High-Resolution Crystal Structure of Human Protease-Activated Receptor 1
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ARTICLE doi:10.1038/nature11701 High-resolution crystal structure of human protease-activated receptor 1 Cheng Zhang1, Yoga Srinivasan2, Daniel H. Arlow3, Juan Jose Fung1{, Daniel Palmer2, Yaowu Zheng2{, Hillary F. Green3, Anjali Pandey4, Ron O. Dror3, David E. Shaw3, William I. Weis1,5, Shaun R. Coughlin2 & Brian K. Kobilka1 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-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 Inactive PAR1 Active PAR1 1,2 a c (GPCRs) that mediate cellular responses to specific proteases . The N Extracellular 60° S coagulation protease thrombin activates the prototypical PAR, PAR1, F L L Thrombin R Extracellular by specific cleavage of the N-terminal exodomain of the receptor to N N R L L F S generate a new N terminus. This new N terminus then functions as a tethered peptide agonist that binds intramolecularly to the seven- C C transmembrane helix bundle of the receptor to affect G-protein Intracellular 1,3–8 G activation (Fig. 1a). In adult mammals, the four members of the i G G q PAR family link tissue injury and local generation of active coagu- 12/13 lation proteases to cellular responses that help to orchestrate hae- 2,9 b ECL2 mostasis, thrombosis, inflammation and perhaps tissue repair . N PARs may also participate in the progression of specific cancers10,11. TM7 TM2 In contrast to a typical receptor–agonist binding interaction, the ECL1 TM1 interaction of PAR1 with its activator, thrombin, is that of a protease Disulphide substrate, with thrombin binding transiently to the receptor, cleaving ECL2 1,3–7,12 it, then dissociating . Proteolytic unmasking of the tethered pep- 90° TM3 tide agonist of the receptor is irreversible, and although a free syn- N thetic hexapeptide with the amino acid sequence of the tethered TM7 agonist (SFLLRN) can activate the receptor with half-maximum effec- m TM6 TM4 tive concentration (EC50) values in the 3–10- M range, the local TM4 concentration of the tethered agonist peptide is estimated to be about 0.4 mM. Accordingly, PAR signalling must be actively terminated13–15 TM5 TM3 and, unlike most other GPCRs that can go though many rounds of TM6 activation by reversible diffusible hormones and neurotransmitters, TM2 TM5 PARs are degraded after a single activation6,13–17. Identification of effec- tive PAR antagonists has been challenging because low molecular mass Figure 1 | PAR1 activation and overall structure of human PAR1 complex compounds must compete with the very high local concentration of with antagonist vorapaxar. a, Thrombin cleaves the PAR1 N terminus and the tethered agonist generated by proteolytic cleavage. exposes a new N-terminal peptide, SFLLRN, which can bind to and activate the Vorapaxar is a highly specific, virtually irreversible PAR1 antago- transmembrane core of PAR1. PAR1 can activate several G proteins including 18 Gi,G12/13 and Gq. b, Overall view of the human PAR1 structure and the nist (Supplementary Fig. 1). In a phase III trial, vorapaxar protected extracellular surface. The receptor is shown as blue ribbon and vorapaxar is patients against recurrent myocardial infarction at a cost of increased shown as green spheres. Monoolein is shown in orange, water in red. The 19,20 bleeding . Given the latter, an antagonist that is reversible in the disulphide bond is shown as a yellow stick. c, Surface view of the ligand-binding setting of bleeding might be desirable. Although the very slow dissoci- pocket viewed from two different perspectives. The vorapaxar-binding pocket is ation rate of vorapaxar from PAR1 probably accounts for its ability to close to the extracellular surface but not well exposed to the extracellular solvent. 1Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA. 2Cardiovascular Research Institute, University of California, San Francisco, 555 Mission Bay Boulevard South, S452P, San Francisco, California 94158, USA. 3D. E. Shaw Research, New York, New York 10036, USA. 4Portola Pharmaceuticals, 270 East Grand Avenue, South San Francisco, California 94080, USA. 5Department 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.). 00 MONTH 2012 | VOL 000 | NATURE | 1 ©2012 Macmillan Publishers Limited. All rights reserved RESEARCH ARTICLE 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- a TM7 L340 nalling but fast enough to allow useful reversal after cessation of drug. A349 TM1 Y350 In an effort to advance our understanding of PAR1 structure and TM6 function and to provide a foundation for discovery of new agents to ECL2 H336 advance the pharmacology of PARs, we obtained the crystal structure of vorapaxar-bound human PAR1. L258 Y353 Y337 H255 L262 TM5 Y183 Crystallization of human PAR1 TM2 F271 To facilitate crystallogenesis, T4 lysozyme (T4L) was inserted into intracellular loop 3 (ICL3) in human PAR1, the N-linked glycosyla- b TM3 Disulphide TM4 tion sites in ECL2 were mutated21, 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- c L263 plementary Fig. 2). The structure of human PAR1–T4L bound to ECL2 L262 vorapaxar was determined to 2.2 A˚ by merging diffraction data sets ECL2 TM7 L258 Y337 from 18 crystals grown in the lipidic cubic phase (Supplementary Figs TM5 3 and 4). Details of data collection and structure refinement are listed F271 TM6 in Supplementary Table 1. H255 Y353 Y337 H255 L332 F274 PAR1 has the expected seven-transmembrane segment bundle L333 TM6 TM3 (Fig. 1b). There are several lipid molecules assigned as monoolein F278 from lipidic cubic phase in the structure (Fig. 1b), but no ordered F271 cholesterol molecules were observed. The remaining N-terminal frag- Y183 TM5 ment Arg 86–Glu 90 and a part of the ICL2 from Gln 209 to Trp 213 Figure 2 | Binding interactions of vorapaxar with human PAR1. are not modelled in the structure because of the weak electron density. a, b, Ligand-binding pocket viewed from the extracellular surface (a) and from There is no clear electron density for residues after Cys 378, and no the side of the transmembrane helix bundle (b). ECL2 is coloured in orange in helix 8 is observed after transmembrane segment 7 (TM7) in the a and b. Ligand vorapaxar is shown as green sticks. Water molecules are shown structure. Whether this reflects a lack of a helix 8 in PAR1 in its native as red spheres. Hydrogen bonds are shown as dotted lines. c, Two residues, state or conditions in the crystal is not known. Leu 262 and Leu 263 in ECL2 (shown as dot surface), which pack against Cys 1753.25 in helix III and Cys 254 in extracellular loop 2 (ECL2) residues His 255, Phe 2715.39 and Tyr 3376.59 (shown in Corey–Pauling–Koltun (CPK) representation), may contribute to the selectivity of vorapaxar for form a conserved disulphide bond (Figs 1b and 2a). Amino-terminal 5.42 5.46 to Cys 254, ECL2 loops outwards in two anti-parallel b strands. This human PAR1. Also shown are Phe 274 and Phe 278 in TM5 (shown as structural feature is found in other peptide receptors, including the dot surface), which may indirectly influence vorapaxar binding by packing interactions with Phe 2715.39. CXCR4 receptor and the opioid receptors22–25, despite absence of amino acid sequence homology among these receptors in ECL2 interactions of the corresponding residues Pro 2825.50, Ile 1903.40 (Supplementary Fig. 5). In contrast to the open, solvent-exposed 6.44 binding pocket observed in the m-opioid receptor (MOR) and other and Phe 322 in the PAR1 differ from those in both active and peptide receptors, access to the vorapaxar-binding pocket is restricted inactive b2AR structures (Fig. 3b). Taken together, these differences by the central location of ECL2 (Fig. 1 and Supplementary Figs 5 and suggest that PAR1 may differ from other family A GPCRs in the 6), which almost completely covers the extracellular-facing surface of mechanism by which signals propagate from the extracellular pep- vorapaxar. ECL2 is anchored in this position by hydrogen bonds tide-binding interface to the cytoplasmic domains that interact with G between His 255 in ECL2 and Tyr 3537.35 in TM7, and between proteins and other signalling molecules. 7.50 Asp 256 in ECL2 and Tyr 95 in the N terminus (Supplementary The NP XXY motif at the end of helix VII observed in most 7.50 Fig.