A Markov State-based Quantitative Kinetic Model of Sodium Release from the Dopamine Transporter

Razavi, Asghar M.; Khelashvili, George; Weinstein, Harel

doi:10.1038/srep40076

Cited by 67 publications

(146 citation statements)

References 74 publications

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“…In dDAT crystal structures, which are all in either an occluded or outward-facing open state, the F325 residue is in a perpendicular state when interacting with either the endogenous substrate dopamine or the inhibitor cocaine (53), while nisoxetine, a selective NET inhibitor in humans that also binds to and inhibits dDAT, contacts F325 with one of its rings to stabilize a parallel-like state of F325 (51). Regarding the possibility that crystallographic conditions could be responsible for the stabilization of F325 in a particular state in the dopamine-bound structure, similar to what we reasoned above based on the QM/MM calculations on the LeuT X-ray structures, we note that significant occupancy of the parallel state was also not seen in our extensive MD simulations of inward-opening in dDAT and the human homologue hDAT (13,14,35). This observation suggests that in DAT, dopamine does in fact stabilize the perpendicular state regardless of intracellular gating configuration.…”

Section: Discussionsupporting

confidence: 75%

“…These results suggest that Na + binds to LeuT-F259W only in the Na1 site, supporting our model that the IO 2 state corresponds to a conformation in which Na2 (Na + in the Na2 site) has been released (23). In light of our recent MSM and TCF analysis in hDAT that revealed an allosteric coupling between inward-opening and Na2 release (13,14,35), these results suggest that the stabilization of F259 in the parallel state promotes release of Na2 through stabilization of the inward-facing intermediate state.…”

Section: Mutations At Position 259 That Favor the Parallel Rotamer Alsupporting

confidence: 84%

“…As Ala demonstrates a substantially greater maximum velocity of transport (V max ) than Leu, we hypothesized that substrate-specific stabilization of the IO 2 state may trigger release of Na + from the Na2 site and allow for transport to proceed, whereas substrate-specific stabilization of the IC state may inhibit Na + release to the extent to which it becomes ratedetermining. This model is consistent with thermodynamic coupling function (TCF) (36) and Markov State Model analyses performed for the human dopamine transporter, which revealed an allosteric coupling between Na + release from the Na2 site and inwardopening (13,14,35).…”

Section: Introductionsupporting

confidence: 82%

“…In addition, we found that the Ala-stabilized IO 2 state was selectively destabilized by high concentrations of Na + , suggesting that this state corresponds to a conformation in which only one Na + binding site is occupied, while the other Na + has been released. Computational (14,17,35) and experimental (17) evidence suggests that it is the Na + in the Na2 site that is released, a process that is thought to be the obligatory first step of the substrate release mechanism. As Ala demonstrates a substantially greater maximum velocity of transport (V max ) than Leu, we hypothesized that substrate-specific stabilization of the IO 2 state may trigger release of Na + from the Na2 site and allow for transport to proceed, whereas substrate-specific stabilization of the IC state may inhibit Na + release to the extent to which it becomes ratedetermining.…”

Section: Introductionmentioning

confidence: 99%

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The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor

Levine¹,

Terry

Khelashvili³

et al. 2019

Preprint

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Neurotransmitter:sodium symporters (NSS) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. While a combination of structural, functional, and computational studies on LeuT, a bacterial NSS homolog, has provided critical insight into the mechanism of sodium-coupled transport, the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of MD simulations, single-molecule FRET imaging, and measurements of Na + binding and substrate transport that reveal an allosteric mechanism in which residues F259 and I359 in the substrate binding pocket couple substrate binding to Na + release from the Na2 site through allosteric modulation of the stability of a partially-open, inward-facing state. We propose a new model for transport selectivity in which the two residues act as a volumetric sensor that inhibits the transport of bulky amino acids. Kirschstein National Research Service Award F31 DA035533 to M.V.L. Computational results utilized in this work were carried out at resources of the Oak Ridge Leadership

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Section: Discussionsupporting

confidence: 75%

Section: Mutations At Position 259 That Favor the Parallel Rotamer Alsupporting

confidence: 84%

Section: Introductionsupporting

confidence: 82%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor

Levine¹,

Terry

Khelashvili³

et al. 2019

Preprint

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show abstract

“…choice of collective variables to discretize the conformational space, number of microstates, or time scales over which transitions are evaluated) has been reduced by machine-learning based automation [156]. Such schemes have provided insight into several biological processes, particular the folding of small proteins, but have only recently been applied to membrane transport [157]. As the time scales accessible to molecular dynamics simulations continue to increase, these methods could find numerous applications in the study of ion transport and other conformational changes associated with membrane protein gating and modulation.…”

Section: Introductionmentioning

confidence: 99%

Permeating disciplines: Overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport

Howard¹,

Carnevale²,

Delemotte³

et al. 2018

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Ion translocation across biological barriers is a fundamental requirement for life. In many cases, controlling this process-for example with neuroactive drugs-demands an understanding of rapid and reversible structural changes in membrane-embedded proteins, including ion channels and transporters. Classical approaches to electrophysiology and structural biology have provided valuable insights into several such proteins over macroscopic, often discontinuous scales of space and time. Integrating these observations into meaningful mechanistic models now relies increasingly on computational methods, particularly molecular dynamics simulations, while surfacing important challenges in data management and conceptual alignment. Here, we seek to provide contemporary context, concrete examples, and a look to the future for bridging disciplinary gaps in biological ion transport. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

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Membrane lipids are both the substrates and a mechanistically responsive environment of TMEM16 scramblase proteins

et al. 2019

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Recent discoveries about functional mechanisms of proteins in the TMEM16 family of phospholipid scramblases have illuminated the dual role of the membrane as both the substrate and a mechanistically responsive environment in the wide range of physiological processes and genetic disorders in which they are implicated. This is highlighted in the review of recent findings from our collaborative investigations of molecular mechanisms of TMEM16 scramblases that emerged from iterative functional, structural, and computational experimentation. In the context of this review, we present new MD simulations and trajectory analyses motivated by the fact that new structural information about the TMEM16 scramblases is emerging from cryo-EM determinations in lipid nanodiscs. Because the functional environment of these proteins in in vivo and in in vitro is closer to flat membranes, we studied comparatively the responses of the membrane to the TMEM16 proteins in flat membranes and nanodiscs. We find that bilayer shapes in the nanodiscs are very different from those observed in the flat membrane systems, but the function-related slanting of the membrane observed at the nhTMEM16 boundary with the protein is similar in the nanodiscs and in the flat bilayers. This changes, however, in the bilayer composed of longer-tail lipids, which is thicker near the phospholipid translocation pathway, which may reflect an enhanced tendency of the long tails to penetrate the pathway and create, as shown previously, a nonconductive environment. These findings support the correspondence between the mechanistic involvement of the lipid environment in the flat membranes, and the nanodiscs.

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A Markov State-based Quantitative Kinetic Model of Sodium Release from the Dopamine Transporter

Cited by 67 publications

References 74 publications

The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor

The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor

Permeating disciplines: Overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport

Membrane lipids are both the substrates and a mechanistically responsive environment of TMEM16 scramblase proteins

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