High-resolution crystal structure of human protease-activated receptor 1

Abstract

Protease-activated receptor 1 (PAR1) is the prototypical member of 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 Å 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.

Figure 1. PAR1 activation and overall structure of human PAR1 complex with antagonist vorapaxar

a, Thrombin cleaves 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 in blue ribbon and vorapaxar is shown as green spheres. Monoolein is shown in orange, water in red. The disulfide 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.

Figure 2. Binding interactions of vorapaxar with human PAR1

a, b, Ligand binding pocket viewed from extracellular surface (a) and from side of transmembrane helix bundle (b). ECL2 is colored in orange in panels a and b. Ligand vorapaxar is shown as green sticks. Water molecules are shown as red spheres. Hydrogen bonds are shown as black dotted lines. c, Two residues L262 and L263 in ICL2 (shown as dot surface), which pack against residues H255, F271^. and Y337^. (shown in CPK representation), may contribute to the selectivity of vorapaxar for human PAR1. Also shown are F274^. and F278^. in TM5 (shown as dot surface), which may indirectly influence vorapaxar binding by packing interactions with F271^..

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 β₂- and β₁-adrenergic receptors (AR), A2A adenosine receptor, dopamine D3 receptor, M2 muscarinic receptor, histamine H1 receptor, µ-opioid receptor, S1P1 receptor and CXCR4 (all in orange). F326^. and F322^. in the F^.xxCF^.xP motif in PAR1 are shown as sticks. This motif is FxxCW^.xP in most other Family A GPCRs. F326^. and F322^. are both in different conformations compared to their counterparts in other GPCRs. b, In the β₂AR, rearrangements of three residues, P^., I^. and F^., are associated with receptor activation. Black arrows indicate changes of these residues in going from inactive (cyan) to active (yellow) β₂AR structures. The counterparts in the inactive state structure of PAR1 (P282^., I190^. and F322^.) are shown in blue. c, DP^.xxY motif in TM7 and sodium binding site in PAR1. Residues D367^., P368^. and Y371^. in DP^.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 dash lines. An Fo-Fc omit electron density map for the putative sodium ion and water molecules contoured at 4σ 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 panel a and in the active structure of β₂AR (in magenta). The C-terminal part of TM7 in PAR1 adopts a conformation more similar to that observed in the active state of the β₂AR.

Figure 4. Collapse of ligand-binding pocket in long-timescale molecular dynamics (MD) simulations of unliganded PAR1

MD 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 MD simulation is shown in gray. 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 MD simulation in the absence of vorapaxar. c Signaling and d cell surface expression for wild-type human PAR1 and PAR1 binding site-mutants. Cos7 cells expressing the indicated receptor constructs were labeled with [³H] myoinositol, pretreated with vehicle or 100nM vorapaxar in DMEM medium containing 0.1% BSA, 20mM HEPES, 0.2% β-hydroxy cyclodextrin (to retain vorapaxar in solution) for 1h, then incubated with vehicle or PAR1 agonist (100µM SFLLRN) for 1h at 37°C. Total [³H] 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 receptor's N terminus. Results are representative of three separate experiments.

Figure 5. Residues important for agonist peptide binding and receptor activation

a, Mutations of residues E260, D256, L96 and E347^. near the extracellular surface have been shown to reduce activation of PAR1 by the free agonist peptide. Among them only E260 is completely exposed to the solvent, while D256 is the most deeply buried, forming H-bond with residue Y95. While none of these amino acids form part of the vorapaxar binding pocket, D256 forms a hydrogen bond with Y95 that may stabilize ECL2 over the vorapaxar binding pocket. Vorapaxar is shown in green. b, c, Superimposition of the unliganded MD simulation model (gray) with the ligand-bound crystal structure (blue). In b, Residues E260, D256, L96 and E347^., which are important for agonist peptide signaling, 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.