Glutamate promotes SSB protein–protein Interactions via intrinsically disordered regions

Козлов, А. Г.; Shinn, Min Kyung; Weiland, Elizabeth; Lohman, Timothy M.

doi:10.1016/j.jmb.2017.07.021

Cited by 51 publications

(107 citation statements)

References 66 publications

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“…Previous studies suggested that the IDLs of adjacent SSB molecules can interact with each other to enhance the cooperativity of ssDNA binding, while the IDL was also proposed to interact with the OB fold Kozlov et al, 2015Kozlov et al, , 2017. Moreover, the central role played by similar low-sequence-complexity, Gln/Gly/Pro-rich IDRs in previously investigated LLPS systems (Bakthavachalu et al, 2018;Mannen et al, 2016;Milovanovic et al, 2018;Sabari et al, 2018) as well as the predicted LLPS propensities suggest a role for the IDL in SSB LLPS ( Fig.…”

Section: Multivalent Interactions Between Ssb Idl Regions Are Requirementioning

confidence: 73%

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Harami

Kovács

Pancsa

et al. 2019

Preprint

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Bacterial single stranded (ss) DNA-binding proteins (SSB) are essential for the replication and maintenance of the genome. SSBs share a conserved ssDNA-binding domain, a less conserved intrinsically disordered linker (IDL) and a highly conserved C-terminal peptide (CTP) motif that mediates a wide array of protein-protein interactions with DNA-metabolizing proteins. Here we show that the E. coli SSB protein forms liquid-liquid phase separated condensates in cellular-like conditions through multifaceted interactions involving all structural regions of the protein. SSB, ssDNA and SSBinteracting molecules are highly concentrated within the condensates, whereas phase separation is overall regulated by the stoichiometry of SSB and ssDNA. Together with recent results on subcellular SSB localization patterns, our results point to a conserved mechanism by which bacterial cells store a pool of SSB and SSB-interacting proteins. Dynamic phase separation enables rapid mobilization of this protein pool to protect exposed ssDNA and repair genomic loci affected by DNA damage. RESULTS SSB forms dynamic LLPS condensates in physiologically relevant conditions in vitroDue to the fact that SSB shares several features with hitherto described LLPS drivers, we sought to determine if LLPS could be a yet undiscovered capability of SSB facilitating its multifaceted roles in genome maintenance. We performed a bioinformatics analysis that revealed that E. coli SSB, in particular its IDL region, shows high propensity for LLPS, according to multiple dedicated sequencebased prediction methods (Bolognesi et al., 2016;Lancaster et al., 2014;Vernon et al., 2018) (Fig. 1B-D). This feature of SSB is universally conserved among bacteria, as the majority of analyzed SSBs (72.1 %) harbored by representative bacterial species from 15 large phylogenetic groups show similar LLPS propensities (Tables S1-2; Fig. 1E).In line with its predicted LLPS propensity, purified E. coli SSB (30 µM; SSB concentrations are expressed as those of tetramer molecules throughout the paper) forms an opaque, turbid solution at low NaCl concentration (50 mM) in vitro at room temperature and also at the native temperature of E. coli cells (37°C) ( Fig. 2A-B). The process is reversible as elevation of NaCl concentration to 200 mM leads to immediate loss of turbidity. The Clconcentration in E. coli cells depends on their environment (Schultz et al., 1962); however, while the exact value is not known, it can be assumed to be in a similar range (5-100 mM) as that measured for the cells of mammalian E. coli hosts, whereas the major metabolic anion in bacterial cells is glutamate (Glu; 100 mM) (Bennett et al., 2009). Strikingly, SSB forms a turbid solution even in 200 mM NaGlu ( Fig. 2A-B). (It must be noted that experiments using NaGlu always contained a fixed amount of 20 mM NaCl, for technical reasons.) Investigation of the protein solution by differential interference contrast (DIC) microscopy after diluting SSB into low-salt buffer revealed the formation of regular spherical drop...

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Section: Multivalent Interactions Between Ssb Idl Regions Are Requirementioning

confidence: 73%

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Harami

Kovács

Pancsa

et al. 2019

Preprint

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“…The N-terminal OB domain mediates both inter-protein interactions to form tetramers (which is referred to as EcSSB henceforth), as well as high-affinity DNA binding. EcSSB was shown to exhibit high cooperativity in certain ssDNA binding conformations, which is eliminated by truncating or replacing the IDL or TIP, as well as by mutating the "bridge interface" that links adjacent SSB tetramers through an evolutionarily conserved surface near the ssDNA-binding site (2,6,8,12,18,(20)(21)(22). EcSSB can bind ssDNA with multiple conformations that wrap the ssDNA substrates to different degrees (8,(23)(24)(25)(26).…”

Section: Introductionmentioning

confidence: 99%

“…Recent single molecule analyses have greatly enhanced the understanding of the binding dynamics of these distinct modes (18,20). These studies have revealed the dynamic equilibrium between well-defined EcSSB functional and structural states (29), and the ability of the tetramer to diffuse quickly along the ssDNA substrate while maintaining its wrapped conformation (30).…”

Section: Introductionmentioning

confidence: 99%

Self-regulation of single-stranded DNA wrapping dynamics byE. coliSSB promotes both stable binding and rapid dissociation

Naufer

Morse

Möller

et al. 2019

Preprint

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E. coli SSB (EcSSB) is a model protein for studying functions of single-stranded DNA (ssDNA) binding proteins (SSBs), which are critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations in order to protect these transiently formed regions during processes such as replication and repair. Using optical tweezers, we measure the binding and wrapping of a single long ssDNA substrate under various conditions and free protein concentrations. We show that EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as protein density on the substrate passes a critical saturation. Increasing free EcSSB concentrations increase the fraction of EcSSBs in less-wrapped conformations, including a previously uncharacterized EcSSB8 bound state in which ~8 nucleotides of ssDNA are bound by a single domain of the tetramer with minimal substrate deformation. When the ssDNA is over-saturated with EcSSB, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped EcSSB-ssDNA complexes. We develop a multi-step kinetic model in which EcSSB tetramers transition through multiple wrapped conformations which are regulated through nearest neighbor interactions and ssDNA occupancy. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers can be rapidly removed from an ssDNA substrate to allow for DNA maintenance and replication functions while still fully protecting ssDNA over a wide range of protein concentrations.

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“…Under low‐salt conditions (<20 m M NaCl, <1 m M MgCl 2 ), the (SSB) 35 mode is a highly cooperative binding mode in which SSB tetramers are clustered on ssDNA. However, highly cooperative binding of E. coli SSB to DNA also occurs at physiological salt and glutamate concentrations …”

Section: Single‐stranded Dna‐binding Proteinmentioning

confidence: 99%

“…However, highly cooperative binding of E. coli SSB to DNA also occurs at physiological salt and glutamate concentrations. 51 The structure of E. coli SSB in complex with dC 35 polymers revealed a "baseball seam" topology of the DNA [ Fig. 4(b)].…”

Section: Single-stranded Dna-binding Proteinmentioning

confidence: 99%

A structural view of bacterial DNA replication

Oakley

2019

Protein Science

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DNA replication mechanisms are conserved across all organisms. The proteins required to initiate, coordinate, and complete the replication process are best characterized in model organisms such as Escherichia coli. These include nucleotide triphosphate‐driven nanomachines such as the DNA‐unwinding helicase DnaB and the clamp loader complex that loads DNA‐clamps onto primer–template junctions. DNA‐clamps are required for the processivity of the DNA polymerase III core, a heterotrimer of α, ε, and θ, required for leading‐ and lagging‐strand synthesis. DnaB binds the DnaG primase that synthesizes RNA primers on both strands. Representative structures are available for most classes of DNA replication proteins, although there are gaps in our understanding of their interactions and the structural transitions that occur in nanomachines such as the helicase, clamp loader, and replicase core as they function. Reviewed here is the structural biology of these bacterial DNA replication proteins and prospects for future research.

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Glutamate promotes SSB protein–protein Interactions via intrinsically disordered regions

Cited by 51 publications

References 66 publications

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Self-regulation of single-stranded DNA wrapping dynamics byE. coliSSB promotes both stable binding and rapid dissociation

A structural view of bacterial DNA replication

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