Protein machines in the molecular arms race

This post was contributed by Clare Sansom, Senior Associate Lecturer at the Department of Biological Sciences

Birkbeck’s Science Week 2015 was held from Monday 23 to Thursday 26 March and included three evenings of public talks by senior researchers. The first two lectures, on the Tuesday, were given by two of the college’s most distinguished women scientists: Helen Saibil from the Department of Biological Sciences and Karen Hudson-Edwards from Earth and Planetary Sciences; they were billed together as a ‘Women in Science Evening’.

Pathways of pore formation – illustration by Adrian HodelThe 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 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.

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 (eg 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.

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.

Find out more

Image Caption: Pathways of pore formation – illustration by Adrian Hodel

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Science Week: Piecing together the jigsaw of climate change and human evolution

This post was contributed by Guy Collender, of Birkbeck’s Department of External Relations.

Dr Phil Hopley, of Birkbeck's Department of Earth and Planetary Sciences

Dr Phil Hopley exhibited replica skulls of our ancestors during Science Week. Photo: Harish Patel

I knew an unusual presentation was in store as soon as I saw six skulls menacingly positioned at the front of the lecture theatre. The exhibits – all different shapes and sizes – immediately caught the audience’s attention, and our questions about their origins were answered in the fascinating hour that followed.

Dr Phil Hopley began Birkbeck’s series of Science Week lectures with a talk on 16 April about the links between climate change and human evolution. He used the skulls – five replicas of our ancestors and one gorilla skull – to illustrate how evolution is all about the changing dimensions of the head as it has become rounder and larger to accommodate a bigger brain over millions of years. In comparison, the gorilla’s skull includes ferocious canines and space for huge powerful jaws – it certainly sent a shiver up my spine being only a few feet away from my seat.

A family tree dating back millions of years
Dr Hopley, of Birkbeck’s Department of Earth and Planetary Sciences, explained how the last common ancestor of chimpanzees and modern humans was on this planet about six of seven million years ago. Both branches of the family tree then developed separately, with chimpanzees on the one hand, and about 20 species of hominins – the ancestors of modern humans – walking on two legs on the other. As the hominins evolved, they became characterised by their tool use, larger brains, language and art, eventually developing into Homo sapiens – our own species. But our ancestral line has not been straightforward, and Dr Hopley highlighted the complexity. He said: “Homo sapiens is the only human species alive today, but for most of human evolution there have been a number of co-existing human species.”

As Dr Hopley explained, hominin fossils have mainly been found in two areas – the Rift Valley in East Africa (dating back five million years), and caves in Southern Africa (dating back 2.5 million years). Yet, hardly surprising, given the awesome amount of time involved, it is very rare to find a whole hominin specimen. What is clear is that the human fossil record is very incomplete, both geographically and temporally, and solving the mystery is a bit like piecing together a jigsaw.

Climate change: from forest to grassland
The question of why our ancestors evolved to become bipedal was then addressed, and this was where Dr Hopley referred to his work studying fossils from caves in South Africa. The study of carbon and oxygen isotypes and climate modelling has shown that the savannah in Africa developed eight million years ago due to the reduction in carbon dioxide and reduction in rainfall. As the grasslands replaced the forests, our ancestors evolved to walk on two feet as they needed to cover large distances to search for food, which wasn’t necessary when they were still living in the forest. Although it’s difficult to build up a comprehensive understanding of how climate change drives evolution, Dr Hopley did present a general conclusion. He said: “Human evolution did occur because of climate change in the broad sense as forests were replaced by savannah.”

I’ve never been to a lecture with skulls on display before and I’ll certainly never forget this one. It was a powerful way to remind us that our common ancestors adapted to the African bush and started walking when the forests began to recede.

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