Structural basis for the activation of proteinase-activated receptors PAR1 and PAR2

Abstract

Members of the proteinase-activated receptor (PAR) subfamily of G protein-coupled receptors (GPCRs) play critical roles in processes like hemostasis, thrombosis, development, wound healing, inflammation, and cancer progression. Comprising PAR1-PAR4, these receptors are specifically activated by protease cleavage at their extracellular amino terminus, revealing a 'tethered ligand' that self-activates the receptor. This triggers complex intracellular signaling via G proteins and beta-arrestins, linking external protease signals to cellular functions. To date, direct structural visualization of these ligand-receptor complexes has been limited. Here, we present structural snapshots of activated PAR1 and PAR2 bound to their endogenous tethered ligands, revealing a shallow and constricted orthosteric binding pocket. Comparisons with antagonist-bound structures show minimal conformational changes in the TM6 helix and larger movements of TM7 upon activation. These findings reveal a common activation mechanism for PAR1 and PAR2, highlighting critical residues involved in ligand recognition. Additionally, the structure of PAR2 bound to a pathway selective antagonist, GB88, demonstrates how potent orthosteric engagement can be achieved by a small molecule mimicking the endogenous tethered ligand's interactions.

Fig. 1. Cryo-EM structure of the PAR1-Gαq-scFv16 tethered peptide agonist complex.

A Cryo-EM density map of PAR1-Gαq-scFv16 with tethered ligand. (PAR1, blue; Gαq, teal; Gβ, yellow; Gγ, purple; scFv16, gray). B Cartoon representation of PAR1-Gαq-scFv16 with tethered ligand bound. Inset: tethered ligand (yellow sticks) is shown within the orthosteric pocket of PAR1 (slabbed surface). The tethered ligand cryo-EM map is shown as blue mesh at 5.0 sigma. C Cartoon representation of PAR1 (blue) with the tethered ligand bound (yellow sticks) viewing from the extracellular side of the membrane with TMs and ECLs labeled. D H-bond interactions between the tethered ligand (yellow sticks) and PAR1 orthosteric residues (orange sticks).

Fig. 2. Cryo-EM structure of the PAR2-Gαq-scFv16 tethered peptide agonist complex.

A Cryo-EM density map of PAR2-Gαq-scFv16 with tethered ligand. (PAR2, salmon; Gαq, teal; Gβ, yellow; Gγ, purple; scFv16, gray) B Cartoon representation of PAR2-Gαq-scFv16 with tethered ligand bound. Inset: tethered ligand (green sticks) is shown within the orthosteric pocket of PAR2 (slabbed surface). The tethered ligand cryo-EM map is shown as blue mesh at 5.0 sigma. C Cartoon representation of PAR2 (salmon) with the tethered ligand bound (green sticks) viewing from the extracellular side of the membrane with TMs and ECLs labeled. D H-bond interactions between the tethered ligand (green sticks) and PAR2 orthosteric residues (yellow sticks).

Fig. 3. Structural Comparison of Active and Inactive States of PAR1.

A Comparison of the tethered ligand-bound to active PAR1 (blue) and the antagonist vorapaxar-bound to inactive PAR1 (pink; PDB 3VW7). Inset: structural superposition illustrating the relative binding positions of vorapaxar (gray sticks) and the tethered peptide (yellow sticks) to PAR1. B Extracellular view of the comparison between tethered ligand-bound active PAR1 (blue tubes) and vorapaxar-bound inactive PAR1 (pink tubes). The tethered peptide is colored in yellow spheres, while vorapaxar is shown in gray spheres. C Key residues conformations in PAR1 bound to the antagonist vorapaxar. D Key residue conformations in PAR1 bound to the tethered ligand.

Fig. 4. Structural Comparison of Active and Inactive States of PAR2.

A Overlay of the tethered ligand-bound active PAR2 (coral) and the antagonist AZ8838-bound inactive PAR2 (yellow; PDB 5NDD). The tethered peptide is colored in green spheres, while AZ8838 is shown in blue spheres. B Rotated extracellular view of the comparison between tethered ligand-bound active PAR2 (coral) and AZ8838-bound inactive PAR2 (yellow). C Conformations of key residues in inactive PAR2 (yellow) bound to the antagonist AZ8838 (blue) compared to active PAR2 (coral). D Detailed view of specific residues involved in H-bond rearrangements in the inactive (yellow) to active conformation (coral) of PAR2. E Detailed view of TM6 and TM7 highlighting residues that propagate agonist binding rearrangements in the active state (coral) compared to relative position in the inactive state (yellow). Red arrows denote steric forces applied by moving residues on neighboring residues.

Fig. 5. Interaction between PAR1 and PAR2 with Gq.

A Structural based alignment (using the receptor chains) of active PAR1-Gαq (blue and green cartoons) and PAR2-Gαq (coral and beige). Comparative analysis of the Gαq conformation in the α5 helix (B) and αN domain (C).

Fig. 6. PAR1 and PAR2 activation and inactivation.

Schematic representation of PAR1 and PAR2 in antagonist-bound (left; A), ligand-free (middle; B), and agonist-bound (right; C) states. The sidechains of two mechanistically important tyrosines are shown in stick representation. Conformational flexibility in TM7, TM5 and TM6 in the three states are indicated with red arrows, while conformational movements of the tyrosines upon agonist and antagonist binding are highlighted with black arrows in (C). The relative location of the PIF/Y, DRF/NRY and DPxxY motifs are indicated with blue arrows.

Fig. 7. Cryo-EM structure of the GB88:PAR2-Gαq-scFv16 complex.

A Cryo-EM density map of the GB88:PAR2-Gαq-scFv16 complex perpendicular to the membrane (B). Model of GB88:PAR2-Gαq-scFv16 complex shown in cartoon representation. C Cartoon representation of PAR2 (green) with GB88 bound (cyan sticks) viewing from the extracellular side of the membrane with TMs and ECLs labeled. D GB88 (cyan sticks) is shown within the orthosteric pocket of PAR2 (slabbed surface). The GB88 cryo-EM map is shown as blue mesh at 5.0 sigma (E). GB88 (cyan) bound to the orthosteric pocket of PAR2 highlighting key residues (yellow) and H-bonds formed between the ligand and receptor. F Structural overlay (receptor not shown) of the GB88 and the tethered agonist structures to demonstrate GB88’s mimicry of the tethered ligand within the orthosteric site. GB88 is shown in cyan with chemical substituents labeled also in cyan. The tethered ligand is shown in green with residues labeled also in green.