Intrinsic conformational plasticity of native EmrE provides a pathway for multidrug resistance

Abstract

EmrE is a multidrug resistance efflux pump with specificity to a wide range of antibiotics and antiseptics. To obtain atomic-scale insight into the attributes of the native state that encodes the broad specificity, we used a hybrid of solution and solid-state NMR methods in lipid bilayers and bicelles. Our results indicate that the native EmrE dimer oscillates between inward and outward facing structural conformations at an exchange rate (k(ex)) of ~300 s(-1) at 37 °C (millisecond motions), which is ~50-fold faster relative to the tetraphenylphosphonium (TPP(+)) substrate-bound form of the protein. These observables provide quantitative evidence that the rate-limiting step in the TPP(+) transport cycle is not the outward-inward conformational change in the absence of drug. In addition, using differential scanning calorimetry, we found that the width of the gel-to-liquid crystalline phase transition was 2 °C broader in the absence of the TPP(+) substrate versus its presence, which suggested that changes in transporter dynamics can impact the phase properties of the membrane. Interestingly, experiments with cross-linked EmrE showed that the millisecond inward-open to outward-open dynamics was not the culprit of the broadening. Instead, the calorimetry and NMR data supported the conclusion that faster time scale structural dynamics (nanosecond-microsecond) were the source and therefore impart the conformationally plastic character of native EmrE capable of binding structurally diverse substrates. These findings provide a clear example how differences in membrane protein transporter structural dynamics between drug-free and bound states can have a direct impact on the physical properties of the lipid bilayer in an allosteric fashion.

Figure 1

Solution NMR spectra of EmrE in the native and substrate-bound forms at 45 °C. ¹H/¹⁵N TROSY-HSQC spectra for native and TPP⁺-bound EmrE are shown in panels A and C, respectively. The Ile methyl groups were imaged using ¹H/¹³C HMQC experiments in the native (B) and TPP⁺-bound states (D).

Figure 2

Native EmrE shows temperature-dependent splitting at backbone amide residues. Trp31 and Gly90 amide backbone cross-peaks of EmrE from ¹H/¹⁵N TROSY-HSQC NMR spectra acquired at several temperatures indicated within the figure. Red spectra correspond to native EmrE, while the black spectra are for the TPP⁺-bound form of the protein.

Figure 3

Temperature-dependent NMR spectra and activation energy. (A) ¹H/¹⁵N TROSY-HSQC spectra in DMPC/DHPC isotropic bicelles that highlight the Trp indole region of native (red) and TPP⁺-bound EmrE (black). (B) One-dimensional experimental (red) and fitted line shapes (blue) for the indole Trp31 residue of native EmrE. The fitted line shapes were obtained from a global fitting procedure that included all resolved residues displaying temperature-dependent peak splitting. (C) Arrhenius plot constructed from all exchange rates reported in SI Table I (i.e., solution NMR, oriented solid-state NMR, and MAS). (D) Model of the inward-open to outward-open conformational change that gives rise to two populations observed in EmrE.

Figure 4

Conformational exchange rate measured in oriented lipid bicelles. Integrals from 1D ¹⁵N PUREX on [U-¹⁵N] EmrE in DMPC/DHPC magnetically aligned bicelles at 37 °C in the (A) native and (B) TPP⁺-bound states. Note the difference in the x-axis between panels A and B. To illustrate this difference, the best fit to the TPP⁺ data is shown in a dashed line with the native protein results in panel A. The k_ex values were obtained by globally fitting PUREX results from two separately prepared samples (SI Figure 11) that gave best fits of 350 ± 60 and 6.5 ± 0.9 s^–1 for the native and TPP⁺-bound forms, respectively.

Figure 5

Tilt angle exchange observed by oriented solid-state NMR. (A) ¹⁵N/¹⁵N PDSD experiments for native and TPP⁺-bound EmrE labeled with [¹⁵N-Thr] at a mixing time of 75 ms and a temperature of 37 °C. The cross-peaks at 155 and 170 ppm have been tentatively assigned to Thr50, which is based on our Val34 assignment in TM2, known helical wheel geometries, and comparison with back-calculated PISEMA spectra from EmrE structural models. (B) TM1 of EmrE from 2I68(50) highlighting Thr18 and Thr19 and the corresponding changes in tilt angle with respect to the lipid bicelle that accompany the conformational exchange between the two populations. The tilt angles for the two TM1 helices of EmrE were calculated to be 16° and 33° relative to the membrane normal (SI Figure 8).

Figure 6

MAS exchange experiments in DMPC lipid bilayers. ¹³C/¹³C PDSD MAS exchange experiments on [¹³C_α,¹⁵N-Leu] EmrE in the native state at a mixing time of 500 ms. Due to the dilute ¹³C labeling of EmrE, the PDSD experiment serves as a ZZ-exchange experiment. The lack of cross-peaks at −22 °C is indicative that the large-scale conformational exchange has been quenched and the cross-peaks observed at 9 °C are not due to magnetization exchange..

Figure 7

Dynamic allostery and the impact on the lipid bilayer phase transition. (A) DSC thermograms for wild-type and CL-EmrE in the absence and presence of TPP⁺ in DMPC lipid bilayers at a lipid/monomer molar ratio of 100/1. Fitted parameters including the temperature, enthalpy, and half-height of the main phase transition are shown in SI Table II. No effect of TPP⁺ alone was observed on the melting profile of DMPC bilayers (SI Figure 12). (B) Model representation of the large- and small-scale conformational rearrangements of EmrE that highlight the faster nanosecond–microsecond motions as the primary driving mechanism of reduced bilayer melting cooperativity. The small-scale transitions persist for both EmrE and CL-EmrE in the absence of substrate. (C) ¹H/¹⁵N TROSY-HSQC spectra of the indole region of EmrE at 45 °C, which shows two populations for substrate-free CL-EmrE that supports halting of the millisecond time scale dynamics.