Glutathione binding to the plant AtAtm3 transporter and implications for the conformational coupling of ABC transporters

Abstract

The ATP Binding Cassette (ABC) transporter of mitochondria (Atm) from Arabidopsis thaliana (AtAtm3) has been implicated in the maturation of cytosolic iron-sulfur proteins and heavy metal detoxification, plausibly by exporting glutathione derivatives. Using single-particle cryo-electron microscopy, we have determined four structures of AtAtm3 in three different conformational states: two inward-facing conformations (with and without bound oxidized glutathione (GSSG)), together with closed and outward-facing states stabilized by MgADP-VO4. These structures not only provide a structural framework for defining the alternating access transport cycle, but also reveal the paucity of cysteine residues in the glutathione binding site that could potentially form inhibitory mixed disulfides with GSSG. Despite extensive efforts, we were unable to prepare the ternary complex of AtAtm3 containing both GSSG and MgATP. A survey of structurally characterized type IV ABC transporters that includes AtAtm3 establishes that while nucleotides are found associated with all conformational states, they are effectively required to stabilize occluded, closed, and outward-facing conformations. In contrast, transport substrates have only been observed associated with inward-facing conformations. The absence of structures with dimerized nucleotide binding domains containing both nucleotide and transport substrate suggests that this form of the ternary complex exists only transiently during the transport cycle.

Structural Basis for Oxidized Glutathione Recognition by the Yeast Cadmium Factor 1

“…Within the P2 pocket, the glycyl carboxylate group of GSSG only forms a single hydrogen bond, pointing towards the possibility for different ligand conformation to be accommodated in the space. This binding mode contrasts significantly from known GSSG transporters like the plant Atm3, which contains multiple polar interactions on all four carboxylate groups of GSSG (25). However, unlike the homodimeric ATM-type GSH transporters that have symmetrical binding pockets, Ycf1 has an asymmetrical binding cavity that permits this bipartite selectivity mechanism in recognizing various substrates (25, 37).…”

“…This binding mode contrasts significantly from known GSSG transporters like the plant Atm3, which contains multiple polar interactions on all four carboxylate groups of GSSG (25). However, unlike the homodimeric ATM-type GSH transporters that have symmetrical binding pockets, Ycf1 has an asymmetrical binding cavity that permits this bipartite selectivity mechanism in recognizing various substrates (25,37). In this way, Ycf1 remains a specific transporter for GSH-adducted molecules while having the flexibility to transport different complexes.…”

“…There, GSH forms contacts in a highly basic pocket and interestingly, GSH binds to the equivalent of the H1 site. On the other hand, the plant mitochondrial transporter ATM3, which is a homodimer and thus has two equivalent halves, displays a perfectly symmetrical binding to both halves of GSSG, contrasting the biased binding mode of Ycf1 (35). Furthermore, the asymmetric ligand binding of GSSG-Ycf1 has been observed in certain ABC transporters where a single binding pocket can accommodate two of the same substrates in a manner like the two identical halves of GSSG.…”

“…Ycf1 purification was conducted as previously described with a slight modification in detergents (35). Briefly, harvested cell pellets were resuspended in cold lysis buffer (50mM Tris-Cl, 300mM NaCl, 2.5μM aprotinin, 2.5μM pepstatin, 6.25μM leupeptin, and 0.5mg/mL 4-benzenesulfonyl fluoride hydrochloride, pH 7.0) at a 3.2 mL/g of cell pellet ratio.…”

“…Besides MRP1, recent structures of GSH ABC transporters like those of ATM-type systems also inform our understanding of GSH and GSSG recognition mechanisms. As homodimers with a symmetrical binding cavity, substrate binding is driven by identical salt bridges formed on both halves of the transporter to the carboxylate ends of GSH or GSSG (25, 26). In contrast, Ycf1 is a monomeric polypeptide with an asymmetric binding cavity, hence, there may be potential differences in its interactions with GSH and GSSG.…”