How we punch our way into cancer cells
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Edible oyster mushrooms have an intriguing secret: they eat spiders and roundworms. And they do so using proteins which can punch their way into cells, leaving tidy but deadly holes. It’s a trick that our immune cells also use to protect us; destroying infected cells, cancerous cells, and bacteria.
Research published today in PLOS Biology by an international team, led by the ARC Imaging Centre at Monash University and Birkbeck College, in London, reveals the molecular process behind the punch.
Using synchrotron light and cryo-electron microscopy, they’ve visualised the action of a protein called pleurotolysin – opening the way to new drug targets and new tools for medicine, agriculture, genetic engineering and nano-engineering.
By taking molecular snapshots, which they’ve turned into a movie, the team have been able to observe 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.
“I never believed I’d be able to see these proteins in action,” says the paper’s lead author Dr Michelle Dunstone. “It’s an amazing mechanism, and also amazing that we now have the technology to see these hole punching proteins at work.”
Using a combination of molecular imaging, along with biophysical and computational experiments, the team have been able to show the way the pleurotolysin protein moves, unfolding and refolding to punch the hole in the target cell.
And in doing so, they’ve also found its Achilles heel. So now they can look at how to block the hole punching mechanism, or introduce it to new places where this function is desirable.
“The next step is to take what we’ve learned from the oyster mushroom proteins and compare them with equivalent proteins across nature,” says Michelle. “We’re particularly interested in this family of proteins in humans, especially perforin, which we believe will behave in the same way.”
There are potential applications in medicine: dampening immune responses in people with autoimmune disease; stopping listeria escaping our immune cells; and preventing malaria from infecting the liver.
In agriculture these proteins could be introduced into plants and crops, helping them to fight off attacks from pests, and reducing the need for pesticides.
“These results are the culmination of over seven years work by from researchers on opposite sides of the world, including thousands of hours by our first authors Natalya Lukoyanova and Stephanie Kondos,” says Michelle.
“We still have a lot of work to do before our ideas reach the clinic or industry but seeing how the machinery works is an important step forward,” says Birkbeck College’s Professor Helen Saibil and co- lead author on the paper.
The surface of the cell is shown by the double horizontal gray envelope across at the bottom. The toxin protein changes its shape, with the red section punching through the cell surface. This intricate machine is just one of 13 identical copies in a ring shape that self-assemble into a giant tunnel that breaches the cell membrane.
- Dr Michelle Dunstone, senior author, Monash University on +61 405 450 410 or email@example.com
- Professor Helen Saibil, senior author, Birkbeck College on +44 20 7631 6820 or firstname.lastname@example.org
- Niall Byrne on +61 417 131 977 or email@example.com (for the ARC Imaging Centre)
- Lucy Handford firstname.lastname@example.org (for Monash University)
- Bryony Merritt email@example.com (for Birkbeck College)
Conformational changes during pore formation by the perforin-related protein pleurotolysin
Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-Electron Microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ~70º opening of the bent and distorted central βsheet of the MACFP domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.
About the work
Humans, animals, plants, fungi, and bacteria all use pore-forming proteins as lethal, cell-killing weapons.
The structure of proteins in this Membrane Attack Complex-Perforin/Cholesterol Dependent Cytolysin family dramatically reorganise as they punch their way into cells.
Using X-ray crystallography and cryo electron microscopy, we’ve visualised for the first time the action of one of these pore-forming proteins—pleurotolysin—found in the edible oyster mushroom.
We show that pleurotolysin assembles into rings of 13 subunits, each of which opens up by about 70º during pore formation. This process is accompanied by refolding of two α-helical regions from each subunit into transmembrane β-hairpins. The hairpins from all 13 subunits assemble into an 80 Å wide β-barrel to create a large channel through the membrane.
We engineered and solved structures of three pleurotolysin variants with pore formation arrested at different intermediate stages. This allowed us to demonstrate a possible trajectory of structural rearrangements during the act of pore formation. We show that the β-barrel channel is formed by a zipper-like, top-down assembly to punch the hole through the membrane.
We hope these results will be relevant to other proteins in the family, including perforin.
This research was funded by:
- European Research Council (Advanced Grant 294408)
- Wellcome Trust (grant 079605/2/06/2)
- Biotechnology and Biological Sciences Research Council (BB/D008573/1 and BB/K01692X/1)
- Leverhulme Trust (RPG-2012- 519)
- National Health and Medical Research Council of Australia (NHMRC)
- Australian Research Council (ARC)
Birkbeck College, London
Natalya Lukoyanova, Irene Farabella, Maya Topf and Helen Saibil
ARC Centre of Excellence in Advanced Molecular Imaging Monash University
Stephanie Kondos, Ruby Law, Cyril Florent Reboul, James Charles Whisstock and Michelle Anne Dunstone
ARC Centre of Excellence in Advanced Molecular Imaging and the Australian Synchrotron
Department of Biochemistry and Molecular Biology, Monash University
Bradley Spicer, Oded Kleifeld, Daouda Traore, Susan Ekkel and Tamas Hatfaludi
Peter MacCallum Cancer Centre
Ilia Voskoboinik and Joseph Trapani
Institute of Structural and Molecular Biology, University College London
Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center
Eileen Hotze and Rodney Tweten
About the Authors
Dr Michelle Dunstone – ARC Centre for Advanced Molecular Imaging and Monash University
Understanding the life and death of cells is crucial information for scientists around the world working to treat illness and disease. Through the process of X-ray crystallography, electron microscopy and biophysical experiments, Dr Michelle Dunstone has been able to make an important contribution to this field of science. She has been able take molecular snapshots that allow scientists to visualise the behaviour of “hole punching” proteins, called pore forming toxins. These toxins are used by our immune system to combat disease and also used by bacteria to cause disease.
Helen Saibil – Birkbeck College, London
Professor Helen Ruth Saibil is a Canadian-British molecular biologist and Professor of Structural Biology at the Department of Crystallography of Birkbeck, University of London. Profess Saibil is a leading expert in the use of single particle cryo-EM to reveal the structure of perforin-like pores. Her research also encompasses molecular chaperones and protein mis-folding.
About the ARC Centre for Advanced Molecular Imaging
The ARC Centre of Excellence for Advanced Molecular Imaging integrates physics, chemistry and biology to unravel the complex molecular interactions that define immunity. The Centre will develop new imaging methods to visualize atomic, molecular and cellular details of how immune proteins interact and affect immune responses.
It will enable Australia to be an international leader in biological imaging, train the next generation of interdisciplinary scientists, and provide new insights into combating common diseases that afflict society.
The highly collaborative Centre brings together biologists, physicists and chemists from five Australian universities, the University of Warwick in the UK, the Australian Nuclear Science and Technology Organisation (ANSTO), synchrotrons in Australia and Germany and several high-tech companies.
It is an Australian Research Council (ARC) Centre of Excellence and funded with more than $39 million over seven years from 2014—$28 million from the ARC and a further $10 million from its partners. The Centre’s Director is Prof James Whisstock of Monash University.