The lectures were all introduced by the Dean of the Faculty of Science, Nicholas Keep who described Saibil, a close colleague, as “our most eminent female scientist”. She came to Birkbeck from Canada via a PhD at King’s College London under the supervision of Nobel laureate Maurice Wilkins and post-doctoral work at Oxford.
Since arriving here in the 1980s she has built up an internationally renowned structural biology lab, focusing in particular on the technique of electron microscopy. She has been a Fellow of the Royal Society since 2006 and of the Academy of Medical Sciences since 2009.
Saibil began her lecture by explaining that proteins can act as little machines, performing mechanical tasks that are essential for the maintenance of life. Her group has been interested for some time in proteins that can punch holes in the walls of cells. This allows the cell contents to leak out in a damaging process known as lysis, and it also allows toxins to enter the cells. These proteins can therefore be thought of as powerful weapons, and they are deployed on both sides of a ‘molecular arms race’: by pathogens and by the immune systems of humans and other animals.
Most soluble proteins fold into a single stable structure that tries, as far as possible, to keep their hydrophobic (“water-hating”) parts – the side chains of certain amino acids – in the interior of the protein, with the hydrophilic (“water-loving”) side chains on the outside, in contact with the watery environment inside or outside cells.
Pore-forming proteins, however, have a ‘Jekyll and Hyde’ like identity: they can form two distinctly different shapes, one as individual, soluble molecules and the other when they associate with each other into membrane-bound rings to form cylindrical pores. These structures, and the conformational change between them, are remarkably similar in proteins from bacteria and from the immune system.
Pore-forming toxins have been found in types of bacteria that are responsible for some deadly human diseases, including meningitis and pneumonia. The structure of a monomeric form of one of these proteins in solution was first solved in 1998, using X-ray crystallography. However, large complexes of many protein molecules are more readily solved by electron microscopy, particularly when those complexes are embedded in membranes.
In 2005 Saibil and her group described structures of the pore-forming toxin pneumolysin from Streptococcus pneumoniae, in complex with a model cell membrane. They found that the proteins formed two distinctly different ring-shaped structures. Initially, they formed into a ring sitting on top of the membrane, which was termed the pre-pore; then they changed shape to burrow part of each protein deep into the membrane and form the pore itself. Each monomer in the pre-pore had a structure that was similar to that of the molecule in solution, but they underwent large structural changes to form the pore.
Schematic illustration of how suilysin, a bacterial cholesterol-dependent cytolysin, drills holes in cell membranes. Image © Adrian Hodel, London Centre for Nanotechnology
Most structures solved by electron microscopy are at lower resolution than those solved by X-ray crystallography, and it is not possible to trace the positions of individual atoms at lower resolutions (e.g. worse than 3 A). Saibil and her colleagues were able to interpret the structure of the proteins making up the pore by fitting pieces of the X-ray structure of the isolated molecule into their electron density.
They found a dramatic change in structure, with the tall, thin protein structure collapsing into an arch and a helical region stretching out to form a long, extended beta hairpin. It is these hairpins that join together to form the walls of the pore. The process of pore forming therefore has three stages: firstly the toxin molecules bind to the surface of their target cells, then they associate into the circular pre-pores and finally they change shape in a concerted manner, punching holes in the cell membranes by ejecting a disc of membrane, letting other toxins in and cell contents out.
Saibil then turned the focus of her talk from attack by bacteria to the human immune system’s defence. Natural killer (NK) cells are specialised lymphocytes (white blood cells) that kill virally-infected and cancerous cells in the bloodstream. They kill on contact with their target cells by releasing a toxic protein into those cells that stimulates those target cells to commit suicide in a process known as programmed cell death or apoptosis. We have only recently learned that the mechanism through which the NK cells work is very similar to the mechanism of the bacterial pore-forming toxins.
Natural killer cells express a protein called perforin that has a similar structure in solution to the bacterial pneumolysin. Although there is very little sequence similarity between these proteins – there is only one amino acid conserved throughout all the known bacterial and vertebrate proteins of this family, a glycine at a critical position for the conformational change – the structures are similar enough to suggest that the proteins all once had a common ancestor.
Saibil and her colleagues used electron microscopy to discover that this protein forms a pore through a similar mechanism to pneumolysin: the helical region that unfolds into the beta hairpin to form the pore forms the core of the molecular machine and is largely unchanged between the structures. There are some differences between the structures, however; in particular, there is no need for the perforin structure to ‘collapse’ as the molecule has ‘arms’ that are long enough to form the hairpin and punch the hole without bending into an arch.
The mechanism through which the NK cells kill their target cells is now quite well understood. When the two cells come into contact they form a temporary structure called an immune synapse that allows the pore to form and proteases called granzymes, which induce apoptosis, to enter the target cells. This YouTube video illustrates the natural killer cells’ mechanism of action, and this one shows a detailed view of the immune synapse. Other, similar proteins have been identified in oyster mushrooms; these form more rigid structures that are easier to work with. Saibil’s group and their collaborators have been able to solve the structure of this protein in intermediate stages of pore formation and are beginning to gain an understanding of exactly how it unfolds.
The pore of the oyster mushroom protein pleurotolysin, a member of the pneumolysin family. Image © Natalya Lukoyanova and Helen Saibil, from Lukoyanova et al., PLoS Biology 13:e1002049
Mutations in perforin that prevent it from functioning cause a rare disease called haemophagocytic lymphohistiocytosis, which is almost invariably fatal in childhood. Understanding the mechanism of action of this important family of protein ‘weapons’ in both attack and defence may help find a cure for this devastating condition, as well as for some commoner disorders of the immune system and important infectious diseases.
This post is cross-posted from the Birkbeck Events blog.
PPS students can learn much more about electron microscopy by taking the second-year module Techniques in Structural Molecular Biology to complete the MSc.