Disordered Protein Sequences Research Articles

Interaction with IP6K1 supports pyrophosphorylation of substrate proteins by the inositol pyrophosphate 5-InsP7.

Inositol pyrophosphates (PP-InsPs) are a sub-family of water soluble inositol phosphates that possess one or more diphosphate groups. PP-InsPs can transfer their β-phosphate group to a phosphorylated Ser residue to generate pyrophosphorylated Ser. This unique post-translational modification occurs on Ser residues that lie in acidic stretches within an intrinsically disordered protein sequence. Serine pyrophosphorylation is dependent on the presence of Mg2+ ions, but does not require an enzyme for catalysis. The mechanisms by which cells regulate PP-InsP-mediated pyrophosphorylation are still unknown. We performed mass spectrometry to identify interactors of IP6K1, an enzyme responsible for the synthesis of the PP-InsP 5-InsP7. Interestingly, IP6K1 interacted with several proteins that are known to undergo 5-InsP7-mediated pyrophosphorylation, including the nucleolar proteins NOLC1, TCOF and UBF1, and AP3B1, the β subunit of the AP3 adaptor protein complex. The IP6K1 interactome also included CK2, a protein kinase that phosphorylates Ser residues prior to pyrophosphorylation. We observe the formation of a protein complex between IP6K1, AP3B1, and the catalytic α-subunit of CK2, and show that disrupting IP6K1 binding to AP3B1 lowers its in vivo pyrophosphorylation. We propose that assembly of a substrate-CK2-IP6K complex would allow for coordinated pre-phosphorylation and pyrophosphorylation of the target serine residue, and provide a mechanism to regulate this enzyme-independent modification.

Predicting Heteropolymer Interactions: Demixing and Hypermixing of Disordered Protein Sequences

Cells contain multiple condensates which spontaneously form due to the heterotypic interactions between their components. Although the proteins and disordered region sequences that are responsible for condensate formation have been extensively studied, the rule of interactions between the components that allow demixing, i.e., the coexistence of multiple condensates, is yet to be elucidated. Here, we construct an effective theory of the interaction between heteropolymers by fitting it to the molecular dynamics simulation results obtained for more than 200 sequences sampled from the disordered regions of human proteins. We find that the sum of amino acid pair interactions across two heteropolymers predicts the Boyle temperature qualitatively well, which can be quantitatively improved by the dimer pair approximation, where we incorporate the effect of neighboring amino acids in the sequences. The improved theory, combined with the finding of a metric that captures the effective interaction strength between distinct sequences, allowed the selection of up to three disordered region sequences that demix with each other in multicomponent simulations, as well as the generation of artificial sequences that demix with a given sequence. The theory points to a generic sequence design strategy to demix or hypermix thanks to the low-dimensional nature of the space of the interactions that we identify. As a consequence of the geometric arguments in the space of interactions, we find that the number of distinct sequences that can demix with each other is strongly constrained, irrespective of the choice of the coarse-grained model. Altogether, we construct a theoretical basis for methods to estimate the effective interaction between heteropolymers, which can be utilized in predicting phase separation properties as well as rules of assignment in the localization and functions of disordered proteins. Published by the American Physical Society 2024

Fuzzy recognition by the prokaryotic transcription factor HigA2 from Vibrio cholerae

Disordered protein sequences can exhibit different binding modes, ranging from well-ordered folding-upon-binding to highly dynamic fuzzy binding. The primary function of the intrinsically disordered region of the antitoxin HigA2 from Vibrio cholerae is to neutralize HigB2 toxin through ultra-high-affinity folding-upon-binding interaction. Here, we show that the same intrinsically disordered region can also mediate fuzzy interactions with its operator DNA and, through interplay with the folded helix-turn-helix domain, regulates transcription from the higBA2 operon. NMR, SAXS, ITC and in vivo experiments converge towards a consistent picture where a specific set of residues in the intrinsically disordered region mediate electrostatic and hydrophobic interactions while “hovering” over the DNA operator. Sensitivity of the intrinsically disordered region to scrambling the sequence, position-specific contacts and absence of redundant, multivalent interactions, point towards a more specific type of fuzzy binding. Our work demonstrates how a bacterial regulator achieves dual functionality by utilizing two distinct interaction modes within the same disordered sequence.

Intrinsic protein disorder is insufficient to drive subnuclear clustering in embryonic transcription factors.

Modern microscopy has revealed that core nuclear functions, including transcription, replication, and heterochromatin formation, occur in spatially restricted clusters. Previous work from our lab has shown that subnuclear high-concentration clusters of transcription factors may play a role in regulating RNA synthesis in the early Drosophila embryo. A nearly ubiquitous feature of eukaryotic transcription factors is that they contain intrinsically disordered regions (IDRs) that often arise from low complexity amino acid sequences within the protein. It has been proposed that IDRs within transcription factors drive co-localization of transcriptional machinery and target genes into high-concentration clusters within nuclei. Here, we test that hypothesis directly, by conducting a broad survey of the subnuclear localization of IDRs derived from transcription factors. Using a novel algorithm to identify IDRs in the Drosophila proteome, we generated a library of IDRs from transcription factors expressed in the early Drosophila embryo. We used this library to perform a high-throughput imaging screen in Drosophila Schneider-2 (S2) cells. We found that while subnuclear clustering does not occur when the majority of IDRs are expressed alone, it is frequently seen in full-length transcription factors. These results are consistent in live Drosophila embryos, suggesting that IDRs are insufficient to drive the subnuclear clustering behavior of transcription factors. Furthermore, the clustering of transcription factors in living embryos was unaffected by the deletion of IDR sequences. Our results demonstrate that IDRs are unlikely to be the primary molecular drivers of the clustering observed during transcription, suggesting a more complex and nuanced role for these disordered protein sequences.

Intrinsic protein disorder is insufficient to drive subnuclear clustering in embryonic transcription factors

Modern microscopy has revealed that core nuclear functions, including transcription, replication, and heterochromatin formation, occur in spatially restricted clusters. Previous work from our lab has shown that subnuclear high-concentration clusters of transcription factors may play a role in regulating RNA synthesis in the early Drosophila embryo. A nearly ubiquitous feature of eukaryotic transcription factors is that they contain intrinsically disordered regions (IDRs) that often arise from low complexity amino acid sequences within the protein. It has been proposed that IDRs within transcription factors drive co-localization of transcriptional machinery and target genes into high-concentration clusters within nuclei. Here, we test that hypothesis directly, by conducting a broad survey of the subnuclear localization of IDRs derived from transcription factors. Using a novel algorithm to identify IDRs in the Drosophila proteome, we generated a library of IDRs from transcription factors expressed in the early Drosophila embryo. We used this library to perform a high-throughput imaging screen in Drosophila Schneider-2 (S2) cells. We found that while subnuclear clustering does not occur when the majority of IDRs are expressed alone, it is frequently seen in full-length transcription factors. These results are consistent in live Drosophila embryos, suggesting that IDRs are insufficient to drive the subnuclear clustering behavior of transcription factors. Furthermore, the clustering of transcription factors in living embryos was unaffected by the deletion of IDR sequences. Our results demonstrate that IDRs are unlikely to be the primary molecular drivers of the clustering observed during transcription, suggesting a more complex and nuanced role for these disordered protein sequences.

The difficulty of aligning intrinsically disordered protein sequences as assessed by conservation and phylogeny.

Intrinsically disordered proteins (IDPs) are proteins that lack a stable 3D structure but maintain a biological function. It has been frequently suggested that IDPs are difficult to align because they tend to have fewer conserved residues compared to ordered proteins, but to our knowledge this has never been directly tested. To compare the alignments of ordered proteins to IDPs, their multiple sequence alignments (MSAs) were assessed using two different methods. The first compared the similarity between MSAs produced using the same sequences but created with Clustal Omega, MAFFT, and MUSCLE. The second assessed MSAs based on how well they recapitulated the species tree. These two methods measure the "correctness" of an MSA with two different approaches; the first method measures consistency while the second measures the underlying phylogenetic signal. Proteins that contained both regions of disorder and order were analyzed along with proteins that were fully disordered and fully ordered, using nucleotide, codon and peptide sequence alignments. We observed that IDPs had less similar MSAs than ordered proteins, which is most likely linked to the lower sequence conservation in IDPs. However, comparisons of tree distances found that trees from the ordered sequence MSAs were not significantly closer to the species tree than those inferred from disordered sequence MSAs. Our results show that it is correct to say that IDPs are difficult to align on the basis of MSA consistency, but that this does not equate with alignments being of poor quality when assessed by their ability to correctly infer a species tree.

Engineered Artificial Membraneless Organelles in Saccharomyces cerevisiae To Enhance Chemical Production

AbstractMicrobial cell factories provide a green and sustainable opportunity to produce value‐added products from renewable feedstock. However, the leakage of toxic or volatile intermediates decreases the efficiency of microbial cell factories. In this study, membraneless organelles (MLOs) were reconstructed in Saccharomyces cerevisiae by the disordered protein sequence A‐IDPs. A regulation system was designed to spatiotemporally regulate the size and rigidity of MLOs. Manipulating the MLO size of strain ZP03‐FM, the amounts of assimilated methanol and malate were increased by 162 % and 61 %, respectively. Furthermore, manipulating the MLO rigidity in strain ZP04‐RB made acetyl‐coA synthesis from oxidative glycolysis change to non‐oxidative glycolysis; consequently, CO2 release decreased by 35 % and the n‐butanol yield increased by 20 %. This artificial MLO provides a strategy for the co‐localization of enzymes to channel C1 starting materials into value‐added chemicals.

Engineered Artificial Membraneless Organelles in Saccharomyces cerevisiae To Enhance Chemical Production.

Microbial cell factories provide a green and sustainable opportunity to produce value-added products from renewable feedstock. However, the leakage of toxic or volatile intermediates decreases the efficiency of microbial cell factories. In this study, membraneless organelles (MLOs) were reconstructed in Saccharomyces cerevisiae by the disordered protein sequence A-IDPs. A regulation system was designed to spatiotemporally regulate the size and rigidity of MLOs. Manipulating the MLO size of strain ZP03-FM, the amounts of assimilated methanol and malate were increased by 162 % and 61 %, respectively. Furthermore, manipulating the MLO rigidity in strain ZP04-RB made acetyl-coA synthesis from oxidative glycolysis change to non-oxidative glycolysis; consequently, CO2 release decreased by 35 % and the n-butanol yield increased by 20 %. This artificial MLO provides a strategy for the co-localization of enzymes to channel C1 starting materials into value-added chemicals.

Field-theoretic formulation for phase behaviors of 20-amino-acid-type intrinsically disordered protein sequences.

Spatial organization of biomolecules through liquid-liquid phase separation (LLPS) has been argued to be essential for cellular function but poses severe challenges for simulation and mathematical modelling due to the large numbers of molecules and interaction types involved. Field theory (FT) models of bio-LLPS, where molecular degrees-of-freedom are traded for a set of fluctuating fields, constitute a promising computational approach since the number of molecules in the system is not a limiting factor in simulation.

Shape Fluctuations of Random Polyampholyte and Intrinsically Disordered Protein Sequences

Charged polymers with quenched charge sequences adopt pearl-necklace structures due to the interplay between charge disorder and thermal fluctuations. Partially globular pearl necklace conformations from random (+/−) sequences are intrinsically heterogeneous and exhibit various structures characterized by different numbers of pearls. On the basis of the molecular dynamics simulations of random model polyampholytes (PAs), we study dynamics of pearls and associated conformational heterogeneity in pearl structures. Fast nucleation/dissolution of small pearls controls the dynamics of the number of pearls, which is well described by first order kinetic equations. Most of the individual sequences we considered have a rather stable number of large pearls. Only a few sequences allow for fast switchings in the number of large pearls following complex processes (trajectories in parameter space) going through states with different numbers of pearls. Processes are most complex in the tail of the switching time distribution. The specific sequences of two intrinsically disordered proteins (IDPs) are studied along the same lines. Our study shows the resemblance in shape fluctuations between the IDP sequences and the fast-switching PA sequences.

Preparation of Bioderived and Biodegradable Surfactants Based on an Intrinsically Disordered Protein Sequence.

Surfactants, block copolymers, and other types of micellar systems are used in a wide variety of biomedical and industrial processes. However, most commonly used surfactants are synthetically derived and pose environmental and toxicological concerns throughout their product life cycle. Because of this, bioderived and biodegradable surfactants are promising alternatives. For biosurfactants to be implemented industrially, they need to be produced on a large scale and also have tailorable properties that match those afforded by the polymerization of synthetic surfactants. In this paper, a scalable and versatile production method for biosurfactants based on a hydrophilic intrinsically disordered protein (IDP) sequence with a genetically engineered hydrophobic domain is used to study variables that impact their physicochemical and self-assembling properties. These amphiphilic sequences were found to self-assemble into micelles over a broad range of temperatures, pH values, and ionic strengths. To investigate the role of the IDP hydrophilic domain on self-assembly, variants with increased overall charges and systematically decreased IDP domain lengths were produced and examined for their sizes, morphologies, and critical micelle concentrations (CMCs). The results of these studies indicate that decreasing the length of the IDP domain and consequently the molecular weight and hydrophilic fraction leads to smaller micelles. In addition, significantly increasing the amount of charged residues in the hydrophilic IDP domain results in micelles of similar sizes but with higher CMC values. This represents an initial step in developing a quantitative model for the future engineering of biosurfactants based on this IDP sequence.

LOCATE reveals electrostatic “islands” and “hotspots” are important for a disordered clock protein's interactions to regulate clock robustness

Many important biological processes are regulated by the circadian molecular clock, a negative feedback loop comprised of disordered proteins. The lack of a fixed conformation means that identifying interaction regions on core clock proteins is untenable using typical biophysical methods. Since disordered proteins often interact through short linear binding motifs (SLiMs), we decided to take a divide-and-conquer approach leveraging synthetic peptide technology. Our method termed Linear mOtif disCovery using rATional dEsign (LOCATE) takes a disordered protein sequence and divides it into a set of short overlapping peptides that can be synthesized and printed in a microarray format.

Mapping disordered protein sequences to their conformational ensembles in live cells

The composition of solutes in the cell routinely fluctuates due to external stress or cell cycle progression. While many proteins may be resilient to these fluctuations, intrinsically disordered protein regions, or IDRs, can be sensitive to changes in their surrounding solution. This is due to a low content of intramolecular bonds and high surface area exposed to their surrounding environment. However, the molecular grammar that dictates this sensitivity in IDRs remains poorly understood. In this project we use Förster Resonance Energy Transfer (FRET) paired with live cell microscopy to characterize the ensemble of a library of synthetic IDR in cells under stressed conditions.

Linking disordered protein sequence and ensemble using interaction maps

Intrinsically disordered protein regions (IDRs) do not have a stable native structure and instead exist in a constantly changing conformational ensemble that can determine their function. While global or average sequence features such as charge or aromatic residue distribution can sometimes predict the structure of IDR ensembles, a complete understanding of how the structure is encoded in IDR sequences remains elusive. We posit that this information is encoded in the intramolecular interactions of these sequences.

Disorder in a two-domain neuronal Ca2+-binding protein regulates domain stability and dynamics using ligand mimicry

Understanding the interplay between sequence, structure and function of proteins has been complicated in recent years by the discovery of intrinsically disordered proteins (IDPs), which perform biological functions in the absence of a well-defined three-dimensional fold. Disordered protein sequences account for roughly 30% of the human proteome and in many proteins, disordered and ordered domains coexist. However, few studies have assessed how either feature affects the properties of the other. In this study, we examine the role of a disordered tail in the overall properties of the two-domain, calcium-sensing protein neuronal calcium sensor 1 (NCS-1). We show that loss of just six of the 190 residues at the flexible C-terminus is sufficient to severely affect stability, dynamics, and folding behavior of both ordered domains. We identify specific hydrophobic contacts mediated by the disordered tail that may be responsible for stabilizing the distal N-terminal domain. Moreover, sequence analyses indicate the presence of an LSL-motif in the tail that acts as a mimic of native ligands critical to the observed order–disorder communication. Removing the disordered tail leads to a shorter life-time of the ligand-bound complex likely originating from the observed destabilization. This close relationship between order and disorder may have important implications for how investigations into mixed systems are designed and opens up a novel avenue of drug targeting exploiting this type of behavior.

Detection of Multisite Phosphorylation of Intrinsically Disordered Proteins Using Quantitative Mass-Spectrometry.

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) within proteins have attracted considerable attention in recent years. Several important biological signaling mechanisms including protein-protein interactions and post-translational modifications can be easily mediated by IDPs and IDRs due to their flexible structure. These regions can encode linear sequences that are indispensable in cell-signaling networks and circuits. For example, the linear multisite phosphorylation networks encoded in disordered protein sequences play a key role in cell-cycle regulation where the phosphorylation of proteins controls the orchestration of all major mechanisms. While elucidating a systems-level understanding of this process and other multisite phosphorylation processes, we extensively used mass-spectrometry and found it to be an ideal tool to identify, characterize, and quantify phosphorylation dynamics within IDPs. Here, we describe a quantitative proteomics method, together with a detailed protocol to analyze dynamic multisite phosphorylation processes within IDPs using an in vitro protein phosphorylation assay with "light" gamma-16O ATP and "heavy" gamma-18O ATP, combined with liquid chromatography mass spectrometry.

A high energy phosphate jump - From pyrophospho-inositol to pyrophospho-serine.

Inositol pyrophosphates (PP-IPs) are a class of energy rich metabolites present in all eukaryotic cells. The hydroxyl groups on these water soluble derivatives of inositol are substituted with diphosphate and monophosphate moieties. Since the discovery of PP-IPs in the early 1990s, enormous progress has been made in uncovering pleiotropic roles for these small molecules in cellular physiology. PP-IPs exert their effect on proteins in two ways - allosteric regulation by direct binding, or post-translational regulation by serine pyrophosphorylation, a modification unique to PP-IPs. Serine pyrophosphorylation is achieved by Mg2+-dependent, but enzyme independent transfer of a β-phosphate from a PP-IP to a pre-phosphorylated serine residue located in an acidic motif, within an intrinsically disordered protein sequence. This distinctive post-translational modification has been shown to regulate diverse cellular processes, including rRNA synthesis, glycolysis, and vesicle transport. However, our understanding of the molecular details of this phosphotransfer from pyrophospho-inositol to generate pyrophospho-serine, is still nascent. This review discusses our current knowledge of protein pyrophosphorylation, and recent advances in understanding the mechanism of this important yet overlooked post-translational modification.

Discovering MoRFs by trisecting intrinsically disordered protein sequence into terminals and middle regions

BackgroundMolecular Recognition Features (MoRFs) are short protein regions present in intrinsically disordered protein (IDPs) sequences. MoRFs interact with structured partner protein and upon interaction, they undergo a disorder-to-order transition to perform various biological functions. Analyses of MoRFs are important towards understanding their function.ResultsPerformance is reported using the MoRF dataset that has been previously used to compare the other existing MoRF predictors. The performance obtained in this study is equivalent to the benchmarked OPAL predictor, i.e., OPAL achieved AUC of 0.815, whereas the model in this study achieved AUC of 0.819 using TEST set.ConclusionAchieving comparable performance, the proposed method can be used as an alternative approach for MoRF prediction.

OPAL+: Length-Specific MoRF Prediction in Intrinsically Disordered Protein Sequences.

Intrinsically disordered proteins (IDPs) contain long unstructured regions, which play an important role in their function. These intrinsically disordered regions (IDRs) participate in binding events through regions called molecular recognition features (MoRFs). Computational prediction of MoRFs helps identify the potentially functional regions in IDRs. In this study, OPAL+, a novel MoRF predictor, is presented. OPAL+ uses separate models to predict MoRFs of varying lengths along with incorporating the hidden Markov model (HMM) profiles and physicochemical properties of MoRFs and their flanking regions. Together, these features help OPAL+ achieve a marginal performance improvement of 0.4-0.7% over its predecessor for diverse MoRF test sets. This performance improvement comes at the expense of increased run time as a result of the requirement of HMM profiles. OPAL+ is available for download at https://github.com/roneshsharma/OPAL-plus/wiki/OPAL-plus-Download.

Interaction modulation through arrays of clustered methyl-arginine protein modifications.

Systematic analysis of human arginine methylation identifies two distinct signaling modes; either isolated modifications akin to canonical post-translational modification regulation, or clustered arrays within disordered protein sequence. Hundreds of proteins contain these methyl-arginine arrays and are more prone to accumulate mutations and more tightly expression-regulated than dispersed methylation targets. Arginines within an array in the highly methylated RNA-binding protein synaptotagmin binding cytoplasmic RNA interacting protein (SYNCRIP) were experimentally shown to function in concert, providing a tunable protein interaction interface. Quantitative immunoprecipitation assays defined two distinct cumulative binding mechanisms operating across 18 proximal arginine-glycine (RG) motifs in SYNCRIP. Functional binding to the methyltransferase PRMT1 was promoted by continual arginine stretches, whereas interaction with the methyl-binding protein SMN1 was arginine content-dependent irrespective of linear position within the unstructured region. This study highlights how highly repetitive modifiable amino acid arrays in low structural complexity regions can provide regulatory platforms, with SYNCRIP as an extreme example how arginine methylation leverages these disordered sequences to mediate cellular interactions.