathways. The structures of rhodopsin (3–7), the β1 - and β2 -adrenergic receptors
(β1 AR and β2 AR) (8–11). the adenosine A2A receptor (A2A R)
(12), the dopamine D3 receptor (13), and the CXCR4 chemokine
receptor (14) show that there is a similar overall architecture
for all family A GPCRs (reviewed in refs. 15–17), and there is
probably a corresponding underlying conservation of the activation process (18, 19). With this wealth of structural information,
the challenge is now to correlate the pharmacological properties
of the hormone-binding receptors with their structure. One functional aspect that still requires a structural explanation is the
phenomenon of basal activity—i.e., most GPCRs show some ability to activate G proteins even in the absence of agonists (20, 21)
—and this will be the focus of this report.
Our current understanding of the structural basis for receptor
activation is based largely on the structures determined for the
dark-state of rhodopsin (R state) (3–5) and its light-activated
conformation, opsin (R* state) (6, 7). Rhodopsin and its close
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homologues are unique amongst GPCRs because they contain
a covalently bound inverse agonist, 11-cis retinal, which is converted by a photon of light to all-trans retinal, which is a full agonist for the receptor. Thus rhodopsin functions as a binary switch,
the photon of light converting an inactive form of receptor to one
that is fully competent to activate G proteins (reviewed in ref. 22).
It is essential that rhodopsin shows no G-protein-coupling activity
in the absence of light and, therefore, it has evolved to have absolutely no basal activity within the lifetime of rhodopsin in the
cell. Consequently, it is accepted that the structure of dark-state
rhodopsin represents the fully inactive state of a GPCR. Upon
activation by light, numerous conformational changes around
the retinal binding pocket result in the cytoplasmic end of transmembrane helix 6 (H6) straightening and moving 6 Å away from
the center of the molecule, accompanied by a smaller movement
and rotation of transmembrane helix 5 (H5). The movements of
H5 and H6 create a cleft in the cytoplasmic face of the opsin that
allows the binding of the C-terminal helix of Gα, thus activating
the G protein.
A key feature of rhodopsin activation is the outward movement of H6 away from H3 (6, 7, 23). In the rhodopsin structure,
there is a salt-bridge R3.50 − E6.30 (Ballesteros-Weinstein numbering system is in superscripts; ref. 24), which links the cytoplasmic
ends of H6 and H3. Both R3.50 and D∕E6.30 are highly conserved
throughout the GPCR superfamily and, prior to the structure
determination of rhodopsin, had already been suggested to provide a constraint that stabilized the inactive state of GPCRs, leading to this interaction being termed the ionic lock (25). In the case
of rhodopsin, the R3.50 − E6.30 salt bridge was proposed to be one
of the major determinants for the stability of the inactive state.
Hormone receptors have a far more complex pharmacology
than does rhodopsin. Many hormone-binding GPCRs can partially activate their respective G proteins in the absence of agonist
ligand (26). The extent of this activity varies among GPCRs and
their subtypes, reflecting different needs for a basal level of signaling in a physiological context. Receptors with no basal activity,
Author contributions: R.M., T.W., C.G.T., and G.F.X.S. designed research; R.M., T.W., P.C.E.,
and M.J.S.-V. performed research; R.M. and A.G.W.L. analyzed data; and R.M., A.G.W.L.,
C.G.T., and G.F.X.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. M.J.L. is a guest editor invited by the Editorial
Board.
Freely available online through the PNAS open access option.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org [PDB ID codes 2YCW (t1118), 2YCX (t148), 2YCY (t468),
2YCZ (t756)].
1
To whom correspondence may be addressed. E-mail: [email protected] and cgt@
mrc-lmb.cam.ac.uk.
2
Present address: Paul Scherrer Institut, Laboratory of Biomolecular Research, OFLC 109,
CH-5232 Villigen PSI, Switzerland.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1100185108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1100185108
such as rhodopsin, seem appropriate for mediating sensory inputs
that control how well we can see in the dark, where any basal
activity would add undesirable noise. In contrast, increased levels
of basal activity may be more useful for the controlled modulation
of an activity such as heart rate that varies over a range of only
two- to fivefold. It is thought that basal activity is caused by a
subset of receptor conformations, which resemble the fully activated agonist-bound state despite the absence of bound agonist,
and that these can bind and activate their respective G proteins.
Recent structures of β1 AR and β2 AR have greatly increased
our understanding of how ligands are recognized and the overall
architecture of the receptors (8–11, 27). However, there are also
many questions that remain unresolved. One feature of all of the
β-receptor structures is that they appear to represent inactive
states (i.e., they are most similar to the structure of dark-state
rhodopsin), yet the ionic lock between helices 3 and 6 is invariably
broken. This observation raises the question of whether the β-receptors can adopt a conformation analogous to dark-state rhodopsin and, if so, how this affects their basal activity. Existing
β-receptor crystal structures cannot address this issue, because
either the cytoplasmic end of H6 is disordered and unresolved
or it is potentially perturbed by the interaction with an antibody
fragment or a T4 lysozyme fusion. Here we present eight structures of the β1 AR, which show that the receptor can indeed form
an inactive state with an ionic lock analogous to the dark-state of
rhodopsin, in addition to an inactive state where the ionic lock is
broken.
Crystallization and Structure Determination. The turkey β1 AR construct that was crystallized contains amino acid deletions in the
flexible regions at the N and C termini and in cytoplasmic loop 3
(CL3), and is called β36 (28). Six thermostabilizing mutations
were introduced to make the receptor more tolerant to shortchain detergents (29), resulting in the β36–m23 construct whose
structure was determined to 2.7-Å resolution with cyanopindolol
bound (11). During the extensive screening required to obtain
these crystals (29), lower resolution datasets were collected from
a variety of crystals containing either bound cyanopindolol (crystals t148, t468, resolution limits 3.25 and 3.15 Å) or iodocyanopindolol (crystal t756, resolution limit 3.65 Å). Cocrystallization
of β36–m23 with the high-affinity antagonist carazolol also led
to crystals suitable for structure determination (crystal t1118,
resolution limit 3.00 Å). All structures were solved by molecular
replacement using the structure of β36–m23 (PDB ID code
2VT4) as a model. There are two monomers (A and B) in the
crystallographic asymmetric unit of each crystal, resulting in a
total of eight structures for β36–m23. Data collection and refinement statistics are shown in Table S1.
Crystal Packing Interactions. Crystals of β36–m23 containing bound
carazolol grew in the monoclinic space group P21 , whereas crystals of β36–m23 with cyanopindolol or iodocyanopindolol bound
grew in the monoclinic space group C2 and displayed significant
variations in lattice parameters (Table S1) as a result of different
cryoprotection protocols (Methods; ref. 28). The packing interactions between receptors in the four crystals are quite different,
although in each case the crystals appear to be built from similar
nonphysiological antiparallel dimers with the dimer interface
comprising H4 and H5 (Fig. S1). Analysis of the crystal packing
interactions (Table S2) shows that the third CL3 region linking
transmembrane helices 5 and 6 is always involved in intermolecular contacts (although to different extents) and suggests that
one of the two distinct conformations of this region (see following
section) is selected by the ability to provide stabilizing crystal contacts (Fig. 1). Both CL3 conformations are observed (in different
monomers) in the carazolol-bound and in the iodocyanopindololbound structures, demonstrating that the different conformations
Moukhametzianov et al.
BIOCHEMISTRY
Results
Fig. 1. Crystal contacts involving CL3. Lattice contacts between different
monomers of the antiparallel crystallographic dimer are compared for different crystal forms. The thickness of the strand representing the Cα backbone
reflects the temperature factor (increasing thickness reflects higher B values).
Residues making crystal contacts are shown explicitly. In the case of t148,
t468, t756 C2 monoclinic crystals, CL3 is in contact with CL3 of the symmetry
related molecule.
are not the result of different crystal growth conditions. In addition, the two conformations show no correlation to the nature of
antagonist ligand. The importance of the CL3 region in forming
lattice contacts probably explains the success of the deletions
made to enhance crystallization (residues 244–271 and 277–278).
The full length CL3 is likely to be conformationally mobile, hindering formation of a stable crystal lattice.
Overall Comparison of the β1 AR Structures
The eight receptor structures, regardless of the type of antagonist
bound, are very similar to the previously reported cyanopindololbound structure with respect to the hydrophobic transmembrane
regions (residues 54–67, 78–101, 114–143, 158–179, 208–234,
289–306, 323–345) with rmsds for Cα atoms ranging from 0.03 to
0.53 Å (Table S3). There are also only minor differences in the
previously defined extracellular and intracellular loops (rmsds in
Cα atoms 0.6–0.7 Å). However, in the case of CL3, there were
substantial differences between some of the monomers. CL3
comprises the cytoplasmic extensions to transmembrane helices
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tion between them. In the previous structure of β1 AR (PDB ID
code 2VT4), 15 residues in the CL3 region (239–283, but note
that residues 244–271 and 277–278 are not in the construct) were
disordered and therefore not modeled (11). Amongst the eight
structures reported here, two distinct conformations of the CL3
region were observed (Fig. 2). Both conformations are clearly in
the inactive R state as neither show the characteristic conformation changes observed in opsin.
Structural Differences in CL3 and H6
Transmembrane helix H6 in the published structure of cyanopindolol-bound β1 AR–M23 (11) extends from residue Glu2856.30 to
Phe3156.60 , with a single kink formed by Pro3056.50 . The cytoplasmic membrane boundary is estimated to be in the region of
Ala288 to Leu289, so the cytoplasmic helical extension of H6
is relatively short (5–6 residues). In one of the two distinct conformations of CL3 observed in the new structures, the conformation of H6 is unchanged but the cytoplasmic extension is longer
by approximately 10 Å, starting at residue Lys276. In the second
conformation, there is a marked kink in the cytoplasmic extension
of H6 at residue Ala2886.33 (t1118, t148) or Lys2876.32 (t756).
Residues 274–286 are still α-helical and the helix is just as long,
but the helix axis is bent by about 35° toward CL2 and rotated
about 10° around the helix axis clockwise if viewed from the
cytoplasmic side. The two conformations are therefore referred
to as “straight” or “bent” accordingly (Fig. 3). The folding of the
Fig. 3. Interhelical polar contacts at the cytoplasmic face of β1 AR–M23.
(A) The structure of the bent conformation is depicted, with the intact ionic
lock between Arg139 and Glu285. (B) The structure of the straight conformation is shown, with the ionic lock broken. Structures are shown as cartoons
in rainbow coloration (N terminus in blue, C terminus in red), except for CL2
and the cytoplasmic extension of H6 which are in gray and residues predicted
to form interhelical hydrogen bonds shown as sticks.
Fig. 2. Two conformations of CL3. (A) Superposition of the straight and bent
H6 conformations of the carazolol-bound structures of β1 AR–M23 (crystal
t1118) colored according to the B factor (increasing values from blue to
red). (B) Close up of the CL3 region from panel A. (C and D) Omit densities
of the CL3 for the bent and straight conformations, respectively (t1118
crystal). Densities are contoured either at (C) 0.8σ or (D) 0.7σ (gray mesh),
along with the Cα trace (red).
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CL3 region is highly conserved within these two conformations
(rmsds in Cα atoms of 0.28–0.39 Å for four structures in the
straight conformation and 0.97–1.21 Å for four structures in
the bent conformation; Fig. 2, Tables S3 and S4).
In structures with H6 in the bent conformation, H5 is extended
by one helical turn relative to H6 in the straight conformation.
The remaining residues Lys240–Ser273 (bent conformation),
Gln237–Arg275 (straight conformation) form an extended loop.
The omit densities for the CL3 region in the carazolol structure
are shown in Fig. 2. The electron density for the bent conformation is systematically better defined in all structures. The straight
conformation has breaks in the density that correspond to the
residues with very high B factors in this region.
The bent conformation of H6 is partially stabilized by van der
Waals interactions primarily involving Met281 and Leu282 with
side chains on H5. The salt bridge between the highly conserved
Arg1393.50 of the E(D)RY motif on H3 and Glu2856.30 on H6
(the ionic lock) will provide additional stabilization. However,
the minimum distance between a nitrogen atom of the guanidinium group of Arg1393.50 and a carboxylate oxygen of Glu2856.30
Moukhametzianov et al.
Discussion
Crystallization of β1 AR–M23 in different crystal forms with three
different ligands has resulted in receptor structures that are
virtually identical except for the different conformations of the
cytoplasmic end of H6. The fact that only two conformations
of H6 are observed, either bent or straight, despite the range
of different crystallization conditions and crystal packing interactions, suggests that these reflect unbiased conformations that
could be adopted in the native cell membranes. This idea is consistent with current thinking that GPCRs are in a finely balanced
dynamic equilibrium between multiple inactive states and multiple activated states, even in the absence of ligands (18, 19).
Recent molecular dynamics simulations performed on the β2 AR
also indicate the coexistence of two different inactive conformations with the ionic lock either intact or disrupted (30). The conformation corresponding to an intact ionic lock in the β2 AR
simulation is broadly similar to that observed in the crystal structures, except that H6 was described as kinking slightly at
Gly2766.38 compared with the more marked kink observed at
Lys2876.32 ∕Ala2886.33 in β1 AR. Given that the bent conformation
is very similar to that observed in rhodopsin (Figs. S2 and S3), this
structure probably represents the most inactive state of receptor
—i.e., the conformation that would be expected if an inverse
agonist were bound (Rinv ). In this conformation, the cytoplasmic
extension of H6 blocks the G-protein peptide binding site observed in the low-pH structure of opsin (7). In the straight conformation, there is still insufficient room for the C-terminal
peptide of the G protein to insert into the receptor, so further
conformational changes upon agonist binding are required for
productive G-protein coupling. Thus transformation of H6 by
the conformational dynamics of the H2866.31 KAL2896.34 region
is one of the essential steps required for G-protein signaling.
Rhodopsin is unusual amongst GPCRs because its basal activity is virtually zero, which has been ascribed in part to the ionic
lock between R3.50 and E6.30 with the distance between the N-O of
the side chains being 2.8–3.2 Å. In contrast, the basal activity of
β1 AR is much higher than rhodopsin and there is a significantly
longer distance between the equivalent side chains of 3.7–3.8 Å.
The difference in the length of the ionic interaction will undoubtedly affect its strength and hence the conformational flexibility
of the region, which could contribute to the higher basal activity
of β1 AR. However, clearly this is not the only structural feature
in GPCRs that affects basal activity. For example, the naturally
occurring polymorphism in human β2 AR, T164I4.56 , is at the
H4–H5 interface and significantly reduces both basal activity
and the agonist-induced activation of the receptor (31). In addition, the recent structure of the dopamine (D3) receptor (D3R)
shows that it contains an intact ionic lock, with the N-O distance
of 2.5 Å (13). There is also no perceptible kink in the cytoplasmic
end of H6 of D3R equivalent to the kink observed in the β1 AR
structures presented here. Thus, although the region of the ionic
lock is conserved, there are clearly subtle variations in the structure between different receptors.
The ability of the HKAL sequence to have two structurally
different conformations constitutes a ligand-independent mechanism that could affect the basal activity of β1 AR and other
GPCRs, depending upon where the equilibrium between the different conformations of the cytoplasmic extension of H6 lies. If
the equilibrium in one GPCR favors the bent conformation with
an intact ionic lock (Rinv ), as is found in rhodopsin, then the basal
activity of the receptor is likely to be low; a strong salt bridge
between R3.50 −D∕E6.30 will produce this situation. If the salt
bridge is weak or absent, the equilibrium will favor formation
of the straight conformation where the ionic lock is open. Once
this state is attained, there are no strong interactions between the
cytoplasmic ends of H3 and H6 that will hinder the movement of
H6 to adopt the conformation (R*) capable of coupling to G
proteins; i.e., the probability of R* formation increases once the
ionic lock is open. Thus the dynamics of the HKAL region can
potentially have an effect on the level of basal activity of a receptor and in this regard it is noteworthy that many constitutively
activating mutations are found here, particularly in position
6.34 (Fig. 4). Thus the HKAL region has probably evolved to
maintain a level of basal activity that is compatible with the physiological function of the receptor, with some receptors having
higher levels of basal activity than others. However, there are
many sites that can affect the basal activity of receptors, and
the importance of the HKAL sequence might not be general
for all rhodopsin-like GPCRs. For example, the M5 muscarinic
receptor, when mutated in the same 6.34 site, was devoid of constitutive activity although this site was still found to be the determinant of M5 receptor affinity to the G protein (32). In contrast,
all 19 amino acids substitutions in this site in the α1B adrenergic
Fig. 4. Sites of constitutively activating mutations (CAMs) in the CL3 region for different GPCRs. The secondary structure of the bent conformation with
the ionic lock present is shown (Left) along with the CL3 region including the ends of TM5 and TM6 enlarged (Middle). The corresponding amino sequence
of β36–m23 is shown for H6, with residues corresponding to CAMs in the β2 adrenergic receptor shown in yellow and the thermostabilizing mutation A282L
shown in red. GPCRs with CAMs at the 6.34 site are shown on the right with reference to their evolutionary relationships. Amino acid residues corresponding to
Leu289 (6.34) in the turkey β1 AR are shown in parentheses.
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BIOCHEMISTRY
(the N-O distance) is consistently between 3.7–3.9 Å in the bent
conformation, which is significantly longer than seen in dark-state
rhodopsin (2.8–3.2 Å; Table S4). An additional potential hydrogen bond is formed between His286 and Gln237 in H5. All these
polar interactions are absent in the straight conformation of H6,
with a minimum N-O distance for the ionic lock of 6.2 Å. In the
straight conformation of H6, there are no hydrogen bonds apparent between the cytoplasmic extension of H6 and the neighboring
H5, although there are van der Waals contacts between Leu282 in
H6 and Ile241 in H5.
receptor have shown different degrees of constitutive activity
(33). In the rat μ-opioid receptor, different substitutions of
Thr6.34 had different effects; the lysine mutant was constitutively
active, whereas the aspartate mutant had lower basal activity and
decreased ability to activate G proteins (34). Several human diseases have also been caused by mutations in this region. Mutation
of this site in the luteinizing hormone receptor gene causes male
gonadotropin-independent precocious puberty (35) and the analogous mutation in the thyrotropin receptor causes hyperfunctioning thyroid adenomas (36). Increased activity of these
receptors could be due to the perturbing effect of the mutations
on the structure and stabilization of the inactive state.
Methods
Purification and Crystallization. For the preparation of β1 AR36–M23 with
either cyanopindolol or iodocyanopindolol bound (crystals t148, t468, and
t756), baculovirus-expressed receptor was solubilized in β-decylmaltoside
and purified by Ni2þ -affinity chromatography and alprenolol sepharose
ligand affinity chromatography, with detergent exchange to octylthioglucoside (0.35%) on the ligand affinity column (28, 37). Crystallization was by
vapor diffusion with addition of receptor (5.5 mg∕mL) to an equal volume
(0.5–1.0 μL) 0.1 M N-(2-acetamido)iminodiacetate-NaOH, pH 6.9–7.3, 29–33%
PEG 600.
For crystallization of β1 AR36–M23 with carazolol bound (crystal t1118),
detergent was exchanged to Hega-10 (0.35%) and receptor was eluted with
50 μM carazolol. Prior to crystallization, ligand and detergent concentrations
were increased to 0.5 mM and 0.5%, respectively. Crystallization was by vapor diffusion with addition of 200 nL receptor (9 mg∕mL) to an equal volume
of precipitant solution (0.1 M Tris • HCl, pH 8.1, 40% PEG 400). Crystals were
cryoprotected by a very short soak in either 60% PEG600 (t148), 40% PEG400
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(t1118), or 35% PEG600 and 5% glycerol (t468). No additional cryoprotectant
was added to crystal t756 (iodocyanopindolol).
Crystallography. Diffraction data for crystals t148 and t756 were collected at
100 K with a MarCCD detector on beamline ID23-2 at the European Synchrotron Radiation Facility (λ ¼ 0.873 Å). Data for crystals t468 and t1118 were
collected at 100 K with a Pilatus detector on beamline X06SA at the Swiss
Light Source (λ ¼ 0.98 and 1.00 Å, respectively). Each dataset was collected
from multiple positions on a single crystal, following extensive screening.
Diffraction data were processed with MOSFLM and SCALA (38). Because
the diffraction was anisotropic, different wedges of data were processed
to different resolutions and consequently the completeness of the highest
resolution shell is low for crystals t148, t468, and t1118 (Table S1). All structures were obtained with molecular replacement using PHASER (39) starting
from the 2VT4 model (11). Refinement was carried out in PHENIX (40) and
REFMAC (41) with application of strict noncrystallographic symmetry averaging between appropriate regions of the two independent monomers in
the asymmetric unit. Structure validation was performed using the MolProbity Web server.
ACKNOWLEDGMENTS. We thank F. Gorrec for his help with crystallization
robotics and F. Marshall, M. Weir, and R. Henderson for helpful comments
on the manuscript. This work was supported by core funding from the Medical Research Council (MRC) and a grant jointly funded by Pfizer Global
Research and Development and MRC Technology. Additional financial support was from a Human Frontier Science Project Program Grant (RG/0052),
a European Commission FP6 specific targeted research project (LSH-20031.1.0-1), a European Synchrotron Radiation Facility (ESRF) long-term proposal
and a Biotechnology and Biological Sciences Research Council Grant (BB/
G003653/1). We would like to thank beamline staff at the ESRF beamline
ID23-2 and at the Swiss Light Source beamline X06SA.
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