The performance of perforin-like proteins

This feature appeared in the May/June 2009 issue of Australian Life Scientist. To subscribe to the magazine, go here.

The great Belgian immunologist Jules Bordet first described the workings of the complement system and how it functions in the innate immune system back it the early 1900s. Bordet was awarded the Nobel Prize in 1919 for this and other discoveries, but since then the actual mechanism behind the lytic factor of complement has not been understood.

In 2007, Professor James Whisstock and his team, including his partner Dr Michelle Dunstone, solved the structure of a perforin protein called Plu-MACPF, and by doing so uncovered the mechanism by which these proteins work. They found that perforins are actually descended from bacterial pore-forming cholesterol cytolysins (CDCs), a specialised family of proteins used by bacteria such as anthrax, Clostridium perfringens and Clostridium difficile to disrupt cell membranes.

They found that the MACPF domain folds in the same way as CDCs and is probably used by vertebrates for defense against infection. The finding was momentous, Whisstock says.

“This project has trotted along for about 10 years,” Whisstock says. “We started working on finding out how perforin-like proteins actually work. When you look at proteins and protein sequences it is actually quite easy to find their relatives using bioinformatic approaches so there are very few surprises, but occasionally, just occasionally, we will solve a structure and the structure remembers its roots long after sequence motifs have disappeared.

“This is what happened to us – we solved the structure, looked at it and went ‘oh my God’ – it’s a bacterial cytolysin. We found that this is actually a common fold found in almost all life, with the exception of viruses and nematodes. Everything has a MACPF protein and they are actually bacterial descendents, the same as bacterial CDCs.”

Whisstock says the discovery raises a couple of very interesting questions, which his lab is now pursuing. “First of all, it is clear that bacteria and mammals use a common mechanism to defend themselves and attack one another.

“Secondly, we can explain how these things work. Complement was discovered over a hundred years ago by Jules Bordet, but the actual mechanism for that lytic factor has been a mystery ever since. Our work addresses that mystery.

“And finally, when you look at the roles of these things there are a lot of immunity proteins involved, but there is also a lot of proteins involved in processes where you don’t expect a MACPF protein to play a part, in developmental biology, in embryonic development, in neural development and so forth.

Then there are these MACPF proteins that perform an unknown role. For example, in Nature the other day a genome-wide association study pulled up one of our MACPF proteins in autism. So suddenly you’ve got this almost entirely new field and it is very rare in one’s career to have that opportunity.” ---PB---

Conformational dynamics

This is all a far cry from when Whisstock did his undergraduate degree at Cambridge University and nothing really interested him overly. Then he did a third-year project with Robin Carrell and Arthur Lesk looking at the shape of proteins. He was intrigued and began a PhD focusing on the conformational dynamics of protein molecules, concentrating on the computational side of things. This was safer, he says, as “I’m an absolute disaster in the lab. I still am – my people won’t let me near it.”

From a computational and modelling background, Whisstock’s interest then moved into x-ray crystallography, and now model organisms. His team is making its first knockout mouse model, and is expanding its work on Drosophila and zebrafish.

“My philosophy is that structural biology is a lovely field but all too often people just focus on structure and miss the biology angle. If you have the capacity and the opportunity to combine biology with structure then it is really exciting. So that’s where I began.”

Australia proved to be his big adventure after seven years in the beautiful but fog-bound city of Cambridge, so he hot-footed it to Australia and suspects he will remain here forever. Accompanying him were two friends from the UK, Steve Bottomley and Rob Pike, who work with him at Monash University, Pike as head of the department and Bottomley as a professor of biochemistry.

At Monash, Whisstock continued his studies into serpins, the superfamily of serine protease inhibitors that perform a number of functions. “They control very important processes in the body, from blood clotting, the inflammatory response, even things like blood pressure,” he says. “These are fundamental cascades which are essential for life.” There are the two classic serpins – anti-trypsin, which is involved in inflammation, controlling inflammatory proteases and tissue remodelling; and anti-thrombin, which controls clotting.

“These proteins are generally involved in highly complex pathways, so they utilise highly complex and dynamic mechanisms to work and some of them are attractive from a therapeutic angle – particularly things like anti-thrombin,” he says.

While his main work has moved onto the perforin-like proteins, he still retains an interest in a serpin called maspin. “Maspin is actually a rather enigmatic member of the super-family in that it is very important for preventing tumour metastasis,” he says.

“In breast and prostate cancer, when maspin is highly expressed the tumours seem to have a very low incidence of metastasis. So my colleague Phil Bird and I are very interested in how maspin works – we have no idea really. There are many different hypotheses and many have been disproved, but there is no real firm understanding yet how it works. We are attacking this from both a structural biology and a cell biology model system based approach.”

Several years ago he worked on cathepsins, or cysteine protease inhibitors, and in particular on a chicken protein called MENT. “We were interested in MENT primarily because it is involved in chromatin compaction and our work has moved on from there to understanding how human proteins related to MENT perform an analogous role.

“There are also a number of serpins that are nuclear, but their role in the nucleus is unclear,” he says. “We’re still continuing to try to unravel what these things are up to. One of the complicating factors is that they have overlapping roles, so if for example you knock out a particular serpin in a mouse, another one is often upregulated and will take its place.

“Because these are conformationally mobile molecules, serpins have this capacity to form disease-linked polymers, and the mechanism by which they do so has long been assumed to be loop A-sheet polymerisation.

“However, this is one of the remarkable things where a hypothesis essentially becomes accepted and the total proof, but we all turned out to be completely wrong. Jim Huntington from Cambridge showed us this in Nature last year. The serpin A-sheet model has served us well but Jim’s new concept for serpin domain swapping has really revolutionised our understanding of disease-linked processes.” ---PB---

Synchrotron science

While primarily working on MACPF domains, Whisstock and his lab recently made headlines for some work they did on the malaria parasite. Led by Professor John Dalton, head of the Institute for the Biotechnology of Infectious Diseases at the University of Technology, Sydney and published in PNAS in February, the team studied an enzyme called PfA-M1, which is part of Plasmodium’s digestive machinery.

Malaria has to break down blood proteins in order to obtain nutrients, and it does this inside its digestive vacuole. The team found, however, that PfA-M1 is located outside the vacuole and thus proves a new potential target for inhibition.

“I’ve always had a protease direction and John Dalton had been working for a while on a family of aminopeptidases involved in digestion and haemoglobin,” Whisstock says. “John had the idea that if you could perhaps inhibit or target these things then there might be a way of starving the parasites.

“John came to us with that idea and said if we are going to do this we need some structural biology so Sheena McGowan from my lab started working on the structural biology. Sheena, John and Don Gardner in Queensland now have a grant on it. I think that this will be a fabulous direction for Sheena to develop her own independent direction.

“Our involvement came about because of the need for and utility of structural biology in drug development.”

The location of the Australian Synchrotron has been an absolute blessing for this sort of work, he says. “It is a fantastic resource. We have some really good collaborations with the staff at the Synchrotron and not having to ship our crystals and lose half of them in US Customs is a very big deal. And we get a lot more synchrotron time now.

“There is a big investment happening at Monash with robotics for x-ray crystallography, so taken together it’s a very exciting time.”