RESUMEN
Intrinsically disordered protein regions exist in a collection of dynamic interconverting conformations that lack a stable 3D structure. These regions are structurally heterogeneous, ubiquitous and found across all kingdoms of life. Despite the absence of a defined 3D structure, disordered regions are essential for cellular processes ranging from transcriptional control and cell signalling to subcellular organization. Through their conformational malleability and adaptability, disordered regions extend the repertoire of macromolecular interactions and are readily tunable by their structural and chemical context, making them ideal responders to regulatory cues. Recent work has led to major advances in understanding the link between protein sequence and conformational behaviour in disordered regions, yet the link between sequence and molecular function is less well defined. Here we consider the biochemical and biophysical foundations that underlie how and why disordered regions can engage in productive cellular functions, provide examples of emerging concepts and discuss how protein disorder contributes to intracellular information processing and regulation of cellular function.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Proteínas Intrínsecamente Desordenadas/metabolismo , Conformación Proteica , Secuencia de Aminoácidos , Sustancias MacromolecularesRESUMEN
Many organisms evolved strategies to survive desiccation. Plant seeds protect dehydrated embryos from various stressors and can lay dormant for millennia. Hydration is the key trigger to initiate germination, but the mechanism by which seeds sense water remains unresolved. We identified an uncharacterized Arabidopsis thaliana prion-like protein we named FLOE1, which phase separates upon hydration and allows the embryo to sense water stress. We demonstrate that biophysical states of FLOE1 condensates modulate its biological function in vivo in suppressing seed germination under unfavorable environments. We find intragenic, intraspecific, and interspecific natural variation in FLOE1 expression and phase separation and show that intragenic variation is associated with adaptive germination strategies in natural populations. This combination of molecular, organismal, and ecological studies uncovers FLOE1 as a tunable environmental sensor with direct implications for the design of drought-resistant crops, in the face of climate change.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Germinación , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Priones/metabolismo , Semillas/crecimiento & desarrollo , Agua/metabolismo , Arabidopsis/genética , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/ultraestructura , Deshidratación , Imagenología Tridimensional , Péptidos y Proteínas de Señalización Intercelular/química , Mutación/genética , Latencia en las Plantas , Plantas Modificadas Genéticamente , Dominios Proteicos , Isoformas de Proteínas/metabolismo , Semillas/ultraestructuraRESUMEN
Stressed cells shut down translation, release mRNA molecules from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramolecular interactions between the intrinsically disordered acidic tracts and the positively charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP clusters drives RNA molecules into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit additional client proteins that promote SG maturation or induce a liquid-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Citoplasma/metabolismo , Células HeLa , Humanos , Conformación de Ácido Nucleico , Orgánulos/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Estrés Fisiológico/genéticaRESUMEN
Proteins such as FUS phase separate to form liquid-like condensates that can harden into less dynamic structures. However, how these properties emerge from the collective interactions of many amino acids remains largely unknown. Here, we use extensive mutagenesis to identify a sequence-encoded molecular grammar underlying the driving forces of phase separation of proteins in the FUS family and test aspects of this grammar in cells. Phase separation is primarily governed by multivalent interactions among tyrosine residues from prion-like domains and arginine residues from RNA-binding domains, which are modulated by negatively charged residues. Glycine residues enhance the fluidity, whereas glutamine and serine residues promote hardening. We develop a model to show that the measured saturation concentrations of phase separation are inversely proportional to the product of the numbers of arginine and tyrosine residues. These results suggest it is possible to predict phase-separation properties based on amino acid sequences.
Asunto(s)
Proteína FUS de Unión a ARN/genética , Proteínas de Unión al ARN/fisiología , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Arginina/química , Simulación por Computador , Células HeLa , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/fisiología , Transición de Fase , Proteínas Priónicas/química , Proteínas Priónicas/genética , Priones/genética , Priones/fisiología , Dominios Proteicos , Proteína FUS de Unión a ARN/fisiología , Proteínas de Unión al ARN/aislamiento & purificación , Células Sf9 , Tirosina/químicaRESUMEN
The low-complexity domain (LCD) of the FUS protein forms concentration-dependent assemblies, including liquid droplets and fibril-based hydrogels. The molecular structures of FUS within different assemblies and their functional relevance are subjects of intense debate. Murray et al. report an atomic-level structural model for FUS LCD fibrils that answers some questions and raises new ones.
Asunto(s)
Proteína FUS de Unión a ARNRESUMEN
Biomolecular condensation underlies the biogenesis of an expanding array of membraneless assemblies, including stress granules (SGs), which form under a variety of cellular stresses. Advances have been made in understanding the molecular grammar of a few scaffold proteins that make up these phases, but how the partitioning of hundreds of SG proteins is regulated remains largely unresolved. While investigating the rules that govern the condensation of ataxin-2, an SG protein implicated in neurodegenerative disease, we unexpectedly identified a short 14 aa sequence that acts as a condensation switch and is conserved across the eukaryote lineage. We identify poly(A)-binding proteins as unconventional RNA-dependent chaperones that control this regulatory switch. Our results uncover a hierarchy of cis and trans interactions that fine-tune ataxin-2 condensation and reveal an unexpected molecular function for ancient poly(A)-binding proteins as regulators of biomolecular condensate proteins. These findings may inspire approaches to therapeutically target aberrant phases in disease.
Asunto(s)
Ataxina-2 , Enfermedades Neurodegenerativas , Humanos , Ataxina-2/genética , Proteína I de Unión a Poli(A) , Enfermedades Neurodegenerativas/metabolismo , Condensados BiomolecularesRESUMEN
The pre-mRNA life cycle requires intron processing; yet, how intron-processing defects influence splicing and gene expression is unclear. Here, we find that TTDN1/MPLKIP, which is encoded by a gene implicated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to spliceosomal function. The conserved TTDN1 C-terminal region directly binds lariat debranching enzyme DBR1, whereas its N-terminal intrinsically disordered region (IDR) binds the intron-binding complex (IBC). TTDN1 loss, or a mutated IDR, causes significant intron lariat accumulation, as well as splicing and gene expression defects, mirroring phenotypes observed in NP-TTD patient cells. A Ttdn1-deficient mouse model recapitulates intron-processing defects and certain neurodevelopmental phenotypes seen in NP-TTD. Fusing DBR1 to the TTDN1 IDR is sufficient to recruit DBR1 to the IBC and circumvents the functional requirement for TTDN1. Collectively, our findings link RNA lariat processing with splicing outcomes by revealing the molecular function of TTDN1.
Asunto(s)
Síndromes de Tricotiodistrofia , Animales , Ratones , Intrones/genética , Síndromes de Tricotiodistrofia/genética , ARN Nucleotidiltransferasas/genética , Empalme del ARNRESUMEN
Gene expression in Arabidopsis is regulated by more than 1,900 transcription factors (TFs), which have been identified genome-wide by the presence of well-conserved DNA-binding domains. Activator TFs contain activation domains (ADs) that recruit coactivator complexes; however, for nearly all Arabidopsis TFs, we lack knowledge about the presence, location and transcriptional strength of their ADs1. To address this gap, here we use a yeast library approach to experimentally identify Arabidopsis ADs on a proteome-wide scale, and find that more than half of the Arabidopsis TFs contain an AD. We annotate 1,553 ADs, the vast majority of which are, to our knowledge, previously unknown. Using the dataset generated, we develop a neural network to accurately predict ADs and to identify sequence features that are necessary to recruit coactivator complexes. We uncover six distinct combinations of sequence features that result in activation activity, providing a framework to interrogate the subfunctionalization of ADs. Furthermore, we identify ADs in the ancient AUXIN RESPONSE FACTOR family of TFs, revealing that AD positioning is conserved in distinct clades. Our findings provide a deep resource for understanding transcriptional activation, a framework for examining function in intrinsically disordered regions and a predictive model of ADs.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Dominios Proteicos , Factores de Transcripción , Activación Transcripcional , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/clasificación , Proteínas de Arabidopsis/metabolismo , Secuencia Conservada/genética , Conjuntos de Datos como Asunto , Regulación de la Expresión Génica de las Plantas/genética , Ácidos Indolacéticos/metabolismo , Proteínas Intrínsecamente Desordenadas , Anotación de Secuencia Molecular , Redes Neurales de la Computación , Proteoma/química , Proteoma/metabolismo , Factores de Transcripción/química , Factores de Transcripción/clasificación , Factores de Transcripción/metabolismo , Activación Transcripcional/genéticaRESUMEN
Xu et al. (2021) uncover a conserved viral mechanism to subvert the innate immune system whereby herpesvirus tegument proteins suppress cGAS:DNA phase separation.
Asunto(s)
ADN , Nucleotidiltransferasas , Citosol , ADN/genética , Nucleotidiltransferasas/genéticaRESUMEN
Intrinsically disordered regions (IDRs) within human proteins play critical roles in cellular information processing, including signaling, transcription, stress response, DNA repair, genome organization, and RNA processing. Here, we summarize current challenges in the field and propose cutting-edge approaches to address them in physiology and disease processes, with a focus on cancer.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Humanos , Proteínas Intrínsecamente Desordenadas/metabolismo , Biofisica , BiologíaRESUMEN
Low-complexity protein domains promote the formation of various biomolecular condensates. However, in many cases, the precise sequence features governing condensate formation and identity remain unclear. Here, we investigate the role of intrinsically disordered mixed-charge domains (MCDs) in nuclear speckle condensation. Proteins composed exclusively of arginine-aspartic acid dipeptide repeats undergo length-dependent condensation and speckle incorporation. Substituting arginine with lysine in synthetic and natural speckle-associated MCDs abolishes these activities, identifying a key role for multivalent contacts through arginine's guanidinium ion. MCDs can synergize with a speckle-associated RNA recognition motif to promote speckle specificity and residence. MCD behavior is tunable through net-charge: increasing negative charge abolishes condensation and speckle incorporation. Contrastingly, increasing positive charge through arginine leads to enhanced condensation, speckle enlargement, decreased splicing factor mobility, and defective mRNA export. Together, these results identify key sequence determinants of MCD-promoted speckle condensation and link the dynamic material properties of speckles with function in mRNA processing.
Asunto(s)
Arginina/metabolismo , Núcleo Celular/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Lisina/metabolismo , Empalme del ARN/genética , ARN Mensajero/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Arginina/genética , Núcleo Celular/genética , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Lisina/genética , Mutación , Fosforilación , Dominios Proteicos , ARN Mensajero/genética , Factores de Empalme Serina-Arginina/genéticaRESUMEN
Intrinsically disordered proteins and protein regions (IDRs) are abundant in eukaryotic proteomes and play a wide variety of essential roles. Instead of folding into a stable structure, IDRs exist in an ensemble of interconverting conformations whose structure is biased by sequence-dependent interactions. The absence of a stable 3D structure, combined with high solvent accessibility, means that IDR conformational biases are inherently sensitive to changes in their environment. Here, we argue that IDRs are ideally poised to act as sensors and actuators of cellular physicochemistry. We review the physical principles that underlie IDR sensitivity, the molecular mechanisms that translate this sensitivity to function, and recent studies where environmental sensing by IDRs may play a key role in their downstream function.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Conformación Proteica , Proteínas Intrínsecamente Desordenadas/metabolismo , Dominios ProteicosRESUMEN
Intrinsically disordered regions (IDRs) are ubiquitous across all domains of life and play a range of functional roles. While folded domains are generally well described by a stable three-dimensional structure, IDRs exist in a collection of interconverting states known as an ensemble. This structural heterogeneity means that IDRs are largely absent from the Protein Data Bank, contributing to a lack of computational approaches to predict ensemble conformational properties from sequence. Here we combine rational sequence design, large-scale molecular simulations and deep learning to develop ALBATROSS, a deep-learning model for predicting ensemble dimensions of IDRs, including the radius of gyration, end-to-end distance, polymer-scaling exponent and ensemble asphericity, directly from sequences at a proteome-wide scale. ALBATROSS is lightweight, easy to use and accessible as both a locally installable software package and a point-and-click-style interface via Google Colab notebooks. We first demonstrate the applicability of our predictors by examining the generalizability of sequence-ensemble relationships in IDRs. Then, we leverage the high-throughput nature of ALBATROSS to characterize the sequence-specific biophysical behavior of IDRs within and between proteomes.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Proteínas Intrínsecamente Desordenadas/química , Conformación Proteica , PolímerosRESUMEN
The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. The auxin response factor (ARF) transcription factor family regulates auxin-responsive gene expression and exhibits nuclear localization in regions of high auxin responsiveness. Here we show that the ARF7 and ARF19 proteins accumulate in micron-sized assemblies within the cytoplasm of tissues with attenuated auxin responsiveness. We found that the intrinsically disordered middle region and the folded PB1 interaction domain of ARFs drive protein assembly formation. Mutation of a single lysine within the PB1 domain abrogates cytoplasmic assemblies, promotes ARF nuclear localization, and results in an altered transcriptome and morphological defects. Our data suggest a model in which ARF nucleo-cytoplasmic partitioning regulates auxin responsiveness, providing a mechanism for cellular competence for auxin signaling.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efectos de los fármacos , Ácidos Indolacéticos/farmacología , Proteínas Intrínsecamente Desordenadas/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Factores de Transcripción/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Unión Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Post-translational modifications of intrinsically disordered regions (IDRs) enable changes in sequence chemistry, which in turn can tune conformational behavior and molecular interactions. In this issue of The EMBO Journal, Gruijs da Silva et al disentangle the effect of hyperphosphorylation on the C-terminal domain of TDP-43, a key IDR implicated in Amyotrophic Lateral Sclerosis (ALS).
Asunto(s)
Esclerosis Amiotrófica Lateral , Proteínas de Unión al ADN , Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/química , Humanos , SolubilidadRESUMEN
The SARS-CoV-2 Nucleocapsid (N) protein is responsible for condensation of the viral genome. Characterizing the mechanisms controlling nucleic acid binding is a key step in understanding how condensation is realized. Here, we focus on the role of the RNA binding domain (RBD) and its flanking disordered N-terminal domain (NTD) tail, using single-molecule Förster Resonance Energy Transfer and coarse-grained simulations. We quantified contact site size and binding affinity for nucleic acids and concomitant conformational changes occurring in the disordered region. We found that the disordered NTD increases the affinity of the RBD for RNA by about 50-fold. Binding of both nonspecific and specific RNA results in a modulation of the tail configurations, which respond in an RNA length-dependent manner. Not only does the disordered NTD increase affinity for RNA, but mutations that occur in the Omicron variant modulate the interactions, indicating a functional role of the disordered tail. Finally, we found that the NTD-RBD preferentially interacts with single-stranded RNA and that the resulting protein:RNA complexes are flexible and dynamic. We speculate that this mechanism of interaction enables the Nucleocapsid protein to search the viral genome for and bind to high-affinity motifs.
Asunto(s)
Proteínas de la Nucleocápside de Coronavirus , ARN Viral , SARS-CoV-2 , Humanos , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas de la Nucleocápside de Coronavirus/metabolismo , COVID-19/virología , Proteínas de la Nucleocápside/química , Unión Proteica , ARN Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismoRESUMEN
Single-stranded DNA (ssDNA) intermediates which emerge during DNA metabolic processes are shielded by replication protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper to direct the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent functional specificity requires knowledge of the structural conformation of ssDNA when RPA-bound. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and double electron-electron resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.
RESUMEN
MOTIVATION: The emergence of high-throughput experiments and high-resolution computational predictions has led to an explosion in the quality and volume of protein sequence annotations at proteomic scales. Unfortunately, sanity checking, integrating, and analyzing complex sequence annotations remains logistically challenging and introduces a major barrier to entry for even superficial integrative bioinformatics. RESULTS: To address this technical burden, we have developed SHEPHARD, a Python framework that trivializes large-scale integrative protein bioinformatics. SHEPHARD combines an object-oriented hierarchical data structure with database-like features, enabling programmatic annotation, integration, and analysis of complex datatypes. Importantly SHEPHARD is easy to use and enables a Pythonic interrogation of largescale protein datasets with millions of unique annotations. We use SHEPHARD to examine three orthogonal proteome-wide questions relating protein sequence to molecular function, illustrating its ability to uncover novel biology. AVAILABILITY AND IMPLEMENTATION: We provided SHEPHARD as both a stand-alone software package (https://github.com/holehouse-lab/shephard), and as a Google Colab notebook with a collection of precomputed proteome-wide annotations (https://github.com/holehouse-lab/shephard-colab).