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Loop-to-helix transition in the structure of multidrug regulator AcrR at
the entrance of the drug-binding cavity
Babu A. Manjasetty
a,b
, Andrei S. Halavaty
c,d,
⇑
, Chi-Hao Luan
d,e
, Jerzy Osipiuk
d,f,g
, Rory Mulligan
d,f,g
,
Keehwan Kwon
d,h
, Wayne F. Anderson
c,d
, Andrzej Joachimiak
d,f,g,
⇑
a
European Molecular Biology Laboratory (EMBL), Grenoble Outstation, 71 avenue des Martyrs, F-38042 Grenoble, France
b
Unit of Virus–Host Cell Interactions (UVHCI), University of Grenoble Alpes, F-38042 Grenoble, France
c
Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611, United States
d
Center for Structural Genomics of Infectious Diseases (CSGID), 303 East Chicago Avenue, Chicago, IL 60626, United States
e
High Throughput Analysis Laboratory, Northwestern University, 2205 Tech Drive, Evanston, IL 60208, United States
f
Computational Institute, The University of Chicago, 5735 South Ellis Avenue, Chicago, IL 60637, United States
g
Structural Biology Center, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439, United States
h
Infectious Diseases, J. Craig Venter Institute, 9714 Medical Center Dr. Rockville, MD 20850, United States
article info
Article history:
Received 1 September 2015
Received in revised form 14 January 2016
Accepted 16 January 2016
Available online 18 January 2016
Keywords:
Transcription regulator
TetR/AcrR
Multidrug resistance
Loop-to-helix transition
abstract
Multidrug transcription regulator AcrR from Salmonella enterica subsp. enterica serovar Typhimurium str.
LT2 belongs to the tetracycline repressor family, one of the largest groups of bacterial transcription fac-
tors. The crystal structure of dimeric AcrR was determined and refined to 1.56 Å resolution. The tertiary
and quaternary structures of AcrR are similar to those of its homologs. The multidrug binding site was
identified based on structural alignment with homologous proteins and has a di(hydroxyethyl)ether
molecule bound. Residues from helices
a
4 and
a
7 shape the entry into this binding site. The structure
of AcrR reveals that the extended helical conformation of helix
a
4 is stabilized by the hydrogen bond
between Glu67 (helix
a
4) and Gln130 (helix
a
7). Based on the structural comparison with the closest
homolog structure, the Escherichia coli AcrR, we propose that this hydrogen bond is responsible for con-
trol of the loop-to-helix transition within helix
a
4. This local conformational switch of helix
a
4 may be a
key step in accessing the multidrug binding site and securing ligands at the binding site. Solution small-
molecule binding studies suggest that AcrR binds ligands with their core chemical structure resembling
the tetracyclic ring of cholesterol.
Ó2016 Published by Elsevier Inc.
1. Introduction
Salmonella is a bacterium that causes one of the most common
enteric infections and is responsible for the widely distributed
foodborne disease – salmonellosis (Herikstad et al., 2002).
Salmonella enterica subsp. enterica serovar Typhimurium str. LT2
is a leading cause of human gastroenteritis, and is used as a mouse
model of human typhoid fever. Salmonella usually enters the host
via the oral route through ingestion of contaminated food of
animal origin (Miller et al., 2010; Swartz, 2002). A variety of food
products have been implicated in the transmission of multidrug-
resistant Salmonella isolates between animals and humans (Alban
et al., 2002; Mead et al., 1999; Scallan et al., 2011; Threlfall et al.,
2003). Many studies have been carried out to shed light on the
spread of multidrug-resistant Salmonella isolates and to better
understand multidrug resistance in salmonellosis (Ajiboye et al.,
2009; Winokur et al., 2000). However, the specific mechanisms
underlying drug resistance of salmonellosis is poorly understood
at the molecular level. Moreover, the treatment options for the
most common foodborne infections become limited as
multidrug-resistant isolates, such as Salmonella, show a significant
level of resistance to the most common antimicrobial drugs
(European Food Safety Authority, 2015). Therefore, the develop-
ment of new antibiotics against salmonellosis is of fundamental
importance and a global health priority.
http://dx.doi.org/10.1016/j.jsb.2016.01.008
1047-8477/Ó2016 Published by Elsevier Inc.
Abbreviations: MDR, multidrug resistance; HTH, helix-turn-helix.
⇑
Corresponding authors at: Biochemistry and Molecular Genetics, Feinberg
School of Medicine, Northwestern University, 303 East Chicago Avenue, Chicago, IL
60611, United States (A.S. Halavaty). Computation Institute, The University of
Chicago, 5735 South Ellis Avenue, Chicago, IL 60637, United States (A. Joachimiak).
E-mail addresses: a-halavaty@northwestern.edu (A.S. Halavaty), andrzejj@anl.
gov (A. Joachimiak).
Journal of Structural Biology 194 (2016) 18–28
Contents lists available at ScienceDirect
Journal of Structural Biology
journal homepage: www.elsevier.com/locate/yjsbi
The tetracycline repressor (TetR) family of transcription regula-
tors (TFRs), named after the protein that regulates genes responsi-
ble for resistance to tetracycline, generally consists of transcription
repressors. Members of the family share high similarity within
N-terminal helix-turn-helix (HTH) DNA-binding motif and control
genes that are involved in multidrug resistance, efflux pumps, vir-
ulence and pathogenicity, catabolic pathways and biosynthesis of
antibiotics in bacteria (Ramos et al., 2005). These regulators bind
with high affinity to their specific operator sequences, typically
10–30-bp in length, residing within the promotor region of
gene(s) that they control. TetRs repress transcription of target
genes by competing with RNA polymerase for binding to the pro-
motor. Derepression occurs when the TetRs bind their cognate
ligands resulting in reducing affinity for DNA (Yu et al., 2010).
Several microbial drug efflux pumps belonging to different
transporter families have been identified and characterized exper-
imentally (Li et al., 2007). Multidrug resistance (MDR) efflux
pumps exhibit multiple functions in mediating antibiotic resis-
tance related to maintaining bacterial physiology. The major efflux
system in Salmonella is AcrAB-TolC, a tripartite multidrug efflux
system belonging to the resistance nodulation-division (RND) fam-
ily (Martinez et al., 2009). The expression level of acrAB is corre-
lated with the efflux function and is regulated by specific
repressors and global regulatory proteins. In particular, most
repressors of the efflux pumps belong to the TetR family. Although
many studies have been conducted on the regulation of efflux
pump expression, much less is known about these mechanisms
in the important clinical isolates. It has been shown that over
expression of MDR pumps, resulting in antibiotic resistance is
mainly due to mutations in specific TetR regulatory genes
(Grkovic et al., 1998; Hagman and Shafer, 1995). In particular,
the over expression of acrAB is due to the mutations within the
repressor AcrR (Olliver et al., 2004). The over expression of acrR
increases organic solvent tolerance (OST) in Escherichia coli (Lee
et al., 2014) and some acrR mutations result in the resistance to
ceftazidime and OST (Tavio et al., 2014; Watanabe and Doukyu,
2012). In Salmonella, an increase in the gene expression of the tran-
scription regulator ramA can be compensated by inactivating
mutations (Abouzeed et al., 2008). These results indicate that reg-
ulation of MDR efflux pumps is an important component to under-
standing the molecular mechanisms of antibiotic resistance (Blair
et al., 2015). Several crystal structures of efflux pump MDR regula-
tors in complex with multiple drugs have been reported (Routh
et al., 2009; Yamasaki et al., 2013). The crystallographic and bio-
chemical studies revealed that members of TFRs are comprised
entirely of
a
-helices and form functional homodimers. However,
the possibility of forming alternative dimers and even tetramers
was recently discussed for the Fad35R regulator from Mycobac-
terium tuberculosis (Singh et al., 2015). In the TetR regulator each
subunit consists of an N-terminal domain with the HTH DNA-
binding motif and a C-terminal domain responsible for dimeriza-
tion and ligand binding. Structure and function analyses of several
TetR proteins suggested a possible regulatory mechanism in which
the biological activity of TFRs is attenuated by a ligand binding
within the C-terminal domain. Comparison of the crystal struc-
tures of TetR complexed with DNA (SimR, TetR, DesT, CgmR, QacR
and FadR) and those with or without ligands suggests that those
small molecules stabilize TetR conformations that are less capable
of binding DNA (Dover et al., 2004; Frenois et al., 2004; Le et al.,
2011a;Orth et al., 2000; Schumacher et al., 2002).
Here, we report the crystallographic analysis of the transcrip-
tion regulator AcrR from S. enterica subsp. enterica serovar Typhi-
murium str. LT2 (StAcrR). StAcrR belongs to the TetR family and
resembles the tertiary and quaternary structures of its homologs.
A large ligand-binding cavity was identified within the
C-terminal domain based on the structural comparisons with
similar TetR proteins. The access to the pocket may be controlled
by conformational changes within the middle portion of helix
a
4.
High-throughput fluorescence-based thermal shift (FTS), fluores-
cence polarization (FP) binding and competition, and in silico dock-
ing data suggest that StAcrR binds ligands similar to the tetracyclic
ring of cholesterol.
2. Materials and methods
2.1. Cloning, expression and purification
The acrR gene of S. enterica subsp. enterica serovar Typhimur-
ium str. LT2 encoding transcriptional repressor AcrR (the UniProt
KnowledgeBase (UniProtKB) ID Q7CR15; residues 1–217) was
amplified by PCR and cloned into the pMCSG7 by the ligation inde-
pendent cloning technique (Schmid-Burgk et al., 2013). The result-
ing plasmid was transformed into BL21(DE3)/MAGIC cells that
grew in the Luria–Bertani medium supplemented with ampicillin
(100
l
gml
1
) and kanamycin (50
l
mml
1
)toOD
600
of 1.0 at
37 °C and 200 rev min
1
aeration. The isopropyl b-
D
-1-
thiogalactopyranoside was added to the cells at final 1 mM concen-
tration for overnight induction at 18 °C and 200 rev min
1
. Cells
were harvested (15,810 RCF; 4 °C), resuspended in 50 mM HEPES
lysis buffer pH 8.0, containing 10 mM imidazole, 500 mM NaCl,
5% (
v
/
v
) glycerol, 10 mM b-mercaptoethanol and 1 mg ml
1
lyso-
zyme and lysed by sonication. Cell debris and soluble fraction were
separated by 27,485 RCF 40-min centrifugation at 4 °C. Super-
natant was loaded on a Ni–NTA column (GE Healthcare, Piscat-
away, NJ), which was washed with the lysis buffer to remove
non-specifically bound E. coli proteins. StAcrR was eluted with
the lysis buffer containing 250 mM imidazole. StAcrR was further
purified on a size-exclusion HILoad
TM
26/60 Superdex
TM
200 column
(GE Healthcare, Piscataway, NJ) pre-equilibrated with 20 mM
HEPES buffer pH 8.0, 250 mM NaCl, 2 mM dithiothreitol). All purifi-
cation steps were performed on the ÄKTAxpress
TM
system
(GE Healthcare, Piscataway, NJ) at 4 °C. After size-exclusion chro-
matography, StAcrR was concentrated using an Amicon Ultra-15
concentrator (Millipore, Billerica, MA, USA) with 10 kDa molecular
weight cut-off. Protein concentration was determined from the
absorbance at 280 nm using a NanoDrop 1000 spectrophotometer
(Thermo Scientific, Hanover Park, IL, USA) and the absorbance of a
0.1% (Abs 0.1% (= 1 g l
1
)) of 0.951 for StAcrR. Purity of StAcrR was
assayed by SDS–PAGE. StAcrR was flash cooled in liquid nitrogen
and stored in 100-
l
l aliquots at 80 °C till further use.
2.2. Crystallization, data collection and structure determination
StAcrR was crystallized by the sitting-drop vapor diffusion tech-
nique by mixing 0.4
l
lof33mgml
1
protein and 0.4
l
l of 200 mM
NaCl, 100 mM Bis–Tris buffer pH 8.5, 25% (w/
v
) PEG 3350 at 12 °C.
Crystals of StAcrR were soaked in the crystallization buffer for cry-
oprotection and flash cooled in liquid nitrogen for data collection
at 100 K. A single-wavelength X-ray diffraction dataset was col-
lected on the Structural Biology Center (SBC) 19-ID beamline
(Rosenbaum et al., 2006) at the Advanced Photon Source (APS),
Argonne National Laboratory (ANL). Diffraction images were pro-
cessed with HKL-3000 (Minor et al., 2006). The StAcrR structure
was determined by molecular replacement using MOLREP (Vagin
and Teplyakov, 2010) integrated into HKL-3000 and the crystal
structure of the transcriptional regulator AcrR from E. coli (EcAcrR;
Protein Data Bank (PDB) ID: 2qop; (Li et al., 2007)) as a template.
The structure was refined with REFMAC v5.5 (Murshudov et al.,
1997, 2011). The Translation/Libration/Screw (TLS) refinement
was applied using each protein chain as one TLS group. Data-
collection and refinement statistics are given in Table 1. Diffraction
B.A. Manjasetty et al. / Journal of Structural Biology 194 (2016) 18–28 19
images for the deposited structure are available at the Center for
Structural Genomics of Infectious Diseases website (http://www.
csgid.org/csgid/pages/home; target IDP02616). Figures were made
using the program PyMOL v1.7.4.1 (Schrödinger, 2010). The StAcrR
structure has been deposited in the PDB with accession code 3lhq.
2.3. Molecular docking
In silico binding analysis of dequalinium, ethidium (Et), profla-
vine (Pf) and rhodamine 6G (R6G) at the multidrug binding pocket
of StAcrR was performed with AutoDock Vina (Trott and Olson,
2010). The selected ligands were reported in the co-crystal struc-
tures of the StAcrR homologs (PDB IDs: 3vw0 (Yamasaki et al.,
2013), 3vvy (Yamasaki et al., 2013), 1qvu (Schumacher et al.,
2002) and 3vvz (Yamasaki et al., 2013), respectively). For docking,
a ligand and StAcrR were prepared with Python Molecular Viewer
(PMV) available with the MGL tools 1.5.4 (Scripps Research Insti-
tute). Firstly, water molecules were removed from the StAcrR
PDB coordinates and polar hydrogen atoms were added to the pro-
tein. The protein was treated rigid including the side chains during
docking. A 30 30 20 Å
3
docking box was prepared to cover all
binding possibilities near the drug recognition residue Glu67 of
StAcrR. Secondly, the ligands were prepared for the docking. The
flexibility of the ligands was applied at their torsional angles. Each
docking run included ten modes. The best mode was selected
based on the binding affinity value and also by viewing the binding
orientation of a ligand with respect to the bound di(hydroxyethyl)
ether molecule in the structure of StAcrR. The selected best mode
was used for the next docking run. For each ligand three additional
runs were carried out. PyMOL v1.7.4.1 (Schrödinger, 2010) was
used to visualize the results.
2.4. Fluorescence-based thermal shift assay
Binding of dequalinium was probed with the FTS approach,
while Et, Pf and R6G were studied with the FP assay (see methods
description below), since they interfered with the FTS method. The
FTS assay was run in 384-well PCR plates using an Echo550 acous-
tic transfer robot (Labcyte, Sunnyvale, CA) for dispensing a
dimethyl sulfoxide stock of ligands to assay plates that contain
10
l
l of a mixture of StAcrR (1
l
g) and 2.5SYPRO Orange fluores-
cence dye (Invitrogen, Carlsbad, CA) in 100 mM HEPES buffer pH
7.5 and 150 mM NaCl. Thermal scanning (from 10 to 80 °Cat
1.5 °Cmin
1
ramp rate) on a real-time PCR machine CFX384
(Bio-Rad Laboratories, Hercules, CA) was coupled with fluores-
cence detection every 10 s.
Dequalinium bound at 40
l
M that prompted us to perform
dose-dependent measurements with its 2.5, 5, 10, 25, 50, 75 and
100
l
M concentrations. Since dequalinium belongs to a 320-
molecule subset of the Spectrum library (Micro Source Discovery,
Gaylordsville, CT, hereafter referred as SPC2-ECH008), we tested
thermal stability of StAcrR against the ligands in the SPC2-
ECH008 library. The best binders were selected based on difference
in melting temperature (
D
T
m
), reduction of the background read-
ing, and shape of the melting curve. The top six hits were further
subjected to a dose-dependent response analysis using their 2.5,
5, 10, 25, 50, 75 and 100
l
M concentrations. Human enolase 1
(1.2
l
gin10
l
l assay mixture) was used as a negative control pro-
tein and tested against the top six binders and dequalinium at 10,
25, 50 and 100
l
M concentrations. FTS data were analyzed with
the in-house ExcelFTS software. The top six hits were also probed
with the in silico binding method performed as described above.
2.5. Fluorescence polarization assay
Twofold dilution series of StAcrR (initial 180
l
M) were used and
each protein concentration was tested in triplicate. Et and R6G
were assayed at 1
l
M each. StAcrR at 40
l
M was completely satu-
rated with bound Pf (tested at 250, 125, 50, or 25 nM). The assay
buffer was 100 mM HEPES pH 7.5 and 150 mM NaCl. Measure-
ments were performed on a Tecan Infinite M1000 plate reader
(Tecan Systems Inc., San Jose, CA) at ambient temperature with
excitation at 470 nm, and emission at 550 nm (Pf and R6G) and
600 nm (Et). Human enolase 1 and diflubenzuron were used as
negative controls. Binding constant (K
d
) for Pf, Et and R6G were
determined from the equation:
y¼AþB=ð1þK
d
=xÞð1Þ
where yis FP in mP units, x– concentration of a protein, A–FPof
free Pf, Et or R6G and B– maximum magnitude of binding response.
The top six binders of StAcrR identified from the FTS analysis
were subjected to a competition FP analysis in the presence of
100 nM Pf using 4, 10 and 20
l
MStAcrR. Samples were incubated
at room temperature for 30 min before reading. Assay was run at
room temperature. This assay allowed estimation of the half max-
imal inhibitory concentration (IC
50
), i.e. relative binding affinity,
for the top six hits.
Table 1
X-ray data-collection and refinement statistics. Values in parentheses are for the
highest resolution shell.
Data-collection statistics
X-ray source APS 19-ID beamline
Wavelength (Å) 0.9792
Resolution range (Å) 37.9–1.56 (1.59–1.56)
Space group P2
1
Cell dimensions
a,b,c(Å) 47.18, 75.82, 55.8
a
,b,
c
(°) 90.0, 108.7, 90.0
Unique reflections 51039 (2219)
Completeness (%) 97.5 (85.1)
R
merge
(%) 5.4 (75.9)
R
meas
(%) 5.9 (84.4)
I/
r
I9.3 (2.12)
Multiplicity 5.1 (4.3)
CC1/2 N/A (0.7)
Refinement statistics
PDB ID 3lhq
Resolution range (Å) 37.9–1.56 (1.60–1.56)
R
work
(%) 15.0 (26.4)
R
free
(%) 19.9 (35.2)
No. atoms
Protein
Chain A1812
Chain B1840
Water 281
1,2-Ethanediol 4
Di(hydroxyethyl)ether 14
Average B(Å
2
)
Protein
Chain A18.2
Chain B18.9
Water 35.2
1,2-Ethanediol 31.6
Di(hydroxyethyl)ether 36.3
R.m.s. deviations
Bond lengths (Å) 0.02
Bond angles (°) 1.64
Ramachandran plot
a
Favored (%) 100.0
Allowed (%) 0.0
Outliers (%) 0.0
a
Ramachandran statistics are based on the PDB and MolProbity (Chen et al.,
2010) validation reports.
20 B.A. Manjasetty et al./ Journal of Structural Biology 194 (2016) 18–28
3. Results and discussion
3.1. Overall structure of StAcrR
The P2
1
asymmetric unit of the StAcrR crystal is comprised of
the chains Aand Bthat form a homodimer (Fig. 1). The two chains
of StAcrR are quite similar with root-mean-square (r.m.s.) devia-
tions of 0.25 Å over 209 C
a
atoms. The overall dimeric structure
exhibits the ‘
X
’ (omega)-shaped form (Yu et al., 2010), which is
typical to the TetR family. Each monomer has two domains: an
N-terminal HTH DNA-binding domain (Met1–Lys55) and a
C-terminal multidrug-binding domain (Ser56–Ala210). The inter-
actions between the domains involve residues from helix
a
1b
and residues from helices
a
4 and
a
6. Helix
a
1 is bent at Ala9
creating two sub-helices
a
1a and
a
1b (Fig. 1). The bending of helix
a
1 is not observed in any structures of the TetR family solved to
date although significantly longer helix
a
1 was reported for the
QacR (PDB ID: 1jt0; (Schumacher et al., 2002)), DesT (PDB ID:
3lsj; (Miller et al., 2010)) and CgmR (PDB ID: 2yvh; (Itou et al.,
2010)) structures. The length of helix
a
1 can vary from 12 to 24
residues. Non-helical N-terminal extension (28 residues) of helix
a
1 has also been reported for a TetR-like repressor SimR from
Streptomyces antibioticus (SaSimR) that was shown to interact with
the minor grove of bound DNA (PDB ID: 3zql; (Le et al., 2011a)).
The bending of helix
a
1inStAcrR may contribute to the stabiliza-
tion of a possible StAcrR-DNA complex. In particular, residues Arg3,
Lys4, Lys6 and Gln7 are near the HTH DNA-binding motif and, thus,
may contribute to the protein–DNA interactions. These residues
also contribute to a strong electropositive surface potential of the
N-terminal region and HTH motif (Fig. 2a). Arg45 from the recog-
nition helix
a
3ofStAcrR is conserved across close homologs in
the AcrR subfamily and most likely is involved in interactions with
the nucleotide bases. Tyr49 from helix
a
3 is also conserved, and
the distance between two adjacent Tyr49 in a regulator’s dimer
is typically used to determine whether the DNA recognition helices
are in a DNA-bound or DNA-unbound form. In StAcrR, this distance
(between the C
a
atoms of both Tyr49) is 39.7 Å, greater than that of
the DNA-bound form (34.0 Å; a distance between two major
grooves of DNA) yet shorter than the DNA-free form (which is
either ligand-bound or apo forms). This distance slightly varies in
the TetR-DNA complex structures. For example, the two Tyr42
are 34.8 Å apart in the E. coli TetR complex with 15-bp DNA
oligonucleotide (PDB ID: 1qpi; (Orth et al., 2000)), while two equiv-
alent Phe41 are 36.8 Å apart in the Thermotoga maritima transcrip-
tional regulator TM1030 complexed with 24-bp DNA
oligonucleotide (PDB ID: 4i6z) (Fig. 2b). Binding of tetracycline to
the E. coli TetR displaces helices
a
4 and
a
6 that consequently drag
its DNA-recognition helices
a
3 3 Å further apart and, thus, disrupt-
ing the protein–DNA interactions (Orth et al., 2000). The Tyr-to-Tyr
distance varies greatly (from 37.0 to 63.4 Å) in known apo or
ligand-bound TFR structures (Cuthbertson and Nodwell, 2013; Yu
et al., 2010). Thus, the StAcrR structure appears to represent one
of the several possible conformations between apo and ligand-
bound forms of TFRs.
The C-terminal domain is comprised of helices
a
4 through
a
9
(Fig. 1). Helix
a
4 is well-ordered and relatively straight. Helices
a
5,
a
6 and
a
7 form a triangle like arrangement, which is found
in other known TFR structures (Ramos et al., 2005). Helices
a
8
and
a
9 contribute to the dimerization interface forming four-
helix bundle (Fig. 1). The StAcrR dimer buries 2020 Å
2
surface
from each monomer as estimated by the PISA server (http://
www.ebi.ac.uk/pdbe/pisa/;(Krissinel and Henrick, 2007)).
3.2. StAcrR multidrug-binding cavity
Similar to the structurally characterized TetR family regulators,
the C-terminal domain of StAcrR contains quite large ligand-
binding cavity (2400 Å
3
; estimated with PDBsum; (de Beer
et al., 2014) that is entirely contained within a single subunit. In
contrast, SaSimR binds bulky simocyclinone D8 (PDB ID: 2y30;
(Le et al., 2011b)), a potent DNA gyrase inhibitor, and its biosyn-
thetic intermediate, simocyclinone C4 (PDB ID: 2y31; (Le et al.,
2011b), using residues from the two subunits of the SaSimR dimer.
This binding mode is possible due to the presence of a 40-residue
insertion within the C-terminal domain of SaSimR; this insertion is
also involved in dimerization of SaSimR. The insertion is absent
between helices
a
8 and
a
9ofStAcrR. The StAcrR multidrug-
binding pocket had an extra electron density, which was inter-
preted as a di(hydroxyethyl)ether molecule (abbreviated as PEG
in the StAcrR crystal structure coordinates), present in the crystal-
lization buffer (Fig. 1). Surface representation of the StAcrR dimer
shows the cavity is widely open to the solvent and is accessible
to ligands through an entry located between helices
a
4 and
a
7
(Fig. 2a). In the StAcrR dimer a solvent accessible tunnel connects
one ligand-binding cavity with another (not shown) similarly to
one found in the crystal structure of a putative transcriptional reg-
ulator YfiR from Bacillus subtilis (PDB ID: 1rkt; (Rajan et al., 2006)).
The surface of the cavity is composed of aromatic residues (Phe114
and Trp63), hydrophobic residues (Ile70, Leu93, Ile92, Ile96,
Met110, Leu133, Cys134, Ser137, Ile141, Met167, Ile171 and
Met175) and polar residues (Glu67, Glu74, Gln130, Glu136,
Arg106, Arg140 and Arg168) (Fig. 2c). A localized negative poten-
tial patch is clearly observed at the entrance of the cavity, while
the visible portion of the pocket itself has positively charged and
neutral patches (Fig. 2a). This charge distribution suggests that
StAcrR may bind ligands that combine neutral and charged moi-
eties. Binding of positively charged Et, Pf and R6G by EcAcrR and
EcAcrR-Glu67Ala mutant has been examined (Su et al., 2007) and
Fig. 1. Ribbon diagram of the StAcrR homodimeric structure. The left monomer is
shown in the two different colors to represent the N-terminal HTH DNA-binding
domain (blue) and a C-terminal small-molecule binding domain (green). The right
monomer is shown in orange. The di(hydroxyethyl)ether (PEG) (depicted as spheres
and colored according to atoms: carbon (gray) and oxygen (red)) bound to the C-
terminal domain’s multidrug-binding site of both monomers is shown. Arg45,
important for DNA binding, and Glu67, important for drug binding are shown in
stick representation. Gln130 that interacts with Glu67 is shown. Distance between
the two Tyr49 is used to measure the distance between the DNA recognition helices
to represent DNA-bound, apo and ligand-bound forms of a transcriptional regulator.
B.A. Manjasetty et al. / Journal of Structural Biology 194 (2016) 18–28 21
Fig. 2. The HTH DNA-binding grove and multidrug-binding pocket. (a) Side view of the electrostatic surface potential of StAcrR with the di(hydroxyethyl)ether molecule
(PEG; carbon atoms (green); oxygen atoms (red)) bound at the ligand-binding pocket. (b) Left panel, side view of the superimposed structures of StAcrR (orange; PEG is in
stick model (orange)), the Thermotoga maritima transcriptional regulator TM1030 (blue) with 24-bp DNA (yellow) oligonucleotide and the TM1030 protein (green; PDB ID:
2iek) with hexaethylene glycol (P6G; green sticks) bound. Right panel, side view of the superimposed structures of StAcrR (as in the left panel), the E. coli TetR (green; PDB ID:
1qpi) with 15-bp DNA (yellow) oligonucleotide and the E. coli TetR (gray; PDB ID: 2tct; (Kisker et al., 1995)) with 7-cholortetracycline (gray sticks) bound. Helices of StAcrR
are labeled and corresponding Tyr (Phe41 in the 1qpi structure) residues that are used to measure the Tyr–Tyr distance in a TetR dimer are shown. Single protomer of a TetR
dimer is shown only. Bottom panel, multiple sequence alignment (area of the N-terminal HTH DNA-binding domain) of the StAcrR (UniProtKB ID: Q7CR15), TM1030
(UniProtKB ID: Q9X0C0) and E. coli TetR (UniProtKB ID: P0ACT4) proteins. The C-terminal portion of the alignment has fewer conserved residues. Figure was generated with
ESPript v2.2 (Gouet et al., 1999) using Similarity Global Score of 0.7, Similarity Diff Score of 0.5 and Similarity Type Risler. (c) Key residues (stick representation) that comprise
the multidrug ligand-binding pocket. The 3
r
OMIT map (magenta mesh) of PEG.
22 B.A. Manjasetty et al./ Journal of Structural Biology 194 (2016) 18–28
it clearly showed the abolishment of the binding of Pf, Et and R6G
by the Glu67Ala mutant of EcAcrR (Li et al., 2007; Su et al., 2007).
The residues within the drug-binding pocket are conserved
between EcAcrR and StAcrR implying that these regulators may
interact with structurally, yet potentially chemically diverse,
ligands. In silico dockings of dequalinium, Et, Pf, R6G to StAcrR
imply that the pocket is capable of accommodating these com-
pounds (Fig. 3). The best binding mode for each ligand was identi-
fied based on its matching position with a PEG molecule bound at
the multidrug-binding site of StAcrR. The AutoDock Vina-derived
binding affinities were 9.3 (rhodamine 6G), 8.8 (dequalinium),
8.4 (ethidium) and 7.0 (proflavine) kcal/mol. The amino groups
of the probed molecules are at an interacting distance with the
carboxyl group of Glu67 (not shown) suggesting that Glu67 has a
critical role in binding of a particular compound and is consistent
with the ligand binding properties of the EcAcrR homolog. In
addition to Glu67, the different cluster of residues that were
involved in interacting with compounds indicates that multidrug
binding nature of the pocket.
3.3. Known cancer, antiviral, antimicrobial and neurological disorder
drugs bind StAcrR; an FTS study
The mean T
m
of StAcrR was 57.5 °C and it increased by 1.6 °C
in the presence of 40
l
M dequalinium. Below 10
l
M, dequalinium
did not notably change T
m
of StAcrR suggesting not very specific
protein–ligand interactions (Fig. 3a right panel). On the other hand,
dequalinium has no effect on thermal stability of a negative control
protein, human enolase 1 (ENO1; Fig. 3a inset), implying that
dequalinium may distinctively bind StAcrR.
We identified another fifteen compounds from the SPC2-
ECH008 library of 320 molecules. Those increased thermal stability
of StAcrR by 1.6–6.2 °C (Tables S1 and S2). Six of those fifteen
resemble the tetracyclic ring of cholesterol. The two TetR-type
transcriptional repressors, KstR and KstR2, were shown to regulate
expression of several genes involved in catabolism of cholesterol in
mycobacteria (Kendall et al., 2010, 2007) and related actino-
mycetes (Van der Geize et al., 2007). The co-crystal structure of
KstR2 and 3a
a
-H-4
a
(3
0
-propanoate)-7ab-methylhexahydro-1,5-in
Fig. 3. AutoDock Vina docking results for dequalinium and the FTS-derived dose-dependent denaturation profile of StAcrR (a). Inset shows dose-dependent FTS data of
human enolase 1 (ENO1), a negative control protein, with dequalinium. Docking results for ethidium (b), proflavine (c) and rhodamine 6G (d). Small molecules are shown in
sticks representation and colored according to atoms: carbon (light gray); oxygen (red); and nitrogen (blue).
B.A. Manjasetty et al. / Journal of Structural Biology 194 (2016) 18–28 23
danedione (HIP)-coenzyme A (HIP-CoA) (PDB ID: 4w97; (Crowe
et al., 2015)) revealed that the HIP moiety of cholesterol binds at
the multidrug-binding site of every KstR2 subunit, while the CoA
portion binds at the dimerization interface. The HIP-(CoA)-like
structures are also present among the StAcrR hits (Table S1).
The top six StAcrR binders showed no effect on thermal stability
of human ENO1 (Fig. S1). Apparent increase in the StAcrR thermal
stability at very low concentrations of fulvestrant, ritonavir and
estramustine suggests that these compounds are distinct binders
(Figs. 4 and 5). In silico calculations may support this notion with
the estimated binding affinities of 9.6 (ritonavir), 8.9 (estra-
mustine) and 8.7 (fulvestrant) kcal/mol. In contrast, prasterone
(8.5 kcal/mol), triclabendazole (7.2 kcal/mol) and idebenone
(6.4 kcal/mol) required higher concentrations to achieved similar
effects with StAcrR. However, in silico-derived affinities should be
considered with caution as they only provide a guidance of how
strong or weak a ligand can bind in a given calculation approach.
Although binding modes for the top ligands cannot be determined
or distinguished from the FTS analysis, the in silico docking data
may yet provide some insights into their coordination in the
multidrug-binding pocket (Figs. 4 and 5). All top compounds are
positioned within the center section of the binding cavity, while
parts of bulky ritonavir and estramustine occupy sites at the entry
into a solvent accessible tunnel that connects the individual
multidrug-binding cavities at the dimerization interface of StAcrR
(Fig. 5). Based on the FTS data and the fact that StAcrR does not
have any further comparable small-molecule binding pocket(s) of
2400 Å
3
, we hypothesized that the top six and perhaps the
Fig. 4. Dose-dependent FTS melting curves (left) and AutoDock Vina docking results (right) for fulvestrant, idebenone and triclabendazole.
24 B.A. Manjasetty et al./ Journal of Structural Biology 194 (2016) 18–28
remaining hits bind in the protein’s multidrug-binding site. None
of the tested by FTS ligands increased thermal stability of human
ENO1 further supporting the hypothesis.
3.4. Size and chemical properties of a ligand affect its affinity to StAcrR:
an FP analysis
Pf had over 10-fold higher affinity to StAcrR than Et and R6G
(Table 2 and Fig. 6) implying that its more compact 3D structure
and chemical properties favor the interactions. No FP increase in
the presence of Pf was observed for human ENO1 (up to 100
l
M)
supporting the specific nature of the StAcrR–Pf interactions.
Diflubenzuron did not compete with Pf for binding (Table 2).
Relative binding affinities (IC
50
) for the top six FTS ligands were
determined from the competition FP assay (Table 2). Perhaps not
surprisingly, comparably small-size idebenone (IC
50
of 3
l
M;
FTS rank 2) and prasterone (IC
50
of 5
l
M; FTS rank 6) are the
top two inhibitors of Pf. Although ritonavir ((IC
50
of 12
l
M; FTS
rank 4) is bulky, presumably three-time surplus of its polar surface
area over that of Pf has greater inhibitory effect than smaller and
more hydrophobic triclabendazole (IC
50
of 26
l
M; FTS rank 3),
estramustine (IC
50
of 18
l
M; FTS rank 5) and dequalinium chlo-
ride (IC
50
of 23
l
M; FTS rank 14) (Table 2). Chlorines within those
Fig. 5. Dose-dependent FTS melting curves (left) and AutoDock Vina docking results (right) for ritonavir, estramustine and prasterone.
B.A. Manjasetty et al. / Journal of Structural Biology 194 (2016) 18–28 25
compounds may contribute to higher IC
50
values, while competing
for positively charged patches within the multidrug-binding
pocket. Fulvestrant, the number 1 FTS hit, had the highest IC
50
of
40
l
M that may be accounted for its complex chemical
composition.
The rear and top sections of the multidrug-binding pocket of
StAcrR are fairly hydrophobic and partially masked by charged
surface-exposed residues of helices
a
4 and
a
7; the bottom section
is charged (Fig. 2c). Thus, accommodation of aliphatic moieties of
some tested ligands would presumably require local structural
rearrangements (see below) to access hydrophobic patches. Thus,
high affinity of Pf and its strongest inhibitors may be explained
by their easy access to and few-to-none structural changes
around/within the pocket upon binding. Although we could not
assess the FTS rank of Pf due to experimental limitations of FTS,
comparing its binding affinity to that of idebenone (FTS rank 2)
and prasterone (FTS rank 9) one proposes, that although Pf is a
strong binder, it may not have a drastic effect on the thermal
stability of StAcrR. Obviously, charged and hydrophobic moieties
of a ligand play a key role in affecting the protein’s thermal stabil-
ity, i.e. the tertiary structure within the multidrug-binding domain
and likely DNA-binding domain of StAcrR.
3.5. Ligand binding necessitates loop-to-helix transition in StAcrR
The search for structural homologs using the Vector Alignment
Search Tool (VAST) (Gibrat et al., 1996) revealed 138 neighbors in
the PDB. The closest structural homologs are EcAcrR (sequence
identity 88.2%; PDB ID: 3bcg; (Gu et al., 2008); r.m.s. deviations
1.2 Å over 203 C
a
atoms) and transcription regulator TtgR from
Pseudomonas putida (PpTtgR; sequence identity 37.4%; PDB ID:
2uxo; (Alguel et al., 2007); r.m.s. deviations 1.9 Å over 198 C
a
atoms). The overall fold is highly conserved, however, the signifi-
cant structural differences are observed in the conformation of
helix
a
4. In the StAcrR structure, helix
a
4 is straight and extended
(Fig. 7), whereas in both crystal forms of apo EcAcrR (EcAcrR-P3
1;
PDB ID: 3bcg (Gu et al., 2008); and EcAcrR-P222
1
; PDB ID: 2qop;
(Li et al., 2007)), helix
a
4 unwinds locally into a loop region (resi-
dues Leu65–Ile70 in EcAcrR-P222
1
and Leu65–Leu73 in EcAcrR-P3
1
that are equivalent to residues Leu65–Leu73 in StAcrR) splitting
helix
a
4 into two sub-helices,
a
4a and
a
4b (Fig. 7). Similar, local
unwinding of helix
a
4 along with its bending is observed in both
chains of the binary tetracycline–PpTtgR complex structure (not
shown), yet the antibiotic was modeled at the ligand-binding site
of one chain only. Thus, comparison of the aforementioned struc-
tures of three homologous regulators suggests that conformation
of helix
a
4 would depend on whether a regulator is in a ligand-
free or ligand-bound form and the size of a ligand may determine
the extent of local alterations in the structure. Additionally, in the
structures of MDR regulator RamR from Salmonella typhimurium
(PDB ID: 3vvy; (Yamasaki et al., 2013)) and transcriptional repres-
sor LfrR from Mycobacterium smegmatis (PDB ID: 2v57; (Bellinzoni
et al., 2009)) helix
a
7 undergoes conformational change in order to
accommodate a drug, ethidium and proflavine, respectively. Thus,
flexibility of helices
a
4 and
a
7 may be an important property that
may be common in the MDR transcription regulators allowing
them to bind ligands of different chemistry and size.
In StAcrR, the ligand recognition residue Glu67 from helix
a
4
interacts with Gln130 from helix
a
7 and, thus, stabilizes the local
tertiary structure. Structural comparison of StAcrR with the two
crystal forms of EcAcrR reveals that Glu67 and Gln130 adopt differ-
ent side chain conformations (Fig. 7). In the EcAcrR-P222
1
crystal
form, Glu67 forms the hydrogen bond with Arg106 (helix
a
6)
whereas Glu67 in the EcAcrR-P3
1
lost that interaction and is
solvent exposed. In the StAcrR structure, the interaction between
Glu67 and Arg106 is not observed. Thus, Glu67 can participate in
Table 2
Competition and binding FP data for StAcrR.
Ligand IC
50
(
l
M)
a
K
d
(
l
M)
b
FTS rank (Table S1)
Et 67.3 ± 4.6 N/A
R6G 43.7 ± 1.3 N/A
Pf 3.9 ± 0.1 N/A
Idebenone 2.8 2
Prasterone 5.4 6
Ritonavir 12.1 4
Estramustine 18.0 5
Dequalinium chloride 22.5 14
Triclabendazole 25.7 3
Fulvestrant 40.1 1
Diflubenzuron N/A Negative control
a
Values obtained in the presence of 10
l
MStAcrR and 100 nM Pf.
b
Values obtained in the presence of 100 nM Pf, 1
l
M Et and 1
l
M R6G.
Fig. 6. The FP binding assay data. (a) FP data for Pf (100 nM), R6G and Et (each
1
l
M) in the presence of StAcrR and human ENO1. (b) Measuring FP with a range of
concentrations of Pf. Red error bars represent standard deviation from the mean
value of independent measurements for each of the StAcrR and ENO1 (not
applicable for panel b) concentrations. Black curves are sigmoidal fittings for each
of the ligands.
26 B.A. Manjasetty et al./ Journal of Structural Biology 194 (2016) 18–28
alternative interactions. Further, the hydrogen-bonding network of
Arg105 with the N-terminal residues Gln14 and Asp18 (helix
a
1b)
is completely retained in the StAcrR and EcAcrR-P222
1
structures
(Fig. 7). This hydrogen bond network seems to be disrupted in
the EcAcrR-P3
1
though the side chain conformations of these
residues have poor fit to the electron density (not shown). Taken
together, we hypothesize that helices
a
4 and
a
7 of the
C-terminal domain function as gatekeepers of the StAcrR
multidrug-binding cavity allowing or preventing the entry of
particular drugs. Apparently, the helices exhibit some level of
conformational diversity, as seen in homologous repressors too,
that is may be crucial for precise recognition and accommodation
of specific drugs, e.g. those identified by the FTS analysis, within
the multidrug-binding cavity of StAcrR.
Acknowledgments
The CSGID project has been funded in whole or in part with
Federal funds from the National Institute of Allergy and Infectious
Diseases, National Institutes of Health, U.S. Department of Health
and Human Services, under Contracts No. HHSN272200700058C
and HHSN272201200026C. We thank Sankar Krishnna for the
human ENO1 sample. The authors wish to thank members of the
Structural Biology Center (SBC) at Argonne National Laboratory
for their help with X-ray diffraction data collection. The operation
of SBC beamlines is supported by the U.S. Department of Energy,
Office of Biological and Environmental Research under contract
DE-AC02-06CH11357.
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