Mechanism of the association between Na+ binding and conformations at the intracellular gate in neurotransmitter:sodium symporters

Stolzenberg, Sebastian; Quick, Matthias; Zhao, Chunfeng; Gotfryd, Kamil; Khelashvili, George; Gether, Ulrik; Loland, Claus J.; Javitch, Jonathan A.; Noskov, Sergei; Weinstein, Harel; Shi, Lei

doi:10.1074/jbc.m114.625343

Mechanism of the association between Na⁺ binding and conformations at the intracellular gate in neurotransmitter:sodium symporters

Journal Article · Mon Apr 13 00:00:00 EDT 2015 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.m114.625343· OSTI ID:1348222

Stolzenberg, Sebastian ^[1]; Quick, Matthias ^[2]; Zhao, Chunfeng ^[3]; Gotfryd, Kamil ^[4]; Khelashvili, George ^[5]; Gether, Ulrik ^[4]; Loland, Claus J. ^[4]; Javitch, Jonathan A. ^[2]; Noskov, Sergei ^[3]; Weinstein, Harel ^[5]; Shi, Lei ^[6]

Weill Medical College of Cornell Univ., New York, NY (United States); Cornell Univ., Ithaca, NY (United States); DOE Office of Scientific and Technical Information (OSTI)
Columbia Univ. College of Physicians and Surgeons, New York, NY (United States); New York State Psychiatric Institute, New York, NY (United States)
Univ. of Calgary, Calgary, AB (Canada)
Univ. of Copenhagen, Copenhagen (Denmark)
Weill Medical College of Cornell Univ., New York, NY (United States)
Weill Medical College of Cornell Univ., New York, NY (United States); National Institutes of Health, Baltimore, MD (United States)

Neurotransmitter:sodium symporters (NSSs) terminate neurotransmission by Na⁺-dependent reuptake of released neurotransmitters. Previous studies suggested that Na⁺-binding reconfigures dynamically coupled structural elements in an allosteric interaction network (AIN) responsible for function-related conformational changes, but the intramolecular pathway of this mechanism has remained uncharted. Here we describe a new approach for the modeling and analysis of intramolecular dynamics in the bacterial NSS homolog LeuT. From microsecond-scale molecular dynamics simulations and cognate experimental verifications in both LeuT and human dopamine transporter (hDAT), we apply the novel method to identify the composition and the dynamic properties of their conserved AIN. In LeuT, two different perturbations disrupting Na+ binding and transport (i.e. replacing Na⁺ with Li⁺ or the Y268A mutation at the intracellular gate) affect the AIN in strikingly similar ways. In contrast, other mutations that affect the intracellular gate (i.e. R5A and D369A) do not significantly impair Na⁺ cooperativity and transport. Our analysis shows these perturbations to have much lesser effects on the AIN, underscoring the sensitivity of this novel method to the mechanistic nature of the perturbation. Notably, this set of observations holds as well for hDAT, where the aligned Y335A, R60A, and D436A mutations also produce different impacts on Na⁺ dependence. Furthermore, the detailed AIN generated from our method is shown to connect Na⁺ binding with global conformational changes that are critical for the transport mechanism. Lastly, that the AIN between the Na⁺ binding sites and the intracellular gate in bacterial LeuT resembles that in eukaryotic hDAT highlights the conservation of allosteric pathways underlying NSS function.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). Oak Ridge Leadership Computing Facility (OLCF); UT-Battelle, LLC, Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1348222

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 22 Vol. 290; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: English

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Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
allosteric regulation
dopamine transporter
metal ion-protein interaction
molecular dynamics
neurotransmitter transport
potential of mean force calculation