Result
The binding modes of µ-opioid receptor with morphine,
fentanyl and
sufentanil
The 3D model of homo sapiens active µ opioid receptor was constructed
based on the crystal structure of mouse active µ opioid receptor (PDB
code: 5c1m)(15 ) and shown inFigure S1A , 91.2% amino acids (270aa/296aa) located in the
additional allowed regions in Ramachandran plot (Figure S1B ).
The molecular docking followed by molecular dynamics simulations
identified the accurate binding modes of µ opioid receptor with
morphine, fentanyl and sufentanil. These three binding modes were
representative structures which accounted for the largest group in
cluster analysis.
As shown in Figure 1A~C, the
protonated nitrogen atom of three
drug molecules forms a strong salt bridge with the carboxyl oxygen atom
of residue D3.32 in μ opioid receptor, though the structure of morphine
is completely different from that of fentanyl and sufentanil. In
addition, the role of H6.52 was identified: the epoxy group of morphine
interacted with the imidazole side chain of H6.52 through the water
bridge. At the same time, the phenolic hydroxyl group of morphine formed
a hydrogen bond with the nitrogen atom of the imidazole ring in H6.52.
In terms of the binding mode of fentanyl and sufentanil with MOR, the
oxygen of amide forms the hydrogen bond network with the side chains of
H6.52 through water bridges. Therefore, these two residues were selected
to mutate to investigate their role in the binding and biased agonism.
Another important residue W6.48 was identified to be a molecular switch
in the activation of MOR. W6.48 was relatively far from the active
pocket and does not have direct interaction with the ligand, while its
role was identified by MD simulations in adjusting the signal
transduction of G-protein or β-arrestin. Therefore, D3.32, H6.52, and
W6.48 were selected to mutate and test their biased signals in order to
investigate their roles in the biased mechanism.
W6.48 is a critical site influencing ligand bias at μ-opioid
receptor
We developed CHO stably expressing MOR (wild-type and D3.32A, W6.48L,
H6.52L mutant) cell lines to explore the role of these three sites in
the interaction between ligand and μ-opioid receptor.
The D3.32A and H6.52L mutation disrupted the binding of fentanyl with
MOR in radioligand binding assay (Figure 1D ), as well as the
binding of the fluorescent ligand with MOR in HTRF binding assay
(Figure 1E ). Both in the cAMP assay and β-arrestin recruitment
assay, the effects of test agonists to stimulate D3.32A and H6.52L
mutant MOR were completely or notably decreased in comparison with
wild-type MOR (Figure S5C, D, G, H ). It did not contradict the
results of binding assays and was in agreement with the binding mode
that these two residues form hydrogen bond network with morphine,
fentanyl, and sufentanil molecules. The D3.32A, and H6.52L mutations
result in the breakdown of the hydrogen bond network, which interfered
the ligand-receptor binding, thus led to completely loss in activity of
MOR.
W6.48L mutation increased the affinity of fentanyl derivatives in
binding with MOR. As shown in radioligand saturation binding curves
(Figure 1D ), the affinity of [3H]fentanyl in binding with
the W6.48L mutant MOR (Kd = 0.23 ± 0.03 nM) was stronger
than that of the wild-type MOR (Kd = 1.07 ± 0.21 nM).
Then we performed HTRF binding assay to test the affinity of a series of
fentanyl derivatives in binding with MOR and determined the differences
between the affinity of W6.48L mutant MOR and wild-type MOR. In HTRF
saturation binding, there was no significant difference observed between
the Kd value of W6.48L mutant MOR and wild-type MOR via
unpaired, two-tailed t-test, which enabled the following determined
Ki and Bmax values of wild-type and
W6.48L mutant MOR to be statistically comparable. The competition
binding curves showed that W6.48L mutation did not block the binding of
test agonists with MOR (Figure 1G ). The corresponding
Ki values of test agonists for wild-type and W6.48L
mutant MOR were shown in Table 1 . Unlike classic MOR ligands
DAMGO and morphine, fentanyl derivatives showed stronger affinity at
W6.48L mutant MOR over the wild-type. Collectively, W6.48L mutation
promoted the binding of fentanyl derivatives with MOR.
We carried out the commercially available cAMP assay (Figure 2A,
C ) and β-arrestin recruitment assay (Figure 2B, D ) to further
investigate if W6.48L mutation influenced the functional activity of
fentanyl derivatives to stimulate MOR-mediated G-protein-dependent
signaling and β-arrestin-dependent signaling. As shown in Table
1 , the potencies of morphine, DAMGO and fentanyl to activate cAMP assay
for W6.48L mutant MOR were lower than wild-type MOR. The efficacies of
fentanyl, sufentanil and remifentanil for W6.48L mutant MOR were
significantly reduced versus the wild-type. It is suggested that W6.48L
mutation decreased the activities of fentanyl derivatives to stimulate
G-protein-dependent signaling. However, remifentanil and 3-methyfentany
showed improved potency and efficacy to promote β-arrestin recruitment
for W6.48L mutant MOR over the wild-type (Table 1 ), which
indicated that W6.48L mutation increase the effects of fentanyl
derivatives to activate β-arrestin-dependent signaling. Altogether,
W6.48L mutation exerted contrary influences to G-protein-dependent
signaling and β-arrestin-dependent signaling, suggesting that W6.48L
changes the ligand bias of fentanyl derivatives at MOR.
To quantify the ligand bias of fentanyl derivatives, the operational
model was applied to calculate the bias factor
ΔΔlog(τ/KA)(21 ,22 ). We found that W6.48L mutation
drove most test agonists relatively bias toward β-arrestin-dependent
signaling except sufentanil (Table 2 ). It is important to note
that W6.48L mutation had comparatively considerate but not identical
impacts on morphine, fentanyl and sufentanil (Figure 2E ). The
mutation reduced the degree of
G-protein-bias for morphine, made the G-protein-bias convert to
β-arrestin-bias for fentanyl, while it made the β-arrestin-bias convert
to G-protein-bias for sufentanil.
W6.48 acts as a structural determinant in adjusting the 3-7
lock
Above studys show that W6.48 is a critical site affecting ligand bias at
MOR, but how the W6.48L mutation changes the ligand bias and why the
mutation have varying impact on the three different ligands remain
unknown. The molecular docking and molecular dynamics (MD) simulations
were used to investigate the biased mechanism of morphine and fentanyl
derivatives to activate MOR-mediated signaling.
The MD simulations showed that, for all of these systems, the
temperature, mass density and volume are relatively stable after 2 ns,
the fluctuations scale became much smaller for both the RMS deviations
of the Cα atoms and potential energy of all of the systems
(Figure S2 ), indicating that all of the molecular systems were
well behaved.
It is reported that the states of
3-7 lock, which was formed by the hydrogen bond between D3.32 and Y7.43,
that impacts the activation of MOR. The distance of 3-7 lock was
monitored to examine if the close or open state of 3-7 lock is relative
to the activation of wild and mutation MOR (Figure 3 ). In terms
of wild type MOR-drug systems, the distance in 3-7 lock of sufentanil
fluctuated from 0.4 to 0.6 nm after simulation of 10 ns, which is
slightly longer than that of morphine and fentanyl’s bound MORs. While
in terms of drug-bound W6.48L mutant MORs, all distances among three
systems tend to be smaller than 0.3 nm, i.e., the 3-7 lock is tightly
closed, i.e. the strong interaction in TM3-TM7 was formed. Combining the
biased signaling assay (Figure 2 ) and the time-evolved distance
changes in 3-7 lock (Figure 3 ), the tight pairs were formed in
morphine and fentanyl bound mutation systems. Correspondingly, the
biased signalling is propitious to β-arresin; while the distance in 3-7
lock is larger than that of morphine and fentanyl’s mutation systmes,
the G-protein biased signaling is enhanced. In this process, 3-7 lock
was adjusted by W6.48, that stabilized Y7.43 by forming paired stable
hydrophobic interaction (Figure 4 ). It was indicated that W6.48
acted as a structural determinant in adjusting the 3-7 lock that
functions as a molecular switch to activate the downstream signaling.
W6.48 and Y7.43 were paired activation switches of ligand
bias at μ-opioid
receptor
By analyzing the processes of morphine, fentanyl and sufentanil
interacting with MOR during the MD simulations as well as the above
results of functional assays, we found that W6.48 and Y7.43 are the
paired activation switches in the biased signal transduction. The
interactions of these two residues in MOR with morphine, fentanyl and
sufentanil were shown in Figure 1A-C . The distances between
W6.48 and the three ligands, together with the distance between Y7.43
and the three ligands were monitored to disclose the molecular mechanism
of biased agonism and elucidate the role of these two residues played in
the biased activation. The time evolved distances as well as the
distance distributions were shown in Figure 4A-F . Furthermore,
the conformational states of the side chains in W6.48 and Y7.43 were
monitored to see how these two residues adjust the biased function of
MOR (Figure 6 ).
In terms of morphine-bound MOR systems, morphine is far away from Y7.43
and does not interact with it in both wild type and mutant MORs
(Figure 1A ). All of the distances were kept stable during the
MD simulations except the distance between W6.48 and Y7.43 in wild-type
morphine-MOR complex, which changes from 0.6 nm to 1.0 nm after the
simulation of 40 ns, and the distance was stable at ca. 0.9 nm upon the
mutation W6.48L. Combining the functional assays, the W6.48L mutation
reduced the degree of G-protein-bias for morphine, W6.48 is the key to
adjust the G-protein bias.
In terms of fentanyl-bound MOR complex, the
phenyl group in fentanyl was
inserted into a small pocket between W6.48 and Y7.43, its phenyl group
forms strong π-π interaction and
this kind of interaction was kept during the whole simulations in both
the wild-type and mutant MORs (Figure 1B ). The distance between
W6.48 and Y7.43 is stable at ca. 1.2 nm after the simulation of 40 ns in
wild type MOR system, while it became stable (ca. 0.8 nm) after the
simulation of 40 ns in the mutant MOR system. Upon the mutation W6.48L
MOR system, Y7.43 approached to L6.48 and formed the stable hydrophobic
network with the phenyl group of fentanyl. Such stable hydrophobic
network is conducive to the biased β-arrestin signal, as demonstrated by
the functional assays, which showed that the biased signal was
strengthened (Figure 4 H and C ).
In terms of sufentanil-bound MORs system, the phenyl group stretches
into the right part of the active site in MOR since the introduction of
methoxymethyl group in sufentanil results in the reorientation of phenyl
group (Figure 1C ). In addition, the phenyl is far away from
Y7.43 in both the wild type and mutant systems. It is quite different
from that of the fentanyl bound MOR system (Figure 4B, C ). In
contrast to the wild type MOR system, W6.48 approached to Y7.43 due to
the formation of hydrophobic network in the W6.48L mutant system
(Figure 4I, L ). The functional assays showed that the signal in
β-arrestin became stronger upon the W6.48L mutation within
fentanyl-bound system and the signal in G-protein pathway is much
stronger than that of in β-arrestin pathway in sufentanil bound W6.48L
MOR system.
All evidences indicated that the stabilization of Y7.43 by ligand and
the stable interactions of ligand with W6.48 and Y7.43 are propitious to
the β-arrestin signal, while the stabilization of W6.48 is propitious to
biased G-protein signal. Correspondingly, both the Y7.43 and W6.48
existed in the gauch- conformational state upon fentanyl
modulated β-arrestin biased signal and existed in the gauch+conformational state upon the sulfentanil modulated G-protein signal
(Figure 6 ).
W6.48L-induced conformational change adjusts MOR signaling
bias
Through further analyzing the states of transmembrane helix in
ligand-MOR systems, we found that morphine, fentanyl, sufentanil induced
the different movement of helices due to the effect of 3-7 lock and the
interactions with D3.32, H6.52, W6.48 and Y7.43. In morphine bound wild
system, Helix 6 (H6) moved toward outside significantly (9.5 Å) and was
far away from Helix 6 (H6). In fentanyl bound W6.48L mutant MOR system,
H6 moved toward H5 and it moved 2.9 Å, while in sufentanil bound W6.48L
mutant MOR, H6 was far away from H5 and it moved outside for 5.9 Å
(Figure 5 ). Collectively, the fentanyl induced the closure of
H6 towards H5 in W6.48L mutant MOR system, correspondingly, it thus
transduced the β-arrestin-bias signal. In contrast to fentanyl,
sufentanil made H6 away from H5, correspondingly, it transduced the
G-protein-bias signal. MD simulation showed that sufentanil induced
larger conformational change on MOR than that of fentanyl. It indicated
that the G-protein biased agonist can induce the larger helix movement
in MOR.