2.3.3. Specific Sequence Features of Processive Linkers

Disordered linkers can also be classified by their amino acid composition [49]. Processive linkers in DLD enzymes may also be under special pressure in this regard, because their potential to interact with the flanking domains and/or with other protein partners, or to undergo regulatory post-translational modifications (PTMs), may be of paramount importance. To assess these features, we analyzed the amino acid composition of disordered linkers in DLD enzymes and compared them to that of DisProt linkers and all disordered regions and annotated disordered linkers in the DisProt database [44] (Figure 6). Our results show that processive linkers have significantly less hydrophobic residues than other linkers and disordered proteins in general, which suggests they have to avoid hydrophobic collapse (cf. restraints on κ value stated above) and/or interactions with partners, which most often is mediated by motifs of hydrophobic character [50]. On the other hand, they are enriched in Pro and Gly (denoted as special residues, Figure 5A only shows P under 'special'), which entails that they have to remain extended and flexible and have a balance in oppositely-charged residues (D + E vs. R + K). Probably also for the same reason, they are, on average, more polar.

**Figure 6.** Special features of amino acid composition of linkers. Amino acid composition of linkers in DLD processive enzymes was analyzed and depicted with reference to similar measures of other data. (**A**) Amino acids of linkers were grouped into five categories and compared to the composition of non-linker (binding domain) regions of DLD enzymes (in Table 1) and also of all disordered linkers and assigned disordered linkers in the DisProt database [44]. (**B**) The abundance of amino acids in linkers and non-linker regions in DLD processive enzymes and in all disordered regions and assigned linker regions in the DisProt database.

A further notable feature of DLD linkers is their enrichment in Ser and Thr, which may be indicative of frequent O-linked glycosylation and/or regulatory phosphorylation. A search in UniProt [51] for post-translational modifications (PTMs) of the DLD linkers shows several such modifications in these enzymes (Table 2).

These modifications may impact their kinetic and structural parameters and may tune their interaction with one of the domains of the flanking domains or with external partners. For example, the linker of cellulase emerges from a point not proximal to the cellulose substrate, rather from a point behind, i.e., the kinetic behavior of the enzyme is fine-tuned by the binding of the linker to the surface of the catalytic domain (see next section). Regulated linker-domain interactions are also instrumental

in MMP-9, in which the linker has two short binding motifs, that bind the catalytic domain of the enzyme [35].


**Table 2.** Additional functions of linkers in DLD processive enzymes. Cases where the linker was shown to bind to its adjacent domain are marked with "+".

The primary function of linkers in DLD processive enzymes is to ensure relatively unrestricted spatial search of domains for binding sites along a multivalent (polymeric) substrate partner. They, however, are also often involved in the regulation of the functioning of the enzyme, as witnessed by additional binding functions and/or PTM events within the linkers themselves (for PTMs, data are either taken from UniProt or from the reference given).
