Docking and homology modeling explain inhibition of the human vesicular glutamate transporters

Abstract

As membrane transporter proteins, VGLUT1-3 mediate the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells. This function is crucial for exocytosis and the role of glutamate as the major excitatory neurotransmitter in the central nervous system. The three transporters, sharing 76% amino acid sequence identity in humans, are highly homologous but differ in regional expression in the brain. Although little is known regarding their three-dimensional structures, hydropathy analysis on these proteins predicts 12 transmembrane segments connected by loops, a topology similar to other members in the major facilitator superfamily, where VGLUT1-3 have been phylogenetically classified. In this work, we present a three-dimensional model for the human VGLUT1 protein based on its distant bacterial homolog in the same superfamily, the glycerol-3-phosphate transporter from Escherichia coli. This structural model, stable during molecular dynamics simulations in phospholipid bilayers solvated by water, reveals amino acid residues that face its pore and are likely to affect substrate translocation. Docking of VGLUT1 substrates to this pore localizes two different binding sites, to which inhibitors also bind with an overall trend in binding affinity that is in agreement with previously published experimental data.

Figure 1.

Multiple sequence alignment between the three human vesicular glutamate transporters, VGLUT1–3, and the glycerol-3-phosphate transporter (GlpT) from E. coli. Solid lines mark the 12 predicted helical regions (H1–H12) and the predicted cytoplasmic loop in VGLUT 1–3. Gray tubes correspond to regions with transmembrane helices in the GlpT crystal structure, connected by cytoplasmic (convex) and periplasmic (concave) loops.

Figure 2.

(A) Proposed topology of VGLUT1 based on transmembrane segment prediction and topology of bacterial MFS proteins. Residues in filled black circles face the center of the pore that separates the N- and C-terminal domains in 3D. The 12 transmembrane helices in this and the following figures are colored according to the grouping of helices in the oxalate transporter (Hirai et al. 2002), based on their symmetrical positions in the structure. Green: straight-spanning peripheral helices that are not involved in defining the pore. Yellow and pink: curved helices, at the interface of the two domains, lining the pore. (B) Packing of the helices viewed from the cytoplasmic side. The 12 helices are numbered sequentially. (C) Cartoon diagram of the human VGLUT1 model. The 12 transmembrane helices form pseudo-symmetrical domains, separated by a pore (gray volume) that is open to the cytoplasmic side. Three arginine residues (R80 from helix 1, R176 from helix 4, and R314 from helix 7) that are exposed to the pore are shown in sticks. The highly variable first and last 60 residues of the N- and C-terminal are not shown.

Figure 3.

C-α RMSDs to the structure of the VGLUT1 model during molecular dynamics simulation (tm = transmembrane).

Figure 4.

(A) Surface display of the VGLUT1 model cut in a plane parallel to the vesicle membrane, with all eight charged residues situated within the hydrophobic region of the membrane displayed. (B–E) The same residues viewed before (green) and after (gray) molecular dynamics simulation. Helices before MD simulation are colored as in ▶, helices after 10 nsec are colored in gray.

Figure 5.

Strong azo dye inhibitors of VGLUT1 docked to the pore of the model (oriented as in ▶). The lowest docked energy conformations are shown. Diameters of different intersections of the pore are labeled along the 27 Å deep cavity. Three arginine residues from VGLUT1 model are shown in green. (A) Evans Blue and (B) Chicago Sky Blue interacting with the central binding site. (C) Trypan blue in the central binding site and (D) Trypan blue in the upper binding site.