Intrinsically unstructured/disordered proteins and domains (IUPs) lack a well-defined three-dimensional structure under native conditions. The IUPred server presents a novel algorithm for predicting such regions from amino acid sequences by estimating their total pairwise interresidue interaction energy, based on the assumption that IUP sequences do not fold due to their inability to form sufficient stabilizing interresidue interactions. Optional to the prediction are built-in parameter sets optimized for predicting short or long disordered regions and structured domains.

The structural states of proteins include ordered globular domains as well as intrinsically disordered protein regions that exist as highly flexible conformational ensembles in isolation. Various computational tools have been developed to discriminate ordered and disordered segments based on the amino acid sequence. However, properties of IDRs can also depend on various conditions, including binding to globular protein partners or environmental factors, such as redox potential. These cases provide further challenges for the computational characterization of disordered segments. In this work we present IUPred2A, a combined web interface that allows to generate energy estimation based predictions for ordered and disordered residues by IUPred2 and for disordered binding regions by ANCHOR2. The updated web server retains the robustness of the original programs but offers several new features. While only minor bug fixes are implemented for IUPred, the next version of ANCHOR is significantly improved through a new architecture and parameters optimized on novel datasets. In addition, redox-sensitive regions can also be highlighted through a novel experimental feature. The web server offers graphical and text outputs, a RESTful interface, access to software download and extensive help, and can be accessed at a new location: http://iupred2a.elte.hu.

Intrinsically disordered proteins and protein regions (IDPs/IDRs) exist without a single well-defined conformation. They carry out important biological functions with multifaceted roles which is also reflected in their evolutionary behavior. Computational methods play important roles in the characterization of IDRs. One of the commonly used disorder prediction methods is IUPred, which relies on an energy estimation approach. The IUPred web server takes an amino acid sequence or a Uniprot ID/accession as an input and predicts the tendency for each amino acid to be in a disordered region with an option to also predict context-dependent disordered regions. In this new iteration of IUPred, we added multiple novel features to enhance the prediction capabilities of the server. First, learning from the latest evaluation of disorder prediction methods we introduced multiple new smoothing functions to the prediction that decreases noise and increases the performance of the predictions. We constructed a dataset consisting of experimentally verified ordered/disordered regions with unambiguous annotations which were added to the prediction. We also introduced a novel tool that enables the exploration of the evolutionary conservation of protein disorder coupled to sequence conservation in model organisms. The web server is freely available to users and accessible at https://iupred3.elte.hu.

Many disordered proteins function via binding to a structured partner and undergo a disorder-to-order transition. The coupled folding and binding can confer several functional advantages such as the precise control of binding specificity without increased affinity. Additionally, the inherent flexibility allows the binding site to adopt various conformations and to bind to multiple partners. These features explain the prevalence of such binding elements in signaling and regulatory processes. In this work, we report ANCHOR, a method for the prediction of disordered binding regions. ANCHOR relies on the pairwise energy estimation approach that is the basis of IUPred, a previous general disorder prediction method. In order to predict disordered binding regions, we seek to identify segments that are in disordered regions, cannot form enough favorable intrachain interactions to fold on their own, and are likely to gain stabilizing energy by interacting with a globular protein partner. The performance of ANCHOR was found to be largely independent from the amino acid composition and adopted secondary structure. Longer binding sites generally were predicted to be segmented, in agreement with available experimentally characterized examples. Scanning several hundred proteomes showed that the occurrence of disordered binding sites increased with the complexity of the organisms even compared to disordered regions in general. Furthermore, the length distribution of binding sites was different from disordered protein regions in general and was dominated by shorter segments. These results underline the importance of disordered proteins and protein segments in establishing new binding regions. Due to their specific biophysical properties, disordered binding sites generally carry a robust sequence signal, and this signal is efficiently captured by our method. Through its generality, ANCHOR opens new ways to study the essential functional sites of disordered proteins. Intrinsically unstructured/disordered proteins (IUPs/IDPs) do not adopt a stable structure in isolation but exist as a highly flexible ensemble of conformations. Despite the lack of a well-defined structure these proteins carry out important functions. Many IUPs/IDPs function via binding specifically to other macromolecules that involves a disorder-to-order transition. The molecular recognition functions of IUPs/IDPs include regulatory and signaling interactions where binding to multiple partners and high-specificity/low-affinity interactions play a crucial role. Due to their specific functional and structural properties, these binding regions have distinct properties compared to both globular proteins and disordered regions in general. Here, we present a general method to identify disordered binding regions from the amino acid sequence. Our method targets the essential feature of these regions: they behave in a characteristically different manner in isolation than bound to their partner protein. This prediction method allows us to compare the binding properties of short and long binding sites. The evolutionary relationship between the amount of disordered binding regions and general disordered regions in various organisms was also analyzed. Our results suggest that disordered binding regions can be recognized even without taking into account their adopted secondary structure or their specific binding partner.

Summary: ANCHOR is a web-based implementation of an original method that takes a single amino acid sequence as an input and predicts protein binding regions that are disordered in isolation but can undergo disorder-to-order transition upon binding. The server incorporates the result of a general disorder prediction method, IUPred and can carry out simple motif searches as well. Availability: The web server is available at http://anchor.enzim.hu. The program package is freely available for academic users. Contact: zsuzsa@enzim.hu