Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR

Research output: Contribution to journalJournal articleResearchpeer-review

Simon Erlendsson, Kamil Gotfryd, Flemming Hofmann Larsen, Jonas Sigurd Mortensen, Michel-Andreas Geiger, Barth-Jan van Rossum, Hartmut Oschkinat, Ulrik Gether, Kaare Teilum, Claus Juul Løland

The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.

Original languageEnglish
Article numbere19314
JournaleLife
Volume6
Number of pages9
ISSN2050-084X
DOIs
Publication statusPublished - 2017

Number of downloads are based on statistics from Google Scholar and www.ku.dk


No data available

ID: 172467398