the idl of e. coli ssb links ssdna and protein binding by ... · 117 or 152 were unable to...
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The IDL of E. coli SSB links ssDNAand protein binding by mediatingprotein–protein interactions
Piero R. Bianco,1* Sasheen Pottinger,1 Hui Yin Tan,1 Trong Nguyenduc,1
Kervin Rex,2 and Umesh Varshney2
1Department of Microbiology and Immunology, Center for Single Molecule Biophysics, University at Buffalo, Buffalo,New York 142142Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India
Received 22 August 2016; Accepted 17 October 2016
DOI: 10.1002/pro.3072Published online proteinscience.org
Abstract: The E. coli single strand DNA binding protein (SSB) is essential to viability where it func-
tions in two seemingly disparate roles: it binds to single stranded DNA (ssDNA) and to target pro-teins that comprise the SSB interactome. The link between these roles resides in a previously
under-appreciated region of the protein known as the intrinsically disordered linker (IDL). We
present a model wherein the IDL is responsible for mediating protein–protein interactions criticalto each role. When interactions occur between SSB tetramers, cooperative binding to ssDNA
results. When binding occurs between SSB and an interactome partner, storage or loading of that
protein onto the DNA takes place. The properties of the IDL that facilitate these interactionsinclude the presence of repeats, a putative polyproline type II helix and, PXXP motifs that may
facilitate direct binding to the OB-fold in a manner similar to that observed for SH3 domain binding
of PXXP ligands in eukaryotic systems.
Keywords: SSB; RecG; RecO; OB-fold; SH3 domain; PXXP motif
Introduction
The Escherichia coli single stranded DNA binding
protein (SSB) is essential to all aspects of DNA
metabolism. Here it performs two seemingly disparate
roles. First, it functions to stabilize single-stranded
DNA (ssDNA) intermediates generated during DNA
processing.1–5 Second, it interacts with as many as 14
proteins in temporal and spatial fashion, to both store
and target enzymes to the DNA when needed.6,7 How
these roles are linked mechanistically is currently not
known.
The protein, like the majority of eubacterial SSBs,
exists as a stable homo-tetramer with a monomer MW
of 18,843 Da.8 Each 178 amino acid length monomer
can be divided into two general domains defined by
proteolytic cleavage: an N-terminal portion comprising
approximately the first 115 residues and a C-terminal
domain that includes residues 116–178.9 The
N-terminal domain contains elements critical to tetra-
mer formation and the oligonucleotide/oligosaccharide
binding-fold (OB-fold) required for binding to ssDNA.10
DNA binding occurs via the wrapping of ssDNA
around the SSB tetramer using an extensive network
of electrostatic and base-stacking interactions with
the phosphodiester backbone and nucleotide bases,
respectively.10–12
The C-terminal domain can be further subdi-
vided into two additional regions: a sequence of �50
amino acids that has been called the intrinsically
Abbreviations: SSB, single stranded DNA binding protein; ssDNA,single stranded DNA; IDL, intrinsically disordered linker; PPII,polyproline type II helix.
Additional Supporting Information may be found in the onlineversion of this article.
Grant sponsor: National Institutes of Health Grant; Grant number:GM100156.
*Correspondence to: Piero R. Bianco, Center for Single MoleculeBiophysics, Department of Microbiology and Immunology, Universityat Buffalo, Buffalo, NY 14214, USA. E-mail: [email protected]
Published by Wiley-Blackwell. VC 2017 The Protein Society PROTEIN SCIENCE 2017 VOL 26:227—241 227
disordered linker (IDL) and the last 8–10 residues
which are known as the acidic tip or C-peptide and
which is very well conserved among eubacterial pro-
teins.1,13–23 While there is extensive knowledge of
the N-terminal domain, there are only a limited
number of studies of the C-terminus. This is due in
part to the fact that it is highly disordered even
when the protein is bound to ssDNA.24 Recent
studies have focused on the acidic tip only as it is
thought to be critical for mediating interactions with
SSB interactome partner proteins.1
The IDL is the least conserved region among
prokaryotic SSB proteins.1,5 Initially, it was proposed
to be non-essential, as a mutant ssb with residues 116-
167 deleted was able to complement an ssb deletion.9
However, truncated ssb genes terminating at residues
117 or 152 were unable to complement the same ssb
deletion. This led to the proposal that the last 20–50
residues of SSB are essential for protein–protein
interactions.9 Subsequent in vitro studies showed that
deletion of the C-terminal 15–20 residues eliminated
interactions with the psi and chi subunits of the DNA
polymerase III complex.18,25 Further work both in vivo
and in vitro using the ssbDC8 gene and SSBDC8 protein
respectively, have shown that the last eight residues are
critical, but not solely responsible, for target protein
binding.7,22 One interpretation is that the acidic tip resi-
dues contact target proteins directly.23,26–28 An alterna-
tive proposal suggests that these last eight residues play
a regulatory role and that target protein binding utilizes
another region of SSB.29
We propose that this other region is the intrinsi-
cally disordered linker. This proposal makes sense
because the IDL is unique to each bacterium, and
may explain species specificity in interactome func-
tion.6,22 What is known about the IDL is limited. As
alluded to above, early studies suggested that it is a
non-essential part of the protein.9 Recently, the
Lohman group found that when the linker is deleted
or its sequence altered, cooperative ssDNA binding
by SSB is affected.23 They proposed a model wherein
the linkers could self-associate so that the linker of
one tetramer binds to that of an adjacent one and,
this would facilitate interactions between acidic tips
and OB cores. This model has at its core, interac-
tions between the acidic tip and OB-folds of adjacent
proteins. The current data show that this interaction
is at its best, only transient.29,30 Furthermore,
recent crystal structures of part of the acidic tip
bound to interactome partners reveal that the tip
does not bind to OB-folds but instead binds to a
hydrophobic pocket that contains a prominent basic
residue.26–28,31
To begin to understand how the linker region of
SSB may link the seemingly disparate activities of
cooperative ssDNA and protein binding, a combinatori-
al strategy was employed. First a series of linker dele-
tion mutants containing the acidic tip was created,
and then utilized in an in vivo binding assay to
monitor the interaction between small (RecO; 27 kDa)
and large (RecG; 76 kDa) interactome partners.22,32
Results show that a full length linker is required to
bind RecG whereas shorter linker SSBs can still bind
to RecO. We then compared the binding results of our
mutants to that of a naturally occurring short-linker
protein, the Thermotoga maritima SSB and also to
E. coli–Mycobacterium tuberculosis hybrid SSB pro-
teins. Collectively, these results reveal novel insights
into the role of the linker in target protein binding as
well as insight into subdomains within the linker
which are critical to IDL function. These include a
section that could adopt a polyproline type II helix,
repeats that impart flexibility and tensile strength
and also PXXP motifs to facilitate binding specificity.
Consequently, a model is proposed whereby the
linker(s) of an SSB tetramer bind to the OB-folds
present in either a neighboring SSB or a member
of the interactome. The common feature in these
interactions are the PXXP motifs in the intrinsically
disordered linker of SSB and the OB-fold of the
target protein. Here binding occurs in a manner analo-
gous to eukaryotic SH3 domains binding PXXP-
containing protein ligands. When binding of a linker to
the OB-fold of another SSB occurs, this results in rapid
and cooperative ssDNA binding, producing a stable
complex that can resist displacement by a variety of
enzymes that might otherwise damage the exposed
portion of the genome. When the IDLs bind to a protein
such as RecG, the helicase is loaded onto the DNA,
leading to the rescue of stalled replication forks. There-
fore, the linker domain of SSB connects the seemingly
disparate roles of ssDNA and target protein binding by
SSB using a common mechanism.
Results
RecO and SSB bind in vivo
The RecO interaction with SSB in vitro has been dem-
onstrated previously.32,33 To determine whether bind-
ing also occurs in vivo, as shown previously for PriA
and RecG, an identical approach was employed.7 To
test this, expression strains were made by transform-
ing high copy number plasmids expressing either wild
type or N-terminally his-tagged SSB into E. coli
strains. Then, high copy number plasmids expressing
his- or wild type RecO were transformed separately
into wild type and his-SSB cells, respectively. One liter
cultures of cells expressing both proteins were grown,
lysed and the cleared cell lysates subjected to Ni-
sepharose chromatography. After extensive washing,
proteins were eluted using a linear, imidazole gradient
as described in the Materials and Methods. The results
show that both SSB and his-RecO coeluted from the
column [Fig. 1(A)]. When the tag was present on the
C-terminus of RecO (i.e., RecO-his), coelution was not
observed, suggesting that this end of the protein being
228 PROTEINSCIENCE.ORG SSB IDL Links ssDNA and Protein Binding
involved in binding to SSB [Fig. 1(B)32,33]. A vanishing-
ly small amount of SSB could be detected and the
resulting analysis shows that binding decreased 67-
fold compared to his-RecO [Fig. 1(E)]. Coelution of SSB
and RecO was observed when the histidine tag was
present on the N-terminus of either protein [Figs.
1(A,C)]. Analysis of the gels shows that when his-RecO
was used, the ratio of RecO to SSB was 1.7 6 0.5 [Fig.
1(A,E)]. When the tag was on SSB, the ratio of
RecO:SSB in the eluted fractions was 1.4 6 0.4 [Fig.
1(C,E)]. Our previous work with RecG and PriA dem-
onstrates that the SSB-partner complexes exist in vivo
and do not form following cell lysis.7 Therefore, the
data presented in Figure 1 show that RecO binds to
SSB in vivo.
RecO-SSB complex formation requires the
presence of the SSB C-terminus
In vitro studies show that SSBDC8, a mutant which
lacks the last eight residues of SSB, does not form a
complex with RecO.32 Previous work has shown that
binding to the RecG and PriA helicases in vivo
requires the presence of the C-terminal eight resi-
dues of SSB.7 To determine whether these residues
are also required for complex formation between
SSB and RecO in vivo, we made dual-expression
strains, replacing wild type SSB with SSBDC8. One
liter cultures of cells expressing SSBDC8 and his-
RecO were grown, lysed and the cleared cell lysates
subjected to Ni Sepharose chromatography as before.
The results show that his-RecO eluted off the col-
umn as expected, but that these fractions contained
little or no SSBDC8 [Fig. 1(D)]. There is however, a
small amount of SSBDC8 found in the fractions elut-
ed from the column at the front end of the peak.
This indicates that there is a weak interaction
between RecO and SSBDC8, similar to what was
observed with RecG.7 Therefore, and as shown previ-
ously for RecG and PriA, binding of RecO requires
that the last 8 amino acids of the acidic tip of SSB
be present.7
SSB linker length appears to be critical
for binding to interactome partners
As the binding of SSB to interactome partners in
vivo is essential to facilitating many DNA metabolic
processes, we wanted to understand the role of the
linker region in target protein binding.7 To do this,
we constructed a series of SSB linker mutants,
designated SSB125, SSB133, SSB145, and SSB155
[Fig. 2(A,B), Supporting Information Fig. S1]. These
are similar to those of the Lohman group, but differ
in the deletion points and the length of the C-
terminal tip retained.23 For the proteins described
herein, the subscript refers to the number of resi-
dues in each protein, counting the first methionine
as position 1. Each protein contains the N-terminal
115 residues of the protein responsible for tetramer
formation and DNA binding, followed by various
length linker regions and the C-terminus (the last
10 amino acids; 50-PMDFDDDIPF-30). Wild type SSB
has a linker 54 residues in length while that of
mutants is 31 (SSB155; SSBD146–167); 21 (SSB145;
SSBD135–167); 6 (SSB133; SSBD124–167); and 0
(SSB125; SSBD115–167). Next, we coexpressed his-
RecO separately with each of the mutants, and
Figure 1. SSB binds to RecO in vivo. The 1-L cultures of cells expressing RecO and SSB were grown to early log phase, IPTG
added to 500 mM and growth continued until early stationary phase. Cells were harvested by centrifugation, lysed and the
cleared cell lysate applied to a 5-mL nickel column as described in the Materials and Methods. Proteins were then eluted using
an imidazole gradient following extensive washing to remove unbound proteins. Aliquots from various fractions throughout the
purification were subjected to electrophoresis. The resulting Coomassie stained, 15% SDS-PAGE gels of the purifications are
presented. (A), hisRecO binds to SSB; (B), RecO-his does not bind to SSB; (C), RecO binds to hisSSB; (D), formation of the
RecO-SSB complex requires the highly conserved and acidic SSB tip.; (E), Analysis of the gels in panels A–C. For each
analysis, only the five central lanes of the peak were analyzed and the ratios averaged. MW, molecular weight marker; WCL,
whole cell lysate; CCL, cleared cell lysate; FT, flow through; numbers, indicate fraction numbers from the peak elution profile.
Proteins were eluted using a linear imidazole gradient from 30 to 500 mM.
Bianco et al. PROTEIN SCIENCE VOL 26:227—241 229
subjected the cleared cell lysates to nickel column
chromatography.
The results show co-elution of his-RecO with
each SSB protein consistent with in vivo complex
formation [Fig. 2(C)]. In the gel shown, we have
normalized each lane to the amount of his-RecO
eluted in the peak fractions from each purification.
When presented in this manner, it is clear that the
average ratio of RecO to SSB is 2.2 for wild type.
This ratio decreases in almost linear fashion as
length of the linker decreases to zero, reaching a
minimum of 1.3 6 0.4 for SSB125. Thus complete
binding to RecO requires a fully intact linker.
Full length SSB binds to RecG whereas linker
mutants do not
Recent work demonstrates that RecG (MW 5 76,430
Da) and SSB, bind to each other in vivo in the
absence of DNA.7 The mass of the RecO protein is
27,391 Da, approximately one third that of RecG. It
is conceivable that the truncated SSB proteins bind
to RecO due to its small size but perhaps binding to
the larger RecG requires a longer linker.
To test this, we co-expressed the histidine
tagged version of each of the SSB mutants with
RecG. As a positive control for the linker mutants,
we repeated the experiment with full length SSB
and RecG. As expected, both his-SSB and RecG
eluted from the column [Fig. 3(A,D)]. In contrast,
when the linker mutants replaced full length SSB,
co-elution to the level of wild type was not observed
[Fig. 3(B,C)]. In fact, binding to RecG was
diminished 10-fold for SSB155 and 26-fold for the
SSB125 mutant. A simple interpretation of these
data is that longer linkers are required to bind
larger interactome partner proteins. However, and
as explained below, this turns out not to be the case.
Key components of the linker are
required for optimal function
To understand why the linker mutants bind to RecO
but not to RecG, two separate sequence analyses
Figure 2. SSB linker length affects binding to RecO. (A), Schematic of the wild type and linker domain mutants. The black line
demarcates the region deleted in each mutant. (B), Sequence alignment of full length and linker domain mutants of E. coli SSB with
T. maritima SSB. The alignment was done using PRALINE multiple sequence alignment and the result shows residues 112–178 (E.
coli numbering)66. The green boxed region indicates the C-terminal tail with invariant residues in red. (C) SSB linker domain mutants
co-elute with his-RecO. The 100 mL cultures were grown to early log phase and expression induced with 500 mM IPTG. Cells were
harvested in early stationary phase by centrifugation and the resulting pellets lysed. The cleared cell lysates were applied to 1mL
nickel columns, extensively washed and eluted with elution buffer as described in the Materials and Methods. A Coomassie stained,
15% SDS-PAGE gel with the apex fraction of each coelution is shown. To allow direct comparison, each lane was normalized to the
amount of RecO loaded. (D), Quantification of SDS-PAGE gels such as those shown in (C).
230 PROTEINSCIENCE.ORG SSB IDL Links ssDNA and Protein Binding
were done. First, we examined the primary amino
acid sequence of SSB focusing on the C-terminal 69
residues. This analysis shows that this region is
over-represented for Gly 24.6%, Gln 17.4%, Pro
14.5%, and to a lesser extent, Ala and Ser [8.7 and
5.8%, respectively; Fig. 4(A)]. The presence and
spacing of these residues in the N-terminal half of
this region, ending at residues 148 and 149 (Phe
and Ser, respectively), is consistent with the forma-
tion of polyproline II helices (PPII) found in proteins
such as collagen.34 However, the spacing of the pro-
line residues in collagen occurs at every fourth posi-
tion which is not the case for SSB. Instead, we
observed that glycine, glutamine and arginine occur
in those positions in SSB. A recent study has shown
that these amino acids can substitute for proline
without disrupting a model PPII helix.35 As one
strand of a collagen fiber is essentially a PPII helix,
we wanted to determine whether this region of SSB
could be modeled on a collagen-like peptide.36,37
Indeed, the SSB sequence can adopt a PPII helix
that superimposes well with the peptide, with an
RMSD 5 0.8 Angstroms for the backbone atoms
(Supporting Information Fig. S3). In contrast, the
C-terminal 30 residues of SSB downstream of F148/
S149 could not be modeled on the collagen peptide
using the same approach (data not shown).
Second, and as the gly, pro and gln residues
appear in clusters in the SSB C-terminal domain
(Fig. 4, lines a and b), we analyzed the entire 69 res-
idue region for repeats using REPRO, which identi-
fies protein sequence repeats using a graph-based
iterative clustering procedure.38 Eleven of the identi-
fied repeats are shown in Figure 4 (lines 1–11; yel-
low box). Some are these are identical (highlighted
in red), while the remaining identified repeats
possess a high degree of similarity with one another
(quasi-repeats), for example, repeats 1 and 10. We
also noticed the presence of seven hydrophilic GGX
repeats (line 11). In addition, of the first ten repeats,
nine cluster in the N-terminal half of the IDL
sequence upstream of F148. Likewise, six of the
seven GGX repeats also occur in this region of the
protein.
Similar repeats containing G, P, and Q have
been identified in several eukaryotic proteins such
as the X-type HMW subunit of wheat gluten, spider
silk protein and x-protein where they have been
shown to confer important structural features and
special functions in addition to conferring elastomer-
ic properties to that region of the protein.39 The
over-representation of gly, gln, pro, and ser residues
and the presence of the repeats, may impart similar
elastomeric properties to the C-terminus of SSB,
Figure 3. Alterations in the SSB linker eliminate binding to RecG. The 1-L cultures of cells expressing RecG and SSB or linker
mutants were grown to early log phase, IPTG added to 500 mM and growth continued until early stationary phase. Cells were
harvested by centrifugation, lysed and the cleared cell lysate applied to a 5-mL nickel column as described in the Materials and
Methods. Proteins were then eluted using an imidazole gradient following extensive washing to remove unbound proteins.
Aliquots from various fractions throughout the purification were subjected to electrophoresis. The resulting Coomassie stained,
12% SDS-PAGE gels of the purifications are presented. (A) RecG binds to full-length SSB as shown previously.7 (B) RecG does
not bind appreciably to SSB125. (C) RecG does not bind appreciably to SSB155. MW, molecular weight marker; WCL, whole cell
lysate; CCL, cleared cell lysate; FT, flow through; numbers, indicate fraction numbers from the peak elution profile.
Bianco et al. PROTEIN SCIENCE VOL 26:227—241 231
enabling it to interact with a large range of partners
of different sizes.
Finally, we also identified three PXXP motifs:
PQQP, PAAP and the similar, PQQS (Fig. 4, motif).
Two of these are deleted in SSB155 and all three are
absent in SSB145. In summary, these analyses sug-
gest that in addition to length, the sequence compo-
sition of the SSB linker is critical to protein–protein
interactions.
TmSSB binds to EcRecO in vivoTo understand whether linker length or sequence
composition is more critical to binding, we decided
to test these independently. First, to evaluate the
role of linker length in protein binding, we selected
an SSB that had a naturally occurring short linker
region relative to the E. coli protein. For this pur-
pose, we selected the Thermotoga maritima SSB
(TmSSB) which has a linker 22 residues in length,
approximately the same as that of SSB145 [Fig. 2(B),
Supporting Information Fig. S1]. In addition, the
last six residues of TmSSB are 50% identical to
that of EcSSB. Correspondingly, we cloned the
Thermotoga RecG protein, which is similar to
EcRecG, but has additional N-terminal residues of
unknown function.40
To test binding, the TmRecG and his-TmSSB
proteins were expressed in E. coli and the resulting
cleared cell lysate subjected to nickel column chro-
matography. The results show that as for the E. coli
proteins, Thermotoga RecG and SSB coelute
although the amount of RecG eluted is significantly
lower [Fig. 5(A)]. The ratio of TmSSB:TmRecG is on
average, 25 whereas for Ec proteins, the average
was 7 [Fig. 5(D)]. Next, we tested binding of TmSSB
to EcRecG. As before, cleared cell lysates were sub-
jected to nickel column chromatography and pro-
teins eluted with a linear imidazole gradient. The
resulting SDS-PAGE gel shows a significant level of
TmSSB eluted and only a small, but detectable level
of EcRecG [Fig. 5(B)]. The ratio of TmSSB: EcRecG
eluted is 39 6 3 and this is comparable to what we
observed for SSB155 where the ratio was 70 6 10
(Fig. 3). This suggests that binding of TmSSB to
EcRecG is impaired.
Next, to test binding to the smaller RecO pro-
tein and as the T. maritima genome does not con-
tain recO, EcRecO had to be used instead. The
results from the nickel column show that the
EcRecO coeluted with TmSSB and the ratio of
SSB:RecO was 3.9 6 0.6 [Fig. 5(C)]. However, the
amount of SSB was in excess of RecO indicating
that binding was not stoichiometric as we observed
for full length E. coli proteins [Fig. 1(E)]. This ratio
is twofold higher than what was observed for the
SSB145 linker mutant [Fig. 2(D)]. Because the
sequences of the linkers in the TmSSB and SSB145
proteins are different while the lengths are similar
[Fig. 2(B)], these data suggest that the sequence of
the linker is more important than length for target
Figure 4. The linker of EcSSB has a specialized sequence composition. The sequence of the C-terminal 69 residues of EcSSB
(top line) contains repetitive elements consisting of glycine, proline and glutamine. Sequence analysis of the protein was done
using REPRO at http://www.ibi.vu.nl/programs/.38 The position of beta sheet 6 is indicated.34,43 Lines a and b illuminate the
over-represented residues in the SSB C-terminus (explained in the next). Lines 1–11 (yellow box) show a subset of the repeated
sequence elements identified using REPRO.35,38 The PXXP motifs were identified by eye. The amino acids spanning the puta-
tive PPII-helix are indicated by the black line and the positions of the deletions in the linker mutants are also shown.
232 PROTEINSCIENCE.ORG SSB IDL Links ssDNA and Protein Binding
protein binding, as suggested by the sequence
analyses.
Binding of SSB to EcRecO requires
more than the acidic tip
To further understand the role of sequence composi-
tion of the linker region in target protein binding,
we utilized a series of five hybrid SSB proteins and
determined their ability to bind EcRecO in vivo.
These hybrid SSBs are combinations of the E. coli
and Mycobacterium tuberculosis proteins and are
shown schematically in Figure 6(A).41,42 The sequen-
ces of each hybrid aligned to full length E. coli SSB
are shown in Supporting Information Figure S2. In
both instances, MtSSB is shown for comparison only
and was not studied here. We selected MtSSB as the
protein has been well studied in vivo and in vitro,
its N-terminal core is similar to that of EcSSB and
the last six residues of the acidic tip are 67% identi-
cal. The hybrid proteins are designated EM followed
by a number. EM-1 and 2 are control proteins with
altered core domains and otherwise E. coli linker
and C-termini, while the test proteins EM-3, 4, and
5 have chimeric linker regions as explained below.
Hybrid proteins EM-1 and EM-2 each have an
E. coli C-terminus and split core domains [Fig.
6(A)]. For EM-1, the N-terminal 73 residues are
Mycobacterial in origin with the remainder of the
protein from E. coli. In contrast, for EM-2, the
C-terminal part of the core, corresponding to resi-
dues 73–131, is from MtSSB. EM-3 is an otherwise
E. coli protein with only part of beta sheet 6 and the
downstream sequence from the Mycobacterium
protein replacing the corresponding region. EM-4
has part of beta sheet 6 as well as the C-terminus
from M. tuberculosis. Finally, EM-5 is an E. coli
protein with only its C-terminal 33 amino acids
replaced with the Mycobacterial sequence. Each of
the hybrids selected forms homotetramers, retains
binding to ssDNA and with the exception of EM-2,
are functional when expressed in E. coli.41,42
To test whether these hybrid proteins could bind
to EcRecO, separate strains expressing his-RecO
and the five hybrid proteins were made, grown,
lysed, and subjected to nickel column chromatogra-
phy as before. Analysis of the resulting gels reveals
some surprising results [Fig. 6(B)]. First, binding of
the EM-1 and EM-2 proteins to RecO is reduced 2.5-
fold. This was surprising given that EM-2 is inactive
in vivo.41,42 Second, and perhaps the most surprising
result is that the efficiency of binding of EM-4 is
reduced 10-fold, while EM-3 and EM-5 bind as well
as wild type E. coli SSB. The failure of EM-4 to bind
is not simply due to a length change because the
EM-5 hybrid is three residues shorter and it retains
wild type levels of binding. Nor is it the presence of
Figure 5. TmSSB forms stable complexes with TmRecG and EcRecO but not EcRecG. The 1-L cultures of cells expressing RecG
(Ec or Tm) with his-TmSSB were grown to early log phase, IPTG added to 500 mM and growth continued until early stationary phase.
Cells were harvested by centrifugation, lysed and the cleared cell lysate applied to a 5-mL nickel column as described in the
Materials and methods. Proteins were then eluted using an imidazole gradient following extensive washing to remove unbound
proteins. Aliquots from various fractions throughout the purification were subjected to electrophoresis. Coomassie stained, 15%
SDS-PAGE gels showing various stages during the purification are presented. (A) TmRecG binds to TmSSB. (B) EcRecG does not
bind appreciably to TmSSB. (C) TmSSB binds to EcRecO. (D), Analysis of the gels in panels A–C. The data for EcSSB/EcRecG are
from column 1, Figure 3(D) and are presented here for comparison. MW, molecular weight marker; WCL, whole cell lysate; CCL,
cleared cell lysate; numbers indicate fraction numbers from the peak elution profile.
Bianco et al. PROTEIN SCIENCE VOL 26:227—241 233
the Mycobacterial terminal eight residues replacing
the E. coli acidic tip, as EM-5 binds well. Instead,
residues 111–131 from MtSSB in combination with
the Mycobacterial C-terminus provide insight into
how the linker may be involved in protein-protein
interactions.
Perturbation of the putative PPII helix-like
region eliminates binding
To gain further insight into the contributing factors
affecting binding in the hybrids, sequence analysis for
composition and repetitive elements was done. Analysis
of the MtSSB sequence reveals it to be over-represented
for Ser (19%), Gly (18%), Ala (14%), and Pro (12%).
Furthermore, and not surprisingly, the REPRO
analysis identified repeats that were different from that
of EcSSB and, in contrast to E. coli, these repeats are
clustered in the C-terminal half of the sequence
(Supporting Information Fig. S4). The clustering within
this region of the C-terminus, prompted us to ascertain
whether a model could be built using collagen as a tem-
plate. Surprisingly, we were able to build a model of the
C-terminal 33 residues of MtSSB using the collagen-
like peptide (Supporting Information Fig. S5). In
contrast, a model of the core-proximal region of the
linker of this SSB could not be built (data not shown).
Next we analyzed hybrid proteins 3, 4, and 5.
Replacement of residues 114–130 as in the EM-3
protein does not significantly alter the over-
representation of amino acids but eliminates five of
the ten repeats identified in EcSSB and only repeats
similar to 2 and 4–7 are retained. In addition, and
Figure 6. The sequence composition of the EcSSB linker influences binding to RecO. (A) Schematic of EcSSB, five Mt-Ec-SSB
hybrid proteins and MtSSB is shown. Residue numbering is from the E. coli sequence with methionine as 1. Numbers
immediately above the hybrid proteins demarcates where the sequence changes from E. coli to M. tuberculosis. (B) Some
hybrid proteins bind to EcRecO while others do not. Quantification of the Coomassie-stained gels of apex fractions from each
coelution of his-RecO and the five hybrid SSB proteins is shown. The 100 mL cultures of his-RecO and each of the five hybrid
were grown separately to early log phase and expression induced with 500 mM IPTG. Cells were harvested in early stationary
phase by centrifugation and the resulting pellets lysed. The cleared cell lysates were applied to 1-mL nickel columns,
extensively washed and eluted with elution buffer as described in the Materials and methods. Eluted fractions were subjected
to electrophoresis in 15% SDS-PAGE gels.
234 PROTEINSCIENCE.ORG SSB IDL Links ssDNA and Protein Binding
in contrast to the wild type protein, the clusters
identified occur in the C-terminal half of the
sequence (data not shown). In addition, while the
region corresponding to the proposed PPII-like helix
is perturbed, the PXXP motifs are retained. Analysis
of EM-4, shows a reduction in Gln to only 2 resi-
dues, Gly and Pro are reduced while Alanine and
Serine are increased. In addition, all of the repeats
present in the wild type E. coli SSB C-terminal 69
residues are eliminated (not shown). Finally, EM-5
retains the PPII-like region but the sequences of the
PXXP motifs are all different. In summary, these
data suggest that when the sequence and/or position
of the proposed PP-II helix are altered it has profound
effects on SSB-protein interactions.
To understand how the changes in sequence in
each of the hybrids relative to wild type E. coli SSB
could lead to the elimination of binding to RecO, we
performed homology modeling of the intact hybrid
proteins using Swiss-model.36,37 In each case, models
of each hybrid were built using subunit 1 of 1QVC
as a template as in this structure, residues 2–145
are visible.43 The resulting Connolly surface repre-
sentations for residues 112 to the C-terminal residue
of each model are shown in Figure 7 with the same
region from subunit 1 from the SSB structure 1QVC
as a reference. When viewed in this way, it is evi-
dent that the N-terminal part of this region presents
as a PPII-like helix with minimal additional second-
ary structure for the EcSSB and EM-3 proteins
(Fig. 7, yellow shaded region). Even though this
region of SSB can adopt different conformations, res-
idues 112–130 still present as a PPII-like helix with
minimal additional secondary structure. In contrast,
the same region in the EM-4protein does not present
as a PPII-like helix but has increased secondary
structure. This results in this region being more
compact and possibly more rigid, stabilized by
hydrogen binding (not shown), resulting in a protein
that cannot bind to RecO. Even though these are
models, they suggest that the changes in primary
amino acid sequence of residues 112–130 leading to
disruption of the putative PPII helix, has profound
effects on the ability of EcSSB to interact with
target proteins.
Discussion
The primary conclusion of this article is that the
intrinsically disordered linker (IDL) connects ssDNA
binding and protein–protein interactions of SSB
using a common mechanism. This mechanism
involves binding of the IDL of an SSB tetramer to
the OB-fold of a nearby protein as explained below.
The importance of the IDL became clear when
sequence analysis revealed several previously unidenti-
fied features that impart specificity, tensile strength,
and flexibility. These properties are conferred on SSB in
the following ways. First, for the linker region to func-
tion correctly, either all, or a subset, of the amino acids
present must be able to adopt a PPII-like helix confor-
mation. This may impart tensile strength when SSB
tetramers associate with one another along a stretch of
ssDNA or when bound to a target protein. Second, the
primary amino acid sequence of the C-terminal 69 resi-
dues arranged in various repeats, imparts flexibility
possibly in a similar fashion to that observed in proteins
such as wheat gluten. This enables SSB to bind to all
interactome partners, regardless of their size or the posi-
tion within multi-subunit complexes and also between
SSB tetramers. Finally, the presence of PXXP motifs
suggests that this region of SSB is directly involved in
protein-protein interactions: either between SSB tet-
ramers during cooperative binding to ssDNA or between
SSB and interactome partners. These interactions
Figure 7. The proposed PPII helix region is important to EcSSB function. Homology modeling of the EM-3 and 4 proteins was
done at Swiss-model in automated mode using PDB file 1QVC as a template.37,39,43 Models were built on the first subunit of
1QVC. As residues 1–111 in each model are identical to 1QVC they are not displayed. Instead, the Connolly surfaces of resi-
dues 110 to the C-terminus of each model are shown with the N-termini at the top left of each image.44,67 For EcSSB this is to
residue 145; for EM-3 it extends to position 149, and for EM-4 to amino acid 161. Colouring is done according to hydrophobici-
ty as indicated and the orientation of each region is approximately the same. A sequence alignment of the hybrids to EcSSB is
shown in Supporting Information Figure S2.
Bianco et al. PROTEIN SCIENCE VOL 26:227—241 235
involve binding between the PXXP regions of SSB and
the OB-fold in the target protein, either another SSB or
an interactome partner.
The in vivo binding studies revealed that SSB
binds to RecO in vivo, similar to what was observed
previously for PriA and RecG.7 Binding was not
observed when SSBDC8 replaced wild type and when
the histidine tag was present on the C-terminus of
RecO, consistent with previous work showing that
this region was involved in SSB binding.32 The in vivo
binding assay was subsequently used to determine
the effects of deletions of residues within the linker on
binding to RecO and separately, to RecG. Results
show that the deletion of 21 residues eliminated bind-
ing to RecG, whereas binding to RecO was only affect-
ed when 42 or more residues were removed. To
determine whether linker length is critical or whether
sequence may be important, we used the Thermotoga
maritima SSB. The IDL in this protein is 16–18 resi-
dues in length, comparable to that in the SSB145
mutant. In vivo binding to EcRecO was observed but
this was reduced compared to full length EcSSB, and
more importantly, to SSB145 as well. Surprisingly,
TmSSB bound to TmRecG but not to EcRecG. Tm/Tm
binding was reduced compared to Ec/Ec binding, but
this was not surprising given these are thermostable
proteins. Collectively, these results suggested that
while the length of the linker is important for protein–
protein interactions, the sequence is more critical, as
illuminated below.
The analysis of the sequence of the C-terminal 69
residues of EcSSB revealed an over-representation of
glycine, glutamine, proline, serine, and alanine. Fur-
ther analysis with REPRO showed the presence of
repeats of the over-represented residues in various com-
binations [Fig. 4(A)]. Similar sequence composition and
repeats have been found in a large number of eukaryot-
ic proteins where they have been shown to confer elas-
tomeric properties to that region of each protein.39
These include the flagelliform silk proteins (repeats of
GPGGx), spider silk proteins (GGX), the X-type HMW
subunit of wheat gluten and x-protein (repeats of
PGQGQQ and GQQ) and dragline protein (repeats of
GPGQQ).44 Each of these repeats occurs in the linker
region of E. coli SSB with most being repeated once and
GGX seven times [Fig. 4(A)]. In the eukaryotic proteins,
the same sequence is typically repeated many times.
The presence of this collection of repeats confers
elastomeric properties such as those observed in the
eukaryotic proteins enabling the C-terminal 69 resi-
dues to adopt different conformations thereby facilitat-
ing a larger range of partner proteins with which SSB
can bind. This sequence composition likely contributes
to the movements of the C-terminal domain of the pro-
tein associated with ssDNA binding.45
Our analysis of the linker region of EcSSB sug-
gests that the amino acids present in the region from
Q117 to Q147 have a propensity to form a PPII-like
helix.46–48 This is supported by modeling which
showed that this region could adopt a structure
resembling a collagen monomer. While this analysis
does not prove the existence of a PPII-helix (experi-
ments are underway to determine this possibility
experimentally), it provides some insight into what
this region of SSB could be doing. First, PP-II helices
are known to bind nucleic acids and to be involved in
protein–protein interactions.34,48,49 Our results
showing that perturbations in the sequence of this
region, as well as in changes in its position relative
to the core of SSB, severely impaired target protein
binding is consistent with a role in protein–protein
interactions. Second, the accessibility of PPII helices
is greatly enhanced by the fact that they are
frequently found either at the amino- or carboxyl ter-
mini of proteins where they form extended structures
that have been described as “sticky arms.”46–48 Third,
PPII-helices are flexible, and this has been suggested
to be the major feature underlying its function in pro-
tein interactions.50
However, this is not the only important compo-
nent of the linker region. The identification of PXXP
motifs in the C-terminus of SSB suggests a possible
mechanism for binding specificity. PXXP motifs are
most well known for their ability to bind structurally
conserved Src homology 3 (SH3) domains. These
domains are �50 residue modules that are ubiquitous
in biological systems and which often occur in signal-
ing and cytoskeletal proteins in eukaryotes.48,51–53
The SH3 domain has a characteristic fold which con-
sists of five or six beta-strands arranged as two tightly
packed anti-parallel beta sheets and is similar in
structure to the OB-fold.54 SH3-like domains have
been identified in several E. coli proteins, including
Exonuclease I, an SSB-interactome partner.14,55 This
prompted us to look at the crystal structures available
of SSB interactome partners to determine whether
they contained an OB-fold. Indeed, each structure
examined contains an OB-fold (not shown and
P.Bianco, manuscript in preparation).
Therefore, since SSB has PXXP motifs and each
partner as well as SSB has an OB-fold, we propose the
following model, shown in schematic fashion in Figure
8. The linker(s) from one SSB tetramer bind to the
OB-fold of the interaction partner. When the partner
is another SSB tetramer, cooperative binding to
ssDNA results with multiple interactions forming a
stable complex wherein the ssDNA is extremely well
protected. In similar fashion, when the nearby protein
is a member of the SSB interactome, storage or load-
ing of that protein on to the DNA results, as shown for
the fork rescue DNA helicase RecG.22,56 While the
OB-fold is most well-known for its ability to bind oligo-
saccharides and single stranded nucleic acids, peptide
binding by an OB-fold is not without precedent. A
scan of the PDB reveals more than fifteen instances
236 PROTEINSCIENCE.ORG SSB IDL Links ssDNA and Protein Binding
including matrix metalloproteinases, Staphylococcal
enterotoxin B, and yeast RNA polymerase II.15,57–59
If the model suggested above is correct, then
there are some obvious predictions. First, loss of
these motifs should eliminate binding to interactome
partners. This was clearly the case for RecG but not
for RecO, indicating that other parts of the IDL may
be important for binding as well. Second, removal of
these motifs should negatively impact cooperative
binding to ssDNA. This is exactly what was
observed by the Lohman group.23 Third, mutation of
the PXXP motifs should negatively impact. Prelimi-
nary data where the motifs were interrupted or
replaced eliminated binding (P. Bianco, manuscript
in preparation). Finally, elimination of the OB-fold
in an interacting partner should eliminate binding.
Consistent, removal of the corresponding regions
in both RecO and RecG eliminate binding entirely
(P. Bianco, unpublished).60
In summary, we propose that the linker region
of E. coli SSB is essential for optimal functioning of
the protein. While previously thought of as being
unimportant, our results show that this is no longer
the case. SSB has two seemingly disparate roles:
it binds rapidly and cooperatively to ssDNA and it
binds to partner proteins that constitute the SSB
interactome (Fig. 8). These two roles are not dispa-
rate but are instead, intimately linked. They are
linked by the properties identified in the IDL that
facilitate protein-protein interactions. When these
interactions occur between an SSB tetramer and an
interactome partner, loading of that protein onto
DNA takes place. When the interactions take place
between SSB tetramers, cooperative ssDNA binding
occurs. When these key elements—the putative
PPII-helix, the repeats and the PXXP motifs are
perturbed, SSB no longer functions correctly.23
Materials and Methods
Chemicals and reagents
All chemicals were reagent grade and were made up
in Nanopure water and passed through 0.2-mm pore
size filters. Ampicillin, IPTG, NaCl, Na2HPO4, tryp-
tone, and yeast extract were from Fisher Scientific
(NJ). Coomassie brilliant blue R-250, kanamycin,
lysozyme, sucrose, tris-base, triton x-100, sodium
dodecyl sulfate were from AMRESCO (OH). EDTA,
NaH2PO4, and nickel sulfate were from J.T. Baker
(NJ). Sodium deoxycholic acid and imidazole were
from Acros Organics (NJ). The infusion kit was from
Clontech (CA). Nonidet P40 was from USB (OH).
Protease and phosphatase inhibitor mini tablets
were from Thermo Scientific (IL). PMSF was from
Omnipur (NJ). The 1 and 5 mL HisTrap FF crude
columns, Ni Sepharose 6 Fast Flow were from GE
Healthcare Life Sciences (NJ). Oligonucleotides were
purchased from IDT (Coralville, IA) and are listed in
Supporting Information Table SI.
Cloning
A pET21a1 plasmid containing the E. coli ssb gene
was obtained from Dr. James Keck (UW-Madison).
The ssb gene was amplified by PCR and subcloned
into pET15b to produce his-SSB. Positive clones
were identified by restriction enzyme mapping and
verified by DNA sequencing. All oligonucleotides
used are listed in Supporting Information Table SI.
To create a series of truncated C-terminal linker
domain SSBs, the region comprising amino acids
113–168 was deleted in separate reactions by infusion
cloning. In these reactions, a single forward primer
(PB513) was used in combination with four reverse
primers (PB514-517) in separate reactions. PCR was
done using the various primer combinations and Infu-
sion to create the final construct. The mutants contain
the SSB N-terminal domain (residues 1–112), various
length linker regions (residues 113–168) and the
C-terminal tail (residues 169–178). The linker domain
mutants constructed were: SSB125 (53 residues delet-
ed; final length: 125aa), SSB133 (48 residues deleted;
final length: 130aa), SSB145 (33 residues deleted; final
length: 145aa) and SSB155 (23 residues deleted; final
length: 155aa). Positive clones were identified by
Figure 8. SSB linkers mediate all protein–protein interac-
tions. Schematics of wild type SSB proteins binding to
ssDNA and to partners. The colouring of SSB monomers is
as follows the core domain in green, linker in blue and acidic
tip in red. Here tetramers have their functional C-terminal
domains exposed in solution. Upon binding to ssDNA the
IDLs of monomers 1 and 2, bind to the OB-folds of mono-
mers 10 and 20, respectively. Concurrently, the linkers of
monomers 10 and 20 bind to the OB-folds of monomers 3 and
4, and their IDLs bind to monomers 30and 40, respectively.
The C-termini of subunits 30 and 40 are available to bind to an
incoming tetramer as shown on the right. On the opposite
side of each tetramer, C-termini are available for binding to
interactome partners such as RecG or RecO (purple and blue
ovals). For simplicity, SSB–SSB interactions are shown in the
top subunits only, and SSB interactome partners binding in
the lower subunits.
Bianco et al. PROTEIN SCIENCE VOL 26:227—241 237
restriction enzyme mapping and verified by DNA
sequencing. The resulting plasmids created were:
pET21a1-SSB125, pET21a1-SSB133, pET21a1-SSB145,
and pET21a1-SSB155. Next, each mutant was cloned
using Infusion technology into pET15b to create
histidine-tagged fusion proteins. Positive clones were
identified by restriction enzyme mapping and verified
by DNA sequencing.
To construct N-terminal his6 tagged truncated
SSBs, primers (PB549 and 550) were used to amplify
truncated ssb genes from the vectors described above
followed by Infusion cloning of the PCR fragments
into pET15b at NdeI and BamHI sites.pET15b-wt-
recO was made by amplifying genomic E. coli recO
from MG1655 using primers (PB458 and 459) and
inserted into pET15b at NcoI and EcoRI sites via Infu-
sion Cloning. To create C-terminal histidine tagged
recO, the recO gene was amplified using primers
(PB462 and 463) and then inserted into pET28a1 at
NcoI and HindIII sites using Infusion Cloning. Posi-
tive clones were identified by restriction enzyme map-
ping and verified by DNA sequencing. The resulting
plasmid was pET28a1-his-rec0.pUC57 plasmids con-
taining the Thermotoga maritima (Tm) ssb and recG
genes were purchased from Genescript. The original
Thermotoga sequences were codon optimized for
expression in E. coli to produce SSB (Q9WZ73); and
RecG (Q9WY48). To create N-terminal his-TmSSB,
the pUC57 Tmssb gene was amplified by PCR using
primers (PB523 and 525) and inserted into NdeI and
BamHI sites of the pET15b vector (Novagen) using
Infusion cloning (Clontech). To create a pET28a1 plas-
mid expressing TmRecG, pUC57 containing TmrecG
gene was amplified by PCR using (PB526 and 528)
and inserted into NcoI and EcoRI sites of the pET28a1
vector using Infusion cloning. Positive clones were
identified by restriction enzyme mapping and
DNA sequencing. The resulting clones were: pET15b-
his-Tmssb, and pET28a1-TmrecG.
Construction of the E. coli–M. tuberculosis hybrid
genes was described previously.41–43 These genes were
amplified by PCR and cloned by Infusion into pET15b.
Positive clones were identified by restriction enzyme
mapping and DNA sequencing. The resulting plas-
mids and strains are listed in Supporting Information
Tables SII and SIII, respectively.
Dual plasmid expression
To facilitate coexpression of his-RecO with potential
binding partners, pET28a1-his-recO was transformed
into Tuner (kDE3) cells. A colony in which high levels
of expression of his-RecO was observed was selected
and made competent. Next, these cells were trans-
formed separately with test plasmids encoding wtSSB,
SSB125, SSB133, SSB145, and SSB155. Next, expression
of his-RecO and the SSB proteins were determined
using 5-mL cultures. Fresh overnights were used to
inoculate LB1 antibiotics (ampicillin and kanamycin,
250 and 25 lg mL21, respectively) and cells grown at
378C for 3 h. IPTG was added to either 100 lM, 500
lM, or 1 mM final, followed by an additional 2 h of
growth. Protein expression levels were assessed using
SDS-PAGE. A colony in which high levels of both his-
RecO and the SSBs were observed was selected and
grown overnight in LB1 antibiotics and 0.2% glucose.
The overnights were used to inoculate 0.1 and 2 L
cultures the following day at a 1:100 ratio.
To facilitate coexpression of TmhisSSB with poten-
tial binding partners, Tuner (kDE3) cells that
expressed his-Tmssb from the pET15b plasmid were
made competent and transformed separately with
pET28a1-TmrecG, pET28a1-EcrecG, or pET28a1-
EcrecO plasmids. Next, expression was determined as
described above except IPTG was used at 500lM. Pro-
tein expression levels were assessed using SDS-PAGE.
A colony in which overexpression of both his-TmSSB
and the target binding proteins was observed was
selected and grown overnight as described above. The
overnights were used to inoculate 1- and 2-L cultures
the following day at a 1:100 ratio.
0.1, 1, and 2 liters cell growth
The 1, 10, and 20 mL of fresh overnight cultures
were added to 100 mL, 1 L, and 2 L of LB, respec-
tively. LB contained ampicillin (250 lg mL21) and
kanamycin (25 lg mL21). Cells were grown at 378C
with vigorous shaking. When the OD600 was between
0.4 and 1, IPTG was added to 500 lM final, and
growth continued for an additional 3 h. Cells were
harvested by centrifugation at 48C and cell pellets
were resuspended in lysis buffer (50 mM Tris-HCl (pH
8.0), 20% sucrose) using 2.05 mL g21 of wet cell
weight. Resuspended cells were frozen at 2208C or
stirred at 48C overnight for lysis the following day.
Elution of proteins using an imidazole gradientThe 100 mL, 1 L, and 2 L cultures were lysed and
the cleared cell lysate was subjected to affinity chro-
matography using a 1 mL or 5 mL HisTrap FF crude
column. To lyse the cells, lysozyme (1 mg mL21
final), benzonase 5 lL, Mg(OAc)2 (4 mM final),
CaCl2 (2 mM final), PMSF (1 mM final), protease
inhibitor mini tablets (50 mL cells/tablet) were
added and mixture stirred for 30 min at 48C. Deoxy-
cholate (0.5% final) and triton x-100 (0.1% final)
were added slowly followed by stirring for 40 min.
Imidazole and NaCl were then added to 30 and
600 mM final, respectively, and stirred for 5 min.
The whole cell lysate of each was centrifuged at
35,000g at 48C for 30 min. The cleared cell lysate
was loaded onto a nickel column equilibrated in
Binding Buffer (20 mM sodium phosphate; 600 mM
NaCl; 30 mM Imidazole; pH 7.4). The column was
sequentially washed with 400 mL binding buffer,
350 mL binding buffer with 0.2% NP40 and 250 mL
binding buffer only. A linear gradient of 30–500 mM
238 PROTEINSCIENCE.ORG SSB IDL Links ssDNA and Protein Binding
of Imidazole was used to elute bound proteins from
the column. Proteins were assessed by SDS-PAGE.
Following electrophoresis, gels were stained with
Coomassie brilliant blue, destained, and photo-
graphed. Protein fractions were pooled and then pre-
cipitated in 70% ammonium sulfate for short term
storage or further processing.
Small scale, step-elution of proteinsCell lysis, centrifugation and buffers were identical
to that of larger cultures with volumes adjusted
accordingly. The cleared cell lysate was loaded onto
the 1 mL HisTrap FF crude column. The column
was washed with 45 mL binding buffer. Proteins
were eluted using 6 mL of Elution buffer containing
500 mM Imidazole. The 1 mL fractions were collect-
ed and aliquots subjected to SDS-PAGE. The majori-
ty of protein(s) eluted in the third fraction.
Analysis of coomassie stained gels
Photographs of stained, SDS-PAGE gels were ana-
lyzed using the Gels Analysis function in Fiji.61,62
Here, the area under each peak was determined from
the densitometric trace of each lane. This value was
then divided by the molecular weight of each protein
to correct for the different amounts of Coomassie dye
being present in bands, assuming each protein stained
equally well with Coomassie. The ratio of protein A:
protein B was then determined as is displayed in vari-
ous graphs throughout the article. Each coelution
experiment was repeated two to five times, with
a minimum of three lanes per gel analyzed and
combined to yield the resulting graphs.
References
1. Shereda RD, Kozlov AG, Lohman TM, Cox MM, Keck
JL (2008) SSB as an organizer/mobilizer of genome
maintenance complexes. Crit Rev Biochem Mol Biol 43:
289–318.2. Chase JW, Williams KR (1986) Single-stranded DNA
binding proteins required for DNA replication. Annu
Rev Biochem 55:103–136.3. Meyer RR, Laine PS (1990) The single-stranded DNA-
binding protein of Escherichia coli. Microbiol Rev 54:
342–380.4. Kowalczykowski SC, Dixon DA, Eggleston AK, Lauder
SD, Rehrauer WM (1994) Biochemistry of homologous
recombination in Escherichia coli. Microbiol Rev 58:
401–465.5. Lohman T, Ferrari M (1994) Escherichia coli single-
stranded DNA-binding protein: multiple DNA-binding
modes and cooperativities. Ann Rev Biochem 63:527–570.6. Costes A, Lecointe F, McGovern S, Quevillon-Cheruel
S, Polard P (2010) The C-terminal domain of the bacte-
rial SSB protein acts as a DNA maintenance hub at
active chromosome replication forks. PLoS Genet 6:
e1001238.7. Yu C, Tan HY, Choi M, Stanenas AJ, Byrd AK, K DR,
Cohan CS, Bianco PR (2016) SSB binds to the RecG
and PriA helicases in vivo in the absence of DNA.
Genes Cells 21:163–184.
8. Sancar A, Williams KR, Chase JW, Rupp WD (1981)Sequences of the ssb gene and protein. Proc Natl AcadSci USA 78:4274–4278.
9. Curth U, Genschel J, Urbanke C, Greipel J (1996) Invitro and in vivo function of the C-terminus of Escheri-
chia coli single-stranded DNA binding protein. NucleicAcids Res 24:2706–2711.
10. Raghunathan S, Kozlov A, Lohman T, Waksman G(2000) Structure of the DNA binding domain of E. coli
SSB bound to ssDNA. Nat Struct Biol 7:648–652.11. Chrysogelos S, Griffith J (1982) Escherichia coli single-
strand binding protein organizes single-stranded DNAin nucleosome-like units. Proc Natl Acad Sci USA 79:5803–5807.
12. Kuznetsov S, Kozlov A, Lohman T, Ansari A (2006)Microsecond dynamics of protein–DNA interactions:direct observation of the wrapping/unwrapping kineticsof single-stranded DNA around the E. coli SSB tetra-mer. J Mol Biol 359:55–65.
13. Umezu K, Kolodner RD (1994) Protein interactions ingenetic recombination in Escherichia coli. Interactionsinvolving RecO and RecR overcome the inhibition ofRecA by single-stranded DNA-binding protein. J BiolChem 269:30005–30013.
14. Genschel J, Curth U, Urbanke C (2000) Interaction ofE. coli single-stranded DNA binding protein (SSB) withexonuclease I. The carboxy-terminus of SSB is the recog-nition site for the nuclease. Biol Chem 381:183–192.
15. Handa P, Acharya N, Varshney U (2001) Chimerasbetween single-stranded DNA-binding proteins fromEscherichia coli and Mycobacterium tuberculosis revealthat their C-terminal domains interact with uracilDNA glycosylases. J Biol Chem 276:16992–16997.
16. Kantake N, Madiraju MV, Sugiyama T, KowalczykowskiSC (2002) Escherichia coli RecO protein anneals ssDNAcomplexed with its cognate ssDNA-binding protein: acommon step in genetic recombination. Proc Natl AcadSci USA 99:15327–15332.
17. Glover B, McHenry C (1998) The chi psi subunits of DNApolymerase III holoenzyme bind to single-strandedDNA-binding protein (SSB) and facilitate replication ofan SSB-coated template. J Biol Chem 273:23476–23484.
18. Witte G, Urbanke C, Curth U (2003) DNA polymeraseIII chi subunit ties single-stranded DNA binding pro-tein to the bacterial replication machinery. NucleicAcids Res 31:4434–4440.
19. Cadman CJ, McGlynn P (2004) PriA helicase and SSBinteract physically and functionally. Nucleic Acids Res32:6378–6387.
20. Shereda RD, Bernstein DA, Keck JL (2007) A centralrole for SSB in Escherichia coli RecQ DNA helicasefunction. J Biol Chem 282:19247–19258.
21. Suski C, Marians KJ (2008) Resolution of convergingreplication forks by RecQ and topoisomerase III. MolCell 30:779–789.
22. Buss J, Kimura Y, Bianco P (2008) RecG interactsdirectly with SSB: implications for stalled replicationfork regression. Nucleic Acids Res 36:7029–7042.
23. Kozlov AG, Weiland E, Mittal A, Waldman V, AntonyE, Fazio N, Pappu RV, Lohman TM (2015) Intrinsicallydisordered C-terminal tails of E. coli single-strandedDNA binding protein regulate cooperative binding tosingle-stranded DNA. J Mol Biol 427:763–774.
24. Savvides SN, Raghunathan S, Futterer K, Kozlov AG,Lohman TM, Waksman G (2004) The C-terminaldomain of full-length E. coli SSB is disordered evenwhen bound to DNA. Protein Sci 13:1942–1947.
25. Kelman Z, Yuzhakov A, Andjelkovic J, O’Donnell M(1998) Devoted to the lagging strand-the subunit of
Bianco et al. PROTEIN SCIENCE VOL 26:227—241 239
DNA polymerase III holoenzyme contacts SSB to pro-mote processive elongation and sliding clamp assembly.EMBO J 17:2436–2449.
26. Bhattacharyya B, George NP, Thurmes TM, Zhou R,Jani N, Wessel SR, Sandler SJ, Ha T, Keck JL (2014)Structural mechanisms of PriA-mediated DNA replica-tion restart. Proc Natl Acad Sci USA 111:1373–1378.
27. Marceau AH, Bahng S, Massoni SC, George NP,Sandler SJ, Marians KJ, Keck JL (2011) Structure ofthe SSB-DNA polymerase III interface and its role inDNA replication. EMBO J 30:4236–4247.
28. Shereda RD, Reiter NJ, Butcher SE, Keck JL (2009)Identification of the SSB binding site on E. coli RecQreveals a conserved surface for binding SSB’s C termi-nus. J Mol Biol 386:612–625.
29. Su XC, Wang Y, Yagi H, Shishmarev D, Mason CE,Smith PJ, Vandevenne M, Dixon NE, Otting G (2014)Bound or free: interaction of the C-terminal domain ofEscherichia coli single-stranded DNA-binding protein(SSB) with the tetrameric core of SSB. Biochemistry53:1925–1934.
30. Shishmarev D, Wang Y, Mason CE, Su XC, Oakley AJ,Graham B, Huber T, Dixon NE, Otting G (2014) Intramo-lecular binding mode of the C-terminus of Escherichia coli
single-stranded DNA binding protein determined bynuclear magnetic resonance spectroscopy. Nucleic AcidsRes 42:2750–2757.
31. Lu D, Keck JL (2008) Structural basis of Escherichia
coli single-stranded DNA-binding protein stimulationof exonuclease I. Proc Natl Acad Sci USA 105:9169–9174.
32. Ryzhikov M, Koroleva O, Postnov D, Tran A, Korolev S(2011) Mechanism of RecO recruitment to DNA bysingle-stranded DNA binding protein. Nucleic Acids Res39:6305–6314.
33. Ryzhikov M, Korolev S (2012) Structural studiesof SSB interaction with RecO. Methods Mol Biol 922:123–131.
34. Adzhubei AA, Sternberg MJ, Makarov AA (2013)Polyproline-II helix in proteins: structure and function.J Mol Biol 425:2100–2132.
35. Brown AM, Zondlo NJ (2012) A propensity scale for type IIpolyproline helices (PPII): aromatic amino acids in proline-rich sequences strongly disfavor PPII due to proline-aromatic interactions. Biochemistry 51:5041–5051.
36. Schwede T, Kopp J, Guex N, Peitsch MC (2003)SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31:3381–3385.
37. Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G,Schmidt T, Kiefer F, Cassarino TG, Bertoni M, Bordoli L,Schwede T (2014) SWISS-MODEL: modelling proteintertiary and quaternary structure using evolutionaryinformation. Nucleic Acids Res 42:W252–W258.
38. George RA, Heringa J (2000) The REPRO server:finding protein internal sequence repeats through theWeb. Trends Biochem Sci 25:515–517.
39. Matsushima N, Yoshida H, Kumaki Y, Kamiya M,Tanaka T, Izumi Y, Kretsinger RH (2008) Flexiblestructures and ligand interactions of tandem repeatsconsisting of proline, glycine, asparagine, serine, and/orthreonine rich oligopeptides in proteins. Curr ProteinPept Sci 9:591–610.
40. Singleton MR, Scaife S, Wigley DB (2001) Structuralanalysis of DNA replication fork reversal by RecG. Cell107:79–89.
41. Rex K, Bharti SK, Sah S, Varshney U (2014) A geneticanalysis of the functional interactions within Mycobacte-
rium tuberculosis single-stranded DNA binding protein.PLoS One 9:e94669.
42. Bharti SK, Rex K, Sreedhar P, Krishnan N, Varshney
U (2011) Chimeras of Escherichia coli and Mycobacteri-
um tuberculosis single-stranded DNA binding proteins:
characterization and function in Escherichia coli. PLoS
One 6:e27216.43. Matsumoto T, Morimoto Y, Shibata N, Kinebuchi T,
Shimamoto N, Tsukihara T, Yasuoka N (2000) Roles of
functional loops and the C-terminal segment of a sin-
gle-stranded DNA binding protein elucidated by X-Ray
structure analysis, J Biochem 127, 329–335.44. Tatham AS, Shewry PR (2000) Elastomeric proteins:
biological roles, structures and mechanisms, Trends
Biochem Sci 25, 567–571.45. Green M, Hatter L, Brookes E, Soultanas P, Scott DJ
(2016) Defining the Intrinsically Disordered C-Termi-
nal Domain of SSB Reveals DNA-Mediated Compac-
tion, J Mol Biol 428, 357–364.46. Matsushima N, Creutz CE, Kretsinger RH (1990) Poly-
proline, beta-turn helices. Novel secondary structures
proposed for the tandem repeats within rhodopsin, syn-
aptophysin, synexin, gliadin, RNA polymerase II, hor-
dein, and gluten, Proteins 7, 125–155.47. Adzhubei AA, Sternberg MJ (1993) Left-handed poly-
proline II helices commonly occur in globular proteins,
J Mol Biol 229, 472–493.48. Kay BK, Williamson MP, Sudol M (2000) The impor-
tance of being proline: the interaction of proline-rich
motifs in signaling proteins with their cognate
domains, FASEB-J 14, 231–241.49. Hicks JM, Hsu VL (2004) The extended left-handed
helix: a simple nucleic acid-binding motif, Proteins 55,
330–338.50. Bochicchio B, Ait-Ali A, Tamburro AM, Alix AJ (2004)
Spectroscopic evidence revealing polyproline II struc-
ture in hydrophobic, putatively elastomeric sequences
encoded by specific exons of human tropoelastin, Bio-
polymers 73, 484–493.51. Dalgarno DC, Botfield MC, Rickles RJ (1997) SH3
domains and drug design: ligands, structure, and bio-
logical function, Biopolymers 43, 383–400.52. Sudol M (1998) From Src Homology domains to other
signaling modules: proposal of the ‘protein recognition
code’, Oncogene 17, 1469–1474.53. Ponting CP, Aravind L, Schultz J, Bork P, Koonin EV
(1999) Eukaryotic signalling domain homologues in
archaea and bacteria. Ancient ancestry and horizontal
gene transfer, Journal of Molecular Biology 289, 729–
745.54. Agrawal V, Kishan RK (2001) Functional evolution of
two subtly different (similar) folds, BMC Struct Biol 1,
5.55. Breyer WA, Matthews BW (2000) Structure of Escheri-
chia coli exonuclease I suggests how processivity is
achieved, Nat Struct Biol 7, 1125–1128.56. Sun Z, Tan HY, Bianco PR, Lyubchenko YL (2015)
Remodeling of RecG Helicase at the DNA Replication
Fork by SSB Protein, Sci Rep 5, 9625.57. Papageorgiou AC, Tranter HS, Acharya KR (1998)
Crystal structure of microbial superantigen staphylo-
coccal enterotoxin B at 1.5 A resolution: implications
for superantigen recognition by MHC class II mole-
cules and T-cell receptors, J Mol Biol 277, 61–79.58. Fernandez-Catalan C, Bode W, Huber R, Turk D,
Calvete JJ, Lichte A, Tschesche H, Maskos K (1998)
Crystal structure of the complex formed by the mem-
brane type 1-matrix metalloproteinase with the tissue
inhibitor of metalloproteinases-2, the soluble progelati-
nase A receptor, EMBO J 17, 5238–5248.
240 PROTEINSCIENCE.ORG SSB IDL Links ssDNA and Protein Binding
59. Cramer P, Bushnell DA, Kornberg RD (2001) Structur-al basis of transcription: RNA polymerase II at 2.8 ang-strom resolution, Science 292, 1863–1876.
60. Lu D, Windsor MA, Gellman SH, Keck JL (2009) Pep-tide inhibitors identify roles for SSB C-terminal resi-dues in SSB/exonuclease I complex formation,Biochemistry 48, 6764–6771.
61. Kozlov AG, Jezewska MJ, Bujalowski W, Lohman TM(2010) Binding specificity of Escherichia coli single-stranded DNA binding protein for the chi subunit ofDNA pol III holoenzyme and PriA helicase., Biochemis-try 49, 3555–3566.
62. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V,Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S,Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K,Tomancak P, Cardona A (2012) Fiji: an open-source platformfor biological-image analysis, Nat Methods 9, 676–682.
63. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, LiW, Lopez R, McWilliam H, Remmert M, Soding J,Thompson JD, Higgins DG (2011) Fast, scalable gener-ation of high-quality protein multiple sequence align-ments using Clustal Omega, Mol Syst Biol 7, 539.
64. Robert X, Gouet P (2014) Deciphering key features inprotein structures with the new ENDscript server,Nucleic Acids Res 42, W320–324.
65. Bella J, Eaton M, Brodsky B, Berman HM (1994)Crystal and molecular structure of a collagen-likepeptide at 1.9 A resolution, Science 266, 75–81.
66. Simossis VA, Heringa J (2005) PRALINE: a multiplesequence alignment toolbox that integrates homology-extended and secondary structure information., NucleicAcids Res 33, W289–294.
67. Connolly ML (1983) Solvent-accessible surfaces of pro-teins and nucleic acids. Science 221, 709–713.
Bianco et al. PROTEIN SCIENCE VOL 26:227—241 241