Scratching the itch with cryo-EM

Abstract

Cryo-EM structures of Mas-related G-protein-coupled receptors (MRGPRs) that are involved in the allergic reaction and itch response reveal structural insights into their activation mechanism, and offer the potential to discover new therapeutic agents for pain and hypersensitivity reactions.

Itch sensation, referred to as pruritus in medical terms, is a common physiological response manifested following exposure to environmental factors such as insect bites and skin-related disease conditions, or as a side effect of clinically prescribed medications¹. Itch sensation cascade can be triggered by multiple factors such as histamines, cytokines and other peptides, and it involves different types of integral membrane protein such as receptors and ion channels¹. A group of Mas-related G-protein-coupled receptor subtype Xs (MRGPRXs) — in particular, MRGPRX2 and MRGPRX4 — have recently emerged as prominent players in mediating the itch response^2–4(Fig. 1a). However, the lack of direct structural visualization of these receptors has limited our understanding of their ligand-recognition and -activation mechanisms until recently. Now, in two papers published in Nature, Cao et al.⁵ and Yang et al.⁶ present a series of cryogenic electron microscopy (cryo-EM) structures of MRGPRX2 and MRGPRX4 in complex with several different ligands and heterotrimeric G proteins, which illuminate ligand binding, activation mechanism and effector coupling.

Fig. 1. The structure and function of MRGPRX2.

a, Schematic representation of MRGPRX2-mediated mast cell degranulation and itch response. Created using BioRender.com. b, Structural snapshot of cortistatin-14-bound MRGPRX2 in complex with heterotrimeric Gα_i proteins determined using cryo-EM. ScFv, single-chain variable fragment. The structural snapshot was designed in ChimeraX using Protein Data Bank ID 7S8M.

The complexes of MRGPRX2, with either the Gα_i or Gα_q heterotrimer, are assembled using standard methodologies that involve recombinant expression and stabilization using a nanobody, and visualized at high resolution by cryo-EM (Fig. 1b). These structures are determined in the presence of different small-molecule agonists that selectively bind to MRGPRX2, or peptide agonists such as neuropeptides cortistatin-14 and substance P, which are also agonists for other G-protein-coupled receptors (GPCRs). An interesting feature in these structures is the shallower binding mode of the agonists where the ligand-binding pocket is mostly solvent exposed, compared to other class-A GPCRs. Moreover, the surface electrostatic potential in the ligand-binding pocket appears to be primarily negatively charged, which rationalizes the binding of polycationic compounds, peptidergic drugs and positively charged endogenous peptides to MRGPRX2, eliciting allergic reactions and the itch response. Another interesting feature observed in MRGPRX2 structures, which is in stark contrast with other class-A GPCRs, is the absence of a disulfide bridge between the extracellular side of transmembrane 3 (TM3) and extracellular loop 2 (ECL2). Instead, a disulfide bridge between the two cysteine residues in TM4 was observed, and its disruption by site-directed mutagenesis negatively impacted agonist-induced Gα_q activation.

Several of the highly conserved motifs in class-A GPCRs — such as the P–I–F motif involving TM3/5/6; the DRY motif in TM3; a key sodium-binding residue in TM3; and the tryptophan toggle switch in TM6 — are either not present in MRGPRX2, or are present in a modified form². This leads to significant differences in the active conformation of MRGPRX2, especially with respect to these motifs, compared to other class-A GPCRs. However, the outward movement of TM6, which is a hallmark of GPCR activation and observed in every GPCR–G-protein complex, also remains conserved in MRGPRX2 structures. In addition, the interface with Gα_i or Gα_q is primarily similar to that observed for other GPCRs. However, some differences are noticeable between Gα_i versus Gα_q engagement with the receptor, particularly with respect to the proximity of the α5-helix in Gα_q to TM6 in the receptor, and the proximity of αN-helix in Gα_i to intracellular loop 2 (ICL2) in the receptor.

MRGPRX4, another subtype of MRGPRX, shows a preference for negatively charged bile acids as agonists — rather than cationic agonists that bind to MRGPRX2 — exhibiting an intriguing example of diversity encoded in the ligand-recognition mechanism of these receptors⁴. As high-affinity agonists for MRGPRX4 were not available to facilitate structural studies, Cao et al.⁵ first identified a specific agonist, referred to as MS47134, using rationale design and optimization and starting with nateglinide — an anti-diabetic drug that binds to MRGPRX4 with low affinity. Subsequently, the authors determined the structure of MRGPRX4 in complex with the Gα_q heterotrimer. Interestingly, the ligand-binding pocket in MRGPRX4 shows a predominantly positive electrostatic potential surface, which is in stark contrast with MRGPRX2, and may explain why MRGPRX4 prefers negatively charged bile acids as endogenous agonists. This is also accompanied by side-chain rearrangement of two negatively charged amino acids in the ligand-binding pocket, which may further prohibit the interaction of cationic agonists, while the interface of Gα_q interaction on MRGPRX4 is mostly similar to that of MRGPRX2.

There are several important implications of these studies not only in terms of understanding GPCR signaling, but also in the integration of these findings in therapeutic design and development. For example, the charge distribution in the ligand-binding pocket of MRGPRX2 and MRGPRX4 now provides a structural basis of distinct ligand preference by these two receptor subtypes, and the shallow binding pocket offers an explanation for the ligand promiscuity exhibited by these receptors. More importantly, high-affinity antagonists and agonists of MRGPRX2 and MRGPRX4 that may be designed based on the structural templates described in these studies, or through high-throughput screening, may be combined with clinically prescribed medicines that elicit allergic reactions and the itch response through these receptors. It is also important to emphasize that although these structures represent a major advance by elucidating the ligand binding and G-protein coupling to MRGPRX2 and MRGPRX4, the functional contribution and structural mechanism of other effectors such as β-arrestins remains to be explored⁷. Considering the emerging role of MRGPR signaling in pain sensation⁸, it would be interesting to explore whether the analgesic effects and pruritus can be pharmacologically separated by using the framework of biased agonism at these receptors^9,10.

In summary, these studies underscore the diversity encoded in ligand recognition and activation mechanisms of GPCRs, and offer previously lacking templates to facilitate structure-guided discovery of novel therapeutic molecules that target MRGPRs.