This study presents evidence for the concerted evolution of LMs and their flanking regions. Although the current knowledge of the complete set of cellular LMs is fragmentary and it is not possible to assess the representativity of the analysed dataset, there are clear trends that are worth considering. LMs are known to be evolutionarily labile modules, which can be easily lost by point mutation 
. Nonetheless, the results of the present study show that LMs, in some cases, determine the conservation of the structural tendency and/or the sequence of the neighbouring amino acids. Here those findings are discussed in the light of the protein interactions mediated by LMs.
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 (
. 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 
. 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 
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 Pdiff
. 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
0.05). 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 
. 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 
A more detailed study of the structural conservation as function of the different types of mutual fit interaction (i.e. α
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 that have
correlation values lower that 0.20 between the
sets. The local sequence conservation could be explained in some cases by the conservation of the local structural tendency (instances in bold in , ). Still, as shown in the Results
(), 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 
. 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 (
sets), even when local structural tendencies between those sequences are not significantly different (e.g. ). Furthermore, these instances have higher chance of co–evolving with the neighbouring residues in comparison to the distant ones ().
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 
. 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 
. 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 
Finally, it is opportune to consider how current LM prediction methods can benefit from these results. A simple sequence analysis, similar to the one described here, would allow the identification of flanking regions with relevant conservation patterns, adding contextual information to already predicted LM instances. This can lead to a more detailed understanding of the role of LMs in determining the protein function. Indeed we consider that the LM field is ready – and has the potential – to go one step further from the timeless binary interactions towards the construction of more dynamic and realistic protein networks.