Structural basis for heavy metal detoxification by an Atm1-type ABC exporter

Abstract

Although substantial progress has been achieved in the structural analysis of exporters from the superfamily of adenosine triphosphate (ATP)-binding cassette (ABC) transporters, much less is known about how they selectively recognize substrates and how substrate binding is coupled to ATP hydrolysis. We have addressed these questions through crystallographic analysis of the Atm1/ABCB7/HMT1/ABCB6 ortholog from Novosphingobium aromaticivorans DSM 12444, NaAtm1, at 2.4 angstrom resolution. Consistent with a physiological role in cellular detoxification processes, functional studies showed that glutathione derivatives can serve as substrates for NaAtm1 and that its overexpression in Escherichia coli confers protection against silver and mercury toxicity. The glutathione binding site highlights the articulated design of ABC exporters, with ligands and nucleotides spanning structurally conserved elements to create adaptable interfaces accommodating conformational rearrangements during the transport cycle.

Fig. 1. Structural representations of NaAtm1

A) Ribbon diagram illustrating the dimeric structure of NaAtm1, viewed normal to the molecular two-fold axis with the membrane spanning domains and NBDs oriented towards the top and bottom, respectively. The approximate position of the membrane is designated by the gray bilayer, with the periplasmic and cytoplasmic facing surfaces towards the top and bottom, respectively. B) Binding sites for GSSG illustrated in the same orientation as (A), with the ligand depicted as space filling models, and the primary and secondary binding sites represented by green and yellow carbons, respectively. C) Representation of NaAtm1 emphasizing the secondary structure arrangement, based on the Sav1866 nomenclature (22). ICL denotes intracellular loop.

Fig. 2. In vitro and in vivo assays of NaAtm1 function

A) Kinetic constants for the ATPase activity of selected substrates derived from Michaelis-Menten type analysis, including the basal rate of ATPase activity. B) Amount of GSSG transported per mg of protein accumulated inside vesicles prepared from E. coli membranes overexpressing wild type or the E523Q Walker B motif ATPase defective mutant. C) Optical density of cells after 12-h growth in the presence of the indicated AgNO₃ concentrations. E. coli strain GG44 (Cu⁺/Ag⁺ sensitive) was transformed either with an empty pET15 plasmid, or a pET15 plasmid encoding NaAtm1. D) Optical density of cells after 12-h growth in the presence of the indicated HgCl₂ concentrations. E. coli strain GG48 (Zn²⁺/Cd²⁺/Hg²⁺ sensitive) was transformed as in (C). The error bars in Figs. 2B-D represent the standard deviations from three independent measurements.

Fig. 3. Binding sites of glutathione derivatives to NaAtm1

A) The primary binding site of GSSG, with the substrate and interacting side chains depicted as bonds and the polypeptide backbone in ribbons. The substrate carbons are colored light gray, while the protein side chain carbons are green. Hydrogen bonds between the protein and ligands are represented by yellow dashes. B) The secondary binding site for GSSG. C) Interactions between the γ-Glu of GSH and surrounding residues of NaAtm1. The carbon atoms of GSH are shaded light gray; while green carbons denote side chains directly interacting with GSH and blue carbons indicate side chains interacting with (green) residues interacting with GSH. D) The ATPase activity (k_cat) of selected mutations in the ligand binding site, with the rate constants in the absence of substrate and in the presence of 10 mM GSH. Error bars represent the standard deviation of 3 independent measurements.

Fig. 4. Structural conservation and the articulated construction of ABC exporters

A) Structural conservation of the individual TM1-2, TM3&6, and TM4-5 elements in the TMDs of ABC exporter structures. The NaAtm1 structure was used for the reference, with the TM1-2 and TM4-5 superpositions horizontally displaced from TM3&6 by 20 Å to the left and right, respectively. The GSSG ligand in the primary binding site is depicted by a space filling model with the carbons colored green. The underlying symmetry in the TMD, first noted in the Sav1866 structure (22), relates TM1-2-3 and TM4-5-6 by a two-fold rotation axis passing between TM3-6 and normal to the plane of the page. B) Variations in the relative orientations of conserved TMD elements observed in the structures of ABC exporters. The TMDs of different exporters were aligned with NaAtm1 using only TM3&6 for the superposition; for clarity, only TM3&6 from NaAtm1, depicted with black ribbons, is shown. Although the individual TM1-2 and TM4-5 elements are structurally conserved (Fig. 4A), significant changes in their relative positions are evident between these structures (especially TM4-5), reflecting tertiary structure changes associated with the transition between inward and outward facing conformations. The binding site for the GSSG ligand spans between these elements, serving to couple ligand binding to changes in TMD and transporter conformation. The Cα traces are colored purple (Sav1866; PDB 2HYD (22)), blue (TM287/288; PDB 3QF4 (24)), black (NaAtm1), green (ABCB10, PDB 4AYT (26)), yellow (mouse Pgp, PDB 3G5U (23)), orange (C. elegans Pgp, PDB 4F4C (25)), and red (MsbA, PDB 3B5W (27)), ordered from outward to inward facing conformations.