title: Spontaneous inward opening of the dopamine transporter is triggered by PIP2-regulated dynamics of the N-terminus.
abstract: We present the dynamic mechanism of concerted motions in a full-length molecular model of the human dopamine transporter (hDAT), a member of the neurotransmitter/sodium symporter (NSS) family, involved in state-to-state transitions underlying function. The findings result from an analysis of unbiased atomistic molecular dynamics simulation trajectories (totaling >14 μs) of the hDAT molecule immersed in lipid membrane environments with or without phosphatidylinositol 4,5-biphosphate (PIP2) lipids. The N-terminal region of hDAT (N-term) is shown to have an essential mechanistic role in correlated rearrangements of specific structural motifs relevant to state-to-state transitions in the hDAT. The mechanism involves PIP2-mediated electrostatic interactions between the N-term and the intracellular loops of the transporter molecule. Quantitative analyses of collective motions in the trajectories reveal that these interactions correlate with the inward-opening dynamics of hDAT and are allosterically coupled to the known functional sites of the transporter. The observed large-scale motions are enabled by specific reconfiguration of the network of ionic interactions at the intracellular end of the protein. The isomerization to the inward-facing state in hDAT is accompanied by concomitant movements in the extracellular vestibule and results in the release of an Na(+) ion from the Na2 site and destabilization of the substrate dopamine in the primary substrate binding S1 site. The dynamic mechanism emerging from the findings highlights the involvement of the PIP2-regulated interactions between the N-term and the intracellular loop 4 in the functionally relevant conformational transitions that are also similar to those found to underlie state-to-state transitions in the leucine transporter (LeuT), a prototypical bacterial homologue of the NSS.
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University (WCMC) , New York, New York 10065, United States.
authors.forename: George
authors.initials: G
authors.lastname: Khelashvili
authors.affiliation: Computational Biophysics Laboratory (GRIB-IMIM), Universitat Pompeu Fabra , Barcelona Biomedical Research Park (PRBB), C/Doctor Aiguader 88, 08003 Barcelona, Spain.
authors.forename: Nathaniel
authors.initials: N
authors.lastname: Stanley
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University (WCMC) , New York, New York 10065, United States.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Sahai
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University (WCMC) , New York, New York 10065, United States.
authors.forename: Jaime
authors.initials: J
authors.lastname: Medina
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University (WCMC) , New York, New York 10065, United States.
authors.forename: Michael V
authors.initials: MV
authors.lastname: LeVine
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University (WCMC) , New York, New York 10065, United States.|HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute of Computational Biomedicine, Weill Cornell Medical College of Cornell University , New York, New York 10065, United States.
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.affiliation: Computational Biophysics Laboratory (GRIB-IMIM), Universitat Pompeu Fabra , Barcelona Biomedical Research Park (PRBB), C/Doctor Aiguader 88, 08003 Barcelona, Spain.|Institució Catalana de Recerca i Estudis Avançats , Passeig Lluis Companys 23, 08010 Barcelona, Spain.
authors.forename: Gianni
authors.initials: G
authors.lastname: De Fabritiis
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University (WCMC) , New York, New York 10065, United States.|HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute of Computational Biomedicine, Weill Cornell Medical College of Cornell University , New York, New York 10065, United States.
authors.forename: Harel
authors.initials: H
authors.lastname: Weinstein
mesh_terms: D000494:Allosteric Regulation; D002412:Cations / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism*; D006801:Humans; D007536:Isomerism; D008567:Membranes, Artificial; D056004:Molecular Dynamics Simulation; D009038:Motion; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry; D017433:Protein Structure, Secondary; D012964:Sodium / Q000378:metabolism; D055672:Static Electricity
chemical_list: D002412:Cations; D050483:Dopamine Plasma Membrane Transport Proteins; D008567:Membranes, Artificial; D019269:Phosphatidylinositol 4,5-Diphosphate; C493214:SLC6A3 protein, human; D012964:Sodium
keywords: LeuT; NSS; Neurotransmitter transporter; allosteric coupling; alternating access; electrostatics; molecular dynamics simulation