Oral Presentation The 5th Prato Conference on Pore Forming Proteins 2021

The structure of human macrophage-expressed gene 1 reveals a novel MACPF architecture. (#30)

Charles Bayly-Jones 1 , Siew Siew Pang 1 , Mazdak Radjainia 2 , Bradley A. Spicer 3 , Ruby H.P. Law 3 , Adrian W. Hodel 4 , Edward S. Parsons 4 , Susan M. Ekkel 3 , Paul J. Conroy 3 , Georg Ramm 2 3 , Hariprasad Venugopal 2 , Phillip I. Bird 3 , Bart W. Hoogenboom 4 , Ilia Voskobonik 5 , Yann Gambin 6 , Emma Sierecki 6 , Michelle A. Dunstone 3 , James C. Whisstock 3 7
  1. Department of Molecular Biology and Biochemistry, Monash University, Melbourne
  2. Ramaciotti Centre for Cryo-Electron Microscopy, Monash University, Clayton, Victoria, Australia
  3. Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia
  4. London Centre for Nanotechnology, London, Greater London, UK
  5. Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia
  6. School of Medical Sciences, UNSW, Sydney, NSW, Australia
  7. ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne

Macrophage Expressed Gene-1 (MPEG1/perforin-2) is an intra-cellular membrane bound protein, that is a close homolog of perforin-1 and components of the membrane attack complex (C6, C7, C8 and C9). Accordingly, it belongs to the MACPF/CDC family of pore forming proteins, which are thought to undergo pore formation via an archetypal three-step mechanism. Initially (1) monomers recognise and bind to their target membrane and (2) subsequently assemble into large oligomeric ring-like species. Transmembrane pore formation occurs (3), driven by a substantial conformational rearrangement of the MACPF domain forming a giant amphipathic β-barrel that spans the membrane bilayer. MACPFs are known to play integral roles in immunity in humans, as well as developmental roles. MPEG1 function however has remained elusive.

MPEG1 localises to the late-endosome and forms pore-like structures on membranes after mild proteolysis. Furthermore, pro-inflammatory molecules such as LPS and TNFα have been shown to trigger expression of MPEG1, as well as regulate the trafficking and distribution of MPEG1 within cells. Lastly, in vitro and in vivo studies illustrate loss of MPEG1 function reduces the ability of parenchymal cells to clear intracellular infections. The current model, hence, postulates MPEG1 is an immune effector that clears intracellular pathogens in the phagolysosome.

Here we present a high resolution cryo-electron microscopy reconstruction of MPEG1 in a soluble and lipid bound prepore state. Moreover, we observe MPEG1 function is triggered by low pH, consistent with its subcellular localisation. Our results indicate that MPEG1 indeed is a pore forming complex – alike to perforin-1 and MAC – and is capable of haemolytic activity at low pH. Moreover, we identify the previously uncharacterised ancillary domain and suggest a mechanism for membrane binding and host cell protection. This study provides insights into the structural architecture of MPEG1 and further reveals functional details supporting a non-canonical mechanism of action.