Glutathione binding to the plant At Atm3 transporter and implications for the conformational coupling of ABC transporters

Elife. 2022 Mar 25;11:e76140. doi: 10.7554/eLife.76140.

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.

Keywords: A. thaliana; ABC transporters; E. coli; alternating access; biochemistry; chemical biology; glutathione transport; molecular biophysics; structural biology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters* / metabolism
  • Adenosine Diphosphate / metabolism
  • Adenosine Triphosphate / metabolism
  • Arabidopsis* / metabolism
  • Binding Sites
  • Cryoelectron Microscopy
  • Glutathione / metabolism
  • Glutathione Disulfide / metabolism
  • Membrane Transport Proteins / metabolism
  • Protein Conformation

Substances

  • ATP-Binding Cassette Transporters
  • Membrane Transport Proteins
  • Adenosine Diphosphate
  • Adenosine Triphosphate
  • Glutathione
  • Glutathione Disulfide