Coupled structural transitions enable highly cooperative regulation of human CTPS2 filaments.
Ontology highlight
ABSTRACT:
SUBMITTER: Lynch EM
PROVIDER: S-EPMC6954954 | biostudies-literature | 2020 Jan
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Rad51 is a core component of the eukaryotic homologous recombination machinery and is responsible for key mechanistic steps during strand invasion. Higher order oligomers of Rad51 display a remarkable degree of structural variation, forming rings, compressed filaments, and elongated filaments. It is unclear whether Rad51 can transition directly between these different oligomeric structures without disassembling first into monomers. We have used single-molecule microscopy to investigate the behavior of human Rad51 assembled on double-stranded DNA. Our results show that human Rad51 can form elongated nucleoprotein filaments on DNA, but ATP hydrolysis causes a decrease in their length without concomitant dissociation of protein. Compressed Rad51 filaments can re-elongate when presented with either ATP or the non-hydrolyzable analog AMP-PNP, and these cycles of elongation and compression are reversible. A Rad51 mutant deficient in ATP hydrolysis is locked into an extended conformation that is incapable of transitioning to a compressed filament. Similarly, wild-type Rad51 bound to DNA in the presence of AMP-PNP was trapped in the elongated state. Proteins incapable of transitioning to the compressed state were also highly resistant to dissociation from the DNA. Taken together, our results indicate that nucleotide hydrolysis by human Rad51 triggers a reversible structural transition leading to filaments with reduced helical pitch.
| S-EPMC2722365 | biostudies-literature
Project description:Translin is a highly conserved RNA- and DNA-binding protein that plays essential roles in eukaryotic cells. Human translin functions as an octamer, but in the octameric crystallographic structure, the residues responsible for nucleic acid binding are not accessible. Moreover, electron microscopy data reveal very different octameric configurations. Consequently, the functional assembly and the mechanism of nucleic acid binding by the protein remain unclear. Here, we present an integrative study combining small-angle X-ray scattering (SAXS), site-directed mutagenesis, biochemical analysis and computational techniques to address these questions. Our data indicate a significant conformational heterogeneity for translin in solution, formed by a lesser-populated compact octameric state resembling the previously solved X-ray structure, and a highly populated open octameric state that had not been previously identified. On the other hand, our SAXS data and computational analyses of translin in complex with the RNA oligonucleotide (GU)12 show that the internal cavity found in the octameric assemblies can accommodate different nucleic acid conformations. According to this model, the nucleic acid binding residues become accessible for binding, which facilitates the entrance of the nucleic acids into the cavity. Our data thus provide a structural basis for the functions that translin performs in RNA metabolism and transport.
| S-EPMC3834833 | biostudies-literature
Project description:BackgroundPRPP synthase (PRPS) transfers the pyrophosphate groups from ATP to ribose-5-phosphate to produce 5-phosphate ribose-1-pyrophosphate (PRPP), a key intermediate in the biosynthesis of several metabolites including nucleotides, dinucleotides and some amino acids. There are three PRPS isoforms encoded in human genome. While human PRPS1 (hPRPS1) and human PRPS2 (hPRPS2) are expressed in most tissues, human PRPS3 (hPRPS3) is exclusively expressed in testis. Although hPRPS1 and hPRPS2 share 95% sequence identity, hPRPS2 has been shown to be less sensitive to allosteric inhibition and specifically upregulated in certain cancers in the translational level. Recent studies demonstrate that PRPS can form a subcellular compartment termed the cytoophidium in multiple organisms across prokaryotes and eukaryotes. Forming filaments and cytoophidia is considered as a distinctive mechanism involving the polymerization of the protein. Previously we solved the filament structures of Escherichia coli PRPS (ecPRPS) using cryo-electron microscopy (cryo-EM) 1.ResultsOrder to investigate the function and molecular mechanism of hPRPS2 polymerization, here we solve the polymer structure of hPRPS2 at 3.08 Å resolution. hPRPS2 hexamers stack into polymers in the conditions with the allosteric/competitive inhibitor ADP. The binding modes of ADP at the canonical allosteric site and at the catalytic active site are clearly determined. A point mutation disrupting the inter-hexamer interaction prevents hPRPS2 polymerization and results in significantly reduced catalytic activity.ConclusionFindings suggest that the regulation of hPRPS2 polymer is distinct from ecPRPS polymer and provide new insights to the regulation of hPRPS2 with structural basis.
| S-EPMC10227994 | biostudies-literature
Project description:The regulation of striated muscle contraction involves cooperative interactions between actin filaments, myosin-S1 (S1), tropomyosin (Tm), troponin (Tn), and calcium. These interactions are modeled by treating overlapping tropomyosins as a continuous flexible chain (CFC), weakly confined by electrostatic interactions with actin. The CFC is displaced locally in opposite directions on the actin surface by the binding of either S1 or Troponin I (TnI) to actin. The apparent rate constants for myosin and TnI binding to and detachment from actin are then intrinsically coupled via the CFC model to the presence of neighboring bound S1s and TnIs. Monte Carlo simulations at prescribed values of the CFC stiffness, the CFC's degree of azimuthal confinement, and the angular displacements caused by the bound proteins were able to predict the stopped-flow transients of S1 binding to regulated F-actin. The transients collected over a large range of calcium concentrations could be well described by adjusting a single calcium-dependent parameter, the rate constant of TnI detachment from actin, k(-I). The resulting equilibrium constant K(B) ? 1/K(I) varied sigmoidally with the free calcium, increasing from 0.12 at low calcium (pCa >7) to 12 at high calcium (pCa <5.5) with a Hill coefficient of ~2.15. The similarity of the curves for excess-actin and excess-myosin data confirms their allosteric relationship. The spatially explicit calculations confirmed variable sizes for the cooperative units and clustering of bound myosins at low calcium concentrations. Moreover, inclusion of negative cooperativity between myosin units predicted the observed slowing of myosin binding at excess-myosin concentrations.
| S-EPMC3509328 | biostudies-literature
Project description:Membrane transporters that clear the neurotransmitter glutamate from synapses are driven by symport of sodium ions and counter-transport of a potassium ion. Previous crystal structures of a homologous archaeal sodium and aspartate symporter showed that a dedicated transport domain carries the substrate and ions across the membrane. Here, we report new crystal structures of this homologue in ligand-free and ions-only bound outward- and inward-facing conformations. We show that after ligand release, the apo transport domain adopts a compact and occluded conformation that can traverse the membrane, completing the transport cycle. Sodium binding primes the transport domain to accept its substrate and triggers extracellular gate opening, which prevents inward domain translocation until substrate binding takes place. Furthermore, we describe a new cation-binding site ideally suited to bind a counter-transported ion. We suggest that potassium binding at this site stabilizes the translocation-competent conformation of the unloaded transport domain in mammalian homologues.DOI: http://dx.doi.org/10.7554/eLife.02283.001.
| S-EPMC4051121 | biostudies-literature
Project description:The three-dimensional (3D) organization of the genome within the cell nucleus contributes to cell-specific gene expression in different cell types1. High-throughput 3CM-bM-^@M-^Sderived methods have revealed that the genome is segmented into contiguous topologically associating domains (TADs), which help to orchestrate gene expression changes during differentiation and development2-5. Using ChIP-Seq, Hi-C and 3D modelling techniques, we reveal that TADs regulate the rapid gene expression changes induced by progestin in T47D breast cancer cells. In response to the hormone, TADs maintain their borders and operate as discrete regulatory units in which the majority of the genes are either transcriptionally activated or repressed. Additionally, the epigenetic signatures of the TADs are coordinately modified by hormone in correlation with the transcriptional changes. Hormone-induced changes in gene activity and chromatin remodelling are accompanied by structural changes that are distinct for activated or repressed TADs. Integrative 3D modelling revealed that TADs are structurally expanded if active and compacted if repressed, and that this is accompanied by differential changes in accessibility. We thus propose that TADs function as M-bM-^@M-^\regulonsM-bM-^@M-^] to enable spatially proximal genes to be coordinately transcribed in response to hormones. T47D-MTVL human breast cancer cells were incubated with the progestin R5020 for different times at 37M-BM-:C and prepared for ChIP-Seq or Hi-C according published protocols
2014-10-06 | E-GEOD-53463 | biostudies-arrayexpress
Project description:The morphological dynamics, instabilities, and transitions of elastic filaments in viscous flows underlie a wealth of biophysical processes from flagellar propulsion to intracellular streaming and are also key to deciphering the rheological behavior of many complex fluids and soft materials. Here, we combine experiments and computational modeling to elucidate the dynamical regimes and morphological transitions of elastic Brownian filaments in a simple shear flow. Actin filaments are used as an experimental model system and their conformations are investigated through fluorescence microscopy in microfluidic channels. Simulations matching the experimental conditions are also performed using inextensible Euler-Bernoulli beam theory and nonlocal slender-body hydrodynamics in the presence of thermal fluctuations and agree quantitatively with observations. We demonstrate that filament dynamics in this system are primarily governed by a dimensionless elasto-viscous number comparing viscous drag forces to elastic bending forces, with thermal fluctuations playing only a secondary role. While short and rigid filaments perform quasi-periodic tumbling motions, a buckling instability arises above a critical flow strength. A second transition to strongly deformed shapes occurs at a yet larger value of the elasto-viscous number and is characterized by the appearance of localized high-curvature bends that propagate along the filaments in apparent "snaking" motions. A theoretical model for the as yet unexplored onset of snaking accurately predicts the transition and explains the observed dynamics. We present a complete characterization of filament morphologies and transitions as a function of elasto-viscous number and scaled persistence length and demonstrate excellent agreement between theory, experiments, and simulations.
| S-EPMC6156685 | biostudies-literature
Project description:Forced expression of pro-neural transcription factors was shown to mediate direct neuronal conversion of human fibroblasts. Since neurons are postmitotic, the conversion efficiency represents an important parameter. Here we present a minimalist approach combining two factor neuronal programming with small molecule-based inhibition of GSK3ß and SMAD signaling, which gives rise to functional neuron-like cells (iNs) of various neurotransmitter phenotypes with an overall yield of up to >200% and a final neuronal purity of up to >80%. Timcourse of reprogramming of fibroblasts towards an neuronal phenotype in two independent fibroblast lines
2012-04-07 | E-GEOD-36246 | biostudies-arrayexpress
Project description:The three-dimensional (3D) organization of the genome within the cell nucleus contributes to cell-specific gene expression in different cell types1. High-throughput 3C–derived methods have revealed that the genome is segmented into contiguous topologically associating domains (TADs), which help to orchestrate gene expression changes during differentiation and development2-5. Using ChIP-Seq, Hi-C and 3D modelling techniques, we reveal that TADs regulate the rapid gene expression changes induced by progestin in T47D breast cancer cells. In response to the hormone, TADs maintain their borders and operate as discrete regulatory units in which the majority of the genes are either transcriptionally activated or repressed. Additionally, the epigenetic signatures of the TADs are coordinately modified by hormone in correlation with the transcriptional changes. Hormone-induced changes in gene activity and chromatin remodelling are accompanied by structural changes that are distinct for activated or repressed TADs. Integrative 3D modelling revealed that TADs are structurally expanded if active and compacted if repressed, and that this is accompanied by differential changes in accessibility. We thus propose that TADs function as “regulons” to enable spatially proximal genes to be coordinately transcribed in response to hormones.
2014-10-06 | GSE53463 | GEO
Project description:At the biochemical level, motor proteins are enzymatic molecules that function by converting chemical energy into mechanical motion. The key element for energy transduction and a major unresolved question common for all motor proteins is the coordination between the chemical and conformational steps in ATP hydrolysis. Here we show time-lapse monitoring of an in vitro ATP hydrolysis reaction by the motor domain of a human Kinesin-5 protein (Eg5) using difference Fourier transform infrared spectroscopy and UV photolysis of caged ATP. In this first continuous observation of a biological reaction coordinate from substrate to product, direct spectral markers for two catalytic events are measured: proton abstraction from nucleophilic water by the catalytic base and formation of the inorganic phosphate leaving group. Simultaneous examination of conformational switching in Eg5, in parallel with catalytic steps, shows structural transitions in solution consistent with published crystal structures of the prehydrolytic and ADP-bound states. In addition, we detect structural modifications in the Eg5 motor domain during bond cleavage between the beta- and gamma-phosphates of ATP. This conclusion challenges mechanochemical models for motor proteins that utilize only two stages of the catalytic cycle to drive force and motion.
| S-EPMC2856982 | biostudies-literature