The substrate-driven transition to an inward-facing conformation in the functional mechanism of the dopamine transporter

Abstract

Background: The dopamine transporter (DAT), a member of the neurotransmitter:Na(+) symporter (NSS) family, terminates dopaminergic neurotransmission and is a major molecular target for psychostimulants such as cocaine and amphetamine, and for the treatment of attention deficit disorder and depression. The crystal structures of the prokaryotic NSS homolog of DAT, the leucine transporter LeuT, have provided critical structural insights about the occluded and outward-facing conformations visited during the substrate transport, but only limited clues regarding mechanism. To understand the transport mechanism in DAT we have used a homology model based on the LeuT structure in a computational protocol validated previously for LeuT, in which steered molecular dynamics (SMD) simulations guide the substrate along a pathway leading from the extracellular end to the intracellular (cytoplasmic) end.

Methodology/principal findings: Key findings are (1) a second substrate binding site in the extracellular vestibule, and (2) models of the conformational states identified as occluded, doubly occupied, and inward-facing. The transition between these states involve a spatially ordered sequence of interactions between the two substrate-binding sites, followed by rearrangements in structural elements located between the primary binding site and the cytoplasmic end. These rearrangements are facilitated by identified conserved hinge regions and a reorganization of interaction networks that had been identified as gates.

Conclusions/significance: Computational simulations supported by information available from experiments in DAT and other NSS transporters have produced a detailed mechanistic proposal for the dynamic changes associated with substrate transport in DAT. This allosteric mechanism is triggered by the binding of substrate in the S2 site in the presence of the substrate in the S1 site. Specific structural elements involved in this mechanism, and their roles in the conformational transitions illuminated here describe, a specific substrate-driven allosteric mechanism that is directly amenable to experiment as shown previously for LeuT.

Figure 1. The substrate binding sites of DAT.

(A) S1,S2-DAT with DA in both the S1 and S2 sites, immersed in a lipid bilayer. The S1 site is located in the middle of the TM bundle and the S2 site is located ∼ 10 Å above the S1 site. (B) DA in the S1 site interacts with residues from TMs1, 3, 6 and 8 (viewing perspective is similar to that in (A)). (C) DA in the S2 site interacts mainly with residues from TMs1, 3 and 10, EL2, and EL4 (viewed from the exit of the extracellular vestibule).

Figure 2. The allosteric effect of S2 on the S1 site.

(A) Change in position of the DA substrate in the S1 site during equilibration of the S1,S2-DAT model. The green trace shows that the Z-coordinate of DA (center of mass) decreases by ∼ 1.5 Å (i.e., DA is shifted downward toward the intracellular side), compared to its position in S1-DAT (orange trace). (B) The positional changes of DA, the rotamer changes of F76^1.42, Y156^3.50 and F320^6.53, and the changes in the composition of the S1 site in S1-DAT (residues rendered in orange) compared to S1,S2-DAT (in green). Note that in S1,S2-DAT, the substrate interacts more (i.e., a higher percentage of time) with S149^3.43, V328^6.61, and G425^8.63, and establishes a new interaction with D421^8.59 (C_α atoms shown as orange spheres) while losing interactions with V152^3.46, G153^3.47 and A423^8.61 (C_α atom shown as green spheres). (C) The residues forming an interaction network involved in conformational transitions between S1-DAT (orange) and S1,S2-DAT (green). Residues I390^EL4 and F391^EL4 are in contact with DA in the S2 site. Note that changes in W84^1.50, L80^1.46, Y156^3.50 and F320^6.53 are coordinated with I390^EL4 and F391^EL4 (Table 1).

Figure 3. The S2-induced transition of DAT to an inward-open conformation is accompanied by water penetration.

(A, B, C) Waters (red balls) gradually penetrate to the S1 site, during the transition from S1-DAT (A) to S1,S2-DAT (B), then to the inward-facing conformation (C). (D) Average number of waters along the membrane normal (the z-axis; z = 0 is at the center of the membrane) in (A)–(C). The insert shows waters accumulated between −5 Å and +3 Å (z-axis) in the corresponding models identified by the colors. (E) DA in the S1 site interacts more favorably with waters in the presence of a substrate in the S2 site (green) than in the absence of a substrate in the S2 site (orange). (F) and (G) show magnified details of (A) and (B), respectively, with the same color coding and water in stick representations.

Figure 4. Conformational changes in the aromatic cluster during DA movement inward from the S1 site.

(A) The cluster of aromatic residues from TMs 1 and 6 shown in S1-DAT is important for conformational transitions; DA in the S1 site is rendered in stick representation. Orange dashed line indicates H-bond of Y335^6.68 to E428^8.66. (B) Time evolution of dihedrals and SASA (bottom panel) during inward pulling in SMD and MD alternation. Time points marked by the A, B, C, D, E, F arrows on the x-axis correspond to the structures shown in the (A)–(F) panels. The lines in the SASA plot are coded in colors corresponding to the residues names in the same colors. (C) The change in the rotamer of F76^1.42 from the conformation in S1-DAT (orange) to the configuration at the time point indicated by the C arrow in (B) (cyan), which allows the downward movement of DA. (D) The subsequent change in the rotamer of F332^6.65 (same color coding as in (C)) as DA moves to the position originally occupied by the sidechain of F332^6.65. (E) When DA is slowly pulled down a bit further in the SMD protocol, its amine forms a new H-bond with the carboxyl oxygen of D421^8.59 which coordinates Na2 in S1-DAT, and its hydroxyl groups forms H-bonds with the sidechain carboxyl group of E428^8.66. Time is indicated by the E arrow in (B). (F) The rotamer of Y335^6.68 changes last, breaking the H-bond between Y335^6.68 and E428^8.66. DA moves to the position originally occupied by the sidechain of Y335^6.68 and E428^8.66. Time is indicated by the F arrow in (B).

Figure 5. The global rearrangement from S1-DAT to the inward-facing conformation.

(A) The global rearrangement of TMs3, 4 and 8 at the extracellular side, from S1-DAT (orange) to the inward-facing conformation (cyan). Proteins are aligned with RMSDTT using the entire models. In the inward-facing conformation, TM3 residues F155^3.49, I159^3.53 and W162^3.56 (in sticks) are in direct contact with DA (in spheres, colored by atom type) in the S2 site. (B) Viewed from the intracellular side, the movement of the 12 intracellular TM segments (rendered in cartoon) can be partitioned into three groups as indicated by the dotted circles (see text). During the transition to the inward-facing conformation, the first group (colored in chocolate brown for S1-DAT and blue for the inward-facing conformation) moves outward; the second group (orange for S1-DAT and cyan for the inward-facing conformation) moves outward and away from the first group; the third group (yellow for S1-DAT and pale cyan for the inward-facing conformation) moves inward. DA is rendered in spheres and colored by atom types. (C) The global movement of selected TMs viewed from the intracellular side (top panel) and parallel to the membrane (bottom panel). During the transition, the extracellular ends of segments TM1b and TM6a do not move, while the intracellular end segments TMs1a and 6b swing away, non-symmetrically, from TMs 3 and 8 to open the substrate translocation pathway. TM1a moves substantially more, thereby distancing itself from TM6b.

Figure 6. Changes in intracellular interaction networks.

Y335^6.68 forms an H-bond with E428^8.66 in S1-DAT (A), and switches its H-bond partner to T62^1.27(NT) in the inward-facing conformation (B).

Figure 7. The endogenous Zn²⁺ binding site.

Extracellular portions of DAT containing the endogenous Zn²⁺ binding site. S1-DAT (orange) and the inward-facing DAT (cyan) are aligned with RMSDTT using the whole structure and rendered in cartoon. The sidechains of Zn²⁺ binding residues H375^EL4a and E396^EL4b are rendered in sticks. In S1-DAT, the average C_α distance between H375^EL4a and E396^El4b is 13 Å (orange dashed line). The distance increases to 15 Å in the inward-facing conformation (blue dashed line).

Figure 8. Cartoon model of a substrate translocation cycle for DAT.

Substrate binding in the outward-facing model (red) promotes the formation of an occluded conformation (orange). The binding of a second substrate (doubly occupied state in green) induces conformational changes in the S1 site and the intracellular side through conserved interaction networks (colored lozenges) positioned between the S2 and S1 sites, which reorganize the interaction network at the intracellular end, eventually leading to the release of substrate in the S1 site from the inward-facing conformation (cyan). Inhibitors that bind to the S1 site block the formation of S1,S2-DAT and thus the translocation. Inhibitors that bind to the S2 site inhibit the release of S1 and act as translocation de-couplers .