Perforin, a 67 kDa multi domain protein, generates membrane-embedded pores through which cytotoxic granzymes enter the cytoplasm of target cells1. The perforin structure comprises a pore-forming membrane attack complex/perforin-like (MACPF) domain, a central disulphide-constrained shelf region (EGF domain), and a lipid calcium (Ca2+) binding C2 domain. The C2 domain, which has a mass of 13 kDa, has three Ca2+ binding sites and requires extracellular concentrations of Ca2+ to mediate the initial binding of perforin to target cell membranes. The high-resolution crystal structure of the perforin monomer has been resolved2. However, the detailed molecular mechanism by which the C2 domain binds to lipid membranes and the role of Ca2+ in this process is still unclear.
In this study, we used the isolated C2 domain and analyzed the interaction with lipid membranes based on titration experiments of the lipid membrane mimics, such as micelles and nanodiscs3, to investigate how the C2 domain interacts with lipids and lipid head groups.