PRELIMINARY STRUCTURAL STUDIES ON THE MULTI-LIGAND-BINDING DOMAIN OF THE TRANSCRIPTION ACTIVATOR, BMRR, FROM BACILLUS-SUBTILIS

In the bacterium Bacillus subtilis, the DNA-binding regulatory protein , BmrR, activates transcription from the multidrug transporter gene, b mr, after binding either rhodamine or tetraphenylphosphonium. These tw o compounds, which have no structural similarity, are also substrates for the bacterial multidrug transporter. BmrR belongs to the MerR fami ly of transcription activators but differs from the other family membe rs in its ability to bind unrelated small molecule activators. As an i nitial step in the elucidation of the mechanism by which BmrR recogniz es rhodamine and tetraphe nylphosphonium and activates transcription, we have crystallized the 144-amino acid-residue carboxy terminal dimer ization/ ligand-binding domain of the BmrR, named the BRC (BmrR C-term inus). Tetragonal crystals of ligand-free BRC take the space group P4( 1)2(1)2, or its enantiomorph P4(3)2(1)2, with unit cell dimensions a = b = 76.3 Angstrom, c = 96.0 Angstrom, alpha = beta = gamma = 90 degre es. Diffraction is observed to at least 2.7 Angstrom resolution at roo m temperature. In addition, we determined the secondary structure cont ent of ligand-free and rhodamine-bound BRC by circular dichroism. In t he ligand-free form, BRC has considerable beta-sheet content (41%) and little alpha-helix structure (13%). After BRC binds rhodamine, its be ta-sheet content increases to 47% while the alpha-helix structure decr eases to 11%. The structure of BRC will provide insight not only into its multidrug recognition mechanism but could as well aid in the eluci dation of the recognition and efflux mechanisms of Bmr and other bacte rial multidrug transporters.