Bálint Mészáros

ANCHOR: web server for predicting protein binding regions in disordered proteins

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

Prediction of Protein Binding Regions in Disordered Proteins

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.

Bioinformatical approaches to characterize intrinsically disordered/unstructured proteins

Intrinsically disordered/unstructured proteins exist without a stable three-dimensional (3D) structure as highly flexible conformational ensembles. The available genome sequences revealed that these proteins are surprisingly common and their frequency reaches high proportions in eukaryotes. Due to their vital role in various biological processes including signaling and regulation and their involvement in various diseases, disordered proteins and protein segments are the focus of many biochemical, molecular biological, pathological and pharmaceutical studies. These proteins are difficult to study experimentally because of the lack of unique structure in the isolated form. Their amino acid sequence, however, is available, and can be used for their identification and characterization by bioinformatic tools, analogously to globular proteins. In this review, we first present a small survey of current methods to identify disordered proteins or protein segments, focusing on those that are publicly available as web servers. In more detail we also discuss approaches that predict disordered regions and specific regions involved in protein binding by modeling the physical background of protein disorder. In our review we argue that the heterogeneity of disordered segments needs to be taken into account for a better understanding of protein disorder.

Disordered binding regions and linear motifs--bridging the gap between two models of molecular recognition.

Intrinsically disordered proteins (IDPs) exist without the presence of a stable tertiary structure in isolation. These proteins are often involved in molecular recognition processes via their disordered binding regions that can recognize partner molecules by undergoing a coupled folding and binding process. The specific properties of disordered binding regions give way to specific, yet transient interactions that enable IDPs to play central roles in signaling pathways and act as hubs of protein interaction networks. An alternative model of protein-protein interactions with largely overlapping functional properties is offered by the concept of linear interaction motifs. This approach focuses on distilling a short consensus sequence pattern from proteins with a common interaction partner. These motifs often reside in disordered regions and are considered to mediate the interaction roughly independent from the rest of the protein. Although a connection between linear motifs and disordered binding regions has been established through common examples, the complementary nature of the two concepts has yet to be fully explored. In many cases the sequence based definition of linear motifs and the structural context based definition of disordered binding regions describe two aspects of the same phenomenon. To gain insight into the connection between the two models, prediction methods were utilized. We combined the regular expression based prediction of linear motifs with the disordered binding region prediction method ANCHOR, each specialized for either model to get the best of both worlds. The thorough analysis of the overlap of the two methods offers a bioinformatics tool for more efficient binding site prediction that can serve a wide range of practical implications. At the same time it can also shed light on the theoretical connection between the two co-existing interaction models.

Proteins with complex architecture as potential targets for drug design: a case study of Mycobacterium tuberculosis.

Lengthy co-evolution of Homo sapiens and Mycobacterium tuberculosis, the main causative agent of tuberculosis, resulted in a dramatically successful pathogen species that presents considerable challenge for modern medicine. The continuous and ever increasing appearance of multi-drug resistant mycobacteria necessitates the identification of novel drug targets and drugs with new mechanisms of action. However, further insights are needed to establish automated protocols for target selection based on the available complete genome sequences. In the present study, we perform complete proteome level comparisons between M. tuberculosis, mycobacteria, other prokaryotes and available eukaryotes based on protein domains, local sequence similarities and protein disorder. We show that the enrichment of certain domains in the genome can indicate an important function specific to M. tuberculosis. We identified two families, termed pkn and PE/PPE that stand out in this respect. The common property of these two protein families is a complex domain organization that combines species-specific regions, commonly occurring domains and disordered segments. Besides highlighting promising novel drug target candidates in M. tuberculosis, the presented analysis can also be viewed as a general protocol to identify proteins involved in species-specific functions in a given organism. We conclude that target selection protocols should be extended to include proteins with complex domain architectures instead of focusing on sequentially unique and essential proteins only.

Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases

Mitogen‐activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less‐characterized disordered regions. We used a structurally consistent model on kinase‐docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under‐explored part of the human proteome and applied experimental tools specifically tailored to detect low‐affinity protein–protein interactions for their validation in vitro and in cell‐based assays. The combined computational and experimental approach enabled the identification of many novel MAPK‐docking motifs that were elusive for other large‐scale protein–protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase‐mediated partnerships evolved over time. The analysis suggests that most human MAPK‐binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK‐binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.

Degrons in cancer

Understanding protein degradation signals and systems reveals targets for cancer therapy. Gloss There are many cellular proteins that need to be eliminated quickly in response to changing conditions in or around the cells. Most of these proteins carry a short functional element in their sequence, called a degron. Degrons are typically composed of 6 to 10 amino acids and are generally located within flexible regions of proteins so that the degrons can easily interact with other proteins. E3 ubiquitin ligases bind to specific degrons, enabling the attachment of multiple copies of ubiquitin to target proteins. The ubiquitin chains are a molecular signal that directs the proteins to the proteasome, where the tagged proteins are chopped up into pieces and recycled. The correct removal of proteins is important for many biological processes, such as regulating transcription and controlling the major steps during cell division. Regulated protein degradation also turns off the activity of some proteins that are activated by transient external signals. The encounter between the E3 ligase and the degron determines whether a protein lives or dies. There are ~600 different E3 ligases that are encoded in the human genome. Each of these E3 ligases targets a different set of proteins and operates under a different condition. This ubiquitin-mediated protein degradation process is regulated at multiple levels. Defects in this system can cause systemic diseases, including cancer. This Review with 8 figures, 1 table, and 360 references describes how mutations that affect ubiquitin-mediated protein degradation system contribute to cancer. Degrons are the elements that are used by E3 ubiquitin ligases to target proteins for degradation. Most degrons are short linear motifs embedded within the sequences of modular proteins. As regulatory sites for protein abundance, they are important for many different cellular processes, such as progression through the cell cycle and monitoring cellular hypoxia. Degrons enable the elimination of proteins that are no longer required, preventing their possible dysfunction. Although the human genome encodes ~600 E3 ubiquitin ligases, only a fraction of these enzymes have well-defined target degrons. Thus, for most cellular proteins, the destruction mechanisms are poorly understood. This is important for many diseases, especially for cancer, a disease that involves the enhanced expression of oncogenes and the persistence of encoded oncoproteins coupled with reduced abundance of tumor suppressors. Loss-of-function mutations occur in the degrons of several oncoproteins, such as the transcription factors MYC and NRF2, and in various mitogenic receptors, such as NOTCH1 and several receptor tyrosine kinases. Mutations eliminating the function of the β-catenin degron are found in many cancers and are considered one of the most abundant mutations driving carcinogenesis. In this Review, we describe the current knowledge of degrons in cancer and suggest that increased research on the “dark degrome” (unknown degron-E3 relationships) would enhance progress in cancer research.

Assessing Conservation of Disordered Regions in Proteins

Intrinsically disordered regions (IDRs) are highly populated in eukaryotic proteomes and serve pivotal, mostly regulatory functions. Many IDRs appear to be functionally conserved and analysis of protein domains indicates high pro- pensity of conserved regions predicted to be disordered. Nevertheless, it is difficult to assess conservation of IDRs in gen- eral due to their fast evolution and low sequence similarity. We propose three measures to evaluate conservation of IDRs: i) similarities of the disorder profiles using different prediction conditions; ii) the conservation of amino acids with pro- pensities for promoting either disorder or order; and iii) the overlap between disordered/ordered regions. These measures are computed on multiple sequence alignments that also include low-complexity regions of proteins. Using three subunits of the Mediator complex of transcription regulation from Homo sapiens and Drosophila melanogaster as an example we show that despite of their sequence dissimilarity IDRs can be conserved and likely carry out the same function in different organisms.

MobiDB 3.0: More annotations for intrinsic disorder, conformational diversity and interactions in proteins

Abstract The MobiDB (URL: mobidb.bio.unipd.it) database of protein disorder and mobility annotations has been significantly updated and upgraded since its last major renewal in 2014. Several curated datasets for intrinsic disorder and folding upon binding have been integrated from specialized databases. The indirect evidence has also been expanded to better capture information available in the PDB, such as high temperature residues in X-ray structures and overall conformational diversity. Novel nuclear magnetic resonance chemical shift data provides an additional experimental information layer on conformational dynamics. Predictions have been expanded to provide new types of annotation on backbone rigidity, secondary structure preference and disordered binding regions. MobiDB 3.0 contains information for the complete UniProt protein set and synchronization has been improved by covering all UniParc sequences. An advanced search function allows the creation of a wide array of custom-made datasets for download and further analysis. A large amount of information and cross-links to more specialized databases are intended to make MobiDB the central resource for the scientific community working on protein intrinsic disorder and mobility.

DIBS: a repository of disordered binding sites mediating interactions with ordered proteins

Motivation Intrinsically Disordered Proteins (IDPs) mediate crucial protein‐protein interactions, most notably in signaling and regulation. As their importance is increasingly recognized, the detailed analyses of specific IDP interactions opened up new opportunities for therapeutic targeting. Yet, large scale information about IDP‐mediated interactions in structural and functional details are lacking, hindering the understanding of the mechanisms underlying this distinct binding mode. Results Here, we present DIBS, the first comprehensive, curated collection of complexes between IDPs and ordered proteins. DIBS not only describes by far the highest number of cases, it also provides the dissociation constants of their interactions, as well as the description of potential post‐translational modifications modulating the binding strength and linear motifs involved in the binding. Together with the wide range of structural and functional annotations, DIBS will provide the cornerstone for structural and functional studies of IDP complexes. Availability and implementation DIBS is freely accessible at http://dibs.enzim.ttk.mta.hu/. The DIBS application is hosted by Apache web server and was implemented in PHP. To enrich querying features and to enhance backend performance a MySQL database was also created.