Dear Editor,
G-protein-coupled receptors (GPCRs) modulate cytoplasmic signaling in response to
extracellular stimuli, and are important therapeutic targets in a wide range of diseases.
Differential ligands binding to receptor promote different conformations of GPCR–G-protein
complex, which can adopt diverse active states. Such ligand-directed biased agonism
is now an important focus in drug discovery. Therefore, structure determination of
GPCR–G-protein complexes in variable activation states is important to elucidate the
mechanisms of signal transduction, and to facilitate drug discovery.
The β2-adrenergic receptor (β2AR), a canonical class A GPCR, is activated by adrenaline
and norepinephrine
1,2
. Recent years, many agonists have been synthesized to stimulate the activation of
β2AR, and some of these ligands have been developed for the clinical treatment of
asthma and chronic obstructive pulmonary diseases
3
. Since the first crystal structure of β2AR bound with the inverse agonist carazolol
was reported
4
, several crystal structures of the β2AR bound with different agonists have been determined.
However, only structure of the BI167107-bound β2AR–Gs complex was determined to date,
which represented the real active-state of β2AR
5
. Whether the observed β2AR–Gs interactions in the complex upon BI167107 binding provide
a general rule for signal transductions from the binding of different agonists to
cyclic adenosine monophosphate (cAMP) accumulation requires further validation, and
also remains a major concern for the pharmacological understanding of β2AR and further
drug development.
Formoterol is a selective, long-acting agonist of β2AR, which is unique as it both
has a long-acting bronchodilator effect (> 12 h) and exhibits a fast onset of action
(1–3 min from inhalation), suggesting that it is effective both as maintenance and
reliever medication
6–8
. Herein, the cryo-EM structure of the formoterol-bound β2AR–Gs complex was determined
with an overall resolution of 3.8 Å. Formoterol was reported to have a weaker affinity
than BI167107 in β2AR binding, and also has lower β2AR activation potency than BI167107
(Fig. 1a). Therefore, comparisons between the structure of the formoterol–β2AR–Gs
complex and the previously reported structure of the BI167107–β2AR–Gs complex will
provide insights into the conformational responses of the β2AR upon binding to agonists
with different potency.
Fig. 1
Cryo-EM structure of human β2AR–Gs complex bound with the agonist formoterol.
a Agonist formoterol has lower activation potency on the β2AR than agonist BI167107.
b Orthogonal view of cryo-EM density map of the formoterol–β2AR–Gs complex. Different
colors are applied for β2AR (cyan), Gαs (blue), Gβ (green), Gγ (purple), and Nb35
(yellow). c Cartoon representation of structure of the β2AR–Gs complex, consisting
of formoterol (red stick)-bound β2AR (cyan) and the Gs complex. d Cryo-EM structure
of β2AR–formoterol (blue) was compared to the crystal structure of inverse agonist
carazolol-bound β2AR-T4L (green). Cytoplasmic view of the superimposed structures
showed significant structural changes. e Structural comparison between formoterol-bound
β2AR (cyan) and BI167107-bound β2AR (orange). Notable differences are observed at
the extracellular side of the receptor. Several residues involved in ligand coordination
adopt different side chain conformations. f Side view of ligand-binding pocket in
the formoterol-bound β2AR structure. Residues within 4 Å are shown in sticks. g Schematic
representation of the interactions between β2AR and the ligand formoterol. h cAMP
accumulation analysis of wild-type β2AR and mutants. Site mutations around the ligand-binding
pocket disrupting the receptor-ligand interactions, resulting in β2AR malfunction
in the cAMP accumulation assay. i Coupling interface between β2AR and Gs heterotrimer.
In comparison with the BI167107–β2AR–Gs complex (gray), the residues (H41, F376 and
R380 in Gs (blue), F139 in β2AR (cyan)) engaged in β2AR–Gs coupling in the formoterol–β2AR–Gs
complex have notable structural changes. Direct interaction is observed between R63
in β2AR and D312 in the Gβ. j A comparison of the Gαs-Ras domain in the formoterol–β2AR–Gs
complex (blue), BI167107–β2AR–Gs complex (orange) and Gαs–GTPγs (green). GTPγs is
shown as balls and sticks. Both the P loop and the β6–α5 loop from the formoterol–β2AR–Gs
complex (blue) stretched away from the guanine nucleotide-binding pocket, when compared
with that in the BI167107–β2AR–Gs complex (orange) and Gαs–GTPγs (green).
First, we optimized the previously reported β2AR construct and obtained an engineered
construct with improved expression in the sf9 insect expression system (Supplementary
Fig. S1). The formoterol–β2AR–Gs complex in lauryl maltose neopentyl glycol (LMNG)
detergent micelles was visualized using a Titan Krios microscope. After imaging and
initial two-dimensional classification, three-dimensional classification yielded a
final map at a global resolution of 3.8 Å (Fig. 1b; Supplementary Figs. S2, S3 and
Table S1). The cryo-EM density map of the formoterol–β2AR–Gs complex exhibits well-resolved
side chains, allowing rotamer placements for most amino acids (Fig. 1b; Supplementary
Fig. S4). As revealed in Fig. 1c, the agonist formoterol is clearly identified in
the orthosteric-binding site on the extracellular side of β2AR. The extensive receptor–Gs
interface in the complex is mainly formed by the α5 helix in the Gαs-Ras domain, which
extends into the transmembrane core of the receptor from the intracellular side. When
compared the structure of formoterol-bound β2AR from cryo-EM complex with that of
carazolol-bound β2AR in an inactive state (PDB: 2RH1), remarkable differences were
observed for TM5, TM6 and ICL2 (Fig. 1d), suggesting that formoterol-bound β2AR is
in an active-state.
When focusing on the structural details of the orthosteric-binding pocket, we found
that the catecholamine phenoxy moiety of formoterol formed hydrogen bonds with Ser2035.42
and Ser2075.46 in TM5 (Fig. 1f, g; Supplementary Fig. S4). The alkylamine and the
β-OH in the middle of formoterol formed polar interactions with Asp1133.32 in TM3
and with Asn3127.39 and Tyr 3167.43 in TM7. Moreover, formoterol formed hydrophobic
interactions with receptors through V1173.36, F1935.32, F2896.51, F2906.52, and Y3087.35,
stabilizing the orthosteric agonist-binding pocket in the active-state (Fig. 1g).
cAMP accumulation assay revealed that mutation of the hydrophobic amino acids F193A,
F289A, F290A, and Y308A in the formoterol-binding pocket decreased the potency of
formoterol (Fig. 1h). Moreover, alanine substitution of residues D113, S203, S207,
N312, and Y316 significantly impaired cAMP signaling (Fig. 1h). All of these results
confirmed that residues involved in interactions between the ligand and β2AR play
important roles in the formoterol-mediated cAMP signaling pathway.
When compared the cryo-EM structure of formoterol-bound β2AR with the crystal structure
of BI167107-bound β2AR (PDB: 3SN6), significant differences were observed for extracellular
regions, which contains the orthosteric ligand-binding pocket of the β2AR. Specifically,
the extracellular top of TM1 extracellular region in formoterol-bound receptor moves
outward by 3.2 Å when measured at the Cα carbon of Val34. ECL3, which connects TM6
and TM7, was also observed to extend slightly into the extracellular side (3.7 Å when
measured at the Cα carbon of Asn301). Another notable difference observed between
the two active-state β2AR structures was the short α-helix inside ECL2, which was
observed to move upward by 4.1 Å when measured at the Cα carbon of Asn183 (Fig. 1e).
It is worth noting that, when compared the crystal structure of BI167107-bound β2AR
to the cryo-EM structure of BI167107-bound β2V2R (PDB: 6NI3), the ligand-binding pocket
in the extracellular region is exactly the same (Supplementary Fig. S6). Thus, the
structural differences observed between the cryo-EM structure of formoterol-bound
β2AR and the crystal structure of BI167107-bound β2AR are not due to the steric restrains
in the crystal lattice, but owing to the binding of different agonists. Taken together,
these structural differences at the extracellular side of the receptors endow β2AR–formoterol
with a slightly larger ligand-binding pocket. There are a total of ten amino acid
residues that interact with formoterol in the orthosteric agonist-binding pocket,
including five hydrophobic residues and five hydrophilic residues (Fig. 1g), compared
with a total of 13 amino acid residues that interact with BI167107
5
(Supplementary Fig. S5b). The decreased number of interacting residues between these
two complexes might contribute to the lower affinity of formoterol versus that of
BI167107
9
. Noteworthy, the side chains of both S2045.43 and N2936.55 rotate away from the formoterol
molecule, which excludes the interactions stabilizing the binding between agonist
and β2AR (Fig. 1e). Considering these observations, we speculate that the lower binding
affinity of formoterol is mainly caused by the enlarged ligand-binding pocket and
the reduced interactions between receptor and agonist due to changes of S2045.43 and
N2936.55.
In the formoterol–β2AR–Gαs complex, the most extensive contacts between the G-protein
and the β2AR are formed by the α5 helix of the Gαs-Ras domain, which inserts into
the intracellular central cavity of the β2AR transmembrane domain, consequently leading
to a 14 Å outward movement of TM6. Briefly, the interfaces are mediated mainly by
extensive hydrophobic interactions (i) between the α5 helix of Gαs and ICL2, TM3,
TM5, TM6 and TM7 of β2AR, and (ii) between the αN helix, αN–β1 loop of Gαs, and ICL2
of β2AR (Fig. 1i; Supplementary Fig. S7). As shown in Fig. 1i, the imidazole ring
of H41 in the αN helix and the phenyl ring of F376 in the α5 helix from Gαs protein
in the formoterol–β2AR–Gs complex rotate away from the hydrophobic pocket compared
with that in the BI167107–β2AR–Gs complex, which might attenuate the hydrophobic interactions
between the αN helix, αN/β1 loop of Gαs and ICL2 of β2AR (Fig. 1i). Since the hydrophobic
pocket between β2AR and Gαs protein is crucial for GDP release and is probably necessary
for the stabilization of the nucleotide-free β2AR–Gs complex, the decreased hydrophobic
interaction in the formoterol-bound β2AR–Gs structure might have an impact on subsequent
signal transduction
5
. Moreover, the side chain of R380 in Gαs protein from the formoterol–β2AR–Gs complex
has a notable rotation away from TM3 relative to that in the BI167107–β2AR–Gs complex.
The side chain rotation increases the distance between R380 in Gαs protein and T136
in β2AR, hence making it impossible to maintain the corresponding polar interaction
found in the BI167107–β2AR–Gs complex.
A new interface absent in the structure of the BI167107–β2AR–Gs complex was observed
between the Gβ protein and ICL1 of β2AR, which is mediated by the charge interaction
between residue R63ECL1 in β2AR and residue D312 in the Gβ protein (Fig. 1i). To be
noted, a similar interface was observed in the interaction between Gβ and class F
GPCR
10
or between Gβ and helix 8 of the class B GPCR
11,12
. Taken together, in comparison to the structure of BI167107-bound β2AR–Gs, the attenuated
hydrophobic interaction between αN–β1 loop of Gα and ICL2 of the receptor, combined
with the disappeared polar interaction between T136 in TM3 and R380 in α5 helix, might
decrease the coupling interaction between β2AR and the Gα-Ras domain. This is consistent
with the observed lower G-protein activation potency of formeterol versus BI167107
(Fig. 1a). Thus, structural comparison between the formoterol- and BI167107-bound
β2AR–Gs complexes provides insights into conformational differences that are responsible
for the distinct cAMP accumulation potency of different agonists.
Owing to the intrinsic flexibility, the density of the α-helical domain (αHD) could
not be well-resolved, and the αHD was, therefore, excluded from the high-resolution
map of the formoterol–β2AR–Gs complex. Superposition of the three Gαs-Ras domains
from our cryo-EM structure of the formoterol–β2AR–Gs complex, a previously reported
crystal structure of the BI167107–β2AR–Gs complex and the crystal structure of the
Gαs-GTPγS complex (PDB:1AZT)
13
revealed pronounced conformational differences for the α5 helix, which displaced toward
the receptor in the two agonist-bound β2AR–Gs complexes versus that in the Gαs–GTPγS
complex (Fig. 1j). In Gαs proteins, β6–α5 loop and β1–α1 loop (P loop) in the Gαs-Ras
domain were reported to interact directly with the guanine ring and the diphosphate
of nucleotide
14
. As nucleotide exchange is the essential step in cAMP accumulation during the signal
transduction of the activated GPCR, conformational changes of these loop regions will
directly affect the potency of GPCR. As shown in Fig. 1j, both P loop and β6−α5 loop
in formoterol–β2AR–Gs displaced outward from the nucleotide-binding site, when compared
with those of BI167107–β2AR–Gs. We suggest that the displacement of the P loop and
β6−α5 loop from the nucleotide-binding site may attenuate their interaction with the
guanine ring and diphosphate in GTP, further decreasing the catalytic efficacy of
Gαs-Ras toward GTP. This might in turn be responsible for the observed lower potency
of β2AR binding to formoterol than that to BI167107 (Fig. 1a).
In summary, here we report the cryo-EM structure of β2AR–Gs complexed with the high-affinity
full agonist formoterol. When compared with the BI167107-bound β2AR–Gs complex, structural
differences were observed at the extracellular side of the receptors, which endow
formoterol-bound β2AR with a slightly larger ligand-binding pocket. Besides, the side
chains of S2045.43 and N2936.55 in formoterol-bound β2AR rotate away from the ligand-binding
pocket, which reduces the interaction between formoterol and β2AR. We suggest that
these structural differences might be responsible for different affinities and activation
potency of agonists formoterol and BI167107, and thus residues involved in these structural
differences might be potential targets for new agonist design and drug development.
Moreover, the influence of attenuated interactions between the Gαs-Ras domain and
β2AR will be transduced to the nucleotide-binding pocket, ultimately leading to a
lower GTP-binding affinity and hydrolytic activity of Gαs. The decreased interactions
between the Gαs-Ras domain and β2AR observed in our structure of the formoterol–β2AR–Gs
complex might in turn be partially responsible for the lower affinity of β2AR for
formoterol, when compared with that of BI167107–β2AR–Gs complex structure
5
. These findings enrich our understanding of ligand-binding interactions and cAMP
accumulation potency, enabling the exploration of new avenues for the development
of innovative drugs targeting β2AR.
Density maps and structure coordinates have been deposited to the Electron Microscopy
Database and the Protein Data Bank with accession numbers EMD-30249 and 7BZ2.
Supplementary information
Supplementary Information