The structure of a minimum amyloid fibril core formed by necroptosis-mediating RHIM of human RIPK3

Abstract

Significance Receptor-interacting protein kinases 3 (RIPK3), a hub player in necrotic cell death, forms amyloid-like polymers in response to upstream mediators. This work, by using cryo-EM and solid-state NMR, reports the structure of human RIPK3 amyloid fibril core. The fibril core is mainly composed of the consensus RHIM domain and exhibits distinctive properties: an exceptionally small fibril core and existence in both handedness. These traits may provide a favorable geometry for the arrangement of kinase domain on the fibril surface to boost RIPK3 phosphorylation and the following activation of downstream MLKL. This work sheds light on how functional fibrils utilize the amyloid state to spatially regulate their functional domains, which is usually not a concern of pathological fibrils. Receptor-interacting protein kinases 3 (RIPK3), a central node in necroptosis, polymerizes in response to the upstream signals and then activates its downstream mediator to induce cell death. The active polymeric form of RIPK3 has been indicated as the form of amyloid fibrils assembled via its RIP homotypic interaction motif (RHIM). In this study, we combine cryogenic electron microscopy and solid-state NMR to determine the amyloid fibril structure of RIPK3 RHIM-containing C-terminal domain (CTD). The structure reveals a single protofilament composed of the RHIM domain. RHIM forms three β-strands (referred to as strands 1 through 3) folding into an S shape, a distinct fold from that in complex with RIPK1. The consensus tetrapeptide VQVG of RHIM forms strand 2, which zips up strands 1 and 3 via heterozipper-like interfaces. Notably, the RIPK3-CTD fibril, as a physiological fibril, exhibits distinctive assembly compared with pathological fibrils. It has an exceptionally small fibril core and twists in both handedness with the smallest pitch known so far. These traits may contribute to a favorable spatial arrangement of RIPK3 kinase domain for efficient phosphorylation.