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The Substrate-Driven Transition to an Inward-Facing Conformation in the Functional Mechanism of the Dopamine Transporter

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  • Jufang Shan
  • Jonathan A Javitch
  • Lei Shi
  • Harel Weinstein

Abstract

Background: The dopamine transporter (DAT), a member of the neurotransmitter:Na+ symporter (NSS) family, terminates dopaminergic neurotransmission and is a major molecular target for psychostimulants such as cocaine and amphetamine, and for the treatment of attention deficit disorder and depression. The crystal structures of the prokaryotic NSS homolog of DAT, the leucine transporter LeuT, have provided critical structural insights about the occluded and outward-facing conformations visited during the substrate transport, but only limited clues regarding mechanism. To understand the transport mechanism in DAT we have used a homology model based on the LeuT structure in a computational protocol validated previously for LeuT, in which steered molecular dynamics (SMD) simulations guide the substrate along a pathway leading from the extracellular end to the intracellular (cytoplasmic) end. Methodology/Principal Findings: Key findings are (1) a second substrate binding site in the extracellular vestibule, and (2) models of the conformational states identified as occluded, doubly occupied, and inward-facing. The transition between these states involve a spatially ordered sequence of interactions between the two substrate-binding sites, followed by rearrangements in structural elements located between the primary binding site and the cytoplasmic end. These rearrangements are facilitated by identified conserved hinge regions and a reorganization of interaction networks that had been identified as gates. Conclusions/Significance: Computational simulations supported by information available from experiments in DAT and other NSS transporters have produced a detailed mechanistic proposal for the dynamic changes associated with substrate transport in DAT. This allosteric mechanism is triggered by the binding of substrate in the S2 site in the presence of the substrate in the S1 site. Specific structural elements involved in this mechanism, and their roles in the conformational transitions illuminated here describe, a specific substrate-driven allosteric mechanism that is directly amenable to experiment as shown previously for LeuT.

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  • Jufang Shan & Jonathan A Javitch & Lei Shi & Harel Weinstein, 2011. "The Substrate-Driven Transition to an Inward-Facing Conformation in the Functional Mechanism of the Dopamine Transporter," PLOS ONE, Public Library of Science, vol. 6(1), pages 1-15, January.
  • Handle: RePEc:plo:pone00:0016350
    DOI: 10.1371/journal.pone.0016350
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    References listed on IDEAS

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    1. Heidi Koldsø & Pernille Noer & Julie Grouleff & Henriette Elisabeth Autzen & Steffen Sinning & Birgit Schiøtt, 2011. "Unbiased Simulations Reveal the Inward-Facing Conformation of the Human Serotonin Transporter and Na+ Ion Release," PLOS Computational Biology, Public Library of Science, vol. 7(10), pages 1-14, October.
    2. Mariarosaria Ferraro & Matteo Masetti & Maurizio Recanatini & Andrea Cavalli & Giovanni Bottegoni, 2016. "Mapping Cholesterol Interaction Sites on Serotonin Transporter through Coarse-Grained Molecular Dynamics," PLOS ONE, Public Library of Science, vol. 11(12), pages 1-24, December.
    3. Michael V LeVine & Harel Weinstein, 2014. "NbIT - A New Information Theory-Based Analysis of Allosteric Mechanisms Reveals Residues that Underlie Function in the Leucine Transporter LeuT," PLOS Computational Biology, Public Library of Science, vol. 10(5), pages 1-15, May.

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