Evolution and viral mimicry of short linear motif-mediated interactions

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.

Evolution and viral mimicry of short linear motif-mediated interactions

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.

Evolution of affinity between p53 transactivation domain and MDM2 across the animal kingdom demonstrates high plasticity of motif-mediated interactions

The interaction between the transcription factor p53 and the ubiquitin ligase MDM2 results in the degradation of p53 and is well-studied in cancer biology and drug development. Available sequence data suggest that both p53 and MDM2-family proteins are present across the animal kingdom. However, the interacting regions are missing in some animal groups, and it is not clear whether MDM2 interacts with, and regulates p53 in all species. We used phylogenetic analyses and biophysical measurements to examine the evolution of affinity between the interacting protein regions: a conserved 12-residue intrinsically disordered binding motif in the p53 transactivation domain (TAD) and the folded SWIB domain of MDM2. The affinity varied significantly across the animal kingdom. The p53TAD/MDM2 interaction among jawed vertebrates displayed high affinity, in particular for chicken and human proteins (K-D around 0.1 & mu;M). The affinity of the bay mussel p53TAD/MDM2 complex was lower (K-D = 15 & mu;M) and those from a placozoan, an arthropod, and a jawless vertebrate were very low or non-detectable (K-D > 100 & mu;M). Binding experiments with reconstructed ancestral p53TAD/MDM2 variants suggested that a micromolar affinity interaction was present in the ancestral bilaterian animal and was later enhanced in tetrapods while lost in other linages. The different evolutionary trajectories of p53TAD/MDM2 affinity during speciation demonstrate high plasticity of motif-mediated interactions and the potential for rapid adaptation of p53 regulation during times of change. Neutral drift in unconstrained disordered regions may underlie the plasticity and explain the observed low sequence conservation in TADs such as p53TAD

Evaluation of affinity-purification coupled to mass spectrometry approaches for capture of short linear motif-based interactions

Low affinity and transient protein-protein interactions, such as short linear motif (SLiM)-based interactions, require dedicated experimental tools for discovery and validation. Here, we evaluated and compared biotinylated peptide pulldown and protein interaction screen on peptide matrix (PRISMA) coupled to massspectrometry (MS) using a set of peptides containing interaction motifs. Eight different peptide sequences that engage in interactions with three distinct protein domains (KEAP1 Kelch, MDM2 SWIB, and TSG101 UEV) with a wide range of affinities were tested. We found that peptide pulldown can be an effective approach for SLiM validation, however, parameters such as protein abundance and competitive interactions can prevent the capture of known interactors. The use of tandem peptide repeats improved the capture and preservation of some interactions. When testing PRISMA, it failed to provide comparable results for model peptides that successfully pulled down known interactors using biotinylated peptide pulldown. Overall, in our hands, we find that albeit more laborious, biotin-peptide pulldown was more successful in terms of validation of known interactions. Our results highlight that the tested affinity-capture MS-based methods for validation of SLiM-based interactions from cell lysates are suboptimal, and we identified parameters for consideration for method development

Conservation of Affinity Rather Than Sequence Underlies a Dynamic Evolution of the Motif-Mediated p53/MDM2 Interaction in Ray-Finned Fishes.

The transcription factor and cell cycle regulator p53 is marked for degradation by the ubiquitin ligase MDM2. The interaction between these 2 proteins is mediated by a conserved binding motif in the disordered p53 transactivation domain (p53TAD) and the folded SWIB domain in MDM2. The conserved motif in p53TAD from zebrafish displays a 20-fold weaker interaction with MDM2, compared to the interaction in human and chicken. To investigate this apparent difference, we tracked the molecular evolution of the p53TAD/MDM2 interaction among ray-finned fishes (Actinopterygii), the largest vertebrate clade. Intriguingly, phylogenetic analyses, ancestral sequence reconstructions, and binding experiments showed that different loss-of-affinity changes in the canonical binding motif within p53TAD have occurred repeatedly and convergently in different fish lineages, resulting in relatively low extant affinities (KD = 0.5 to 5 μM). However, for 11 different fish p53TAD/MDM2 interactions, nonconserved regions flanking the canonical motif increased the affinity 4- to 73-fold to be on par with the human interaction. Our findings suggest that compensating changes at conserved and nonconserved positions within the motif, as well as in flanking regions of low conservation, underlie a stabilizing selection of functional affinity in the p53TAD/MDM2 interaction. Such interplay complicates bioinformatic prediction of binding and calls for experimental validation. Motif-mediated protein-protein interactions involving short binding motifs and folded interaction domains are very common across multicellular life. It is likely that the evolution of affinity in motif-mediated interactions often involves an interplay between specific interactions made by conserved motif residues and nonspecific interactions by nonconserved disordered regions

Conservation of Affinity Rather Than Sequence Underlies a Dynamic Evolution of the Motif-Mediated p53/MDM2 Interaction in Ray-Finned Fishes

The transcription factor and cell cycle regulator p53 is marked for degradation by the ubiquitin ligase MDM2. The interaction between these 2 proteins is mediated by a conserved binding motif in the disordered p53 transactivation domain (p53TAD) and the folded SWIB domain in MDM2. The conserved motif in p53TAD from zebrafish displays a 20-fold weaker interaction with MDM2, compared to the interaction in human and chicken. To investigate this apparent difference, we tracked the molecular evolution of the p53TAD/MDM2 interaction among ray-finned fishes (Actinopterygii), the largest vertebrate clade. Intriguingly, phylogenetic analyses, ancestral sequence reconstructions, and binding experiments showed that different loss-of-affinity changes in the canonical binding motif within p53TAD have occurred repeatedly and convergently in different fish lineages, resulting in relatively low extant affinities (KD = 0.5 to 5 mu M). However, for 11 different fish p53TAD/MDM2 interactions, nonconserved regions flanking the canonical motif increased the affinity 4- to 73-fold to be on par with the human interaction. Our findings suggest that compensating changes at conserved and nonconserved positions within the motif, as well as in flanking regions of low conservation, underlie a stabilizing selection of "functional affinity" in the p53TAD/MDM2 interaction. Such interplay complicates bioinformatic prediction of binding and calls for experimental validation. Motif-mediated protein-protein interactions involving short binding motifs and folded interaction domains are very common across multicellular life. It is likely that the evolution of affinity in motif-mediated interactions often involves an interplay between specific interactions made by conserved motif residues and nonspecific interactions by nonconserved disordered regions

Identification of motif-based interactions between SARS-CoV-2 protein domains and human peptide ligands pinpoint antiviral targets

Abstract The virus life cycle depends on host-virus protein-protein interactions, which often involve a disordered protein region binding to a folded protein domain. Here, we used proteomic peptide phage display (ProP-PD) to identify peptides from the intrinsically disordered regions of the human proteome that bind to folded protein domains encoded by the SARS-CoV-2 genome. Eleven folded domains of SARS-CoV-2 proteins were found to bind 281 peptides from human proteins, and affinities of 31 interactions involving eight SARS-CoV-2 protein domains were determined (K D ∼ 7-300 μM). Key specificity residues of the peptides were established for six of the interactions. Two of the peptides, binding Nsp9 and Nsp16, respectively, inhibited viral replication. Our findings demonstrate how high-throughput peptide binding screens simultaneously identify potential host-virus interactions and peptides with antiviral properties. Furthermore, the high number of low-affinity interactions suggest that overexpression of viral proteins during infection may perturb multiple cellular pathways

Large-scale phage-based screening reveals extensive pan-viral mimicry of host short linear motifs

Abstract Viruses mimic host short linear motifs (SLiMs) to hijack and deregulate cellular functions. Studies of motif-mediated interactions therefore provide insight into virus-host dependencies, and reveal targets for therapeutic intervention. Here, we describe the pan-viral discovery of 1712 SLiM-based virus-host interactions using a phage peptidome tiling the intrinsically disordered protein regions of 229 RNA viruses. We find mimicry of host SLiMs to be a ubiquitous viral strategy, reveal novel host proteins hijacked by viruses, and identify cellular pathways frequently deregulated by viral motif mimicry. Using structural and biophysical analyses, we show that viral mimicry-based interactions have similar binding strength and bound conformations as endogenous interactions. Finally, we establish polyadenylate-binding protein 1 as a potential target for broad-spectrum antiviral agent development. Our platform enables rapid discovery of mechanisms of viral interference and the identification of potential therapeutic targets which can aid in combating future epidemics and pandemics