Blocking dynamics of the SMR transporter EmrE impairs efflux activity. Biophys J 2014 Aug 05;107(3):613-620
Date
08/08/2014Pubmed ID
25099800Pubmed Central ID
PMC4129506DOI
10.1016/j.bpj.2014.06.030Scopus ID
2-s2.0-84905640039 (requires institutional sign-in at Scopus site) 17 CitationsAbstract
EmrE is a small multidrug resistance transporter that has been well studied as a model for secondary active transport. Because transport requires the protein to convert between at least two states open to opposite sides of the membrane, it is expected that blocking these conformational transitions will prevent transport activity. We have previously shown that NMR can quantitatively measure the transition between the open-in and open-out states of EmrE in bicelles. Now, we have used the antiparallel EmrE crystal structure to design a cross-link to inhibit this conformational exchange process. We probed the structural, dynamic, and functional effects of this cross-link with NMR and in vivo efflux assays. Our NMR results show that our antiparallel cross-link performs as predicted: dramatically reducing conformational exchange while minimally perturbing the overall structure of EmrE and essentially trapping EmrE in a single state. The same cross-link also impairs ethidium efflux activity by EmrE in Escherichia coli. This confirms the hypothesis that transport can be inhibited simply by blocking conformational transitions in a properly folded transporter. The success of our cross-linker design also provides further evidence that the antiparallel crystal structure provides a good model for functional EmrE.
Author List
Dutta S, Morrison EA, Henzler-Wildman KAAuthor
Emma A. Morrison PhD Assistant Professor in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceAntiporters
Biological Transport
Escherichia coli
Escherichia coli Proteins
Ethidium
Molecular Dynamics Simulation
Molecular Sequence Data
Protein Binding
