1
2
Glutathione binding to the plant
At
Atm3 transporter and implications for
3
the conformational coupling of ABC transporters
4
5
Chengcheng Fan
1
,2
and Douglas C. Rees
1,*
6
1
Division of Chemistry and Chemical Engineering,
Howard Hughes Medical Institute,
California
7
Institute of Technology, Pasadena, CA 91125
8
2
P
resent
address
:
Division of Biology and Biological Engineering
, California Institute of
9
Technology, Pasadena, CA 91125
10
*Correspond
ing author
:
dcrees@caltech.edu
11
12
13
2
Abstract
14
The ATP Binding Cassette (ABC) transporter of mitochondria (Atm) from
Arabidopsis thaliana
15
(
At
Atm3) has been implicated in the maturation of cytosolic iron
-
sulfur proteins and heavy
16
metal detoxification, plausibly by exporting glutathione derivatives. Using
single
-
particle cryo
-
17
electron microscopy, we have determined
four
structures of
At
Atm3 in
th
ree different
18
conformational states
: two inward
-
facing conformations (with and without bound oxidized
19
glutathione (GSSG)), together with closed and outward
-
facing states stabilized by MgADP
-
VO
4
.
20
These structures not only provide a structural framework for
defining the alternating access
21
transport cycle, but also
reveal
the paucity of cysteine residues
in the
glutathione
binding site
22
that could potentially form inhibitory mixed disulfides with
GSSG
.
Despite extensive efforts, we
23
were unable to prepare the te
rnary complex of
At
A
tm
3
containing both
GSSG and MgATP.
A
24
survey of
structurally characterized
t
ype IV ABC transporter
s
that includes
At
Atm3
establishes
25
that while nucleotides
are found associated with all
conformational state
s
, they are effectively
26
required to stabilize occluded
, closed
,
and outward
-
facing conformations. In contrast,
transport
27
substrate
s have only been observed associated with inward
-
facing conformations. The
28
absence of structures
with
dimerized
nucleotide binding domains
containing
both nucleotide
29
and
transport substrate
suggests that this
form of the
ternary complex exists only transiently
30
during the transport cycle.
31
32
3
Introduction
33
The ATP Binding Cassette (ABC) transporter of mitochondria (Atm) family plays a vital
34
(Leighton and Schatz, 1995)
, but enigmatic, role b
roadly related to transition metal
35
homeostasis in eukaryotes
(Lill et al., 2014)
.
The best characterized member is
36
Saccharomyces cerevisiae
Atm1
(
Sc
Atm1)
present in the
inner
membrane of
mitochondri
a
37
(Leig
hton and Schatz, 1995)
and required for formation of cytosolic iron
-
sulfur cluster
38
containing proteins
(Kispal et al., 1999)
.
Defects in
Sc
Atm1 lead to an overaccumulation of iron
39
in the mitochondria
(Kispal et al., 1997)
. Atm1 is proposed to transport a sulfur containing
40
intermediate
(Kispal et al., 1999)
that may also include
iron
(Pandey et al., 2019)
.
It is also
41
likely to transport a similar sulfur containing species from the mitochondria
that is
required for
42
the cytoplasmic thiolation of tRNA
(Pandey et al., 2018)
. While the precise substrate that is
43
transported remains unknown, derivatives of glutathione have been implicated
based on their
44
ability to stim
ulate the ATPase activity of Atm1
(Kuhnke et al., 2006)
.
45
Structures
for Atm family members
are currently available for
Sc
Atm1
(Srinivasan et al.,
46
2014)
, the bacterial homolog
Na
Atm1
fr
om
Novosphingobium aromaticivorans
(Lee et al., 2014)
47
and human ABCB6
(Wang et al., 2020)
;
the pairwise
sequence identities
between these
48
homologous transporters range from
40% to 46%
.
These proteins occur as homodimers
of
49
half
-
transporters
, where each
half
-
transporter
c
ontains a transmembrane domain (TMD)
50
followed by the canonical nucleotide binding domain (NBD) that defines the ABC
transporter
51
family.
Each
TMD consist
s
of six transmembrane helices
(TMs)
that exhibit the exporter type I
52
fold first observed for Sav1866
(Dawson and Locher, 2006)
; a recent re
-
classification now
53
identifies this group as type IV ABC transporters
(Tho
mas et al., 2020)
. The translocation of
54
substrates across the membrane proceeds through an alternating access mechanism involving
55
the ATP dependent interconversion between inward
-
and outward
-
facing
conformational states
.
56
Among the Atm1 family,
these
conformations
have been most extensively characterized for
57
4
Na
Atm1
and
include
the
occluded and closed states that provide a structural framework for the
58
unidirectional
transport cycle
(Fan et al., 2020)
. Structures of
Sc
A
tm1 with reduced glutathione
59
(GSH)
(Srinivasan et al., 2014)
, and
of
Na
Atm1
bound to
reduced
(GSH)
, oxidized (GSSG)
60
and metal
lated (GS
-
Hg
-
SG)
(Lee et al., 2014)
,
have defined the gene
ral
substrate
binding site
61
in the TMD
for the
transport substrate
s
.
62
Plants have been found to have large numbers of transporters
(Hwang et al., 2016)
,
63
including
Arabidopsis
with
three
Atm orthologues,
At
A
tm
1,
At
A
tm
2, and
At
A
tm
3
(Chen et al.,
64
2007)
.
Of these,
At
A
t
m3
(also known as ABCB25)
rescues the
Sc
A
tm1 phenotype
(Chen et al.,
65
2007)
, and has been shown to be associated with maturation of cytosolic
iron
-
sulfur
proteins
66
(Kushnir et al., 2001)
, confer
resistance to heavy metals such as
cadmium
and
lead
(Kim et al.,
67
2006)
,
and participate in the formation of molybdenum
-
cofactor containing enzymes
(Bernard
68
et al., 2009; Teschner et al., 2010)
.
Unlike yeast, defects in
At
Atm
3 are not associated with
69
iron accumulation in mitochondria
(Bernard et al., 2009)
. While the physiological
substrate is
70
unknown,
At
A
tm
3 has been shown to transport oxidized glutathione and glutathione polysulfide
71
(
GSSSG
)
, with the persulfidated species perhaps relevant to cytosolic iron
-
sulfur cluster
72
assembly
(Schaedler et al., 2014)
.
The ability of
At
A
tm
3 to export oxidized glutathione has
73
been implicated in helping stabilize against excessive glutathione oxidation in the mitochondria
74
and thereby
serving to
maintain a suitable reduction potential
(Marty et al., 2019)
.
75
To help address the functional role(s) of Atm transporters, we have determined
76
structure
s
of
At
A
tm
3 in multiple
conformational states by single
-
particle
cryo
-
electron
77
microscopy (
cryo
EM
)
. These structures not only provide a structural framework for defining the
78
alternating access transport cycle, but
they
also
illuminate
an
unappreciated
feature of the
79
glutathione b
inding site, namely the paucity of cysteine residues that could potentially form
80
inhibitory
mixed disulfides
during the transport cycle. A survey of
structurally characterized
81
members of the
type IV
family of
ABC
transporters, including the Atm
1
family, es
tablishes that
82
5
nucleotides are
effectively
required for the stabilization of the
occluded,
closed,
and outward
-
83
facing conformations.
In contrast to the
nucleotide states
,
transport substrate
s and related
84
inhibitors have only been observed associated with
inward
-
facing conformational states. The
85
absence of structures
with
dimerized
nucleotide binding domains
containing both nucleotide
86
and
transport substrate
suggests that this
form of the
ternary complex exists only transiently
87
during the transport cycle.
88
89
Results
90
At
Atm3 contains an N
-
terminal mitochondrial targeting sequence
that
directs
the
91
translated
protein to the mitochondria
, where it is
cleaved following delivery
to
the inner
92
membrane.
Since this
targeting sequence consists of ~80 residues and is anticipated to be
93
poorly ordered
, we
generated
three different
N
-
terminal
truncation mutants
of
At
Atm3
through
94
deletion of
60, 70
or
80 residues
to identify the
best
-
behaved
construct
.
Together
with
the w
ild
95
type
construct
, these three variants
were
heterologously
over
expressed
in
E.
coli
. The
96
construct
with
the
80 amino acid
s
deletion
showed
the highest
expression level and
97
proportionally less
aggregation by size exclusion chromatography
(Figure
1
-
figure
supplement
98
1
)
and hence was used for further
functional and structural
studies
.
99
100
ATPase activities
101
Using the 80
-
residue truncation construct,
At
Atm3
was
purified in
the
detergent dodecyl
-
102
-
D
-
maltoside (DDM) and reconstituted in
to
nanodiscs formed
from
the
membrane scaffolding
103
protein (MSP)
1D1
and
the lipid
1
-
palmitoyl
-
2
-
oleoyl
-
glycero
-
3
-
phosphocholine (POPC)
. The
104
ATPase activity of this construct was measured
as a function of
MgATP
concentration
in the
105
absence
and presence of
either 2.5 mM
GSSG
or 10 mM
GSH,
which approximate the
ir
106
physiological concentration
s
in
E. coli
(Bennett et al., 2009)
. The rate of ATP hydrolysis was
107
6
determined by measuring phosphate release
using
a molybdate based colorimetric ATPase
108
activity assay
(Chifflet, 1988)
.
T
he basal ATPase activity, measured in the absence of
109
glutathione derivatives, was significantly higher in detergent than in n
anodiscs (104
versus
7.7
110
nmol min
-
1
mg
-
1
, respectively
; Figures 1ab
)
,
while the
apparent K
m
s
for MgATP
were
within a
111
factor of two
(
~0.
16
mM
and
0.
08
mM, respectively)
.
The
ATPase
activit
y
of
At
Atm3
is
112
stimulated by both 2.5 mM GSSG and 10 mM GSH
, but the extent of stimulation depends
113
strongly
on the solubilization conditions. In nanodiscs, the ATPase rates increase to 32 and
39
114
nmol min
-
1
mg
-
1
with
2.5 mM
GSSG
and
10 mM
GS
H
, respectively, for an overall increase of 4
-
115
5x above the basal rate.
The ATPase rates for
At
Atm3 in DDM also increase with GSSG and
116
GSH,
to 1
17
and 154 nmol min
-
1
mg
-
1
, respectively
.
B
ecause of the higher basal ATPase rate
117
in detergent, however
, the stimulation effect is significantly less pronounced, corresponding to
118
only
a
~50%
increase
for GSSG
stimulation
. Little change is observed for the K
m
s of MgATP
119
between
the presence
and absence
of glutathione derivatives
for either detergent solubilized
120
or nanodisc reconstituted
At
Atm3
(Figure 1)
.
121
122
Inward
-
facing
, nucleotide
-
free
conformation
al states
123
T
o
map out the transport cycle, we attempted to
capture
At
Atm3 in
distinct liganded
124
conformational states
using
single
-
particle cryoE
M
.
We
first determined
the
structure of
125
At
Atm3
reconstituted in nanodiscs at 3.4 Å resolution
in the
absence of
either
nucleotide or
126
transport
substrate
(Figure 2a
and
2
-
figure supplement 1
)
.
This structure revealed an inward
-
127
facing conformation for
At
Atm3
similar to those observed for
the
inward
-
facing conformations
128
for
Sc
Atm1
(PDB ID
: 4myc
)
and
Na
Atm1
(PDB ID
:
6
vqu
)
with
overall
alignment
rmsds
for the
129
dimer
of 2.
6
Å
(Figure
2
-
figure supplement 2ab
)
and
2
.1 Å
(Figure
2
)
, respectively
, and
half
-
130
transporter alignment
rmsds of 2.
3
Å and 2.
0
Å
(Figure
2
-
figure supplement 2
c
)
, respectively.
131
The primary distinction between these
structures
is the presence of a
~20 amino acid loop
132
7
between TM1 and TM2
of
At
Atm3
that
would be positioned in the intermembrane space and
is
133
absent from
the structures of
ABCB7
(Jumper et
al., 2021; Varadi et al., 2022)
, ABCB6
(Song
134
et al., 2021)
,
Sc
Atm1
(Srinivasan et al., 2014)
and
Na
Atm1
(Lee et al., 2014)
(Figure
2
-
figure
135
supplement 3
)
.
While the functional
relevance
of this
loop in
At
Atm3 is not known, s
tructural
136
characterization of PglK, a lipid
-
linked oligosaccharide flippase
,
revealed a
comparably
137
positioned
external helix between TM1 and TM2
that
was
implicated
in substrate flipping
138
(Perez et al., 2015)
,
suggestive that the
corresponding
loop
could also
have
a
fun
ctional or
139
structural
role
in
At
Atm3.
140
To further look at substrate binding, we
determin
ed a
3.6 Å resolution
single
-
particle
141
cryoEM
structure of
At
Atm3
purified in DDM
with
bound
GSSG
(Figure 2b
and
2
-
figure
142
supplement 4
)
.
Although the overall resolution of the reconstruction was moderate (Figure
2
-
143
figure supplement 4d
), we were able to model the GSSG molecul
e
into the
density map.
In this
144
structure,
At
Atm3
adopts an inward
-
facing conformation
,
with an overall
alignment
rmsd
to the
145
ligand
-
free inward
-
facing structure
of
2.9
Å
(Figure
2
-
figure supplement 5
a
)
and
a
146
corresponding
half
-
transporter
alignment
rmsd of
1.6
Å
(Figure
2
-
figure supplement 5
b
)
. The
147
main difference between the two structures is the
exten
t
of
NBD dimer
separation
(Figure
2
-
148
figure supplement 5
a
)
,
where
the GSSG bound structure presents
a more closed NBD dimer
149
relative
to the
substrate
-
free struc
t
ure
.
As previously noted
with
Na
Atm1
(Fan et al.
, 2020)
,
the
150
TM6
helices
in these inward
-
facing structures
of
At
Atm3
adopt
a kinked
conformation
including
151
residues 429
-
438
(Figure 2c
d
)
.
This
opens
the backbone hydrogen bonding interactions
to
152
create
the binding site for GSSG (Figure 2e)
with
binding pocket
residues identified by
153
PDBePISA
(Krissinel and Henrick, 2007)
.
The binding mode of GSSG in this
At
Atm3 inward
-
154
facing
conformation is similar to
that observed in
the inward
-
facing structure of the GSSG
155
bound
Na
Atm1
(Lee et al., 2014)
.
156
157
8
MgADP
-
VO
4
stabilized closed
and
outward
-
facing conformation
158
MgADP
-
VO
4
has been
found to be a potent inhibitor of
multiple
ATPases
through
159
formation of
a stable
species
resembling
an intermediate
state during
ATP hydrolysis
(Crans et
160
al., 2004; Davies and Hol, 2004)
.
W
e determine
d
two
structures of
At
Atm3
stabilized with
161
MgADP
-
VO
4
, one
in
the
closed conformation
with
At
Atm3 reconstituted in nanodiscs
at
3.9
Å
162
resolution
(Figure
2
f
and
2
-
figure
supplement 6
)
,
and
the other
in
the
outward
-
facing
163
conformation with
At
Atm3 in DDM
at 3.
8
Å resolution
(Figure
2
g
and
2
-
figure supplement 7
).
164
These
two structures share an overall
alignment
rmsd of
1.
7
Å
with the primary difference
165
being
a
change in separation of the TM helices
surrounding the translocation pathway
on the
166
side of the transporter facing the intermembrane space
(Figure
2
-
figure supplement 8
)
. As a
167
result of these changes in the TMDs, access
to
the intermembrane
space
is either
blocked
in
168
the closed conformation
(Figure
2
f
)
or
is open
in the outward
-
facing conformation
(Figure
2
g
)
.
169
The changes in the TMDs are reflected in the conformations of TM6, which in the closed
170
structure presents
a
kinked conformation (Figure 2
h
), in
contrast to the straight conformation in
171
the outward
-
facing structure
that has the backbone hydrogen bonding interaction restored in
172
the helices
(Figure 2
i
).
Further
, the loops between TM1 and TM2 that are characteristic of
the
173
At
Atm3 transporter are
bette
r
ordered in the closed conformation than in the outward
-
facing
174
conformation (Figure
2
fg
and
2
-
figure supplement 6
-
7
).
In contrast to the variation in the TMDs,
175
the
dimerized NBDs
are virtually
identical
in these two structures
with an overall alignment
176
rmsd of 0.8 Å
(Figure
2
fg
and
2
-
figure supplement 8
).
177
178
Discussion
179
The plant mitochondrial Atm3 transporter has been implicated in a diverse set of
180
functions
associated with
transition metal homeostasis that are reflective of the roles that have
181
been
described
for the
broader
Atm1 transporter family.
To provide a general framework for
182
9
addressing the detailed function of this transporter in plants
, we
have
structurally and
183
functionally characterized
Atm3
from
Arabidopsis
thaliana
.
We first identified a
construct of
184
At
Atm3 with the mitochondrial targeting sequence deleted that expressed well in
E. coli
(Figure
185
1
-
figure supplement 1
)
.
Following purification, the
ATPase activities of
At
Atm3 were measured
186
in both detergent and MSP nanodiscs as a function of
MgATP concentration
s
(Figure 1).
187
Overall,
the ATPase rate
measured
in detergent is
about 5
-
fold
greater than
that measured in
188
nanodiscs
, perhaps
indic
a
tive
of
a more
tightly
coupled ATPase activity in
a
membrane
-
like
189
environment
.
Both GSH and GSSG stimulat
e the ATPase activity
by increasing
V
max
, with little
190
change observed in the K
m
for
Mg
ATP.
The ability of GSSG to stimulate the ATPase activity of
191
At
Atm3 agrees with previous reports
(Schaedler et al., 2014)
, while the stimulation
we observe
192
with
10 mM GSH
differs
from
the lack
of stimulation
notes
with
1.7 and 3.3 mM GSH
in that
193
work
.
T
h
is
discrepancy
may
reflect
the higher GSH concentration utilized in the present
studies,
194
as well as
differences in
other experimental conditions
including the
use of a
Lactococcus
195
lactis
expression system and ∆60 N
-
terminal truncation by
(Schaedler et al., 2014)
, compared
196
to the
E. coli
expression system and the ∆80 N
-
terminal truncation employed in the present
197
work.
198
ABC transporters are typically envisioned as utilizing an ‘alternating access’ mechanism, in
199
which the substrate
-
binding site transitions between
in
ward
-
and
out
ward
-
facing
200
conformations coupled to the binding and hydrolysis of ATP.
In an idealized two
-
s
tate model,
201
ABC transporters only adopt these two limiting conformations, but structural characterizations
202
of ABC transporters in the presence of nucleotides and substrate analogs have identified a
203
variety of intermediates, including occluded (
with a ligan
d binding
cavity
exhibiting little or no
204
access to either side of the membrane
)
and
closed (no ligand binding cavity) conformations.
205
The most extensive analysis of the conformational states of an Atm1 type exporter has been
206
detailed for
Na
Atm1 and assigned
to various states in the transport cy
cle
(Fan et al., 2020; Lee
207
10
et al., 2014)
.
In
the present
work,
we
have
determined
four
structures of
At
Atm3 in
three
208
different conformational states
by single particle cryo
-
EM
: two
inward
-
facin
g conformations
209
(with and without bound GSSG
)
(Figure 2ab)
, together with
closed and outward
-
facing
states
210
stabilized by MgADP
-
VO
4
(Figure
2
fg
)
.
The
parallels
between
the structurally characterized
211
conformations of
At
Atm3 and
Na
Atm1 support the idea that these conformational states are
212
relevant to the transport cycle, and not simply an artifact of the
specific conditions used to
213
prepare each sample.
The conformations observed for
At
Atm3 and
Na
Atm1
d
o
not completely
214
correspond
, however;
most notably,
the
outward
-
facing conformation observed
for
At
Atm3
had
215
not been previously observed with
Na
Atm1
(Fan et al., 2020; Lee et al., 2014)
, while
the
216
occluded conformations found with
Na
Atm1 were not observed for
At
Atm3.
217
The
closed conformation stabilized by
MgADP
-
VO
4
has been observed for both
Na
Atm1
218
(Fan et al., 2020)
and
At
Atm3.
In
comparing
the closed and
the ou
t
ward
-
facing conformation
s
219
of
At
Atm3
,
the arrangement
s
of the NBDs are superimposable,
with
the major differences
220
between
the two
conformation
al states
involving
the local conformation of TM6s.
I
n the
closed
221
conformation
, the TM6s
adopt a
kinked conformation
at residues
438
-
441
that
eliminate
s
the
222
substrate binding
cavity
, whereas the TM6s in the outward
-
facing conformation present
223
straight
TM6s
(Figure 2hi)
.
The
TM6 helical kink in the closed conformation is adjacent to, but
224
distinct from, the helical kink present at residues 429
-
438 in the inward
-
facing conformation. By
225
eliminating the substrate binding cavity, the presence of the closed conformation in a post
-
AT
P
226
hydrolysis state enforces un
i
directionality of the transport process by precluding the uptake of
227
substrate from the outside
.
We note
that
MgADP
-
VO
4
was observed to
stabilize two different
228
conformational state
s
for
At
Atm3,
the
closed state in
nanodiscs
and
the
outward
-
facing
229
conformation in
detergent
(Figure 2fg). This contrasts with our previous observation
s
of
230
Na
Atm1, where
the
closed conformation was observed in nanodiscs
(Fan et al., 2020)
.
The
231
underlying basis for these differences is not
known but
may reflect differences in the
232
11
conformational stabilization of the membrane spanning regions between detergents and
233
nanodiscs.
Structural d
ifferences
between
detergent
s
and
nanodiscs have been pre
viously
234
reported for
MsbA under
MgADP
-
VO
4
stabiliz
ing
conditions
(Mi et al., 2017; Ward et al., 2007)
,
235
and in the
functional analysis of other membrane protein
s
(Hanelt et al., 2013)
.
236
In contrast
to
differences in the structures of the TMDs between
the closed an
d outward
-
237
facing conformations
,
the TMDs in the
inward
-
facing conformation structures of
At
Atm3
are
238
similar
.
T
he primary difference
s
between the two structures of inward
-
facing conformations of
239
At
A
tm
3
are
in the relative positioning of the NBDs which are more widely separated in the apo
240
structure relative to the GSSG bound structure
. Similar substrate induced NBD movements
241
have been
previously observed in MRP1
(Johnson and Chen, 2017)
and ABCB1
(Barbieri et
242
al., 2021)
.
243
The conformational changes in the TMD
s
underlying the transport cycle are associated with
244
changes in the extent of kinking
of TM6 and the positioning of TM4
-
TM5 relative to the core
245
formed by the remaining four TM helices. As noted for
Na
Atm1, we observed kinked TM6s in
246
the inward
-
facing and closed state of
At
Atm3 (Figure 2cd
h
), but not the outward
-
facing
247
conformation (Figure
2
i
). These conformational changes lead to changes in the volume of the
248
central cavity
forming
the glutathione binding site.
U
sing
the program
CastP
(Tian et al., 2018)
249
with a probe radius of 2.5 Å
,
the
cavity volumes
of the
inward
-
facing apo and GSSG bound
250
structure
s
were measured to
be
~6,500 Å
3
(Figure
3
a) and ~4,300 Å
3
(Figure
3
b),
respectively
,
251
while
the closed conformation
exhibits
a cavity
volume
of
~300 Å
3
(Figure
3
c), and the
252
outward
-
facing conformation has a cavity
volume
of ~5,700 Å
3
(Figure
3
d).
We also
measured
253
the accessible solvent areas (ASA) of the key residues
forming the binding site
for
GSSG
in
254
the different conformational states
using Areaimol in CCP4
(Winn et al., 2011)
;
the ASA of the
255
inward
-
facing, inward
-
facing with GSSG bound, closed and outward
-
facing structures are
256
~1,500
Å
2
, ~1,100
Å
2
, ~900
Å
2
, and ~1,300 Å
2
, which are also highly correlated with the cav
ity
257
12
volume calculations.
Most of the binding pocket residues remain exposed in all conformations
258
with a few having large relative changes than others
(Figure
3
-
figure supplement 1
).
Further,
259
the
cavity volume measurements
are
comparable to th
ose calculated
for
Na
Atm1
(Fan et al.,
260
2020)
.
The similarities in conformational states between
Na
Atm1 and
At
Atm3
indicate
these
261
transporters follow the same basic mechanism, in which
straightening of TM6s
in the transition
262
from inward
-
to outward
-
conformation
leads to the release of substrate to the opposite side of
263
the membrane
.
Following substrate
release,
the
transporter resets to the inward
-
facing
264
conformation through the
closed conformation
adopted after ATP hydrolysis; the
decreased
265
size
of
the substrate binding cavity helps enforce substrate release and unidirectionality of
266
substrate transport.
267
The binding pocket for
GSSG
identified in this work
primarily consists of residues from
268
TM5 and TM6, with additional contributions from residues in
TM3 and TM4
(Figure
4
-
figure
269
supplement 1
)
. The
GSSG
binding site
for
At
Atm3
largely overlaps with that identified
270
previously for
Na
Atm1
(Lee et al., 2014)
and for the binding of reduced GSH to
Sc
Atm1
271
(Srinivasan et al., 2014)
.
Inspection of a sequence alignment of
Atm1 homologs
(Figure
4
-
272
figure supplement 1
)
reveals that
those
residues forming the glutathione binding site are
273
largely conserved, particularly if they are involved in polar interactions. A striking feature is the
274
stretch of residues from P432 to
R
441 in the middle of TM6 (
At
Atm3 sequence numbering)
275
with sequence PL
NFLGSVYR with a high degree of sequence conservatio
n
.
P
432
is
276
associated with
the TM6 kink in
inward
-
facing conformations that permits
formation of
277
hydrogen bonds between exposed peptide groups with GSSG
(Lee et al., 2014)
; as TM6
278
straightens in the occluded and outward
-
facing
conformations, these peptide groups are no
279
longer available to bind the
transport substrate
(
Fan et al., 2020)
.
A
sequence alignment of
the
280
structurally characterized
At
Atm3,
Na
Atm1,
Sc
Atm1 and human ABCB
7
and ABCB
6
281
transporters establishes
that residues in the binding pockets are conserved, including T317,
282