Supercoiled DNA and non-equilibrium formation of protein complexes: A quantitative model of the nucleoprotein ParBS partition complex

ParABS, the most widespread bacterial DNA segregation system, is composed of a centromeric sequence, parS, and two proteins, the ParA ATPase and the ParB DNA binding proteins. Hundreds of ParB proteins assemble dynamically to form nucleoprotein parS-anchored complexes that serve as substrates for ParA molecules to catalyze positioning and segregation events. The exact nature of this ParBS complex has remained elusive, what we address here by revisiting the Stochastic Binding model (SBM) introduced to explain the non-specific binding profile of ParB in the vicinity of parS. In the SBM, DNA loops stochastically bring loci inside a sharp cluster of ParB. However, previous SBM versions did not include the negative supercoiling of bacterial DNA, leading to use unphysically small DNA persistences to explain the ParB binding profiles. In addition, recent super-resolution microscopy experiments have revealed a ParB cluster that is significantly smaller than previous estimations and suggest that it results from a liquid-liquid like phase separation. Here, by simulating the folding of long (≥ 30 kb) supercoiled DNA molecules calibrated with realistic DNA parameters and by considering different possibilities for the physics of the ParB cluster assembly, we show that the SBM can quantitatively explain the ChIP-seq ParB binding profiles without any fitting parameter, aside from the supercoiling density of DNA, which, remarkably, is in accord with independent measurements. We also predict that ParB assembly results from a non-equilibrium, stationary balance between an influx of produced proteins and an outflux of excess proteins, i.e., ParB clusters behave like liquid-like protein condensates with unconventional “leaky” boundaries.

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A quantitative view of strategies to engineer cell-selective ligand binding

10.1101/2020.11.25.398974 ◽

2020 ◽

Author(s):

Zhixin Cyrillus Tan ◽

Brian Orcutt-Jahns ◽

Aaron S. Meyer

Keyword(s):

Specific Binding ◽

Quantitative Model ◽

Design Guidelines ◽

Target Cells ◽

List Type ◽

Binding Model ◽

Selective Ligand ◽

Unique Cell ◽

Development Literature ◽

Enhanced Selectivity

AbstractA critical property of many therapies is their selective binding to specific target populations. Exceptional specificity can arise from high-affinity binding to unique cell surface targets. In many cases, however, therapeutic targets are only expressed at subtly different levels relative to off-target cells. More complex binding strategies have been developed to overcome this limitation, including multi-specific and multi-valent molecules, but these create a combinatorial explosion of design possibilities. Therefore, guiding strategies for developing cell-specific binding are critical to employ these tools. Here, we extend a multi-valent binding model to multi-ligand and multi-receptor interactions. Using this model, we explore a series of mechanisms to engineer cell selectivity, including mixtures of molecules, affinity adjustments, and valency changes. Each of these strategies maximizes selectivity in distinct cases, leading to synergistic improvements when used in combination. Finally, we identify situations in which selectivity cannot be derived through passive binding alone to highlight areas in need of new developments. In total, this work uses a quantitative model to unify a comprehensive set of design guidelines for engineering cell-specific therapies.Summary pointsAffinity, valency, and other alterations to target cell binding provide enhanced selectivity in specific situations.Evidence for the effectiveness and limitations of each strategy are abundant within the drug development literature.Combining strategies can offer enhanced selectivity.A simple, multivalent ligand-receptor binding model can help to direct therapeutic engineering.

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Bacterial DNA topoisomerase I can relax positively supercoiled DNA containing a single-stranded loop

Journal of Molecular Biology ◽

10.1016/0022-2836(85)90075-0 ◽

1985 ◽

Vol 185 (3) ◽

pp. 625-637 ◽

Cited By ~ 115

Author(s):

Karla Kirkegaard ◽

James C. Wang

Keyword(s):

Topoisomerase I ◽

Dna Topoisomerase I ◽

Dna Topoisomerase ◽

Supercoiled Dna ◽

Bacterial Dna

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Metallocluster transactions: dynamic protein interactions guide the biosynthesis of Fe–S clusters in bacteria

Biochemical Society Transactions ◽

10.1042/bst20180365 ◽

2018 ◽

Vol 46 (6) ◽

pp. 1593-1603 ◽

Cited By ~ 9

Author(s):

Chenkang Zheng ◽

Patricia C. Dos Santos

Keyword(s):

Protein Interactions ◽

Protein Complexes ◽

Specific Protein ◽

Biological Synthesis ◽

Cluster Assembly ◽

Sequence Motifs ◽

Protein Protein Interactions ◽

Cysteine Desulfurase ◽

Wide Range ◽

Domains Of Life

Iron–sulfur (Fe–S) clusters are ubiquitous cofactors present in all domains of life. The chemistries catalyzed by these inorganic cofactors are diverse and their associated enzymes are involved in many cellular processes. Despite the wide range of structures reported for Fe–S clusters inserted into proteins, the biological synthesis of all Fe–S clusters starts with the assembly of simple units of 2Fe–2S and 4Fe–4S clusters. Several systems have been associated with the formation of Fe–S clusters in bacteria with varying phylogenetic origins and number of biosynthetic and regulatory components. All systems, however, construct Fe–S clusters through a similar biosynthetic scheme involving three main steps: (1) sulfur activation by a cysteine desulfurase, (2) cluster assembly by a scaffold protein, and (3) guided delivery of Fe–S units to either final acceptors or biosynthetic enzymes involved in the formation of complex metalloclusters. Another unifying feature on the biological formation of Fe–S clusters in bacteria is that these systems are tightly regulated by a network of protein interactions. Thus, the formation of transient protein complexes among biosynthetic components allows for the direct transfer of reactive sulfur and Fe–S intermediates preventing oxygen damage and reactions with non-physiological targets. Recent studies revealed the importance of reciprocal signature sequence motifs that enable specific protein–protein interactions and consequently guide the transactions between physiological donors and acceptors. Such findings provide insights into strategies used by bacteria to regulate the flow of reactive intermediates and provide protein barcodes to uncover yet-unidentified cellular components involved in Fe–S metabolism.

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A particle size threshold governs diffusion and segregation of PAR-3 during cell polarization

10.1101/2021.06.07.447386 ◽

2021 ◽

Author(s):

Yiran Chang ◽

Danie J Dickinson

Keyword(s):

Cell Biology ◽

Protein Complexes ◽

Quantitative Model ◽

Cell Polarization ◽

Protein Distribution ◽

Cell Stage ◽

Viscous Forces ◽

Dependent Protein ◽

Regulate Protein ◽

Size Threshold

Regulation of subcellular components' localization and motion is a critical theme in cell biology. Cells use the actomyosin cortex to regulate protein distribution on the plasma membrane, but the interplay between membrane binding, cortical movements and protein distribution remains poorly understood. In a polarizing one-cell stage Caenorhabditis elegans embryo, actomyosin flows transport PAR protein complexes into an anterior cortical domain to establish the anterior-posterior axis of the animal. Oligomerization of a key scaffold protein, PAR-3, is required for aPAR cortical localization and segregation. Although PAR-3 oligomerization is essential for polarization, it remains unclear how oligomer size contributes to aPAR segregation because PAR-3 oligomers are a heterogeneous population of many different sizes. To address this question, we engineered PAR-3 to defined sizes. We report that PAR-3 trimers are necessary and sufficient for PAR-3 function during polarization and later embryo development, while larger PAR-3 clusters are dispensable. Quantitative analysis of PAR-3 diffusion showed that PAR-3 clusters larger than a trimer are transported by viscous forces without being physically captured by the actomyosin cortex. Our study provides a quantitative model for size-dependent protein transportation of membrane proteins by cortical flow.

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Multi-omics profiling of U2AF1 mutants dissects pathogenic mechanisms affecting RNA granules in myeloid malignancies

10.1101/2021.04.22.441020 ◽

2021 ◽

Author(s):

Giulia Biancon ◽

Poorval Joshi ◽

Joshua T Zimmer ◽

Torben Hunck ◽

Yimeng Gao ◽

...

Keyword(s):

Rna Binding ◽

Specific Binding ◽

Aberrant Splicing ◽

Binding Model ◽

Full Spectrum ◽

Myeloid Malignancies ◽

Rna Granules ◽

Rna Biology ◽

Site Recognition

AbstractSomatic mutations in splicing factors are of significant interest in myeloid malignancies and other cancers. U2AF1, together with U2AF2, is essential for 3’ splice site recognition. U2AF1 mutations result in aberrant splicing, but the molecular mechanism and the full spectrum of consequences on RNA biology have not been fully elucidated to date. We performed multi-omics profiling of in vivo RNA binding, splicing and turnover for U2AF1 S34F and Q157R mutants. We dissected specific binding signals of U2AF1 and U2AF2 and showed that U2AF1 mutations individually alter U2AF1-RNA binding, resulting in defective U2AF2 recruitment. We demonstrated a complex relationship between differential binding and splicing, expanding upon the currently accepted loss-of-binding model. Finally, we observed that U2AF1 mutations increase the formation of stress granules in both cell lines and primary acute myeloid leukemia samples. Our results uncover U2AF1 mutation-dependent pathogenic RNA mechanisms and provide the basis for developing targeted therapeutic strategies.

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Myosins generate contractile force and maintain organization in the cytokinetic contractile ring

10.1101/2021.05.02.442363 ◽

2021 ◽

Author(s):

Zachary A. McDargh ◽

Shuyuan Wang ◽

Harvey F. Chin ◽

Sathish Thiyagarajan ◽

Erdem Karatekin ◽

...

Keyword(s):

Fission Yeast ◽

Striated Muscle ◽

Protein Complexes ◽

Myosin Ii ◽

Force Production ◽

Super Resolution ◽

Building Blocks ◽

Contractile Ring ◽

Ring Model ◽

Self Assembling

During cytokinesis, cells assemble an actomyosin contractile ring whose tension constricts and divides cells, but the ring tension was rarely measured. Actomyosin force generation is well understood for the regular sarcomeric architecture of striated muscle, but recent super-resolution studies of fission yeast contractile rings revealed organizational building blocks that are not sarcomeres but irregularly positioned plasma membrane-anchored protein complexes called nodes. Here, we measured contractile ring tensions in fission yeast protoplast cells. The myosin II isoforms Myo2 and Myp2 generated the tension, with a ~2-fold greater contribution from Myo2. Simulations of a molecularly detailed ring model revealed a sliding node mechanism for tension, where nodes hosting tense actin filaments were pulled bidirectionally around the ring. Myo2 and Myp2 chaperoned self-assembling components into the ring organization, and anchored the ring against bridging instabilities. Thus, beyond force production, Myo2 and Myp2 are the principal organizers, bundlers and anchors of the contractile ring.

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Structure and promoter characterization of the gene encoding the large subunit (R1 protein) of mouse ribonucleotide reductase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.90.23.11322 ◽

1993 ◽

Vol 90 (23) ◽

pp. 11322-11326 ◽

Cited By ~ 20

Author(s):

S Björklund ◽

K Hjortsberg ◽

E Johansson ◽

L Thelander

Keyword(s):

Ribonucleotide Reductase ◽

Genomic Library ◽

Protein Complexes ◽

Specific Binding ◽

Transcriptional Start Site ◽

Large Subunit ◽

Mobility Shift ◽

Direct Role ◽

Encoding Gene ◽

R1 Gene

Mammalian ribonucleotide reductase (EC 1.17.4.1) is composed of two nonidentical subunits, proteins R1 and R2, both required for enzyme activity. The structure of the genomic mouse ribonucleotide reductase R1 gene was compiled from a number of overlapping lambda clones isolated from a Charon 4A mouse sperm genomic library. The R1-encoding gene covers 26 kb and consists of 19 exons. All exon-intron boundaries were located by dideoxynucleotide sequencing, showing that intron 7 starts with the variant GC instead of GT. About 3.5 kb of DNA from the 5'-flanking region of the R1-encoding gene were cloned and sequenced, and the transcriptional start site was determined by nuclease S1 mapping of RNA. DNase I footprinting assays on the R1 promoter identified two nearly identical 23-bp-long protein-binding regions. Three protein complexes binding to one of the 23-mer regions were resolved and partially identified by using gel-retardation mobility-shift assays and UV crosslinking. One complex most likely contained Sp1, and another complex showed S-phase-specific binding, suggesting a direct role in the cell-cycle-dependent R1 gene expression.

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Specific binding sites for oestrogen—receptor-protein complexes in human myometrial nuclei

Biochemical Society Transactions ◽

10.1042/bst0130177 ◽

1985 ◽

Vol 13 (1) ◽

pp. 177-178

Author(s):

ALAN SCOBBIE ◽

ROBIN LEAKE

Keyword(s):

Binding Sites ◽

Oestrogen Receptor ◽

Protein Complexes ◽

Specific Binding ◽

Receptor Protein ◽

Specific Binding Sites

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Nuclear Pore Scaffold Structure Analyzed by Super-Resolution Microscopy and Particle Averaging

Science ◽

10.1126/science.1240672 ◽

2013 ◽

Vol 341 (6146) ◽

pp. 655-658 ◽

Cited By ~ 274

Author(s):

Anna Szymborska ◽

Alex de Marco ◽

Nathalie Daigle ◽

Volker C. Cordes ◽

John A. G. Briggs ◽

...

Keyword(s):

Protein Complexes ◽

Super Resolution ◽

Molecular Organization ◽

Nuclear Pore ◽

Protein Assemblies ◽

Large Protein ◽

Whole Cells ◽

Molecular Machinery ◽

Scaffold Structure ◽

Super Resolution Microscopy

Much of life’s essential molecular machinery consists of large protein assemblies that currently pose challenges for structure determination. A prominent example is the nuclear pore complex (NPC), for which the organization of its individual components remains unknown. By combining stochastic super-resolution microscopy, to directly resolve the ringlike structure of the NPC, with single particle averaging, to use information from thousands of pores, we determined the average positions of fluorescent molecular labels in the NPC with a precision well below 1 nanometer. Applying this approach systematically to the largest building block of the NPC, the Nup107-160 subcomplex, we assessed the structure of the NPC scaffold. Thus, light microscopy can be used to study the molecular organization of large protein complexes in situ in whole cells.

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