Structural contributions to multidrug recognition in
the multidrug resistance (MDR) gene regulator, BmrR
Sharrol Bachas, Christopher Eginton, Drew Gunio, and Herschel Wade1
Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, MD 21205
Edited* by William F. DeGrado, University of Pennsylvania, Philadelphia, PA, and approved May 23, 2011 (received for review March 28, 2011)
Current views of multidrug (MD) recognition focus on large drug-
binding cavities with flexible elements. However, MD recognition
in BmrR is supported by a small, rigid drug-binding pocket. Here, a
detailed description of MD binding by the noncanonical BmrR
protein is offered through the combined use of X-ray and solution
studies. Low shape complementarity, suboptimal packing, and
efficient burial of a diverse set of ligands is facilitated by an aro-
matic docking platform formed by a set of conformationally fixed
aromatic residues, hydrophobic pincer pair that locks the different
drug structures on the adaptable platform surface, and a trio of
acidic residues that enables cation selectivity without much regard
to ligand structure. Within the binding pocket is a set of BmrR-
derived H-bonding donor and acceptors that solvate a wide range
of ligand polar substituent arrangements in a manner analogous to
aqueous solvent. Energetic analyses of MD binding by BmrR are
consistent with structural data. A common binding orientation
for the different BmrR ligands is in line with promiscuous allosteric
ligand binding ∣ ligand responsive transcription factor ∣
molecular recognition ∣ multidrug resistance ∣ multispecificity
efflux can extend broad chemoprotection to drug-targeted cells,
which in turn are rendered resistant to lethal doses of multiple,
unrelated drug therapies. The transport of antimicrobial andanti-
fungal agents has been established as a primary cause of multi-
drug resistance (MDR) in pathogenic strains of Escherichia coli,
Staphylococcus aureus, Pseudomonas aeruginosa, and Candida
albicans (3). In human cells, increased export of chemicals by
P-glycoprotein (Pgp) and numerous MDR-associated proteins
can also confer simultaneous resistance to a multitude of antitu-
mor agents and has been linked to chemotherapy failure (4).
MDR-related transport is controlled largely by two cellular func-
tions, namely those enacted by drug-responsive gene regulators
and efflux pumps (3). Quorum sensing regulators are also known
to influence MD efflux pump expression (5). For the former,
promiscuous chemical recognition facilitates broad cellular
responses that target drug-like compounds for export while leav-
ing native biological ligands untouched. Elucidating the basis of
MDR functions requires detailed descriptions of MD recogni-
tion. To date, an incomplete understanding of how MDR proteins
interact with structurally and chemically diverse drugs continue to
hinder efforts to evade, inhibit and control MDR activities.
Crystallographic studies of drug binding by MDR proteins
have enabled improved descriptions of MD recognition (6, 7).
Indeed, details made available through structural data connect
MD recognition to well-known physical and chemical features of
binding. Moreover, contributions to multispecificity are now
more recognizable due to the increasing availability of X-ray
structures of MDR systems with different architectures and bind-
ing-site designs. However, both multispecificity and partial selec-
tivity remain poorly defined due to insufficient analyses of MD
interactions with individual MDR proteins. Current views of
MD recognition are dominated by the canonical “multisite” mod-
el, which highlights structural and biochemical aspects of drug
ultidrug (MD) efflux protects nearly all living cells against a
barrage of cytotoxic chemicals (1, 2). High levels of MD
binding by regulators and transporters. Binding plasticity and
adaptability are central to this model and, in many cases, are
believed to derive largely from large drug-binding cavities com-
posed of distinct, overlapping “minipockets” and flexible protein
elements (SI Appendix, Fig. S1). For QacR (7), TtgR (8), AcrB
(9), Pgp (10), and other characterized MDR systems, these
features are present and appear to be critical for multifaceted
binding, which includes the accommodation of dissimilar drug
structures and bound configurations. Descriptions of MD recog-
nition are currently limited to the canonical model. However,
recent data suggest that it offers only a partial picture of multi-
specific drug binding.
BmrR is a gene regulator with a verified MDR role in Bacillus
subtilis. It controls the expression of the Bmr efflux pump in
response to a diverse array of cationic antibiotics, dyes, and dis-
infectants, which are also transported by Bmr and other efflux
pumps (11). Although BmrR has been widely viewed as a proto-
type for investigating MD recognition, only a limited number of
binding studies have been reported. Importantly, the data avail-
able include crystal structures of three BmrR-drug complexes
that suggest possible departures from the canonical MD-binding
model wherein MD recognition occurs in a small, rigid drug
pocket (12, 13). Irreconcilable differences between BmrRand the
canonical view suggest alternative binding models; one has been
proposed based on the limited amount of data available (14). The
model considers two binding components (Fig. 1A). One, coined
the “hydrophobic (Hb) slot,” provides a common anchoring point
for different drugs through contacts with rigid, nonpolar ligand
moieties. The second, called the “hydrophilic (Hp) cavity,” offers
extended binding versatility due to its concave structure and
solvent exposure. The Hb slot–Hp cavity combination appears to
provide another biological solution to MD recognition. The pre-
sent study replaces this model with one that provides a molecular
depiction of MD recognition in BmrR. Interestingly, the Hb slot–
Hp cavity elements are retained.
Because of its noncanonical features, BmrR offers an alterna-
tive framework to investigate MD recognition. To better under-
stand MD binding by the small, rigid BmrR pocket, solution and
crystallographic approaches have been employed to investigate
BmrR interactions with medically important ligands, including
puromycin (PUR), ethidium (ET), tetracycline (TET), 4-amino-
qualdine (4AQ), kanamycin (KAN), and acetylcholine (Ach)
(Fig. 1B). Due to the broad range of structural, chemical, and
binding properties exhibited, the selected probe set facilitates
the most extensive analyses of MD recognition for BmrR to date.
Furthermore, by combining structural with solution-binding data
Author contributions: S.B. and H.W. designed research; S.B., C.E., and D.G. performed
research; S.B., C.E., and D.G. contributed new reagents/analytic tools; H.W. analyzed
data; and S.B. and H.W. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org (PDB ID codes 3Q5P, 3Q5R, 3Q5S, 3Q3D, 3Q2Y, 3Q1M).
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/
11046–11051 ∣ PNAS ∣ July 5, 2011 ∣ vol. 108 ∣ no. 27www.pnas.org/cgi/doi/10.1073/pnas.1104850108
mode of binding may be important for allosteric regulation of the
promiscuous BmrR switch.
The combination of solution binding and X-ray studies has elu-
cidated key features of MD recognition in a system that presents
a binding scheme very different to views of the currently domi-
nant, canonical MD-binding model. Although BmrR does not
employ a large drug-binding cavity or flexible protein elements,
its drug recognition features highlight key issues regarding the
recognition of structurally and chemically diverse compounds.
Our current knowledge of MD recognition remains rudimentary.
The discovery of additional structural solutions to MD recogni-
tion will be important toward better understanding the functions
of systems that influence drug action.
Protein Preparation. The His-tagged version of the bmrR gene was amplified
by PCR from B. subtilis genome using gene specific primers, subcloned into
the pBad vector and transformed into E. coli BL21(DE3) cells. Cells were har-
vested by centrifugation and the pellet was resuspended in lysis buffer
(30 mM phosphate, 1.5 mM EDTA, 5% glycerol, 200 mM imidazole, 100 μM
protease inhibitor cocktail, 50 μg∕mL lysozyme, and 100 uM PMSF). BmrR was
then purified by HisTrap HP, Heparin HP, and HiTrap Q HP (GE Healthcare)
chromatography. BmrR was subsequently concentrated and subjected to
gel filtration on a Superdex 200 (GE Healthcare) in a buffer containing
10 mM Hepes, 300 mM NaCl, 500 μM EDTA, and 10% glycerol.
Crystallization and Data Collection. Crystals of BmrR bound to a 23 bp DNA
duplex containing the bmr promoter sequence were produced as described
previously (15). Ligand complexes were produced by soaking preexisting
crystals (24 h) in solutions containing 1 M sodium malonate pH 7.0, 0.05%
jeffamine, and 2 mM of each ligand: 4-aminoquinaldine, ethidium bromide,
puromycin, tetracycline, and kanamycin A (Sigma Aldrich). The ligand-soaked
crystals were stabilized and cryoprotected using a solution containing 1 M
sodium malonate, 0.05% jeffamine, and 30% glycerol. These crystals were
flash frozen in liquid nitrogen. All diffraction data were collected at the
Stanford Synchotron Radiation Light Source, beamline 9-2.
Data Reduction and Refinement. The diffraction data were indexed, reduced,
and scaled using the program HKL 2000. After scaling, phases were obtained
by molecular replacement by rigid-body refinement using CNS (17) and the
structure of BmrR bound to TPP and DNA (accession code IR8E, 2.4 Å) (16) as a
starting model. At this point, 5.0% of the data were removed to be used for
cross-validation (RFREE). Several rounds of simulated annealing, B-factor re-
finement, positional refinement, and model building were carried out using
CNS (17) and Coot (37). After the refinements converged in CNS, the structure
was further refined using the program Refmac5 (CCP4 program suite) (18).
The ligands were then modeled into the excess density observed in the BmrR
drug pocket, resulting in a 1–2% decrease in RFREE. Fixed translation libration
screw (TLS) parameters were determined using the TLS motion detection
server (38) and then used in the subsequent rounds of model building and
structure refinement. In the last stages of the refinements, waters were
added to models. The criteria used to judge their validity included H-bonding
geometries and B factors (less than 80). The quality of the final structures
were validated using the Molprobity server (39), which shows 97% of all re-
sidues in the most favored region of the Ramachadran plot; no residues were
found in an unfavorable conformation.
ACKNOWLEDGMENTS. S.B. is a National Science Foundation (NSF) graduate
research fellowship awardee (DGE-0707427). H.W. is a recipient of The
Arnold and Mabel Beckman Young Investigator Award. This work is also
supported by a NSF CAREER Award (MCB-0953430). Intensity data was
collected at the Sanford Synchrotron Laboratory, beamline 9-2.
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