Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

NMR characterization of the RIP homotypic interaction motif in the host adapter protein, RIPK3, and the viral inhibitor of necroptosis, M45 (#348)

Nikhil R Varghese 1 , Chi LL Pham 1 , Gustavo A Titaux-Delgado 2 , Paula Polonio 2 , Karyn L Wilde 3 , Miguel Mompeán 2 , Margaret Sunde 1
  1. School of Medical Sciences, Sydney Nano and Sydney Infectious Diseases, University of Sydney, Sydney, NSW, Australia
  2. "Rocasolano" Institute of Physical Chemistry, Spanish National Research Council, Serrano 116, Madrid, Spain
  3. National Deuteration Facility, Australian Nuclear Science and Technology Organisation (ANSTO), Kirrawee, NSW, Australia

Necroptosis is a form of programmed cell death that functions to commit cells to death during viral infections when apoptosis pathways are inhibited [1]. Necroptosis relies on the formation of a functional amyloid signalling complex comprised of the receptor interacting protein kinase 3 (RIPK3) and other host adapter proteins [2, 3]. These structured protein assemblies are stabilised by interactions between RIP Homotypic Interaction Motifs (RHIMs) in the component proteins [3]. Previous structural studies of RIPK3 through ssNMR and Cryo-EM have revealed the structure of the amyloid core in both, heteromeric assemblies with RIPK1 and homomeric RIPK3 assemblies [3, 4, 5].

The work presented here expands on the existing knowledge of the RHIM interactions of RIPK3 by probing the dynamics of homomeric RIPK3 assembly through solution NMR. The results presented here establish that the RHIM of RIPK3 is an intrinsically disordered protein motif, which challenges dominant thinking, and that exchange dynamics between free and amyloid-bound RIPK3 monomers involve a 20-residue stretch outside the RHIM that is absent in previous cryo-EM or ssNMR structural models of RIPK3 assemblies. The murine cytomegalovirus is also known to encode a viral RHIM containing protein, M45, which can interact with RIPK3 and enable viral evasion of necroptosis [6, 7]. This work also utilises solution NMR to characterise the homomeric assembly dynamics of the viral M45 protein, further expanding our understanding of viral RHIM-mediated interactions that underpin the viral inhibition of necroptosis.

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