Cross-Linked α-Synuclein as Inhibitor of Amyloid Formation.

The aggregation and amyloid formation of α-synuclein is associated with Parkinson's disease and other synucleinopathies. In its native, monomeric form α-synuclein is an intrinsically disordered protein represented by highly dynamic conformational ensembles. Inhibition of α-synuclein aggregation using small molecules, peptides, or proteins has been at the center of interest in recent years. Our aim was to explore the effects of cross-linking on the structure and aggregation/amyloid formation properties of α-synuclein. Comparative analysis of available high-resolution amyloid structures and representative structural models and MD trajectory of monomeric α-synuclein revealed that potential cross-links in the monomeric protein are mostly incompatible with the amyloid forms and thus might inhibit fibrillation. Monomeric α-synuclein has been intramolecularly chemically cross-linked under various conditions using different cross-linkers. We determined the location of cross-links and their frequency using mass spectrometry and found that most of them cannot be realized in the amyloid structures. The inhibitory potential of cross-linked proteins has been experimentally investigated using various methods, including thioflavin-T fluorescence and transmission electron microscopy. We found that conformational constraints applied by cross-linking fully blocked α-synuclein amyloid formation. Moreover, DTSSP-cross-linked molecules exhibited an inhibitory effect on the aggregation of unmodified α-synuclein as well.

BeStSel: webserver for secondary structure and fold prediction for protein CD spectroscopy.

Circular dichroism (CD) spectroscopy is widely used to characterize the secondary structure composition of proteins. To derive accurate and detailed structural information from the CD spectra, we have developed the Beta Structure Selection (BeStSel) method (PNAS, 112, E3095), which can handle the spectral diversity of ß-structured proteins. The BeStSel webserver provides this method with useful accessories to the community with the main goal to analyze single or multiple protein CD spectra. Uniquely, BeStSel provides information on eight secondary structure components including parallel ß-structure and antiparallel ß-sheets with three different groups of twist. It overperforms any available method in accuracy and information content, moreover, it is capable of predicting the protein fold down to the topology/homology level of the CATH classification. A new module of the webserver helps to distinguish intrinsically disordered proteins by their CD spectrum. Secondary structure calculation for uploaded PDB files will help the experimental verification of protein MD and in silico modelling using CD spectroscopy. The server also calculates extinction coefficients from the primary sequence for CD users to determine the accurate protein concentrations which is a prerequisite for reliable secondary structure determination. The BeStSel server can be freely accessed at https://bestsel.elte.hu.

Disordered-Ordered Protein Binary Classification by Circular Dichroism Spectroscopy.

Intrinsically disordered proteins lack a stable tertiary structure and form dynamic conformational ensembles due to their characteristic physicochemical properties and amino acid composition. They are abundant in nature and responsible for a large variety of cellular functions. While numerous bioinformatics tools have been developed for in silico disorder prediction in the last decades, there is a need for experimental methods to verify the disordered state. CD spectroscopy is widely used for protein secondary structure analysis. It is usable in a wide concentration range under various buffer conditions. Even without providing high-resolution information, it is especially useful when NMR, X-ray, or other techniques are problematic or one simply needs a fast technique to verify the structure of proteins. Here, we propose an automatized binary disorder-order classification method by analyzing far-UV CD spectroscopy data. The method needs CD data at only three wavelength points, making high-throughput data collection possible. The mathematical analysis applies the k-nearest neighbor algorithm with cosine distance function, which is independent of the spectral amplitude and thus free of concentration determination errors. Moreover, the method can be used even for strong absorbing samples, such as the case of crowded environmental conditions, if the spectrum can be recorded down to the wavelength of 212 nm. We believe the classification method will be useful in identifying disorder and will also facilitate the growth of experimental data in IDP databases. The method is implemented on a webserver and freely available for academic users.

Cellular Chaperone Function of Intrinsically Disordered Dehydrin ERD14.

Disordered plant chaperones play key roles in helping plants survive in harsh conditions, and they are indispensable for seeds to remain viable. Aside from well-known and thoroughly characterized globular chaperone proteins, there are a number of intrinsically disordered proteins (IDPs) that can also serve as highly effective protecting agents in the cells. One of the largest groups of disordered chaperones is the group of dehydrins, proteins that are expressed at high levels under different abiotic stress conditions, such as drought, high temperature, or osmotic stress. Dehydrins are characterized by the presence of different conserved sequence motifs that also serve as the basis for their categorization. Despite their accepted importance, the exact role and relevance of the conserved regions have not yet been formally addressed. Here, we explored the involvement of each conserved segment in the protective function of the intrinsically disordered stress protein (IDSP) A. thaliana's Early Response to Dehydration (ERD14). We show that segments that are directly involved in partner binding, and others that are not, are equally necessary for proper function and that cellular protection emerges from the balanced interplay of different regions of ERD14.

Interplay of Structural Disorder and Short Binding Elements in the Cellular Chaperone Function of Plant Dehydrin ERD14.

Details of the functional mechanisms of intrinsically disordered proteins (IDPs) in living cells is an area not frequently investigated. Here, we dissect the molecular mechanism of action of an IDP in cells by detailed structural analyses based on an in-cell nuclear magnetic resonance experiment. We show that the ID stress protein (IDSP) A. thaliana Early Response to Dehydration (ERD14) is capable of protecting E. coli cells under heat stress. The overexpression of ERD14 increases the viability of E. coli cells from 38.9% to 73.9% following heat stress (50 °C × 15 min). We also provide evidence that the protection is mainly achieved by protecting the proteome of the cells. In-cell NMR experiments performed in E. coli cells show that the protective activity is associated with a largely disordered structural state with conserved, short sequence motifs (K- and H-segments), which transiently sample helical conformations in vitro and engage in partner binding in vivo. Other regions of the protein, such as its S segment and its regions linking and flanking the binding motifs, remain unbound and disordered in the cell. Our data suggest that the cellular function of ERD14 is compatible with its residual structural disorder in vivo.

PhaSePro: the database of proteins driving liquid-liquid phase separation.

Membraneless organelles (MOs) are dynamic liquid condensates that host a variety of specific cellular processes, such as ribosome biogenesis or RNA degradation. MOs form through liquid-liquid phase separation (LLPS), a process that relies on multivalent weak interactions of the constituent proteins and other macromolecules. Since the first discoveries of certain proteins being able to drive LLPS, it emerged as a general mechanism for the effective organization of cellular space that is exploited in all kingdoms of life. While numerous experimental studies report novel cases, the computational identification of LLPS drivers is lagging behind, and many open questions remain about the sequence determinants, composition, regulation and biological relevance of the resulting condensates. Our limited ability to overcome these issues is largely due to the lack of a dedicated LLPS database. Therefore, here we introduce PhaSePro (https://phasepro.elte.hu), an openly accessible, comprehensive, manually curated database of experimentally validated LLPS driver proteins/protein regions. It not only provides a wealth of information on such systems, but improves the standardization of data by introducing novel LLPS-specific controlled vocabularies. PhaSePro can be accessed through an appealing, user-friendly interface and thus has definite potential to become the central resource in this dynamically developing field.

DisProt: intrinsic protein disorder annotation in 2020.

The Database of Protein Disorder (DisProt, URL: https://disprot.org) provides manually curated annotations of intrinsically disordered proteins from the literature. Here we report recent developments with DisProt (version 8), including the doubling of protein entries, a new disorder ontology, improvements of the annotation format and a completely new website. The website includes a redesigned graphical interface, a better search engine, a clearer API for programmatic access and a new annotation interface that integrates text mining technologies. The new entry format provides a greater flexibility, simplifies maintenance and allows the capture of more information from the literature. The new disorder ontology has been formalized and made interoperable by adopting the OWL format, as well as its structure and term definitions have been improved. The new annotation interface has made the curation process faster and more effective. We recently showed that new DisProt annotations can be effectively used to train and validate disorder predictors. We believe the growth of DisProt will accelerate, contributing to the improvement of function and disorder predictors and therefore to illuminate the 'dark' proteome.

Intrinsically Disordered Linkers Impart Processivity on Enzymes by Spatial Confinement of Binding Domains.

(1) Background: Processivity is common among enzymes and mechanochemical motors that synthesize, degrade, modify or move along polymeric substrates, such as DNA, RNA, polysaccharides or proteins. Processive enzymes can make multiple rounds of modification without releasing the substrate/partner, making their operation extremely effective and economical. The molecular mechanism of processivity is rather well understood in cases when the enzyme structurally confines the substrate, such as the DNA replication factor PCNA, and also when ATP energy is used to confine the succession of molecular events, such as with mechanochemical motors. Processivity may also result from the kinetic bias of binding imposed by spatial confinement of two binding elements connected by an intrinsically disordered (ID) linker. (2) Method: By statistical physical modeling, we show that this arrangement results in processive systems, in which the linker ensures an optimized effective concentration around novel binding site(s), favoring rebinding over full release of the polymeric partner. (3) Results: By analyzing 12 such proteins, such as cellulase, and RNAse-H, we illustrate that in these proteins linker length and flexibility, and the kinetic parameters of binding elements, are fine-tuned for optimizing processivity. We also report a conservation of structural disorder, special amino acid composition of linkers, and the correlation of their length with step size. (4) Conclusion: These observations suggest a unique type of entropic chain function of ID proteins, that may impart functional advantages on diverse enzymes in a variety of biological contexts.

Disordered Regions of Mixed Lineage Leukemia 4 (MLL4) Protein Are Capable of RNA Binding.

Long non-coding RNAs (lncRNAs) are emerging as important regulators of cellular processes and are extensively involved in the development of different cancers; including leukemias. As one of the accepted methods of lncRNA function is affecting chromatin structure; lncRNA binding has been shown for different chromatin modifiers. Histone lysine methyltransferases (HKMTs) are also subject of lncRNA regulation as demonstrated for example in the case of Polycomb Repressive Complex 2 (PRC2). Mixed Lineage Leukemia (MLL) proteins that catalyze the methylation of H3K4 have been implicated in several different cancers; yet many details of their regulation and targeting remain elusive. In this work we explored the RNA binding capability of two; so far uncharacterized regions of MLL4; with the aim of shedding light to the existence of possible regulatory lncRNA interactions of the protein. We demonstrated that both regions; one that contains a predicted RNA binding sequence and one that does not; are capable of binding to different RNA constructs in vitro. To our knowledge, these findings are the first to indicate that an MLL protein itself is capable of lncRNA binding.

DisProt 7.0: a major update of the database of disordered proteins.

The Database of Protein Disorder (DisProt, URL: www.disprot.org) has been significantly updated and upgraded since its last major renewal in 2007. The current release holds information on more than 800 entries of IDPs/IDRs, i.e. intrinsically disordered proteins or regions that exist and function without a well-defined three-dimensional structure. We have re-curated previous entries to purge DisProt from conflicting cases, and also upgraded the functional classification scheme to reflect continuous advance in the field in the past 10 years or so. We define IDPs as proteins that are disordered along their entire sequence, i.e. entirely lack structural elements, and IDRs as regions that are at least five consecutive residues without well-defined structure. We base our assessment of disorder strictly on experimental evidence, such as X-ray crystallography and nuclear magnetic resonance (primary techniques) and a broad range of other experimental approaches (secondary techniques). Confident and ambiguous annotations are highlighted separately. DisProt 7.0 presents classified knowledge regarding the experimental characterization and functional annotations of IDPs/IDRs, and is intended to provide an invaluable resource for the research community for a better understanding structural disorder and for developing better computational tools for studying disordered proteins.