RESUMO
The compaction of chromatin into mitotic chromosomes is essential for faithful transmission of the genome during cell division. In eukaryotes, chromosome morphogenesis is regulated by the condensin complex, though the exact mechanism used to target condensin to chromatin and initiate condensation is not understood. Here, we reveal that condensin contains an intrinsically disordered region (IDR) that modulates its association with chromatin in early mitosis and exhibits phase separation. We describe DNA-binding motifs within the IDR that, upon deletion, inflict striking defects in chromosome condensation and segregation, ill-timed condensin turnover on chromatin, and cell death. Importantly, we demonstrate that the condensin IDR can impart cell cycle regulatory functions when transferred to other subunits within the complex, indicating its autonomous nature. Collectively, our study unveils the molecular basis for the initiation of chromosome condensation in early mitosis and how this process ultimately promotes genomic stability and faultless cell division.
Assuntos
Adenosina Trifosfatases , Proteínas de Ligação a DNA , Mitose , Complexos Multiproteicos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Complexos Multiproteicos/metabolismo , Adenosina Trifosfatases/metabolismo , Cromatina/metabolismo , DNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Cromossomos/metabolismo , Ligação Proteica , Segregação de Cromossomos , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Biomolecular condensates can influence cellular function in a number of ways, including by changing the structural dynamics and conformational equilibria of the molecules partitioned within them. Here we use methyl transverse relaxation optimized spectroscopy (methyl-TROSY) NMR in conjunction with 2'-O-methyl labeling of RNA to characterize the thermodynamics and kinetics of RNA-RNA base pairing in condensates formed by the C-terminal intrinsically disordered region of CAPRIN1, an RNA-binding protein involved in RNA transport, translation, and stability. CAPRIN1 condensates destabilize RNA-RNA base pairing, resulting from a â¼270-fold decrease and a concomitant â¼15-fold increase in the on- and off-rates for duplex formation, respectively. The â¼30-fold slower diffusion of RNA single strands within the condensed phase partially accounts for the reduced on-rate, but the further â¼9-fold reduction likely reflects shedding of CAPRIN1 chains that are interacting with the RNA prior to hybridization. Our study emphasizes the important role of protein solvation in modulating nucleic acid recognition processes inside condensates.
Assuntos
Hibridização de Ácido Nucleico , RNA , Termodinâmica , RNA/química , Cinética , Conformação de Ácido Nucleico , Pareamento de Bases , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Separação de FasesRESUMO
The ability to sense and respond to osmotic fluctuations is critical for the maintenance of cellular integrity. We used gene co-essentiality analysis to identify an unappreciated relationship between TSC22D2, WNK1, and NRBP1 in regulating cell volume homeostasis. All of these genes have paralogs and are functionally buffered for osmo-sensing and cell volume control. Within seconds of hyperosmotic stress, TSC22D, WNK, and NRBP family members physically associate into biomolecular condensates, a process that is dependent on intrinsically disordered regions (IDRs). A close examination of these protein families across metazoans revealed that TSC22D genes evolved alongside a domain in NRBPs that specifically binds to TSC22D proteins, which we have termed NbrT (NRBP binding region with TSC22D), and this co-evolution is accompanied by rapid IDR length expansion in WNK-family kinases. Our study reveals that TSC22D, WNK, and NRBP genes evolved in metazoans to co-regulate rapid cell volume changes in response to osmolarity.
Assuntos
Tamanho Celular , Proteína Quinase 1 Deficiente de Lisina WNK , Humanos , Animais , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/genética , Evolução Molecular , Células HEK293 , Ligação Proteica , Família Multigênica , Pressão OsmóticaRESUMO
In his commentary in this issue of Molecular Cell,1 Struhl reasons that the term "intrinsically disordered regions" represents a vague and confusing concept for protein function. However, the term "intrinsically disordered" highlights the important physicochemical characteristic of conformational heterogeneity. Thus, "intrinsically disordered" is the counterpart to the term "folded, " with neither term having specific functional implications.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/metabolismo , Conformação ProteicaRESUMO
Poly(ADP-ribose) polymerase 1 (PARP1) is one of the first responders to DNA damage and plays crucial roles in recruiting DNA repair proteins through its activity - poly(ADP-ribosyl)ation (PARylation). The enrichment of DNA repair proteins at sites of DNA damage has been described as the formation of a biomolecular condensate. However, it is not understood how PARP1 and PARylation contribute to the formation and organization of DNA repair condensates. Using recombinant human PARP1 in vitro, we find that PARP1 readily forms viscous biomolecular condensates in a DNA-dependent manner and that this depends on its three zinc finger (ZnF) domains. PARylation enhances PARP1 condensation in a PAR chain-length dependent manner and increases the internal dynamics of PARP1 condensates. DNA and single-strand break repair proteins XRCC1, LigIII, Polß, and FUS partition in PARP1 condensates, although in different patterns. While Polß and FUS are both homogeneously mixed within PARP1 condensates, FUS enrichment is greatly enhanced upon PARylation whereas Polß partitioning is not. XRCC1 and LigIII display an inhomogeneous organization within PARP1 condensates; their enrichment in these multiphase condensates is enhanced by PARylation. Functionally, PARP1 condensates concentrate short DNA fragments and facilitate compaction of long DNA and bridge DNA ends. Furthermore, the presence of PARP1 condensates significantly promotes DNA ligation upon PARylation. These findings provide insight into how PARP1 condensation and PARylation regulate the assembly and biochemical activities in DNA repair foci, which may inform on how PARPs function in other PAR-driven condensates.
RESUMO
Nucleosomes, the basic structural units of chromatin, hinder recruitment and activity of various DNA repair proteins, necessitating modifications that enhance DNA accessibility. Poly(ADP-ribosyl)ation (PARylation) of proteins near damage sites is an essential initiation step in several DNA-repair pathways; however, its effects on nucleosome structural dynamics and organization are unclear. Using NMR, cryoelectron microscopy (cryo-EM), and biochemical assays, we show that PARylation enhances motions of the histone H3 tail and DNA, leaving the configuration of the core intact while also stimulating nuclease digestion and ligation of nicked nucleosomal DNA by LIG3. PARylation disrupted interactions between nucleosomes, preventing self-association. Addition of LIG3 and XRCC1 to PARylated nucleosomes generated condensates that selectively partition DNA repair-associated proteins in a PAR- and phosphorylation-dependent manner in vitro. Our results establish that PARylation influences nucleosomes across different length scales, extending from the atom-level motions of histone tails to the mesoscale formation of condensates with selective compositions.
Assuntos
Nucleossomos , Poli ADP Ribosilação , Nucleossomos/genética , Poli ADP Ribosilação/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Microscopia Crioeletrônica , Condensados Biomoleculares , Reparo do DNA , Histonas/genética , Histonas/metabolismo , DNA/genética , DNA/metabolismo , Dano ao DNA , Poli(ADP-Ribose) Polimerase-1/metabolismoRESUMO
SUMMARY: The Local Disordered Region Sampling (LDRS, pronounced loaders) tool is a new module developed for IDPConformerGenerator, a previously validated approach to model intrinsically disordered proteins (IDPs). The IDPConformerGenerator LDRS module provides a method for generating all-atom conformations of intrinsically disordered protein regions at N- and C-termini of and in loops or linkers between folded regions of an existing protein structure. These disordered elements often lead to missing coordinates in experimental structures or low confidence in predicted structures. Requiring only a pre-existing PDB or mmCIF formatted structural template of the protein with missing coordinates or with predicted confidence scores and its full-length primary sequence, LDRS will automatically generate physically meaningful conformational ensembles of the missing flexible regions to complete the full-length protein. The capabilities of the LDRS tool of IDPConformerGenerator include modeling phosphorylation sites using enhanced Monte Carlo-Side Chain Entropy, transmembrane proteins within an all-atom bilayer, and multi-chain complexes. The modeling capacity of LDRS capitalizes on the modularity, the ability to be used as a library and via command-line, and the computational speed of the IDPConformerGenerator platform. AVAILABILITY AND IMPLEMENTATION: The LDRS module is part of the IDPConformerGenerator modeling suite, which can be downloaded from GitHub at https://github.com/julie-forman-kay-lab/IDPConformerGenerator. IDPConformerGenerator is written in Python3 and works on Linux, Microsoft Windows, and Mac OS versions that support DSSP. Users can utilize LDRS's Python API for scripting the same way they can use any part of IDPConformerGenerator's API, by importing functions from the "idpconfgen.ldrs_helper" library. Otherwise, LDRS can be used as a command line interface application within IDPConformerGenerator. Full documentation is available within the command-line interface as well as on IDPConformerGenerator's official documentation pages (https://idpconformergenerator.readthedocs.io/en/latest/).
Assuntos
Proteínas Intrinsicamente Desordenadas , Software , Biblioteca Gênica , Proteínas de Membrana , DocumentaçãoRESUMO
Accurate potential energy models of proteins must describe the many different types of noncovalent interactions that contribute to a protein's stability and structure. Pi-pi contacts are ubiquitous structural motifs in all proteins, occurring between aromatic and nonaromatic residues and play a nontrivial role in protein folding and in the formation of biomolecular condensates. Guided by a geometric criterion for isolating pi-pi contacts from classical molecular dynamics simulations of proteins, we use quantum mechanical energy decomposition analysis to determine the molecular interactions that stabilize different pi-pi contact motifs. We find that neutral pi-pi interactions in proteins are dominated by Pauli repulsion and London dispersion rather than repulsive quadrupole electrostatics, which is central to the textbook Hunter-Sanders model. This results in a notable lack of variability in the interaction profiles of neutral pi-pi contacts even with extreme changes in the dielectric medium, explaining the prevalence of pi-stacked arrangements in and between proteins. We also find interactions involving pi-containing anions and cations to be extremely malleable, interacting like neutral pi-pi contacts in polar media and like typical ion-pi interactions in nonpolar environments. Like-charged pairs such as arginine-arginine contacts are particularly sensitive to the polarity of their immediate surroundings and exhibit canonical pi-pi stacking behavior only if the interaction is mediated by environmental effects, such as aqueous solvation.
RESUMO
The AlphaFold Protein Structure Database contains predicted structures for millions of proteins. For the majority of human proteins that contain intrinsically disordered regions (IDRs), which do not adopt a stable structure, it is generally assumed that these regions have low AlphaFold2 confidence scores that reflect low-confidence structural predictions. Here, we show that AlphaFold2 assigns confident structures to nearly 15% of human IDRs. By comparison to experimental NMR data for a subset of IDRs that are known to conditionally fold (i.e., upon binding or under other specific conditions), we find that AlphaFold2 often predicts the structure of the conditionally folded state. Based on databases of IDRs that are known to conditionally fold, we estimate that AlphaFold2 can identify conditionally folding IDRs at a precision as high as 88% at a 10% false positive rate, which is remarkable considering that conditionally folded IDR structures were minimally represented in its training data. We find that human disease mutations are nearly fivefold enriched in conditionally folded IDRs over IDRs in general and that up to 80% of IDRs in prokaryotes are predicted to conditionally fold, compared to less than 20% of eukaryotic IDRs. These results indicate that a large majority of IDRs in the proteomes of human and other eukaryotes function in the absence of conditional folding, but the regions that do acquire folds are more sensitive to mutations. We emphasize that the AlphaFold2 predictions do not reveal functionally relevant structural plasticity within IDRs and cannot offer realistic ensemble representations of conditionally folded IDRs.
Assuntos
Proteínas Intrinsicamente Desordenadas , Humanos , Proteínas Intrinsicamente Desordenadas/química , Eucariotos/metabolismo , Conformação ProteicaRESUMO
The intrinsically disordered 4E-BP2 protein regulates mRNA cap-dependent translation through interaction with the predominantly folded eukaryotic initiation factor 4E (eIF4E). Phosphorylation of 4E-BP2 dramatically reduces the level of eIF4E binding, in part by stabilizing a binding-incompatible folded domain. Here, we used a Rosetta-based sampling algorithm optimized for IDRs to generate initial ensembles for two phospho forms of 4E-BP2, non- and 5-fold phosphorylated (NP and 5P, respectively), with the 5P folded domain flanked by N- and C-terminal IDRs (N-IDR and C-IDR, respectively). We then applied an integrative Bayesian approach to obtain NP and 5P conformational ensembles that agree with experimental data from nuclear magnetic resonance, small-angle X-ray scattering, and single-molecule Förster resonance energy transfer (smFRET). For the NP state, inter-residue distance scaling and 2D maps revealed the role of charge segregation and pi interactions in driving contacts between distal regions of the chain (â¼70 residues apart). The 5P ensemble shows prominent contacts of the N-IDR region with the two phosphosites in the folded domain, pT37 and pT46, and, to a lesser extent, delocalized interactions with the C-IDR region. Agglomerative hierarchical clustering led to partitioning of each of the two ensembles into four clusters with different global dimensions and contact maps. This helped delineate an NP cluster that, based on our smFRET data, is compatible with the eIF4E-bound state. 5P clusters were differentiated by interactions of C-IDR with the folded domain and of the N-IDR with the two phosphosites in the folded domain. Our study provides both a better visualization of fundamental structural poses of 4E-BP2 and a set of falsifiable insights on intrachain interactions that bias folding and binding of this protein.
Assuntos
Fator de Iniciação 4E em Eucariotos , Proteínas Intrinsicamente Desordenadas , Teorema de Bayes , Análise por Conglomerados , AlgoritmosRESUMO
The Local Disordered Region Sampling (LDRS, pronounced loaders) tool, developed for the IDPConformerGenerator platform (Teixeira et al. 2022), provides a method for generating all-atom conformations of intrinsically disordered regions (IDRs) at N- and C-termini of and in loops or linkers between folded regions of an existing protein structure. These disordered elements often lead to missing coordinates in experimental structures or low confidence in predicted structures. Requiring only a pre-existing PDB structure of the protein with missing coordinates or with predicted confidence scores and its full-length primary sequence, LDRS will automatically generate physically meaningful conformational ensembles of the missing flexible regions to complete the full-length protein. The capabilities of the LDRS tool of IDPConformerGenerator include modeling phosphorylation sites using enhanced Monte Carlo Side Chain Entropy (MC-SCE) (Bhowmick and Head-Gordon 2015), transmembrane proteins within an all-atom bilayer, and multi-chain complexes. The modeling capacity of LDRS capitalizes on the modularity, ability to be used as a library and via command-line, and computational speed of the IDPConformerGenerator platform.
RESUMO
The structural characterization of proteins with a disorder requires a computational approach backed by experiments to model their diverse and dynamic structural ensembles. The selection of conformational ensembles consistent with solution experiments of disordered proteins highly depends on the initial pool of conformers, with currently available tools limited by conformational sampling. We have developed a Generative Recurrent Neural Network (GRNN) that uses supervised learning to bias the probability distributions of torsions to take advantage of experimental data types such as nuclear magnetic resonance J-couplings, nuclear Overhauser effects, and paramagnetic resonance enhancements. We show that updating the generative model parameters according to the reward feedback on the basis of the agreement between experimental data and probabilistic selection of torsions from learned distributions provides an alternative to existing approaches that simply reweight conformers of a static structural pool for disordered proteins. Instead, the biased GRNN, DynamICE, learns to physically change the conformations of the underlying pool of the disordered protein to those that better agree with experiments.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas , Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Espectroscopia de Ressonância Magnética , Conformação Proteica , Proteínas Intrinsicamente Desordenadas/químicaRESUMO
As phase separation is found in an increasing variety of biological contexts, additional challenges have arisen in understanding the underlying principles of condensate formation and function. We spoke with researchers across disciplines about their views on the ever-changing landscape of biomolecular condensates.
Assuntos
Condensados Biomoleculares , Pesquisadores , Humanos , BiologiaRESUMO
O-GlcNAc transferase (OGT) is an essential glycosylating enzyme that catalyzes the addition of N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. The enzyme glycosylates a broad range of peptide sequences and the prediction of glycosylation sites has proven challenging. The lack of an experimentally verified set of polypeptide sequences that are not glycosylated by OGT has made prediction of legitimate glycosylation sites more difficult. Here, we tested a number of intrinsically disordered protein regions as substrates of OGT to establish a set of sequences that are not glycosylated by OGT. The negative data set suggests an amino acid compositional bias for OGT targets. This compositional bias was validated by modifying the amino acid composition of the protein fused in sarcoma (FUS) to enhance glycosylation. NMR experiments demonstrate that the tetratricopeptide repeat region of OGT can bind FUS and that glycosylation-promoting mutations enhance binding. These results provide evidence that the tetratricopeptide repeat region recognizes disordered segments of substrates with particular compositions to promote glycosylation, providing insight into the broad specificity of OGT.
Assuntos
N-Acetilglucosaminiltransferases , Aminoácidos/metabolismo , Glicosilação , Mutação , N-Acetilglucosaminiltransferases/metabolismo , Humanos , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biologia Computacional , Imageamento por Ressonância MagnéticaRESUMO
We consider a generic representation problem of internal coordinates (bond lengths, valence angles, and dihedral angles) and their transformation to 3-dimensional Cartesian coordinates of a biomolecule. We show that the internal-to-Cartesian process relies on correctly predicting chemically subtle correlations among the internal coordinates themselves, and learning these correlations increases the fidelity of the Cartesian representation. We developed a machine learning algorithm, Int2Cart, to predict bond lengths and bond angles from backbone torsion angles and residue types of a protein, which allows reconstruction of protein structures better than using fixed bond lengths and bond angles or a static library method that relies on backbone torsion angles and residue types in a local environment. The method is able to be used for structure validation, as we show that the agreement between Int2Cart-predicted bond geometries and those from an AlphaFold 2 model can be used to estimate model quality. Additionally, by using Int2Cart to reconstruct an IDP ensemble, we are able to decrease the clash rate during modeling. The Int2Cart algorithm has been implemented as a publicly accessible python package at https://github.com/THGLab/int2cart.
Assuntos
Algoritmos , Proteínas , Proteínas/química , Aprendizado de MáquinaRESUMO
Biomolecular condensates concentrate proteins, nucleic acids, and small molecules and play an essential role in many biological processes. Their formation is tuned by a balance between energetically favorable and unfavorable contacts, with charge-charge interactions playing a central role in some systems. The positively charged intrinsically disordered carboxy-terminal region of the RNA-binding protein CAPRIN1 is one such example, phase separating upon addition of negatively charged ATP or high concentrations of sodium chloride (NaCl). Using solution NMR spectroscopy, we measured residue-specific near-surface electrostatic potentials (ÏENS) of CAPRIN1 along its NaCl-induced phase separation trajectory to compare with those obtained using ATP. In both cases, electrostatic shielding decreases ÏENS values, yet surface potentials of CAPRIN1 in the two condensates can be different, depending on the amount of NaCl or ATP added. Our results establish that even small differences in ÏENS can significantly affect the level of protein enrichment and the mechanical properties of the condensed phase, leading, potentially, to the regulation of biological processes.
Assuntos
Hidrodinâmica , Proteínas Intrinsicamente Desordenadas , Proteínas de Ligação a RNA , Trifosfato de Adenosina , Proteínas Intrinsicamente Desordenadas/química , Proteínas de Ligação a RNA/química , Cloreto de Sódio/metabolismo , Eletricidade EstáticaRESUMO
The spatial and temporal organization of interactions between proteins underlie the regulation of most cellular processes. The requirement for such interactions to be specific predisposes a view that protein-protein interactions are relatively static and are formed through the stable complementarity of the interacting partners. A growing body of reports indicate, however, that many interactions lead to fuzzy complexes with an ensemble of conformations in dynamic exchange accounting for the observed binding. Here, we discuss how NMR has facilitated the characterization of these discrete, dynamic complexes and how such characterization has aided the understanding of dynamic, condensed phases of phase-separating proteins with exchanging multivalent interactions.
Assuntos
Fenômenos Bioquímicos , Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/químicaRESUMO
The precise regulation of gene expression is fundamental to neurodevelopment, plasticity and cognitive function. Although several studies have profiled transcription in the developing human brain, there is a gap in understanding of accompanying translational regulation. In this study, we performed ribosome profiling on 73 human prenatal and adult cortex samples. We characterized the translational regulation of annotated open reading frames (ORFs) and identified thousands of previously unknown translation events, including small ORFs that give rise to human-specific and/or brain-specific microproteins, many of which we independently verified using proteomics. Ribosome profiling in stem-cell-derived human neuronal cultures corroborated these findings and revealed that several neuronal activity-induced non-coding RNAs encode previously undescribed microproteins. Physicochemical analysis of brain microproteins identified a class of proteins that contain arginine-glycine-glycine (RGG) repeats and, thus, may be regulators of RNA metabolism. This resource expands the known translational landscape of the human brain and illuminates previously unknown brain-specific protein products.