Background

MRGPRX2 is a G-protein-coupled receptor (GPCR) primarily expressed in skin mast cells. It serves as a key mediator of non-IgE-mediated mast cell activation and has been defined as the molecular basis of type VII hypersensitivity in the new classification of allergic reactions. This receptor can be activated by various endogenous substances (such as the neuropeptide substance P and the antimicrobial peptide LL-37) as well as exogenous compounds (like opioid drugs). Through the Gq protein signaling pathway, it triggers calcium influx and mast cell degranulation, leading to the release of inflammatory mediators such as histamine. This process results in symptoms like skin redness, wheals, and itching. Clinical studies have demonstrated that in conditions such as chronic spontaneous urticaria and atopic dermatitis, the expression levels of MRGPRX2 and its ligands (e.g., LL-37) are significantly elevated and positively correlated with disease severity, underscoring its critical pathological role in allergic inflammatory diseases.

Technical difficulty

· Protein Purification Stage: The GPCR complex is unstable and prone to dissociation.

· Sample Preparation (Cryo-grid Preparation): The majority of protein particles resided on the continuous carbon support film of the grids, rather than being embedded in the thin vitreous ice layer within the holes.

· Structure Determination Stage: The cryo-EM dataset exhibited a severe preferred orientation problem.

Solution

· Utilized NanoBit technology to engineer the GPCR protein, thereby enhancing complex stability.

· Optimized the sample preparation process by adjusting protein concentration and testing different grid types, ultimately achieving a high proportion of particles embedded in the vitreous ice.

· Addressed the preferred orientation issue by employing graphene oxide grids and collecting tilt-series data, which yielded a sufficient number of protein particles in various orientations.