Browse
Submit Data
Databases
API
Help

Dataset Information

22 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Analyzing conformational changes in the transport cycle of EmrE.

ABSTRACT: The small multidrug resistance transporters represent a unique model system for studying the mechanism of secondary active transport and membrane protein evolution. However, this seemingly simple protein has been highly controversial. Recent studies have provided experimental evidence that EmrE exists as an asymmetric dimer that exchanges between identical inward-facing and outward-facing states. Re-examination of the published literature in light of these findings fills in many details of the microscopic steps in the transport cycle. Future work will need to examine how the symmetry observed in vitro affects EmrE function in the asymmetric environment of its native Escherichia coli membrane.

SUBMITTER: Henzler-Wildman K

PROVIDER: S-EPMC3288627 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Analyzing conformational changes in the transport cycle of EmrE.

Henzler-Wildman Katherine K

Current opinion in structural biology 20111116 1

The small multidrug resistance transporters represent a unique model system for studying the mechanism of secondary active transport and membrane protein evolution. However, this seemingly simple protein has been highly controversial. Recent studies have provided experimental evidence that EmrE exists as an asymmetric dimer that exchanges between identical inward-facing and outward-facing states. Re-examination of the published literature in light of these findings fills in many details of the m ...[more]

PMID: 22100111

Similar Datasets

Electrostatic lock in the transport cycle of the multidrug resistance transporter EmrE.

Project description:EmrE is a small, homodimeric membrane transporter that exploits the established electrochemical proton gradient across the Escherichia coli inner membrane to export toxic polyaromatic cations, prototypical of the wider small-multidrug resistance transporter family. While prior studies have established many fundamental aspects of the specificity and rate of substrate transport in EmrE, low resolution of available structures has hampered identification of the transport coupling mechanism. Here we present a complete, refined atomic structure of EmrE optimized against available cryo-electron microscopy (cryo-EM) data to delineate the critical interactions by which EmrE regulates its conformation during the transport process. With the model, we conduct molecular dynamics simulations of the transporter in explicit membranes to probe EmrE dynamics under different substrate loading and conformational states, representing different intermediates in the transport cycle. The refined model is stable under extended simulation. The water dynamics in simulation indicate that the hydrogen-bonding networks around a pair of solvent-exposed glutamate residues (E14) depend on the loading state of EmrE. One specific hydrogen bond from a tyrosine (Y60) on one monomer to a glutamate (E14) on the opposite monomer is especially critical, as it locks the protein conformation when the glutamate is deprotonated. The hydrogen bond provided by Y60 lowers the [Formula: see text] of one glutamate relative to the other, suggesting both glutamates should be protonated for the hydrogen bond to break and a substrate-free transition to take place. These findings establish the molecular mechanism for the coupling between proton transfer reactions and protein conformation in this proton-coupled secondary transporter.

| S-EPMC6094130 | biostudies-literature

Conformational changes in the yeast mitochondrial ABC transporter Atm1 during the transport cycle.

Project description:[Figure: see text].

| S-EPMC8694623 | biostudies-literature

Structure, dynamics, and substrate-induced conformational changes of the multidrug transporter EmrE in liposomes.

Project description:EmrE, a member of the small multidrug transporters superfamily, extrudes positively charged hydrophobic compounds out of Escherichia coli cytoplasm in exchange for inward movement of protons down their electrochemical gradient. Although its transport mechanism has been thoroughly characterized, the structural basis of energy coupling and the conformational cycle mediating transport have yet to be elucidated. In this study, EmrE structure in liposomes and the substrate-induced conformational changes were investigated by systematic spin labeling and EPR analysis. Spin label mobilities and accessibilities describe a highly dynamic ligand-free (apo) conformation. Dipolar coupling between spin labels across the dimer reveals at least two spin label populations arising from different packing interfaces of the EmrE dimer. One population is consistent with antiparallel arrangement of the monomers, although the EPR parameters suggest deviations from the crystal structure of substrate-bound EmrE. Resolving these discrepancies requires an unusual disposition of TM3 relative to the membrane-water interface and a kink in its backbone that enables bending of its C-terminal part. Binding of the substrate tetraphenylphosphonium changes the environment of spin labels and their proximity in three transmembrane helices. The underlying conformational transition involves repacking of TM1, tilting of TM2, and changes in the backbone configurations of TM3 and the adjacent loop connecting it to TM4. A dynamic apo conformation is necessary for the polyspecificity of EmrE allowing the binding of structurally diverse substrates. The flexibility of TM3 may play a critical role in movement of substrates across the membrane.

| S-EPMC2924113 | biostudies-literature

New free-exchange model of EmrE transport.

Project description:EmrE is a small multidrug resistance transporter found in Escherichia coli that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton and drug. The functional consequence of this finding is an exceptionally promiscuous transporter: not only can EmrE export diverse drug substrates, it can couple antiport of a drug to either one or two protons, performing both electrogenic and electroneutral transport of a single substrate. We present a free-exchange model for EmrE antiport that is consistent with these results and recapitulates ?pH-driven concentrative drug uptake. Kinetic modeling suggests that free exchange by EmrE sacrifices coupling efficiency but boosts initial transport speed and drug release rate, which may facilitate efficient multidrug efflux.

| S-EPMC5703289 | biostudies-literature

Relative Principal Components Analysis: Application to Analyzing Biomolecular Conformational Changes.

Project description:A new method termed "Relative Principal Components Analysis" (RPCA) is introduced that extracts optimal relevant principal components to describe the change between two data samples representing two macroscopic states. The method is widely applicable in data-driven science. Calculating the components is based on a physical framework that introduces the objective function (the Kullback-Leibler divergence) appropriate for quantifying the change of the macroscopic state affected by the changes in the microscopic features. To demonstrate the applicability of RPCA, we analyze the thermodynamically relevant conformational changes of the protein HIV-1 protease upon binding to different drug molecules. In this case, the RPCA method provides a sound thermodynamic foundation for analyzing the binding process and thus characterizing both the collective and the locally relevant conformational changes. Moreover, the relevant collective conformational changes can be reconstructed from the informative latent variables to exhibit both the enhanced and the restricted conformational fluctuations upon ligand association.

| S-EPMC6728065 | biostudies-literature

Ligand-induced conformational changes of the multidrug resistance transporter EmrE probed by oriented solid-state NMR spectroscopy.

Project description: Not available

| S-EPMC3876743 | biostudies-literature

Protonation-dependent conformational dynamics of the multidrug transporter EmrE.

Project description:The small multidrug transporter from Escherichia coli, EmrE, couples the energetically uphill extrusion of hydrophobic cations out of the cell to the transport of two protons down their electrochemical gradient. Although principal mechanistic elements of proton/substrate antiport have been described, the structural record is limited to the conformation of the substrate-bound state, which has been shown to undergo isoenergetic alternating access. A central but missing link in the structure/mechanism relationship is a description of the proton-bound state, which is an obligatory intermediate in the transport cycle. Here we report a systematic spin labeling and double electron electron resonance (DEER) study that uncovers the conformational changes of EmrE subsequent to protonation of critical acidic residues in the context of a global description of ligand-induced structural rearrangements. We find that protonation of E14 leads to extensive rotation and tilt of transmembrane helices 1-3 in conjunction with repacking of loops, conformational changes that alter the coordination of the bound substrate and modulate its access to the binding site from the lipid bilayer. The transport model that emerges from our data posits a proton-bound, but occluded, resting state. Substrate binding from the inner leaflet of the bilayer releases the protons and triggers alternating access between inward- and outward-facing conformations of the substrate-loaded transporter, thus enabling antiport without dissipation of the proton gradient.

| S-EPMC4747756 | biostudies-literature

Resolution of angstrom-scale protein conformational changes by analyzing fluorescence anisotropy.

Project description:Conformational changes within typical protein molecules are rapid and small, making their quantitative resolution challenging. These changes generally involve rotational motions and may thus be resolved by determining changes in the orientation of a fluorescent label that assumes a unique orientation in each conformation. Here, by analyzing fluorescence intensities collected using a polarization microscope at a rate of 50 frames per second, we follow the changes of 10-16° in the orientation of a single bifunctional rhodamine molecule attached to a regulator of conductance to K+ (RCK) domain of the MthK channel, and thus, the transitions between its three conformational states, with effective standard deviation (?) of 2-5°. Based on available crystal structures, the position of the fluorophore's center differs by 3.4-8.1?Å among the states. Thus, the present approach allows the resolution of protein conformational changes involving ångström-scale displacements.

| S-EPMC6822697 | biostudies-literature

A mass spectrometry based transport assay for studying EmrE transport of unlabeled substrates.

Project description:Membrane transporters are an important class of proteins which remain challenging to study. Transport assays are crucial to developing our understanding of such proteins as they allow direct measurement of their transport activity. However, currently available methods for monitoring liposomal loading of organic substrates primarily rely on detection of radioactively or fluorescently labeled substrates. The requirement of a labeled substrate significantly restricts the systems and substrates that can be studied. Here we present a mass spectrometry based detection method for liposomal uptake assays that eliminates the need for labeled substrates. We demonstrate the efficacy of the assay with EmrE, a small multidrug resistance transporter found in E. coli that has become a model transport system for the study of secondary active transport. Furthermore, we develop a method for differentiation between bound and transported substrate, enhancing the information gained from the liposomal uptake assay. The transport assay presented here is readily applicable to other transport systems and substrates.

| S-EPMC5938110 | biostudies-literature

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

Project description: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.

| S-EPMC6805155 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data