Vrije Universiteit Brussel
Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-
coupled receptor activation
Staus, Dean P; Strachan, Ryan T; Manglik, Aashish; Pani, Biswaranjan; Kahsai, Alem W;
Kim, Tae Hun; Wingler, Laura M; Ahn, Seungkirl; Chatterjee, Arnab; Masoudi, Ali; Kruse,
Andrew C; Pardon, Els; Steyaert, Jan; Weis, William I; Prosser, R Scott; Kobilka, Brian K;
Costa, Tommaso; Lefkowitz, Robert J
Published in:
Nature
DOI:
10.1038/nature18636
Publication date:
2016
Document Version:
Final published version
Link to publication
Citation for published version (APA):
Staus, D. P., Strachan, R. T., Manglik, A., Pani, B., Kahsai, A. W., Kim, T. H., ... Lefkowitz, R. J. (2016).
Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor
activation. Nature, 535(7612), 448-452. https://doi.org/10.1038/nature18636
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00 MONTH 2016 | VOL 000 | NATURE | 1
LETTER
doi:10.1038/nature18636
Allosteric nanobodies reveal the dynamic range and
diverse mechanisms of G-protein-coupled receptor
activation
Dean P. Staus
1
*, Ryan T. Strachan
2
*, Aashish Manglik
3
*, Biswaranjan Pani
1
, Alem W. Kahsai
1
, Tae Hun Kim
4
, Laura M. Wingler
1
,
Seungkirl Ahn
1
, Arnab Chatterjee
1
, Ali Masoudi
1
, Andrew C. Kruse
5
, Els Pardon
6,7
, Jan Steyaert
6,7
, William I. Weis
3,8
,
R. Scott Prosser
4
, Brian K. Kobilka
3
, Tommaso Costa
9
& Robert J. Lefkowitz
1,10,11
G-protein-coupled receptors (GPCRs) modulate many
physiological processes by transducing a variety of extracellular
cues into intracellular responses. Ligand binding to an extracellular
orthosteric pocket propagates conformational change to the receptor
cytosolic region to promote binding and activation of downstream
signalling effectors such as G proteins and β-arrestins. It is well
known that different agonists can share the same binding pocket
but evoke unique receptor conformations leading to a wide range
of downstream responses (‘efficacy’)
1
. Furthermore, increasing
biophysical evidence, primarily using the β
2
-adrenergic receptor
(β
2
AR) as a model system, supports the existence of multiple active
and inactive conformational states
2–5
. However, how agonists with
varying efficacy modulate these receptor states to initiate cellular
responses is not well understood. Here we report stabilization of
two distinct β
2
AR conformations using single domain camelid
antibodies (nanobodies)—a previously described positive allosteric
nanobody (Nb80)
6,7
and a newly identified negative allosteric
nanobody (Nb60). We show that Nb60 stabilizes a previously
unappreciated low-affinity receptor state which corresponds to
one of two inactive receptor conformations as delineated by X-ray
crystallography and NMR spectroscopy. We find that the agonist
isoprenaline has a 15,000-fold higher affinity for β
2
AR in the
presence of Nb80 compared to the affinity of isoprenaline for β
2
AR
in the presence of Nb60, highlighting the full allosteric range of a
GPCR. Assessing the binding of 17 ligands of varying efficacy to
the β
2
AR in the absence and presence of Nb60 or Nb80 reveals large
ligand-specific effects that can only be explained using an allosteric
model which assumes equilibrium amongst at least three receptor
states. Agonists generally exert efficacy by stabilizing the active
Nb80-stabilized receptor state (R
80
). In contrast, for a number of
partial agonists, both stabilization of R
80
and destabilization of
the inactive, Nb60-bound state (R
60
) contribute to their ability to
modulate receptor activation. These data demonstrate that ligands
can initiate a wide range of cellular responses by differentially
stabilizing multiple receptor states.
The allosteric behaviour of GPCRs is responsible for the complex
signalling properties associated with these important regulators of
human physiology. GPCR allostery, defined here as a linkage between
the extracellular orthosteric ligand pocket and the intracellular
G-protein-binding pocket, has long been analysed by pharmacological
methods
8–10
(see Supplementary Information). Conformational
changes within a GPCR induced by agonist binding can enhance the
affinity and binding of intracellular signalling transducers, such as
G proteins and β -arrestins. Conversely, transducer coupling further
enhances agonist affinity, resulting in the formation of the ternary
complex of receptor, intracellular signalling transducer, and ligand
(Fig. 1a). The conceptual framework of the ternary complex model
equates the magnitude of these affinity changes with the strength
of transducer activation in cells
11,12
, as demonstrated for several
GPCR systems
13–16
. However, the structural basis underlying these
allosteric relationships and how they relate to ligand efficacy is not
well understood.
Ligand-dependent GPCR activation has traditionally been
conceptualized as a conversion between a single inactive and a
single active receptor state. However, recent studies using various
spectroscopic techniques have identified multiple inactive and active
receptor states, suggesting that the mechanisms underlying receptor
activation may be more complex than previously thought
2–5
. To better
understand how ligands with varying efficacies may differentially
regulate these conformations, we sought to develop reagents to
stabilize specific inactive and active conformations of the β
2
AR. We
and others have previously used Nb80, a Gs mimetic nanobody, to
stabilize an active conformation of the β
2
AR
6,7
. Indeed, competition
radioligand binding assays using iodinated cyanopindolol ([
125
I]CYP)
and β
2
AR reconstituted into high-density lipoprotein (HDL) particles
(nanodiscs) demonstrated that Nb80 increases the affinity of the agonist
isoprenaline by 75-fold (Fig. 1b), which is similar, but not identical, to
the 33-fold increase seen in the presence of purified heterotrimeric Gs
(Fig. 1b). To investigate the pharmacological properties of the inactive
receptor, we identified a nanobody (Nb60) that preferentially bound
inverse-agonist-bound β
2
AR
7
. Remarkably, though the affinity of the
receptor for agonist in the absence of Gs or Nb80 was presumed to
reflect the pharmacological properties of the inactive state, the presence
of Nb60 reduced agonist affinity by approximately 70-fold (Fig. 1b).
The effects of Nb60 and Nb80 on radiotracer affinity were negligible
and could not account for the large changes in affinity (Extended Data
Table 1).
To further quantify the allosteric effects of Nb60, Nb80, and Gs on
agonist binding, we measured isoprenaline affinity with radioligand
competition binding over a range of nanobody/Gs concentrations
(Extended Data Fig. 1). As the concentration of allosteric modulator
increases, the effect on isoprenaline affinity becomes saturable,
reaching two opposite plateau values with Nb60 and Nb80 or Gs
(Fig. 1c). This is a hallmark pattern of true allosteric interactions, with
1
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
2
Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599,
USA.
3
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.
4
Department of Chemistry, University of Toronto, University of
Toronto Mississauga, 3359 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada.
5
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston,
Massachusetts 02115, USA.
6
Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.
7
Structural Biology Research Center, VIB, Pleinlaan 2, 1050 Brussels,
Belgium.
8
Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
9
Department of Pharmacology, Istituto Superiore di Sanità, Rome 00161,
Italy.
10
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
11
Howard Hughes Medical Institute, Chevy Chase, Maryland 20815-6789, USA.
*These authors contributed equally to this work.
© 2016 Macmillan Publishers Limited. All rights reserved
2 | NATURE | VOL 000 | 00 MONTH 2016
Letter
reSeArCH
the net log-change in isoprenaline affinity at saturating concentration
of nanobody/Gs gauging the extent of cooperativity between nanobody
and agonist binding (negative for Nb60 or positive for Nb80 and Gs)
(see Supplementary Information). As predicted by the ternary complex
model, this coupling energy, termed α , must be constant—the effect
of bound nanobody on agonist binding is reciprocal to the effect of
bound agonist on nanobody binding. To verify such a prediction, we
measured the affinity of Nb60 for the β
2
AR in the absence and presence
of agonist (isoprenaline) using isothermal titration calorimetry
(Fig. 1d, e). As expected, the affinity of Nb60 for β
2
AR decreased in the
presence of isoprenaline. Consistent with its preference for the inactive
state, Nb60 dose-dependently increased binding of the radiolabelled
inverse antagonist [
3
H]ICI-118,551 to the β
2
AR, whereas binding was
decreased in the presence of Nb80 (Fig. 1f). Together, these data show
that Nb60 and Nb80 are potent allosteric modulators that can be used
to stabilize inactive and active β
2
AR states.
The decrease in isoprenaline affinity observed in the presence of
Nb60 reveals a previously unappreciated ‘very-low-affinity’ state
(K
VL
) in competition binding experiments. The affinity of agonist
for an uncoupled GPCR has traditionally been referred to as the
‘low-affinity’ (K
L
) state; however, our results show that K
L
values
reflect binding of an ensemble of conformations that exchange rapidly
over the course of the binding reaction. This phenomenon is probably
conserved among GPCRs, as a similar K
VL
state has been observed
with the A
2A
adenosine receptor using an antibody fragment
17
. This
conformational heterogeneity is consistent with recent spectroscopic
and computational studies, which have shown that the β
2
AR exists in
multiple inactive, intermediate, and active conformations that exchange
within milliseconds
2–5,18
. To assess which receptor state Nb60 stabilizes,
we conducted
19
F fluorine NMR spectroscopy of β
2
AR labelled with a
trifluoroacetanilide probe at the endogenous residue C265 located at
the cytoplasmic end of transmembrane 6 (TM6). As shown previously,
the unliganded β
2
AR exists in two equally distributed inactive states
(termed S1 and S2) that exchange on a fast timescale (700 ± 137 μ s), and
complete conversion into the active S4 state requires both agonist and
transducer binding
5
(Fig. 2a, b). Using structural insights from double
electron–electron resonance studies, S1 was identified as an inactive
state with an interaction between TM3 residue R131
3.50
(superscripts
indicate Ballesteros–Weinstein numbering for GPCRs
19
) and E268
6.30
in TM6 (ref. 5), commonly termed the ionic lock. The ionic lock has
previously been shown to be important in maintaining the inactive con-
formation of β
2
AR, as charge-neutralizing mutations at these positions
increase receptor constitutive activity
20
. Additionally, the S2 conforma-
tion was also identified as an inactive state but with a disengaged ionic
lock (Fig. 2b). The binding of G protein or other positive allosteric
modulators such as Nb80 lowers the energy of the active receptor states,
driving the receptor from S2 towards active conformations
5
. The
19
F
NMR spectra showed that the addition of Nb60 to β
2
AR bound to
the inverse agonist carazolol shifted the S1–S2 equilibrium towards
the inactive S2 state (Fig. 2c), providing a mechanism for its negative
cooperative effects on isoprenaline affinity. Given the broad NMR line
shape (Fig. 3c, red line) of the β
2
AR when bound to carazolol and Nb60,
we conducted Carr–Purcell–Meiboom–Gill (CPMG)
21
relaxation
dispersion measurements to measure potential conformational
heterogeneity. We found that the β
2
AR when bound to Nb60 and
carazolol interconverts (860 ± 530 s
−1
) between S1 and S2, but is predom-
inantly found (75–90%) in the S2 inactive state (Extended Data Fig. 1d).
To further decipher how Nb60 induces a negative cooperative effect
on agonist binding, we determined a 3.2 Å X-ray crystal structure of
a ternary complex comprised of β
2
AR, Nb60, and the inverse agonist
carazolol (Fig. 2d–f, Extended Data Fig. 2 and Extended Data Table 2).
The complementary determining region 3 (CDR3) of Nb60 inserts
into a similar β
2
AR allosteric pocket as G protein and Nb80, located
between the cytoplasmic ends of TM3, TM4, and TM6 (Fig. 2d, e).
We found that T102 and Y106 in Nb60 bridge an interaction between
residues R131
3.50
and E268
6.30
. This interaction does not exist in the
absence of Nb60 (Protein Data Bank (PDB) accession code 2RH1),
indicating that Nb60 stabilizes an inactive conformation through inter-
actions with the β
2
AR ionic lock (Fig. 2e). However, this polar network
appears ‘disengaged’ compared to the fully closed ionic lock in the
β
1
-adrenergic receptor (PDB accession code 2YCW)
22
(Fig. 2e), thus
supporting the
19
F NMR data showing that Nb60 specifically stabilizes
the S2 inactive state. The insertion of Nb60 F103 into a hydrophobic
pocket in the β
2
AR may also contribute to the affinity and/or negative
allosteric properties of Nb60 (Extended Data Fig. 2c). Confirming the
importance of T102 and F103 for the β
2
AR–Nb60 interaction, alanine
mutations at these positions inhibited Nb60 binding to the β
2
AR and
the negative cooperative effects on isoprenaline binding (Extended
Log (Nb/Gs) [M]
Log (iso) [M]
–12 –11 –10 –9 –8 –7 –6 –5 –4 –3
0
25
50
75
100
125
β
2
AR HDL
+ Nb60
+ Nb80
+ Gs
–11 –10 –9 –8 –7 –6
–2
–1
0
1
2
Nb60
Nb80
Gs
–5
kcal mol
–1
of Nb60
–10
–15
–20
–25
–30
0
0.5
1.0
1.5 2.0
Molar ratio (Nb60/β
2
AR iso)
0
20
40 60 80
Time (min)
–0.2
0.0
0.2
0.4
0.6
μcal s
–1
Iso
Nb60 K
d
= 1.27 μM
kcal mol
–1
of Nb60
0
–2
–4
–6
–8
–10
–12
0
0.5 1.0
1.5
2.0
2.5 3.0
Molar ratio (Nb60/β
2
AR DMSO)
DMSO
0
20 40 60 80
Time (min)
0
0.1
0.2
0.3
0.4
0.5
μcal s
–1
Log (Nb) [M]
–11 –10 –9 –8 –7 –6
–5
0.00
0.25
0.50
0.75
1.00
1.25
Nb60
Nb80
ab c
def
Nb60 K
d
= 56.8 nM
R
RL
L
RT
Ternary
complex
model
Normalized [
125
I]CYP
specic binding
Normalized ΔlogIC
50
Relative [
3
H]ICI binding
L
L
L
T
T
T
T
RLT
Figure 1 | Allosteric nanobodies have opposing effects on agonist
affinity for the β
2
AR. a, Schematic of the ternary complex model. Ligand
(L) affinity to receptor (R) increases in the presence of transducer (T), this
allosteric linkage is denoted by dashed line with arrows. b, Compared to
the absence of modulator, Nb60 decreases isoprenaline affinity (negative
cooperativity) and Nb80 and Gs increases affinity (positive cooperativity)
as assessed by radioligand competition assays using β
2
AR HDL particles.
c, The effects of Nb60 and Nb80 or Gs on isoprenaline affinity are
saturable functions of their concentration. d, e, The affinity of Nb60 for
unliganded β
2
AR (d), represented by a tight isotherm sigmoidal binding
curve
23
, is reduced in the presence of isoprenaline (iso) (e), as determined
by isothermal titration calorimetry. f, Nb60 dose-dependently increases
and Nb80 decreases the binding of the radiolabelled antagonist [
3
H]ICI-
118,551 to the β
2
AR. All radioligand binding studies represent a minimum
of three independent experiments with deviation shown as the standard
error.
© 2016 Macmillan Publishers Limited. All rights reserved
00 MONTH 2016 | VOL 000 | NATURE | 3
Letter
reSeArCH
Data Fig. 2e, f). Other than changes within the ionic lock, the overall
structure of β
2
AR bound to Nb60 is highly similar to the previously
determined inactive β
2
AR structure bound to carazolol alone (root
mean squared deviation (r.m.s.d.) of 0.3 Å for the transmembrane
domains and orthosteric binding pocket, Fig. 2f). Taken together, the
pharmacological, biophysical, and crystallographic studies show that
Nb60 exerts its negative allosteric effect on agonist binding by stabiliz-
ing the S2 inactive β
2
AR conformation.
Our observation that isoprenaline bound to the Nb80-stabilized
active β
2
AR with approximately 15,000-fold greater affinity than to
the Nb60-stabilized inactive β
2
AR (Fig. 1b, c) provides a measurement
of the full allosteric power of an agonist to activate a GPCR. The large
free energy difference (− 24 kJmol
−1
) between these states is probably
important for GPCR function, allowing agonist-stimulated activity to
be significantly higher than that of the basal activity. As simulated for
a full agonist in Fig. 3a, the overall affinity shift from inactive to active
receptor (black arrow) is comprised of two components, the negative
cooperative (α ) effects of Nb60 (α Nb60, blue) and positive effects of
Nb80 (α Nb80, red). Given the complexities and limitations of using
NMR and crystallography to gain mechanistic insights into ligand
activation of a GPCR, we used a pharmacological approach to quantify
α Nb60 and α Nb80 for 17 β
2
AR ligands of varying efficacy (Fig. 3b and
Extended Data Fig. 3).
We first identified a significant positive correlation (r = 0.8514,
P = 0.004) between α Nb80 values (K
L
/K
H
ratios) and the relative
intrinsic efficacies (τ values) of various ligands obtained from cellular
G-protein assays (Extended Data Fig. 4a, b). This finding provides
additional support for the claim that Nb80 exerts allosteric effects that
mimic those of a G protein. It also confirms our previous reports that
ligand efficacy is not a product of modified downstream signalling
events but is rather achieved at the level of ternary complex interactions,
reflecting the allosteric interactions between different ligands and
transducers
12
. Interestingly, we find no significant correlation between
Nb60
β
2
AR
β
2
AR + Cz
S1
S2
S2
S1
TM1
TM2
TM4
TM3
TM5
Gs
S4
TM2
TM4
TM7
TM3
R131
E268
TM5
S1
TM6
S2
Inactive
TM3
ab
c
ef
β
2
AR–Cz–Nb60
β
2
AR–Cz
Carazolol
β
1
AR–Cz
TM6
TM3
Nb60
CDR3
Y106
E268
6.40
T102
R131
3.50
d
E268
6.30
R131
3.50
β
2
AR–Cz–Nb60 β
2
AR–Cz
Extracellula
r
Intracellular
R139
3.50
E285
6.30
TM6
TM3
TM6
TM6
TM1
TM2
TM3
TM4
TM7
Helix8
S1
S2
S4
TM6
ECL2
TM5
Active
TM1
TM7
–60.0–61.0–62.0
19
F chemical shift (p.p.m.)
β
2
AR + Cz + Nb60
–60.0–61.0–62.0
19
F chemical shift (p.p.m.)
TM3
Figure 2 | Nb60 stabilizes the S2 inactive state by coordinating the β
2
AR
ionic lock. a, b, Cartoon depicting a side (a) or cytoplasmic (b) view of
the β
2
AR transmembranes (TM). Conversion from the two inactive states
(S1 and S2) to the active S4 state requires both agonist and transducer
(G protein) binding and is represented by a 14 Å outward movement of
TM6. c,
19
F NMR spectroscopy of the β
2
AR with the antagonist carazolol
(Cz) ± Nb60. d, The 3.2 Å structure of the β
2
AR bound to carazolol (Cz)
and Nb60 (β
2
AR–Cz–Nb60). e, Coordination of β
2
AR ionic lock (R131
and E268) by Nb60 CDR3 residues T102 and Y106. For comparison,
a disengaged and fully formed ionic lock are shown by the β
2
AR–Cz
(PDB accession code 2RH1) and β
1
AR–Cz (PDB accession code 2YCZ),
respectively. Hydrogen bonds are shown as black dotted lines. f, Overlay of
β
2
AR–Cz and β
2
AR–Cz–Nb60 structures.
Log (drug) [M]
Normalized c.p.m.
–14 –12 –10 –8 –6 –4 –2
0
25
50
75
100
125
Nb80
Nb60
αNb80 αNb60
–5 –4 –3 –2 –1 0 1 2
–2
–1
0
1
2
3
4
Adr
Noradr
Isoe
Iso
Hbi
Fen
Clen
Salb
Proc
Zint
Salm
Form
Alp
Pin
Caraz
Carv
ICI
R = –0.7417
P = 0.0013
a
b
c
–4–2024
ICI
Carv
Caraz
Alp
Pin
Proc
Form
Salm
Zint
Hbi
Clen
Salb
Iso
Fen
Isoe
Adr
Noradr
Full agonist
Partial agonist
Antagonist
Log
α
Nb60
Log αNb80
Log
α
αNb80 αNb60
Figure 3 | Nb60 and Nb80 have varying effects on the affinity
of different β
2
AR ligands. a, Schematic depicting the use of equilibrium
radioligand binding studies to quantify the cooperativity
(α ) between Nb60 or Nb80 binding and ligand affinity (see Methods
and Supplementary Information). c.p.m., counts per minute.
b, Cooperativity values for Nb60 (α Nb60) and Nb80 (α Nb80) for β
2
AR
ligands with varying efficacies. Ligands are ordered by magnitude
of α Nb80. c, Correlation plot of α Nb60 and α Nb80; regression
shown as solid red line with 95% confidence interval (dotted red line).
All α values derived from at least three independent radioligand
binding experiments with the deviation depicted as standard error.
Adr, adrenaline; alp, alprenolol; carv, carvedilol; caraz, carazolol;
clen, clenbuterol; fen, fenoterol; form, formoterol; hbi, hydroxybenzyl
isoproterenol; ICI, ICI-118,551; iso, isoprenaline; isoe, isoetharine;
pin, pindolol; proc, procaterol; salb, salbutamol; salm, salmeterol; zint,
zinterol.
© 2016 Macmillan Publishers Limited. All rights reserved
4 | NATURE | VOL 000 | 00 MONTH 2016
Letter
reSeArCH
α Nb60 and ligand efficacy, providing the first evidence, to our
knowledge, that ligands perceive the Nb60 and Nb80 stabilized recep-
tor states differently (Extended Data Fig. 4c). We observed a signifi-
cant negative correlation (r = 0.7417, P = 0.0013) between α Nb60 and
α Nb80 values (Fig. 3c) across all ligands, suggesting these nanobodies
stabilize functionally opposite conformations. However, the relation-
ship between α Nb60 and α Nb80 was unexpectedly complex (Fig. 3b).
For example, several full agonists exhibited comparable levels of positive
(Nb80) and negative (Nb60) cooperativity (noradrenaline, adrenaline,
and isoprenaline), whereas some partial agonists displayed patterns
with surprising discrepancies (clenbuterol, salbutamol, and zinterol)
(Fig. 3b). Importantly, for a subset of these ligands we confirmed that
the allosteric effects of Nb80 are consistent with those elicited by the
physiological transducer heterotrimeric Gs (Extended Data Fig. 5a, b),
Moreover, the surprisingly divergent, ligand-specific effects of Nb60
were also observed with another inactive state-stabilizing nanobody,
A11 (Extended Data Fig. 5c). Together, these data indicate that the
ligand-specific effects of Nb80 and Nb60 are not nanobody-specific,
but rather reflect how ligands perceive specific receptor conformations.
We next tested two different allosteric models to try to explain how
the different conformations stabilized by Nb60 and Nb80 can generate
the observed cooperativities. First we tested whether the dynamics of
receptor states can be sufficiently modelled as a simple interconversion
between two allosteric conformations, despite biophysical evidence for
multiple inactive and active receptor states. Accordingly, we attempted
to fit the experimentally observed α Nb60 and α Nb80 values for all
ligands using the two-state model of receptor activation (Fig. 4a). In
this simulation, the equilibrium constant J represents the distribution
of inactive (R
60
) and active (R
80
) receptor states in the absence of
ligand. The variable β describes the effect that each ligand has on
the distribution of receptor states (J), with agonists displaying larger
β values (that is, they stabilize more R
80
relative to R
60
). As shown in
Fig. 4a, the theoretical curve generated by varying β in the two-state
model (dotted black line) failed to accurately predict the experimen-
tally determined α Nb60 and α Nb80 values for 30% of ligands tested,
consisting primarily of partial agonists (dotted red oval). A different
equilibrium J constant would be required to explain the cooperativity of
these ligands with the same two-state model. These findings argue that
ligands must modulate more than these two states to control receptor
activation.
Biophysical evidence supports the existence of at least three predomi-
nant receptor states; therefore we hypothesized that a three-state model
(Fig. 4b) may better explain the experimentally observed α Nb60 and
α Nb80 values. In this model, the equilibria of R
80
(J
1
) and R
60
(J
2
) can
be regulated separately by ligands, described by the allosteric factors
β
1
and β
2
, respectively. We found that the observed cooperativities
for 12 ligands (Fig. 4b, class I, orange), which encompasses all the full
agonists, can be predicted if these ligands primarily stabilize the active
R
80
state, while having negligible effects on the inactive R
60
state. The
efficacy of these ligands is thus directly proportionally related to their
effect on the R
80
equilibrium (β
1
), such that highly efficacious ligands
have a large β
1
(adrenaline), whereas partial agonists have a lower β
1
(clenbuterol). In contrast, the observed cooperativity of other partial
agonists (classes II and III) could only be predicted by differentially
modulating the R
80
and R
60
equilibria, suggesting these ligands regulate
multiple receptor states to control receptor activation. For example,
even though clenbuterol (class I), zinterol (class II), and procaterol
(class III) are similar partial agonists (Extended Data Fig. 4a), the
mechanism by which they promote receptor activation varies based
on their ability to differentially stabilize and destabilize the active (R
80
)
and inactive (R
60
) states, respectively (Fig. 4b). Importantly, divergences
in receptor activation mechanisms can only be uncovered by studying
R
60
R
80
R
60
R
80
EJ
J
Two-state model of receptor activation
Ligand
Inactive
state
Active
state
4
Noradr
Isoe
Fen
Salb
–4 –3 –2 –1 0
–2
–1
0
1
2
3
Adr
Iso
Hbi
Clen
Proc
Zint
Salm
Form
Alp
Pin
Caraz
Carv
ICI
R
60
R
R
80
J
2
J
1
R
60
R
R
80
III
R
60
R
R
80
E
2
J
2
II
R
60
R
R
80
E
2
J
2
I
Three-state model of receptor activation
Inactive states
Active
state
–4 –3 –2 –1 0
–2
–1
0
1
2
3
4
I
II
III
E
1
J
1
E
1
J
1
E
2
J
2
E
1
J
1
Salb
Clen
Zint
Salm
Form
Proc
ab
Log αNb60
Log αNb80
Log
α
Nb60
Log αNb80
Noradr
Adr
Isoe
Iso
Fen
Pin
Alp
Carv
ICI
Cara
z
Hbi
Figure 4 | β
2
AR agonists differentially stabilize receptor states to
regulate receptor activation. a, b, Illustration of a two-state (a), or
three-state (b), model of receptor activation describing the effect of β
2
AR
ligands on receptor conformations stabilized by Nb60 (R
60
) or Nb80 (R
80
).
The equilibrium (J) between receptor states can be influenced by ligand
binding through the allosteric factor β . The theoretical cooperativity (α )
between nanobody and ligand binding derived from the two-state model
(dashed black line) fails to predict the observed α values for a subset of
ligands (dashed red oval). However, the observed cooperativity values can
be accurately predicted using an allosteric model in which ligands can
differentially modulate three independent receptor states (three-state).
Certain ligands (orange) primarily stabilize the active R
80
state, whereas
others (purple or green) can stabilize R
80
but simultaneously destabilize
the inactive R
60
state. All α values are derived from at least three
independent radioligand binding experiments with the deviation depicted
as standard error.
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