Orchestrating serine/threonine phosphorylation and elucidating downstream effects by short linear motifs

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.

Single-molecule analysis of specificity and multivalency in binding of short linear substrate motifs to the APC/C

Robust regulatory signals in the cell often depend on interactions between short linear motifs (SLiMs) and globular proteins. Many of these interactions are poorly characterized because the binding proteins cannot be produced in the amounts needed for traditional methods. To address this problem, we developed a single-molecule off-rate (SMOR) assay based on microscopy of fluorescent ligand binding to immobilized protein partners. We used it to characterize substrate binding to the Anaphase-Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that triggers chromosome segregation. We find that SLiMs in APC/C substrates (the D box and KEN box) display distinct affinities and specificities for the substrate-binding subunits of the APC/C, and we show that multiple SLiMs in a substrate generate a high-affinity multivalent interaction. The remarkably adaptable substrate-binding mechanisms of the APC/C have the potential to govern the order of substrate destruction in mitosis.

Docking to a Basic Helix Promotes Specific Phosphorylation by G1-Cdk1

Cyclins are the activators of cyclin-dependent kinase (CDK) complex, but they also act as docking scaffolds for different short linear motifs (SLiMs) in CDK substrates and inhibitors. According to the unified model of CDK function, the cell cycle is coordinated by CDK both via general CDK activity thresholds and cyclin-specific substrate docking. Recently, it was found that the G1-cyclins of S. cerevisiae have a specific function in promoting polarization and growth of the buds, making the G1 cyclins essential for cell survival. Thus, while a uniform CDK specificity of a single cyclin can be sufficient to drive the cell cycle in some cells, such as in fission yeast, cyclin specificity can be essential in other organisms. However, the known G1-CDK specific LP docking motif, was not responsible for this essential function, indicating that G1-CDKs use yet other unknown docking mechanisms. Here we report a discovery of a G1 cyclin-specific (Cln1,2) lysine-arginine-rich helical docking motif (the K/R motif) in G1-CDK targets involved in the mating pathway (Ste7), transcription (Xbp1), bud morphogenesis (Bud2) and spindle pole body (Spc29, Spc42, Spc110, Sli15) function of S. cerevisiae. We also show that the docking efficiency of K/R motif can be regulated by basophilic kinases such as protein kinase A. Our results further widen the list of cyclin specificity mechanisms and may explain the recently demonstrated unique essential function of G1 cyclins in budding yeast.

MotSASi: Functional Short Linear Motifs (SLiMs) prediction based on genomic single nucleotide variants and structural data

Short linear motifs (SLiMs) are key to cell physiology mediating reversible protein-protein interactions. Precise identification of SLiMs remains a challenge, being the main drawback of most bioinformatic prediction tools their low specificity (high number of false positives). An important, usually overlooked, aspect is the relation between SLiMs mutations and disease. The presence of variants in each residue position can be used to assess the relevance of the corresponding residue(s) for protein function, and its (in)tolerance to change. In the present work, we combined sequence variant information and structural analysis of the energetic impact of single amino acid substitution (SAS) in SLiM-Receptor complex structure, and showed that it significantly improves prediction of true functional SLiMs. Our strategy is based on building a SAS tolerance matrix that shows, for each position, whether one of the possible 19 SAS is tolerated or not. Herein we present the MotSASi strategy and analyze in detail 4 SLiMs involved in intracellular protein trafficking. Our results show that inclusion of variant and sequence information significantly improves both prediction of true SLiMs and rejection of false positives, while also allowing better classification of variants inside SLiMs, a results with a direct impact in clinical genomics.

Co-translational assembly counteracts promiscuous interactions

During the co-translational assembly of protein complexes, a fully synthesized subunit engages with the nascent chain of a newly synthesized interaction partner. Such events are thought to contribute to productive assembly, but their exact physiological relevance remains underexplored. Here, we examined structural motifs contained in nucleoporins for their potential to facilitate co-translational assembly. We experimentally tested candidate structural motifs and identified several previously unknown co-translational interactions. We demonstrate by selective ribosome profiling that domain invasion motifs of beta-propellers, coiled-coils, and short linear motifs act as co-translational assembly domains. Such motifs are often contained in proteins that are members of multiple complexes (moonlighters) and engage with closely related paralogs. Surprisingly, moonlighters and paralogs assembled co-translationally in only one but not all of the relevant assembly pathways. Our results highlight the regulatory complexity of assembly pathways.

Anchored and labile pools of Calcineurin are enabled by a disordered AKAP scaffold

Signalling requires precise spatial and temporal regulation of molecular interactions, which is frequently orchestrated by disordered scaffolding proteins, such as A-kinase anchoring protein 5 (AKAP5). AKAP5 contains multiple Short Linear Motifs (SLiMs) that assemble the necessary components, including the phosphatase Calcineurin, which is anchored via a well-characterised PxIxIT SLiM. Here we show, using a combination of biochemical and biophysical approaches, that Calcineurin also recognises additional lower-affinity SLiMs C-terminal to the PxIxIT motif. Moreover, we demonstrate that the assembly is in reality a complex system in which AKAP SLiMs spanning a wide affinity range act cooperatively to maintain distinct pools of anchored and more loosely held enzyme, analogous to the well-understood transcription factor search complexes on DNA, and compatible with the requirement for both stable anchoring and responsive downstream signalling. We conclude that the AKAP5 C-terminus is enriched in lower-affinity/mini-SLiMs that cooperate to maintain a structurally disordered but tightly regulated signalosome.

Peptide array–based interactomics

The analysis of protein-protein interactions (PPIs) is essential for the understanding of cellular signaling. Besides probing PPIs with immunoprecipitation-based techniques, peptide pull-downs are an alternative tool specifically useful to study interactome changes induced by post-translational modifications. Peptides for pull-downs can be chemically synthesized and thus offer the possibility to include amino acid exchanges and post-translational modifications (PTMs) in the pull-down reaction. The combination of peptide pull-down and analysis of the binding partners with mass spectrometry offers the direct measurement of interactome changes induced by PTMs or by amino acid exchanges in the interaction site. The possibility of large-scale peptide synthesis on a membrane surface opened the possibility to systematically analyze interactome changes for mutations of many proteins at the same time. Short linear motifs (SLiMs) are amino acid patterns that can mediate protein binding. A significant number of SLiMs are located in regions of proteins, which are lacking a secondary structure, making the interaction motifs readily available for binding reactions. Peptides are particularly well suited to study protein interactions, which are based on SLiM-mediated binding. New technologies using arrayed peptides for interaction studies are able to identify SLIM-based interaction and identify the interaction motifs. Graphical abstract

Short Linear Motifs Characterizing Snake Venom and Mammalian Phospholipases A2

Snake venom phospholipases A2 (PLA2s) have sequences and structures very similar to those of mammalian group I and II secretory PLA2s, but they possess many toxic properties, ranging from the inhibition of coagulation to the blockage of nerve transmission, and the induction of muscle necrosis. The biological properties of these proteins are not only due to their enzymatic activity, but also to protein–protein interactions which are still unidentified. Here, we compare sequence alignments of snake venom and mammalian PLA2s, grouped according to their structure and biological activity, looking for differences that can justify their different behavior. This bioinformatics analysis has evidenced three distinct regions, two central and one C-terminal, having amino acid compositions that distinguish the different categories of PLA2s. In these regions, we identified short linear motifs (SLiMs), peptide modules involved in protein–protein interactions, conserved in mammalian and not in snake venom PLA2s, or vice versa. The different content in the SLiMs of snake venom with respect to mammalian PLA2s may result in the formation of protein membrane complexes having a toxic activity, or in the formation of complexes whose activity cannot be blocked due to the lack of switches in the toxic PLA2s, as the motif recognized by the prolyl isomerase Pin1.