István Simon

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.

PDB_TM: selection and membrane localization of transmembrane proteins in the protein data bank

PDB_TM is a database for transmembrane proteins with known structures. It aims to collect all transmembrane proteins that are deposited in the protein structure database (PDB) and to determine their membrane-spanning regions. These assignments are based on the TMDET algorithm, which uses only structural information to locate the most likely position of the lipid bilayer and to distinguish between transmembrane and globular proteins. This algorithm was applied to all PDB entries and the results were collected in the PDB_TM database. By using TMDET algorithm, the PDB_TM database can be automatically updated every week, keeping it synchronized with the latest PDB updates. The PDB_TM database is available at http://www.enzim.hu/PDB_TM.

Close encounters of the third kind: disordered domains and the interactions of proteins

Protein–protein interactions are thought to be mediated by domains, which are autonomous folding units of proteins. Recently, a second type of interaction has been suggested, mediated by short segments termed linear motifs, which are related to recognition elements of intrinsically disordered regions. Here, we propose a third kind of protein–protein recognition mechanism, mediated by disordered regions longer than 20–30 residues. Bioinformatics predictions and well‐characterized examples, such as the kinase‐inhibitory domain of Cdk inhibitors and the Wiskott–Aldrich syndrome protein (WASP)‐homology domain 2 of actin‐binding proteins, show that these disordered regions conform to the definition of domains rather than motifs, i.e., they represent functional, evolutionary, and structural units. Their functions are distinct from those of short motifs and ordered domains, and establish a third kind of interaction principle. With these points, we argue that these long disordered regions should be recognized as a distinct class of biologically functional protein domains.

Malleable machines take shape in eukaryotic transcriptional regulation.

Transcriptional control requires the spatially and temporally coordinated action of many macromolecular complexes. Chromosomal proteins, transcription factors, co-activators and components of the general transcription machinery, including RNA polymerases, often use structurally or stoichiometrically ill-defined regions for interactions that convey regulatory information in processes ranging from chromatin remodeling to mRNA processing. Determining the functional significance of intrinsically disordered protein regions and developing conceptual models of their action will help to illuminate their key role in transcription regulation. Complexes comprising disordered regions often display short recognition elements embedded in flexible and sequentially variable environments that can lead to structural and functional malleability. This provides versatility to recognize multiple targets having different structures, facilitate conformational rearrangements and physically communicate with many partners in response to environmental changes. All these features expand the capacities of ordered complexes and give rise to efficient regulatory mechanisms.

Rod models of DNA: sequence-dependent anisotropic elastic modelling of local bending phenomena

Local bending phenomena can be predicted by elastic models that incorporate sequence-dependent anisotropic-bendability (SDAB). SDAB models consider DNA to be an initially straight, segmented, elastic rod, in which the flexibility of each segment is greater towards the major groove than it is in other directions. While local bending can be predicted by static-geometry models as well, SDAB models, in addition, qualitatively explain such phenomena as the affinity of protein binding and kinking. A set of prediction tools is available at http:/(/)www.icgeb.trieste.it/dna.

PDBTM: Protein Data Bank of transmembrane proteins after 8 years

The PDBTM database (available at http://pdbtm.enzim.hu), the first comprehensive and up-to-date transmembrane protein selection of the Protein Data Bank, was launched in 2004. The database was created and has been continuously updated by the TMDET algorithm that is able to distinguish between transmembrane and non-transmembrane proteins using their 3D atomic coordinates only. The TMDET algorithm can locate the spatial positions of transmembrane proteins in lipid bilayer as well. During the last 8 years not only the size of the PDBTM database has been steadily growing from ∼400 to 1700 entries but also new structural elements have been identified, in addition to the well-known α-helical bundle and β-barrel structures. Numerous ‘exotic’ transmembrane protein structures have been solved since the first release, which has made it necessary to define these new structural elements, such as membrane loops or interfacial helices in the database. This article reports the new features of the PDBTM database that have been added since its first release, and our current efforts to keep the database up-to-date and easy to use so that it may continue to serve as a fundamental resource for the scientific community.

BiSearch: primer-design and search tool for PCR on bisulfite-treated genomes

Bisulfite genomic sequencing is the most widely used technique to analyze the 5-methylation of cytosines, the prevalent covalent DNA modification in mammals. The process is based on the selective transformation of unmethylated cytosines to uridines. Then, the investigated genomic regions are PCR amplified, subcloned and sequenced. During sequencing, the initially unmethylated cytosines are detected as thymines. The efficacy of bisulfite PCR is generally low; mispriming and non-specific amplification often occurs due to the T richness of the target sequences. In order to ameliorate the efficiency of PCR, we developed a new primer-design software called BiSearch, available on the World Wide Web. It has the unique property of analyzing the primer pairs for mispriming sites on the bisulfite-treated genome and determines potential non-specific amplification products with a new search algorithm. The options of primer-design and analysis for mispriming sites can be used sequentially or separately, both on bisulfite-treated and untreated sequences. In silico and in vitro tests of the software suggest that new PCR strategies may increase the efficiency of the amplification.

The role of hydrophobic microenvironments in modulating pKa shifts in proteins

The screened Coulomb potential (SCP) method, combined with a quantitative description of the microenvironments around titratable groups, based on the Hydrophobic Fragmental Constants developed by Rekker, has been applied to calculate the pKa values of groups embedded in extremely hydrophobic microenvironments in proteins. This type of microenvironment is not common; but constitutes a small class, where the proteins architecture has evolved to lend special properties to the embedded residue. They are of significant interest because they are frequently important in catalysis and in proton and electron transfer reactions. In the SCP treatment these special cases are treated locally and therefore do not affect the accuracy of the pKa values calculated for other residues in less hydrophobic environments. Here the calibration of the algorithm is extended with the help of earlier results from lysozyme and of three mutants of staphylococcal nuclease (SNase) that were specially designed to measure the energetics of ionization of titratable groups buried in extremely hydrophobic microenvironments. The calibrated algorithm was subsequently applied to a fourth mutant of SNase and then to a very large dimeric amine oxidase of 1284 residues, where 334 are titratable. The observed pKa shifts of the buried residues are large (up to 4.7 pK units), and all cases are well reproduced by the calculations with a root mean square error of 0.22. These results support the hypothesis that protein electrostatics can only be described correctly and self‐consistently if the inherent heterogeneity of these systems is properly accounted for. Proteins 2002;48:283–292.

Dynamic protein–DNA recognition: beyond what can be seen

Traditionally, specific DNA recognition is thought to rely on static contacts with the bases or phosphates. Recent results, however, indicate that residues far outside the binding context can crucially influence selectivity or binding affinity via transient, dynamic interactions with the DNA binding interface. These regions usually do not adopt a well-defined structure, even when bound to DNA, and thus form a fuzzy complex. Here, we propose the existence of a dynamic DNA readout mechanism, wherein distant segments modulate conformational preferences, flexibility or spacing of the DNA binding motifs or serve as competitive partners. Despite their low sequence similarity, these intrinsically disordered regions are often conserved at the structural level, and exploited for regulation of the transcription machinery via protein-protein interactions, post-translational modifications or alternative splicing.