The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na+/sugar symport

Abstract

Membrane transporters that use energy stored in sodium gradients to drive nutrients into cells constitute a major class of proteins. We report the crystal structure of a member of the solute sodium symporters (SSS), the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT). The approximately 3.0 angstrom structure contains 14 transmembrane (TM) helices in an inward-facing conformation with a core structure of inverted repeats of 5 TM helices (TM2 to TM6 and TM7 to TM11). Galactose is bound in the center of the core, occluded from the outside solutions by hydrophobic residues. Surprisingly, the architecture of the core is similar to that of the leucine transporter (LeuT) from a different gene family. Modeling the outward-facing conformation based on the LeuT structure, in conjunction with biophysical data, provides insight into structural rearrangements for active transport.

Fig. 1

Structure of vSGLT. (A) Topology. The structure is colored as a rainbow from the N-terminus (red) to C-terminius (purple). The blue and red trapeziums represent the inverted topology of TM2-TM6 and TM7-TM11. The grey hexagon with red trim represents the galactose. Residues involved in sugar recognition, gate residues, and a proposed Na⁺ site are shown in cyan, gray, and yellow circles. (B) Structure viewed in the membrane plane. The coloring scheme and numbering of α helices is the same as in Fig1A. Bound galactose is shown as black and red spheres for the C and O atoms. The proposed Na⁺ ion is colored as a blue sphere. (C) Structure viewed from the intracellular side.

Fig. 1

Structure of vSGLT. (A) Topology. The structure is colored as a rainbow from the N-terminus (red) to C-terminius (purple). The blue and red trapeziums represent the inverted topology of TM2-TM6 and TM7-TM11. The grey hexagon with red trim represents the galactose. Residues involved in sugar recognition, gate residues, and a proposed Na⁺ site are shown in cyan, gray, and yellow circles. (B) Structure viewed in the membrane plane. The coloring scheme and numbering of α helices is the same as in Fig1A. Bound galactose is shown as black and red spheres for the C and O atoms. The proposed Na⁺ ion is colored as a blue sphere. (C) Structure viewed from the intracellular side.

Fig. 1

Structure of vSGLT. (A) Topology. The structure is colored as a rainbow from the N-terminus (red) to C-terminius (purple). The blue and red trapeziums represent the inverted topology of TM2-TM6 and TM7-TM11. The grey hexagon with red trim represents the galactose. Residues involved in sugar recognition, gate residues, and a proposed Na⁺ site are shown in cyan, gray, and yellow circles. (B) Structure viewed in the membrane plane. The coloring scheme and numbering of α helices is the same as in Fig1A. Bound galactose is shown as black and red spheres for the C and O atoms. The proposed Na⁺ ion is colored as a blue sphere. (C) Structure viewed from the intracellular side.

Fig. 2

Galactose binding site. (A) Overview of the galactose and proposed Na⁺-binding site viewed in the membrane plane maintaining the same color scheme as in Figure 1. (B) Hydrophobic gate residues (viewed from the extracellular side). The intracellular (Y263) and extracellular (M73, Y87 and F424) gates are shown as spheres, and the galactose is shown as sticks. (C) The galactose-binding site (same view as in B), with the extracellular hydrophobic gate residues removed to view the galactose-binding site. Residues in the galactose-binding site are displayed as sticks colored by atom type. Hydrogen bonds are depicted as black dashed lines. (D) D-galactose transport by vSGLT mutants in proteoliposomes. The results are expressed as % Control of that measured for p3C423 in 100 mM NaCl (~1.2 nmoles min⁻¹ mg⁻¹ protein). Standard error of the mean (SEM) is displayed for each experiment.

Fig. 2

Galactose binding site. (A) Overview of the galactose and proposed Na⁺-binding site viewed in the membrane plane maintaining the same color scheme as in Figure 1. (B) Hydrophobic gate residues (viewed from the extracellular side). The intracellular (Y263) and extracellular (M73, Y87 and F424) gates are shown as spheres, and the galactose is shown as sticks. (C) The galactose-binding site (same view as in B), with the extracellular hydrophobic gate residues removed to view the galactose-binding site. Residues in the galactose-binding site are displayed as sticks colored by atom type. Hydrogen bonds are depicted as black dashed lines. (D) D-galactose transport by vSGLT mutants in proteoliposomes. The results are expressed as % Control of that measured for p3C423 in 100 mM NaCl (~1.2 nmoles min⁻¹ mg⁻¹ protein). Standard error of the mean (SEM) is displayed for each experiment.

Fig. 2

Galactose binding site. (A) Overview of the galactose and proposed Na⁺-binding site viewed in the membrane plane maintaining the same color scheme as in Figure 1. (B) Hydrophobic gate residues (viewed from the extracellular side). The intracellular (Y263) and extracellular (M73, Y87 and F424) gates are shown as spheres, and the galactose is shown as sticks. (C) The galactose-binding site (same view as in B), with the extracellular hydrophobic gate residues removed to view the galactose-binding site. Residues in the galactose-binding site are displayed as sticks colored by atom type. Hydrogen bonds are depicted as black dashed lines. (D) D-galactose transport by vSGLT mutants in proteoliposomes. The results are expressed as % Control of that measured for p3C423 in 100 mM NaCl (~1.2 nmoles min⁻¹ mg⁻¹ protein). Standard error of the mean (SEM) is displayed for each experiment.

Fig. 2

Galactose binding site. (A) Overview of the galactose and proposed Na⁺-binding site viewed in the membrane plane maintaining the same color scheme as in Figure 1. (B) Hydrophobic gate residues (viewed from the extracellular side). The intracellular (Y263) and extracellular (M73, Y87 and F424) gates are shown as spheres, and the galactose is shown as sticks. (C) The galactose-binding site (same view as in B), with the extracellular hydrophobic gate residues removed to view the galactose-binding site. Residues in the galactose-binding site are displayed as sticks colored by atom type. Hydrogen bonds are depicted as black dashed lines. (D) D-galactose transport by vSGLT mutants in proteoliposomes. The results are expressed as % Control of that measured for p3C423 in 100 mM NaCl (~1.2 nmoles min⁻¹ mg⁻¹ protein). Standard error of the mean (SEM) is displayed for each experiment.

Fig. 3

The proposed Na⁺ binding site. (A) Residues in the Na⁺-binding site are displayed as sticks colored by atom type, and the helices are colored as in Figure 1. (B) Superposition of the proposed Na⁺ site of vSGLT on to the 2^nd Na⁺ site from the LeuT structure (green). Alignment was performed using only helices 2, and 9 from vSGLT, and the corresponding helices from LeuT.

Fig. 3

The proposed Na⁺ binding site. (A) Residues in the Na⁺-binding site are displayed as sticks colored by atom type, and the helices are colored as in Figure 1. (B) Superposition of the proposed Na⁺ site of vSGLT on to the 2^nd Na⁺ site from the LeuT structure (green). Alignment was performed using only helices 2, and 9 from vSGLT, and the corresponding helices from LeuT.

Fig. 4

Alternating accessibility. (A) Slice through surface of the outward-facing model viewed from the membrane plane showing the extracellular cavity (blue mesh). (B) Slice through surface of the inward-facing structure of vSGLT in the membrane plane showing the intracellular cavity (blue mesh). Helices showing structural rearrangement are colored orange, green and blue for helices TM3, TM7 and TM11; helices with little movements are colored white. The surface is shown in beige. Galactose is shown as black and red spheres for the C and O atoms, respectively. Y263 and the Na⁺ ion are colored as grey and blue sphere, respectively.