**2. Intrinsically Disordered Regions Facilitate LLPS**

An interesting feature of proteins that undergo LLPS is they frequently contain long segments that lack well-defined three-dimensional structure. These segments are typically termed intrinsically disordered regions (IDRs), or intrinsically disordered proteins (IDPs), because they have no single equilibrium structure; instead, they exist as broad structural (or population) ensembles or they exchange between multiple conformations rapidly. IDRs are usually defined as being approximately 30 amino acids or longer [21], and their distinguishing characteristic is a relative paucity of hydrophobic amino acids to drive folding into a narrow conformational landscape.

The sequence composition of IDRs can vary, but is commonly disproportionately represented by only a few amino acids (i.e., low-complexity). Some low-complexity sequences are called yeast prion-like because they are compositionally very similar to the domains that enable certain yeast proteins to form self-propagating amyloid fibers. Yeast prion domains (and prion-like domains (PrLDs)) are usually very rich in hydrophilic amino acids (e.g., asparagine, glutamine, serine, and tyrosine). Other low-complexity sequences may disproportionately contain charged amino acids, such as the arginine/glycine repeats (RGG or GRG), which occur in several IDRs within liquid phase-separating proteins. Repeating (or spatially distributed) motifs of a subset of amino acids are also common to IDRs [6]. IDRs of MLO-forming proteins are also enriched in amino acids that can form π–π interactions, in which induced electrostatic interactions occur between sp<sup>2</sup> hybridized atoms [22]. These interactions can also involve the backbone amide bonds, which are accessible due to the non-folded arrangement of IDRs.

The significance of IDRs in liquid phase-separating proteins is they enable diverse networks of transient interactions with moderate affinities (i.e., reversible, due partly to entropic penalties of IDRs adopting binding conformations). Relative to folded domains, IDRs have greater accessible conformational space and flexibility for forming molecular contacts. The frequent presence of repetitive motifs can enable numerous low-affinity interactions with the potential for high-avidity binding [23]. IDRs can therefore support multivalent interactions, meaning they can form multiple molecular contacts with a potential variety of binding partners. Thus, an MLO may emerge from a continuous network of IDRs forming inter-protein (or RNA-protein) multivalent contacts.
