Evolution and viral mimicry of short linear motif-mediated interactions

Abstract: Proteins are one of the most fundamental building blocks of life and their interactions regulate every cellular process. Historically they have been conceptualized as predominantly folded entities with well-defined secondary and tertiary structures. However, in recent decades, up to 50% of the human proteome has been shown to contain long disordered sequences that are flexible and unstructured in their natural environment. These intrinsically disordered regions exhibit low levels of sequence conservation and are enriched in short linear motifs (SLiMs). SLiMs are typically less then 10 amino acids long and serve as docking sites recognized by various globular domains. They exhibit a high degree of sequence degeneracy and evolutionary plasticity, allowing for rapid de novo emergence. SLiMs play crucial roles in a variety of cellular processes including cellular signalling, trafficking, transcriptional modulation, and protein degradation. Because they are small and degenerate, located in disordered regions, and form relatively weak interactions, they are difficult to identify using conventional high-throughput methods such as mass spectrometry and the yeast two-hybrid system. The same attributes that make them difficult to identify also make them ideal targets for viral SLiM mimicry, of which several examples have been described to date.To address the elusive nature of SLiMs, we have developed a novel approach for the discovery of motif-mediated interactions at the proteome scale using proteomic peptide phage display. We constructed two separate phage libraries with either human or viral disordered regions displayed on their surface. These libraries were then subjected to phage display selections against over 300 globular domains, resulting in the identification of more than 1,700 potential novel interactions. We validated a subset of these interactions with affinity measurements and GST-pulldown assays, solved the crystal structure of human globular domains in complex with viral linear motifs, and demonstrated that the gained knowledge can be applied to design peptidomimetic inhibitors of viral replication. In addition, we demonstrated that direct binding of viral SLiMs to the N-terminal domain of clathrin disrupts cellular trafficking and identified the C-terminal domain of polyadenylate-binding protein 1 as a novel target for viral SLiM mimicry. Furthermore, we demonstrated that SARS-CoV-2 viral proteins possess both SLiMs that bind to human proteins, and globular domains that recognize human SLiMs, showcasing the versatility of SLiM-mediated interactions. Finally, we examined the evolutionary trajectory of the interaction between the SWIB domain of MDM2 and the SLiM of p53 TAD to describe an example of the extraordinary evolutionary plasticity of SLiM-mediated interactions.Overall, the research presented in this thesis created the basis for an atlas of human motif-mediated interactions, yielded an extensive dataset of potential and validated cases of viral SLiM mimicry, and expanded our understanding of motif-mediated interactions from an evolutionary perspective.