Biochemical and Biophysical Research Communications 534 (2021) 47e52

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

A novel protein-engineered dsDNA-binding protein (HU-Simulacrum)

inspired by HU, a nucleoid-associated DNABII protein
Bhishem Thakur**, Archit Gupta, Purnananda Guptasarma*
Centre for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education and Research (IISER),
Mohali, Knowledge City, Sector-81, SAS Nagar, Punjab, 140306, India

a r t i c l e i n f o a b s t r a c t

Article history: HU, a DNA-binding protein, has a helical N-terminal region (NTR) of ~44 residues and a beta strand- and
Received 4 November 2020 IDR-rich C-terminal region (CTR) of ~46 residues. CTR binds to DNA through (i) a clasp (two arginine/
Accepted 21 November 2020 lysine-rich, IDR-rich beta hairpins that bind to phosphate groups in the minor groove), (ii) a flat sur-
Available online 10 December 2020
face (comprising four antiparallel beta strands that abut the major groove), and (iii) a charge cluster (two
lysine residues upon a short C-terminal helix). HU forms a dimer displaying extensive inter-subunit CTR-
CTR contacts. A single-chain simulacrum of these contacts (HU-Simul) incorporating all DNA-binding
elements was created by fusing together the CTRs of Escherichia coli HU-A and Thermus thermophilus
HU. HU-Simul is monomeric, binds to dsDNA and cruciform DNA, but not to ssDNA.
© 2020 Elsevier Inc. All rights reserved.

1. Introduction
HU is a bacterial histone-like nucleoid-associated protein (NAP)
[1]. It is classified as a DNABII protein, together with integration
host factor (IHF) and SCOP1 transcription factor 1 [1e3]. Unlike
other DNABII proteins, HU interacts with DNA in a non-sequence-
specific manner; it binds to dsRNA, ssDNA, nicked DNA, bent
DNA, and cruciform DNA, displaying the highest affinity for cruci-
form DNA [1,4,5]. In E.coli, HU exists as two homologous isoforms,
HU-A and HU-B, encoded by the hupA and hupB genes [6]. Most
other bacteria have only one HU gene. HU forms dimeric structures.
In E.coli, these can be either homodimers [HU-AA, HU-BB] or het-
erodimers [HU-AB]. HU dimers also associate into tetramers and
octamers, principally through interactions involving residues in the
N-terminal half of the protein [7,8]. The predominant mode of HU’s
interaction with DNA involves clasping of the minor groove by two
largely-unstructured beta-hairpin loops that emerge from a pair of
antiparallel beta strands present in each monomer. The loops clasp
the minor groove from either side [9]. In E. coli HU, the DNA-binding
beta-hairpin loop shows no electron density during X-ray crystal-
lography, suggesting that it is completely unstructured in the
* Corresponding author.
** Corresponding author.
E-mail addresses: bhishamthakur05@gmail.com (B. Thakur), guptasarma@
iisermohali.ac.in (P. Guptasarma).
absence of bound DNA [8]. However, the structure of this loop in a
DNA-bound state has been determined through the determination
of the structure of a homologous HU (from Anabaena) in a DNA-
bound state [8]. From the structure of Anabaena HU, it is evident
that the two beta hairpin loops from the two HU monomers
embrace DNA in the minor groove, whereas the antiparallel beta
strands that give rise to these beta hairpins touch (or abut) the
major groove. The interactions are shown in Fig. 1A. The ‘head-to-
head and tail-to-tail’ associated HU dimer also has an alpha helical
core, consisting of the interacting alpha helical regions of each of
the two HU monomers (shown in green and cyan colours) which
interact to generate dimeric HU. The helical regions are present in
the N-terminal half of HU’s polypeptide chain (in the E. coli struc-
ture, this region spans residues 1-44), displaying no interactions
whatsoever with DNA. The polypeptide chains of HU and its variant
isoforms thus consists of two equally-sized regions: (i) an N-ter-
minal region (NTR) which is predominantly helical, and which does
not interact with DNA, and (ii) a C-terminal region (CTR) dominated
by beta strands and intrinsically disordered regions (IDRs), which is
involved in the binding of DNA through interactions mediated by
three sites on the CTR: a double-hairpin clasp that clasps the minor
groove, the antiparallel beta strands that contact the major groove,
and a lysine cluster that binds to other regions of DNA [10].
Unfolding of HU involves distinct unfolding intermediates,
rather than a two-state transition [11]. Together with the evidence
of extensive NTR-NTR ‘head-to-head’ and CTR-CTR ‘tail-to-tail’
inter-subunit contacts, and evidence of only NTR-CTR intra-subunit

https://doi.org/10.1016/j.bbrc.2020.11.088
0006-291X/© 2020 Elsevier Inc. All rights reserved.
B. Thakur, A. Gupta and P. Guptasarma Biochemical and Biophysical Research Communications 534 (2021) 47e52

Fig. 1. Association of HU with dsDNA:Panel A. Structure of the Anabaena HU dimer (PDB ID: 1P51) bound to a synthetic 20-mer stretch of dsDNA. Sites of canonical (large oval) and
non-canonical (small oval) interactions with DNA are shown.Panel B.An imagined structure of the HU dimer shown in Panel A. The NTR which is the core helical region (not involved
in contacts with DNA) is shown removed through excision of coordinates from the PDB file. The imagined structure retains both canonical and non-canonical DNA-contacting sites.
Panel C. A schematic showing the imagined structure from Panel B (with the coordinates of both the NTR and the DNA removed), with a cartoon helical linker (shown in black)
joining the C-terminal of one chain (cyan) to the N-terminal of the other chain (green) to generate what could be HU-Simul, with or without the helical linker retaining hundred
percent helicity in the construct. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

contacts (but no NTR-CTR inter-subunit contacts) in HU’s three-
dimensional structure, it seemed to us that the non-two-state
unfolding of HU indicates that the HU molecule consists of two
distinct and potentially-autonomous structural entities: (a) the
NTR-NTR contacts generating the helical core of the HU dimers, and
(b) the CTR-CTR contacts generating the DNA-binding regions of
the molecule. In other words, instead of dissociating and then
unfolding through a non-two-state transition, HU probably displays
unfolding of the helical core (generated by inter-subunit NTR-NTR
contacts) independently of the unfolding of the beta-strand and
IDR-rich core (generated by inter-subunit CTR-CTR contacts).
To examine whether these two cores might fold and assemble
autonomously, and specifically, to further examine whether the
CTR-CTR core can fold and assemble autonomously by tying two
CTR polypeptides together into a single chain through a suitable
linker peptide, we decided to do the following experiment. We
decided to use the CTR [consisting only of residues 44-90 (shown
schematically in Fig. 1B), starting from just before the first residue
of the anti-parallel beta strands containing the beta hairpin, and
ending at the C-terminal end of HU’s C-terminal helix], and join two
copies of the CTR (either derived from the same HU, or from two
different HUs with slightly-different sequences) by a structured
(preferably-helical) linker peptide, as shown schematically in
Fig. 1C.
The purpose of attempting this reconstruction was to examine
whether an engineered protein created through such a fusion
would fold and display DNA-binding in the complete absence of
HU’s NTR regions, and function as a single-chain simulacrum of the
DNA-binding regions of HU (HU-Simul, or mini-HU). To lend
additional stability to the construct, we decided that one of the
anti-parallel pair of beta strands would be derived from a ther-
mophilic HU. Therefore, we decided to fuse the CTR of E. coli HU-A
discussed above with the CTR of T. thermophiles HU [12] into a
construct of 116 residues [including the 47 residues of E. coli HU
CTR, a 14 residues-long linker, the 47 residues of the T. thermophilus
HU CTR, and an affinity tag of 8 residues]. Here, we report that the
construct, HU-Simul, turned out to be monomeric, folded, and
DNA-binding, binding to both dsDNA and cruciform DNA, but not to
ssDNA. Folded HU-Simul can thus be called a chimeric, intra-chain,
non-naturally-occurring, hetero-dimer-simulating, monomeric,
simulacrum of the HU DNABII archetype, consisting of two 8
residues-long helices in each CTR, one presumed 14 residues-long
helical linker, some beta strands and about ~50% randomly coiled
48
secondary structural content, since the beta hairpin loops are
predominantly unstructured.
2. Material and methods
Protein Design. A comparison was carried out to examine
whether key hydrophobic residues (Phe47, Phe50, Phe79 and
Pro77) are conserved at analogous positions in the antiparallel beta
strands of E. coli HU-A and T. thermophilus HU. Alignment of the
structures and sequences of the CTRs of E.coli HU-A and T.thermo-
philus HU was performed to ensure that residues responsible for
DNA binding as well as other inter-subunit contacts between HU
monomers are conserved between the two CTRs. The structures of
the monomers of both proteins were visually examined using
PYMOL software (Supplementary Figs. 1A and 1B). The structures
were superimposed (Supplementary Fig. 1C) by TM-ALIGN soft-
ware [13] and visualized using PYMOL software. Using the TM-
ALIGN software, CTRs of the monomers were superimposed
(Supplementary Fig. 1D) to confirm structural similarity. The se-
quences of the polypeptide chains were also superimposed
(Supplementary Fig. 2), using Clustal Omega software [14]. After
confirming that the similarity of the E. coli CTR (EcCTR) and the
T. thermophilus CTR (TtCTR) is sufficient to indicate a likelihood of
their assembling through hetero-meric EcCTR-TtCTR association,
the two CTRs were notionally fused through the insertion of a
random helical linker (with a length of 14 amino acid residues and
the sequence N- TRKSPQREGQEGPA-C, derived from a bacterial GAF
domain), to bridge the C-terminus of the E. coli CTR and the N-
terminus of the T. thermophilus CTR, as shown schematically in
Fig. 1C, and Supplementary Fig. 1E.
Protein Structure Prediction. The final protein sequence (N-
EcCTR-linker-TtCTR-C) was submitted for structural modelling by
the Robetta software for ab initio protein structural modeling [15].
The structure predicted by Robetta (Fig. 2A) was docked with DNA
(Fig. 2B) using NPDock (Nucleotide protein docking) rigid body
docking software [16]. The DNA used for the docking was the 20-
mer sequence derived from the structure of PDB ID: 1P51 for
DNA-bound Anabaena HU [8].
Cloning and protein expression. The synthetic gene encoding N-
EcCTR-linker-TtCTR-C (HU-Simul) was produced through splicing-
by-overlap-extension PCR (SOE-PCR) and cloned in the pET-23a
vector between Nde-1 and Xho-1 restriction sites in the MCS re-
gion of the vector, for which first suitable primers were used for
B. Thakur, A. Gupta and P. Guptasarma
Fig. 2. Panel A. Structure of HU-Simul predicted by the protein structure prediction software, Robetta. Panel B.The predicted mode of docking of the structure shown in panel A with
20-mer DNA predicted by NPDock. Panel C. SDS-PAGE showing the level of induction of HU-Simul.
generation of the fragments encoding EcCTR and TtCTR, and then
the amplified fragments were joined through SOE-PCR using other
suitable designed primers. Positive clones were prepared in E. coli
XL-1 Blue and sequenced. The pET-23a plasmid was then trans-
formed into Rosetta (DE3) cells for protein expression under IPTG
induction. Cells were induced with 1 mM of IPTG after reaching
O.D. of 0.6 at 600 nm, allowed to produce protein at 37
C for 4 h,
after which cells were harvested by centrifugation at 8000 rpm for
10 min. The cells were pelleted, re-suspended in lysis buffer
(20 mM PBS, pH7.4, with 10 mM imidazole, 1 M Nacl), sonicated,
centrifuged at 11000 rpm for 40 min and the clear protein-
containing supernatant was loaded on Ni-NTA chromatographic
media (15 ml column, Qiagen) equilibrated with lysis buffer.
Loosely bound proteins were removed with 20 mM PBS, containing
20 mM imidazole and 2 M NaCl. Tightly-bound EcCTR-linker-TtCTR
protein (HU-Simul) was eluted with 20 mM PBS containing
250 mM imidazole without NaCl.
Preparation of 20-mer dsDNA substrate. Oligonucleotides [Strand
1: GTTCAATTGTTGTTAACTTG and Strand 2: CAAGTTAACAA-
CAATTGAAC] displaying base pair complementarity were sourced
from IDT or Sigma and reconstituted in water. A 50 mM stock of
dsDNA was made by mixing equal volumes from 100 mM stock
solutions of each of the above oligonucleotides. The mixture was
heated at 80
C and then cooled to room temperature over a period
of a hour to allow Strand 1 to anneal with Strand 2.
Preparation of 4-way junction (4WJ) cruciform substrate. Four-
way junction (4WJ) DNA was sourced in the form of four inde-
pendent oligonucleotides from IDT or Sigma through contract
synthesis, and assembled into 4WJ DNA through addition of the
following four oligonucleotide strands to each other in equimolar
amounts (Strand 1 : 50 -CCCTATAACCCCTGCATTGAATTCCTGTCTGA-
TAA-3’; Strand 2 : 50 -GTAGTCGTGATAGGTGCAGGGGTTATAGGG-3’;
Strand 3 : 50 -AACAGTAGCTCTTAATTCGAGCTCGCGCCCTATCACGA
CTA-3’; Strand 4 : 50 -TTTATCAGACTGGAATTCAAGCGCGAGCTCG
AATAAGAGCTACTGT-30 ).
Establishment of oligomeric status. Gel filtration chromatography
was performed to determine oligomeric status on Akta Purifier 10
(GE), using Superdex-75 [10/300 GL columns]. Protein eluted from
the Qiagen Ni-NTA column was loaded after equilibration with PBS
(pH7.4). Eluted fractions were collected in 1 ml volumes. Moni-
toring of elution was done by detecting UV absorption at 215 nm
(the construct lacks tryptophan residues). Standard calibration was
performed. Dynamic light scattering was performed to determine
hydrodynamic volume, oligomeric status and population size
49
Biochemical and Biophysical Research Communications 534 (2021) 47e52
homogeneity on a Dawn 8þ QUELS dynamic light scattering
workstation (Wyatt), after filtration of the sample and buffer
through a 0.02 mm syringe filter, using a baseline collected with PBS
buffer (pH 7.4). Data was analysed using ASTRA software after
subtracting buffer scattering data from the sample signal. Glutar-
aldehyde crosslinking. Protein fractions from gel filtration were
attempted to be cross-linked using different concentrations of
glutaraldehyde, followed by SDS-PAGE analyses.
Circular Dichroism (CD) spectroscopy. CD spectra were collected
on a Chirascan CD spectrometer (Applied Photophysics), using cu-
vettes of 0.2 cm path length, and protein concentrations of
~0.15 mg/ml. Spectra were recorded spectra in the wavelength
range of 190e250 nm. Thermodynamic stability was assessed
through heating from 25 to 90
C at 4
C/min, collecting one
spectrum/min. Thermokinetic stability was assessed by heating at
90
C for 10 min, collecting one spectrum every 2 min.
Electrophoretic mobility shift assay (EMSA). Binding to dsDNA,
ssDNA and cruciform DNA was assessed by incubating a fixed
amount of DNA with increasing concentrations of HU-Simul and
separating samples on a 2% agarose gel to detect signs of a shift in
electrophoretic mobility of DNA due to protein binding. DNA con-
centrations of 4 mM for dsDNA and 8 mM for ssDNA were used. As
the protein lacks tryptophan residues, protein concentrations were
estimated through the Bradford assay (not accurate). The stock
concentration of the protein was ~0.6 mM and concentrations used
for successive lanes increased by an increment of 1 ml (1.2 mM in the
first capillary with an increment of 0.6 mM in successive capillaries).
The protein and DNA were kept at room temperature for 30 min to
maximize the interaction between them, after mixing. Binding of
protein to DNA was established by appearance of a smeared band of
lower mobility on the 2% agarose gel.
Biolayer interferometry for distinguishing the affinity for ss vs
dsDNA. The interaction of ssDNA and dsDNA with HU-Simul was
done on a BLItz Biolayer Interferometry instrument (ForteBio). HU-
Simul was immobilized on a Ni-NTA biosensor through dipping of
the sensor in 600 nM protein, followed by BLI analyses using 10 and
20 mM dsDNA and ssDNA, respectively.
3. Results and discussion
3.1. Bioinformatics-based analyses of HU-Simul
Supplementary Figs. 1A and 1B, respectively, show the struc-
tures of the monomers of E. coli HU-A (in green) and T. Thermophilus
B. Thakur, A. Gupta and P. Guptasarma
HU (in red). Supplementary Fig. 1C shows that the structures are
highly superimposable (RMSD of superimposition of ~1.07 Å).
Supplementary Fig. 1D shows that the two protein CTRs are also
highly superimposable. Supplementary Fig. 1E presents a schematic
generated to show the likely geometry of heteromeric CTR-CTR
associations between EcCTR and TtCTR, using known dimeric HU
structure(s) to generate an idea of what the HU Simulacrum (HU-
Simul) could look like. The linker peptide (with high helix-forming
propensity, homologous to the helix at the C-terminus of the GAF
domain) is shown as a cartoon (in black) in Supplementary Fig. 1E,
distorted to schematically indicate potential for distortion in the
physical protein construct. Supplementary Fig. 2 shows sequences
of E. coli HU-A and T. Thermophilus HU, as well as an alignment of
the two sequences, along with the NTRs (in black) and the CTRs of
E. coli (in cyan) and T. thermophiles (in red), respectively. The CTR
sequences forming strands 1 and 2 are also shown. Fig. 2A shows
the structure of HU-Simul (i.e., EcCTR-linker-TtCTR) predicted by
Rosetta, with a similar packing of hydrophobic side chains between
antiparallel beta strands of the CTRs as seen in HU dimers, at the
interface between CTRs. Fig. 2B shows the modelled structure of the
protein docked with 20-mer DNA, in which HU-Simul binds to DNA
through a clasping of dsDNA similar to HU, with two beta hairpin
loops wrapped around the DNA backbone from opposite sides,
Fig. 3. Panel A. Gel filtration chromatogram of HU-Simul. Panel B. Glutaraldehyde-treated samples of HU-Simul derived from gel filtration chromatography, analysed on reducing
SDS-PAGE (15%), with details of lanes mentioned in the panel (PMWM stands for protein molecular weight markers). Panel C. Correlation function of HU-Simul size distribution.
Panel D. Size distribution of HU-Simul as a function of hydrodynamic radius.
50
Biochemical and Biophysical Research Communications 534 (2021) 47e52
around the minor groove, and antiparallel beta strands abutting the
major grove.
3.2. Construction and production of HU-Simul
The 116 residues-long sequence of HU-Simul was determined and
established to be N-MVGFGSFKANHRAERTGRNPQTGKEIKIAAANVP
AFVSGKALKDEVQTRKSPQREGQEGPATGFGTFEVRKRKARTGVKPGT-
KEKIKIPATQYPAFKPGKALKDKVKKLEHHHHHH-C, with predicted
molecular weight 12828.75 Da, and isoelectric point (pI) of 10.54
(note: more basic than HU, due to removal of the NTR). Fig. 2C shows
that HU-Simul is expressed in abundance after IPTG induction, and
that it displays anomalous mobility (i.e., higher apparent molecular
weight) than the calculated size of ~12.8 kDa.
3.3. Monomeric status of HU-Simul
The oligomeric nature of the protein was first examined through
size exclusion (gel filtration) chromatography on a 24 ml Superdex-
75 column. Fig. 3A shows that the protein elutes predominantly at
~12.2 ml, corresponding to a size somewhat smaller than ~20 kDa,
suggesting that it is a monomer since a dimer is larger than ~25
kDA. Folded HU-Simul is likely to display two long unstructured
B. Thakur, A. Gupta and P. Guptasarma Biochemical and Biophysical Research Communications 534 (2021) 47e52

Fig. 4. Panel A. A 2% agarose gel EMSA assays in which dsDNA (20-mer) was present in lanes 1-5 at 4 mM, ssDNA (20-mer) was present in lanes 7-10 at 8 mM, and HU-Simul was
present in lanes 1 and 7 at 0.12 mM, in lanes 2 and 8 at 0.72 mM, in lanes 3 and 9 at 1.32 mM, and in lanes 4 and 10 at 2.02 mM concentration. Panel B. BLI sensorgram involving binding
of HU-Simul to the Ni-NTA biosensor before addition of dsDNA. Panel C.BLI sensorgram involving binding of HU-Simul to the Ni-NTA biosensor before addition of ssDNA.

beta hair pin loops containing intrinsically-disordered regions
(IDRs) in solution in the absence of DNA. Thus, the protein’s
behaviour during gel filtration only approximates to expectations of
elution by a ~12.8 kDa protein, with the possibility of the protein
appearing to be somewhat bigger. There appears to be no attendant
aggregation, since no population of protein is seen eluting at
smaller elution volumes. Protein fractions were subjected to cross-
linking attempts through use of a range of glutaraldehyde con-
centrations, to examine existence of oligomeric forms (note: HU is
crosslinked into dimers and higher forms). However, as Fig. 3B
shows, there is no evidence of oligomerization, and only a single
(monomeric) population of the protein is observed on SDS-PAGE
for all glutaraldehyde concentrations used. The diffused smears
below the main bands are typically seen due to internal (intra-
chain) crosslinks that do not allow unfolding to the same degree in
the entire glutaraldehyde-treated population. HU-Simul was also
subjected to dynamic light scattering (DLS) studies in which the
mono exponential decay of the scatter correlation function is
indicative of homogeneity of molecular conformations. Fig. 3C
shows the scatter correlation function representing distribution of
scattering intensity with time, used to calculate the diffusion con-
stant that yields a molecule’s hydrodynamic radius. The molecular
size plotted in terms of the weight fraction of population, yields an
average estimated hydrodynamic radius of only ~2.56 nm for the
majority of the HU-Simul population, as shown in Fig. 3D. HU-
Simul is thus firmly established to be monomeric by three
different approaches.

51
B. Thakur, A. Gupta and P. Guptasarma
3.4. Structure and thermal stability of HU-Simul
Supplementary Figure 3A shows the CD spectrum of HU-Simul
which displays both a pronounced negative band at ~198 nm (a
signature of high content of randomly-coiled secondary structure)
as well as significant negative band-like features in the 210-230
range (signatures of some beta sheet and alpha helical content). The
ellipticity measured per randomly-coiled residue tends to be be-
tween 5 and 10 times higher than that for a beta sheet, but com-
parable to that for an alpha helix. Thus, the relative intensities of
the 210e230 nm feature, and the 198 nm feature, in the CD spec-
trum of HU-Simul suggest significant beta sheet content in addition
to randomly coiled content, and little alpha helical content. This
indicates that the 14 residues-long linker did not form a helix, since
if it had done so, together with the 8 residue helices at the end of
each CTR, greater ellipticity should have been seen at 208 nm.
Unfortunately, the CD spectra could not be collected down to lower
wavelengths (e.g., 180 nm). The spectra presented display only raw
ellipticity on the y-axis, instead of mean residue ellipticity because
we would like to be more sure of protein concentrations (note: raw
ellipticity is acceptable where absolute structural content is not
sought to be determined). Heating between 25
C and 89
C, with
temperature intervals of 4
C increased the intensity of the negative
210e230 nm band, suggesting that no heat-induced unfolding oc-
curs (consistent with the presence of a CTR derived from
T. Thermophilus). A high degree of kinetic thermal stability of the
folded structure of HU-Simul was suggested by spectral shape
remaining unaltered by 8 min of exposure to 90
C. as shown in
Supplementary Fig. 3B.
3.5. Binding of HU-Simul to dsDNA and cruciform DNA established
by EMSA and BLI
The DNA ligand binding preference of HU-Simul towards double
stranded DNA (dsDNA) and single stranded DNA (ssDNA) was
checked through the electrophoretic mobility shift assay (EMSA)
using 2% agarose gels. DNA-protein complexes can manifest in
electrophoresis either as sharp bands of poorer mobility, or as
smeared bands representing a range of poorer mobilities and
binding and dissociation occurring over the time-scale of electro-
phoresis. Fig. 4A shows weakened-band smears with dsDNA with
HU-Simul in the first four lanes of the agarose gel. However, no such
are seen with ssDNA in the last four lanes. The control 20-mer
dsDNA and 20-mer ssDNA bands appear sharp and display the
expected mobilities.
The binding of HU-Simul to 20-mer dsDNA and ssDNA was also
examined through biolayer interferometry (BLI), with the protein
being immobilized on a Ni-NTA biosensor through its 6xHis affinity
tag and then allowed to interact with DNA. Loading of mass results
in changes in the interference pattern of incident light which are
plotted on a sensorgram. Fig. 4B and C displays BLI sensorgrams of
HU-Simul with dsDNA, and ssDNA, respectively. In both, binding of
HU-Simul to the Ni-NTA sensor is seen as a large increase in signal
eliciting no dissociation in the experiment shown in Fig. 4B, but
eliciting dissociation in Fig. 4C (manifested as a reduction in signal),
bring both experiments to the same signal strength (5.2 units) after
immobilization of HU-Simul upon Ni-NTA. Thereafter, addition of
dsDNA elicits a further increase in signal (Fig. 4B), but ssDNA does
not (Fig. 4C), supporting the conclusion from the EMSA data in
Fig. 4A. HU-Simul was also observed to bind to cruciform four-way
junction (4WJ) DNA with the appearance of a smear along with two
high molecular weight bands (data not shown). We have also
confirmed using CD spectra between 200 and 250 nm (of HU-
Simul, dsDNA, and mixtures) that conformational changes occur
through binding of dsDNA by HU-Simul.
52
Biochemical and Biophysical Research Communications 534 (2021) 47e52
4. Conclusion
To try and obtain a novel DNA-binding protein inspired by the
structure of HU, but with a higher isoelectric point, and a mono-
meric nature, we attempted to create a simulacrum of HU’s DNA
binding properties by notionally excising the CTRs of E. coli HU-A
and T. thermophiles HU away from their respective NTRs (which
are not as basic, and which are responsible for higher-order oligo-
merization) and fusing them through a linker region of 14 amino
acids. We created the simulacrum physically through molecular
genetic approaches and found that it is indeed monomeric and
binds to both dsDNA and cruciform DNA. Unlike HU, it does not
appear to bind to ssDNA to a detectable degree.
Acknowledgements
BT thanks the Council of Scientific and Industrial Research
(CSIR), New Delhi, for a doctoral fellowship. AG thanks the
Department of Biotechnology, Government of India, for a doctoral
fellowship. PG thanks the Ministry of Human Resource Develop-
ment (MHRD), Government of India, for a Centre of Excellence
Grant (MHRD-14-0064) in Protein Science, Design and Engineering.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2020.11.088.
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