Carnivorous mushrooms reveal human immune trick

MELBOURNE, THURSDAY 5 FEBRUARY 2015: 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.


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You can read the full paper online at:

Background Information


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.