Structural basis for Ca²⁺-mediated interaction of the perforin C2 domain with lipid membranes

Natural killer cells and cytotoxic T-lymphocytes deploy perforin and granzymes to kill infected host cells. Perforin, secreted by immune cells, binds target membranes to form pores that deliver pro-apoptotic granzymes into the target cell. A crucial first step in this process is interaction of its C2 domain with target cell membranes, which is a calcium-dependent event. Some aspects of this process are understood, but many molecular details remain unclear. To address this, we investigated the mechanism of Ca²⁺ and lipid binding to the C2 domain by NMR spectroscopy and x-ray crystallography. Calcium titrations, together with do decylphosphocholine micelle experiments, confirmed that multiple Ca²⁺ ions bind within the calcium-binding regions, activating perforin with respect to membrane binding. We have also determined the affinities of several of these binding sites and have shown that this interaction causes a significant structural rearrangement in CBR1. Thus, it is proposed that Ca²⁺ binding at the weakest affinity site triggers changes in the C2 domain that facilitate its interaction with lipid membranes.