Crystal structures of bacterial small multidrug resistance transporter EmrE in complex with structurally diverse substrates

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.

Activating alternative transport modes in a multidrug resistance efflux pump to confer chemical susceptibility

“…Comparing the NMR CSP induced by TPP + and harmane with S64V-EmrE reveals that both induce CSPs in the middle of transmembrane helix 1 and 3, the regions of EmrE in close proximity to E14. This data shows that both substrates can interact with the known binding site at E14, as visualized in recent monobody-assisted crystal structures of EmrE with each of these ligands 24 . However, the magnitude and extent of the CSPs are strikingly different for these two substrates.…”

Cotranslational folding and assembly of the dimeric E. coli inner membrane protein EmrE

“…(a) The EmrE dimer (left) and one monomer (right) (10) (PDB 7MH6). E 14 , S 64 , G 90 , and G 97 are shown in spacefill and the other residues mutated in TMH3 are indicated in stick representation.…”

“…Many soluble cytoplasmic proteins can form both homo- and heterodimers while one of the partner proteins is still being translated (3). Here, we wanted to ascertain whether this is possible also for EmrE that assembles into an anti-parallel 4+4 TMH homodimer in the inner membrane (10, 26, 27), Fig. 2a.…”

“…A recent FPA analysis of EmrE suggested that there may be long-range cotranslational interactions between a conserved Glu residue (E 14 ) in the middle of transmembrane helix 1 (TMH1) and unidentified residues in TMH2 and TMH3 during membrane insertion (7), and hence that the monomer might start to fold cotranslationally. Further, given the extensive inter-subunit packing interactions between the two monomers in the EmrE dimer (10), we speculated that cotranslational dimerization of EmrE might be possible to observe by FPA.…”

Cotranslational folding and assembly of the dimeric Escherichia coli inner membrane protein EmrE

“…A recent FPA analysis of EmrE suggested that there may be long-range cotranslational interactions between a conserved Glu residue (E 14 ) in the middle of TMH1 and unidentified residues in TMH2 and TMH3 during membrane insertion ( 7 ) and hence, that the monomer might start to fold cotranslationally. Further, given the extensive intersubunit packing interactions between the two monomers in the EmrE dimer ( 10 ), we speculated that cotranslational dimerization of EmrE might be possible to observe by FPA.…”