Journal Article

X-ray structures of LeuT in substrate-free outward-open and apo inward-open states

Gouaux, Eric, Krishnamurthy, Harini

2012

Overview

Neurotransmitter sodium symporters are integral membrane proteins that remove chemical transmitters from the synapse and terminate neurotransmission mediated by serotonin, dopamine, noradrenaline, glycine and GABA (γ-aminobutyric acid). Crystal structures of the bacterial homologue, LeuT, in substrate-bound outward-occluded and competitive inhibitor-bound outward-facing states have advanced our mechanistic understanding of neurotransmitter sodium symporters but have left fundamental questions unanswered. Here we report crystal structures of LeuT mutants in complexes with conformation-specific antibody fragments in the outward-open and inward-open states. In the absence of substrate but in the presence of sodium the transporter is outward-open, illustrating how the binding of substrate closes the extracellular gate through local conformational changes: hinge-bending movements of the extracellular halves of transmembrane domains 1, 2 and 6, together with translation of extracellular loop 4. The inward-open conformation, by contrast, involves large-scale conformational changes, including a reorientation of transmembrane domains 1, 2, 5, 6 and 7, a marked hinge bending of transmembrane domain 1a and occlusion of the extracellular vestibule by extracellular loop 4. These changes close the extracellular gate, open an intracellular vestibule, and largely disrupt the two sodium sites, thus providing a mechanism by which ions and substrate are released to the cytoplasm. The new structures establish a structural framework for the mechanism of neurotransmitter sodium symporters and their modulation by therapeutic and illicit substances. The X-ray crystal structure of LeuT, the bacterial homologue of the neurotransmitter sodium symporter family, is reported in the outward-open and inward-open states. Ins and outs of neurotransmitter channels Chemical neurotransmission in the central nervous system is terminated through re-uptake of neurotransmitters from the synapse into surrounding neuronal and glial cells. Chemical transmitters are removed from the synaptic cleft by neurotransmitter sodium symporters (NSSs). The X-ray crystal structures of LeuT, a bacterial homologue of the NSS family, have now been determined in the outward-open and inward-open states. The structure of the outward-open state illustrates how substrate binding leads to the closure of the extracellular gate. The switch to the inward-open form requires large-scale conformational changes in the membrane protein. These structures establish a framework for the mechanism of neurotransmitter sodium symporters and their modulation by therapeutic and illicit drugs.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/378/2587 / 631/378/548/1964 / 631/45/535 / 631/57/2272/2273 / Animals / Antibodies, Monoclonal - immunology / Antibody Specificity - immunology / Apoproteins - chemistry / Apoproteins - genetics / Apoproteins - immunology / Apoproteins - metabolism / Bacterial Proteins - chemistry / Bacterial Proteins - genetics / Bacterial Proteins - immunology / Bacterial Proteins - metabolism / Binding Sites / Biochemistry / Biological and medical sciences / Crystalline structure / Crystallography, X-Ray / Fundamental and applied biological sciences. Psychology / Humanities and Social Sciences / Immunoglobulin Fab Fragments - immunology / Ions - chemistry / Models, Molecular / Molecular biophysics / Movement / multidisciplinary / Mutation / Protein Conformation / Proteins / Science / Science (multidisciplinary) / Sodium / Sodium - chemistry / Sodium - metabolism / Structure in molecular biology / Structure-Activity Relationship / Substrate Specificity / Substrates