Systematic discovery of protein interaction interfaces using AlphaFold and experimental validation.

Structural resolution of protein interactions enables mechanistic and functional studies as well as interpretation of disease variants. However, structural data is still missing for most protein interactions because we lack computational and experimental tools at scale. This is particularly true for interactions mediated by short linear motifs occurring in disordered regions of proteins. We find that AlphaFold-Multimer predicts with high sensitivity but limited specificity structures of domain-motif interactions when using small protein fragments as input. Sensitivity decreased substantially when using long protein fragments or full length proteins. We delineated a protein fragmentation strategy particularly suited for the prediction of domain-motif interfaces and applied it to interactions between human proteins associated with neurodevelopmental disorders. This enabled the prediction of highly confident and likely disease-related novel interfaces, which we further experimentally corroborated for FBXO23-STX1B, STX1B-VAMP2, ESRRG-PSMC5, PEX3-PEX19, PEX3-PEX16, and SNRPB-GIGYF1 providing novel molecular insights for diverse biological processes. Our work highlights exciting perspectives, but also reveals clear limitations and the need for future developments to maximize the power of Alphafold-Multimer for interface predictions.

A truncated PACT protein resulting from a frameshift mutation reported in movement disorder DYT16 triggers caspase activation and apoptosis.

Protein Activator (PACT) activates the interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR) in response to stress signals. Oxidative stress and endoplasmic reticulum (ER) stress causes PACT-mediated PKR activation, which leads to phosphorylation of translation initiation factor eIF2α, inhibition of protein synthesis, and apoptosis. A dominantly inherited form of early-onset dystonia 16 (DYT16) has been identified to arise due to a frameshift (FS) mutation in PACT. To examine the effect of the resulting truncated mutant PACT protein on the PKR pathway, we examined the biochemical properties of the mutant protein and its effect on mammalian cells. Our results indicate that the FS mutant protein loses its ability to bind dsRNA as well as its ability to interact with PKR while surprisingly retaining the ability to interact with PACT and PKR-inhibitory protein TRBP. The truncated FS mutant protein, when expressed as a fusion protein with a N-terminal fluorescent mCherry tag aggregates in mammalian cells to induce apoptosis via activation of caspases both in a PKR- and PACT-dependent as well as independent manner. Our results indicate that interaction of FS mutant protein with PKR inhibitor TRBP can dissociate PACT from the TRBP-PACT complex resulting in PKR activation and consequent apoptosis. These findings are relevant to diseases resulting from protein aggregation especially since the PKR activation is a characteristic of several neurodegenerative conditions.