*3.1. Regulation of IDP Activity through Structural Changes*

Intrinsically disordered proteins acting in intracellular pathways contain conserved motifs for interaction with nucleic acids and other proteins, and frequently form low-affinity complexes advantageous to processes like signal transduction [184]. The recognition elements (being often called "SLiMs"—short linear motifs ranging from 3 to 10 amino acids [189–191]) determine a pivotal feature associated with disordered proteins, that is their binding promiscuity, which is carried out through "one-to-many" and "many-to-one" mechanisms [192]. Interestingly, many IDPs are able to adopt ordered structures when interacting with certain targets, characterizing the coupled folding and binding phenomenon. Also, structural polymorphisms can emerge from the IDP conformational landscape in cases where the same disordered protein can assume different defined structures as it binds to different targets. Some of the recognition elements are also targeted for post-translational modifications by regulatory enzymes, enabling disordered-to-ordered transitions in IDPs. However, induced folding in IDPs is not mandatory for activity, as many regions that remain disordered upon partner binding are important to function, constituting "fuzzy" complexes [193,194].

#### 3.1.1. Coupled Folding and Binding

The folding induction by partner interaction is possibly one of the most reported characteristics of IDPs, despite not being a phenomenon absolutely widespread across this class of proteins (considering the fuzzy complexes), nor a completely understood process. There are numerous examples of disordered-to-ordered transitions in proteins implicated in the regulation of gene expression, like transcription factors that assume folded motifs when interacting with DNA. One particular example is the leucine zipper protein GCN4, which presents a basic region that is unstructured in the absence of DNA but becomes a stable helical structure when interacting to its cognate AP-1 site [195]. The transition begins with transient nascent helical forms, observed in the unbound state, that interact with DNA and lead to dramatic structural changes, explained by a reduction of the entropic cost of DNA binding due to restriction of the conformational space accessible to the basic region [8,196]. Thus, the induced folding is usually explained by loss of conformational entropy (from the unbound IDP state) upon target binding and compensatory favorable contributions from reduction in exposed hydrophobic surfaces and enhanced electrostatic interactions [196]. Exhaustive kinetic studies are necessary for the investigation on what order the events of binding and folding occur, and segregate

them in schemes of induced fit (IF, when the IDP binds to a partner and then folds) or conformational selection (CS, when the partner only binds to IDPs in a certain conformation) [197]. However, trying to define the coupled folding and binding in two categories may offer a rather simplistic explanation for the IDPs behavior, since the mechanisms can overlap depending upon the system conditions [197,198].
