Structural Studies of an Anti-necroptosis Viral:Human Functional Hetero-amyloid M45:RIPK3 using SSNMR

The formation of RIP-homotypic interaction motif (RHIM)-based heteromeric amyloid assemblies between effector proteins such as RIPK1, ZBP1, or TRIF and the kinase RIPK3 serve as regulating signals for the necroptosis process, a key element of innate immune defense. Murine cytomegalovirus (MCMV) expresses the M45-encoded viral inhibitor of RIP activation (vIRA) which inhibits necroptosis in a RHIM-dependent manner. A pivotal question is how viral M45 forms hetero-amyloids with RIPK3 to effectively create an inhibitory assembly. We report a novel high-resolution structure of the M45:RIPK3 complex where M45 and RIPK3 alternately stack in an amyloid-state structure. Mutagenesis of the residues flanking the IQIG tetrad in M45 results in specific impacts on co-assembly with RIPK3, indicating an extended interface in the heteromeric fibrils. Other key interactions support the formation of stable viral:host fibrils. The M45: RIPK3 hetero-amyloid is likely to act as an anti-necroptotic signal by competing with formation of other pro-necroptotic species and introducing a barrier to RIPK3 autophosphorylation.