Crystal structures of CmeR‐bile acid complexes from Campylobacter jejuni

Abstract

The TetR family of transcription regulators are diverse proteins capable of sensing and responding to various structurally dissimilar antimicrobial agents. Upon detecting these agents, the regulators allow transcription of an appropriate array of resistance markers to counteract the deleterious compounds. Campylobacter jejuni CmeR is a pleiotropic regulator of multiple proteins, including the membrane-bound multidrug efflux transporter CmeABC. CmeR represses the expression of CmeABC and is induced by bile acids, which are substrates of the CmeABC tripartite pump. The multiligand-binding pocket of CmeR has been shown to be very extensive and consists of several positively charged and multiple aromatic amino acids. Here we describe the crystal structures of CmeR in complexes with the bile acids, taurocholate and cholate. Taurocholate and cholate are structurally related, differing by only the anionic charged group. However, these two ligands bind distinctly in the binding tunnel. Taurocholate spans the novel bile acid binding site adjacent to and without overlapping with the previously determined glycerol-binding site. The anionic aminoethanesulfonate group of taurocholate is neutralized by a charge-dipole interaction. Unlike taurocholate, cholate binds in an anti-parallel orientation but occupies the same bile acidbinding site. Its anionic pentanoate moiety makes a water-mediated hydrogen bond with a cationic residue to neutralize the formal negative charge. These structures underscore the promiscuity of the multifaceted binding pocket of CmeR. The capacity of CmeR to recognize bile acids was confirmed using isothermal titration calorimetry and fluorescence polarization. The results revealed that the regulator binds these acids with dissociation constants in the micromolar region.

Crystal structure of the transcriptional regulator Rv1219c of Mycobacterium tuberculosis

“…A similar characteristic for the TetR-family regulators that recognize negatively charged antimicrobials has also been observed with TtgR 27 and CmeR. 28 In this case, positively charged histidines or lysines within the ligand-binding pockets are critical for the binding. In the case of Rv1219c, the predicted substrates are either neutral or positively charged.…”

The TetR Family of Regulators

“…This is also seen for the ligand-binding pockets of the majority of TFRs (e.g., ActR, CmeR, and QacR [53,54,56]). In contrast, the SimR ligand-binding cavity is composed of residues from both monomers such that each binds either the aminocoumarin or the angucyclinone moiety of the simocyclinone ligand (50).…”

“…This is also the case for many TFRs (e.g., SimR, CmeR, and MphR [50,56,59]), but different drug-binding stoichiometries are seen in some others. In the case of ActR, each ActR dimer is capable of binding either two molecules of actinorhodin or four molecules of (S)-2,4-dinitrophenyl acetate [(S)-DNPA] (54).…”

“…Similar to the case of EthR, the structure of CmeR shows how two structurally similar molecules can fit very differently into the same binding pocket (56). The structure of CmeR has been solved in complex with two structurally similar bile acids, taurocholate and cholate.…”

“…For example, ActR, QacR, SmeT, TetR, and TtgR all have a "side entry" opening distal to the dimerization interface that is believed to be the site of access for different ligands (22,(51)(52)(53)(54). Ligands appear to enter CmeR, CgmR, HrtR, LfrR, and SimR via an entry point closer to the "front" of the protein (43,46,50,55,56). Finally, DesT, EthR, and FadR exhibit a relative "top entry" (44,57,58).…”