We previously reported the X-ray structures of wild-type Escherichia coli AcrB, a proton motive force-dependent multidrug efflux pump, and its N109A mutant. These structures presumably reflect the resting state of AcrB, which can bind drugs. After ligand binding, a proton may bind to an acidic residue(s) in the transmembrane domain, i.e., Asp407 or Asp408, within the putative network of electrostatically interacting residues, which also include Lys940 and Thr978, and this may initiate a series of conformational changes that result in drug expulsion. Herein we report the X-ray structures of four AcrB mutants, the D407A, D408A, K940A, and T978A mutants, in which the structure of this tight electrostatic network is expected to become disrupted. These mutant proteins revealed remarkably similar conformations, which show striking differences from the previously known conformations of the wild-type protein. For example, the loop containing Phe386 and Phe388, which play a major role in the initial binding of substrates in the central cavity, becomes prominently extended into the center of the cavity, such that binding of large substrate molecules may become difficult. We believe that this new conformation may mimic, at least partially, one of the transient conformations of the transporter during the transport cycle.

The AcrAB multidrug efflux pump, which belongs to the resistance nodulation division (RND) family, recognizes and extrudes a wide range of antibiotics and chemotherapeutic agents and causes the intrinsic antibiotic resistance in Escherichia coli. The expression of AcrAB is controlled by the transcriptional regulator AcrR, whose open reading frame is located 141 bp upstream of the acrAB operon. To understand the structural basis of AcrR regulation, we have determined the crystal structure of AcrR to 2.55-A resolution, revealing a dimeric two-domain molecule with an entirely helical architecture similar to members of the TetR family of transcriptional regulators. Each monomer of AcrR forms a multientrance pocket of 350 A(3) in the ligand-binding domain. The ligand-binding pocket is surrounded with mostly hydrophobic residues. In addition, a completely buried negatively charged glutamate, expected to be critical for drug binding, is located at the center of the binding pocket. The crystal structure provides novel insight into the mechanisms of ligand binding and AcrR regulation.

The CmeABC multidrug efflux pump, which belongs to the resistance-nodulation-division (RND) family, recognizes and extrudes a broad range of antimicrobial agents and is essential for Campylobacter jejuni colonization of the animal intestinal tract by mediating the efflux of bile acids. The expression of CmeABC is controlled by the transcriptional regulator CmeR, whose open reading frame is located immediately upstream of the cmeABC operon. To understand the structural basis of CmeR regulation, we have determined the crystal structure of CmeR to 2.2 A resolution, revealing a dimeric two-domain molecule with an entirely helical architecture similar to members of the TetR family of transcriptional regulators. Unlike the rest of the TetR regulators, CmeR has a large center-to-center distance (54 A) between two N termini of the dimer, and a large flexible ligand-binding pocket in the C-terminal domain. Each monomer forms a 20 A long tunnel-like cavity in the ligand-binding domain of CmeR and is occupied by a fortuitous ligand that is identified as glycerol. The binding of glycerol to CmeR induces a conformational state that is incompatible with target DNA. As glycerol has a chemical structure similar to that of potential ligands of CmeR, the structure obtained mimics the induced form of CmeR. These findings reveal novel structural features of a TetR family regulator, and provide new insight into the mechanisms of ligand binding and CmeR regulation.

The Escherichia coli AcrR multidrug‐binding protein represses transcription of acrAB and is induced by many structurally unrelated cytotoxic compounds. The crystal structure of AcrR in space group P2221 has been reported previously. This P2221 structure has provided direct information about the multidrug‐binding site and important residues for drug recognition. Here, a crystal structure of this regulator in space group P31 is presented. Comparison of the two AcrR structures reveals possible mechanisms of ligand binding and AcrR regulation.

This paper describes the cloning, expression, purification and preliminary X-ray data analysis of the AcrR regulatory protein. The Escherichia coli AcrR is a member of the TetR family of transcriptional regulators. It regulates the expression of the AcrAB multidrug transporter. Recombinant AcrR with a 6ÂHis tag at the C-terminus was expressed in E. coli and purified by metalaffinity chromatography. The protein was crystallized using hanging-drop vapor diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source. The best crystal diffracted to 2.5 Å . The space group was determined to be P3 2 , with unit-cell parameters a = b = 46.61, c = 166.16 Å .