Asymmetric protonation of glutamate residues drives a preferred transport pathway in EmrE.

EmrE is an Escherichia coli multidrug efflux pump and member of the small multidrug resistance (SMR) family that transports drugs as a homodimer by harnessing energy from the proton motive force. SMR family transporters contain a conserved glutamate residue in transmembrane 1 (Glu14 in EmrE) that is required for binding protons and drugs. Yet the mechanism underlying proton-coupled transport by the two glutamate residues in the dimer remains unresolved. Here, we used NMR spectroscopy to determine acid dissociation constants (pKa ) for wild-type EmrE and heterodimers containing one or two Glu14 residues in the dimer. For wild-type EmrE, we measured chemical shifts of the carboxyl side chain of Glu14 using solid-state NMR in lipid bilayers and obtained unambiguous evidence on the existence of asymmetric protonation states. Subsequent measurements of pKa values for heterodimers with a single Glu14 residue showed no significant differences from heterodimers with two Glu14 residues, supporting a model where the two Glu14 residues have independent pKa values and are not electrostatically coupled. These insights support a transport pathway with well-defined protonation states in each monomer of the dimer, including a preferred cytoplasmic-facing state where Glu14 is deprotonated in monomer A and protonated in monomer B under pH conditions in the cytoplasm of E. coli Our findings also lead to a model, hop-free exchange, which proposes how exchangers with conformation-dependent pKa values reduce proton leakage. This model is relevant to the SMR family and transporters comprised of inverted repeat domains.

Site-specific resolution of anionic residues in proteins using solid-state NMR spectroscopy.

NMR spectroscopy is commonly used to infer site-specific acid dissociation constants (pKa) since the chemical shift is sensitive to the protonation state. Methods that probe atoms nearest to the functional groups involved in acid/base chemistry are the most sensitive for determining the protonation state. In this work, we describe a magic-angle-spinning (MAS) solid-state NMR approach to measure chemical shifts on the side chain of the anionic residues aspartate and glutamate. This method involves a combination of double quantum spectroscopy in the indirect dimension and REDOR dephasing to provide a sensitive and resolved view of these amino acid residues that are commonly involved in enzyme catalysis and membrane protein transport. To demonstrate the applicability of the approach, we carried out measurements using a microcrystalline soluble protein (ubiquitin) and a membrane protein embedded in lipid bilayers (EmrE). Overall, the resolution available from the double quantum dimension and confidence in identification of aspartate and glutamate residues from the REDOR filter make this method the most convenient for characterizing protonation states and deriving pKa values using MAS solid-state NMR.

Compositional Analysis of Complex Mixtures using Automatic MicroED Data Collection.

Quantitative analysis of complex mixtures, including compounds having similar chemical properties, is demonstrated using an automatic and high throughput approach to microcrystal electron diffraction (MicroED). Compositional analysis of organic and inorganic compounds can be accurately executed without the need of diffraction standards. Additionally, with sufficient statistics, small amounts of compounds in mixtures can be reliably detected. These compounds can be distinguished by their crystal structure properties prior to structure solution. In addition, if the crystals are of good quality, the crystal structures can be generated on the fly, providing a complete analysis of the sample. MicroED is an effective method for analyzing the structural properties of sub-micron crystals, which are frequently found in small-molecule powders. By developing and using an automatic and high throughput approach to MicroED, and with the use of SerialEM for data collection, data from thousands of crystals allow sufficient statistics to detect even small amounts of compounds reliably.

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.

Bioinformatic and biochemical characterizations of C-S bond formation and cleavage enzymes in the fungus Neurospora crassa ergothioneine biosynthetic pathway.

Ergothioneine is a histidine thiol derivative. Its mycobacterial biosynthetic pathway has five steps (EgtA-E catalysis) with two novel reactions: a mononuclear nonheme iron enzyme (EgtB) catalyzed oxidative C-S bond formation and a PLP-mediated C-S lyase (EgtE) reaction. Our bioinformatic and biochemical analyses indicate that the fungus Neurospora crassa has a more concise ergothioneine biosynthetic pathway because its nonheme iron enzyme, Egt1, makes use of cysteine instead of γ-Glu-Cys as the substrate. Such a change of substrate preference eliminates the competition between ergothioneine and glutathione biosyntheses. In addition, we have identified the N. crassa C-S lyase (NCU11365) and reconstituted its activity in vitro, which makes the future ergothioneine production through metabolic engineering feasible.