Biomolecules at Kenyon XX HHMI at Kenyon xx Jmol Home xx Biology Dept xx COMMENTS and CORRECTIONS

B. subtilis Multidrug Transporter 

Activation, N terminus

Jake Calcei '09 and Ben Alexander '09


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.

Back to Top