Dynamics underlie the drug recognition mechanism by the efflux transporter EmrE

The multidrug efflux transporter EmrE from Escherichia coli requires anionic residues in the substrate binding pocket for coupling drug transport with the proton motive force. Here, we show how protonation of a single membrane embedded glutamate residue (Glu14) within the homodimer of EmrE modulates the structure and dynamics in an allosteric manner using NMR spectroscopy. The structure of EmrE in the Glu14 protonated state displays a partially occluded conformation that is inaccessible for drug binding by the presence of aromatic residues in the binding pocket. Deprotonation of a single Glu14 residue in one monomer induces an equilibrium shift toward the open state by altering its side chain position and that of a nearby tryptophan residue. This structural change promotes an open conformation that facilitates drug binding through a conformational selection mechanism and increases the binding affinity by approximately 2000-fold. The prevalence of proton-coupled exchange in efflux systems suggests a mechanism that may be shared in other antiporters where acid/base chemistry modulates access of drugs to the substrate binding pocket.

experiments are displayed with no error bars.Data obtained in isotropic bicelles were replotted from Thomas et al. 1 and Shcherbakov et al. 2 .b. 1 H/ 13 C HMQC spectra of 13 C-Ile methyl labeled EmrE at pH 5 (black) and pH 9 (red) in DMPC/DHPC isotropic bicelles at 15 °C.Peak labels indicate the assigned isoleucine residue with the monomer in superscript.

c.
Tryptophan fluorescence spectra of an EmrE mutant with a single Trp63 (W31F/W45F/W76F) in DMPC/DHPC isotropic bicelles at 25 °C.Spectra in black circles and red squares correspond to data collected at pH 5 and pH 9, respectively.Data are presented as mean values ± s.d.among three replicates from one independent experiment.The same trend was observed in other independent experiments.
d.The location of the two Trp63 residues highlighted in the NMR structure of proton-bound EmrE.Superscripts "A" or "B" refer to the corresponding monomer in EmrE.
e. Overlays of 13 C/ 15 N MAS correlation spectra of [ 13 C, TPP at natural abundance, while control spectra in black were performed with perdeuterated TPP.Superscripts "A" or "B" for indicated residues refer to the corresponding monomer.1.10 * Structural ensembles were derived using unrestrained MD simulations in the absence of NMR constraints.The structures of the ensemble correspond to snapshots selected from the simulation that were in best agreement with the experimental data.Note that 95.5% to 96.2% of the distance restraints were satisfied for the protonbound EmrE ensemble and 94.2% to 94.9% for the TPP-bound ensemble.Most violations likely reflect an imprecise location of the MTSL tag in PRE experiments (i.e., constraints implemented from the sulfur of cysteine, the CG1 of isoleucine, or the CG of leucine).** Pairwise r.m.s.deviation was calculated among 10 refined structures in each ensemble.The ensembles were determined using unrestrained all-atom MD simulations in DMPC lipid bilayers by selecting snapshots with the fewest violations of experimental constraints.

Figure 4 .
13 C/ 13 C MAS solid-state NMR spectra of EmrE in the protonbound and TPP-bound forms.a, b.13  C/ 13 C PDSD spectra of EmrE L51I /EmrE heterodimers where either EmrE L51I (a) or EmrE (b) was isotopically enriched with 1,3-13 C glycerol (left) or 2-13 C glycerol (right).Spectra in panel "a" were acquired with a 1 sec mixing time, while spectra in panel "b" were acquired with a 0.5 sec mixing time.Assigned cross-peaks are labeled in each of the panels, where superscripts "A" or "B" refer to the corresponding monomer.The 1D 13 C slices in panel "a" were extracted from the 13 C/ 15 N MAS correlation spectra recorded on the EmrE L51I /EmrE heterodimer, where EmrE L51I was isotopically labeled.c.13  C/ 13 C PDSD spectra with a 1 sec mixing time obtained using dynamic nuclear polarization at ~100 K.The sample was comprised of 13 C-Tyr ( 13 C a,b ) labeled EmrE mixed with natural abundance EmrE E14Q in the presence of TPP at pH 6.2.The spectrum on the left corresponds to addition of 13 C-labeled TPP while the spectrum on the right corresponds to addition of TPP at natural abundance.Cross-peaks highlighted by a dashed blue box show an intermolecular contact between TPP and Tyr60 of monomer A.selective amino acid labeling is 15 N-Ile, 15 N-Leu, 15 N-Met, 15 N-Phe, 15 N-Thr, 15 N-Trp, 15 N-Tyr, and 15 N-Val.In each panel, the assignments are indicated in a monomer specific manner as indicated by "A" or "B" superscripts.Glu14, and "D" corresponds to drug.The upper and lower states denote inward-open and outward-open conformations, respectively.The dotted boxes correspond to structures solved in this work.Since wild-type EmrE is a homodimer, structures solved can be viewed as cytoplasmic or periplasmic facing states.Bottom: Box and whisker plot of scaled violations calculated for each structure relative to experimental distance restraints after each MD simulation round in explicit lipid bilayers for proton-bound EmrE (left) and TPP-bound EmrE (right).The grey boxes correspond to the standard deviation, error bars are three times the standard deviation, and grey diamonds are all points outside the error bar range.Scaled violations are calculated as in Equation 1. b. Left: Side views of the proton-bound EmrE L51I /EmrE heterodimer structure (top) and the TPP-bound EmrE/EmrE E14Q heterodimer structure (bottom) in a cartoon representation.Monomers A and B are displayed in pink and blue, respectively, and TPP is displayed in green.Middle, right: backbone overlay of the 10 lowest violated structures from MD simulations deposited in the PDB with the same view and coloring as in the left panels.e, f.Display of the chi2 angles as a function of the simulation time initiated from the NMR structure (e) or X-ray structure (f).The three replicate runs are displayed in different colors and are the same data as in panel (c, d).a, b.
H/ 15 N TROSY spectra (a) and 1 H/ 13 C HMQC spectra (b) of PRE samples of EmrE L51I /EmrE heterodimers used to derive distance constraints for the proton-bound form.In each panel, the MTSL oxidized spectrum is displayed in red, while the reduced spectrum is H/ 1 H/ 13 C HSQC-NOESY spectra of EmrE/EmrE E14Q heterodimers bound to TPP at pH 5.8 where either EmrE (c) or EmrE E14Q (d) was isotopically enriched with -13 CH3 methyl groups at isoleucine, leucine, and valine.Strips correspond to 1 H/ 1 H spectral slices at the 15 N]-EmrE (black) and the heterodimers [ 13 C, 15 N]-EmrE L51I /EmrE (red, left panel) or [ 13 C, 15 N]-EmrE/EmrE L51I (red, right panel) in DMPC lipid bilayers at pH 5.0.Superscripts "A" or "B" refer to the corresponding monomer A or B, respectively.f.Overlays of 13 C/ 15 N MAS correlation spectra of [ 13 C, 15 N]-EmrE (black) and the heterodimers [ 13 C, 15 N]-EmrE/EmrE E14Q (red, left panel) or [ 13 C, 15 N]-EmrE E14Q /EmrE (red, right panel) in DMPC lipid bilayers bound to TPP at pH 5.0.Superscripts "A" or "B" refer to the corresponding monomer A or B, respectively.a, b. 1 1 indicated 13 C frequency in the indirect dimension.Labeled "TPP" cross-peaks indicate intermolecular NOEs between monomer A or B and TPP.Spectra in blue were performed with

Table 1 . NMR and refinement statistics for protein structures
1H/ 15 N TROSY spectra (a) and 1 H/ 13 C HMQC spectra (b) of PRE experiments on EmrE/EmrE E14Q heterodimers bound to TPP.In each panel, the MTSL oxidized spectrum is displayed in red and the reduced spectrum is displayed in blue.The sample nomenclature is as follows: "Intra" refers to intramolecular PRE constraints where MTSL is covalently attached at the indicated cysteine residue with the corresponding monomer displayed in parentheses.For example, "Intra I5C (A)" corresponds to 15 N/ 13 C labeled EmrE with the MTSL tag at Cys5 and mixed with unlabeled EmrE E14Q ."Inter" refers to intermolecular constraints.For example, "Inter C39 (A to B)" corresponds to 15 N/ 13 C labeled EmrE E14Q (monomer B) mixed with MTSL labeled EmrE at Cys39 (monomer A). c. MAS 13 C/ 15 N correlation spectra of the side chain tryptophan region for wild-type EmrE bound to TPP at low (top) and high pH (bottom) values.Labeled Trp63 signals of Trp63 for monomer A and B are colored and highlighted in rectangular boxes in pink and blue, respectively.Superscripts "A" or "B" refer to the corresponding monomer.d. 1 H/ 13 C HMQC spectra of 13 C-Ile methyl labeled EmrE bound to TPP at the indicated pH values in DMPC/DHPC isotropic bicelles at 37 °C.Peak labels indicate the assigned isoleucine residue with monomer A or B displayed in the superscript.e. Structural views of our NMR TPP-bound conformation of EmrE/EmrE E14Q heterodimer (left) and the X-ray structure of EmrE in complex with TPP crystallized at pH 6.5 (right).TPP is colored in green with monomer B of EmrE displayed as a grey cartoon representation.The dashed yellow lines indicate the closest distance between the heavy atoms of Ile68 in monomer B and TPP (in Å).Water radial distribution function (RDF) surrounding Glu14 of monomer A derived from MD simulations on proton-bound EmrE (left), EmrE deprotonated at Glu14 of monomer A (middle), and EmrE bound to TPP at low pH (right).Analyses were performed after 30 nsec f. Left, middle: our TPP-bound EmrE structure (left) and a representative snapshot from MD simulations starting from this structure (middle), where Glu14 of monomer A is deprotonated and Glu14 of monomer B is protonated.Right: r.m.s.d. of TPP for the MD simulation plotted as a frequency distribution and calculated from the phosphorus atom after aligning the protein backbone to the starting structure.c.