The adenosine receptors and β-adrenoceptors (βARs) are G protein-coupled receptors
(GPCRs) that activate intracellular G proteins upon binding agonist such as adenosine
1
or noradrenaline
2
, respectively. GPCRs have similar structures consisting of 7 transmembrane helices
that contain well-conserved sequence motifs, suggesting that they are probably activated
by a common mechanism
3,4
. Recent structures of βARs highlight residues in transmembrane region 5 that initially
bind specifically to agonists rather than to antagonists, suggesting an important
role in agonist-induced activation of receptors
5-7
. Here we present two crystal structures of the thermostabilised human adenosine A2A
receptor (A2AR-GL31) bound to its endogenous agonist adenosine and the synthetic agonist
NECA. The structures represent an intermediate conformation between the inactive and
active states, because they share all the features of GPCRs that are thought to be
in a fully activated state, except that the cytoplasmic end of transmembrane helix
6 partially occludes the G protein binding site. The adenine substituent of the agonists
bind in a similar fashion to the chemically-related region of the inverse agonist
ZM241385
8
. Both agonists contain a ribose group, not found in ZM241385, which extends deep
into the ligand binding pocket where it makes polar interactions with conserved residues
in H7 (Ser2777.42 and His2787.43; superscripts refer to Ballesteros-Weinstein numbering
9
) and non-polar interactions with residues in H3. In contrast, the inverse agonist
ZM241385 does not interact with any of these residues and comparison with the agonist-bound
structures suggests that ZM241385 sterically prevents the conformational change in
H5 and therefore it acts as an inverse agonist. Comparison of the agonist-bound structures
of A2AR with the agonist-bound structures of β-adrenoceptors suggests that the contraction
of the ligand binding pocket caused by the inward motion of helices 3, 5 and 7 may
be a common feature in the activation of all GPCRs.
In the simplest model for the conformational dynamics of GPCRs
10
there is an equilibrium between two states, R and R*. The inactive state R preferentially
binds inverse agonists and the activated state R* preferentially binds agonists
11
. Only R* can couple and activate G proteins. Although there are far more complex
schemes
12
describing intermediates between R and R*, studies on rhodopsin have indicated that
there is only one major conformational change that significantly alters the structure
of the receptor
3
. Thus the structures of dark-state rhodopsin
13,14
and of opsin
15,16
are considered to be representative structures for the R and R* state, respectively.
Structures of 6 different GPCRs
8,13,17-21
in conformations closely approximating to the R state have now been determined and
it is clear that they are similar to each other, with RMSDs between any pair of structures
in the transmembrane domains being less than 3 Å. As observed in light-activation
of rhodopsin, the major structural difference between R and R* is the movement of
the cytoplasmic ends of helices 5 and 6 away from the receptor core by 5-6 Å, opening
up a cleft in the centre of the helix bundle where the C-terminus of a G protein can
bind
16
. Recently, the structure of an agonist-bound β-adrenoceptor (β2AR) was determined
in complex with an antibody fragment (nanobody Nb80)
5
. This structure of β2AR is very similar to the structure of opsin, which suggests
that the nanobody mimicked the action of a G protein by maintaining the receptor structure
in an activated state. Given the structural similarities between opsin and the β2AR-Nb80
complex, it is likely that the structures of the R* states of other GPCRs are also
highly similar. This is consistent with the same heterotrimeric G proteins being able
to couple to multiple different receptors
22
. However, do the conserved structures of R and R* imply that all agonists activate
the receptors in an identical fashion? The recent structures of a thermostabilised
β1AR bound to 4 different agonists suggested that a defining feature of agonist binding
to this receptor is the formation of a hydrogen bond with Ser5.46 on transmembrane
helix 5 that accompanies the contraction of the ligand binding pocket
7
. Here we describe two structures of the adenosine A2A receptor (A2AR) bound to two
different agonists, which suggests that the initial action of agonist binding to A2AR
has both similarities and differences compared to agonist binding in βARs.
The native human A2AR when bound to its endogenous agonist adenosine or to the high-affinity
synthetic agonist NECA is unstable in detergent, so crystallization and structure
determination relied on using a thermostabilised construct (A2AR-GL31) that contained
four point mutations, which dramatically improved its thermostability. Pharmacological
analysis showed that the mutant receptor bound the five antagonists tested with greatly
reduced affinity (1.8 - 4.3 log units), whereas four agonists bound with similar affinity
to the wild-type receptor (Supplementary Fig. 1). However, A2AR-GL31 is only weakly
activated by the agonist CGS21680 (Supplementary Fig. 2), which suggests that the
thermostabilising mutations might also decouple high-affinity agonist binding from
the formation of R*. The conformation of GL31 is not consistent with it being in the
fully-activated G protein coupled state, because we do not observe a 42-fold increase
in affinity for NECA binding measured for Gαs-coupled A2AR
23
. These data all suggest that A2AR-GL31 is in an intermediate conformation between
R and R*, which is consistent with the structural analysis presented below.
The two structures we have determined are of A2AR-GL31 bound to adenosine and NECA
with resolutions of 3.0 Å and 2.6 Å, respectively (Supplementary Table 1). Global
alignments of the A2AR-GL31 structures with A2A-T4L (A2AR with T4 lysozyme inserted
into inner loop 3) bound to the inverse agonist ZM241385 were performed based on those
residues in the region of the ligand binding pocket that show the closest structural
homology (Fig. 1 and Supplementary Text). This gave an rmsd in Cα positions of 0.66
Å for the 96 atoms selected, which include all residues involved in binding either
adenosine or NECA, with the exception of those in H3. Using this transformation, the
adenine-like moiety of the two ligands superimposes almost exactly (rmsd 0.56 Å).
The most significant differences between the two structures are seen in a distortion
and a 2 Å shift primarily along the helical axis of H3, a bulge in H5 (resulting from
non-helical backbone conformation angles of residues Cys185 and Val186) that shifts
residues into the binding pocket by up to 2 Å and also a change in conformation of
the cytoplasmic ends of H5, H6 and H7 (Fig. 1). Comparison of the A2AR-GL31 structure
with the agonist-bound β2AR-Nb80 complex indicates that these differences are similar
to the conformational changes in the β2AR that are proposed to be responsible for
the formation of the R* state
5
. However, it is unlikely that the structure of A2AR-GL31 represents the fully activated
state, because comparison with opsin bound to the C-terminal peptide of the G protein
transducin shows that there is insufficient space in A2AR-GL31 for the C-terminus
of the G protein to bind (Supplementary Fig. 3). This is based on the assumption that
all G proteins bind and activate GPCRs in a similar fashion, but given the highly-conserved
structures of both G proteins and GPCRs this seems a reasonable hypothesis.
The fact that the structure of A2AR-GL31 represents an agonist-binding state is consistent
with how A2AR-GL31 was engineered. Thermostabilising mutations were selected by heating
the NECA-bound detergent-solubilised receptor, so the mutations are anticipated to
stabilize the agonist-bound state either by stabilizing helix-helix interactions and/or
biasing the conformational equilibrium between the agonist-bound R* state and the
agonist bound R-state
24-26
. The two most thermostabilising mutations, L48A and Q89A, are in regions of the receptor
that are involved in transitions between R and R*, providing a possible explanation
for their thermostabilising effect (Supplementary Fig. 4). The other two mutations,
A54L and T65A, are at the receptor-lipid interface and the reason for their thermostabilising
effect is unclear. Although the overall shape of the ligand binding pockets of A2AR
and β2AR are different, the structural similarities with the β2AR-Nb80
5
and the structural differences to ZM241385-bound A2A-T4L
8
indicate that the structure of the binding pocket in A2AR-GL31 is a good representation
of the agonist-bound binding pocket of the wild-type receptor (Fig.1).
Adenosine and NECA bind to A2AR-GL31 in a virtually identical fashion, in addition,
the adenine ring in the agonists interacts with A2AR in a similar way to the chemically-related
triazolotriazine ring of the inverse agonist ZM241385 (Fig. 2). Thus the hydrogen
bonds between exocyclic adenosine N6 (Supplementary Fig. 5) with both Glu169 in extracellular
loop 2 (EL2) and Asn2536.55 in H6 are similar, with the significant π-stacking interaction
with Phe168 in EL2 also conserved. One of the major structural differences between
ZM241385 and the agonists is the presence of a furan substituent on C20 of triazolotriazine
in the inverse agonist, whilst agonists contain a ribose substituent linked to N9
of adenine (Fig. 2 and Supplementary Fig. 5). In ZM241385, the furan group forms a
hydrogen bond with Asn2536.55 in H6 and van der Waals contacts with other residues
in H3, H5 and H6
8
. In contrast, the ribose moiety in agonists forms hydrogen bonds with Ser2777.42
and His2787.43 in H7, in addition to van der Waals interactions with other residues
in H3 and H6 (Fig. 2). In particular, Val843.32 has to shift its position upon agonist
binding due to a steric clash with the ribose ring, which may contribute to the 2
Å shift observed in H3 (Fig. 3). These differences in binding between ZM241385 and
either adenosine or NECA suggest that the residues that bind uniquely to agonists
(Ser2777.42 and His2787.43) play a key role in the activation of the receptor, as
previously shown by mutagenesis studies
27,28
. This is analogous to the situation in the activation of β1AR, where only full agonists
cause the rotamer conformation changes of Ser5.46 in H5, whereas the inverse agonist
ICI118551 prevents receptor activation by sterically blocking the rotamer change
7,29
. However, the details of the activation differ in that the critical residues that
bind agonists and not antagonists are in H5 in β1AR, but in H7 in A2AR (Fig. 4).
Adenosine and NECA activate the A2AR through interactions with H3 and H7 that are
absent in the interactions between the receptor and the inverse agonist ZM241385 (Fig.
2). The inward shift of H7, the movement of H3 and the consequent formation of a bulge
in H5 are all observed in the structure of agonist-bound A2AR-GL31 and β2AR-Nb80 (Fig.1).
The formation of the bulge in H5 of the β2AR-Nb80 structure was linked to a series
of conformational changes that generates the 60° rotation of H6 about Phe2826.44,
resulting in the cytoplasmic end of H6 moving out from the receptor centre and opening
the cleft where the C terminus of a G protein is predicted to bind as observed in
opsin
5,6
. There are analogous side chain movements in A2AR-GL31 that result in a 40° rotation
of H6, but the cytoplasmic end of H6 remains partially occluding the G protein-binding
cleft (Supplementary Fig. 3), perhaps because the fully active conformation requires
the binding of G proteins to stabilize it. Interestingly, the structure of β2AR
6
with a covalently bound agonist is also not in the fully activated R* conformation,
which is only seen after the nanobody Nb80 is bound
5
. The importance of the bulge in H5 in the activation of A2AR is highlighted by how
inverse agonists bind. Formation of the H5 bulge results in the inward movement of
Cys1855.46 (Cβ moves by 4 Å), which in turn causes the movement of Val186 and ultimately
a shift of His2506.52 by 2 Å into the ligand binding pocket thereby sterically blocking
the binding of ZM241385 (Supplementary Fig. 4). Hence, when the inverse agonist binds,
it is anticipated that the H5 bulge is unlikely to form due to the opposite series
of events and hence the formation of the R* state is inhibited.
Thus in both βARs and A2AR, the formation of the H5 bulge seems to be a common action
of agonists, whereas inverse agonists seem to prevent its formation. However, the
energetic contributions to its formation may be different between the two receptors.
In the βARs there is a major contribution from direct interaction between the agonist
and Ser5.46, while in the A2AR, the major interaction appears to come from polar interactions
involving residues in H7 combined with interactions between the agonist and H3. Despite
these differences, agonist binding to both receptors involves strong attractive non-covalent
interactions that pull the extracellular ends of H3, H5 and H7 together, which is
the necessary prerequisite to receptor activation.
While this manuscript was in review, a related manuscript appeared
30
, describing the structure of the A2A-T4L chimera bound to the agonist UK432097, which
is identical to NECA except for two large substituents on the adenine ring. The structure
of UK432097-bound A2A-T4L is very similar to the structures presented here in the
transmembrane regions (rmsd 0.6 Å), although there are differences in the extracellular
surface due to the bulky extensions of UK432097 interacting with the extracellular
loops and the absence of density for residues 149-157. Xu et al. conclude that the
structure of UK432097-bound A2A-T4L is in an “active state configuration”, whereas
we conclude that the NECA- and adenosine-bound structures are best defined as representing
an intermediate state between R and R*.
METHODS SUMMARY
Expression, purification and crystallization
The thermostabilised A2AR-GL31 construct contains amino acid residues 1-316 of the
human A2AR, four thermostabilising point mutations (L48A2.46, A54L2.52, T65A2.63 and
Q89A3.37) and the mutation N154A to remove a potential N-glycosylation site. A2AR-GL31
was expressed in insect cells using the baculovirus expression system and purified
in the detergent octylthioglucoside using Ni2+-NTA affinity chromatography and size
exclusion chromatography (see Online Methods). The purified receptor was crystallized
in the presence of cholesteryl hemisuccinate by vapour diffusion, with the conditions
described in Online Methods.
Data collection, structure solution and refinement
Diffraction data were collected in multiple wedges (20° per wedge) from a single cryo-cooled
crystal (100 K) for the GL31-NECA complex at beamline ID23-2 at ESRF, Grenoble, France
and from 4 crystals for the GL31-adenosine complex, at beamline I24 at Diamond, Harwell,
UK. The structures were solved by molecular replacement using the ZM241385-bound A2A-T4L
structure (PDB code 3EML)
8
as a model (see Online Methods). Data collection and refinement statistics are presented
in Supplementary Table 1 and omit densities for the ligands are shown in Supplementary
Fig. 6.
Supplementary Material
1