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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.

Protein-protein interactions (PPIs) orchestrate a variety of cellular events, ranging from signal transduction, scaffolding to subcellular localisation. A subclass of PPIs is mediated by short linear motifs, which are short amino acid stretches found in the intrinsically disordered regions of the proteome. Regulation of these interactions, which determines which proteins interact, as well as when and where interactions occur, is vital for performing cellular tasks. Phosphorylation can act as cue for this regulation, creating, breaking or fine-tuning a given interaction site. Disease-associated mutations may, in turn, deregulate motif-based PPIs. The Ivarsson lab established proteomic peptide-phage display (ProP-PD) for the discovery of binding peptides and motifs of protein domains. I extended the approach to kinase domains to assess their peptide binding properties and uncovered potential docking interactions of CASK and MAPK8.I further investigated the modulation of motif-based PPIs by phosphorylation guided either by bioinformatic predictions or by phosphomimetic ProP-PD. This led to unravelling of phospho-modulated binding motifs in the cytoplasmic tails of coronavirus host receptors. In addition, I developed an improved phosphomimetic phage library combining the intrinsically disordered regions of the human proteome with functionally prioritised phosphosites. Screening protein domains against the phosphomimetic library suggested novel interaction partners and their phospho-modulation. I demonstrated the dependency of clathrin binding on S839 HURP phosphorylation, which was further found to be required for the mitotic function of HURP. Lastly, I assessed, with the genetic variation phage library, whether mutational ProP-PD is suited to capture changes in motif-based PPIs as a consequence of disease-associated mutation. The method can with high confidence identify PPI-disruptive mutations, such as the P348L SQSTM1 mutation that diminishes binding to KEAP1 and a R

Cellular function is based on protein-protein interactions. A large proportion of these interactions involves the binding of short linear motifs (SLiMs) by folded globular domains. These interactions are regulated by post-translational modifications, such as phosphorylation, that create and break motif binding sites or tune the affinity of the interactions. In addition, motif-based interactions are involved in targeting serine/threonine kinases and phosphatases to their substrate and contribute to the specificity of the enzymatic actions regulating which sites are phosphorylated. Here, we review how SLiM-based interactions assist in determining the specificity of serine/threonine kinases and phosphatases, and how phosphorylation, in turn, affects motif-based interactions. We provide examples of SLiM-based interactions that are turned on/off, or are tuned by serine/threonine phosphorylation and exemplify how this affects SLiM-based protein complex formation.

The spike protein of SARS-CoV-2 binds the angiotensin-converting enzyme 2 (ACE2) on the host cell surface and subsequently enters host cells through receptor-mediated endocytosis. Additional cell receptors may be directly or indirectly involved, including integrins. The cytoplasmic tails of ACE2 and integrins contain several predicted short linear motifs (SLiMs) that may facilitate internalization of the virus as well as its subsequent propagation through processes such as autophagy. Here, we measured the binding affinity of predicted interactions between SLiMs in the cytoplasmic tails of ACE2 and integrin beta(3) with proteins that mediate endocytic trafficking and autophagy. We validated that a class I PDZ-binding motif mediated binding of ACE2 to the scaffolding proteins SNX27, NHERF3, and SHANK, and that a binding site for the clathrin adaptor AP2 mu 2 in ACE2 overlaps with a phospho-dependent binding site for the SH2 domains of Src family tyrosine kinases. Furthermore, we validated that an LC3-interacting region (LIR) in integrin beta(3) bound to the ATG8 domains of the autophagy receptors MAP1LC3 and GABARAP in a manner enhanced by LIR-adjacent phosphorylation. Our results provide molecular links between cell receptors and mediators of endocytosis and autophagy that may facilitate viral entry and propagation.