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ARTICLE
doi:10.1038/nature11701
High-resolution crystal structure of
human protease-activ ated receptor 1
Cheng Zhang
1
, Yoga Srinivasan
2
, Daniel H. Arlow
3
, Juan Jose Fung
1
{, Daniel Palmer
2
, Yaowu Zheng
2
{, Hillary F. Green
3
,
Anjali Pandey
4
, Ron O. Dror
3
, David E. Shaw
3
, William I. Weis
1,5
, Shaun R. Coughlin
2
& Brian K. Kobilka
1
Protease-activated receptor 1 (PAR1) is the prototypical memberof a family of G-protein-coupled receptors that mediate
cellular responses to thrombin and related proteases. Thrombin irreversibly activates PAR1 by cleaving the
amino-terminal exodomain of the receptor, which exposes a tethered peptide ligand that binds the heptahelical
bundle of the receptor to affect G-protein activation. Here we report the 2.2 A
˚
resolution crystal structure of human
PAR1 bound to vorapaxar, a PAR1 antagonist. The structure reveals an unusual mode of drug binding that explains how a
small molecule binds virtually irreversibly to inhibit receptor activation by the tethered ligand of PAR1. In contrast to
deep, solvent-exposed binding pockets observed in other peptide-activated G-protein-coupled receptors, the
vorapaxar-binding pocket is superficial but has little surface exposed to the aqueous solvent. Protease-activated
receptors are important targets for drug development. The structure reported here will aid the development of
improved PAR1 antagonists and the discovery of antagonists to other members of this receptor family.
Protease-activated receptors (PARs) are G-protein-coupled receptors
(GPCRs) that mediate cellular responses to specific proteases
1,2
. The
coagulation protease thrombin activates the prototypical PAR, PAR1,
by specific cleavage of the N-terminal exodomain of the receptor to
generate a new N terminus. This new N terminus then functions as a
tethered peptide agonist that binds intramolecularly to the seven-
transmembrane helix bundle of the receptor to affect G-protein
activation
1,3–8
(Fig. 1a). In adult mammals, the four members of the
PAR family link tissue injury and local generation of active coagu-
lation proteases to cellular responses that help to orchestrate hae-
mostasis, thrombosis, inflammation and perhaps tissue repair
2,9
.
PARs may also participate in the progression of specific cancers
10,11
.
In contrast to a typical receptor–agonist binding interaction, the
interaction of PAR1 with its activator, thrombin, is that of a protease
substrate, with thrombin binding transiently to the receptor, cleaving
it, then dissociating
1,3–7,12
. Proteolytic unmasking of the tethered pep-
tide agonist of the receptor is irreversible, and although a free syn-
thetic hexapeptide with the amino acid sequence of the tethered
agonist (SFLLRN) can activate the receptor with half-maximum effec-
tive concentration (EC
50
) values in the 3–10-mM range, the local
concentration of the tethered agonist peptide is estimated to be about
0.4 mM. Accordingly, PAR signalling must be actively terminated
13–15
and, unlike most other GPCRs that can go though many rounds of
activation by reversible diffusible hormones and neurotransmitters,
PARs are degraded after a single activation
6,13–17
. Identification of effec-
tive PAR antagonists has been challenging because low molecular mass
compounds must compete with the very high local concentration of
the tethered agonist generated by proteolytic cleavage.
Vorapaxar is a highly specific, virtually irreversible PAR1 antago-
nist
18
(Supplementary Fig. 1). In a phase III trial, vorapaxar protected
patients against recurrent myocardial infarction at a cost of increased
bleeding
19,20
. Given the latter, an antagonist that is reversible in the
setting of bleeding might be desirable. Although the very slow dissoci-
ation rate of vorapaxar from PAR1 probably accounts for its ability to
1
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.
2
Cardiovascular Research Institute, University of California, San Francisco, 555
Mission Bay Boulevard South, S452P, San Francisco, California 94158, USA.
3
D. E. Shaw Research, New York, New York 10036, USA.
4
Portola Pharmaceuticals, 270 East Grand Avenue, South San
Francisco, California 94080, USA.
5
Department of Structural Biology, Stanford University School of Medicine, 299 Campus Drive, Stanford, California 94305, USA. {Present addresses: ProNovus
Bioscience, LLC. 544 E Weddell Drive, Sunnyvale, California 94089, USA (J.J.F.); Northeast Normal University, Changchun, Jilin 130024, China (Y.Z.).
S
F
L
L
R
N
Thrombin
G
i
G
q
G
12/13
Extracellular
Intracellular
Inactive PAR1 Active PAR1
N
C
C
N R L L F S
a
b
c
60°
90°
ECL2
TM4
TM3
TM5
TM1
TM2
TM3
TM4
TM5
TM6
TM7
ECL1
Disulphide
Extracellular
N
N
ECL2
TM6
TM2
TM7
Figure 1
|
PAR1 activation and overall structure of human PAR1 complex
with antagonist vorapaxar. a, Thrombin cleaves the PAR1 N terminus and
exposes a new N-terminal peptide, SFLLRN, which can bind to and activate the
transmembrane core of PAR1. PAR1 can activate several G proteins including
G
i
,G
12/13
and G
q
. b, Overall view of the human PAR1 structure and the
extracellular surface. The receptor is shown as blue ribbon and vorapaxar is
shown as green spheres. Monoolein is shown in orange, water in red. The
disulphide bond is shown as a yellow stick. c, Surface view of the ligand-binding
pocket viewed from two different perspectives. The vorapaxar-binding pocket is
close to the extracellular surface but not well exposed to the extracellular solvent.
20/27 DECEMBER 2012 | VOL 492 | NATURE | 387
Macmillan Publishers Limited. All rights reserved
©2012
inhibit receptor activation by its tethered agonist peptide, it may be
possible to develop a drug with an off rate slow enough to block sig-
nalling but fast enough to allow useful reversal after cessation of drug.
In an effort to advance our understanding of PAR1 structure and
function and to provide a foundation for discovery of new agents to
advance the pharmacology of PARs, we obtained the crystal structure
of vorapaxar-bound human PAR1.
Crystallization of human PAR1
To facilitate crystallogenesis, T4 lysozyme (T4L) was inserted into
intracellular loop 3 (ICL3) in human PAR1, the N-linked glycosyla-
tion sites in ECL2 were mutated
21
, and the N-terminal exodomain was
removed by site-specific cleavage at a tobacco etch virus (TEV) prote-
ase site introduced between amino acids 85 and 86 (ref. 4; Sup-
plementary Fig. 2). The structure of human PAR1–T4L bound to
vorapaxar was determined to 2.2 A
˚
by merging diffraction data sets
from 18 crystals grown in the lipidic cubic phase (Supplementary Figs
3 and 4). Details of data collection and structure refinement are listed
in Supplementary Table 1.
PAR1 has the expected seven-transmembrane segment bundle
(Fig. 1b). There are several lipid molecules assigned as monoolein
from lipidic cubic phase in the structure (Fig. 1b), but no ordered
cholesterol molecules were observed. The remaining N-terminal frag-
ment Arg 86–Glu 90 and a part of the ICL2 from Gln 209 to Trp 213
are not modelled in the structure because of the weak electron density.
There is no clear electron density for residues after Cys 378, and no
helix 8 is observed after transmembrane segment 7 (TM7) in the
structure. Whether this reflects a lack of a helix 8 in PAR1 in its native
state or conditions in the crystal is not known.
Cys 175
3.25
in helix III and Cys 254 in extracellular loop 2 (ECL2)
form a conserved disulphide bond (Figs 1b and 2a). Amino-terminal
to Cys 254, ECL2 loops outwards in two anti-parallel b strands. This
structural feature is found in other peptide receptors, including the
CXCR4 receptor and the opioid receptors
22–25
, despite absence of
amino acid sequence homology among these receptors in ECL2
(Supplementary Fig. 5). In contrast to the open, solvent-exposed
binding pocket observed in the m-opioid receptor (MOR) and other
peptide receptors, access to the vorapaxar-binding pocket is restricted
by the central location of ECL2 (Fig. 1 and Supplementary Figs 5 and
6), which almost completely covers the extracellular-facing surface of
vorapaxar. ECL2 is anchored in this position by hydrogen bonds
between His 255 in ECL2 and Tyr 353
7.35
in TM7, and between
Asp 256 in ECL2 and Tyr 95 in the N terminus (Supplementary
Fig. 6), and by extensive interactions with vorapaxar (Fig. 2). The
covered vorapaxar-binding pocket in PAR1 more closely resembles
rhodopsin and the lipid-activated sphingosine-1-phosphate receptor
(S1P
1
) than other peptide-activated GPCRs (Supplementary Fig. 5).
Divergence of PAR1 from other family A GPCRs
Members of the family A GPCRs share a set of conserved amino acids
that are thought to be important in signal transduction
26,27
. Specific
residues in the highly conserved FXXCWXP motif (in which X
denotes any amino acid) in TM6, and the NPXXY motif in TM7
undergo structural rearrangements during activation of the b
2
adre-
nergic receptor (b
2
AR) (Fig. 3b, d). However, based on a phylogenetic
analysis of amino acid sequences
28
, PAR1 is a more distant relative of
the family A GPCRs that have been crystallized thus far. PAR1
belongs to the d-subfamily, which includes the glycoprotein receptors,
the purinergic receptors and the olfactory receptors
28
. The tryptophan
residue in FXXCW
6.48
XP proposed to act as a toggle switch during
activation in some GPCRs
28
is replaced by Phe
6.48
in all PARs (Fig. 3a).
Phe
6.44
, also highly conserved in family A GPCRs, is Phe
6.44
in PAR1,
but Tyr
6.44
in PAR2 and Ala
6.44
in PAR4. When comparing inactive
and active states of the b
2
AR, changes in packing interactions in-
volving Pro
5.50
, Ile
3.40
and Phe
6.44
seem to have a role in structural
changes needed to accommodate G-protein binding
29–31
. Packing
interactions of the corresponding residues Pro 282
5.50
, Ile 190
3.40
and Phe 322
6.44
in the PAR1 differ from those in both active and
inactive b
2
AR structures (Fig. 3b). Taken together, these differences
suggest that PAR1 may differ from other family A GPCRs in the
mechanism by which signals propagate from the extracellular pep-
tide-binding interface to the cytoplasmic domains that interact with G
proteins and other signalling molecules.
The NP
7.50
XXY motif at the end of helix VII observed in most
family A GPCRs is DP
7.50
XXY in PAR1. This region undergoes
structural rearrangement after activation of the b
2
AR. In PAR1,
Asp 367
7.49
and Tyr 371
7.53
form hydrogen bonds with residues in
TM2 and TM1 (Fig. 3c). The hydrogen-bonding network associated
with Asp 367
7.49
is extensive and includes several water molecules and
a putative sodium ion. Na
1
, rather than a water molecule, was
assigned to this region of electron density as it has five oxygen neigh-
bours and short distances to its oxygen ligands (average refined dis-
tance 2.4 A
˚
), both consistent with known Na
1
–oxygen interactions
32
,
and it interacts with two acidic side chains that, assuming deproto-
nated states, would repel one another without charge neutralization
provided by the Na
1
. The sodium ion also interacts with a conserved
Asp 148
2.50
in TM2 and Ser 189
3.39
in TM3, with two water molecules
nearby (Fig. 3c). Na
1
is an allosteric modulator for several family A
GPCRs such as the a
2A
adrenergic receptor, A
2A
adenosine receptor,
m- and d-opioid receptors and D
2
dopamine receptor
33–37
. The con-
served Asp
2.50
is necessary for sodium sensitivity of the a
2A
adrenergic
receptor
37
and D
2
dopamine receptor
36,38
. In PAR1, Asp 367
7.49
might
be expected to form a stronger hydrogen-bonding network and
sodium coordination site than asparagine residues found in most
other family A GPCRs. This more stable network may contribute
to the unusual position of the cytoplasmic end of TM7 that is dis-
placed inward towards TM2. This position is more similar to the
a
c
b
a
L340
L340
Y337
Y337
F271
F271
Y183
Y183
Y353
Y353
A349
A349
H336
H336
L258
L258
TM6
TM6
TM5
TM5
TM7
TM7
TM4
TM4
TM3
TM3
H255
H255
ECL2
ECL2
H255
H255
Y337
Y337
F271
F271
Y183
Y183
Y353
Y353
L332
L332
L333
L333
TM6
TM6
TM5
TM5
TM7
TM7
TM3
TM3
L258
L258
L262
L262
Y350
Y350
L263
L263
L262
L262
H255
H255
ECL2
ECL2
TM5
TM5
ECL2
ECL2
TM2
TM2
TM1
TM1
TM6
TM6
Disulphide
Disulphide
F274
F274
F278
F278
F271
F271
L340
Y337
F271
Y183
Y353
A349
H336
L258
TM6
TM5
TM7
TM4
TM3
H255
ECL2
H255
Y337
F271
F271
Y183
Y353
L332
L333
TM6
TM5
TM7
TM3
L258
L262
Y350
L263
L262
H255
F271
ECL2
TM5
ECL2
Y337
TM2
TM1
TM6
Disulphide
F274
F278
F271
Figure 2
|
Binding interactions of vorapaxar with human PAR1.
a, b, Ligand-binding pocket viewed from the extracellular surface (a) and from
the side of the transmembrane helix bundle (b). ECL2 is coloured in orange in
a and b. Ligand vorapaxar is shown as green sticks. Water molecules are shown
as red spheres. Hydrogen bonds are shown as dotted lines. c, Two residues,
Leu 262 and Leu 263 in ECL2 (shown as dot surface), which pack against
residues His 255, Phe 271
5.39
and Tyr 337
6.59
(shown in Corey–Pauling–Koltun
(CPK) representation), may contribute to the selectivity of vorapaxar for
human PAR1. Also shown are Phe 274
5.42
and Phe 278
5.46
in TM5 (shown as
dot surface), which may indirectly influence vorapaxar binding by packing
interactions with Phe 271
5.39
.
RESEARCH ARTICLE
388 | NATURE | VOL 492 | 20/27 DECEMBER 2012
Macmillan Publishers Limited. All rights reserved
©2012
active b
2
AR bound with either nanobody 80 or heterotrimeric G
protein
29,31
(Fig. 3d).
Structural insights into binding properties of vorapaxar
Vorapaxar binds in an unusual location very close to the extracellular
surface of PAR1. By contrast, ligands for other GPCRs penetrate more
deeply into the transmembrane core (Fig. 1 and Supplementary Figs 5
and 7). The vorapaxar-binding pocket, composed of residues from
TM3, TM4, TM5, TM6 and TM7 as well as ECL2 and ECL3, forms a
tunnel across the receptor with one end open between TM4 and TM5
and the other between TM6 and TM7 occupied by the ethyl carbamate
tail of vorapaxar (Figs 1 and 2). There is only a small opening in the
extracellular surface between ECL2 and ECL3. Details of interactions
between vorapaxar and PAR1 are illustrated in Fig. 2 and Sup-
plementary Fig. 8.
Vorapaxar shows high selectivity for human PAR1 over human
PAR2 and PAR4, and mouse PAR1 in functional assays (Sup-
plementary Fig. 9A, B). The structural basis for this selectivity is not
readily apparent from the crystal structure. Nearly all the residues that
interact with vorapaxar in human PAR1 are conserved in human
PAR2, human PAR4 and mouse PAR1 (Supplementary Fig. 10).
Residues Leu 262 and Leu 263, which are involved in weak hydro-
phobic interactions with vorapaxar in human PAR1, are alanine
and asparagine, respectively, in human PAR4, and Leu 263 is a
methionine in mouse PAR1 (Fig. 2c and Supplementary Fig. 10).
These differences by themselves would not be expected to explain
the high selectivity of vorapaxar. However, Leu 262 and Leu 263 pack
against other amino acids that have more extensive interactions with
vorapaxar. Leu 262 interacts with His 255 in ECL2 and Leu 263 inter-
acts with Phe 271
5.39
at the top of TM5 and Tyr 337
6.59
at the top of
TM6 (Fig. 2c). These interactions may influence ligand-binding
selectivity indirectly by contributing to the overall structure and
stability of the binding pocket. Amino acid differences between
PAR1, PAR2 and PAR4 more distant from the ligand-binding pocket
may also contribute to subtype-specific binding of vorapaxar.
Phe 274
5.42
is Leu in PAR2 and PAR4, and Phe 278
5.46
is Val in
PAR2 and Gly in PAR4. Although neither Phe 274
5.42
nor Phe 278
5.46
directly contact vorapaxar, Phe 278
5.46
packs against Phe 274
5.42
,which
in turn packs against Phe 271
5.39
in the binding pocket (Fig. 2c).
In human PAR2 and PAR4, ECL3 connecting TM6 and TM7 is one
residue shorter than it is in PAR1 (Supplementary Fig. 9C).
Tyr 337
6.59
at the carboxy-terminal end of TM6 forms a strong hydro-
gen bond with vorapaxar (Fig. 2 and Supplementary Fig. 9D).
Another residue, Tyr 353
7.35
, which forms the base of the ligand-
binding pocket together with Tyr 183
3.33
, is located at the N-
terminal end of TM7 (Fig. 2 and Supplementary Fig. 9D). A shorter
ECL3 in human PAR2 and PAR4 may change the relative position of
these amino acids, thereby altering the overall geometry of the binding
pocket. Although the length of ECL3 in mouse and human PAR1 is
the same, four of the eight amino acids are different (Supplementary
Fig. 9C). These differences may affect the structure of ECL3 and
thereby influence interactions between vorapaxar in Tyr 337
6.59
and
Tyr 353
7.35
. Alternatively, these differences could have an effect on the
mechanism by which vorapaxar gains access to the binding pocket.
a
TM5
TM6
F326
6.48
F322
6.44
b
F322
6.44
F326
6.48
I190
3.40
P282
5.50
c
N185
3.35
S189
3.39
D148
2.50
N120
1.50
D367
7.49
Y371
7.53
P368
7.50
TM3
TM2
TM7
TM1 TM7
TM6
TM5
C-terminal
d
Figure 3
|
Structure motifs in PAR1 compared with other family A GPCRs.
a, Superimposition of TM5 and TM6 of human PAR1 (in blue) with those of
other GPCRs including b
2
AR and b
1
AR, A
2A
adenosine receptor, dopamine D
3
receptor, M2 muscarinic receptor, histamine H1 receptor, m-opioid receptor,
S1P
1
and CXCR4 (all in orange). Phe 326
6.48
and Phe 322
6.44
in the
F
6.44
XXC(F)
6.48
XP motif in PAR1 are shown as sticks. This motif is
FXXC(W)
6.48
XP in most other family A GPCRs. Phe 326
6.48
and Phe 322
6.44
are
both in different conformations compared to their counterparts in other
GPCRs. b, In the b
2
AR, rearrangements of three residues, Pro
5.50
, Ile
3.40
and
Phe
6.44
, are associated with receptor activation. Black arrows indicate changes
of these residues going from inactive (cyan) to active (yellow) b
2
AR structures.
The counterparts in the inactive state structure of PAR1 (Pro 282
5.50
, Ile 190
3.40
and Phe 322
6.44
) are shown in blue. c,DP
7.50
XXY motif in TM7 and sodium-
binding site in PAR1. Residues Asp 367
7.49
, Pro 368
7.50
and Tyr 371
7.53
in the
DP
7.50
XXY motif are shown as cyan sticks. This motif is normally NPXXY in
most other family A GPCRs. Sodium is shown as a purple sphere and water
molecules are shown as red spheres. Polar interactions are shown as black
dashed lines. An F
o
2 F
c
omit electron-density map for the putative sodium ion
and water molecules contoured at 4s is shown as purple mesh.
d, Superimposition of the C-terminal part of TM7 in the structure of human
PAR1 (blue), in the inactive structures of other GPCRs (all in orange)
mentioned in a and in the active structure of b
2
AR (in magenta). The
C-terminal part of TM7 in PAR1 adopts a conformation more similar to that
observed in the active state of the b
2
AR.
ARTICLE RESEARCH
20/27 DECEMBER 2012 | VOL 492 | NATURE | 389
Macmillan Publishers Limited. All rights reserved
©2012
Structural insights into vorapaxar inhibition of PAR1
Although this structure is compatible with the very slow dissociation
rate of vorapaxar, it does not provide insight into the mechanism by
which vorapaxar or an agonist peptide gains access to the binding
pocket. None of the three openings to the vorapaxar-binding pocket is
large enough to accommodate the passage of the ligand. We thus
wondered whether the unliganded recep tor might have a more open
structure, similar to that observed for opioid receptors, with unique
interactions between vorapaxar and PAR1 causing an otherwise open
binding pocket to close after vorapaxar binding. To investigate this
issue, we performed long-timescale molecular dynamics simulations
of PAR1 with and without vorapaxar bound. Intriguingly, removal of
the ligand did not lead to a more open binding pocket, but instead to
one that was even more closed (Fig. 4a, b, Supplementary Fig. 11 and
Supplementary Table 2). The extracellular end of TM6 moved about
4A
˚
inward towards TM4, bringing ECL3 in full contact with ECL2
and completely occluding the binding pocket. By contrast, in a similar
study on the MOR, the binding pocket remained open when the
ligand was removed (Supplementary Fig. 11). The collapse of the
vorapaxar-binding pocket may reflect the fact that both vorapaxar
and its binding pocket are uncharged, whereas in the opioid receptors
and many other family A GPCRs, the charged residue Asp
3.32
helps to
keep the binding pocket hydrated after the ligand is removed.
It is interesting to speculate that vorapaxar, a highly lipophilic
molecule, may access the binding pocket through the lipid bilayer,
possibly between TM6 and TM7. This is similar to the binding
mode proposed for retinal to rhodopsin and the lipid sphingosine-1-
phosphate (S1P) to the S1P
1
receptor
39,40
(Supplementary Fig. 5).
To understand the ability of vorapaxar to inhibit agonist binding
and activation, we examined the functional consequences of mutating
four aromatic amino acids that form strong interactions with vora-
paxar: Tyr 183
3.33
, Tyr 353
7.35
, Phe 271
5.39
and Tyr 337
6.59
(Fig. 2a, b
and Supplementary Fig. 3C). Three of these (Tyr 353
7.35
, Phe 271
5.39
and Tyr 337
6.59
) assume substantially different positions in simula-
tions of unliganded PAR1 (Fig. 4a). Tyrosine residues Tyr 183
3.33
in
TM3 and Tyr 353
7.35
in TM7 are linked to each other by a hydrogen
bond and form the base of the binding ‘tunnel’, and are part of a
hydrophobic cage that surrounds the ligand. Tyr 353
7.35
also forms
a hydrogen bond with His 255, the most deeply buried amino acid in
ECL2 (Fig. 2a and Supplementary Fig. 6). This interaction of His 255
with Tyr 353
7.35
contributes to the closed conformation of ECL2
over the ligand-binding pocket. Phe 271
5.39
interacts with the fluoro-
phenyl ring and Tyr 337
6.59
forms a strong hydrogen bond with the
pyridine ring of vorapaxar.
Mutation of Tyr 337
6.59
to phenylalanine and Tyr 353
7.35
to alanine
led to a reduction in cell surface expression, making it difficult to
interpret the associated reduction in agonist peptide activation
(Fig. 4c, d). Mutation of Phe 271
5.39
to alanine was associated with
enhanced cell surface expression, but reduced activation by agonist
peptide. There was little effect of this mutation on maximal inhibition
by vorapaxar. Although not conclusive, this result suggests that
Phe 271
5.39
may have a role in both peptide and vorapaxar binding.
Of interest, mutation of Tyr 183
3.33
exhibited enhanced response to the
agonist peptide and loss of inhibition by vorapaxar (Fig. 4c). This result
suggests a possible role for Tyr 183
3.33
in maintaining the receptor in an
inactive state and indicates that interactions between Tyr 183
3.33
and
vorapaxar may further stabilize an inactive conformation.
Activation of PAR1 by the agonist peptide
Our structure is consistent with datafrom mutagenesis studies thatsug-
gest that the PAR1-tethered agonist peptide may activate the heptahe-
lical bundle of the receptor by interacting with superficial structures
rather than penetrating deeply into the transmembrane core
8,41–45
.
Glu 260, a solvent-exposed residue in ECL2 in both vorapaxar-bound
and unliganded PAR1 (Fig. 5), is of particular interest, as evidence from
mutagenesis studies suggests an interaction with Arg 46 in the tethered
peptide SFLLRN
8,44
. Substitution of Glu 260 with arginine markedly
reduces activation of PAR1 by a peptide with the native tethered ligand
sequence (SFLLRN) but facilitates activation by SFLLEN. Arginine
substitution of Glu 264, which is surface-exposed and near Glu 260 in
the structure, also facilitates activation by SFLLEN.
Mutation of other residues near the extracellular surface, including
Leu96Ala (N terminus), Asp256Ala (ECL2) and Glu347
7.29
Ala/Gln
(ref. 45) (Fig. 5a), markedly reduces activation of PAR1 by the peptide
Y353
H336
Y337
L340
TM3
TM7
TM6
F271
TM5
c
ba
Y183
TM4
TM1
PAR1–vorapaxar
PAR1 no ligand
Vorapaxar
PAR1
PAR1(Y183A)
PAR1(F271A)
PAR1(Y337F)
PAR1(Y353A)
0
5,000
10,000
15,000
20,000
25,000
Vehicle
SFLLRN
SFLLRN + VOR (100 nM)
**
*
*
NS NS
[
3
H]-inositol phosphates (c.p.m.)
Untransfected
PAR1
PAR1(Y183A)
PAR1(F271A)
PAR1(Y337F)
PAR1(Y353A)
0.0
0.5
1.0
1.5
2.0
Surface receptor A
405 nm
d
Figure 4
|
Collapse of ligand-binding pocket in long-timescale molecular
dynamics simulations of unliganded PAR1. Molecular dynamics simulations
were performed on PAR1 from which vorapaxar had been removed. The
vorapaxar-bound PAR1 crystal structure is shown in blue and the unliganded
structure obtained from molecular dynamics simulation is shown in grey.
a, The largest differences between vorapaxar-bound and unliganded PAR1 are
at the extracellular end of TM6 and in ECL3. Residues involved in vorapaxar
binding are shown as sticks. b, Surface view showing collapse of the ligand-
binding pocket during molecular dynamics simulation in the absence of
vorapaxar. c, d, Signalling (c) and cell surface (d) expression for wild-type
human PAR1 and binding-site mutants. Cos7 cells expressing the indicated
receptor constructs were labelled with [
3
H]-myoinositol, pretreated with
vehicle or 100 nM vorapaxar in DMEM medium containing 0.1% BSA, 20 mM
HEPES and 0.2% b-hydroxy cyclodextrin (to retain vorapaxar in solution) for
1 h, then incubated with vehicle or PAR1 agonist (100 mM SFLLRN) for 1 h at
37 uC. Total [
3
H]-inositol-phosphate accumulation was measured. Surface
expression of receptors in cells transfected in parallel was assessed by measuring
binding of anti-Flag antibody to an epitope displayed at the N terminus of the
receptor. Error bars, mean 6 s.e. Results are representative of three separate
experiments. NS, not significant.
RESEARCH ARTICLE
390 | NATURE | VOL 492 | 20/27 DECEMBER 2012
Macmillan Publishers Limited. All rights reserved
©2012
agonist. However, these mutations have only a small effect on agonist
peptide binding, with only Asp256Ala resulting in a more than tenfold
loss of binding affinity
43
. The positions of these residues do not change
substantially when comparing vorapaxar-bound and unliganded PAR1
(Fig. 5b). Of interest, only Glu 347
7.29
is surface exposed (Fig. 5a, b),
suggesting that Leu 96 and Asp 256 may not interact directly with the
agonist peptide or that these amino acids are more exposed than would
appear from the molecular dynamics model of the unliganded receptor.
In the inactive structure, Asp 256 forms a hydrogen bond with Tyr 95
and helps to stabilize interactions between the C-terminal end of the
N terminus and ECL2 (Fig. 5a).
Interestingly, substitution of human PAR1 sequence Asn 259–
Ala 268, the region of ECL2 implicated in tethered ligand binding,
with the cognate Xenopus ECL2 sequence results in an approximately
tenfold increase in basal activity
46
. Figure 5c shows the position of
amino acids that differ between human and Xenopus receptors in
ECL2 in both the crystal structure and the unliganded molecular
dynamics simulation. The superficial location of these activating
mutations suggests that very superficial interactions between the
tethered agonist peptide and the extracellular loops may be sufficient
to activate PAR1. Taken together, these mutagenesis studies suggest
that the PAR1 agonist peptide may activate PAR1 by binding more
superficially than do agonist peptides for opioid receptors. Alterna-
tively, the tethered peptide may bind in a sequential manner, initially
to the extracellular loops but penetrating more deeply into the core of
the receptor through a sequence of conformational intermediates.
Conclusion
The unusual mode of activation and the paucity of pharmacological
tools have made PAR1 one of the more challenging GPCRs to cha-
racterize and a difficult target for drug development. The crystal
structure offers insights into the very high affinity interaction with
the antagonist vorapaxar. This structure will provide a template for
the development of PAR1 antagonists with better drug properties and
the development of antagonists for other PAR subtypes to probe their
biological roles. The mechanism of activation of PAR1 remains poorly
understood. Molecular dynamics simulations of an unliganded recep-
tor together with the location of amino acids known to influence
agonist peptide activity suggest that activation of PAR1 by its agonist
peptide may involve superficial interactions with extracellular loops.
Future efforts will focus on an active-state structure of PAR1 bound to
its tethered agonist peptide.
METHODS SUMMARY
The human PAR1–T4L fusion protein was expressed in Sf9 insect cells and
purified by nickel-affinity chromatography, Flag M1 antibody affinity chromato-
graphy followed by size exclusion chromatography. PAR1–T4L crystals were
grown using the in meso crystallization method. The diffraction data were col-
lected from 18 crystals at the GM/CA@APS beamline in the Argonne National
Laboratory. The structure was solved by molecular replacement and refined in
Phenix. Refinement statistics are provided in Supplementary Table 1. All-atom
molecular dynamics simulations were performed on Anton
47
with lipids and
water molecules represented explicitly. Phosphoinositide hydrolysis assays were
done in Cos7 cells transfected with wild-type and mutant PAR1. More details are
provided in Methods.
Full Methods and any associated references are available in the online version of
the paper.
Received 6 August; accepted 22 October 2012.
Published online 9 December 2012.
1. Vu, T. K., Hung, D. T., Wheaton, V. I. & Coughlin, S. R. Molecular cloning of a
functional thrombin receptor reveals a novel proteolytic mechanism of receptor
activation. Cell 64, 1057–1068 (1991).
2. Coughlin, S. R. Thrombin signalling and protease-activated receptors. Nature 407,
258–264 (2000).
3. Vu, T. K., Wheaton, V. I., Hung, D. T., Charo, I. & Coughlin, S. R. Domains specifying
thrombin-receptor interaction. Nature 353, 674–677 (1991).
4. Chen, J., Ishii, M., Wang, L., Ishii, K. & Coughlin, S. R. Thrombin receptor activation.
Confirmation of the intramolecular tethered liganding hypothesis and discovery of
an alternative intermolecular liganding mode. J. Biol. Chem. 269, 16041–16045
(1994).
E260
D256
L96
Y95
E347
ECL2
ECL3
TM6
TM7
TM1
a
E260
D256
TM6
TM1
L96
TM4
TM5
TM3
ECL2
ECL3
Y266
E264
T261
L262
E260
S259
TM4
TM5
ECL2
b
ECL2
E347
7.29
E264
PAR1–vorapaxar PAR1 no li
g
and
c
Figure 5
|
Residues important for agonist peptide binding and receptor
activation. a, Mutations of residues Glu 260, Asp 256, Leu 96 and Glu 347
7.29
near the extracellular surface have been shown to reduce activation of PAR1 by
the free agonist peptide. Among them only Glu 260 is completely exposed to the
solvent, whereas Asp 256 is the most deeply buried, forming a hydrogen bond
with residue Tyr 95. Although none of these amino acids forms part of the
vorapaxar-binding pocket, Asp 256 forms a hydrogen bond with Tyr 95 that
may stabilize ECL2 over the vorapaxar-binding pocket. Vorapaxar is shown in
green. b, c, Superimposition of the unliganded molecular dynamics simulation
model (grey) with the ligand-bound crystal structure (blue). In b, Residues
Glu 260, Asp 256, Leu 96 and Glu 347
7.29
, which are important for agonist
peptide signalling, are in similar positions in both structures. c, The positions of
residues that differ between human and Xenopus PAR1 in ECL2. Substitution
of these residues in human PAR1 with corresponding residues from Xenopus
PAR1 results in increased basal activity.
ARTICLE RESEARCH
20/27 DECEMBER 2012 | VOL 492 | NATURE | 391
Macmillan Publishers Limited. All rights reserved
©2012
5. Liu, L. W., Vu, T. K., Esmon, C. T. & Coughlin, S. R. The region of the thrombin
receptor resembling hirudin binds to thrombin and alters enzyme specificity.
J. Biol. Chem. 266, 16977–16980 (1991).
6. Ishii, K., Hein, L., Kobilka, B. & Coughlin, S. R. Kinetics of thrombin receptorcleavage
on intact cells. Relation to signaling. J. Biol. Chem. 268, 9780–9786 (1993).
7. Mathews, I. I. et al. Crystallographic structures of thrombin complexed with
thrombin receptor peptides: existence of expected and novel binding modes.
Biochemistry 33, 3266–3279 (1994).
8. Gerszten, R. E. et al. Specificity of the thrombin receptor for agonist peptide is
defined by its extracellular surface. Nature 368, 648–651 (1994).
9. Coughlin, S. R. Protease-activated receptors in hemostasis, thrombosis and
vascular biology. J. Thromb. Haemost. 3, 1800–1814 (2005).
10. Garcı
´
a-Lo
´
pez, M. T., Gutierrez-Rodriguez, M. & Herranz, R. Thrombin-activated
receptors: promising targets for cancer therapy? Curr. Med. Chem. 17, 109–128
(2010).
11. Ramachandran, R., Noorbakhsh, F., Defea, K. & Hollenberg, M. D. Targeting
proteinase-activated receptors: therapeutic potential and challenges. Nature Rev.
Drug Discov. 11, 69–86 (2012).
12. Ishii, K. et al. Determinants of thrombin receptor cleavage. Receptor domains
involved, specificity, and role of the P3 aspartate. J. Biol. Chem. 270,
16435–16440 (1995).
13. Shapiro, M. J., Trejo, J., Zeng, D. & Coughlin, S. R. Role of the thrombin receptor’s
cytoplasmic tail in intracellular trafficking. Distinct determinants for agonist-
triggered versus tonic internalization and intracellular localization. J. Biol. Chem.
271, 32874–32880 (1996).
14. Trejo, J. & Coughlin, S. R. The cytoplasmic tails of protease-activated receptor-1
and substance P receptor specify sorting to lysosomes versus recycling. J. Biol.
Chem. 274, 2216–2224 (1999).
15. Trejo, J., Hammes, S. R. & Coughlin, S. R. Termination of signaling by protease-
activated receptor-1 is linked to lysosomal sorting. Proc. Natl Acad. Sci. USA 95,
13698–13702 (1998).
16. Shapiro, M. J. & Coughlin, S. R. Separate signals for agonist-independent and
agonist-triggered trafficking of protease-activated receptor 1. J. Biol. Chem. 273,
29009–29014 (1998).
17. Hein, L., Ishii, K., Coughlin, S. R. & Kobilka, B. K. Intracellular targeting and
trafficking of thrombin receptors. A novel mechanism for resensitization of a G
protein-coupled receptor. J. Biol. Chem. 269, 27719–27726 (1994).
18. Chackalamannil, S. et al. Discovery of potent orally active thrombin receptor
(protease activated receptor 1) antagonists as novel antithrombotic agents. J. Med.
Chem. 48, 5884–5887 (2005).
19. Morrow, D. A. et al. Vorapaxar in the secondary prevention of atherothrombotic
events. N. Engl. J. Med. 366, 1404–1413 (2012).
20. Kosoglou, T. et al. Pharmacodynamics and pharmacokinetics of the novel PAR-1
antagonist vorapaxar (formerly SCH 530348) in healthy subjects. Eur. J. Clin.
Pharmacol. 68, 249–258 (2012).
21. Soto, A. G. & Trejo, J. N-linked glycosylation of protease-activated receptor-1
second extracellular loop: a critical determinant for ligand-induced receptor
activation and internalization. J. Biol. Chem. 285, 18781–18793 (2010).
22. Wu, B. et al. Structures of the CXCR4 chemokine GPCR with small-molecule and
cyclic peptide antagonists. Science 330, 1066–1071 (2010).
23. Manglik, A. et al. Crystal structure of the micro-opioid receptor bound to a
morphinan antagonist. Nature 485, 321–326 (2012).
24. Wu, H. et al. Structure of the human k-opioid receptor in complex with JDTic.
Nature 485, 327–332 (2012).
25. Granier, S. et al. Structure of the d-opioid receptor bound to naltrindole. Nature
485, 400–404 (2012).
26. Smit, M. J. et al. Pharmacogenomic and structural analysis of constitutive g
protein-coupled receptor activity. Annu. Rev. Pharmacol. Toxicol. 47, 53–87 (2007).
27. Schwartz, T. W., Frimurer, T. M., Holst, B., Rosenkilde, M. M. & Elling, C. E. Molecular
mechanism of 7TM receptor activation—a global toggle switch model. Annu. Rev.
Pharmacol. Toxicol. 46, 481–519 (2006).
28. Fredriksson, R., Lagerstrom, M. C., Lundin, L. G. & Schioth, H. B. The G-protein-
coupled receptors in the human genome form five main families. Phylogenetic
analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272
(2003).
29. Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the b
2
adrenoceptor. Nature 469, 175–180 (2011).
30. Rosenbaum, D. M. et al. Structure and function of an irreversible agonist-b
2
adrenoceptor complex. Nature 469, 236–240 (2011).
31. Rasmussen, S. G. et al. Crystal structure of the b
2
adrenergic receptor–Gs protein
complex. Nature 477, 549–555 (2011).
32. Harding, M. M. Metal-ligand geometry relevant to proteins and in proteins: sodium
and potassium. Acta Crystallogr. D 58, 872–874 (2002).
33. Horstman, D. A. et al. An aspartate conserved among G-protein receptors confers
allosteric regulation of a
2
-adrenergic receptors by sodium. J. Biol. Chem. 265,
21590–21595 (1990).
34. Costa, T., Lang, J., Gless, C. & Herz, A. Spontaneous association between opioid
receptors and GTP-binding regulatory proteins in native membranes: specific
regulation by antagonists and sodium ions. Mol. Pharmacol. 37, 383–394 (1990).
35. Gao, Z. G. & Ijzerman, A. P. Allosteric modulation of A
2A
adenosine receptors by
amiloride analogues and sodium ions. Biochem. Pharmacol. 60, 669–676 (2000).
36. Selent, J., Sanz, F., Pastor, M. & De Fabritiis, G. Induced effects of sodium ions on
dopaminergic G-protein coupled receptors. PLOS Comput. Biol. 6, e1000884
(2010).
37. Wilson, M. H., Highfield, H. A. & Limbird, L. E. The role of a conserved inter-
transmembrane domain interface in regulating a
2a
-adrenergic receptor
conformational stability and cell-surface turnover. Mol. Pharmacol. 59, 929–938
(2001).
38. Neve, K. A. et al. Modeling and mutational analysis of a putative sodium-binding
pocket on the dopamine D2 receptor. Mol. Pharmacol. 60, 373–381 (2001).
39. Palczewski, K. et al. Crystal structure of rhodopsin: A G protein-coupled receptor.
Science 289, 739–745 (2000).
40. Hanson, M. A. et al. Crystal structure of a lipid G protein-coupled receptor. Science
335, 851–855 (2012).
41. Lerner, D. J., Chen, M., Tram, T. & Coughlin, S. R. Agonist recognition by proteinase-
activated receptor 2 and thrombin receptor. Importance of extracellular loop
interactions for receptor function. J. Biol. Chem. 271, 13943–13947 (1996).
42. Nanevicz, T. et al. Mechanisms of thrombin receptor agonist specificity. Chimeric
receptors and complementary mutations identify an agonist recognition site.
J. Biol. Chem. 270, 21619–21625 (1995).
43. Blackhart, B. D. et al. Extracellular mutations of protease-activated receptor-1
result in differential activation by thrombin and thrombin receptor agonist
peptide. Mol. Pharmacol. 58, 1178–1187 (2000).
44. Seeley, S. et al. Structural basis for thrombin activation of a protease-activated
receptor: inhibition of intramolecular liganding. Chem. Biol. 10, 1033–1041
(2003).
45. Bahou, W. F., Kutok, J. L., Wong, A., Potter, C. L. & Coller, B. S. Identification of a novel
thrombin receptor sequence required for activation-dependent responses. Blood
84, 4195–4202 (1994).
46. Nanevicz, T., Wang, L., Chen, M., Ishii, M. & Coughlin, S. R. Thrombin receptor
activating mutations. Alteration of an extracellular agonist recognition domain
causes constitutive signaling. J. Biol. Chem. 271, 702–706 (1996).
47. Shaw, D. E. et al. Millisecond-scale molecular dynamicssimulations on Anton. Proc.
Conf. High Performance Computing Networking, Storage Analysis Article no. 39
(2009).
Supplementary Information is available in the online version of the paper.
Acknowledgements We acknowledge support from the National Institutes of Health,
grants NS028471 (B.K.K.), and HL44907 and HL65590 (S.R.C.), and from the Mathers
Foundation (B.K.K. and W.I.W.).
Author Contributions C.Z. optimized the construct, expressed and purified human
PAR1–T4L for crystallization, developed the purification procedure, performed
crystallization trials, optimizedcrystallization conditions, collected diffraction data, and
solved and refined the structure. Y.S. helped design and make constructs for
baculoviral expression, and executed and analysed cell-based functional assays of
wild-type and mutant PAR1. D.H.A. designed, performed and analysed molecular
dynamics simulations. J.J.F. developed the initial expression and purification protocol
for PAR1. D.P. helped design, execute and analyse platelet function studies. Y.Z. helped
design and make constructs for baculoviral PAR expression. H.F.G. performed and
analysed molecular dynamics simulations, and assisted with manuscript preparation.
A.P. made vorapaxar. R.O.D. oversaw, designed and analysed molecular dynamics
simulations, and assisted with manuscript preparation. D.E.S. oversaw molecular
dynamics simulations and analysis. W.I.W. assisted with X-ray diffraction data
processing and crystal structure refinement. S.R.C. and B.K.K. initiated the project,
planned and analysed experiments, and supervised the research and wrote the
manuscript with C.Z.
Author Information Atomic coordinates and structure factors for PAR1 are deposited
in the Protein Data Bank under accession code 3VW7. Reprints and permissions
information is available at www.nature.com/reprints. The authors declare no
competing financial interests. Readers are welcome to comment on the online version
of the paper. Correspondence and requests for materials should be addressed to S.R.C.
(shaun.coughlin@ucsf.edu) or B.K.K. (kobilka@stanford.edu).
RESEARCH ARTICLE
392 | NATURE | VOL 492 | 20/27 DECEMBER 2012
Macmillan Publishers Limited. All rights reserved
©2012
METHODS
PAR1–T4L expression and purification. To facilitate crystallogenesis, a human
PAR1 construct was generated with several modifications. A TEV protease-
recognition site was introduced after residue Pro 85, two N-linked glycosylation
sites in ECL2 were removed by mutation (Asn250Gly and Asn259Ser), and the C
terminus was truncated after residue Ser 395. T4L residues 2–161 (ref. 48) were
inserted into the third intracellular loop between residues Ala 301 and Ala 303,
with only one residue Val 302 removed. To facilitate purification, an N-terminal
Flag epitope was inserted after a signal peptide and a C-terminal deca-histidine
tag was introduced. The final crystallization construct PAR1–T4L is shown in
Supplementary Fig. 2.
The modified PAR1 was expressed in Sf9 cells using the pFastBac baculovirus
system (Invitrogen). The ligand vorapaxar was added at 100 nM to the cells
during expression. The cells were infected with baculovirus at 27 uC for 48 h
before collection. To purify the receptor, infected cells were lysed by osmotic
shock in low-salt buffer containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA,
100 nM vorapaxar and 2 mg ml
21
iodoacetamide. Iodoacetamide was used to
alkylate reactive cysteines to prevent nonspecific oligomerization. The protein
was further extracted from cell membranes using a glass dounce homogenizer in
buffer containing 20 mM Tris-HCl, pH 7.5, 350 mM NaCl, 1% dodecyl maltoside
(DDM), 0.03% cholesterol hemisuccinate (CHS), 0.2% sodium cholate, 10% gly-
cerol, 2 mg ml
21
iodoacetamide and 100 nM vorapaxar. Cell debris was removed
by high-speed centrifugation. From this point, 1 m M vorapaxar was added in all
the following buffers used for purification. Nickel-NTA agarose resin was added
to the supernatant after homogenization and stirred for 1 h at 4 uC. The resin was
then washed three times in batch with buffer comprised of 20 mM HEPES,
pH 7.5, 350 mM NaCl, 0.1% DDM, 0.02% CHS and 1 mM vorapaxar, and trans-
ferred to a glass column. The bound receptor was eluted with buffer containing
300 mM imidazole and loaded onto an anti-Flag M1 affinity column. After
extensive washing with buffer comprised of 20 mM HEPES, pH 7.5, 350 mM
NaCl, 0.1% DDM, 0.02% CHS, 1 mM vorapaxar and 2 mM Ca
21
, the receptor
was eluted from M1 resin using the same buffer without Ca
21
but with 200 mg
ml
21
Flag peptide and 5 mM EDTA. To remove extra N-terminal residues and
the Flag epitope, TEV protease was added to the receptor and the cleavage
reaction run at room temperature overnight. Size exclusion chromatography
was used to obtain the final monodisperse receptor preparation. Purified
PAR1–T4L was concentrated to 40–50 mg ml
21
using 100 kDa cut-off Vivaspin
concentrators for crystallization.
Crystallization. As for other T4L-fused GPCRs crystallized so far, in meso crys-
tallization was used to obtain PAR1–T4L crystals
49,50
. The protein was mixed with
monoolein and cholesterol (10:1 by mass) using the two syringe mixing method
by weight of 1:1.5 (protein:lipid). After a clear lipidic cubic phase formed, the mix
was dispensed onto glass plates in 20–40 nl drops overlaid with 700 nl precipitant
solution using a Gryphon LCP robot. Crystals appeared in two days in 0.1–0.2 M
sodium chloride, 100 mM sodium phosphate, pH 6.0–6.5, 25–35% PEG300, and
grew to full size after 1 week (Supplementary Fig. 3).
Data collection and structure determination. Crystals were collected and frozen
in liquid nitrogen. Date collection was performed at beamline 23-ID of GM/
CA@APS at the Advanced Photon Source. Microbeams of 10 or 20 mM diameter
were used to acquire all diffraction data. Owing to radiation damage, only 5–20
degrees of rotation data were collected from each crystal. All data were processed
with the HKL2000 package
51
. A 2.2-A
˚
data set was obtained by merging diffrac-
tion data from 18 crystals. The space group was determined to be P2
1
2
1
2
1
.
Molecular replacement was performed using the program Phaser
52
in Phenix
53
,
with the CXCR4 structure (PDB accession 3ODU) as the search model. The
seven-transmembrane helices without any loops, and the T4L in the CXCR4
structure, were used as independent search models. The initial structure model
was completed and improved through iterative refinement in Phenix
53
and man-
ual rebuilding of all the loops and several parts in the transmembrane region in
Coot
54
. Model refinement in Phenix and manual adjustment in Coot was per-
formed to improve the model. The final structure was determined at 2.2-A
˚
reso-
lution. The quality of the structure was assessed using Molprobity
55
. Data
processing and structure refinement statistics are shown in Supplementary
Table 1. Figures were prepared with PyMol
56
.
Phosphoinositide hydrolysis assays and cell-surface expression level. The
QuikChange site-directed mutagenesis kit (Agilent) was used to generate human
PAR1 mutants and all mutants were fully sequenced. Cos7 cells were transiently
transfected using Fugene HD with either empty vector or wild-type human PAR1
and mutants in the mammalian expression vector pBJ1 and signalling assays were
performed as described in ref. 46. In brief, Cos7 cells expressing wild-type or
mutant human PAR1 were labelled with [
3
H]-myoinositol, then incubated with
vehicle or 100 nM vorapaxar in DMEM medium containing 0.1% BSA, 20 mM
HEPES, 0.2% 2-hydroxypropyl-b-cyclodextrin (to retain vorapaxar in solution)
for 1 h at 37 uC. Agonist (100 mM SFLLRN or 10 nM thrombin for PAR1 or other
PAR agonists as indicated) was added and incubation continued for 1 h. The total
amount of accumulated [
3
H]-inositol phosphates accumulated was determined
as in ref. 46. Cos7 cells transfected with empty vector had little response to PAR
agonists, and treatment with vorapaxar alone did not affect phosphoinositide
hydrolysis (Supplementary Fig. 9A).
Surface expression of receptors was measured as described in refs 13 and 16.
Cos7 cells were transiently transfected with empty pBJ1 or pBJ1 directing expres-
sion of N-terminal Flag-tagged versions of wild-type human PAR1 or mutants.
After 48 h, cells were washed once with serum-free medium containing 0.1% BSA
and 20 mM HEPES, then incubated with 3 mgml
21
Flag M1 antibody (Sigma) for
1 h at 4 uC in the same medium. The cells were then washed twice with PBS
containing Ca
21
and Mg
21
to remove unbound antibody and fixed with 2%
paraformaldehyde for 5 min. The cells were then washed twice with PBS with
Ca
21
and Mg
21
, and incubated with goat anti-mouse horseradish peroxidase
(HRP)-conjugated secondary antibody, washed and developed with one-step
ABTS HRP substrate (Pierce). The absorbance at 405 nm was measured as indica-
tion of cell surface receptor expression levels.
Platelet signalling assays. Washed human platelets were prepared and PAR1-
dependent responses were measured as described in ref. 57. In brief, acid-citrate-
dextrose anti-coagulated human blood samples (60 ml per donor) were obtained
from AllCells. Blood was centrifuged without braking at 250g at 37 uC for 15 min.
The upper platelet-rich plasma phase was collected, incubated at 37 uC for 10 min
in the presence of prostacyclin (PGI
2
, 0.5 mM), and centrifuged at 2,200g for
15 min. The pellet was resuspended in complete Tyrode’s solution (134 mM
NaCl, 12 mM NaHCO
3
, 2.9 mM KCl, 0.34 mM Na
2
HPO
4
, 1.0 mM MgCl
2
,
10 mM HEPES, 0.9% (w/v) dextrose, pH 7.4) containing 2 mM CaCl
2
, 0.35%
(w/v) bovine serum albumin (BSA), 10 U ml
21
heparin, and 0.5 mM PGI
2
. The
platelet suspension was incubated for 10 min at 37 uC then centrifuged at 1,900g
for 8 min. This wash step was repeated and the final pellet resuspended in
Tyrode’s buffer supplemented with BSA and 0.02 U ml
21
apyrase. Platelets were
incubated at 37 uC for 30 min to allow recovery from the effects of PGI
2
, counted
using a Hemavet FS950 (Drew Scientific) and diluted to 300,000 cells per micro-
litre in Tyrode’s solution.
To antagonize PAR1, vorapaxar or vehicle (2% (w/v) 2-hydroxypropyl-b-
cyclodextrin in dimethylsulphoxide (DMSO)) were added to platelet suspensions
that were then incubated for 1 h at 37 uC before addition of agonists. The final
concentrations of 2-hydroxypropyl-b-cyclodextrin and DMSO in platelet sus-
pensions were 0.002% and 0.1%, respectively. Where reversibility was evaluated,
platelets were washed twice with Tyrode’s buffer containing BSA and PGI
2
after
vorapaxar treatment then diluted for cell activation assays as above.
For flow cytometric analysis of platelet activation, platelets suspended in
Tyrode’s solution containing 2 mM CaCl
2
, 0.35% BSA and 0.02 U ml
21
apyrase
were incubated with agonist and antibody simultaneously. Fluor-conjugated
antibodies directed against P-selectin (phycoerythrin (PE)-conjugate of AK-4;
Ebiosciences; 1:25 dilution) and the activated conformation of integrin a
IIb
b
3
(FITC-conjugate of PAC-1, BD Biosciences; 1:25 dilution) were used. After
15 min at 37 uC, the platelet suspension was diluted with PBS and platelet-bound
antibody measured using an Accuri C6 flow cytometer (Accuri). Samples from at
least three different donors were analysed, each in triplicate.
Molecular dynamics simulation methods. In all simulations, the receptor was
embedded in a hydrated lipid bilayer with all atoms, including those in the lipids
and water, represented explicitly. Simulations were performed on Anton
47
,a
special-purpose computer designed to accelerate molecular dynamics simula-
tions by orders of magnitude.
System set-up and simulation protocol. Simulations of PAR1 were based on the
crystal structure of the PAR1–vorapaxar complex. The crystallized construct has
T4L inserted into ICL3 in place of residue 302. For the simulations, the T4L
portion was omitted, and residue 302 was modelled in. The unresolved segment
of ICL2 (residues 209–213) was also modelled in. Residues 209 and 213 were
added manually, and residues 210–212 were modelled in using Prime
(Schro¨dinger LLC). The Refine Loops tool in Prime, with default settings, was
then used to refine residues 209–213.
The simulation of the MOR dimer was based on the crystal structure of MOR
bound to the irreversible antagonist b-funaltrexamine (PDB accession 4DKL).
Both monomers of the crystallographic dimer were included in the simulation,
but b-funaltrexamine was deleted from the binding pocket. As with PAR1, the
T4L sequence was omitted in our simulations. Side chains for residue Met 65
1.29
,
Thr 67
1.31
, Lys 260
ICL3
and Arg 263
ICL3
were not fully resolved in the crystal
structure, so they were modelled in by hand, with rotamers chosen to avoid
any clashes with resolved residues.
For both PAR1 and MOR, hydrogens were added to the crystal structures using
Maestro (Schro¨dinger LLC), as described in previous work
58
. Histidines were
ARTICLE RESEARCH
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©2012
singly protonated on the epsilon nitrogen. All other titratable residues were left in
their dominant protonation state for pH 7.0, except for Asp 367
7.49
in PAR1 and
Asp 114
2.50
in MOR, which were protonated, and Asp 148
2.50
in PAR1, which was
protonated in certain simulations. PAR1 was simulated both with and without the
crystallographic sodium ion by Asp 148
2.50
(Supplementary Table 2); Asp 148
2.50
was not protonated in simulations that included this ion, but was protonated
otherwise. The conserved aspartate at position 2.50 is known to be protonated in
rhodopsin
59
, and the residue at position 7.49 is most often an (uncharged) aspara-
gine residue in family A GPCRs (the ‘N’ of the NPXXY motif).
Prepared protein structures were inserted into an equilibrated POPC bilayer as
described previously
32
. Sodium and chloride ions were added to neutralize the net
charge of the system and to create a 150-mM solution.
Simulations of the PAR1 receptor initially measured 88.9 3 88.9 3 88.7 A
˚
and
contained 174 lipid molecules, and approximately 13,152 water molecules, for a
total of ,67,500 atoms. When the crystallographic sodium ion near Asp 148
2.50
was included, the simulation contained 32 sodium ions and 36 chloride ions.
When the crystallographic sodium ion was not included, the system contained 31
sodium ions and 36 chloride ions. To simulate the unliganded PAR1 receptor,
vorapaxar was deleted from the binding pocket. Simulations of the MOR dimer
initially measured 100.0 3 100.0 3 89.0 A
˚
and contained 204 lipid molecules, 19
sodium ions, 43 chloride ions, and approximately 16,654 water molecules, for a
total of ,86,700 atoms.
All simulations were equilibrated using Anton in the NPT ensemble at 310 K
(37 uC) and 10
5
Pa with 5 kcal mol
21
A
˚
22
harmonic position restraints applied to
all non-hydrogen atoms of the protein and the ligand; these restraints were
tapered off linearly over 50 ns. All bond lengths to hydrogen atoms were con-
strained using M-SHAKE
60
. A RESPA integrator
61
was used with a time step of
2 fs, and long-range electrostatics were computed every 6 fs. Production simula-
tions were initiated from the final snapshot of the corresponding equilibration
runs, with velocities sampled from the Boltzmann distribution at 310 K, using the
same integration scheme, long-range electrostatics method, temperature and
pressure. For PAR1, Van der Waals and short-range electrostatic interactions
were cut-off at 10.3 A
˚
and long-range electrostatic interactions were computed
using the k-space Gaussian split Ewald method
62
with a 32 3 32 3 32 grid,
s 5 2.27 A
˚
, and s
s
5 1.59 A
˚
. For MOR, van der Waals and short-range electro-
static interactions were cut-off at 10.16 A
˚
and long-range electrostatic interac-
tions were computed using the k-space Gaussian split Ewald method with a
64 3 64 3 64 grid, s 5 2.25 A
˚
, and s
s
5 1.55 A
˚
.
We performed two vorapaxar-bound PAR1 simulations and four unliganded
PAR1 simulations, and results were consistent across each set. The two receptors
in our MOR dimer simulation also exhibited consistent behaviour. The simu-
lation protocol we followed has been validated in previous simulations of
GPCRs
63,64
. Nevertheless, it is possible that different behaviour might have been
observed in even longer simulations, with different force field parameters, or with
a different choice of simulation conditions.
Force field parameters. We used the CHARMM27 parameter set for protein
molecules and salt ions, with the CHARMM TIP3P water model
65
; protein para-
meters incorporated CMAP terms
66
and modified charges on the Asp, Glu and
Arg side chains
67
. We used a modified CHARMM lipid force field
68
. Force field
parameters for vorapaxar were obtained from the CHARMM ParamChem web
server
69
, version 0.9.6 b.
Analysis protocols. Trajectory snapshots, each containing a record of all
atom positions at a particular instant in time, were saved every 180 ps during
production simulations. Time series data shown in Supplementary Fig. 11 were
smoothed by applying a 9.9-ns (55-snapshot) running average. VMD was used to
visualize trajectories
70
.
48. Rosenbaum, D. M. et al. GPCR engineering yields high-resolution structural
insights into b
2
-adrenergic receptor function. Science 318, 1266–1273 (2007).
49. Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic
mesophases. Nature Protocols 4, 706–731 (2009).
50. Caffrey, M. Crystallizing membrane proteins for structure determination: use of
lipidic mesophases. Annu. Rev. Biophys. 38, 29–51 (2009).
51. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in
oscillation mode. Methods Enzymol. 276, 307–326 (1997).
52. McCoy, A. J. et al. Phasercrystallographic software. J. Appl. Crystallogr. 40, 658–674
(2007).
53. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for
macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).
54. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta
Crystallogr. D. 60, 2126–2132 (2004).
55. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular
crystallography. Acta Crystallogr. D 66, 12–21 (2010).
56. The. PyMOL Molecular Graphics System v.1.5.0.4 (Schro
¨
dinger, LLC, 2010).
57. Cornelissen, I. et al. Roles and interactions among protease-activated receptors
and P2ry12 in hemostasis and thrombosis. Proc. Natl Acad. Sci. USA 107,
18605–18610 (2010).
58. Dror, R. O. et al. Identification of two distinct inactive conformations of the b
2
-
adrenergic receptor reconciles structural and biochemical observations. Proc. Natl
Acad. Sci. USA 106, 4689–4694 (2009).
59. Fahmy, K. et al. Protonation states of membrane-embedded carboxylic acid
groups in rhodopsin and metarhodopsin II: a Fourier-transform infrared
spectroscopy study of site-directed mutants. Proc. Natl Acad. Sci. USA 90,
10206–10210 (1993).
60. Kra
¨
utler, V., Gunsteren, W. F. v. & Hu
¨
nenberger, P. H. A fast SHAKE algorithm to
solve distance constraint equations for small molecules in molecular dynamics
simulations. J. Comput. Chem. 22, 501–508 (2001).
61. Tuckerman, M., Berne, B. J. & Martyna, G. J. Reversible multiple time scale
molecular dynamics. J. Chem. Phys. 97, 1990–2001 (1992).
62. Shan, Y., Klepeis, J. L., Eastwood, M. P., Dror, R. O. & Shaw, D. E. Gaussian split
Ewald: A fast Ewald mesh method for molecular simulation. J. Chem. Phys. 122,
54101 (2005).
63. Dror, R. O. et al. Activation mechanism of the b
2
-adrenergic receptor. Proc. Natl
Acad. Sci. USA 108, 18684–18689 (2011).
64. Dror, R. O. et al. Pathway and mechanism of drug binding to G-protein-coupled
receptors. Proc. Natl Acad. Sci. USA 108, 13118–13123 (2011).
65. MacKerell, A. D. Jr, Bashford, D. & Bellott, M. All-atom empirical potential for
molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102,
3586–3616 (1998).
66. Mackerell, A. D. Jr, Feig, M. & Brooks, C. L. III. Extending the treatment of backbone
energetics in protein force fields: limitations of gas-phase quantum mechanics in
reproducing protein conformational distributions in molecular dynamics
simulations. J. Comput. Chem. 25, 1400–1415 (2004).
67. Piana, S., Lindorff-Larsen, K. & Shaw, D. E. How robust are protein folding
simulations with respect to force field parameterization? Biophys. J. 100, L47–L49
(2011).
68. Klauda, J. B. et al. Update of the CHARMM all-atom additive force field for lipids:
validation on six lipid types. J. Phys. Chem. B 114, 7830–7843 (2010).
69. Vanommeslaeghe, K. et al. CHARMM general force field: a force field for drug-like
molecules compatible with the CHARMM all-atom additive biological force fields.
J. Comput. Chem. 31, 671–690 (2010).
70. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol.
Graph. 14, 33–38 (1996).
RESEARCH ARTICLE
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