Fan and Rees. eLife 2022;11:e76140. DOI: https://doi.org/10.7554/eLife.76140
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Glutathione binding to the plant
At
Atm3 transporter and implications
for the conformational coupling of
ABC transporters
Chengcheng Fan
†
, Douglas C Rees*
Division of Chemistry and Chemical Engineering, Howard Hughes Medical Institute,
California Institute of Technology, Pasadena, United States
Abstract
The ATP binding cassette (ABC) transporter of mitochondria (Atm) from
Arabidopsis
thaliana
(
At
Atm3) 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
At
Atm3 in three different confor
-
mational states: two inward-
facing conformations (with and without bound oxidized glutathione
[GSSG]), together with closed and outward-
facing states stabilized by MgADP-
VO
4
. 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
At
Atm3 containing both GSSG and MgATP. A survey of structurally characterized
type IV ABC transporters that includes
At
Atm3 establishes that while nucleotides are found associ-
ated 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.
Editor's evaluation
Mitochondrial glutathione is an important line of defence against free radical production. The ATP
binding cassette (ABC) transporter Atm3 exports oxidized glutathione out of the mitochondria to
help maintain a suitable reducing environment. In this study, the authors have biochemically char
-
acterized Atm3 and determined four cryo-
EM structures exhibiting three different conformational
states, revealing new insights into the transport mechanism. This well-
executed study will be of
broad interest to the membrane biology and transport communities.
Introduction
The ATP binding cassette (ABC) transporter of mitochondria (Atm) family plays a vital (
Leighton and
Schatz, 1995
), but enigmatic, role broadly related to transition metal homeostasis in eukaryotes (
Lill
et al., 2014
). The best characterized member is
Saccharomyces cerevisiae
Atm1 (
Sc
Atm1) present in
the inner membrane of mitochondria (
Leighton and Schatz, 1995
) and required for the formation
of cytosolic iron-
sulfur cluster containing proteins (
Kispal et al., 1999
). Defects in
Sc
Atm1 lead to an
overaccumulation of iron in the mitochondria (
Kispal et al., 1997
). Atm1 is proposed to transport a
RESEARCH ARTICLE
*For correspondence:
dcrees@caltech.edu
Present address:
†
Division
of Biology and Biological
Engineering, California Institute
of Technology, Pasadena, United
States
Competing interest:
The authors
declare that no competing
interests exist.
Funding:
See page 14
Received:
06 December 2021
Preprinted:
14 December 2021
Accepted:
23 March 2022
Published:
25 March 2022
Reviewing Editor:
David Drew,
Stockholm University, Sweden
Copyright Fan and Rees. This
article is distributed under the
terms of the Creative Commons
Attribution License, which
permits unrestricted use and
redistribution provided that the
original author and source are
credited.
Research article
Biochemistry and Chemical Biology | Structural Biology and Molecular Biophysics
Fan and Rees. eLife 2022;11:e76140. DOI: https://doi.org/10.7554/eLife.76140
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sulfur containing intermediate (
Kispal et al., 1999
) that may also include iron (
Pandey et al., 2019
).
It is also likely to transport a similar sulfur containing species from the mitochondria that is required
for the cytoplasmic thiolation of tRNA (
Pandey et al., 2018
). While the precise substrate that is trans-
ported remains unknown, derivatives of glutathione have been implicated based on their ability to
stimulate the ATPase activity of Atm1 (
Kuhnke et al., 2006
).
Structures for Atm family members are currently available for
Sc
Atm1 (
Srinivasan et al., 2014
), the
bacterial homolog
Na
Atm1 from
Novosphingobium aromaticivorans
(
Lee et al., 2014
) and human
ABCB6 (
Wang et al., 2020
); the pairwise sequence identities between these homologous trans-
porters range from 40% to 46%. These proteins occur as homodimers of half-
transporters, where
each half-
transporter contains a transmembrane domain (TMD) followed by the canonical nucleotide
binding domain (NBD) that defines the ABC transporter family. Each TMD consists of six transmem-
brane helices (TMs) that exhibit the exporter type I fold first observed for Sav1866 (
Dawson and
Locher, 2006
); a recent re-
classification now identifies this group as type IV ABC transporters (
Thomas
et al., 2020
). The translocation of substrates across the membrane proceeds through an alternating
access mechanism involving the ATP-
dependent interconversion between inward- and outward-
facing
conformational states. Among the Atm1 family, these conformations have been most extensively char
-
acterized for
Na
Atm1 and include the occluded and closed states that provide a structural framework
for the unidirectional transport cycle (
Fan et al., 2020
). Structures of
Sc
Atm1 with reduced gluta-
thione (GSH) (
Srinivasan et al., 2014
), and of
Na
Atm1 complexed with reduced (GSH), oxidized
(GSSG), and metallated (GS-
Hg-
SG) (
Lee et al., 2014
), have defined the general substrate binding
site in the TMD for the transport substrates.
Plants have been found to have large numbers of transporters (
Hwang et al., 2016
), including
Arabidopsis
with three Atm orthologues,
At
Atm1,
At
Atm2, and
At
Atm3 (
Chen et al., 2007
). Of
these,
At
Atm3 (also known as ABCB25) rescues the
Sc
Atm1 phenotype (
Chen et al., 2007
), and has
been shown to be associated with maturation of cytosolic iron-
sulfur proteins (
Kushnir et al., 2001
),
confer resistance to heavy metals such as cadmium and lead (
Kim et al., 2006
), and participate in
the formation of molybdenum-
cofactor containing enzymes (
Bernard et al., 2009
;
Teschner et al.,
2010
). Unlike yeast, defects in
At
Atm3 are not associated with iron accumulation in mitochondria
(
Bernard et al., 2009
). While the physiological substrate is unknown,
At
Atm3 has been shown to
transport GSSG and glutathione polysulfide, with the persulfidated species perhaps relevant to cyto-
solic iron-
sulfur cluster assembly (
Schaedler et al., 2014
). The ability of
At
Atm3 to export GSSG has
been implicated in helping stabilize against excessive glutathione oxidation in the mitochondria and
thereby serving to maintain a suitable reduction potential (
Marty et al., 2019
).
To help address the functional role(s) of Atm transporters, we have determined structures of
At
Atm3 in multiple conformational states by single-
particle cryo-
electron microscopy (cryoEM). These
structures not only provide a structural framework for defining the alternating access transport cycle,
but they also illuminate an unappreciated feature of the glutathione binding site, namely the paucity
of cysteine residues that could potentially form inhibitory mixed disulfides during the transport cycle.
A survey of structurally characterized members of the type IV family of ABC transporters, including the
Atm1 family, establishes that nucleotides are effectively required for the stabilization of the occluded,
closed, and outward-
facing conformations. In contrast to the nucleotide states, transport substrates
and related inhibitors have only been observed associated with inward-
facing conformational states.
The absence of structures with dimerized NBDs containing both nucleotide and transport substrate
suggests that this form of the ternary complex exists only transiently during the transport cycle.
Results
At
Atm3 contains an N-
terminal mitochondrial targeting sequence that directs the translated protein to the
mitochondria, where it is cleaved following delivery to the inner membrane. Since this targeting sequence
consists of ~80 residues and is anticipated to be poorly ordered, we generated three different N-
terminal
truncation mutants of
At
Atm3 through deletion of 60, 70, or 80 residues to identify the best-
behaved
construct. Together with the wild-
type construct, these three variants were heterologously overexpressed
in
Escherichia coli
. The construct with the 80 amino acids deletion showed the highest expression level and
proportionally less aggregation by size exclusion chromatography (
Figure 1—figure supplement 1
) and
hence was used for further functional and structural studies.
Research article
Biochemistry and Chemical Biology | Structural Biology and Molecular Biophysics
Fan and Rees. eLife 2022;11:e76140. DOI: https://doi.org/10.7554/eLife.76140
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ATPase activities
Using the 80-
residue truncation construct,
At
Atm3 was purified in the detergent
n
- dodecyl-
β
-D-
maltoside (DDM) and reconstituted into nanodiscs formed from the membrane scaffolding protein
(MSP) 1D1 and the lipid 1-
palmitoyl-
2- oleoyl-
glycero-
3- phosphocholine (POPC). The ATPase activity
of this construct was measured as a function of MgATP concentration in the absence and presence of
either 2.5 mM GSSG or 10 mM GSH, which approximate their physiological concentrations in
E. coli
(
Bennett et al., 2009
). The rate of ATP hydrolysis was determined by measuring phosphate release
using a molybdate-
based colorimetric ATPase activity assay (
Chifflet et al., 1988
). The basal ATPase
activity, measured in the absence of glutathione derivatives, was significantly higher in detergent than
in nanodiscs (104 vs. 7.7 nmol/min/mg, respectively;
Figure 1ab
), while the apparent K
m
s for MgATP
were within a factor of two (~0.16 and 0.08 mM, respectively). The ATPase activity of
At
Atm3 is stim-
ulated by both 2.5 mM GSSG and 10 mM GSH, but the extent of stimulation depends strongly on
the solubilization conditions. In nanodiscs, the ATPase rates increase to 32 and 39 nmol/min/mg with
2.5 mM GSSG and 10 mM GSH, respectively, for an overall increase of 4–5× above the basal rate. The
ATPase rates for
At
Atm3 in DDM also increase with GSSG and GSH, to 117 and 154 nmol/min/mg,
respectively. Because of the higher basal ATPase rate in detergent, however, the stimulation effect
is significantly less pronounced, corresponding to only an ~50% increase for GSSG stimulation. Little
change is observed for the K
m
s of MgATP between the presence and absence of glutathione deriva-
tives for either detergent solubilized or nanodisc reconstituted
At
Atm3 (
Figure 1
).
Inward-facing, nucleotide-free conformational states
To map out the transport cycle, we attempted to capture
At
Atm3 in distinct liganded conformational
states using single-
particle cryoEM. We first determined the structure of
At
Atm3 reconstituted in
nanodiscs at 3.4 Å resolution in the absence of either nucleotide or transport substrate (
Figure 2a
and
Figure 2—figure supplement 1
). This structure revealed an inward-
facing conformation for
At
Atm3 similar to those observed for the inward-
facing conformations for
Sc
Atm1 (PDB ID: 4myc) and
Na
Atm1 (PDB ID: 6vqu) with overall alignment root mean square deviations (rmsds) for the dimer of
ab
05
10
0
60
120
180
[MgA
TP] (mM)
AT
Pase activity
(nmole/min/mg protei
n)
V
max
(nmol/min/mg
)K
m
(mM)
No substrate1
04.1 ± 0.90
.16 ± 0.01
2.5 mM GSSG
117 ± 10
.13 ± 0.01
10 mM GSH1
54 ± 10
.14 ± 0.01
no substrate
10 mM GSH
2.5 mM GSSG
05
10
0
15
30
45
[MgA
TP] (mM)
ATPase activity
(nmole/min/mg protei
n)
V
max
(nmol/min/mg)K
m
(mM)
No substrate
7.7 ± 0.40
.08 ± 0.02
2.5 mM GSSG
32.3 ± 0.90
.05 ± 0.01
10 mM GSH
39 ± 10
.06 ± 0.01
no substrate
10 mM GSH
2.5 mM GSSG
R
2
= 1.00
R
2
= 0.99
R
2
= 0.99
R
2
= 0.84
R
2
= 0.93
R
2
= 0.93
Figure 1.
ATPase activities of
At
Atm3. ATPase activities measured in (
a
) the detergent
n
- dodecyl-
β
-
D-
maltoside (DDM) and (
b
) nanodiscs formed by
membrane scaffolding proteins (MSP) and the lipid 1-
palmitoyl-
2- oleoyl-
glycero-
3- phosphocholine (POPC). The ATPase activities were measured in the
absence of substrate (
●
), at 2.5 mM GSSG (
■
) and 10 mM GSH (
▲
). The corresponding values of V
max
and K
m
in different substrate conditions derived
from fitting to the Michaelis-
Menten equations are indicated. Each condition was measured three times with the individual data points displayed.
The online version of this article includes the following source data and figure supplement(s) for figure 1:
Source data 1.
Numerical data for the graphs depicted in
Figure 1a and b
.
Figure supplement 1.
At
Atm3 constructs.
Research article
Biochemistry and Chemical Biology | Structural Biology and Molecular Biophysics
Fan and Rees. eLife 2022;11:e76140. DOI: https://doi.org/10.7554/eLife.76140
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a
b
cd
90º
90º
intermembrane
space
matrix
intermembrane
space
matrix
f
g
hi
90º
90º
intermembrane
space
matrix
intermembrane
space
matrix
e
N434
N434
F435
F435
GSSG
L436
L436
G437
G437
S438
S438
R441
R441
R328
R328
Q390
Q390
N387
N387
T317
T317
L383
L383
R324
R324
Q380
Q380
T379
T379
L386
L386
Inward-facing conformation
Inward-facing conformation
Closed conformation
Outward-facing conformation
Inward-facing
Inward-facing + GSSG
Closed
Outward-facing
Figure 2.
Structures of
At
Atm3. (
a
) Inward-
facing conformation in the apo state. (
b
) Inward-
facing conformation with oxidized glutathione (GSSG)
bound. (
c
) TM6s (residues 416–460) in the inward-
facing conformation. (
d
) TM6s in the GSSG-
bound inward-
facing conformation. The location of GSSG
is indicated. (
e
) Residues important in stabilizing GSSG binding site, identified by PDBePISA (
Krissinel and Henrick, 2007
). (
f
) Closed conformation
with MgADP- VO
4
bound. (
g
) Outward-
facing conformation with MgADP-
VO
4
bound. (
h
) TM6s in the closed conformation. (
i
) TM6s in the outward-
facing
conformation.
Figure 2 continued on next page
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Fan and Rees. eLife 2022;11:e76140. DOI: https://doi.org/10.7554/eLife.76140
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2.6 Å (
Figure 2—figure supplement 2a, b
) and 2.1 Å (
Figure 2
), respectively, and half-
transporter
alignment rmsds of 2.3 and 2.0 Å (
Figure 2—figure supplement 2c
), respectively. The primary distinc-
tion between these structures is the presence of an approximately 20 amino acid loop between TM1
and TM2 of
At
Atm3 that would be positioned in the intermembrane space and is absent from the
structures of ABCB7 (
Jumper et al., 2021
;
Varadi et al., 2022
), ABCB6 (
Song et al., 2021
),
Sc
Atm1
(
Srinivasan et al., 2014
), and
Na
Atm1 (
Lee et al., 2014
;
Figure 2—figure supplement 3
). While the
functional relevance of this loop in
At
Atm3 is not known, structural characterization of PglK, a lipid-
linked oligosaccharide flippase, revealed a comparably positioned external helix between TM1 and
TM2 that was implicated in substrate flipping (
Perez et al., 2015
), suggestive that the corresponding
loop could also have a functional or structural role in
At
Atm3.
To further look at substrate binding, we determined a 3.6 Å resolution single-
particle cryoEM structure
of
At
Atm3 purified in DDM with bound GSSG (
Figure 2b
and
Figure 2—figure supplement 4
). Although
the overall resolution of the reconstruction was moderate (
Figure 2—figure supplement 4d
), we were
able to model the GSSG molecule into the density map. In this structure,
At
Atm3 adopts an inward-
facing
conformation, with an overall alignment rmsd to the ligand-
free inward-
facing structure of 2.9 Å (
Figure 2—
figure supplement 5a
) and a corresponding half-
transporter alignment rmsd of 1.6 Å (
Figure 2—figure
supplement 5b
). The main difference between the two structures is the extent of NBD dimer separation
(
Figure 2—figure supplement 5a
), where the GSSG-
bound structure presents a more closed NBD dimer
relative to the substrate-
free structure. As previously noted with
Na
Atm1 (
Fan et al., 2020
), the TM6s in these
inward-
facing structures of
At
Atm3 adopt a kinked conformation including residues 429–438 (
Figure 2cd
).
This opens the backbone hydrogen bonding interactions to create the binding site for GSSG (
Figure 2e
)
with binding pocket residues identified by PDBePISA (
Krissinel and Henrick, 2007
). The binding mode of
GSSG in this
At
Atm3 inward-
facing conformation is similar to that observed in the inward-
facing structure of
the GSSG- bound
Na
Atm1 (
Lee et al., 2014
).
MgADP-VO
4
stabilized closed and outward-facing conformational
states
MgADP- VO
4
has been found to be a potent inhibitor of multiple ATPases through formation of a
stable species resembling an intermediate state during ATP hydrolysis (
Crans et al., 2004
;
Davies and
Hol, 2004
). We determined two structures of
At
Atm3 stabilized with MgADP-
VO
4
, one in the closed
conformation with
At
Atm3 reconstituted in nanodiscs at 3.9 Å resolution (
Figure 2f
and
Figure 2—
figure supplement 6
) and the other in the outward-
facing conformation with
At
Atm3 in DDM at
3.8 Å resolution (
Figure 2g
and
Figure 2—figure supplement 7
). These two structures share an
overall alignment rmsd of 1.7 Å with the primary difference being a change in separation of the TMs
surrounding the translocation pathway on the side of the transporter facing the intermembrane space
(
Figure 2—figure supplement 8
). As a result of these changes in the TMDs, access to the inter
-
membrane space is either blocked in the closed conformation (
Figure 2f
) or is open in the outward-
facing conformation (
Figure 2g
). The changes in the TMDs are reflected in the conformations of TM6,
which in the closed structure presents a kinked conformation (
Figure 2h
), in contrast to the straight
conformation in the outward-
facing structure that has the backbone hydrogen bonding interaction
restored in the helices (
Figure 2i
). Further, the loops between TM1 and TM2 that are characteristics
The online version of this article includes the following figure supplement(s) for figure 2:
Figure supplement 1.
Single-
particle cryo-
electron microscopy (cryoEM) structure of
At
Atm3 in the inward-
facing conformation.
Figure supplement 2.
Structural alignment of
At
Atm3 to other ATM transporters.
Figure supplement 3.
Half-
transporter comparison of transporters in the ATM family.
Figure supplement 4.
Single-
particle cryo-
electron microscopy (cryoEM) structure of
At
Atm3 in the inward-
facing conformation with oxidized
glutathione (GSSG) bound.
Figure supplement 5.
Structural alignment of
At
Atm3 in the inward-
facing conformation.
Figure supplement 6.
Single-
particle cryo-
electron microscopy (cryoEM) structure of
At
Atm3 in the closed conformation.
Figure supplement 7.
Single-
particle cryo-
electron microscopy (cryoEM) structures of
At
Atm3 in the outward-
facing conformation.
Figure supplement 8.
Structural alignment of
At
Atm3 in the closed and outward-
facing conformation.
Figure 2 continued