AlphaFold2 modeling and molecular dynamics simulations of an intrinsically disordered protein.

We use AlphaFold2 (AF2) to model the monomer and dimer structures of an intrinsically disordered protein (IDP), Nvjp-1, assisted by molecular dynamics (MD) simulations. We observe relatively rigid dimeric structures of Nvjp-1 when compared with the monomer structures. We suggest that protein conformations from multiple AF2 models and those from MD trajectories exhibit a coherent trend: the conformations of an IDP are deviated from each other and the conformations of a well-folded protein are consistent with each other. We use a residue-residue interaction network (RIN) derived from the contact map which show that the residue-residue interactions in Nvjp-1 are mainly transient; however, those in a well-folded protein are mainly persistent. Despite the variation in 3D shapes, we show that the AF2 models of both disordered and ordered proteins exhibit highly consistent profiles of the pLDDT (predicted local distance difference test) scores. These results indicate a potential protocol to justify the IDPs based on multiple AF2 models and MD simulations.

Hydrodynamic Radii of Intrinsically Disordered Proteins: Fast Prediction by Minimum Dissipation Approximation and Experimental Validation.

The diffusion coefficients of globular and fully unfolded proteins can be predicted with high accuracy solely from their mass or chain length. However, this approach fails for intrinsically disordered proteins (IDPs) containing structural domains. We propose a rapid predictive methodology for estimating the diffusion coefficients of IDPs. The methodology uses accelerated conformational sampling based on self-avoiding random walks and includes hydrodynamic interactions between coarse-grained protein subunits, modeled using the generalized Rotne-Prager-Yamakawa approximation. To estimate the hydrodynamic radius, we rely on the minimum dissipation approximation recently introduced by Cichocki et al. Using a large set of experimentally measured hydrodynamic radii of IDPs over a wide range of chain lengths and domain contributions, we demonstrate that our predictions are more accurate than the Kirkwood approximation and phenomenological approaches. Our technique may prove to be valuable in predicting the hydrodynamic properties of both fully unstructured and multidomain disordered proteins.

Structural studies of intrinsically disordered MLL-fusion protein AF9 in complex with peptidomimetic inhibitors.

AF9 (MLLT3) and its paralog ENL(MLLT1) are members of the YEATS family of proteins with important role in transcriptional and epigenetic regulatory complexes. These proteins are two common MLL fusion partners in MLL-rearranged leukemias. The oncofusion proteins MLL-AF9/ENL recruit multiple binding partners, including the histone methyltransferase DOT1L, leading to aberrant transcriptional activation and enhancing the expression of a characteristic set of genes that drive leukemogenesis. The interaction between AF9 and DOT1L is mediated by an intrinsically disordered C-terminal ANC1 homology domain (AHD) in AF9, which undergoes folding upon binding of DOT1L and other partner proteins. We have recently reported peptidomimetics that disrupt the recruitment of DOT1L by AF9 and ENL, providing a proof-of-concept for targeting AHD and assessing its druggability. Intrinsically disordered proteins, such as AF9 AHD, are difficult to study and characterize experimentally on a structural level. In this study, we present a successful protein engineering strategy to facilitate structural investigation of the intrinsically disordered AF9 AHD domain in complex with peptidomimetic inhibitors by using maltose binding protein (MBP) as a crystallization chaperone connected with linkers of varying flexibility and length. The strategic incorporation of disulfide bonds provided diffraction-quality crystals of the two disulfide-bridged MBP-AF9 AHD fusion proteins in complex with the peptidomimetics. These successfully determined first series of 2.1-2.6 Å crystal complex structures provide high-resolution insights into the interactions between AHD and its inhibitors, shedding light on the role of AHD in recruiting various binding partner proteins. We show that the overall complex structures closely resemble the reported NMR structure of AF9 AHD/DOT1L with notable difference in the conformation of the ß-hairpin region, stabilized through conserved hydrogen bonds network. These first series of AF9 AHD/peptidomimetics complex structures are providing insights of the protein-inhibitor interactions and will facilitate further development of novel inhibitors targeting the AF9/ENL AHD domain.

Copper(II) coordination to the intrinsically disordered region of SARS-CoV-2 Nsp1.

The intrinsically disordered C-terminal peptide region of severe acute respiratory syndrome coronavirus 2 nonstructural protein-1 (Nsp1-CT) inhibits host protein synthesis by blocking messenger RNA (mRNA) access to the 40S ribosome entrance tunnel. Aqueous copper(II) ions bind to the disordered peptide with micromolar affinity, creating a possible strategy to restore protein synthesis during host infection. Electron paramagnetic resonance (EPR) and tryptophan fluorescence measurements on a 10-residue model of the disordered protein region (Nsp1-CT10), combined with advanced quantum mechanics calculations, suggest that the peptide binds to copper(II) as a multidentate ligand. Two optimized computational models of the copper(II)-peptide complexes were derived: One corresponding to pH 6.5 and the other describing the complex at pH 7.5 to 8.5. Simulated EPR spectra based on the calculated model structures are in good agreement with experimental spectra.

Synthetic intrinsically disordered protein fusion tags that enhance protein solubility.

We report the de novo design of small (<20 kDa) and highly soluble synthetic intrinsically disordered proteins (SynIDPs) that confer solubility to a fusion partner with minimal effect on the activity of the fused protein. To identify highly soluble SynIDPs, we create a pooled gene-library utilizing a one-pot gene synthesis technology to create a large library of repetitive genes that encode SynIDPs. We identify three small (<20 kDa) and highly soluble SynIDPs from this gene library that lack secondary structure and have high solvation. Recombinant fusion of these SynIDPs to three known inclusion body forming proteins rescue their soluble expression and do not impede the activity of the fusion partner, thereby eliminating the need for removal of the SynIDP tag. These findings highlight the utility of SynIDPs as solubility tags, as they promote the soluble expression of proteins in E. coli and are small, unstructured proteins that minimally interfere with the biological activity of the fused protein.

Isolation of nucleic acids using liquid-liquid phase separation of pH-sensitive elastin-like polypeptides.

Extraction of nucleic acids (NAs) is critical for many methods in molecular biology and bioanalytical chemistry. NA extraction has been extensively studied and optimized for a wide range of applications and its importance to society has significantly increased. The COVID-19 pandemic highlighted the importance of early and efficient NA testing, for which NA extraction is a critical analytical step prior to the detection by methods like polymerase chain reaction. This study explores simple, new approaches to extraction using engineered smart nanomaterials, namely NA-binding, intrinsically disordered proteins (IDPs), that undergo triggered liquid-liquid phase separation (LLPS). Two types of NA-binding IDPs are studied, both based on genetically engineered elastin-like polypeptides (ELPs), model IDPs that exhibit a lower critical solution temperature in water and can be designed to exhibit LLPS at desired temperatures in a variety of biological solutions. We show that ELP fusion proteins with natural NA-binding domains can be used to extract DNA and RNA from physiologically relevant solutions. We further show that LLPS of pH responsive ELPs that incorporate histidine in their sequences can be used for both binding, extraction and release of NAs from biological solutions, and can be used to detect SARS-CoV-2 RNA in samples from COVID-positive patients.

Predicting condensate formation of protein and RNA under various environmental conditions.

BACKGROUND: Liquid-liquid phase separation (LLPS) by biomolecules plays a central role in various biological phenomena and has garnered significant attention. The behavior of LLPS is strongly influenced by the characteristics of RNAs and environmental factors such as pH and temperature, as well as the properties of proteins. Recently, several databases recording LLPS-related biomolecules have been established, and prediction models of LLPS-related phenomena have been explored using these databases. However, a prediction model that concurrently considers proteins, RNAs, and experimental conditions has not been developed due to the limited information available from individual experiments in public databases. RESULTS: To address this challenge, we have constructed a new dataset, RNAPSEC, which serves each experiment as a data point. This dataset was accomplished by manually collecting data from public literature. Utilizing RNAPSEC, we developed two prediction models that consider a protein, RNA, and experimental conditions. The first model can predict the LLPS behavior of a protein and RNA under given experimental conditions. The second model can predict the required conditions for a given protein and RNA to undergo LLPS. CONCLUSIONS: RNAPSEC and these prediction models are expected to accelerate our understanding of the roles of proteins, RNAs, and environmental factors in LLPS.

Adenosine Triphosphate: The Primordial Molecule That Controls Protein Homeostasis and Shapes the Genome-Proteome Interface.

Adenosine triphosphate (ATP) acts as the universal energy currency that drives various biological processes, while nucleic acids function to store and transmit genetic information for all living organisms. Liquid-liquid phase separation (LLPS) represents the common principle for the formation of membrane-less organelles (MLOs) composed of proteins rich in intrinsically disordered regions (IDRs) and nucleic acids. Currently, while IDRs are well recognized to facilitate LLPS through dynamic and multivalent interactions, the precise mechanisms by which ATP and nucleic acids affect LLPS still remain elusive. This review summarizes recent NMR results on the LLPS of human FUS, TDP-43, and the viral nucleocapsid (N) protein of SARS-CoV-2, as modulated by ATP and nucleic acids, revealing the following: (1) ATP binds to folded domains overlapping with nucleic-acid-binding interfaces; (2) ATP and nucleic acids interplay to biphasically modulate LLPS by competitively binding to overlapping pockets of folded domains and Arg/Lys within IDRs; (3) ATP energy-independently induces protein folding with the highest efficiency known so far. As ATP likely emerged in the prebiotic monomeric world, while LLPS represents a pivotal mechanism to concentrate and compartmentalize rare molecules for forming primordial cells, ATP appears to control protein homeostasis and shape genome-proteome interfaces throughout the evolutionary trajectory, from prebiotic origins to modern cells.

Toward Synthetic Intrinsically Disordered Polypeptides (IDPs): Controlled Incorporation of Glycine in the Ring-Opening Polymerization of N-Carboxyanhydrides.

Intrinsically disordered proteins (IDPs) do not have a well-defined folded structure but instead behave as extended polymer chains in solution. Many IDPs are rich in glycine residues, which create steric barriers to secondary structuring and protein folding. Inspired by this feature, we have studied how the introduction of glycine residues influences the secondary structure of a model polypeptide, poly(l-glutamic acid), a helical polymer. For this purpose, we carried out ring-opening copolymerization with γ-benzyl-l-glutamate and glycine N-carboxyanhydride (NCA) monomers. We aimed to control the glycine distribution within PBLG by adjusting the reactivity ratios of the two NCAs using different reaction conditions (temperature, solvent). The relationship between those conditions, the monomer distributions, and the secondary structure enabled the design of intrinsically disordered polypeptides when a highly gradient microstructure was achieved in DMSO.

Protein misfolding and amyloid nucleation through liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) is an emerging phenomenon in cell physiology and diseases. The weak multivalent interaction prerequisite for LLPS is believed to be facilitated through intrinsically disordered regions, which are prevalent in neurodegenerative disease-associated proteins. These aggregation-prone proteins also exhibit an inherent property for phase separation, resulting in protein-rich liquid-like droplets. The very high local protein concentration in the water-deficient confined microenvironment not only drives the viscoelastic transition from the liquid to solid-like state but also most often nucleate amyloid fibril formation. Indeed, protein misfolding, oligomerization, and amyloid aggregation are observed to be initiated from the LLPS of various neurodegeneration-related proteins. Moreover, in these cases, neurodegeneration-promoting genetic and environmental factors play a direct role in amyloid aggregation preceded by the phase separation. These cumulative recent observations ignite the possibility of LLPS being a prominent nucleation mechanism associated with aberrant protein aggregation. The present review elaborates on the nucleation mechanism of the amyloid aggregation pathway and the possible early molecular events associated with amyloid-related protein phase separation. It also summarizes the recent advancement in understanding the aberrant phase transition of major proteins contributing to neurodegeneration focusing on the common disease-associated factors. Overall, this review proposes a generic LLPS-mediated multistep nucleation mechanism for amyloid aggregation and its implication in neurodegeneration.

Functional in vitro diversity of an intrinsically disordered plant protein during freeze-thawing is encoded by its structural plasticity.

Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration-sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra- and inter-monomeric helix-helix interactions, demonstrate how oligomerization is driven by an α-helical molecular recognition feature (α-MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right-handed coiled-coils might be a recurring theme for homo- and hetero-oligomerization of LEA proteins.

How an intrinsically disordered regulatory subunit assembles a PP1:eIF2 complex.

Fatalska et al.1 use an interdisciplinary strategy to elucidate how an intrinsically disordered regulatory subunit of protein phosphatase 1 binds trimeric eIF2 and positions the phosphatase-substrate complex for dephosphorylation. As validation, they show that a disease mutation abolishes the interaction.

HYPK: A marginally disordered protein sensitive to charge decoration.

Intrinsically disordered proteins (IDPs) that lie close to the empirical boundary separating IDPs and folded proteins in Uversky's charge-hydropathy plot may behave as "marginal IDPs" and sensitively switch conformation upon changes in environment (temperature, crowding, and charge screening), sequence, or both. In our search for such a marginal IDP, we selected Huntingtin-interacting protein K (HYPK) near that boundary as a candidate; PKIα, also near that boundary, has lower secondary structure propensity; and Crk1, just across the boundary on the folded side, has higher secondary structure propensity. We used a qualitative Förster resonance energy transfer-based assay together with circular dichroism to simultaneously probe global and local conformation. HYPK shows several unique features indicating marginality: a cooperative transition in end-to-end distance with temperature, like Crk1 and folded proteins, but unlike PKIα; enhanced secondary structure upon crowding, in contrast to Crk1 and PKIα; and a cross-over from salt-induced expansion to compaction at high temperature, likely due to a structure-to-disorder transition not seen in Crk1 and PKIα. We then tested HYPK's sensitivity to charge patterning by designing charge-flipped variants including two specific sequences with identical amino acid composition that markedly differ in their predicted size and response to salt. The experimentally observed trends, also including mutants of PKIα, verify the predictions from sequence charge decoration metrics. Marginal proteins like HYPK show features of both folded and disordered proteins that make them sensitive to physicochemical perturbations and structural control by charge patterning.

Disordered clock protein interactions and charge blocks turn an hourglass into a persistent circadian oscillator.

Organismal physiology is widely regulated by the molecular circadian clock, a feedback loop composed of protein complexes whose members are enriched in intrinsically disordered regions. These regions can mediate protein-protein interactions via SLiMs, but the contribution of these disordered regions to clock protein interactions had not been elucidated. To determine the functionality of these disordered regions, we applied a synthetic peptide microarray approach to the disordered clock protein FRQ in Neurospora crassa. We identified residues required for FRQ's interaction with its partner protein FRH, the mutation of which demonstrated FRH is necessary for persistent clock oscillations but not repression of transcriptional activity. Additionally, the microarray demonstrated an enrichment of FRH binding to FRQ peptides with a net positive charge. We found that positively charged residues occurred in significant "blocks" within the amino acid sequence of FRQ and that ablation of one of these blocks affected both core clock timing and physiological clock output. Finally, we found positive charge clusters were a commonly shared molecular feature in repressive circadian clock proteins. Overall, our study suggests a mechanistic purpose for positive charge blocks and yielded insights into repressive arm protein roles in clock function.

Quantum Defect Sensitization via Phase-Changing Supercharged Antibody Fragments.

Quantum defects in single-walled carbon nanotubes promote exciton localization, which enables potential applications in biodevices and quantum light sources. However, the effects of local electric fields on the emissive energy states of quantum defects and how they can be controlled are unexplored. Here, we investigate quantum defect sensitization by engineering an intrinsically disordered protein to undergo a phase change at a quantum defect site. We designed a supercharged single-chain antibody fragment (scFv) to enable a full ligand-induced folding transition from an intrinsically disordered state to a compact folded state in the presence of a cytokine. The supercharged scFv was conjugated to a quantum defect to induce a substantial local electric change upon ligand binding. Employing the detection of a proinflammatory biomarker, interleukin-6, as a representative model system, supercharged scFv-coupled quantum defects exhibited robust fluorescence wavelength shifts concomitant with the protein folding transition. Quantum chemical simulations suggest that the quantum defects amplify the optical response to the localization of charges produced upon the antigen-induced folding of the proteins, which is difficult to achieve in unmodified nanotubes. These findings portend new approaches to modulate quantum defect emission for biomarker sensing and protein biophysics and to engineer proteins to modulate binding signal transduction.

Evolutionary analyses of intrinsically disordered regions reveal widespread signals of conservation.

Intrinsically disordered regions (IDRs) are segments of proteins without stable three-dimensional structures. As this flexibility allows them to interact with diverse binding partners, IDRs play key roles in cell signaling and gene expression. Despite the prevalence and importance of IDRs in eukaryotic proteomes and various biological processes, associating them with specific molecular functions remains a significant challenge due to their high rates of sequence evolution. However, by comparing the observed values of various IDR-associated properties against those generated under a simulated model of evolution, a recent study found most IDRs across the entire yeast proteome contain conserved features. Furthermore, it showed clusters of IDRs with common "evolutionary signatures," i.e. patterns of conserved features, were associated with specific biological functions. To determine if similar patterns of conservation are found in the IDRs of other systems, in this work we applied a series of phylogenetic models to over 7,500 orthologous IDRs identified in the Drosophila genome to dissect the forces driving their evolution. By comparing models of constrained and unconstrained continuous trait evolution using the Brownian motion and Ornstein-Uhlenbeck models, respectively, we identified signals of widespread constraint, indicating conservation of distributed features is mechanism of IDR evolution common to multiple biological systems. In contrast to the previous study in yeast, however, we observed limited evidence of IDR clusters with specific biological functions, which suggests a more complex relationship between evolutionary constraints and function in the IDRs of multicellular organisms.

Intrinsic disorder in the human vitreous proteome.

The vitreous is a vital component of the eye, occupying a substantial portion of its volume and maintaining its structure. This study delves into the presence and significance of intrinsically disordered proteins (IDPs) within the vitreous, utilizing a dataset of 1240 vitreous proteins previously discovered in the vitreous proteome by Murthy et al.in five healthy subjects. The results indicate that 26.9 % of vitreous proteins are highly disordered, 68.8 % possess moderate disorder, and only 4.3 % are highly ordered. A complex interaction network among these proteins suggests their biological importance, and approximately 25 % may undergo liquid-liquid phase separation (LLPS). These findings offer new perspectives on the vitreous' molecular composition and behavior, potentially impacting our understanding of eye-related diseases, physiological changes such as vitreous syneresis. Further research is needed to translate these insights into clinical applications, although the intrinsic protein disorder and its association with LLPS appears to play a role in vitreous proteome function.

Order wrapped in chaos: On the roles of intrinsically disordered proteins and RNAs in the arrangement of the mitochondrial enzymatic machines.

The analysis of cryo-electron tomography images of human and rat mitochondria revealed that the mitochondrial matrix is at least as crowded as the cytosol. To mitigate the crowding effects, metabolite transport in the mitochondria primarily occurs through the intermembrane space, which is significantly less crowded. The scientific literature largely ignores how enzyme systems and metabolite transport are organized in the crowded environment of the mitochondrial matrix. Under crowded conditions, multivalent interactions carried out by disordered protein regions (IDRs), may become extremely important. We analyzed the human mitochondrial proteome to determine the presence and physiological significance of IDRs. Despite mitochondrial proteins being generally more ordered than cytosolic or overall proteome proteins, disordered regions plays a significant role in certain mitochondrial compartments and processes. Even in highly ordered enzyme systems, there are proteins with long IDRs. Some IDRs act as binding elements between highly ordered subunits, while the roles of others are not yet established. Mitochondrial systems, like their bacterial ancestors, rely less on IDRs and more on RNA for LLPS compartmentalization. More evolutionarily advanced subsystems that enable mitochondria-cell interactions contain more IDRs. The study highlights the crucial and often overlooked role played by IDRs and non-coding RNAs in mitochondrial organization.

Neurofilament Biophysics: From Structure to Biomechanics.

Neurofilaments (NFs) are multisubunit, neuron-specific intermediate filaments consisting of a 10-nm diameter filament "core" surrounded by a layer of long intrinsically disordered protein (IDP) "tails." NFs are thought to regulate axonal caliber during development and then stabilize the mature axon, with NF subunit misregulation, mutation, and aggregation featuring prominently in multiple neurological diseases. The field's understanding of NF structure, mechanics, and function has been deeply informed by a rich variety of biochemical, cell biological, and mouse genetic studies spanning more than four decades. These studies have contributed much to our collective understanding of NF function in axonal physiology and disease. In recent years, however, there has been a resurgence of interest in NF subunit proteins in two new contexts: as potential blood- and cerebrospinal fluid-based biomarkers of neuronal damage, and as model IDPs with intriguing properties. Here, we review established principles and more recent discoveries in NF structure and function. Where possible, we place these findings in the context of biophysics of NF assembly, interaction, and contributions to axonal mechanics.

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.