Featured Article
How Glutamate Promotes Liquid-liquid Phase Separation and DNA Binding Cooperativity of E. coli SSB Protein

https://doi.org/10.1016/j.jmb.2022.167562Get rights and content

Highlights

  • •

    Liquid-liquid phase separation of E. coli SSB protein is promoted by KGlu, but inhibited by KCl.

  • •

    KGlu promotes NNN cooperativity and collapse of single molecules of SSB-coated polymeric ssDNA.

  • •

    SSB LLPS and NNN cooperativity both require its intrinsically disordered linker (IDL) within its C-terminal tails.

  • •

    KGlu interacts unfavorably with amide groups within the SSB IDL, whereas KCl interacts favorably.

  • •

    ssDNA binding inhibits SSB phase separation.

Abstract

E. coli single-stranded-DNA binding protein (EcSSB) displays nearest-neighbor (NN) and non-nearest-neighbor (NNN)) cooperativity in binding ssDNA during genome maintenance. NNN cooperativity requires the intrinsically-disordered linkers (IDL) of the C-terminal tails. Potassium glutamate (KGlu), the primary E. coli salt, promotes NNN-cooperativity, while KCl inhibits it. We find that KGlu promotes compaction of a single polymeric SSB-coated ssDNA beyond what occurs in KCl, indicating a link of compaction to NNN-cooperativity. EcSSB also undergoes liquid–liquid phase separation (LLPS), inhibited by ssDNA binding. We find that LLPS, like NNN-cooperativity, is promoted by increasing [KGlu] in the physiological range, while increasing [KCl] and/or deletion of the IDL eliminate LLPS, indicating similar interactions in both processes. From quantitative determinations of interactions of KGlu and KCl with protein model compounds, we deduce that the opposing effects of KGlu and KCl on SSB LLPS and cooperativity arise from their opposite interactions with amide groups. KGlu interacts unfavorably with the backbone (especially Gly) and side chain amide groups of the IDL, promoting amide-amide interactions in LLPS and NNN-cooperativity. By contrast, KCl interacts favorably with these amide groups and therefore inhibits LLPS and NNN-cooperativity. These results highlight the importance of salt interactions in regulating the propensity of proteins to undergo LLPS.

Introduction

Single stranded (ss) DNA binding proteins (SSBs) are essential in all kingdoms of life. SSBs bind ssDNA intermediates formed transiently during genome maintenance to protect them from degradation and inhibit DNA secondary structures.1, 2, 3, 4 Escherichia coli SSB (EcSSB) also serves as a central hub for binding numerous metabolic proteins (SSB interacting proteins – SIPs) involved in replication, recombination and repair.5

EcSSB functions as a homo-tetramer (Figure 1(A)),3, 6 with each subunit (177 amino acids (aa)) composed of two domains (Figure 1(B)): a structured N-terminal DNA binding domain (DBD) (residues 1–112), and a C-terminal domain (residues 113–177, Figure 1(D)) composed of a flexible, intrinsically disordered linker (IDL) [56aa] and a nine residue “acidic tip” (Figure 1(B)). This acidic tip is conserved among many bacterial SSBs and is the primary site of interaction with the SIPs.5, 7, 8, 9, 10, 11, 12, 13 EcSSB binds ssDNA in two major modes referred to as (SSB)35 and (SSB)65, where the subscripts denote the average number of ssDNA nucleotides occluded.14, 15 The relative stabilities of these modes depend on salt concentration and type, and protein to DNA ratio (binding density),14, 16, 17, 18, 19, 20, 21, 22, 23, 24 as well as applied force.25, 26, 27, 28

In the (SSB)65 mode, favored at [NaCl] > 0.20 M or [Mg2+] > 10 mM at 25˚C, the ssDNA wraps around all four subunits of the tetramer6 with a ∼65 nucleotide occluded site size. The topology of ssDNA wrapping in the (SSB)65 binding mode follows the seams on a baseball such that ssDNA enters and exits the tetramer in close proximity. On long ssDNA, the (SSB)65 mode displays “limited” cooperativity between adjacent tetramers.16, 24, 29 In this mode SSB can diffuse along ssDNA destabilizing DNA hairpins and promoting RecA filament formation.25, 30

In the (SSB)35 mode, favored at [NaCl] < 10 mM or [MgCl2] < 1 mM, and high SSB to DNA ratios,14, 15, 18 ssDNA interacts with only two subunits on average with a ∼35 nucleotide occluded site size. In this mode SSB binds ssDNA with unlimited nearest-neighbor (NN) cooperativity favoring formation of long protein clusters.17, 18, 20, 22, 24, 31, 32 Based on structural considerations it was suggested that SSB NN cooperativity might be promoted by interactions of adjacent tetramers through the L45 loops within the tetrameric core6 as well as directly through the residues of the core not involved in DNA binding.24, 33 In this mode SSB can diffuse along ssDNA25, 26 and undergo direct or intersegment transfer between separate ssDNA molecules34 or between distant sites on the same DNA molecule.35 The ability to undergo direct transfer appears to play a role in SSB recycling during replication.34, 36

Another level of non-nearest-neighbor (NNN) cooperativity has been identified recently for SSB bound to polymeric ssDNA.23, 28, 32 This NNN cooperativity occurs between SSB tetramers distantly bound to polymeric ssDNA and results in compaction/condensation of nucleoprotein complexes. Such interactions require the IDL23, 32 and are promoted by glutamate and acetate salts.23, 28, 32

EcSSB also undergoes liquid–liquid phase separation (LLPS) under solution conditions that mimic the E. coli environment. The intrinsically disordered C-terminal tails of SSB are essential for LLPS, which is suppressed by ssDNA.37 Here we explore the ability of EcSSB to undergo LLPS as a function of temperature, salt type and concentration. We also explore how modifications within the IDL affect LLPS. We show that elevated concentrations of potassium glutamate (KGlu), the primary monovalent salt in E. coli,38, 39 promotes LLPS whereas KCl has the opposite effect. A similar observation was first made by Harami et al. using NaGlu and NaCl.37 We present a thermodynamic analysis of interactions of KCl with protein model compounds and compare these with results for KGlu40 showing that these large opposing effects of KGlu and KCl on SSB LLPS likely result from their opposite interactions with backbone (especially G) and side chain amides of the SSB IDL in solution. We therefore propose that these amide groups interact with one another in the condensed phase, reducing or eliminating their interactions with water and salt ions. These amide-amide interactions, favored by KGlu and disfavored by KCl, appear to be important contributors to LLPS.

SSB LLPS, like NNN cooperative interactions of DNA-bound SSB, is a highly cooperative process. We find that conditions that promote LLPS also promote NNN cooperativity of SSB binding to ssDNA and conclude that similar cooperative interactions of tail residues drive these two processes mediated by ssDNA.

Section snippets

Constructing phase diagrams using turbidity measurements

Liquid-liquid phase separation (LLPS) of EcSSB, recently observed in the absence of ssDNA, is inhibited by binding of ssDNA.37 We refer to this phenomenon as LLPS, or simply phase separation (PS), although it has also been referred to as phase separation aided percolation (PSP).41 LLPS of SSB is promoted by KGlu and requires the intrinsically disordered tails of SSB.37 Here, we explore how KGlu and KCl affect phase separation by determining their effect on the temperature corresponding to the

Discussion

The formation of membraneless biomolecular condensates (phase separation) is a well-recognized, biologically important process.54, 55, 56, 57 Phase separation has been studied most widely and thought to be most relevant in eukaryotic systems. This is due to the fact that intrinsically disordered regions (IDRs) of proteins, that are effective drivers of phase separation, make up ∼40% of eukaryotic proteomes, but less than ∼5% of bacterial proteomes.58 However, it has been argued that

Reagents and buffers

Buffers were prepared with reagent grade chemicals and distilled water treated with a Milli Q (Millipore, Bedford, MA) water purification system. Buffer T is 10 mM Tris, pH 8.1 (25 °C), 0.1 mM Na3EDTA, Buffer P is 10 mM phosphate, pH 7.5 (25 °C), 0.1 mM Na3EDTA. The final concentrations of monovalent salts (KGlu, KCl and NaCl, Sigma-Aldrich, reagent grade) in the solutions were achieved by mixing with solutions of Buffer T or Buffer P containing 1 M KGlu, 2 M KCl or 2 M NaCl. Single molecule

CRediT authorship contribution statement

Alexander G. Kozlov: Conceptualization, Data curation, Formal analysis, Investigation, Writing – original draft, Methodology. Xian Cheng: Conceptualization, Data curation, Formal analysis, Investigation. Hongshan Zhang: Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing. Min Kyung Shinn: Data curation, Formal analysis, Investigation. Elizabeth Weiland: Investigation. Binh Nguyen: Data curation, Formal analysis, Investigation. Irina A. Shkel: Formal analysis,

Acknowledgements

We thank R. Galletto for use of his Cary-100 spectrophotometer for turbidity measurements and J. Incicco and A. Soranno for help with imaging experiments. We thank R.Pappu for stimulating discussions, advice, enlightening comments and encouragement, and K. Mersch for discussions and comments on the ms. This research was supported in part by the NIH (R01 GM30498 and R35 GM136632 to TML) (R35 GM118100 to MTR), and NSF (CAREER award 1453358 to IJF).

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Cover Illustration: E. coli SSB protein, an essential replication and repair protein, can form a biomolecular condensate via phase separation in the absence of DNA. Phase separation is promoted by potassium glutamate (KGlu), which is the major physiological monovalent salt in bacteria, however, single stranded DNA binding to SSB inhibits this phase separation. However, when SSB is bound to polymeric single stranded DNA, KGlu promotes cooperative binding of SSB on the DNA and compaction of

References (99)

  • W. Bujalowski et al.

    Limited co-operativity in protein-nucleic acid interactions. A thermodynamic model for the interactions of Escherichia coli single strand binding protein with single-stranded nucleic acids in the “beaded”, (SSB)65 mode

    J. Mol. Biol.

    (1987)
  • A.G. Kozlov et al.

    Intrinsically Disordered C-terminal Tails of E. coli Single Stranded DNA Binding Protein Regulate Cooperative Binding to Single Stranded DNA

    J. Mol. Biol.

    (2015)
  • K. Dubiel et al.

    Structural Mechanisms of Cooperative DNA Binding by Bacterial Single-Stranded DNA-Binding Proteins

    J. Mol. Biol.

    (2019)
  • K.S. Lee et al.

    Ultrafast Redistribution of E. coli SSB along Long Single-Stranded DNA via Intersegment Transfer

    J. Mol. Biol.

    (2014)
  • B. Richey et al.

    Variability of the intracellular ionic environment of Escherichia coli. Differences between in vitro and in vivo effects of ion concentrations on protein-DNA interactions and gene expression

    J. Biol. Chem.

    (1987)
  • X. Cheng et al.

    Basis of Protein Stabilization by K Glutamate: Unfavorable Interactions with Carbon, Oxygen Groups

    Biophys J.

    (2016)
  • M.C. Cohan et al.

    Making the Case for Disordered Proteins and Biomolecular Condensates in Bacteria

    Trends Biochem. Sci.

    (2020)
  • D.F. Lyons et al.

    Structural and hydrodynamic analysis of a novel drug delivery vector: ELP[V5G3A2-150]

    Biophys. J.

    (2013)
  • A.E. Posey et al.

    Phase Separation of Intrinsically Disordered Proteins

    Intrinsically Disordered Proteins

    (2018)
  • A.G. Kozlov et al.

    Regulation of single-stranded DNA binding by the C termini of Escherichia coli single-stranded DNA-binding (SSB) protein

    J. Biol. Chem.

    (2010)
  • S. Alberti et al.

    Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates

    Cell

    (2019)
  • M.C. Cohan et al.

    Uncovering Non-random Binary Patterns Within Sequences of Intrinsically Disordered Proteins

    J. Mol. Biol.

    (2022)
  • P.A. Chong et al.

    RGG/RG Motif Regions in RNA Binding and Phase Separation

    J. Mol. Biol.

    (2018)
  • S. Boyko et al.

    Liquid-liquid phase separation of tau protein: The crucial role of electrostatic interactions

    J. Biol. Chem.

    (2019)
  • T.J. Nott et al.

    Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles

    Mol. Cell

    (2015)
  • K.A. Burke et al.

    Residue-by-Residue View of In Vitro FUS Granules that Bind the C-Terminal Domain of RNA Polymerase II

    Mol. Cell

    (2015)
  • B.D. Mason et al.

    Liquid-Liquid Phase Separation of a Monoclonal Antibody and Nonmonotonic Influence of Hofmeister Anions

    Biophys. J.

    (2010)
  • D.J. Deredge et al.

    The glutamate effect on DNA binding by pol I DNA polymerases: osmotic stress and the effective reversal of salt linkage

    J. Mol. Biol.

    (2010)
  • S. Cayley et al.

    Characterization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity. Implications for protein-DNA interactions in vivo

    JMolBiol

    (1991)
  • E. Antony et al.

    Plasmodium falciparum SSB tetramer binds single-stranded DNA only in a fully wrapped mode

    J. Mol. Biol.

    (2012)
  • J. Dam et al.

    Calculating sedimentation coefficient distributions by direct modeling of sedimentation velocity concentration profiles

    Methods Enzymol.

    (2004)
  • M.M. Soniat et al.

    Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination

    Method Enzymol.

    (2017)
  • J.W. Chase et al.

    Single-stranded DNA binding proteins required for DNA replication

    AnnuRevBiochem

    (1986)
  • R.R. Meyer et al.

    The single-stranded DNA-binding protein of Escherichia coli

    Microbiol. Rev.

    (1990)
  • T.M. Lohman et al.

    Escherichia coli single-stranded DNA-binding protein: multiple DNA-binding modes and cooperativities

    Annu. Rev. Biochem.

    (1994)
  • M.S. Wold

    Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism

    Annu. Rev. Biochem.

    (1997)
  • R.D. Shereda et al.

    SSB as an organizer/mobilizer of genome maintenance complexes

    Crit. Rev. Biochem. Mol. Biol.

    (2008)
  • S. Raghunathan et al.

    Structure of the DNA binding domain of E-coli SSB bound to ssDNA

    Nature Struct. Biol.

    (2000)
  • J. Genschel et al.

    Interaction of E-coli single-stranded DNA binding protein (SSB) with exonuclease I. The carboxy-terminus of SSB is the recognition site for the nuclease

    Biol. Chem.

    (2000)
  • A.G. Kozlov et al.

    Binding specificity of Escherichia coli single-stranded DNA binding protein for the chi subunit of DNA pol III holoenzyme and PriA helicase

    Biochemistry

    (2010)
  • A.H. Marceau et al.

    Structure of the SSB-DNA polymerase III interface and its role in DNA replication

    EMBO J.

    (2011)
  • M. Ryzhikov et al.

    Structural studies of SSB interaction with RecO

    Methods Mol. Biol.

    (2012)
  • M.K. Shinn et al.

    Are the intrinsically disordered linkers involved in SSB binding to accessory proteins?

    Nucleic Acids Res.

    (2019)
  • W. Bujalowski et al.

    Escherichia coli single-strand binding protein forms multiple, distinct complexes with single-stranded DNA

    Biochemistry

    (1986)
  • S. Chrysogelos et al.

    Escherichia coli single-strand binding protein organizes single-stranded DNA in nucleosome-like units

    Proc. Natl. Acad. Sci. U. S. A.

    (1982)
  • J.D. Griffith et al.

    Visualization of SSB-ssDNA complexes active in the assembly of stable RecA-DNA filaments

    Cold Spring Harb. Symp. Quant. Biol.

    (1984)
  • L. Hamon et al.

    High-resolution AFM imaging of single-stranded DNA-binding (SSB) protein–DNA complexes

    Nucleic Acids Res.

    (2007)
  • S. Suksombat et al.

    Structural dynamics of E. coli single-stranded DNA binding protein reveal DNA wrapping and unwrapping pathways

    Elife

    (2015)
  • K. Hatch et al.

    Measurement of the salt-dependent stabilization of partially open DNA by Escherichia coli SSB protein

    Nucleic Acids Res.

    (2008)

    • Cited by (0)

    Cover Illustration: E. coli SSB protein, an essential replication and repair protein, can form a biomolecular condensate via phase separation in the absence of DNA. Phase separation is promoted by potassium glutamate (KGlu), which is the major physiological monovalent salt in bacteria, however, single stranded DNA binding to SSB inhibits this phase separation. However, when SSB is bound to polymeric single stranded DNA, KGlu promotes cooperative binding of SSB on the DNA and compaction of single DNA molecules. The non-physiological salt, KCl, inhibits both phase separation and DNA collapse. The equilibria in the presence of KGlu are proposed to be used to regulate SSB function in genome maintenance. The cover refers to the JMB article in this issue by A.G. Kozlov et al., “How Glutamate Promotes Liquid-liquid Phase Separation and DNA Binding Cooperativity of E. coli SSB Protein,” Volume xxx, Issue xx. The figure was made by Kacey Mersch.

    View full text
    © 2022 Elsevier Ltd. All rights reserved.