DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

Abstract

Neurotransmitter:sodium symporters (NSSs) 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. Results from many structural, functional, and computational studies on LeuT, a bacterial NSS homolog, have provided critical insight into the mechanism of sodium-coupled transport, but the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of molecular dynamics simulations, single-molecule fluorescence resonance energy transfer (smFRET) imaging, and measurements of Na+ binding and substrate transport that reveals an allosteric substrate specificity mechanism. In this mechanism, residues F259 and I359 in the substrate binding pocket couple the binding of substrate to Na+ release from the Na2 site by allosterically modulating the stability of a partially open, inward-facing state. We propose a model for transport selectivity in which residues F259 and I359 act as a volumetric sensor that inhibits the transport of bulky amino acids.

Authors:
 [1];  [1];  [1];  [1];  [2];  [2]; ORCiD logo [1]; ORCiD logo [1]
  1. Weill Cornell Medicine, New York, NY (United States)
  2. Columbia Univ., New York, NY (United States); New York State Psychiatric Inst. New York, NY (United States)
Publication Date:
Research Org.:
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). Oak Ridge Leadership Computing Facility; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). National Energy Research Scientific Computing Center (NERSC) (OLCF); Univ. of California, Oakland, CA (United States); UT-Battelle LLC/ORNL, Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Advanced Scientific Computing Research (ASCR); National Institutes of Health (NIH); National Science Foundation (NSF)
OSTI Identifier:
1565765
Grant/Contract Number:  
AC02-05CH11231; AC05-00OR22725; R21-MH099491; U54-GM087510; P01-DA012408; R01-DA041510; F31-DA035533; ACI-1053575; R01-GM116961
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 116; Journal Issue: 32; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES

Citation Formats

LeVine, Michael V., Terry, Daniel S., Khelashvili, George, Siegel, Zarek S., Quick, Matthias, Javitch, Jonathan A., Blanchard, Scott C., and Weinstein, Harel. The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor. United States: N. p., 2019. Web. doi:10.1073/pnas.1903020116.
LeVine, Michael V., Terry, Daniel S., Khelashvili, George, Siegel, Zarek S., Quick, Matthias, Javitch, Jonathan A., Blanchard, Scott C., & Weinstein, Harel. The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor. United States. https://doi.org/10.1073/pnas.1903020116
LeVine, Michael V., Terry, Daniel S., Khelashvili, George, Siegel, Zarek S., Quick, Matthias, Javitch, Jonathan A., Blanchard, Scott C., and Weinstein, Harel. Fri . "The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor". United States. https://doi.org/10.1073/pnas.1903020116. https://www.osti.gov/servlets/purl/1565765.
@article{osti_1565765,
title = {The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor},
author = {LeVine, Michael V. and Terry, Daniel S. and Khelashvili, George and Siegel, Zarek S. and Quick, Matthias and Javitch, Jonathan A. and Blanchard, Scott C. and Weinstein, Harel},
abstractNote = {Neurotransmitter:sodium symporters (NSSs) 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. Results from many structural, functional, and computational studies on LeuT, a bacterial NSS homolog, have provided critical insight into the mechanism of sodium-coupled transport, but the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of molecular dynamics simulations, single-molecule fluorescence resonance energy transfer (smFRET) imaging, and measurements of Na+ binding and substrate transport that reveals an allosteric substrate specificity mechanism. In this mechanism, residues F259 and I359 in the substrate binding pocket couple the binding of substrate to Na+ release from the Na2 site by allosterically modulating the stability of a partially open, inward-facing state. We propose a model for transport selectivity in which residues F259 and I359 act as a volumetric sensor that inhibits the transport of bulky amino acids.},
doi = {10.1073/pnas.1903020116},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 32,
volume = 116,
place = {United States},
year = {2019},
month = {7}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 4 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Thermodynamic Coupling Function Analysis of Allosteric Mechanisms in the Human Dopamine Transporter
journal, January 2018

  • LeVine, Michael V.; Cuendet, Michel A.; Razavi, Asghar M.
  • Biophysical Journal, Vol. 114, Issue 1
  • DOI: 10.1016/j.bpj.2017.10.030

Spontaneous Inward Opening of the Dopamine Transporter is Triggered by PIP2-Regulated Dynamics of the N-Terminus
journal, February 2016


X-ray structures of LeuT in substrate-free outward-open and apo inward-open states
journal, January 2012


Ultra-stable organic fluorophores for single-molecule research
journal, January 2014

  • Zheng, Qinsi; Juette, Manuel F.; Jockusch, Steffen
  • Chem. Soc. Rev., Vol. 43, Issue 4
  • DOI: 10.1039/c3cs60237k

Simple Allosteric Model for Membrane Pumps
journal, August 1966


Scalable molecular dynamics with NAMD
journal, January 2005

  • Phillips, James C.; Braun, Rosemary; Wang, Wei
  • Journal of Computational Chemistry, Vol. 26, Issue 16, p. 1781-1802
  • DOI: 10.1002/jcc.20289

Molecular Determinants for Substrate Interactions with the Glycine Transporter GlyT2
journal, November 2017


Spontaneous Inward Opening of the Dopamine Transporter Is Triggered by PIP 2 -Regulated Dynamics of the N-Terminus
journal, August 2015


Glu-311 in External Loop 4 of the Sodium/Proline Transporter PutP Is Crucial for External Gate Closure
journal, January 2016

  • Bracher, Susanne; Guérin, Kamila; Polyhach, Yevhen
  • Journal of Biological Chemistry, Vol. 291, Issue 10
  • DOI: 10.1074/jbc.M115.675306

The Allostery Landscape: Quantifying Thermodynamic Couplings in Biomolecular Systems
journal, November 2016

  • Cuendet, Michel A.; Weinstein, Harel; LeVine, Michael V.
  • Journal of Chemical Theory and Computation, Vol. 12, Issue 12
  • DOI: 10.1021/acs.jctc.6b00841

Computational approaches to detect allosteric pathways in transmembrane molecular machines
journal, July 2016

  • Stolzenberg, Sebastian; Michino, Mayako; LeVine, Michael V.
  • Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1858, Issue 7
  • DOI: 10.1016/j.bbamem.2016.01.010

The role of transmembrane segment 5 (TM5) in Na2 release and the conformational transition of neurotransmitter:sodium symporters toward the inward-open state
journal, March 2017

  • Stolzenberg, Sebastian; Li, Zheng; Quick, Matthias
  • Journal of Biological Chemistry, Vol. 292, Issue 18
  • DOI: 10.1074/jbc.M116.757153

Chloride binding site of neurotransmitter sodium symporters
journal, May 2013

  • Kantcheva, A. K.; Quick, M.; Shi, L.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 21
  • DOI: 10.1073/pnas.1221279110

High-Affinity Adaptors for Switchable Recognition of Histidine-Tagged Proteins
journal, July 2005

  • Lata, Suman; Reichel, Annett; Brock, Roland
  • Journal of the American Chemical Society, Vol. 127, Issue 29
  • DOI: 10.1021/ja050690c

Allosteric modulation of human dopamine transporter activity under conditions promoting its dimerization
journal, June 2017

  • Cheng, Mary Hongying; Garcia-Olivares, Jennie; Wasserman, Steven
  • Journal of Biological Chemistry, Vol. 292, Issue 30
  • DOI: 10.1074/jbc.M116.763565

Unlocking the molecular secrets of sodium-coupled transporters
journal, May 2009

  • Krishnamurthy, Harini; Piscitelli, Chayne L.; Gouaux, Eric
  • Nature, Vol. 459, Issue 7245
  • DOI: 10.1038/nature08143

How structural elements evolving from bacterial to human SLC6 transporters enabled new functional properties
journal, March 2018


Identification of a second substrate-binding site in solute-sodium symporters.
journal, March 2015

  • Li, Zheng; Lee, Ashely S. E.; Bracher, Susanne
  • Journal of Biological Chemistry, Vol. 290, Issue 12
  • DOI: 10.1074/jbc.a114.584383

Single-molecule imaging of non-equilibrium molecular ensembles on the millisecond timescale
journal, February 2016

  • Juette, Manuel F.; Terry, Daniel S.; Wasserman, Michael R.
  • Nature Methods, Vol. 13, Issue 4
  • DOI: 10.1038/nmeth.3769

Anton, a special-purpose machine for molecular dynamics simulation
conference, January 2007

  • Shaw, David E.; Chao, Jack C.; Eastwood, Michael P.
  • Proceedings of the 34th annual international symposium on Computer architecture - ISCA '07
  • DOI: 10.1145/1250662.1250664

Neurotransmitter transporters as molecular targets for addictive drugs
journal, June 1998


Unbiased Simulations Reveal the Inward-Facing Conformation of the Human Serotonin Transporter and Na+ Ion Release
journal, October 2011


Conformational transitions of the sodium-dependent sugar transporter, vSGLT
journal, March 2018

  • Paz, Aviv; Claxton, Derek P.; Kumar, Jay Prakash
  • Proceedings of the National Academy of Sciences, Vol. 115, Issue 12
  • DOI: 10.1073/pnas.1718451115

The bright future of single-molecule fluorescence imaging
journal, June 2014

  • Juette, Manuel F.; Terry, Daniel S.; Wasserman, Michael R.
  • Current Opinion in Chemical Biology, Vol. 20
  • DOI: 10.1016/j.cbpa.2014.05.010

Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters
journal, July 2005

  • Yamashita, Atsuko; Singh, Satinder K.; Kawate, Toshimitsu
  • Nature, Vol. 437, Issue 7056
  • DOI: 10.1038/nature03978

Ion-Controlled Conformational Dynamics in the Outward-Open Transition from an Occluded State of LeuT
journal, September 2012


X-ray structures of Drosophila dopamine transporter in complex with nisoxetine and reboxetine
journal, May 2015

  • Penmatsa, Aravind; Wang, Kevin H.; Gouaux, Eric
  • Nature Structural & Molecular Biology, Vol. 22, Issue 6
  • DOI: 10.1038/nsmb.3029

A Competitive Inhibitor Traps LeuT in an Open-to-Out Conformation
journal, December 2008


Rotamers: To be or not to be?
journal, March 1993

  • Schrauber, Hannelore; Eisenhaber, Frank; Argos, Patrick
  • Journal of Molecular Biology, Vol. 230, Issue 2
  • DOI: 10.1006/jmbi.1993.1172

Common Folds and Transport Mechanisms of Secondary Active Transporters
journal, May 2013


Substrate-induced Unlocking of the Inner Gate Determines the Catalytic Efficiency of a Neurotransmitter:Sodium Symporter
journal, September 2015

  • Billesbølle, Christian B.; Krüger, Mie B.; Shi, Lei
  • Journal of Biological Chemistry, Vol. 290, Issue 44
  • DOI: 10.1074/jbc.M115.677658

Neurotransmitter and psychostimulant recognition by the dopamine transporter
journal, May 2015

  • Wang, Kevin H.; Penmatsa, Aravind; Gouaux, Eric
  • Nature, Vol. 521, Issue 7552
  • DOI: 10.1038/nature14431

The LeuT-fold neurotransmitter:sodium symporter MhsT has two substrate sites
journal, August 2018

  • Quick, Matthias; Abramyan, Ara M.; Wiriyasermkul, Pattama
  • Proceedings of the National Academy of Sciences, Vol. 115, Issue 34
  • DOI: 10.1073/pnas.1717444115

Anton, a special-purpose machine for molecular dynamics simulation
journal, June 2007

  • Shaw, David E.; Chao, Jack C.; Eastwood, Michael P.
  • ACM SIGARCH Computer Architecture News, Vol. 35, Issue 2
  • DOI: 10.1145/1273440.1250664

Conformational dynamics of ligand-dependent alternating access in LeuT
journal, April 2014

  • Kazmier, Kelli; Sharma, Shruti; Quick, Matthias
  • Nature Structural & Molecular Biology, Vol. 21, Issue 5
  • DOI: 10.1038/nsmb.2816

Detection of allosteric signal transmission by information-theoretic analysis of protein dynamics
journal, February 2012

  • Pandini, Alessandro; Fornili, Arianna; Fraternali, Franca
  • The FASEB Journal, Vol. 26, Issue 2
  • DOI: 10.1096/fj.11-190868

A Simple Method for Automated Equilibration Detection in Molecular Simulations
journal, March 2016


Update of the CHARMM All-Atom Additive Force Field for Lipids: Validation on Six Lipid Types
journal, June 2010

  • Klauda, Jeffery B.; Venable, Richard M.; Freites, J. Alfredo
  • The Journal of Physical Chemistry B, Vol. 114, Issue 23
  • DOI: 10.1021/jp101759q

Allosteric Mechanisms of Molecular Machines at the Membrane: Transport by Sodium-Coupled Symporters
journal, February 2016


Mechanism of chloride interaction with neurotransmitter:sodium symporters
journal, August 2007

  • Zomot, Elia; Bendahan, Annie; Quick, Matthias
  • Nature, Vol. 449, Issue 7163
  • DOI: 10.1038/nature06133

The Allostery Landscape: Quantifying Thermodynamic Couplings in Biomolecular Systems
journal, February 2017


Density‐functional thermochemistry. III. The role of exact exchange
journal, April 1993

  • Becke, Axel D.
  • The Journal of Chemical Physics, Vol. 98, Issue 7, p. 5648-5652
  • DOI: 10.1063/1.464913

Structural basis for recognition of diverse antidepressants by the human serotonin transporter
journal, January 2018


Comparative Protein Structure Modeling Using Modeller
journal, September 2006


A rapid, sensitive, and specific method for the determination of protein in dilute solution
journal, December 1973


X-ray structure of dopamine transporter elucidates antidepressant mechanism
journal, September 2013

  • Penmatsa, Aravind; Wang, Kevin H.; Gouaux, Eric
  • Nature, Vol. 503, Issue 7474
  • DOI: 10.1038/nature12533

Computational characterization of structural dynamics underlying function in active membrane transporters
journal, April 2015


Two Na + Sites Control Conformational Change in a Neurotransmitter Transporter Homolog
journal, November 2015

  • Tavoulari, Sotiria; Margheritis, Eleonora; Nagarajan, Anu
  • Journal of Biological Chemistry, Vol. 291, Issue 3
  • DOI: 10.1074/jbc.M115.692012

Identification of a Second Substrate-binding Site in Solute-Sodium Symporters
journal, November 2014

  • Li, Zheng; Lee, Ashley S. E.; Bracher, Susanne
  • Journal of Biological Chemistry, Vol. 290, Issue 1
  • DOI: 10.1074/jbc.M114.584383

A partially-open inward-facing intermediate conformation of LeuT is associated with Na+ release and substrate transport
journal, January 2018


Accurate and efficient integration for molecular dynamics simulations at constant temperature and pressure
journal, October 2013

  • Lippert, Ross A.; Predescu, Cristian; Ierardi, Douglas J.
  • The Journal of Chemical Physics, Vol. 139, Issue 16
  • DOI: 10.1063/1.4825247

Transport domain unlocking sets the uptake rate of an aspartate transporter
journal, February 2015

  • Akyuz, Nurunisa; Georgieva, Elka R.; Zhou, Zhou
  • Nature, Vol. 518, Issue 7537
  • DOI: 10.1038/nature14158

Coupled Global and Local Changes Direct Substrate Translocation by Neurotransmitter-Sodium Symporter Ortholog LeuT
journal, August 2013


Conformational cycle and ion-coupling mechanism of the Na+/hydantoin transporter Mhp1
journal, September 2014

  • Kazmier, K.; Sharma, S.; Islam, S. M.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 41
  • DOI: 10.1073/pnas.1410431111

Optimization of the Additive CHARMM All-Atom Protein Force Field Targeting Improved Sampling of the Backbone ϕ, ψ and Side-Chain χ 1 and χ 2 Dihedral Angles
journal, August 2012

  • Best, Robert B.; Zhu, Xiao; Shim, Jihyun
  • Journal of Chemical Theory and Computation, Vol. 8, Issue 9
  • DOI: 10.1021/ct300400x

Computation-guided analysis of paroxetine binding to hSERT reveals functionally important structural elements and dynamics
journal, December 2019


X-ray structures and mechanism of the human serotonin transporter
journal, April 2016

  • Coleman, Jonathan A.; Green, Evan M.; Gouaux, Eric
  • Nature, Vol. 532, Issue 7599
  • DOI: 10.1038/nature17629

Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue
journal, April 2011

  • Zhao, Yongfang; Terry, Daniel S.; Shi, Lei
  • Nature, Vol. 474, Issue 7349
  • DOI: 10.1038/nature09971

Single-molecule dynamics of gating in a neurotransmitter transporter homologue
journal, May 2010

  • Zhao, Yongfang; Terry, Daniel; Shi, Lei
  • Nature, Vol. 465, Issue 7295
  • DOI: 10.1038/nature09057

CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM Simulations Using the CHARMM36 Additive Force Field
journal, December 2015

  • Lee, Jumin; Cheng, Xi; Swails, Jason M.
  • Journal of Chemical Theory and Computation, Vol. 12, Issue 1
  • DOI: 10.1021/acs.jctc.5b00935

Monitoring the function of membrane transport proteins in detergent-solubilized form
journal, February 2007

  • Quick, M.; Javitch, J. A.
  • Proceedings of the National Academy of Sciences, Vol. 104, Issue 9
  • DOI: 10.1073/pnas.0609573104

Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer
journal, February 2017

  • Adhikary, Suraj; Deredge, Daniel J.; Nagarajan, Anu
  • Proceedings of the National Academy of Sciences, Vol. 114, Issue 10
  • DOI: 10.1073/pnas.1613293114

Comparative Protein Structure Modeling Using MODELLER
journal, November 2007

  • Eswar, Narayanan; Webb, Ben; Marti-Renom, Marc A.
  • Current Protocols in Protein Science, Vol. 50, Issue 1
  • DOI: 10.1002/0471140864.ps0209s50

State-dependent Conformations of the Translocation Pathway in the Tyrosine Transporter Tyt1, a Novel Neurotransmitter:Sodium Symporter from Fusobacterium nucleatum
journal, June 2006

  • Quick, Matthias; Yano, Hideaki; Goldberg, Naomi R.
  • Journal of Biological Chemistry, Vol. 281, Issue 36
  • DOI: 10.1074/jbc.M602438200

Canonical dynamics: Equilibrium phase-space distributions
journal, March 1985


Dynamical networks in tRNA:protein complexes
journal, April 2009

  • Sethi, A.; Eargle, J.; Black, A. A.
  • Proceedings of the National Academy of Sciences, Vol. 106, Issue 16
  • DOI: 10.1073/pnas.0810961106

A smooth particle mesh Ewald method
journal, November 1995

  • Essmann, Ulrich; Perera, Lalith; Berkowitz, Max L.
  • The Journal of Chemical Physics, Vol. 103, Issue 19
  • DOI: 10.1063/1.470117

An Intracellular Interaction Network Regulates Conformational Transitions in the Dopamine Transporter
journal, April 2008

  • Kniazeff, Julie; Shi, Lei; Loland, Claus J.
  • Journal of Biological Chemistry, Vol. 283, Issue 25
  • DOI: 10.1074/jbc.M800475200

Residues crucial for maintaining short paths in network communication mediate signaling in proteins
journal, January 2006

  • del Sol, Antonio; Fujihashi, Hirotomo; Amoros, Dolors
  • Molecular Systems Biology, Vol. 2, Issue 1
  • DOI: 10.1038/msb4100063

Ab Initio Calculation of Vibrational Absorption and Circular Dichroism Spectra Using Density Functional Force Fields
journal, November 1994

  • Stephens, P. J.; Devlin, F. J.; Chabalowski, C. F.
  • The Journal of Physical Chemistry, Vol. 98, Issue 45, p. 11623-11627
  • DOI: 10.1021/j100096a001

Neurotransmitter transporters and their impact on the development of psychopharmacology: Neurotransmitter transporters
journal, January 2006


The Role of Local Hydration and Hydrogen-Bonding Dynamics in Ion and Solute Release from Ion-Coupled Secondary Transporters
journal, March 2011

  • Zhao, Chunfeng; Noskov, Sergei Yu.
  • Biochemistry, Vol. 50, Issue 11
  • DOI: 10.1021/bi101454f

VMD: Visual molecular dynamics
journal, February 1996


A Markov State-based Quantitative Kinetic Model of Sodium Release from the Dopamine Transporter
journal, January 2017

  • Razavi, Asghar M.; Khelashvili, George; Weinstein, Harel
  • Scientific Reports, Vol. 7, Issue 1
  • DOI: 10.1038/srep40076

Substrate-dependent proton antiport in neurotransmitter:sodium symporters
journal, December 2009

  • Zhao, Yongfang; Quick, Matthias; Shi, Lei
  • Nature Chemical Biology, Vol. 6, Issue 2
  • DOI: 10.1038/nchembio.284

Anton, a special-purpose machine for molecular dynamics simulation
journal, July 2008

  • Shaw, David E.; Chao, Jack C.; Eastwood, Michael P.
  • Communications of the ACM, Vol. 51, Issue 7
  • DOI: 10.1145/1364782.1364802

Ultra-stable organic fluorophores for single-molecule research
journal, January 2014

  • Zheng, Qinsi; Juette, Manuel F.; Jockusch, Steffen
  • Chem. Soc. Rev., Vol. 43, Issue 4
  • DOI: 10.1039/C3CS60237K

Detection of Allosteric Signal Transmission by Information-Theoretic Analysis of Protein Dynamics
journal, January 2012

  • Pandini, Alessandro; Fornili, Arianna; Fraternali, Franca
  • Biophysical Journal, Vol. 102, Issue 3
  • DOI: 10.1016/j.bpj.2011.11.1237

Works referencing / citing this record:

The Amino Terminus of LeuT Changes Conformation in an Environment Sensitive Manner
journal, December 2019


Transmembrane helix 6b links proton and metal release pathways and drives conformational change in an Nramp-family transition metal transporter
journal, December 2019

  • Bozzi, Aaron T.; McCabe, Anne L.; Barnett, Benjamin C.
  • Journal of Biological Chemistry, Vol. 295, Issue 5
  • DOI: 10.1074/jbc.ra119.011336

Transmembrane helix 6b links proton and metal release pathways and drives conformational change in an Nramp-family transition metal transporter
journal, January 2020