Oral Presentation The 6th Prato Conference on Pore Forming Proteins 2025

The structural basis of perforin and perforin-2 oligomerisation (123555)

Josh Benton 1 , Rex Anane 1 , Johnny Nguyen 1 , Charles Bayly-Jones 1 , Ruby Law 1 , James Whisstock 1
  1. Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia

Perforin and perforin-2 (MPEG1) are pore-forming proteins of the MACPF family that function cooperatively in mammalian host defence. Perforin-2, the evolutionary precursor to perforin, is an ancient pore-forming protein that is deployed to acidic phagolysosomes to damage engulfed microbes and/or release antigens into the cytosol for presentation on MHC I (1-4). Cross-presented antigens can then prime cytotoxic T cells to kill virally infected or cancerous cells harbouring the presented antigens. Cytotoxic T cells kill their targets by releasing membranolytic perforin and pro-apoptotic granzyme B into an immune synapse formed with the target.  

 

Structural studies on perforin-2 to date have focussed on ‘complete’ prepore and acid-activated pore states (5-7). How perforin-2 monomers oligomerise into these structures is unknown. Using cryo-electron microscopy (cryo-EM), we have solved sub-3 Å resolution structures of a wild-type, transmembrane perforin-2 monomer, dimer, trimer and complete prepore. These prepore intermediates provide high-resolution, step-by-step insights into the mechanism of perforin-2 oligomerisation – the first insights of their kind for a MACPF protein. We discover that perforin-2 undergoes relatively small conformational changes during oligomerisation, centred on β-strand 1 of its MACPF β-sheet and transmembrane hairpin 2 (TMH2).

 

We then compare the mechanism of perforin-2 oligomerisation to that of perforin. Our 3.15 Å resolution cryo-EM structure of a wild-type human perforin trimeric prepore appears vertically compressed relative to the previously solved crystal structure of monomeric perforin (8). Combined with molecular dynamics simulations, these data may suggest that oligomerisation-induced vertical compression primes protomers for pore formation.

 

The prepore intermediates of human perforin and perforin-2 were solved without TMH-lock mutation, providing near-native insight into their oligomerisation mechanisms. Collectively, the perforin and perforin-2 prepore intermediate structures elucidate key conformational transitions that occur from monomer to prepore, revealing how each system primes for pore formation.

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