β-Adrenergic receptors (βARs) are G protein-coupled receptors (GPCRs) that activate
intracellular G proteins upon binding catecholamine agonist ligands such as adrenaline
and noradrenaline1,2. Synthetic ligands have been developed that either activate or
inhibit βARs for the treatment of asthma, hypertension or cardiac dysfunction. These
ligands are classified as either full agonists, partial agonists or antagonists, depending
on whether the cellular response is similar to that of the native ligand, reduced
or inhibited, respectively. However, the structural basis for these different ligand
efficacies is unknown. Here we present four crystal structures of the thermostabilised
turkey (Meleagris gallopavo) β1-adrenergic receptor (β1AR-m23) bound to the full agonists
carmoterol and isoprenaline and the partial agonists salbutamol and dobutamine. In
each case, agonist binding induces a 1 Å contraction of the catecholamine binding
pocket relative to the antagonist bound receptor. Full agonists can form hydrogen
bonds with two conserved serine residues in transmembrane helix 5 (Ser5.42 and Ser5.46),
but partial agonists only interact with Ser5.42 (superscripts refer to Ballesteros-Weinstein
numbering3). The structures provide an understanding of the pharmacological differences
between different ligand classes, illuminating how GPCRs function and providing a
solid foundation for the structure-based design of novel ligands with predictable
efficacies.
Determining how agonists and antagonists bind to the β receptors has been the goal
of research for more than 20 years4-11. Although the structures of the homologous
β1 and β2 receptors12-15 show how some antagonists bind to receptors in the inactive
state16, structures with agonists bound are required to understand subsequent structural
transitions involved in activation. GPCRs exist in an equilibrium between an inactive
state (R) and an activated state (R*) that can couple and activate G proteins17. The
binding of a full agonist, such as adrenaline or noradrenaline, is thought to increase
the probability of the receptor converting to R*, with a conformation similar to that
of opsin18,19. In the absence of any ligand, the βARs exhibit a low level of constitutive
activity, indicating that there is always a small proportion of the receptor in the
activated state, with the β2AR showing a 5-fold higher level of basal activity than
the β1AR20. Basal activity of β2AR is important physiologically, as shown by the T164I4.56
human polymorphism that reduces the basal activity of β2AR to levels similar to β1AR21
and whose expression has been associated with heart disease22.
As a first step towards understanding how agonists activate receptors, we have determined
the structures of β1AR bound to 4 different agonists. Native turkey β1AR is unstable
in detergent23, so crystallization and structure determination relied on using a thermostabilised
construct (β1AR-m23) that contained six point mutations, which dramatically improved
its thermostability24. In addition, the thermostabilising mutations altered the equilibrium
between R and R*, so that the receptor was preferentially in the R state24. However,
it could still couple to G proteins after activation by agonists13 (Supplementary
Fig. 1, Supplementary Tables 1-3), although the activation energy barrier is predicted
to be considerably higher than for the wild-type receptor25. Here we report structures
of β1AR-m23 (see Methods) bound to r-isoprenaline (2.85 Å resolution), r,r-carmoterol
(2.6 Å resolution), r-salbutamol (3.05 Å resolution) and r-dobutamine (two independent
structures at 2.6 Å and 2.5 Å resolution) (Supplementary Table 5). The overall structures
of β1AR-m23 bound to the agonists are very similar to the structure with the bound
antagonist cyanopindolol13, as expected for a receptor mutant stabilised preferentially
in the R state. None of the structures show the outward movement of the cytoplasmic
end of transmembrane helix H6 by 5-6 Å that is observed during light activation of
rhodopsin18,19,26. This suggests that the structures represent an inactive, non-signaling
state of the receptor formed on initial agonist binding.
All four agonists bind in the catecholamine pocket in a virtually identical fashion
(Fig. 1). The secondary amine and β-hydroxyl groups shared by all the agonists (except
for dobutamine, which lacks the β-hydroxyl; see Supplementary Figure 4) form potential
hydrogen bonds with Asp1213.32 and Asn3297.39, while the hydrogen bond donor/acceptor
group equivalent to the catecholamine meta-hydroxyl (m-OH) generally forms a hydrogen
bond with Asn3106.55. In addition, all the agonists can form a hydrogen bond with
Ser2115.42, as seen for cyanopindolol13, and they also induce the rotamer conformation
change of Ser2125.43 so that it makes a hydrogen bond with Asn3106.55. The major difference
between the binding of full agonists compared to the partial agonists is that only
full agonists make a hydrogen bond to the side chain of Ser2155.46 as a result of
a change in side chain rotamer. All of these amino acid residues involved in the binding
of the catecholamine headgroups to β1AR are fully conserved in both β1 and β2 receptors
(Fig. 2). Furthermore, the role of many of these amino acid residues in ligand binding
is supported by extensive mutagenesis studies on β2AR that were performed before the
first β2AR structure was determined27. Thus it was predicted that Asp1133.32, Ser2035.42,
Ser2075.46, Asn2936.55 and Asn3127.39 in β2AR were all involved in agonist binding4,5,7-9
(Fig. 3). Inspection of the region outside the catecholamine binding pocket in the
structures with bound dobutamine and carmoterol allows the identification of non-conserved
residues that interact with these ligands (Fig. 2 and Supplementary Figure 7), which
may contribute to the subtype specificity of these ligands10,28.
There are three significant differences in the β1AR catecholamine binding pocket when
full agonists are bound compared to when an antagonist is bound, namely the rotamer
conformation changes of side chains Ser2125.43 and Ser2155.46 (Fig.3) and the contraction
of the catecholamine binding pocket by ~1 Å, as measured between the Cα atoms of Asn3297.39
and Ser2115.42 (Fig. 4). So why should these small changes increase the probability
of R* formation? Agonist binding has not changed the conformation of transmembrane
helix H5 below Ser2155.46, although significant changes in this region are predicted
once the receptor has reached the fully activated state18,19. The only effect that
the agonist-induced rotamer conformation change of Ser2155.46 appears to have is to
break the van der Waals interaction between Val1724.56 and Ser2155.46, thus reducing
the number of interactions between H4 and H5. As there is only a minimal interface
between transmembrane helices H4 and H5 in this region (Supplementary Table 8 and
Supplementary Fig. 8), this loss of interaction may be significant in the activation
process. In this regard, it is noteworthy that the naturally occurring polymorphism
in β2AR at the H4-H5 interface, T164I4.56, converts a polar residue to a hydrophobic
residue as seen in β1AR (Val1724.56), which results in both reduced basal activity
and reduced agonist stimulation21. This supports the hypothesis that the extent of
interaction between H4 and H5 could affect the probability of a receptor transition
into the activated state.
In contrast to the apparent weakening of helix-helix interactions by the agonist-induced
rotamer conformation change of Ser2155.46, the agonist-induced rotamer conformation
change of Ser2125.43 probably results in the strengthening of interactions between
H5 and H6. Upon agonist binding, Ser2125.43 forms a hydrogen bond with Asn3106.55
(Fig. 3) and, in addition, hydrogen bond interactions to Ser2115.43 and Asn3106.55mediated
by the ligand serve to bridge H5 and H6. The combined effects of strengthening the
H5-H6 interface and weakening the H4-H5 interface could facilitate the subsequent
movements of H5 and H6, as observed in the activation of rhodopsin.
Stabilisation of the contracted catecholamine binding pocket is probably the most
important role of bound agonists in the activation process (Fig. 4). This probably
requires strong hydrogen bonding interactions between the catechol (or equivalent)
moiety and both H5 and H6, and strong interactions between the secondary amine and
β-hydroxyl groups in the agonist and the amino acid side chains in helices H3 and
H7. Reduction in the strength of these interactions is likely to reduce the efficacy
of a ligand29. Both salbutamol and dobutamine are partial agonists of β1AR-m23 (Supplementary
Table 3) and human β1AR. In the case of salbutamol, there are only two predicted hydrogen
bonds between the headgroup and H5/H6, compared to 3-4 potential hydrogen bonds for
isoprenaline and carmoterol. Dobutamine lacks the β-hydroxyl group, which similarly
reduces the number of potential hydrogen bonds to H3/H7 from 3-4 seen in the other
agonists to only 2. We propose that this weakening of agonist interactions with H5/H6
for salbutamol and H3/H7 for dobutamine is a major contributing factor in making these
ligands partial agonists rather than full agonists.
The agonist-bound structures of β1AR suggest there are three major determinants that
dictate the efficacy of any ligand; ligand-induced rotamer conformational changes
of (i) Ser2125.43 and (ii) Ser2155.46 and (iii) stabilization of the contracted ligand
binding pocket. The full agonists studied here achieve all three. The partial agonists
studied here do not alter the conformation of Ser2155.46 and may be less successful
than isoprenaline or carmoterol at stabilizing the contracted catecholamine binding
pocket due to reduced numbers of hydrogen bonds between the ligand and the receptor.
The antagonist cyanopindolol acts as a very weak partial agonist and none of the three
agonist-induced changes are observed. In contrast to partial agonists, neutral antagonists
or very weak partial agonists such as cyanopindolol may also have a reduced ability
to contract the binding pocket due to the greater distance between the secondary amine
and the catechol moiety (or equivalent). For example, the number of atoms in the linker
between the secondary amine and the headgroup of cyanopindolol is 4 whereas the agonists
in this study only contain 2 (Fig. 1 and Supplementary Fig. 4). A ligand with a sufficiently
bulky headgroup that binds with high-affinity and which actively prevents any spontaneous
contraction of the binding pocket and/or Ser5.46 rotamer change, would be predicted
to act as a full inverse agonist. This is indeed what is observed in the recently
determined structure15 of β2AR bound to the inverse agonist ICI 118,551.
The significant structural similarities amongst GPCRs suggests that similar agonist-induced
conformational changes to those we have observed here may also be applicable to many
other members of the GPCR superfamily, though undoubtedly there will be many subtle
variations on this theme.
METHODS SUMMARY
Expression, purification and crystallization
The β44-m23 construct was expressed in insect cells, purified in the detergent Hega-10
and crystallized in the presence of cholesterol hemisuccinate (CHS), following previously
established protocols30. Crystals were grown by vapour diffusion, with the conditions
shown in Supplementary Table 4.
Data collection, structure solution and refinement
Diffraction data were collected from a single cryo-cooled crystal (100 K) of each
complex in multiple wedges at beamline ID23-2 at ESRF, Grenoble. The structures were
solved by molecular replacement using the β1AR structure13 (PDB code 2VT4) as a model
(see Online Methods). Data collection and refinement statistics are presented in Supplementary
Table 5.
Supplementary Material
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