Maureen Leninger

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

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 (kex) 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.

Protonation of a glutamate residue modulates the dynamics of the drug transporter EmrE

Secondary active transport proteins play a central role in conferring bacterial multidrug resistance. In this work, we investigated the proton-coupled transport mechanism for the Escherichia coli drug efflux pump EmrE using nuclear magnetic resonance (NMR) spectroscopy. Our results show that the global conformational motions necessary for transport are modulated in an allosteric fashion by the protonation state of a membrane-embedded glutamate residue. These observations directly correlate with the resistance phenotype for EmrE and the E14D mutant as a function of pH. Furthermore, our results support a model in which the pH gradient across the inner membrane of E. coli may be used on a mechanistic level to shift the equilibrium of the transporter in favor of an inward-open resting conformation poised for drug binding.

NMR Spectroscopy Approach to Study the Structure, Orientation, and Mechanism of the Multidrug Exporter EmrE

Multidrug exporters are a class of membrane proteins that remove antibiotics from the cytoplasm of bacteria and in the process confer multidrug resistance to the organism. This chapter outlines the sample preparation and optimization of oriented solid-state NMR experiments applied to the study of structure and dynamics for the model transporter EmrE from the small multidrug resistance (SMR) family.

Assessing Interactions Between a Polytopic Membrane Protein and Lipid Bilayers Using Differential Scanning Calorimetry and Solid-State NMR

It is known that the lipid composition within a cellular membrane can influence membrane protein structure and function. In this Article, we investigated how structural changes to a membrane protein upon substrate binding can impact the lipid bilayer. To carry out this study, we reconstituted the secondary active drug transporter EmrE into a variety of phospholipid bilayers varying in headgroup and chain length and carried out differential scanning calorimetry (DSC) and solid-state NMR experiments. The DSC results revealed a difference in cooperativity of the lipid phase transition for drug-free EmrE protonated at glutamic acid 14 (i.e., proton-loaded form) and the tetraphenylphosphonium (TPP+) bound form of the protein (i.e., drug-loaded form). To complement these findings, we acquired magic-angle-spinning (MAS) spectra in the presence and absence of TPP+ by directly probing the phospholipid headgroup using 31P NMR. These spectra showed a reduction in lipid line widths around the main phase transition for samples where EmrE was bound to TPP+ compared to the drug free form. Finally, we collected oriented solid-state NMR spectra on isotopically enriched EmrE that displayed chemical shift perturbations to both transmembrane and loop residues upon TPP+ binding. All of these results prompt us to propose a mechanism whereby substrate-induced changes to the structural dynamics of EmrE alters the surrounding lipids within the bilayer.