Abstract
Proteins from the bacterial small multidrug resistance (SMR) family are proton-coupled exporters of diverse antiseptics and antimicrobials, including polyaromatic cations and quaternary ammonium compounds. The transport mechanism of the Escherichia coli transporter, EmrE, has been studied extensively, but a lack of high-resolution structural information has impeded a structural description of its molecular mechanism. Here, we apply a novel approach, multipurpose crystallization chaperones, to solve several structures of EmrE, including a 2.9 Å structure at low pH without substrate. We report five additional structures in complex with structurally diverse transported substrates, including quaternary phosphonium, quaternary ammonium, and planar polyaromatic compounds. These structures show that binding site tryptophan and glutamate residues adopt different rotamers to conform to disparate structures without requiring major rearrangements of the backbone structure. Structural and functional comparison to Gdx-Clo, an SMR protein that transports a much narrower spectrum of substrates, suggests that in EmrE, a relatively sparse hydrogen bond network among binding site residues permits increased sidechain flexibility.
Highlights
The small multidrug resistance (SMR) family of microbial membrane proteins is a wellstudied family composed of primitive dual-topology proton-coupled transporters
We noticed that the interface between Gdx-Clo and monobody L10 is limited to a 9-residue stretch of loop 1 that is relatively well-conserved among SMR proteins (Figure 1A)
These observations suggested that conservative mutagenesis of EmrE loop 1 to introduce the Gdx-Clo residues might permit monobody L10 binding in order to facilitate crystallization of EmrE
Summary
The small multidrug resistance (SMR) family of microbial membrane proteins is a wellstudied family composed of primitive dual-topology proton-coupled transporters. The SMR family has two major physiological subtypes that can be distinguished based on sequence(Kermani et al, 2020). Representatives of the “Gdx” (guanidinium export) subtype 43 export a bacterial metabolite, guanidinium ion (Gdm+), in exchange for two protons(Kermani et al, 2018; Nelson et al, 2017). Representatives of the “Qac” (quaternary ammonium compound) subtype are proton-coupled exchangers of quaternary ammoniums and other hydrophobic, cationic compounds. Since the first quaternary ammonium antiseptics were introduced approximately one hundred years ago, proteins from the Qac cluster have been closely associated with the spread of multidrug resistance elements (Gillings, 2017; Pal et al, 2015; Russell, 2002; Zhu et al, 2017). Many bacteria possess SMR proteins belonging to both subtypes.
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