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BmrR, A Transcription Activator in Bacillus spp.

Emma R. Cummins '13 and Kari N. W. Deininger '13


I. Introduction

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).

BmrR 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 and 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 domain, referred to the BmrR C terminus (BRC) , which shares no homology with MerR family proteins or any other protein in current databases. The BmrR C terminus is the site of ligand binding within BmrR.

This 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.

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II. General Structure Overview

When 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 , the carboxy-terminal 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. (1)

The DNA 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. Within the DNA-binding 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 binding domain is a distorted eight-strand β-barrel which contains the hydrophobic core that functions as the active site for ligand-binding. The linker domain is an 11-turn-α-helix that connects the DNA binding domain and the drug binding domain. (1)

All 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 α6 of the drug binding domain interact with helices α3 and the C terminus of α1 of the DNA binding domain of the other monomer. The two monomers also dimerize through the wedging of the loop connecting β10 and β11 in the ligand-binding domain into the space between helices α3 and α4 of the DNA-binding domain of the other monomer. The linker domain α5 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. The α5 helix also makes van der Waals contacts with the α3 helix of the DNA-binding domain and the loop connecting β10 and β11 in the ligand- binding domain of the other monomer. There 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 proteins. (1) 

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III. DNA-Binding Domain

The 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 α2 and the turn between them. The 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 guanine 3. Van der waals contacts occur between Arg 23 and cytosine 9', thymine 7' and Tyr 24 and adenine 2, guanine 3 . Residues Ser 18 and Lys 20 may also participate in bonding, yet their side chains are disordered. Helix α1 makes a hydrogen bond between Gly 9 and the phosphate of cytosine 9' and van der Waals interactions between Ile 8 and the deoxyribose ring of cytosine 9'. (1)

Wing W1 is made up of β2 and β3 and the connecting loop 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 Arg-43 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. (1)

Wing 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). There are very few contacts between W2 and the DNA which are between Lys-60 and guanine 3 and Leu-66 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. (1)

Even 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. DNA 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. This abnormal structure of DNA is stabilized by interactions between Tyr-24, Tyr-25, Lys-60, and the N terminus of helix α4 with the phosphate backbone of the DNA. (1)

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)

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IV. Ligand-Binding Domain

The drug-binding 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 molecule participate in electrostatic interactions with negatively charged residues Asp-47 from the DNA-binding domain and Glu-253 and Glu-266 from the ligand-binding domain. The active site Glu-253 residue is buried within the hydrophobic core and is stabilized by hydrogen bonds from neighboring hydrophobic residues (Tyr 152, Tyr 187, and Tyr 229) in α2, 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 and Tyr-268 that surround the active site. The resulting arrangement 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)

Drug binding induces local conformational changes to the ligand-binding 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. Helix 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. Upon drug binding, 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)

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V. BmrR Function

Upon 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)

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VI. References

1. Heldwein, Ekaterina E. Zheleznova and 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., Schlundiner, Shimon, 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., Markham, Penelope 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., Markham, Penelope 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 6:2465-2468.

5. Markham, Penelope N., Ahmed, Maqbool, and 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.

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