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Conformational changes in dopamine transporter intracellular regions upon cocaine binding and dopamine translocation.

ABSTRACT: The dopamine transporter (DAT), a member of the neurotransmitter:sodium symporter family, mediates the reuptake of dopamine at the synaptic cleft. DAT is the primary target for psychostimulants such as cocaine and amphetamine. We previously demonstrated that cocaine binding and dopamine transport alter the accessibility of Cys342 in the third intracellular loop (IL3). To study the conformational changes associated with the functional mechanism of the transporter, we made cysteine substitution mutants, one at a time, from Phe332 to Ser351 in IL3 of the background DAT construct, X7C, in which 7 endogenous cysteines were mutated. The accessibility of the 20 engineered cysteines to polar charged sulfhydryl reagents was studied in the absence and presence of cocaine or dopamine. Of the 11 positions that reacted with methanethiosulfonate ethyl ammonium, as evidenced by inhibition of ligand binding, 5 were protected against this inhibition by cocaine and dopamine (S333C, S334C, N336C, M342C and T349C), indicating that reagent accessibility is affected by conformational changes associated with inhibitor and substrate binding. In some of the cysteine mutants, transport activity is disrupted, but can be rescued by the presence of zinc, most likely because the distribution between inward- and outward-facing conformations is restored by zinc binding. The experimental data were interpreted in the context of molecular models of DAT in both the inward- and outward-facing conformations. Differences in the solvent accessible surface area for individual IL3 residues calculated for these states correlate well with the experimental accessibility data, and suggest that protection by ligand binding results from the stabilization of the outward-facing configuration. Changes in the residue interaction networks observed from the molecular dynamics simulations also revealed the critical roles of several positions during the conformational transitions. We conclude that the IL3 region of DAT undergoes significant conformational changes in transitions necessary for both cocaine binding and substrate transport.

SUBMITTER: Dehnes Y

PROVIDER: S-EPMC4058377 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Conformational changes in dopamine transporter intracellular regions upon cocaine binding and dopamine translocation.

Dehnes Yvette Y Shan Jufang J Beuming Thijs T Shi Lei L Weinstein Harel H Javitch Jonathan A JA

Neurochemistry international 20140224

The dopamine transporter (DAT), a member of the neurotransmitter:sodium symporter family, mediates the reuptake of dopamine at the synaptic cleft. DAT is the primary target for psychostimulants such as cocaine and amphetamine. We previously demonstrated that cocaine binding and dopamine transport alter the accessibility of Cys342 in the third intracellular loop (IL3). To study the conformational changes associated with the functional mechanism of the transporter, we made cysteine substitution mu ...[more]

PMID: 24576496

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Project description:Protein-protein interactions represent difficult but increasingly important targets for the design of therapeutic compounds able to interfere with biological processes. Recently, fragment-based strategies have been proposed as attractive approaches for the elaboration of protein-protein surface inhibitors from fragment-like molecules. One major challenge in targeting protein-protein interactions is related to the structural adaptation of the protein surface upon molecular recognition. Methods capable of identifying subtle conformational changes of proteins upon fragment binding are therefore required at the early steps of the drug design process. In this report we present a fast NMR method able to probe subtle conformational changes upon fragment binding. The approach relies on the comparison of experimental fragment-induced Chemical Shift Perturbation (CSP) of amine protons to CSP simulated for a set of docked fragment poses, considering the ring-current effect from fragment binding. We illustrate the method by the retrospective analysis of the complex between the anti-apoptotic Bcl-xL protein and the fragment 4'-fluoro-[1,1'-biphenyl]-4-carboxylic acid that was previously shown to bind one of the Bcl-xL hot spots. The CSP-based approach shows that the protein undergoes a subtle conformational rearrangement upon interaction, for residues located in helices [Formula: see text]2, [Formula: see text]3 and the very beginning of [Formula: see text]5. Our observations are corroborated by residual dipolar coupling measurements performed on the free and fragment-bound forms of the Bcl-xL protein. These NMR-based results are in total agreement with previous molecular dynamic calculations that evidenced a high flexibility of Bcl-xL around the binding site. Here we show that CSP of protein amine protons are useful and reliable structural probes. Therefore, we propose to use CSP simulation to assess protein conformational changes upon ligand binding in the fragment-based drug design approach.

Dataset Information

Conformational changes in dopamine transporter intracellular regions upon cocaine binding and dopamine translocation.

Publications

Conformational changes in dopamine transporter intracellular regions upon cocaine binding and dopamine translocation.

Similar Datasets

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