Hole-punching proteins in pathogen attack and immune defence

The way in which protein machines punch holes in cell membranes to destroy cells has been uncovered by Professor Helen Saibil’s research group at the Institute of Structural and Molecular Biology (ISMB), working in collaboration with other scientists in the UK and Australia.

The way in which protein machines punch holes in cell membranes to destroy cells has been uncovered by Professor Helen Saibil’s research group at the Institute of Structural and Molecular Biology (ISMB) at Birkbeck, working in collaboration with scientists at the London Centre for Nanotechnology, Monash University, Australia, and the University of Leicester. The team has published two papers: one in the journal PLOS Biology on 5 February 2015, and the other in eLife on 4 December 2014.

This hole-punching activity is used by many pathogenic organisms, such as bacteria and parasites, to gain access to our cells. Our immune system uses a similar protein machine to defend us against invaders and kill infected cells.

In the eLife paper, the team revealed the mechanics of how a bacterial toxin drills holes through a membrane. Professor Saibil’s group used electron microscopy to determine 3D structures that reveal details of how the proteins are constructed, in a series of frozen snapshots of different steps in the assembly process. They observed dramatic shape changes when the proteins assemble into rings on a membrane surface and punch holes. To complement these structural snapshots, the London Centre for Nanotechnology group used atomic force microscopy (AFM) to record a movie of what happens when the toxins are added to a membrane. AFM repeatedly scans the surface to produce a moving image that refreshes fast enough to show how the toxins move over the membrane and then cut holes in the membrane as they sink in.

Unlike drills from a DIY kit, which twist and grind their way through a surface, these bacterial nanodrills do not contain rotating parts. Rather, the toxin molecules assemble on the membrane surface into arcs and rings. These assemblies then form blades that slice down into the cell membrane, creating holes by ejecting the encircled patches of membrane.

In the PLOS Biology paper, the ISMB and Monash teams studied the action of a protein called pleurotolysin, isolated from oyster mushrooms. Pleurotolysin is a member of the subgroup of pore-forming proteins related to perforin, which is deployed by the human immune system for defence. The mushroom protein was used to produce rings on the membrane surface, at various intermediate stages during formation of the pore channel. By taking molecular snapshots with X-ray crystallography and electron microscopy, the team synthesised a molecular movie of the hole-punching protein as it latches onto, and puts a hole in the target cell – either killing the cell directly or providing a passage for other proteins that can kill it. Using a combination of molecular imaging, along with biophysical and computational experiments, the team have been able to show the way the pleurotolysin protein reorganises its structure, unfolding and extruding parts of the protein to punch the hole in the target cell.

Together, the findings give a detailed view of how these pore-forming proteins drill holes in cell membranes. These discoveries provide a basis for the development of new drugs that can target bacterial toxins and help treat diseases such as pneumonia, meningitis and septicaemia. Structures of the perforin related pores can similarly help in developing drugs to control the immune response, for example in autoimmune diseases, or to block cell entry of parasites such as malaria.

[Image: Structure of the Pleurotolysin pore (from the oyster mushroom), looking down the channel formed by the ring of protein. The protein molecules are in rainbow colours and the electron microscopy map is shown as a semi-transparent white surface.]