Structural aspects of the pore forming protein perforin

The Membrane Attack Complex/PerForin (MACPF) / (Cholesterol Dependent Cytolysin (CDC) superfamily encompasses two branches of pore forming proteins (PFPs). The CDC branch is a well-described family of pore forming toxins secreted by pathogenic Gram-positive bacteria. The MACPF branch includes effectors of the vertebrate immune system as well as fungal defence toxins and animal venoms. Both MACPF proteins and CDCs share the ability to form giant membrane embedded pores and a common evolutionary ancestry as they all possess the common MACPF/CDC fold that constitutes the pore forming component of the protein. As such, the common fold shared by CDCs and MACPF PFPs suggests these proteins use a common mechanism to perforate membranes. Structural and biophysical studies have contributed much to our understanding of the basic mechanism of CDC pore formation. In contrast, less is known about the MACPF branch of the family. Furthermore, the lack of sub-nanometer resolution structure of any MACPF/CDC pore limits our knowledge of the structural basis for pore formation. In order to address these problems, the CDC model of pore formation was first re- examined employing molecular modelling techniques to understand how the toxins change shape to form pores. The CDC pore transmembrane region was suggested to adopt a novel structure in order to span the membrane. In addition, cryo-Electron Microscopy (cryo-EM) guided modelling resulted in an improved model of the CDC pore consistent with new experimental data. In the second part of this thesis, a structural model of the MACPF immune effector C9 in the oligomeric form was built based on the cryo-EM maps determined by collaborators. These data permitted an improved understanding of the molecular mechanisms underlying MAC assembly and MACPF pore formation. Collectively, the results presented in this thesis present a new understanding of the common pore forming mechanism utilised by important bacterial toxins and effectors of the vertebrate immune system. This study further highlights the variations of this common mechanism within both branches of the superfamily.