Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

The murine cytomegalovirus M45 protein interacts with necroptosis-associated proteins to form heteromeric amyloid assemblies. (#283)

Chi L. L. Pham 1 , Nirukshan Shanmugam 1 , Merryn Strange 1 , Ailis O'Carroll 2 , James W. Brown 2 , Emma Sierecki 2 , Yann Gambin 2 , Megan Steain 3 , Margie Sunde 1
  1. Discipline of Pharmacology, School of Medical Sciences, The University of Sydney, Camperdown, NSW, Australia
  2. EMBL Australia Node in Single Molecule Sciences, School of Medical Sciences, University of New South Wales, NSW, Australia
  3. Infectious Diseases and Immunology, Central Clinical School, School of Medical Sciences, The University of Sydney, Camperdown, NSW, Australia

The RIP homotypic interaction motif (RHIM) is an amino acid sequence of 18 residues with a conserved tetrad (V/I)-Q-(V/I/L/C)-G core sequence. It was first identified as the protein sequence required for the interaction of two receptor interacting protein kinases, RIPK1 and RIPK3 (1).  This RHIM:RHIM  interaction results in the assembly of a necrosome with a functional heteromeric amyloid core, that activates the programmed cell death response necroptosis (1,2).  TIR-domain-containing adaptor-inducing interferon-B (TRIF) and the Z-DNA binding protein 1 (ZBP1) are two additional RHIM-containing proteins that also interact with RIPK3 to mediate necroptosis via two other distinct pathways (3).  Murine cytomegalovirus expressing the RHIM-containing M45 protein can suppress host cell necroptosis in both mouse and human as an evading strategy to sustain infection.  An intact RHIM of the M45 protein is necessary for the interaction with RIPK1, RIPK3 and ZBP1 to inhibit necroptosis.

Here, we have utilised a number of biophysical techniques firstly, to characterise the homomeric amyloid assembly of the N-terminal 90 residues of the M45 protein containing the RHIM sequence.  We showed that the M45 protein interacts with human RHIM-containing proteins to form heteromeric amyloid assemblies with altered morphologies. Mutation of the tetrad core sequence of M45 protein attenuates homo- and hetero-amyloid assembly, suggesting that the amyloidogenic nature of M45 RHIM is essential for its biological function. M45 preferentially interact with RIPK3 and ZBP1 over RIPK1.  Based on our results and the common tetrad core sequence IQIG of M45 and the adapter proteins RIPK1 and ZBP1, suggested that M45 may mimic the interaction of these adapter proteins with RIPK3.  Thus, sequestering RIPK3 into forming alternative heteromeric amyloid assemblies that may be incapable of activating RIPK3 and/or downstream signalling events leading to cell death (4).

(1) Li et al., Cell (2012), 150 339-50

(2) Mompean et al., Cell (2018), https://doi.org/10.1016/j.cell.2018.03.032

(3) Baker et al. Semin. Cell. Dev. Biol. (2018), https://doi.org/10.1016/j.semcdb.2018.05.004

(4) Pham et al., EMBO Report (2018), https://doi.org/10.15252/embr.201846518