The C-terminal self-binding helical peptide of human estrogen-related receptor γ can be druggably targeted by a novel class of rationally designed peptidic antagonists.

Orphan nuclear estrogen-related receptor γ (ERRγ) has been recognized as a potential therapeutic target for cancer, inflammation and metabolic disorder. The ERRγ contains a regulatory AF2 helical tail linked C-terminally to its ligand-binding domain (LBD), which is a self-binding peptide (SBP) and serves as molecular switch to dynamically regulate the receptor alternation between active and inactive states by binding to and unbinding from the AF2-binding site on ERRγ LBD surface, respectively. Traditional ERRγ modulators are all small-molecule chemical ligands that can be classified into agonists and inverse agonists in terms of their action mechanism; the agonists stabilize the AF2 in ABS site with an agonist conformation, while the inverse agonists lock the AF2 out of the site to largely abolish ERRγ transcriptional activity. Here, a class of ERRγ peptidic antagonists was described to compete with native AF2 for the ABS site, thus blocking the active state of AF2 binding to ERRγ LBD domain. Self-inhibitory peptide was derived from the SBP-covering AF2 region and we expected it can rebind potently to the ABS site by reducing its intrinsic disorder and entropy cost upon the rebinding. Hydrocarbon stapling was employed to do so, which employed an all-hydrocarbon bridge across the [i, i + 4]-anchor residue pair in the N-terminal, middle or C-terminal region of the self-inhibitory peptide. As might be expected, it is revealed that the stapled peptides are good binders of ERRγ LBD domain and can effectively compete with the native AF2 helical tail for ERRγ ABS site, which exhibit a basically similar binding mode with AF2 to the site and form diverse noncovalent interactions with the site, thus conferring stability and specificity to the domain-peptide complexes.

Methodological approaches to studying phase separation and HIV-1 replication: Current and future perspectives.

This article reviews tried-and-tested methodologies that have been employed in the first studies on phase separating properties of structural, RNA-binding and catalytic proteins of HIV-1. These are described here to stimulate interest for any who may want to initiate similar studies on virus-mediated liquid-liquid phase separation. Such studies serve to better understand the life cycle and pathogenesis of viruses and open the door to new therapeutics.

A suicidal and extensively disordered luciferase with a bright luminescence.

Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis, GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here, we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 Å and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate-binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase.

A Comparative Experimental and Computational Study on the Nature of the Pangolin-CoV and COVID-19 Omicron.

The relationship between pangolin-CoV and SARS-CoV-2 has been a subject of debate. Further evidence of a special relationship between the two viruses can be found by the fact that all known COVID-19 viruses have an abnormally hard outer shell (low M disorder, i.e., low content of intrinsically disordered residues in the membrane (M) protein) that so far has been found in CoVs associated with burrowing animals, such as rabbits and pangolins, in which transmission involves virus remaining in buried feces for a long time. While a hard outer shell is necessary for viral survival, a harder inner shell could also help. For this reason, the N disorder range of pangolin-CoVs, not bat-CoVs, more closely matches that of SARS-CoV-2, especially when Omicron is included. The low N disorder (i.e., low content of intrinsically disordered residues in the nucleocapsid (N) protein), first observed in pangolin-CoV-2017 and later in Omicron, is associated with attenuation according to the Shell-Disorder Model. Our experimental study revealed that pangolin-CoV-2017 and SARS-CoV-2 Omicron (XBB.1.16 subvariant) show similar attenuations with respect to viral growth and plaque formation. Subtle differences have been observed that are consistent with disorder-centric computational analysis.

Structural Properties of Rat Intestinal Fatty Acid-Binding Protein with its Dynamics: Insights into Intrinsic Disorder.

BACKGROUND: The rat intestinal fatty acid-binding protein (I-FABP) is expressed in the small intestine and is involved in the absorption and transport of dietary fatty acids. It is used as a marker for intestinal injury and is associated with various gastrointestinal disorders. I-FABP has been studied extensively using conventional experimental and computational techniques. However, the detection of intrinsically disordered regions requires the application of special sampling molecular dynamics simulations along with certain bioinformatics because conventional computational and experimental studies face challenges in identifying the features of intrinsic disorder. METHOD: Replica exchange molecular dynamics simulations were conducted along with bioinformatics studies to gain deeper insights into the structural properties of I-FABP. Specifically, the Cα and Hα chemical shift values werecalculated, and the findings were compared to the experiments. Furthermore, secondary and tertiary structure properties were also calculated, and the protein was clustered using k-means clustering. The end-to-end distance and radius of gyration values were reported for the protein in an aqueous solution medium. In addition, its disorder tendency was studied using various bioinformatics tools. RESULTS AND CONCLUSION: It was reported that I-FABP is a flexible protein with regions that demonstrate intrinsic disorder characteristics. This flexibility and intrinsic disorder characteristics of I-- FABP may be related to its nature in ligand binding processes.

When an underdog becomes a major player: the role of protein structural disorder in the Atg8 conjugation system.

The noncanonical ubiquitin-like conjugation cascade involving the E1 (Atg7), E2 (Atg3, Atg10), and E3 (Atg12-Atg5-Atg16 complex) enzymes is essential for incorporation of Atg8 into the growing phagophore via covalent linkage to PE. This process is an indispensable step in autophagy. Atg8 and E1-E3 enzymes are the first subset from the core autophagy protein machinery structures that were investigated in earlier studies by crystallographic analyses of globular domains. However, research over the past decade shows that many important functions in the conjugation machinery are mediated by intrinsically disordered protein regions (IDPRs) - parts of the protein that do not adopt a stable secondary or tertiary structure, which are inherently dynamic and well suited for protein-membrane interactions but are invisible in protein crystals. Here, we summarize earlier and recent findings on the autophagy conjugation machinery by focusing on the IDPRs. This summary reveals that IDPRs, originally considered dispensable, are in fact major players and a driving force in the function of the autophagy conjugation system. Abbreviation: AD, activation domain of Atg7; AH, amphipathic helix; AIM, Atg8-family interacting motif; CL, catalytic loop (of Atg7); CTD, C-terminal domain; FR, flexible region (of Atg3 or Atg10); GUV, giant unilammelar vesicles; HR, handle region (of Atg3); IDPR, intrinsically disordered protein region; IDPs: intrinsically disordered proteins; LIR, LC3-interacting region; NHD: N-terminal helical domain; NMR, nuclear magnetic resonance; PE, phosphatidylethanolamine; UBL, ubiquitin like.

Taxonomy-specific assessment of intrinsic disorder predictions at residue and region levels in higher eukaryotes, protists, archaea, bacteria and viruses.

Intrinsic disorder predictors were evaluated in several studies including the two large CAID experiments. However, these studies are biased towards eukaryotic proteins and focus primarily on the residue-level predictions. We provide first-of-its-kind assessment that comprehensively covers the taxonomy and evaluates predictions at the residue and disordered region levels. We curate a benchmark dataset that uniformly covers eukaryotic, archaeal, bacterial, and viral proteins. We find that predictive performance differs substantially across taxonomy, where viruses are predicted most accurately, followed by protists and higher eukaryotes, while bacterial and archaeal proteins suffer lower levels of accuracy. These trends are consistent across predictors. We also find that current tools, except for flDPnn, struggle with reproducing native distributions of the numbers and sizes of the disordered regions. Moreover, analysis of two variants of disorder predictions derived from the AlphaFold2 predicted structures reveals that they produce accurate residue-level propensities for archaea, bacteria and protists. However, they underperform for higher eukaryotes and generally struggle to accurately identify disordered regions. Our results motivate development of new predictors that target bacteria and archaea and which produce accurate results at both residue and region levels. We also stress the need to include the region-level assessments in future assessments.

Vitamin K Epoxide Reductase Complex-Protein Disulphide Isomerase Assemblies in the Thiol-Disulphide Exchange Reactions: Portrayal of Precursor-to-Successor Complexes.

The human Vitamin K Epoxide Reductase Complex (hVKORC1), a key enzyme that converts vitamin K into the form necessary for blood clotting, requires for its activation the reducing equivalents supplied by its redox partner through thiol-disulphide exchange reactions. The functionally related molecular complexes assembled during this process have never been described, except for a proposed de novo model of a 'precursor' complex of hVKORC1 associated with protein disulphide isomerase (PDI). Using numerical approaches (in silico modelling and molecular dynamics simulation), we generated alternative 3D models for each molecular complex bonded either covalently or non-covalently. These models differ in the orientation of the PDI relative to hVKORC1 and in the cysteine residue involved in forming protein-protein disulphide bonds. Based on a comparative analysis of these models' shape, folding, and conformational dynamics, the most probable putative complexes, mimicking the 'precursor', 'intermediate', and 'successor' states, were suggested. In addition, we propose using these complexes to develop the 'allo-network drugs' necessary for treating blood diseases.

Assessment of Disordered Linker Predictions in the CAID2 Experiment.

Disordered linkers (DLs) are intrinsically disordered regions that facilitate movement between adjacent functional regions/domains, contributing to many key cellular functions. The recently completed second Critical Assessments of protein Intrinsic Disorder prediction (CAID2) experiment evaluated DL predictions by considering a rather narrow scenario when predicting 40 proteins that are already known to have DLs. We expand this evaluation by using a much larger set of nearly 350 test proteins from CAID2 and by investigating three distinct scenarios: (1) prediction residues in DLs vs. in non-DL regions (typical use of DL predictors); (2) prediction of residues in DLs vs. other disordered residues (to evaluate whether predictors can differentiate residues in DLs from other types of intrinsically disordered residues); and (3) prediction of proteins harboring DLs. We find that several methods provide relatively accurate predictions of DLs in the first scenario. However, only one method, APOD, accurately identifies DLs among other types of disordered residues (scenario 2) and predicts proteins harboring DLs (scenario 3). We also find that APOD's predictive performance is modest, motivating further research into the development of new and more accurate DL predictors. We note that these efforts will benefit from a growing amount of training data and the availability of sophisticated deep network models and emphasize that future methods should provide accurate results across the three scenarios.

Protein ensemble modeling and analysis with MMMx.

Proteins, especially of eukaryotes, often have disordered domains and may contain multiple folded domains whose relative spatial arrangement is distributed. The MMMx ensemble modeling and analysis toolbox (https://github.com/gjeschke/MMMx) can support the design of experiments to characterize the distributed structure of such proteins, starting from AlphaFold2 predictions or folded domain structures. Weak order can be analyzed with reference to a random coil model or to peptide chains that match the residue-specific Ramachandran angle distribution of the loop regions and are otherwise unrestrained. The deviation of the mean square end-to-end distance of chain sections from their average over sections of the same sequence length reveals localized compaction or expansion of the chain. The shape sampled by disordered chains is visualized by superposition in the principal axes frame of their inertia tensor. Ensembles of different sizes and with weighted conformers can be compared based on a similarity parameter that abstracts from the ensemble width.

Synergy of Mutation-Induced Effects in Human Vitamin K Epoxide Reductase: Perspectives and Challenges for Allo-Network Modulator Design.

The human Vitamin K Epoxide Reductase Complex (hVKORC1), a key enzyme transforming vitamin K into the form necessary for blood clotting, requires for its activation the reducing equivalents delivered by its redox partner through thiol-disulfide exchange reactions. The luminal loop (L-loop) is the principal mediator of hVKORC1 activation, and it is a region frequently harbouring numerous missense mutations. Four L-loop hVKORC1 mutants, suggested in vitro as either resistant (A41S, H68Y) or completely inactive (S52W, W59R), were studied in the oxidised state by numerical approaches (in silico). The DYNASOME and POCKETOME of each mutant were characterised and compared to the native protein, recently described as a modular protein composed of the structurally stable transmembrane domain (TMD) and the intrinsically disordered L-loop, exhibiting quasi-independent dynamics. The DYNASOME of mutants revealed that L-loop missense point mutations impact not only its folding and dynamics, but also those of the TMD, highlighting a strong mutation-specific interdependence between these domains. Another consequence of the mutation-induced effects manifests in the global changes (geometric, topological, and probabilistic) of the newly detected cryptic pockets and the alternation of the recognition properties of the L-loop with its redox protein. Based on our results, we postulate that (i) intra-protein allosteric regulation and (ii) the inherent allosteric regulation and cryptic pockets of each mutant depend on its DYNASOME; and (iii) the recognition of the redox protein by hVKORC1 (INTERACTOME) depend on their DYNASOME. This multifaceted description of proteins produces "omics" data sets, crucial for understanding the physiological processes of proteins and the pathologies caused by alteration of the protein properties at various "omics" levels. Additionally, such characterisation opens novel perspectives for the development of "allo-network drugs" essential for the treatment of blood disorders.

Phosphorylation regulates viral biomolecular condensates to promote infectious progeny production.

Biomolecular condensates (BMCs) play important roles in diverse biological processes. Many viruses form BMCs which have been implicated in various functions critical for the productive infection of host cells. The adenovirus L1-52/55 kilodalton protein (52K) was recently shown to form viral BMCs that coordinate viral genome packaging and capsid assembly. Although critical for packaging, we do not know how viral condensates are regulated during adenovirus infection. Here we show that phosphorylation of serine residues 28 and 75 within the N-terminal intrinsically disordered region of 52K modulates viral condensates in vitro and in cells, promoting liquid-like properties. Furthermore, we demonstrate that phosphorylation of 52K promotes viral genome packaging and the production of infectious progeny particles. Collectively, our findings provide insights into how viral condensate properties are regulated and maintained in a state conducive to their function in viral progeny production. In addition, our findings have implications for antiviral strategies aimed at targeting the regulation of viral BMCs to limit viral multiplication.

DisoFLAG: accurate prediction of protein intrinsic disorder and its functions using graph-based interaction protein language model.

Intrinsically disordered proteins and regions (IDPs/IDRs) are functionally important proteins and regions that exist as highly dynamic conformations under natural physiological conditions. IDPs/IDRs exhibit a broad range of molecular functions, and their functions involve binding interactions with partners and remaining native structural flexibility. The rapid increase in the number of proteins in sequence databases and the diversity of disordered functions challenge existing computational methods for predicting protein intrinsic disorder and disordered functions. A disordered region interacts with different partners to perform multiple functions, and these disordered functions exhibit different dependencies and correlations. In this study, we introduce DisoFLAG, a computational method that leverages a graph-based interaction protein language model (GiPLM) for jointly predicting disorder and its multiple potential functions. GiPLM integrates protein semantic information based on pre-trained protein language models into graph-based interaction units to enhance the correlation of the semantic representation of multiple disordered functions. The DisoFLAG predictor takes amino acid sequences as the only inputs and provides predictions of intrinsic disorder and six disordered functions for proteins, including protein-binding, DNA-binding, RNA-binding, ion-binding, lipid-binding, and flexible linker. We evaluated the predictive performance of DisoFLAG following the Critical Assessment of protein Intrinsic Disorder (CAID) experiments, and the results demonstrated that DisoFLAG offers accurate and comprehensive predictions of disordered functions, extending the current coverage of computationally predicted disordered function categories. The standalone package and web server of DisoFLAG have been established to provide accurate prediction tools for intrinsic disorders and their associated functions.

Leucine Motifs Stabilize Residual Helical Structure in Disordered Proteins.

Many examples are known of regions of intrinsically disordered proteins that fold into α-helices upon binding to their targets. These helical binding motifs (HBMs) can be partially helical also in the unbound state, and this so-called residual structure can affect binding affinity and kinetics. To investigate the underlying mechanisms governing the formation of residual helical structure, we assembled a dataset of experimental helix contents of 65 peptides containing HBM that fold-upon-binding. The average residual helicity is 17% and increases to 60% upon target binding. The helix contents of residual and target-bound structures do not correlate, however the relative location of helix elements in both states shows a strong overlap. Compared to the general disordered regions, HBMs are enriched in amino acids with high helix preference and these residues are typically involved in target binding, explaining the overlap in helix positions. In particular, we find that leucine residues and leucine motifs in HBMs are the major contributors to helix stabilization and target-binding. For the two model peptides, we show that substitution of leucine motifs to other hydrophobic residues (valine or isoleucine) leads to reduction of residual helicity, supporting the role of leucine as helix stabilizer. From the three hydrophobic residues only leucine can efficiently stabilize residual helical structure. We suggest that the high occurrence of leucine motifs and a general preference for leucine at binding interfaces in HBMs can be explained by its unique ability to stabilize helical elements.

Biological importance of arginine: A comprehensive review of the roles in structure, disorder, and functionality of peptides and proteins.

Arginine shows Jekyll and Hyde behavior in several respects. It participates in protein folding via ionic and H-bonds and cation-pi interactions; the charge and hydrophobicity of its side chain make it a disorder-promoting amino acid. Its methylation in histones; RNA binding proteins; chaperones regulates several cellular processes. The arginine-centric modifications are important in oncogenesis and as biomarkers in several cardiovascular diseases. The cross-links involving arginine in collagen and cornea are involved in pathogenesis of tissues but have also been useful in tissue engineering and wound-dressing materials. Arginine is a part of active site of several enzymes such as GTPases, peroxidases, and sulfotransferases. Its metabolic importance is obvious as it is involved in production of urea, NO, ornithine and citrulline. It can form unusual functional structures such as molecular tweezers in vitro and sprockets which engage DNA chains as part of histones in vivo. It has been used in design of cell-penetrating peptides as drugs. Arginine has been used as an excipient in both solid and injectable drug formulations; its role in suppressing opalescence due to liquid-liquid phase separation is particularly very promising. It has been known as a suppressor of protein aggregation during protein refolding. It has proved its usefulness in protein bioseparation processes like ion-exchange, hydrophobic and affinity chromatographies. Arginine is an amino acid, whose importance in biological sciences and biotechnology continues to grow in diverse ways.

Intrinsic disorder of a nucleoplasmin-like histone chaperone specifies its discrete nuclear and nucleolar functions.

Nucleoplasmin (NPM) histone chaperones regulate distinct processes in the nucleus and nucleolus. While intrinsically disordered regions (IDRs) are hallmarks of NPMs, it is not clear whether all NPM functions require these unstructured features. We assessed the importance of IDRs in a yeast NPM-like protein and found that regulation of rDNA copy number and genetic interactions with the nucleolar RNA surveillance machinery require the highly conserved FKBP prolyl isomerase domain, but not the NPM domain or IDRs. By contrast, transcriptional repression in the nucleus requires IDRs. Furthermore, multiple lysines in polyacidic serine/lysine motifs of IDRs are required for both lysine polyphosphorylation and NPM-mediated transcriptional repression. These results demonstrate that this NPM-like protein relies on IDRs only for some of its chromatin-related functions.

Biophysical characterization of the Plasmodium falciparum circumsporozoite protein's N-terminal domain.

The circumsporozoite protein (CSP) is the main surface antigen of the Plasmodium sporozoite (SPZ) and forms the basis of the currently only licensed anti-malarial vaccine (RTS,S/AS01). CSP uniformly coats the SPZ and plays a pivotal role in its immunobiology, in both the insect and the vertebrate hosts. Although CSP's N-terminal domain (CSPN ) has been reported to play an important role in multiple CSP functions, a thorough biophysical and structural characterization of CSPN is currently lacking. Here, we present an alternative method for the recombinant production and purification of CSPN from Plasmodium falciparum (PfCSPN ), which provides pure, high-quality protein preparations with high yields. Through an interdisciplinary approach combining in-solution experimental methods and in silico analyses, we provide strong evidence that PfCSPN is an intrinsically disordered region displaying some degree of compaction.

Hierarchical Assembly of Intrinsically Disordered Short Peptides.

The understanding on how short peptide assemblies transit from disorder to order remains limited due to the lack of atomistic structures. Here we report cryo-EM structure of the nanofibers short intrinsically disordered peptides (IDPs). Upon lowering pH or adding calcium ions, the IDP transitions from individual nanoparticles to nanofibers containing an aromatic core and a disordered periphery comprised of 2 to 5 amino acids. Protonating the phosphate or adding more metal ions further assembles the nanofibers into filament bundles. The assemblies of the IDP analogs with controlled chemistry, such as phosphorylation site, hydrophobic interactions, and sequences indicate that metal ions interact with the flexible periphery of the nanoparticles of the IDPs to form fibrils and enhance the interfibrillar interactions to form filament bundles. Illustrating that an IDP self-assembles from disorder to order, this work offers atomistic molecular insights to understand assemblies of short peptides driven by noncovalent interactions.

Uncovering the structural stability of Magnaporthe oryzae effectors: a secretome-wide in silico analysis.

Rice blast, caused by the ascomycete fungus Magnaporthe oryzae, is a deadly disease and a major threat to global food security. The pathogen secretes small proteinaceous effectors, virulence factors, inside the host to manipulate and perturb the host immune system, allowing the pathogen to colonize and establish a successful infection. While the molecular functions of several effectors are characterized, very little is known about the structural stability of these effectors. We analyzed a total of 554 small secretory proteins (SSPs) from the M. oryzae secretome to decipher key features of intrinsic disorder (ID) and the structural dynamics of the selected putative effectors through thorough and systematic in silico studies. Our results suggest that out of the total SSPs, 66% were predicted as effector proteins, released either into the apoplast or cytoplasm of the host cell. Of these, 68% were found to be intrinsically disordered effector proteins (IDEPs). Among the six distinct classes of disordered effectors, we observed peculiar relationships between the localization of several effectors in the apoplast or cytoplasm and the degree of disorder. We determined the degree of structural disorder and its impact on protein foldability across all the putative small secretory effector proteins from the blast pathogen, further validated by molecular dynamics simulation studies. This study provides definite clues toward unraveling the mystery behind the importance of structural distortions in effectors and their impact on plant-pathogen interactions. The study of these dynamical segments may help identify new effectors as well.Communicated by Ramaswamy H. Sarma.

Explored secretome of M. oryzae for intrinsic disorder in effectorsClassified intrinsic disorder into six categoriesNoted varying degrees of disorder in apoplastic vs. cytoplasmic effectorsFound a correlation between intrinsic disorder and flexibilityDemonstrated flexibility patterns through molecular dynamics simulationsRevealed that intrinsic disorder influences effector interactionsIdentified an exceptional 100% disordered effector defying observed trends.

PICKLE RELATED 2 is a Neofunctionalized Gene Duplicate Under Positive Selection With Antagonistic Effects to the Ancestral PICKLE Gene on the Seed Transcriptome.

The evolution and diversification of proteins capable of remodeling domains has been critical for transcriptional reprogramming during cell fate determination in multicellular eukaryotes. Chromatin remodeling proteins of the CHD3 family have been shown to have important and antagonistic impacts on seed development in the model plant, Arabidopsis thaliana, yet the basis of this functional divergence remains unknown. In this study, we demonstrate that genes encoding the CHD3 proteins PICKLE (PKL) and PICKLE-RELATED 2 (PKR2) originated from a duplication event during the diversification of crown Brassicaceae, and that these homologs have undergone distinct evolutionary trajectories since this duplication, with PKR2 fast evolving under positive selection, while PKL is subject to purifying selection. We find that the rapid evolution of PKR2 under positive selection reduces the encoded protein's intrinsic disorder, possibly suggesting a tertiary structure configuration which differs from that of PKL. Our whole genome transcriptome analysis in seeds of pkr2 and pkl mutants reveals that they act antagonistically on the expression of specific sets of genes, providing a basis for their differing roles in seed development. Our results provide insights into how gene duplication and neofunctionalization can lead to differing and antagonistic selective pressures on transcriptomes during plant reproduction, as well as on the evolutionary diversification of the CHD3 family within seed plants.