Identification of an Alternating-Access Dynamics Mutant of EmrE with Impaired Transport

Publication date: Available online 31 May 2019Source: Journal of Molecular BiologyAuthor(s): Chao Wu, Samantha A. Wynne, Nathan E. Thomas, Eva-Maria Uhlemann, Christopher G. Tate, Katherine A. Henzler-WildmanAbstractProteins that perform active transport must alternate the access of a binding site, first to one side of a membrane and then to the other, resulting in the transport of bound substrates across the membrane. To better understand this process, we sought to identify mutants of the small multidrug resistance transporter EmrE with reduced rates of alternating access. We performed extensive scanning mutagenesis by changing every amino acid residue either to Val, Ala or Gly, and then screening the drug resistance phenotypes of the resulting mutants. We identified EmrE mutants that had impaired transport activity but retained the ability to bind substrate and further tested their alternating access rates using NMR. Ultimately, we were able to identify a single mutation, S64 V, which significantly reduced the rate of alternating access but did not impair substrate binding. Six other transport-impaired mutants did not have reduced alternating access rates, highlighting the importance of other aspects of the transport cycle to achieve drug resistance activity in vivo. To better understand the transport cycle of EmrE, efforts are now underway to determine a high-resolution structure using the S64 V mutant identified here.Graphical abstract
Source: Journal of Molecular Biology - Category: Molecular Biology Source Type: research