Pre-pore structure of perforin
Pore structure of perforin
Pores of perforin on giant unilamellar vesicles (red fluorescing phosphatidylethanolamine) ...
Pore-forming proteins are ubiqitous in living organisms, and possess the characteristic that they are synthesised in a soluble form and then on contact with a membrane refold to a bilayer-inserted, lesion-generating form. They usually achieve this alongside oligomerisation into a ring-shaped assembly, which defines the pore. In humans, the pore-forming proteins perforin and the complement membrane attack complex (MAC) are involved in cellular and innate immunity, and especially in immune surveillance concerning parasitic infection of cells, including viruses, and malignancy. In bacteria, the cholesterol-dependent cytolysins (CDCs) are central to the way that disease is generated, facilitating infection and mediating inflammation and other downstream effects. Human pathogenic bacteria that produce CDCs include Streptococcus pneumoniae (producing pneumolysin), and Listeria monocytogenes (producing listeriolysin). In single-celled parasites such as Plasmodium, the causative agent of malaria, pore-forming proteins are involved in the invasion of cells and tissues and mediate vital stages in transfer between cell and host types.
It has recently become clear that perforin and MAC-forming proteins (the MACPF proteins), the CDCs and the proteins from Plasmodium that form pores are all evolutionarily related. We have studied the CDCs and perforin in some detail already and are expanding our programme extensively in this area. Our aims are to understand the molecular basis for pore formation by these related proteins, how their common features are deployed in different contexts and how their biological function is determined by features unique to specific members of the MACPF/CDC superfamily of proteins. We thus seek to describe how pores form and how that contributes to infection and immunity. We are doing this using a range of functional assays, X-ray crystallography and cryo-electron microscopy and tomography. This collection of techniques will allow us to reach an understanding of MACPF/CDC mechanism stretching from the chemical to the cellular level. Recent papers in this area include one revealing the structural diversity of perforin pores (Praper et al., 2011); another paper currently under re-submission describes a novel activity for perforin that would explain its role in delivering apoptotic enzymes into target cells.
Cell and tissue culture, protein expression and purification. Cryo-electron microscopy and threep-dimensional image reconstruction and cryo-tomography. X-ray crystallography, structure solution and atomic model building.
Protein Science & Structural Biology and Immunology & Infectious Disease
Project reference number: 88
| Name | Department | Institution | Country | |
|---|---|---|---|---|
| Dr Robert JC Gilbert | Structural Biology | Oxford University | UK | gilbert@strubi.ox.ac.uk |
1999. Two structural transitions in membrane pore formation by pneumolysin, the pore-forming toxin of Streptococcus pneumoniae. Cell, 97 (5), pp. 647-55. Read abstract | Read more
The human pathogen Streptococcus pneumoniae produces soluble pneumolysin monomers that bind host cell membranes to form ring-shaped, oligomeric pores. We have determined three-dimensional structures of a helical oligomer of pneumolysin and of a membrane-bound ring form by cryo-electron microscopy. Fitting the four domains from the crystal structure of the closely related perfringolysin reveals major domain rotations during pore assembly. Oligomerization results in the expulsion of domain 3 from its original position in the monomer. However, domain 3 reassociates with the other domains in the membrane pore form. The base of domain 4 contacts the bilayer, possibly along with an extension of domain 3. These results reveal a two-stage mechanism for pore formation by the cholesterol-binding toxins. Hide abstract
2002. Pore-forming toxins. Cell. Mol. Life Sci., 59 (5), pp. 832-44. Read abstract | Read more
Pore-forming toxins are widely distributed proteins which form lesions in biological membranes. In this review, bacterial pore-forming toxins are treated as a paradigm and discussed in terms of the structural principles on which they work. Then, a large family of bacterial toxins, the cholesterol-binding toxins, are analyzed in depth to provide an overview of the processes involved in pore formation. The ways in which the cholesterol-binding toxins (cholesterol-dependent cytolysins) interact with membranes and form pores, the structure of the monomeric soluble and oligomeric pore-forming states, and the effects of the toxin on membrane structure are discussed. By surveying the range of work which has been done on cholesterol-binding toxins, a working model is elaborated which reconciles two current, apparently diametrically opposed, models for their mechanism. Hide abstract
2005. Inactivation and activity of cholesterol-dependent cytolysins: what structural studies tell us. Structure, 13 (8), pp. 1097-106. Read abstract | Read more
The homologous bacterially expressed cholesterol-dependent cytolysins (CDCs) form pores via oligomerization; this must occur preferentially once the target membrane has been engaged. Conformational changes in CDCs then drive partition from an aqueous environment to a lipidic one. This review addresses how premature oligomerization is prevented, how conformational changes are triggered, and how cooperativity between subunits brings about new functionality absent from isolated protomers. Variations are found in the answers provided by the CDCs to these issues. Some toxins use pH as a trigger of activity, but recent results have shown that dimerization in solution is an alternative way of preventing premature oligomerization, in particular for the CDC from Clostridium perfringens, perfringolysin. More controversially, there is still no resolution to the debate as to whether incomplete (arciform) oligomers form pores: recent results again suggest that they do. Hide abstract
2011. Human perforin employs different avenues to damage membranes. J. Biol. Chem., 286 (4), pp. 2946-55. Read abstract | Read more
Perforin (PFN) is a pore-forming protein produced by cytotoxic lymphocytes that aids in the clearance of tumor or virus-infected cells by a mechanism that involves the formation of transmembrane pores. The properties of PFN pores and the mechanism of their assembly remain unclear. Here, we studied pore characteristics by functional and structural methods to show that perforin forms pores more heterogeneous than anticipated. Planar lipid bilayer experiments indicate that perforin pores exhibit a broad range of conductances, from 0.15 to 21 nanosiemens. In comparison with large pores that possessed low noise and remained stably open, small pores exhibited high noise and were very unstable. Furthermore, the opening step and the pore size were dependent on the lipid composition of the membrane. The heterogeneity in pore sizes was confirmed with cryo-electron microscopy and showed a range of sizes matching that observed in the conductance measurements. Furthermore, two different membrane-bound PFN conformations were observed, interpreted as pre-pore and pore states of the protein. The results collectively indicate that PFN forms heterogeneous pores through a multistep mechanism and provide a new paradigm for understanding the range of different effects of PFN and related membrane attack complex/perforin domain proteins observed in vivo and in vitro. Hide abstract
2005. Structural basis of pore formation by the bacterial toxin pneumolysin. Cell, 121 (2), pp. 247-56. Read abstract | Read more
The bacterial toxin pneumolysin is released as a soluble monomer that kills target cells by assembling into large oligomeric rings and forming pores in cholesterol-containing membranes. Using cryo-EM and image processing, we have determined the structures of membrane-surface bound (prepore) and inserted-pore oligomer forms, providing a direct observation of the conformational transition into the pore form of a cholesterol-dependent cytolysin. In the pore structure, the domains of the monomer separate and double over into an arch, forming a wall sealing the bilayer around the pore. This transformation is accomplished by substantial refolding of two of the four protein domains along with deformation of the membrane. Extension of protein density into the bilayer supports earlier predictions that the protein inserts beta hairpins into the membrane. With an oligomer size of up to 44 subunits in the pore, this assembly creates a transmembrane channel 260 A in diameter lined by 176 beta strands. Hide abstract