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J Biol Chem. 2015 Jan 2;290(1):127-41. doi: 10.1074/jbc.M114.584383. Epub 2014 Nov 14.

Identification of a second substrate-binding site in solute-sodium symporters.

Author information

1
From the Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, New York, New York 10065.
2
the Center for Molecular Recognition and Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York 10032.
3
the Ludwig Maximilian University of Munich, Biocentre, Microbiology, Grosshaderner Strasse 2-4, Martinsried, D-82152, Germany.
4
the Department of Physiology, UCLA, Los Angeles, California 90095.
5
the Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bellary Road, Bangalore-560065, Karnataka, India.
6
the Department of Physiology, UCLA, Los Angeles, California 90095, the Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bellary Road, Bangalore-560065, Karnataka, India.
7
the Center for Molecular Recognition and Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York 10032, the Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032, and mq2102@columbia.edu.
8
From the Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, New York, New York 10065, the Institute for Computational Biomedicine, Weill Cornell Medical College, Cornell University, New York, New York 10021 les2007@med.cornell.edu.

Abstract

The structure of the sodium/galactose transporter (vSGLT), a solute-sodium symporter (SSS) from Vibrio parahaemolyticus, shares a common structural fold with LeuT of the neurotransmitter-sodium symporter family. Structural alignments between LeuT and vSGLT reveal that the crystallographically identified galactose-binding site in vSGLT is located in a more extracellular location relative to the central substrate-binding site (S1) in LeuT. Our computational analyses suggest the existence of an additional galactose-binding site in vSGLT that aligns to the S1 site of LeuT. Radiolabeled galactose saturation binding experiments indicate that, like LeuT, vSGLT can simultaneously bind two substrate molecules under equilibrium conditions. Mutating key residues in the individual substrate-binding sites reduced the molar substrate-to-protein binding stoichiometry to ~1. In addition, the related and more experimentally tractable SSS member PutP (the Na(+)/proline transporter) also exhibits a binding stoichiometry of 2. Targeting residues in the proposed sites with mutations results in the reduction of the binding stoichiometry and is accompanied by severely impaired translocation of proline. Our data suggest that substrate transport by SSS members requires both substrate-binding sites, thereby implying that SSSs and neurotransmitter-sodium symporters share common mechanistic elements in substrate transport.

KEYWORDS:

Amino Acid Transport; Computational Biology; Equilibrium Binding; Galactose; Membrane Transport; Molecular Dynamics; Scintillation Proximity Assay; Solute-Sodium Symporter; Structural Alignment; Transporter

PMID:
25398883
PMCID:
PMC4281715
DOI:
10.1074/jbc.M114.584383
[Indexed for MEDLINE]
Free PMC Article

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