Journal Article

X-ray structure of LeuT in an inward-facing occluded conformation reveals mechanism of substrate release

Weinstein, Harel, Nissen, Poul, Mortensen, Jonas S., Khelashvili, George, Loland, Claus J., Gotfryd, Kamil, Boesen, Thomas, Blanchard, Scott C., Quick, Matthias, LeVine, Michael V., Gourdon, Pontus, Javitch, Jonathan A., Terry, Daniel S., Missel, Julie W., Gether, Ulrik

2020

Overview

Neurotransmitter:sodium symporters (NSS) are conserved from bacteria to man and serve as targets for drugs, including antidepressants and psychostimulants. Here we report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na + /substrate-bound, inward-facing occluded conformation. To obtain this structure, we were guided by findings from single-molecule fluorescence spectroscopy and molecular dynamics simulations indicating that L -Phe binding and mutation of the conserved N-terminal Trp8 to Ala both promote an inward-facing state. Compared to the outward-facing occluded conformation, our structure reveals a major tilting of the cytoplasmic end of transmembrane segment (TM) 5, which, together with release of the N-terminus but without coupled movement of TM1, opens a wide cavity towards the second Na + binding site. The structure of this key intermediate in the LeuT transport cycle, in the context of other NSS structures, leads to the proposal of an intracellular release mechanism of substrate and ions in NSS proteins. Neurotransmitter:sodium symporters (NSS) serve as targets for drugs including antidepressants and psychostimulants. Here authors report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na + /substrate-bound, inward-facing occluded conformation which is a key intermediate in the LeuT transport cycle.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 631/114 / 631/378 / 631/45 / 631/535 / 631/57 / 82/83 / Amino Acid Substitution / Antidepressants / Aquifex / Bacteria - chemistry / Bacteria - genetics / Bacteria - metabolism / Bacterial Proteins - chemistry / Bacterial Proteins - genetics / Bacterial Proteins - metabolism / Binding Sites / Conformation / Crystallography, X-Ray / Fluorescence / Fluorescence spectroscopy / Humanities and Social Sciences / Leucine - metabolism / Molecular dynamics / Molecular Dynamics Simulation / multidisciplinary / Mutagenesis, Site-Directed / Mutation / N-Terminus / Neurotransmitters / Plasma Membrane Neurotransmitter Transport Proteins - chemistry / Plasma Membrane Neurotransmitter Transport Proteins - genetics / Plasma Membrane Neurotransmitter Transport Proteins - metabolism / Protein Conformation / Science / Science & Technology - Other Topics / Science (multidisciplinary) / Sodium / Substrates / Transport