Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their functionality through a quantitative description of the evolutionary conservation of disordered segments involved in binding, and investigated the structural implications of flexibility in terms of conformational stability and interface formation. We conclude that the functional role of intrinsically disordered protein segments in RNA-binding is two-fold: first, these regions establish extended, conserved electrostatic interfaces with RNAs via induced fit. Second, conformational flexibility enables them to target different RNA partners, providing multi-functionality, while also ensuring specificity. These findings emphasize the functional importance of intrinsically disordered regions in RNA-binding proteins.

Edited by Wilhelm JustProteins may undergo adaptive structural transitions to accommodate to their cellular milieu and respond to external signals. Modulation of conformational ensembles can rewire the intra-or intermolecular interaction networks and shift between different functional states. Adaptive conformational transitions are associated with protein fuzziness, which enables (a) rewiring interaction networks via alternative motifs, (b) new functional features via allosteric motifs, (c) functional switches upon post-translational modifications, or (d) regulation of higher-order organizations. We propose that all these contextdependent functional changes are intertwined with structural multiplicity or dynamic disorder in protein assemblies and can only be described by stochastic structure-function relationships.

BackgroundAnalyzing the amino acid sequence of an intrinsically disordered protein (IDP) in an evolutionary context can yield novel insights on the functional role of disordered regions and sequence element(s). However, in the case of many IDPs, the lack of evolutionary conservation of the primary sequence can hamper the study of functionality, because the conservation of their disorder profile and ensuing function(s) may not appear in a traditional analysis of the evolutionary history of the protein.ResultsHere we present DisCons (Disorder Conservation), a novel pipelined tool that combines the quantification of sequence- and disorder conservation to classify disordered residue positions. According to this scheme, the most interesting categories (for functional purposes) are constrained disordered residues and flexible disordered residues. The former residues show conservation of both the sequence and the property of disorder and are associated mainly with specific binding functionalities (e.g., short, linear motifs, SLiMs), whereas the latter class correspond to segments where disorder as a feature is important for function as opposed to the identity of the underlying sequence (e.g., entropic chains and linkers). DisCons therefore helps with elucidating the function(s) arising from the disordered state by analyzing individual proteins as well as large-scale proteomics datasets.ConclusionsDisCons is an openly accessible sequence analysis tool that identifies and highlights structurally disordered segments of proteins where the conformational flexibility is conserved across homologs, and therefore potentially functional. The tool is freely available both as a web application and as stand-alone source code hosted at http://pedb.vib.be/discons.

Although improved strategies for the detection and analysis of evolutionary couplings (ECs) between protein residues already enable the prediction of protein structures and interactions, they are mostly restricted to conserved and well-folded proteins. Whereas intrinsically disordered proteins (IDPs) are central to cellular interaction networks, due to the lack of strict structural constraints, they undergo faster evolutionary changes than folded domains. This makes the reliable identification and alignment of IDP homologs difficult, which led to IDPs being omitted in most large-scale residue co-variation analyses. By preforming a dedicated analysis of phylogenetically widespread bacterial IDP–partner interactions, here we demonstrate that partner binding imposes constraints on IDP sequences that manifest in detectable interprotein ECs. These ECs were not detected for interactions mediated by short motifs, rather for those with larger IDP–partner interfaces. Most identified coupled residue pairs reside close (<10 Å) to each other on the interface, with a third of them forming multiple direct atomic contacts. EC-carrying interfaces of IDPs are enriched in negatively charged residues, and the EC residues of both IDPs and partners preferentially reside in helices. Our analysis brings hope that IDP–partner interactions difficult to study could soon be successfully dissected through residue co-variation analysis.