B.
subtilis Multidrug Transporter
Activation, N terminus
Jake Calcei '09 and Ben Alexander '09
Contents:
I. Introduction
A group
of DNA binding proteins known as the MerR family are known for their
characteristic N-terminal helix-turn-helix domain that binds to DNA in
the
activation of transcription. The
N-terminal domain of the protein is connected to the C-terminus through
a
number of coiled-coils. The C-terminal domain acts as a
coactivator for
transcription by serving as a ligand binding site.
These transcription regulating proteins that
belong to the MerR family are commonly found in bacteria binding to a
suboptimal (greater than 17 bp) region of the -35 to -10 promoter
elements
{2}. Certain
members of the MerR
family bind to slightly different length suboptimal regions of the DNA
of the
specific bacteria where they are located {2,4,5}.
The
member of the MerR family that is found in the bacteria Bacillus
subtilis
is MtaN, multidrug transporter activation, N terminus, which is a
global
transcriptional activator that interacts with other members of the MerR
family including
BmrR and BltR in promoting transcription of the bmr,
blt,
ydfK
genes, and
its own gene, mta {1,2}.
MtaN
shares structural similarities with BmrR along with other MerR family
members
that allow the proteins to undertake certain conformational changes as
well as
forcing the DNA to change conformation during the protein-DNA binding
process. The structural similarities
among the different members of the MerR family are due to a
conservation of
certain sequences in their secondary structures {4}.
II. General Structure
MtaN
is an N-terminal DNA binding domain that
takes on a dimeric structure that binds conformationally changed DNA
.
The asymmetric subunit of MtaN contains single subunit
of the MtaN
protein bound to an oligodeoxynucleotide
26-base pairs in length
.
This 109 residue truncated mutant contains
only the N-terminal
DNA binding domain of the Mta protein as well as
its dimerization
domain which are
connected by a hinge region
.
The structure of a single subunit in the dimer is composed of five
α-helices and three antiparallel β-sheets
. The monomer takes on a winged
helix-turn-helix structure
with
the α5
helix
projecting outwards
.
α5
is an 8-turn helix that
makes up the dimerization domain as it interacts with the α5’
domain on the
second subunit
.
The other chains, α-helices (1-4) and
β-sheets (1-3) of subunit
1 and subunit
2, make up the DNA-binding domain of the mutant
protein
{4}.
III. DNA Binding Domain
The DNA binding domain of each MtaN monomer is
a winged helix turn helix motif
which
binds the mta promoter region of
26 bp DNA. The
winged helix
turn helix
consists of α helices α3
and α4
which make up wing
2 (W2)
and
antiparallel β sheets β1,
β2,
and
β3
which make up wing
1 (W1)
. α1
serves as the structural support of the body of
the DNA binding domain.
This domain is stabilized by the 23
hydrophobic amino acids
that
make
up the hydrophobic core in each subunit. It is interesting to
note that 21 of the 23 amino acids
that make up the hydrophobic core of the MtaN DNA binding domain are conserved
across the MerR family of binding proteins. Additional
structural support is provided by a salt bridge connecting OD2 of Asp-23
and NH1
of Arg-39
{3,4}.
The binding
interactions
that
stabilize the MtaN to the DNA include H-bonds between the protein side
chains and the DNA, protein backbone amide-DNA interactions, and van
der Waals contacts. When the MtaN dimer binds DNA, it
induces a bend
in the DNA of 47° at the central TpT base pair
step, where weak
and unfavorable Watson and Crick hydrogen
bonds are formed between Thy-1'
and Ade-1
as
a result of the bending . The
DNA of the mta
promoter is distorted in comparison with the B-form DNA,
causing the overall length of the helix to be shortened by 5.9
angstroms. This bend is stabilized by interactions with
the MtaN residues Ser-15
and Tyr-22
as
well as Tyr-38
of W1
,
and Lys-56
and Leu-62
of W2
.
A number of other
interactions contribute to the protein-DNA
binding, including the sequence specific binding of Arg-17 with Gua-4
and Thy-5 {3,4}.
IV. Dimerization Domain
The
dimerization domain
consists of an 8-turn α5 helix
that forms an anti parallel
coiled coil with α5’ of the other subunit
.
The interface between α5 and
α5’ is hydrophobic and consists of Leu-80, Leu-87,
Met-94, Ile-98, Ile-101, Leu-105, and the methylene
carbons of Lys-84
and Lys-91 on the symmetric locations of both helices
.
Similarly, α5
also interacts with α3’
at the contacts of Phe-54'
and Thr-104,
Ile-58'
and Ile
101, and a van der Waals
interaction between Glu-57'
and Met-97
.
In
addition to dimerization, α5
is also
responsible for conformational DNA bending.
Residues 71-75 of the N-terminal of α5,
act as a hinge
and rotate the DNA
binding domains towards each other 11°
{3}.
V.
MtaN Function
MtaN
is a member of the MerR DNA distortion mechanism family of
transcription factors. The protein induces a conformational change in
suboptimal 19 bp spacer between -10 and -35 promoter sequence {4}. This
results in promoter DNA distortion and transcriptional
activation {2,4}. MtaN refers to the N-terminal domain of
the Mta regulatory protein. Evidence has been shown that Mta is a
global regulator of the multidrug transporters Bmr and Blt. When
separated from the C-terminal domain, expression of the N-terminal
domain (MtaN) constitutively activates the
bmr, blt, mta,
and ydfk
genes {1}. This supports the widely supported prediction that most, if
not all, MerR family proteins are activators that act on similarly
structured promoters {2}.
VI.
References
1.
Baranova,Natalya
N., Antoine Danchin and Alexander A. Neyfakh. 1999. Mta, a global
MerR-type regulator of the Bacillus subtilis multidrug-efflux
transporters. Molecular Microbiology 31:
1549-1559.
2.
Brown, Nigel L., Jivko
V. Stoyanov, Stephen P. Kidd, and Jon L.
Hobman. 2003. The MerR family of transcriptional regulators. FEMS
Microbiology Reviews 27: 145-163.
3. Godsey, Michael H., Natalya N.
Baranova, Alexander A.
Neyfakh, and Richard G. Brennan. 2001. Crystal Structure of MtaN, a
Global
Multidrug Transporter Gene Activator. Journal of Biological Chemistry
276:
47178-47184.
4. Newberry, Kate J., and
Richard G. Brennan. 2004.
The structural
mechanism for transcription activation by MerR family member multidrug
transporter
activation, N terminus. Journal of Biological Chemistry
279:
20356-20362.
5.
Outten,
Caryn E., F. Wayne Outten, and Thomas V. O’Halloran. 1999.
DNA distortion
mechanism for transcriptional activation by ZntR, a Zn(II)-responsive
MerR homologue
in Escherichia
coli. Journal
of Biological Chemistry 274: 37517–37524.
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