The pore forming calcium-dependent protein, perforin, is essential for the cytolytic function of cytotoxic lymphocytes, which are at the frontline of immune defence [Brennan et al., CDD 2010]. Cytotoxic lymphocytes store cytotoxic effector molecules, including perforin and pro-apoptotic serine proteases granzymes, in secretory granules. Cytotoxic lymphocytes form a tight contact (immunological synapse) with virus-infected or tumour target cells, which triggers rapid exocytic trafficking and membrane fusion of secretory granules at the synapse, resulting in the release of perforin and granzymes into the synaptic cleft. Perforin remains inert at the acidic pH of the granules [Brennan et al., Immunity 2011], but when it reaches the neutral pH and high calcium conditions of the immune synapse, it binds to target cell lipid membranes through its C2 domain. Once bound, perforin undergoes a dramatic conformational change and oligomerises into large transmembrane pores that penetrate the lipid bilayer to allow the entry of granzymes, which trigger apoptotic cell death.

Although an elevated calcium concentration is essential for pore formation, the exact mechanism of perforin membrane binding and the triggering mechanism for pore formation are unknown. Here, we have revealed an exciting and unique mechanism governing C2 domain membrane binding of perforin. We found that in the presence of calcium, an outward-orientated loop sweeps across the C2 domain and triggers conformational changes that expose and permit four key hydrophobic residues to interact with the lipid membrane of a target cell. This mechanism is independent of membrane phospholipid composition, and is required to anchor perforin to the lipid membrane in the correct orientation for subsequent oligomerisation and pore formation. Contrary to previous ideas, these movements and membrane binding do not trigger irreversible conformational changes in the membrane-piercing domain of perforin. Rather, subsequent monomer-monomer interactions at the lipid membrane surface propagate formation of perforin transmembrane pores.