Inducing conformational preference of the membrane protein transporter EmrE through conservative mutations.

Transporters from bacteria to humans contain inverted repeat domains thought to arise evolutionarily from the fusion of smaller membrane protein genes. Association between these domains forms the functional unit that enables transporters to adopt distinct conformations necessary for function. The small multidrug resistance (SMR) family provides an ideal system to explore the role of mutations in altering conformational preference since transporters from this family consist of antiparallel dimers that resemble the inverted repeats present in larger transporters. Here, we show using NMR spectroscopy how a single conservative mutation introduced into an SMR dimer is sufficient to change the resting conformation and function in bacteria. These results underscore the dynamic energy landscape for transporters and demonstrate how conservative mutations can influence structure and function.

Magnetization transfer in liposome and proteoliposome samples that mimic the protein and lipid composition of myelin.

Although magnetization transfer (MT) has been widely used in brain MRI, for example in brain inflammation and multiple sclerosis, the detailed molecular origin of MT effects and the role that proteins play in MT remain unclear. In this work, a proteoliposome model system was used to mimic the myelin environment and to examine the roles of protein, cholesterol, brain cerebrosides, and sphingomyelin embedded in the liposome matrix. Exchange parameters were determined using a double-quantum filter experiment. The goal was to determine the relative contributions to exchange and MT of cerebrosides, sphingomyelin, cholesterol, and proteins in 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers. The main finding was that cerebrosides produced the strongest exchange effects, and that these were even more pronounced than those found for proteins. Sphingomyelin (which also has exchangeable groups at the head of the fatty acid chains, albeit closer to the lipid acyl chains) and cholesterol showed only minimal transfer. Overall, the extracted exchange rates appeared much smaller than commonly assumed for -OH and -NH groups.

Multiple frequency saturation pulses reduce CEST acquisition time for quantifying conformational exchange in biomolecules.

Exchange between conformational states is required for biomolecular catalysis, allostery, and folding. A variety of NMR experiments have been developed to quantify motional regimes ranging from nanoseconds to seconds. In this work, we describe an approach to speed up the acquisition of chemical exchange saturation transfer (CEST) experiments that are commonly used to probe millisecond to second conformational exchange in proteins and nucleic acids. The standard approach is to obtain CEST datasets through the acquisition of a series of 2D correlation spectra where each experiment utilizes a single saturation frequency to 1H, 15N or 13C. These pseudo 3D datasets are time consuming to collect and are further lengthened by reduced signal to noise stemming from the long saturation pulse. In this article, we show how usage of a multiple frequency saturation pulse (i.e., MF-CEST) changes the nature of data collection from series to parallel, and thus decreases the total acquisition time by an integer factor corresponding to the number of frequencies in the pulse. We demonstrate the applicability of MF-CEST on a Src homology 2 (SH2) domain from phospholipase Cγ and the secondary active transport protein EmrE as model systems by collecting 13C methyl and 15N backbone datasets. MF-CEST can also be extended to additional sites within proteins and nucleic acids. The only notable drawback of MF-CEST as applied to backbone 15N experiments occurs when a large chemical shift difference between the major and minor populations is present (typically greater than ~ 8 ppm). In these cases, ambiguity may arise between the chemical shift of the minor population and the multiple frequency saturation pulse. Nevertheless, this drawback does not occur for methyl group MF-CEST experiments or in cases where somewhat smaller chemical shift differences occur are present.

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.

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.

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 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 wild-type 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.

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 (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.

Regioselectivity of the oxidative C-S bond formation in ergothioneine and ovothiol biosyntheses.

Ergothioneine (5) and ovothiol (8) are two novel thiol-containing natural products. Their C-S bonds are formed by oxidative coupling reactions catalyzed by EgtB and OvoA enzymes, respectively. In this work, it was discovered that in addition to catalyzing the oxidative coupling between histidine and cysteine (1 → 6 conversion), OvoA can also catalyze a direct oxidative coupling between hercynine (2) and cysteine (2 → 4 conversion), which can shorten the ergothioneine biosynthetic pathway by two steps.