Identification of a Second Substrate-binding Site in Solute-Sodium Symporters

• DOI: 10.1074/jbc.m114.584383
• Publication Date: 2014-11-15T07:02:25Z
• Authors: Zheng Li; Ashley S.E. Lee; Susanne Bracher; Heinrich Jung; Aviv Paz; Jay P. Kumar; Jeff Abramson; Matthias Quick; Lei Shi
• Keywords: Protein Folding; Binding Sites; Escherichia coli Proteins; Molecular Sequence Data; Sodium; Galactose; Biological Transport; Molecular Dynamics Simulation; Plasma Membrane Neurotransmitter Transport Proteins; Sodium-Glucose Transport Proteins; Protein Structure, Secondary; Recombinant Proteins; Protein Structure, Tertiary; Molecular Docking Simulation; Kinetics; Amino Acid Transport Systems, Neutral; Escherichia coli; Amino Acid Sequence; Sequence Alignment; Protein Binding

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.