LMs are mainly found in intrinsically unstructured regions of proteins [5]. Disordered regions allow a thermodynamical control of the affinity and specificity of protein interactions. They favour transient, that is to say low affinity, and conditional interactions, often depending on a previous modification like a phosphorylation [6]. Hence the localisation of LMs in disordered regions suits dynamic regulation of protein networks, where a rapid but deterministic response is needed [7]. Indeed, LM–mediated interactions allow the emergence of several regulatory modes (i.e. sequential, mutually exclusive and cooperative) frequently observed in signalling, vesicular trafficking and transcription pathways [8].

Function of LMs strongly depends on the context. An instance of the KDEL motif, which is an endoplasmic reticulum retrieving signal, is likely to be functional only if present in protein sequences known to localise to the ER or Golgi apparatus. On one hand, the context defines the natural constraints that act on LMs and therefore provides “rules” that can be applied to evaluate the reliability of a newly predicted pattern or instance. For example the domain masking strategy, which is used to discard instances occurring in protein regions inaccessible for interaction like globular domains or coiled coils [3], [9], [10], [11].

On the other hand, the context can also give detailed information about the mode of action of LMs. The role of the local amino acid composition in determining specificity of LM interactions has been experimentally studied at the interactome level [12], [13], [14]. At the structural level, unstructured regions flanking LMs have been observed to undergo disorder to order transition upon binding [15], forming either -helices [16] or additional strands that join a sheet of the partner [17]. This coincides with the observation that two thirds of LMs bind to their partners by mutual fit, meaning that they acquire a fixed structure upon binding to a well structured template [1]. Furthermore, a recent survey of 3D structures of protein–peptide complexes has estimated that neighbouring residues account for 20% of the global binding energy of peptide–mediated interactions. They are thought to improve the interaction affinity with the native partner or to impede non–native interactions [18].

The evolutionary context of LMs has also been studied and used in predictive methods. Convergent evolution of LMs is at the basis of discovery algorithms like SLiMFinder [19] and DILIMOT [20], which search for over–represented motifs in unrelated proteins with a common functional attribute. Additionally, conservation of LMs in closely and distantly related proteins has been used to improve the identification of functional instances of known LM patterns [11], [21], [22], [23]. Methods for de novo discovery, have also benefited from the evolutionary signal provided by analysing patterns of conservation. SLiMFinder uses global or local sequence conservation to improve confidence in motif predictions [9], [24]; DILIMOT takes into account conservation of the motif in orthologs as part of the scoring scheme [10].

It is clear that LM predictions from the current generation of predictors require experimental validation to be considered genuine. The methods are often working at the limits of signal to noise and are dependent on the information content of the bioinformatics databases being used for LM prediction [3], [25], [26]. Nevertheless, LM prediction methods could be valuable tools for the study of high dimensional systems like the protein signalling networks. Therefore it is necessary to move from the identification of a LM in a protein towards the prediction of the role of that instance inside the functional framework of the protein, e.g. its network of interactors.

In the first section of the Results it was shown that, for some instances, the conservation of the LM is associated with the maintenance of the structural tendency of the surrounding residues. What is the meaning of this conservation? As mentioned in the Introduction, two thirds of the LM–mediated interactions lead to the formation of secondary structure elements (–helices or –strands) [1]. If the LM functionality is to be maintained, the structural properties of the neighbouring amino acids that allow such disorder/order transition are likely to be conserved. This local propensity would be reflected by the corresponding IUPred values and hence the low observed in the sets would indicate the conservation of such propensity.

However, the conservation of the local structural tendency could also indicate the maintenance of the local disorder. Several studies on protein–protein interactions have drawn attention to the importance of intrinsic disorder in the formation of protein complexes [6], [35], [36], [37]. If the local disorder provides the flexibility required to bind different patterns, it is not surprising to observe the conservation of this structural tendency in the regions involved in such interactions. Previous work by [38] has connected the conservation of predicted disordered regions in eukaryotic proteins with DNA/RNA binding domains. The conservation of disorder around LMs would extend this result to a broader set of biological processes.

The instances of the molecular hub p53 exemplify the double meaning of the structural conservation measured by the IU P_diff. For three out of four of the p53 instances in the dataset (TRG_NES_CRM1_1, 339–352; MOD_SUMO, 385–388; MOD_PIKK_1, 12–18), the presence of the instance coincides with the conservation of the local structural tendency. They belong to the group of instances that have a significantly different distribution of the between and sets (p-value0.05). Those instances are located in the C and N terminal regions of P53, which are disordered modules known to bind different partners by acquiring different conformations [39]. Additionally, the MOD_SUMO and the MOD_PIKK_1 (but not the TRG_NES_CRM1_1) occur in predicted α–MoREs, disordered regions having propensities to form α–helix upon molecular recognition [16].

A more detailed study of the structural conservation as function of the different types of mutual fit interaction (i.e. α–helix formation, augmentation or irregular topology) may be interesting. It would shade light on the specific requirements of each conformation. This would require the definition of a more elaborated metric for the local structure conservation than the . However, independently from its specific meaning, the structural tendency conservation around the LM suggests the occurrence of overlapping interaction surfaces. Those clustered overlaps are likely to entail different regulatory mechanisms for the spatial or temporal isolation of the mutually exclusive interactions.

In the second and third part of the Results it was shown that the presence of some LM instances is accompanied by the conservation of the amino acids flanking the motif. This is the case for 42% of the instances in Table 2 that have correlation values lower that 0.20 between the and sets. The local sequence conservation could be explained in some cases by the conservation of the local structural tendency (instances in bold in Table 2, Figure 3C and D). Still, as shown in the Results (Figure 2), sequence identity does not seem to be a requirement for the maintenance of the local order/disorder tendency. Indeed, it has been recently demonstrated by nuclear magnetic resonance spectroscopy that intrinsically disordered regions can maintain their dynamic behaviour despite low sequence similarity [40]. Yet there must be a functional meaning for the local sequence conservation associated with these instances, especially considering that it allows to discriminate sequences with and without the motif ( and sets), even when local structural tendencies between those sequences are not significantly different (e.g. Figure 3A and B). Furthermore, these instances have higher chance of co–evolving with the neighbouring residues in comparison to the distant ones (Figure 4A).

It is likely that the flanking regions of those instances are related with the regulation of the LM or with the regulation of another interaction, which is functionally connected to the one mediated by the motif. This is the case of the LIG_AP2alpha_1 in positions 324–328 of amphiphysin (P49418, correlation 0.03), which is involved in clathrin coated vesicle formation. Phosphorylation of amphiphysin by Cdk5 in S276, S285 and T310 has been shown to directly regulate the intramolecular interaction in amphiphysin, which in turn regulates dynamin-dependent endocytosis [41], [42]. Likewise, other instances with correlation between −0.05 and 0.16 (LIG_SH3_1 P10636 565–572, LIG_COP1 P17535 241–248) have experimentally verified phosphorylation sites in their flanking regions: T561 for P17535 and S251, S255 and S259 for P17535 [25]. Those phosphorylation site are likely to regulate the local protein conformation and activity, as recently shown in a phosphoproteomic analysis of the mouse brain cytosol [43].

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