Agonists and Antagonists of Protease-Activated Receptor 2 Discovered within a DNA-Encoded Chemical Library Using Mutational Stabilization of the Target.

The discovery of ligands via affinity-mediated selection of DNA-encoded chemical libraries is driven by the quality and concentration of the protein target. G-protein-coupled receptors (GPCRs) and other membrane-bound targets can be difficult to isolate in their functional state and at high concentrations, and therefore have been challenging for affinity-mediated selection. Here, we report a successful selection campaign against protease-activated receptor 2 (PAR2). Using a thermo-stabilized mutant of PAR2, we conducted affinity selection using our >100-billion-compound DNA-encoded library. We observed a number of putative ligands enriched upon selection, and subsequent cellular profiling revealed these ligands to comprise both agonists and antagonists. The agonist series shared structural similarity with known agonists. The antagonists were shown to bind in a novel allosteric binding site on the PAR2 protein. This report serves to demonstrate that cell-free affinity selection against GPCRs can be achieved with mutant stabilized protein targets.

Structural insight into allosteric modulation of protease-activated receptor 2.

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.

Structures of dimeric GIT1 and trimeric beta-PIX and implications for GIT-PIX complex assembly.

GIT (G protein-coupled receptor kinase-interacting protein) and PIX (p21-activated kinase-interacting exchange factor) family proteins integrate signaling pathways involving Arf and Rho family GTPases. GIT1 and beta-PIX form a constitutively associated complex that acts as a scaffold to allow the formation of large multiprotein assemblies that regulate synaptogenesis, cell polarity and cell migration among other physiological processes. Complex formation is mediated by the GIT binding domain (GBD) in beta-PIX, which recognizes the Spa homology domain of GIT1. Both binding domains are adjacent to predicted coiled-coil segments that allow homo-oligomerization of GIT1 and beta-PIX, respectively. Oligomerization of GIT and PIX proteins is important for their physiological functions, and deletion of the coiled-coil domains interferes with correct subcellular localization and the GEF (guanine nucleotide exchange factor) activity of PIX. We have solved the crystal structures of the CC domains of GIT1 and beta-PIX and determined the stoichiometry of complex formation between the two proteins in order to understand the molecular architecture of the GIT1-beta-PIX complex. The crystal structure of the CC domain of GIT1 solved at 1.4 A resolution shows a dimeric, parallel CC that spans 67 A in length. Unexpectedly, and in contrast to prevalent dimeric models, the structure of the CC region of beta-PIX determined at 2.8 A resolution, combined with hydrodynamic studies, reveals that this protein forms a parallel trimer. Furthermore, we demonstrate that dimeric GIT and trimeric PIX form an unusual high-affinity heteropentameric complex in which each Spa homology domain of the GIT1 dimer recognizes one GBD of the beta-PIX trimer, leaving one GBD unoccupied. These results can serve as a basis to better understand oligomerization-dependent GIT1-beta-PIX-regulated signaling events and provide an insight into the architecture of large signaling complexes involving GIT1 and beta-PIX.

Signal recognition particle receptor exposes the ribosomal translocon binding site.

Signal sequences of secretory and membrane proteins are recognized by the signal recognition particle (SRP) as they emerge from the ribosome. This results in their targeting to the membrane by docking with the SRP receptor, which facilitates transfer of the ribosome to the translocon. Here, we present the 8 angstrom cryo-electron microscopy structure of a "docking complex" consisting of a SRP-bound 80S ribosome and the SRP receptor. Interaction of the SRP receptor with both SRP and the ribosome rearranged the S domain of SRP such that a ribosomal binding site for the translocon, the L23e/L35 site, became exposed, whereas Alu domain-mediated elongation arrest persisted.

The structure of the mammalian signal recognition particle (SRP) receptor as prototype for the interaction of small GTPases with Longin domains.

The eukaryotic signal recognition particle (SRP) and its receptor (SR) play a central role in co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum. The SR is a heterodimeric complex assembled by the two GTPases SRalpha and SRbeta, which is membrane-anchored. Here we present the 2.45-A structure of mammalian SRbeta in its Mg2+ GTP-bound state in complex with the minimal binding domain of SRalpha termed SRX. SRbeta is a member of the Ras-GTPase superfamily closely related to Arf and Sar1, while SRX belongs to the SNARE-like superfamily with a fold also known as longin domain. SRX binds to the P loop and the switch regions of SRbeta-GTP. The binding mode and structural similarity with other GTPase-effector complexes suggests a co-GAP (GTPase-activating protein) function for SRX. Comparison with the homologous yeast structure and other longin domains reveals a conserved adjustable hydrophobic surface within SRX which is of central importance for the SRbeta-GTP:SRX interface. A helix swap in SRX results in the formation of a dimer in the crystal structure. Based on structural conservation we present the SRbeta-GTP:SRX structure as a prototype for conserved interactions in a variety of GTPase regulated targeting events occurring at endomembranes.

Signal recognition particle mediates post-translational targeting in eukaryotes.

Signal recognition particle (SRP) plays a central role in the delivery of classical secretory and membrane proteins to the endoplasmic reticulum (ER). All nascent chains studied to date dissociate from SRP once released from the ribosome, thereby supporting a strictly cotranslational mode of action for eukaryotic SRP. We now report a novel post-translational function for SRP in the targeting of tail-anchored (TA) proteins to the ER. TA proteins possess a hydrophobic membrane insertion sequence at their C-terminus such that it can only emerge from the ribosome after translation is terminated. We show that SRP can associate post-translationally with this type of ER-targeting signal, and deliver newly synthesised TA proteins to the ER membrane by a pathway dependent upon GTP and the SRP receptor. We find that dependency upon this SRP-dependent route is precursor specific, and propose a unifying model to describe the biogenesis of TA proteins in vivo.