A Transcription Activator in Bacillus
Emma R. Cummins '13 and Kari N. W. Deininger '13
BmrR is a transcription
activator of the multidrug efflux transporter gene bmr
in the gram positive prokaryote Bacillus
subtilis. Multidrug efflux
transporters export a variety of toxins from within the cytoplasm
across the cell membrane in exchange for protons across the
transporter. The multidrug efflux transporter bmr
confers the cell with resistance to a variety of
structurally different substrates, including rhodamine and
tetraphenylphosphonium (TPP)(2). Transcription of bmr
is stimulated when BmrR is bound to any one of its several hydrophobic
cationic ligand targets, including TPP and rhodamine (3).
is part of the MerR family of transcriptional regulators which
regulates cellular responses to stressors caused by exposure to either
toxic compounds or free oxygen radicals in bacteria (1). BmrR shares
common homologies with the MerR family in its helix-turn-helix
DNA binding domain
its broad substrate
specificity (3). Broad
substrate specificity is a quality that is unique to both multidrug
efflux transporters and their regulators, which contrasts drastically
with the chemical specificity of many other ligand-binding proteins
(4). Unique to the BmrR protein is its carboxy-terminal
referred to the BmrR C terminus (BRC)
which shares no homology with MerR family proteins or any other protein
databases. The BmrR C terminus is the site of ligand binding within
tutorial aims to highlight the structure and explain the function of
the various domains of BmrR as well as provide a glimpse into each
domain’s role in the overall function of BmrR within the cell.
Back to Top
General Structure Overview
two monomers of BmrR bind to the promoter of the gene bmr,
they form a dimer with an overall structure that resembles a butterfly.
Each monomer of BmrR has three distict domains: the amino-terminal
DNA binding domain
drug binding domain
and the linker
between the amino-terminal and carboxy-terminal domains
The DNA binding domain shares homology with that of other MerR family
proteins while the ligand-binding domain is unique to the protein,
which allows for binding to specific coactivators.
binding domain is composed of
a four α-helix bundle and a
three-stranded antiparallel β-sheet with a topology of β1-α1-α2-β2-β3-α3-α4.
domain topology, there are conserved hydrophobic residues
throughout the MerR family. This suggests that the DNA-binding domains
of MerR family proteins share similar folding structure. The drug
is a distorted
eight-strand β-barrel which contains the hydrophobic core that
functions as the active site for ligand-binding.
an 11-turn-α-helix that connects the DNA binding domain and
the drug binding domain.
three domains participate in dimerization by burying 5,800 Å
of accessible surface area (ASA). The ligand-binding domain packs
tightly against the DNA-binding domain of the other monomer during
dimerization, burying 2,000 Å of ASA per monomer. Each
monomer has helix
of the drug
binding domain interact with helices α3
and the C terminus of α1
the DNA binding domain of the other monomer.
also dimerize through the wedging of the loop
β11 in the
ligand-binding domain into the space between helices
α3 and α4
of the DNA-binding domain of the other monomer.
helices participate in
dimerization by forming an antiparallel coiled coil, which buries 900
Å of ASA per monomer. This binding structure is thought to be
a common feature of MerR family proteins.
helix also makes van der
Waals contacts with the α3 helix
the DNA-binding domain and the loop
connecting β10 and
β11 in the ligand-
binding domain of the other monomer.
is very little
structural change when BmrR momomers dimerize with one another and when
the BmrR dimer binds to its substrate, such as TPP. This lack of
conformational change is uncommon compared to other substrate-binding
Back to Top
DNA-binding domain of BmrR is made up of a helix-turn-helix (HTH) motif
and two wings (W1 and W2). The HTH
motif is made up of helices α1
and the turn between them.
recognition helices of each
monomer contact two consecutive major
grooves of the DNA which
results in the helical axis alligning itself almost perpendicularly to
the DNA helical axis. The majority
of the HTH contacts are made by the residues from α2.
Hydrogen bonds occur between Ala 21 and guanine
4; Tyr 25 and
der waals contacts
23 and cytosine 9', thymine 7'
24 and adenine 2,
guanine 3 .
Ser 18 and Lys
20 may also participate in bonding,
yet their side chains are disordered.
Helix α1 makes a hydrogen bond between Gly
and the phosphate of cytosine 9'
and van der
Waals interactions between Ile 8 and the
deoxyribose ring of
W1 is made up of β2 and
between them. Like the HTH motif, there are many interactions between
the DNA and W1, including both hydrogen bonds and van der Waals
contacts. Hydrogen bonds are formed between Ser-41
and cytosine 8';
Tyr-42 and cytosine 9' and 10'; Arg 43 and thymine 7', cytosine 8'.
Van der Waals contacts
are formed between Tyr-42
and guanine 10
and cytosine 8'. Arg
43 makes a hydrogen bond to Asp 26,
which correctly positions Arg 23
and allows for the reading of the DNA to take place.
W2 is the last DNA binding
element that helps with DNA-protein
interaction. W2 is not a loop but another HTH motif that is made up of helices
α3 and α4
which are less crossed than the
major groove HTH motif and has Cα atoms superimposed
(residues 9-26 and 56-73).
are very few
W2 and the DNA which are between Lys-60 and
and adenine 2. The α4 helix
also has a dipole moment
which contributes to the binding of the electropositive N terminus and
the phosphate of adenine 2.
with the many contacts between BmrR and DNA, RNA polymerase is still
able to bind to the DNA. This is possible because of the structure
changes that occur in the DNA when the BmrR-TPP complex binds. When the
complex binds, the promoter
is bent by approximately 50 degrees away
from BmrR and toward the major groove.
is able to bend
this much because the A-T base pairs that surround the bmr
promoter break and shift toward the 3’ end of the strand.
This shift creates ripples in both strands of DNA which causes the DNA
to bunch up in the middle.
of DNA is
stabilized by interactions between Tyr-24,
and the N
terminus of helix α4
with the phosphate backbone of the DNA.
distortion serves a very important mechanistic purpose in the
binding of the BmrR complex. In a traditional sigma-regulated promoter,
the 17-bp spacer between the -10 and -35 elements in the B-DNA
conformation results in each binding element
exposed on the same side of the DNA. In contrast, a 19-bp spacer
between the -10 and -35 elements results in each element being exposed
on opposite sides of the B-DNA, which makes it impossible for
RNA-Polymerase to bind to
each element at the same time. The BmrR-ligand-induced base unpairing
and sliding shortens the B-DNA by 2-bp, which results in local
untwisting which brings the -10 and -35 elements onto the same side of
the DNA. The resulting promoter rearrangement is similar to the 17-bp
transcription ready spacer in traditional sigma promoters.(1)
Back to Top
pocket is formed by Ile-23 and Val-28
in α1, Tyr-51 and Ala-53 in β3, Tyr-68
and Ile-71 in β4, and Ile136 from β7 that flank the
hydrophobic core active site.
The positively charged phosphorous of the TPP
participate in electrostatic interactions with negatively charged
from the DNA-binding domain and Glu-253
from the ligand-binding domain.
active site Glu-253
residue is buried within the hydrophobic core and is stabilized by
hydrogen bonds from
hydrophobic residues (Tyr 152,
Tyr 187, and Tyr 229) in
in the connecting loop between β3 and β4, and in
α3. The phenyl rings on the TPP molecule participate in
van der Waals contacts with hydrophobic residues Phe-224
that surround the active site.
of the negatively charged acidic
residue surrounded by aromatic residues leads to favorable binding of
hydrophobic cationic drugs such as TPP. It is unclear how Glu-266
contributes to drug binding because of its disordered structure. (1,3,5)
binding induces local
conformational changes to the
domain but does not greatly alter the overall protein structure. Helices
α1 and α2
along with the segment connecting
the two helices change conformation upon binding.
α1 rotates ~10° about its amino terminus which
unwinds its carboxyl terminus (Asn-30). The segment connecting
α1 and α2 (comprised of Phe-31-Ser-32-Tyr-33)
unfolds and extends away from the binding site so that the Tyr-33 is
translocated 11.5 Å from its location in the unbound complex.
This Tyr-33 residue is a
key phenyl residue that helps to stabilize
Glu-253 within the hydrophobic core through a bond which is broken
when Tyr-33 is translocated. A water molecule occupies the space left
by Tyr-33 and helps to stabilize Glu-134.
α2 becomes disordered, an action that is necessary for
binding. In the unbound state, α2 blocks access to the
hydrophobic core in addition to stabilizing Glu-134. The helix must
unfold to allow for ligand binding. This ligand-induced helix to coil
transition by BCR is quite uncommon and contrasts with other proteins.
(1, 3, 5)
Back to Top
ligand binding, BmrR is converted into an activator of transcription
for the gene bmr
which codes for the multidrug efflux pump bmr. The mechanism behind
BmrR activation is similar to other MerR family proteins. After drug
binding, the unwinding and relocation of α2 helix serves as a
signal between the two active domains in BmrR which may trigger
activator behavior. Unwinding the spacer region on the bmr
promoter allows for BmrR to bind to its newly-exposed binding site and
successful recruitment of transcription elements to initiate
transcription. (1, 3)
Back to Top
1. Heldwein, Ekaterina E. Zheleznova
Brennan, Richard G. 2001. Crystal structure of the transcription
activator BmrR bound to DNA and a drug. Nature 409:378-381.
2. Klyachko, Katya A.,
and Neyfakh, Alexander A. 1997. Mutations affecting substrate
specificity of the Bacillus
subtilis multidrug transporter
bmr. Journal of Bacteriology 179:2189-2193.
3. Zheleznova, Ekaternia E.,
N., Neyfakh, Alexander A., and Brennan, Richard G. 1999. Structural
basis of multidrug recognition of by BmrR, a transcription activator of
a multidrug transporter. Cell 96:353-362.
4. Zheleznova, Ekaternia E.,
N., Neyfakh, Alexander A., and Brennan, Richard G. 1997. Preliminary
structural studies on the multi-ligand-binding domain of the
transcription activator, BrmR from Bacillus
subtilis. Protein Science
5. Markham, Penelope N., Ahmed,
Neyfakh, Alexander A. 1995. The drug-binding activity of the
multidrug-responding transcriptional regulator BmrR resdies in its
C-terminal domain. Journal of Bacteriology 178:1473-1475.
Back to Top