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Bound or free: interaction of the C-terminal domain of Escherichia coli single-stranded DNA-binding protein (SSB) with the tetrameric core of SSB

Su, Xun-Cheng; Wang, Yao; Yagi, Hiromasa; Shishmarev, Dmitry; Mason, Claire E; Smith, Paul; Vandevenne, Marylene; Dixon, Nicholas; Otting, Gottfried

Description

Single-stranded DNA (ssDNA)-binding protein (SSB) protects ssDNA from degradation and recruits other proteins for DNA replication and repair. Escherichia coli SSB is the prototypical eubacterial SSB in a family of tetrameric SSBs. It consists of a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain). The eight-residue C-terminal segment of SSB (C-peptide) mediates the binding of SSB to many different SSB-binding proteins. Previously published...[Show more]

dc.contributor.authorSu, Xun-Cheng
dc.contributor.authorWang, Yao
dc.contributor.authorYagi, Hiromasa
dc.contributor.authorShishmarev, Dmitry
dc.contributor.authorMason, Claire E
dc.contributor.authorSmith, Paul
dc.contributor.authorVandevenne, Marylene
dc.contributor.authorDixon, Nicholas
dc.contributor.authorOtting, Gottfried
dc.date.accessioned2015-12-10T23:07:54Z
dc.identifier.issn0006-2960
dc.identifier.urihttp://hdl.handle.net/1885/63061
dc.description.abstractSingle-stranded DNA (ssDNA)-binding protein (SSB) protects ssDNA from degradation and recruits other proteins for DNA replication and repair. Escherichia coli SSB is the prototypical eubacterial SSB in a family of tetrameric SSBs. It consists of a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain). The eight-residue C-terminal segment of SSB (C-peptide) mediates the binding of SSB to many different SSB-binding proteins. Previously published nuclear magnetic resonance (NMR) data of the monomeric state at pH 3.4 showed that the C-peptide binds to the OB-domain at a site that overlaps with the ssDNA binding site, but investigating the protein at neutral pH is difficult because of the high molecular mass and limited solubility of the tetramer. Here we show that the C-domain is highly mobile in the SSB tetramer at neutral pH and that binding of the C-peptide to the OB-domain is so weak that most of the C-peptides are unbound even in the absence of ssDNA. We address the problem of determining intramolecular binding affinities in the situation of fast exchange between two states, one of which cannot be observed by NMR and cannot be fully populated. The results were confirmed by electron paramagnetic resonance spectroscopy and microscale thermophoresis. The C-peptide-OB-domain interaction is shown to be driven primarily by electrostatic interactions, so that binding of 1 equiv of (dT)35 releases practically all C-peptides from the OB-domain tetramer. The interaction is much more sensitive to NaCl than to potassium glutamate, which is the usual osmolyte in E. coli. As the C-peptide is predominantly in the unbound state irrespective of the presence of ssDNA, long-range electrostatic effects from the C-peptide may contribute more to regulating the activity of SSB than any engagement of the C-peptide by the OB-domain.
dc.publisherAmerican Chemical Society
dc.rightshttp://www.sherpa.ac.uk/romeo/issn/0006-2960/..."author can archive post-print (ie final draft post-refereeing) If mandated by funding agency or employer/ institution" from SHERPA/RoMEO site (as at 18/10/18).
dc.sourceBiochemistry
dc.titleBound or free: interaction of the C-terminal domain of Escherichia coli single-stranded DNA-binding protein (SSB) with the tetrameric core of SSB
dc.typeJournal article
local.description.notesImported from ARIES
local.description.notesThis work was supported by the Australian Research Council (DP0877658 and DP0984797 to N.E.D. and DP110102737 and DP120100561 to G.O.) and the National Science Foundation of China (21073101 to X.-C.S.).
local.identifier.citationvolume53
dc.date.issued2014
local.identifier.absfor030403 - Characterisation of Biological Macromolecules
local.identifier.ariespublicationu4005981xPUB765
local.type.statusAccepted Version
local.contributor.affiliationSu, Xun-Cheng, College of Physical and Mathematical Sciences, ANU
local.contributor.affiliationWang, Yao, University of Wollongong
local.contributor.affiliationYagi, Hiromasa, College of Physical and Mathematical Sciences, ANU
local.contributor.affiliationShishmarev, Dmitry S., College of Physical and Mathematical Sciences, ANU
local.contributor.affiliationMason, Claire E, University of Wollongong
local.contributor.affiliationSmith, Paul, College of Physical and Mathematical Sciences, ANU
local.contributor.affiliationVandevenne, Marylene, University of Sydney
local.contributor.affiliationDixon, Nicholas, College of Physical and Mathematical Sciences, ANU
local.contributor.affiliationOtting, Gottfried, College of Physical and Mathematical Sciences, ANU
dc.relationhttp://purl.org/au-research/grants/arc/DP0877658
dc.relationhttp://purl.org/au-research/grants/arc/DP0984797
dc.relationhttp://purl.org/au-research/grants/arc/DP110102737
dc.relationhttp://purl.org/au-research/grants/arc/DP120100561
local.bibliographicCitation.issue12
local.bibliographicCitation.startpage1925
local.bibliographicCitation.lastpage1934
local.identifier.doi10.1021/bi5001867
local.identifier.absseo920109 - Infectious Diseases
dc.date.updated2015-12-10T09:02:55Z
local.identifier.scopusID2-s2.0-84898080409
local.identifier.thomsonID000333776300006
dcterms.accessRightsOpen Access
CollectionsANU Research Publications

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