| Membrane attack is an important mechanism in mammalian immune defense against invading pathogens and infected host cells. Protection against Gram-negative bacteria in human blood depends on the activation of the terminal pathway of the complement system. Activation of this pathway leads to formation of a multi-protein complex, called the membrane-attack complex (MAC), which forms ~100-Å wide pores in membranes leading to lysis of the targeted cells. Our goal is to understand the mechanism of MAC formation and membrane perforation by the complement proteins. MAC formation involves several large, multi-domain complement proteins. It starts with cleavage of C5 into C5b in the complement cascade. C5b binds sequentially the homologous proteins C6, C7, C8 and multiple copies of C9, which form the actual membrane pore. Similar to bacterial pore-forming proteins, these soluble mammalian proteins are thought to undergo conformational changes when oligomerizing into a membrane-bound complex that perforates the membrane. Recently, we published the structure of the central MACPF domain of human C8, which is the domain responsible for membrane insertion (Hadders, Beringer and Gros, Science 317, 1552-1554, 2007). The structure reveals that the mammalian MACPFs are structurally homologous to bacterial cholesterol-dependent cytolysins, which are beta-barrel pore-forming proteins. This insight and the structural data are instrumental to study the underlying mechanisms by which MAC is formed and perforates the membrane. Here we propose to study the steps of complex formation and membrane perforation. We will express full-length proteins C6, C7, C8 and C9 and generate C5b by proteolysis from plasma purified C5. We will study membrane insertion of specifically labeled C7, C8 and C9 by fluorescence spectroscopy. The conformational changes will be studied by comparing full-length proteins in their free state versus proteins in complex, using protein crystallography. In addition, we will study the mechanism of host cell protection by the surface protein CD59, which blocks membrane insertion of C8 and C9. Finally, we will study the C9 pore. The predicted beta-barrel wall of the C9 pore will be tested by fluorescence using pyrene stacking. Reconstruction of (negatively stained) electron-microscopy images will be performed to map available structures of domains into the complete pore. Taken together these studies provide detailed insights into the MAC structure and the underlying mechanisms of formation and membrane perforation. These insights may be instrumental in designing new therapeutics to prevent undue MAC formation, as is the case in e.g. paroxysmal nocturnal hemoglobinuria and hyperacute rejection of transplanted organs. |
