EP67 decapeptide preferentially activates human C3aR, revealing hook-like binding via cryo-EM structures.

The complement cascade is a critical component of innate immunity, essential for combating pathogens. Key players are the anaphylatoxins C3a and C5a, which trigger robust inflammatory responses by activating their respective G protein-coupled receptors, C3aR and C5aR1. While these receptors are vital for immune defense, uncontrolled activation can lead to excessive inflammation. Developing immunostimulants that can elicit a beneficial immune response without inducing detrimental inflammation is a significant therapeutic challenge. Peptides derived from anaphylatoxins, such as EP67, have shown promise in preclinical antiviral and antibacterial contexts, yet their precise molecular mechanisms and receptor targets have remained largely undefined.

Researchers conducted comprehensive pharmacological profiling of the decapeptide EP67 on both human and mouse C3aR and C5aR1 receptors. They utilized transducer-coupling assays to assess receptor activation. Subsequently, they determined four distinct cryo-electron microscopy (cryo-EM) structures of C3aR and C5aR1 in complex with EP67 to elucidate the molecular details of their interactions. To validate key binding interactions and corroborate structural observations, including the engagement of a critical activation switch, site-directed mutagenesis studies were performed on the receptors.

The study revealed that EP67 preferentially activates human C3aR in transducer-coupling assays, indicating a selective agonistic effect. Cryo-EM structures provided unprecedented molecular detail, showing that EP67 adopts a distinctive hook-like conformation. This hook-like structure allows EP67 to bind within the orthosteric pocket of both C3aR and C5aR1, a binding mode analogous to that of the carboxyl terminus of the natural ligands C3a and C5a. Site-directed mutagenesis confirmed the critical interactions identified in the structural models, validating the engagement of a key receptor activation switch. Most notably, the researchers observed that EP67 induces distinct conformational changes at the TM7-Helix8 interface for C3aR compared to C5aR1.