The emergence of bacterial multidrug resistance (MDR) poses a serious threat to human health. One key factor underlying MDR is membrane bound transporters that extrude multiple, chemical diverse drugs from the bacterial cell. The structural mechanism by which these proteins recognize dissimilar drugs is completely unknown, primarily because they are integral membrane proteins and thus more difficult to purify. Bacteria also have a second class of multidrug binding proteins that is central to their multidrug resistant phenotypes. These cytosolic proteins are transcription regulators of the multidrug transporter genes. One regulator from Bacillus subtilis is BmrR. BmrR dramatically increases transcription of the multidrug transporter gene, bmr, only after binding drugs that are Bmr substrates but have invaded the cytosol. Thus, BmrR acts as a second line of defence against drugs from reaching their cellular targets. Structures of BmrR-Drug and BmrR-DNA+Drug complexes will also reveal the transcription regulation mechanism of the MerR family member, the class to which BmrR belongs. A second multidrug binding regulatory protein is QacR from Staphylococcus aureus. QacR represses the qacA and multidrug transporter gene and belongs to the TetR/CamR family. Drugs, which are also substrates of the QacA transporter, induce QacR and derepress the qacA gene thereby providing the bacterium with the more transporters to fend off potentially lethal drug doses. Structural studies will unveil the underpinnings of the multidrug binding and transcription repression mechanisms of QacR. Interestingly, QacR and BmrR display overlapping drug binding specificities and structures of their same-drug complexes will reveal the similarities and differences of their multidrug binding mechanisms. This grant proposal has four specific aims. To crystallize and determine the structures of the C-terminal, multidrug binding domain of BmrR, the so named BRC, bound to a number of drugs that display a wide range of binding affinities. BRC offers the advantages of high resolution, which will greatly aid the analysis of the drug binding mechanism of BrnrR. To crystallize and determine the x-ray structures of BmrRDrug-DNA and BmrR-DNA complexes. To crystallize and determine the x-ray structures of the B. subtilis global MD regulator, MtaN and its DNA complexes. To crystallize and determine the x-ray structures of QacR-drug and QacR-DNA complexes. The broad goals of this work are to provide a complete understanding of the mechanisms of multidrug binding by BrnrR, MtaN and QacR and gene regulation of these MerR and TetR/CamR family members. These data will be key to the future structure-based drug design of novel drugs against pathogenic bacteria.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI048593-03
Application #
6632454
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Perdue, Samuel S
Project Start
2001-03-01
Project End
2005-02-28
Budget Start
2003-03-01
Budget End
2004-02-29
Support Year
3
Fiscal Year
2003
Total Cost
$289,250
Indirect Cost
Name
Oregon Health and Science University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
Peters, Kate M; Brooks, Benjamin E; Schumacher, Maria A et al. (2011) A single acidic residue can guide binding site selection but does not govern QacR cationic-drug affinity. PLoS One 6:e15974
Kumaraswami, Muthiah; Newberry, Kate J; Brennan, Richard G (2010) Conformational plasticity of the coiled-coil domain of BmrR is required for bmr operator binding: the structure of unliganded BmrR. J Mol Biol 398:264-75
Hassan, Karl A; Xu, Zhiqiang; Watkins, Ryan E et al. (2009) Optimized production and analysis of the staphylococcal multidrug efflux protein QacA. Protein Expr Purif 64:118-24
Schumacher, Maria A; Piro, Kevin M; Xu, Weijun et al. (2009) Molecular mechanisms of HipA-mediated multidrug tolerance and its neutralization by HipB. Science 323:396-401
Kumaraswami, Muthiah; Schuman, Jason T; Seo, Susan M et al. (2009) Structural and biochemical characterization of MepR, a multidrug binding transcription regulator of the Staphylococcus aureus multidrug efflux pump MepA. Nucleic Acids Res 37:1211-24
Newberry, Kate J; Huffman, Joy L; Miller, Marshall C et al. (2008) Structures of BmrR-drug complexes reveal a rigid multidrug binding pocket and transcription activation through tyrosine expulsion. J Biol Chem 283:26795-804
Peters, Kate M; Schuman, Jason T; Skurray, Ronald A et al. (2008) QacR-cation recognition is mediated by a redundancy of residues capable of charge neutralization. Biochemistry 47:8122-9
Brooks, Benjamin E; Piro, Kevin M; Brennan, Richard G (2007) Multidrug-binding transcription factor QacR binds the bivalent aromatic diamidines DB75 and DB359 in multiple positions. J Am Chem Soc 129:8389-95
Schumacher, Maria A; Allen, Gregory S; Diel, Marco et al. (2004) Structural basis for allosteric control of the transcription regulator CcpA by the phosphoprotein HPr-Ser46-P. Cell 118:731-41
Newberry, Kate J; Brennan, Richard G (2004) The structural mechanism for transcription activation by MerR family member multidrug transporter activation, N terminus. J Biol Chem 279:20356-62

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