Article

https://doi.org/10.1038/s41467-023-40521-5

Cryo-EM structures of human zinctransporter ZnT7 reveal the mechanismof Zn2+ uptake into the Golgi apparatus
Received: 14 March 2022

Check for updates

1234567890():,;

1234567890():,;

Accepted: 27 July 2023

Han Ba Bui1,2,12, Satoshi Watanabe 1,2,3,12, Norimichi Nomura 4, Kehong Liu4,
Tomoko Uemura4, Michio Inoue1, Akihisa Tsutsumi5, Hiroyuki Fujita6,
Kengo Kinoshita7,8, Yukinari Kato9, So Iwata 4, Masahide Kikkawa 5 &Kenji Inaba 1,2,3,10,11

Zinc ions (Zn2+) are vital to most cells, with the intracellular concentrations of
Zn2+ being tightly regulated by multiple zinc transporters located at the plasmaand organelle membranes. We herein present the 2.2-3.1 Å-resolution cryo-EM
structures of a Golgi-localized human Zn2+/H+ antiporter ZnT7 (hZnT7) in Zn2+bound and unbound forms. Cryo-EM analyses show that hZnT7 exists as a
dimer via tight interactions in both the cytosolic and transmembrane (TM)domains of two protomers, each of which contains a single Zn2+-binding site inits TM domain. hZnT7 undergoes a TM-helix rearrangement to create anegatively charged cytosolic cavity for Zn2+ entry in the inward-facing conformation and widens the luminal cavity for Zn2+ release in the outward-facing
conformation. An exceptionally long cytosolic histidine-rich loop characteristic of hZnT7 binds two Zn2+ ions, seemingly facilitating Zn2+ recruitment to
the TM metal transport pathway. These structures permit mechanisms of
hZnT7-mediated Zn2+ uptake into the Golgi to be proposed.

Zinc ions (Zn2+) are an essential trace element that plays vital roles inthe structure and function of various proteins, acting as either acofactor essential for enzymatic reactions or as a structural element
stabilizing protein folding. About 10% of the mammalian proteome is
known to bind Zn2+1. Cellular zinc homeostasis involves the opposing
actions of two families of zinc transporters, SLC30 (ZnTs) and SLC39(ZIPs)2. The SLC30 family proteins transport Zn2+ from the cytosol tothe extracellular space or intracellular compartments, thereby reducing cytosolic Zn2+ concentrations. In human cells, the SLC30 (ZnTs)

family consists of 10 homologs, named ZnT1 to ZnT10, which primarily
regulate the dynamics of intracellular and extracellular Zn2+3,4. One ofthese proteins, human ZnT7 (hZnT7), localizes in the Golgi
membrane5–8 and transports Zn2+ from the cytosol into the Golgi lumen
for incorporation into newly synthesized zinc enzymes3,4. The presence of ZnT7 and other Golgi-resident ZnTs maintains the labile Zn2+
concentration in the Golgi at around 25 nM or higher9. ZnT7 functions
as a Zn2+/H+ antiporter and uses proton motive force to transport Zn2+
from the cytosol to the Golgi lumen. Our latest study showed that1

Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan. 2Department of Molecular and ChemicalLife Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8577, Japan. 3Department of Chemistry, Graduate School of Science,
Tohoku University, Sendai, Miyagi 980-8578, Japan. 4Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
5
Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 6Advanced Research Laboratory, Canon Medical
Systems Corporation, Otawara 324-8550, Japan. 7Department of System Bioinformatics, Graduate School of Information Sciences, Tohoku University, Sendai,
Miyagi 980-8579, Japan. 8Department of Integrative Genomics, Tohoku Medical Megabank Organization, Tohoku University, Sendai, Miyagi 980-8573, Japan.
9
Graduate School of Medicine, Tohoku University, Sendai 980-8575, Japan. 10Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan.
11Core Research for Evolutional Science and Technology (CREST), Japan Agency for Medical Research and Development (AMED), Chiyoda-ku, Tokyo, Japan.
12
These authors contributed equally: Han Ba Bui, Satoshi Watanabe.
e-mail: kenji.inaba.a1@tohoku.ac.jp

Nature Communications | (2023)14:4770

1

Articlehttps://doi.org/10.1038/s41467-023-40521-5

hZnT7 localizes at the proximal side of the Golgi and contributes to theregulation of the intracellular localization and trafﬁc of ERp44, an ERGolgi cycling chaperone10.
Physiologically, hZnT7 plays essential roles in dietary zincabsorption and regulation of body adiposity11. Decrease of cellular Zn2+
in the epithelium of the prostate was shown to be involved in thedevelopment of prostate cancer in mice, with apoptosis being prevented in TRAMP/Znt7-/- mice12. Therefore, a null mutation in the Znt7
gene accelerated prostate tumor formation in mice12. In addition, adeﬁciency in ZnT7 reduced lipid synthesis in adipocytes by inhibiting
insulin-dependent Akt activation and glucose uptake13.
Despite progress in studies of the physiology of ZnT7, its structure and Zn2+ transport mechanism remain to be elucidated. Structures
have been determined for several zinc transporters belonging to the
Cation Diffusion Facilitator (CDF) superfamily, including Escherichia
coli YiiP (EcYiiP)14,15, Shewanella oneidensis YiiP (soYiiP)16–18 and human
ZnT8 (hZnT8)19, all of which belong to the SLC30 family. Cryo-EM
analysis of hZnT8, a Zn2+/H+ antiporter localized to the insulin secretory granules of pancreatic β cells, revealed its overall architecture andZn2+-binding sites19. Amino acid sequence alignment (Fig. 1) suggests
signiﬁcant differences between hZnT7 and hZnT8, especially in theiroverall architecture, length of their cytosolic histidine-rich loops (Hisloop), and numbers of Zn2+-binding sites. Because of its physiologicalimportance, hZnT7 is a particular target of structural and mechanistic
studies.
This study presents high-resolution cryo-electron microscopy(cryo-EM) structures of hZnT7 in both its Zn2+-bound and -unboundstates. A monoclonal antibody Fab fragment that speciﬁcally and tightly
binds to native-state hZnT7 was prepared, and the structures of thehZnT7-Fab complex were determined by single-particle cryo-EM analysis. Consequently, ﬁve different types of hZnT7 conformations were
illuminated; Zn2+-unbound homodimer with both protomers in
outward-facing (OF) form, Zn2+-unbound heterodimer comprising protomers in OF and inward-facing (IF) forms individually, and three typesof Zn2+-bound dimers with different Zn2+-coordination structures andtransmembrane (TM) helix arrangements. Determinations of these distinct structures provided essential insight into mechanisms of hZnT7mediated Zn2+ transport into the Golgi lumen. Notably, His164, a Hisresidue within the His-loop, transiently engages in Zn2+ coordination at
the TM Zn2+-binding site, followed by the replacement with His240, oneof the His-residues that constitute a zinc-binding HDHD motif in the TM
domain, suggesting the role of the His-loop for efﬁcient Zn2+ recruitment to the Zn2+ transport pathway. The present ﬁndings reveal thestructural and mechanistic features of hZnT7 in comparison with thoseof other Zn2+ transporters of known structure, and have physiologicalimplications for Zn2+ homeostasis in the Golgi apparatus.

Fig. 1 | Sequence alignment of representative ZnT-family members of known
structure. Amino acid sequences of human ZnT7 (hZnT7, UniProt ID: Q8NEW0)and human ZnT8 (hZnT8, UniProt ID: Q8IWU4), Escherichia coli YiiP (EcYiiP, UniProt
ID: P69380) and Shewanella oneidensis YiiP (SoYiiP, UniProt ID: Q8E919) werealigned. Highly conserved, similar, and weakly conserved residues are colored
black, light blue, and rose, respectively. The α-helix and β-strand elements of hZnT7
are indicated by solid and broken green lines above the amino acid sequence,
respectively. The histidine-rich loop (His-loop) is indicated by a broken purple line

above the sequence. HDHD-motif residues present in TM2 and TM5 are indicatedby light green boxes. Zn2+-binding residues present in the cytosolic domain of
hZnT8 and Ec/SoYiiP are indicated by red and violet boxes, respectively; and histidine residues in the His-loop of ZnT7 are indicated by blue boxes. Green stars
indicate residues involved in Zn2+ binding. The red triangle indicates His164. TM,
transmembrane helix; α and β, alpha-helix and beta-strand in the cytosolic domain,
respectively.

Nature Communications | (2023)14:4770

2

Articlehttps://doi.org/10.1038/s41467-023-40521-5

Resultspresent cryo-EM analysis revealed that the His-loop ﬂanked by TM4
and TM5 is located on the cytosolic side (Fig. 2a, b). While the major
portion of the His-loop was invisible probably due to its highly ﬂexible
nature, signiﬁcant density was seen near the C-terminal end of TM4(residues 159-163) and the N-terminal end of TM5 (residues 229-232),
enabling the model building for the corresponding His-loop segments (Fig. 2e).
The cytosolic domain (CTD) of hZnT7 consists of residues 302376 and follows the C-terminus of TM6 via a short linker composed ofresidues Met294 to Leu301. Cryo-EM analysis revealed that the CTD
comprises two α helices (α1 and α2) and four β-strands (β1a, β1b, β2and β3) (Figs. 1 and 2b), with the latter providing a contact surface fordimerization (Fig. 2f). An EQ linker (Glu323 and Gln324) ﬂanked by β1a
and β1b is conserved among hZnT7 orthologues and participates in ahydrogen bond network, along with neighboring residues of anotherprotomer (Fig. 2f). The main chain of Glu323 and the side chain of
His325 form hydrogen bonds to Tyr368 of another protomer. Tyr98 ishydrogen bonded to Gln366 (α2-β3 loop) and forms a π-π stack with
Trp327 (β1b) at the TMD-CTD interface. Additionally, TM3 interacts
with TM6 near the cytosolic end as also seen in hZnT8 and bacterial
YiiP (EcYiiP and SoYiiP) (Supplementary Fig. 8a-d), via polar interactions between Arg102 (TM3) and Gln295 (TM6-CTD linker) (Supplementary 7c and 8d). The Fab fragment is speciﬁcally bound to the α1β1a and β2-α2 loops at the end of the hZnT7 CTD (Supplementary
Fig. 9). No direct interactions are made between the Fab fragment andthe TMD or the TMD-CTD intermediate regions of hZnT7. It seems
unlikely that Fab binding considerably affects the overall fold and TM
helix arrangement of hZnT7.

Structure of Zn2+-unbound hZnT7 homodimer
We employed single-particle cryo-electron microscopy (cryo-EM)
analysis to determine the structure of the 74-kDa hZnT7 homodimer.
After data processing of the repeated 2D- and 3D classiﬁcations, adensity map of the hZnT7 homodimer was obtained at ~12 Å resolution,
which did not allow de novo model building. This problem was overcome by preparing monoclonal antibody Fab fragments against
recombinant hZnT7 to increase the molecular size and thereby generate higher-resolution cryo-EM maps (see “Methods” section). After
thorough screening by ELISA and size-exclusion chromatography(SEC), four candidate monoclonal antibodies were selected, their Fabfragments (Fab#1, #3, #4 and #5) were generated, and four types of
hZnT7-Fab complexes were prepared (Supplementary Fig. 1a-c). Twoof these complexes, hZnT7-Fab#1 and hZnT7-Fab#3, yielded highquality two-dimensional class averages by negative-stain EM measurements, showing that a Fab fragment symmetrically bound to each
subunit of the hZnT7 homodimer (Supplementary Fig. 1d). The hZnT7
proteins in complex with Fab#1 and Fab#3 were analyzed by Talos
Arctica Cryo-Transmission Electron Microscopy (Cryo-TEM), yielding
5.0 Å- and 7.7 Å-resolution density maps, respectively (Supplementary
Fig. 2a, b). These results indicated that Fab#1 was the optimal binder of
hZnT7 for cryo-EM analysis. The hZnT7-Fab#1 complex was therefore
assessed by high-end Cryo-TEM with a Gatan K3 BioQuantum directelectron detector.
To prepare Zn2+-unbound hZnT7 uniformly, we puriﬁed hZnT7
throughout in the presence of 1 mM EDTA, and then assessed thestructure of the hZnT7-Fab#1 complex using the JEOL CRYO ARM
300II. As a result, we determined the cryo-EM structure of the complex
with a nominal resolution of 2.2 Å (Fig. 2a, Supplementary Fig. 3a-c, eleft and Supplementary Table 1), which showed clear side-chain density for both the transmembrane domain (TMD) and cytosolic domain(CTD) (Supplementary Fig. 4a). Thus, hZnT7 was found to adopt a“mushroom”-shaped dimeric architecture with the TMDs and CTDs of
two protomers tightly packed (Fig. 2c), which is distinguishable fromthe “V”-shaped dimers of hZnT8 and EcYiiP with less tight interactions
between the TMDs of two protomers (Supplementary Fig. 5)14,19. On theother hand, SoYiiP forms a dimer via tight inter-protomer
interactions16–18, similar to hZnT7 (Supplementary Fig. 5). The TMD
of each hZnT7 protomer contains six transmembrane helices (TM1bTM6), with both the amino and carboxyl termini exposed to thecytosol (Fig. 2b, c) and forms a Zn2+ transport pathway like that of thehZnT8 and bacterial YiiP protomer14,19. TM helices are tightly bundled
at the cytosolic half of TMD in both protomers, whereas they arewidely opened to encompass a deep cavity at the Golgi luminal half,
forming a solvent-accessible passageway from the luminal side (Fig. 2cd). These structural features are typical of the outward-facing (OF)form of membrane transporters. Thus, the major portion of Zn2+unbound hZnT7 has been found to form a homodimer composed of
two OF protomers (named OF-OF homodimer). Structural alignmentsof hZnT7 with hZnT8 and YiiP show that TM1 and TM2 of hZnT7 moveaway from the rest of TMs to greater extent than those of hZnT8 and
YiiP, forming the wider luminal cavity in the OF protomer (Supplementary Fig. 6a).
At the dimer interface of hZnT7, TM2 of one protomer tightlyinteracts with TM3 of another protomer near the luminal side (Fig. 2cand Supplementary Fig. 7a). In line with this, TM2 and TM3 containnumerous aromatic residues that interdigitate with each other at the
dimer interface (Supplementary Fig. 7a). This mode of interaction is
not seen at the dimer interface of hZnT8 or EcYiiP, likely leading to theless tight interactions between their TMDs (Supplementary Fig. 5 and8b, c). Being unique to hZnT7, a membrane-parallel short α-helix(TM1a) is juxtaposed to the N-terminus of TM1b (Fig. 2b, c and Supplementary Fig. 8a), potentially contributing to maintaining or controlling the TM-helix arrangement from the outside. Additionally, theNature Communications | (2023)14:4770

Structure of Zn2+-unbound hZnT7 heterodimer
It is notable that 3D variability analysis yielded a minor hZnT7 heterodimer composed of inward-facing (IF) and OF protomers at 2.8 Å
resolution (Fig. 3a, b, Supplementary Fig. 3a, d, e right and Supplementary Table 1). The density map showed no Zn2+ bound to either
protomer. Thus, some portion of Zn2+-unbound hZnT7 has been foundto form a heterodimer composed of IF and OF protomers (named IFOF heterodimer). Notably, a similar heterogeneous conformation was
also observed in the structure of hZnT819. TMs 1-6 of the OF protomer
in the heterogeneous dimer are virtually superimposable with those of
the OF protomer in the homogenous dimer (Supplementary Fig. 10a),suggesting that the structure of one protomer is hardly affected by the
conformation of another protomer. In the IF protomer, TMs 2-6 aretightly bundled at the luminal half of the TMD, blocking the passage ofsolvent from the Golgi luminal side (Fig. 3c), whereas TM1b and TM4
are located more separately from the rest of TMs to create the wider
cytosolic cavity in the IF protomer (Fig. 3d), while the OF protomer
create the wider Golgi luminal cavity (Fig. 3e). During the transition
from the OF to IF conformations, indeed, TM1 and TM2 considerably
incline, and TM4 undergoes parallel shift so that the Zn2+ transport
pathway opens at the cytosolic side concomitant with closing at theluminal side (Fig. 3f). By contrast, TM3, TM6, and the CTD remain
almost static during this transition (Fig. 3f, Supplementary Fig. 11 andSupplementary Movie 1).
We superimposed the TMD of hZnT7, hZnT8 and SoYiiP in their IF
conformations and found marginal differences between them, except
for the orientations of TM1 and TM5 (Supplementary Fig. 6b). In
hZnT7, TM1 swings away from TM2 while TM5 is located closer to thecenter of the TMD on the cytosolic side, compared to those of hZnT8(Supplementary Fig. 6b left). Similar observations were made in comparison between hZnT7 and SoYiiP (Supplementary Fig. 6b right).
Thus, hZnT7 appears to exert a different mechanism of action to open
the cytosolic gate in the IF conformation than hZnT8 and SoYiiP, asfurther described in the next section. In line with this, we observed thedifferent movements of TM4 and TM5 between hZnT7, hZnT8 andbacterial YiiP during the conversion between the OF and IF
3

Articlehttps://doi.org/10.1038/s41467-023-40521-5

a

b

hZnT7

c

hZnT7

1b’

Membrane

1b

4’
5’

TMD
2

2

3’

5’

4’

4

6’

1a’

1a
1a’

2’

TMD

1b

90o

4

4’

1a
β1b

5’

TM2-TM3
loop

α1

β1a
α2

6’
OF

Golgi luminal
cavityGolgi lumen

OF

β3
W327

Outward-facing Outward-facing
(OF)
(OF)
TM2-TM3
loop
H325

e

Y98

Q324
3

β1a

K339

Q370

H356
β3 L352

β2
His-loopsegment

Q324

β3 L367

H161
Cytosol

W327

Q366
Y368

E323

1b

Y98

H325

4
5

Membrane

1a

1b

CTD

f

d

3’

β2

CTD

Outward-facing Outward-facing
(OF)
(OF)

5

2

6

His-loopsegment

TM2-TM3
loop

6

3

2’

Cytosol
1a’

1b’

5
3

1b’

His-loopsegment

Golgi luminal
cavityGolgi lumen

6’ 3

D372

α2

Fig. 2 | Cryo-EM structure of the Zn2+-unbound hZnT7 homodimer composed of
the outward-facing (OF) protomers (OF-OF form). a Side view of the cryo-EM
map of Zn2+-unbound hZnT7 homodimer. Chain A (violet), chain B (green), TMs 4-5(yellow) and His-loop segment (magenta) are highlighted. Numbers indicate thetransmembrane (TM) helix number from the N-terminus. TMD, transmembrane
domain; CTD, cytosolic domain. b Cartoon representation of the hZnT7 homodimer in the same orientation as in (a). Both left and right protomers adopt an OF
conformation. TM1 to TM3 and TM6 are shown in green or violet, respectively,
while TM4 and TM5 are both in yellow. Visible regions of the histidine-rich loop
(His-loop) are shown in magenta. Numbers indicate the TM helix number from the
N-terminus. The black dashed-line squares indicate the regions that are highlighted
in panel (f). TMD, transmembrane domain; CTD, cytosolic domain. c Golgi luminal

view of the TMD of hZnT7, where the CTD and TM loops are removed for clarity.
The red double-headed arrows indicate the proximity between TM2 and TM3’ (orTM2’ and TM3) at the dimer interface. Numbers indicate the TM helix number fromthe N-terminus. d Coronal section of the electrostatic potential surface map of an
hZnT7 protomer in the OF form. Surface colors indicate Coulombic potentials (red,negative; white, neutral; blue, positive). e Zoomed-in view of the visible segments ofthe His-loop near the C-terminus of TM4 and the N-terminus of TM5. TM4 and TM5
are represented by yellow ribbon. The density map is shown by blue mesh at acontour level of 0.17σ. f Zoomed-in view of the CTD-CTD interface in the OF-OF
homodimer of hZnT7. Yellow dotted lines indicate interactions formed between
polar residues.

conformations. As aforementioned, TM4 of hZnT7 shifts outward via
parallel movement, whereas its TM5 moves only slightly (Fig. 3f). In
hZnT8 and bacterial YiiP, both TM4 and TM5 swing using their luminal
ends as pivot points, resulting in a wider cytosolic cavity in the IF
conformation14,19 (Supplementary Fig. 12).

sampled by exogenously adding 200 µM or 300 µM Zn2+ to the above
hZnT7 preparation, generated two types of dimeric structures: 2.9 Åresolution cryo-EM structure of a heterogenous dimer with one protomer in Zn2+-bound IF conformation and the other in Zn2+-bound OF
conformation (named IF/Zn-OF/Zn heterodimer or Zn2+ state 2) (Fig. 4a
middle, Supplementary Fig. 14a middle and 15a, c, e and Supplementary Table 1), and 3.1 Å-resolution one of a homogenous dimer withboth protomers in Zn2+-bound OF conformation (named OF/Zn-OF/Zn
homodimer or Zn2+ state 3) (Fig. 4a right, Supplementary Fig. 14a rightand 15b, d, f and Supplementary Table 1). The cross sections of theelectrostatic potential surface demonstrate that the IF protomers of
the Zn2+ states 1 and 2 have an even wider and deeper cytosolic cavity
than the Zn2+-unbound IF protomer (Fig. 4b), whereas the OF protomerof the Zn2+ state 3 possesses a closed cytosolic cavity (Fig. 4b). Notably,
the cytosolic cavity of the Zn2+ state 1 is strongly negatively charged
with the open cytosolic cavity (Fig. 4c) compared with that of the Zn2+-

Structures of Zn2+-bound hZnT7 dimers
We next determined the cryo-EM structure of Zn2+-bound hZnT7 incomplex with Fab#1. To this end, we prepared Zn2+-bound hZnT7 in
two different ways and found that these two preparations generated
different dimeric structures of hZnT7. Concretely, the sample puriﬁed
throughout in buffer containing 10 μM Zn2+ yielded a 2.7 Å-resolution
cryo-EM structure of a heterogeneous hZnT7 dimer composed of Zn2+bound IF and Zn2+-unbound OF protomers (named IF/Zn-OF heterodimer or Zn2+ state 1) (Fig. 4a left, Supplementary Fig. 13 and 14a left
and Supplementary Table 1). Another hZnT7 preparation, which was

Nature Communications | (2023)14:4770

4

Articlehttps://doi.org/10.1038/s41467-023-40521-5

a

b

hZnT7
6’

c

hZnT7
Golgi lumen

3

Membrane

1b’
1b
4
2

4’
5’

TMD

2’
4’

5
2

6’

1b’
1a’

3’

5’

4

TMD

3

1b

1b’

His-loopsegment

90o
6

Cytosoliccavity1a

1aTM2-TM3loop
β1b

His-loopsegment

TM2-TM3
loop

β2

α1

CTD

β1a
β3

α2

Inward-facing(IF)

Outward-facing
(OF)

2’

3

1a’
CytosoliccavityGolgi luminal
cavity65

1a’

Inward-facing
(IF)

4

4’
2
5’

CTD

6’

1b

IF

1a

OF

Outward-facing
(OF)

d

e
Golgi lumen

Golgi luminal
cavityGolgi lumen

Cytosolic cavity(Closed)Membrane3’

Membrane

TM4’
TM5’

80o

TM1b’

Cytosol

CTD’Cytosoliccavity(Closed)Cytosol

TM1a’

OF

IF

f

G o l g i l u m i n a l vi e w
6

3

1b’
1a’

C y t o s o l i c vi e w

Side view
5’

5

1a
5’

2’

6’

2

3’

1b

4’
4
1b’
1b’
4

4’

2
3’
5’

6’

2’
1b
1a

1a’

3
6

5
3’

6’

2’

1a’

OF-OF homodimer (Zn2+-unbound): ◉TM1’ to TM6’ and ◉TM1 to TM6
IF-OF heterodimer (Zn2+-unbound): ◉TM1’-TM2’; ◉TM3’-TM6’; ◉TM4’-TM5’ (in IF) and ◉TM1 to TM6 (in OF)

unbound IF protomer (Fig. 3d), which seems key to efﬁcient recruitment of Zn2+ to the metal transport pathway in the TMD.
Superimposition of the Zn2+ states 1 and 2 reveals that there is nolarge conformational difference in the TMD between these two (Supplementary Fig. 10b). Similarly, the Zn2+-unbound heterodimer also
shows good structural alignment with the Zn2+ states 1 and 2 (Supplementary Fig. 10c). The TMD of the Zn2+-unbound homodimer also

Nature Communications | (2023)14:4770

displayed a highly similar TM-helix arrangement to that of Zn2+-boundhomodimer (Supplementary Fig. 10d). However, the IF protomer of theZn2+ state 1 has a notable structural feature in the TMD; TM5 is kinked
at Thr245 by 67o compared to that of the Zn2+-unbound IF protomer(Fig. 5a-c and Supplementary Movie 2). As a result, His240 is moved
away from the Zn2+-binding site in the TMD (Fig. 5b, c). Instead, His164
contained in the His-loop is located nearby Zn2+ to be coordinated to5Articlehttps://doi.org/10.1038/s41467-023-40521-5

Fig. 3 | Cryo-EM structure of the Zn2+-unbound hZnT7 heterodimer composedof the inward-facing (IF) and OF protomers (IF-OF form). a Side view of the cryoEM map of Zn2+-unbound hZnT7 heterodimer. Chain A (violet), chain B (green), TMs
4-5 (yellow) and histidine-rich segment (magenta) are highlighted. Numbers indicate the transmembrane (TM) helix number from the N-terminus. TMD, transmembrane domain; CTD, cytosolic domain. b Cartoon representation of the hZnT7
heterodimer in the same orientation as in (a). Left and right protomers adopt IF and
OF conformations, respectively. TM1 to TM3 and TM6 are shown in green or violet,
respectively, while TM4 and TM5 are both in yellow. Visible regions of the histidinerich loop (His-loop) are shown in magenta. Numbers indicate the TM helix number
from the N-terminus. TMD, transmembrane domain; CTD, cytosolic domain.
c Golgi luminal view of the TMD of hZnT7, where the CTD and TM loops are

removed for clarity. The red double-headed arrows indicate the proximity between
TM2 and TM3’ (or TM2’ and TM3) at the dimer interface. Numbers indicate the TM
helix number from the N-terminus. d Coronal section of the electrostatic potential
map of the IF protomer in the Zn2+-unbound hZnT7 heterodimer. Surface colors
indicate Coulombic potentials (red, negative; white, neutral; blue, positive).
e Coronal section of the electrostatic potential map of the OF protomer in the Zn2+unbound hZnT7 heterodimer. f Superimposition of the Zn2+-unbound OF-OF
homodimer (gray) and IF-OF heterodimer (colored as indicated) of hZnT7 viewed
from the Golgi lumen (left), cytosol (middle), and side (right). The cytosolic domain
and TM loops are omitted for clarity. Numbers indicate the TM helix number fromthe N-terminus. Dark blue arrows indicate the movements of TM helices during theconversion from the OF to IF conformations in the left protomer.

the metal ion (Fig. 5e). In this connection, a His-loop segment consisting of residues Val158 to Gly165 is inserted into the cytosolic cavity(Fig. 5e). Of further note, the kinked segment of TM5 gets disordered in
the IF protomer of the Zn2+ state 2, and His240 moves back to becoordinated to Zn2+ in place of His164 (Fig. 5d). These structural ﬁndings suggest a unique mechanism of the Zn2+ uptake by hZnT7; His164
located near the N-terminus of the His-loop serves to recruit Zn2+ to thenegatively charged cytosolic cavity via the bending of TM5, followedby the exchange of a Zn2+-coordinating residue from His164 to His240
at the TM Zn2+-binding site (for more details, see the next section andDiscussion).

the neighboring protomer, whereas bacterial YiiP forms a binuclear
zinc coordination with the (HHD)2 motif at the CTD dimer interface(Supplementary Fig. 17c). These Zn2+-binding residues in the CTDs ofbacterial YiiP and hZnT8 are replaced by other residues in hZnT7(Supplementary Fig. 18). Despite the absence of Zn2+ binding sites in its
CTD, hZnT7 forms a stable dimer. Thus, it seems that Zn2+ ions boundto the TMD-CTD interface and CTD are not always necessary for the
dimerization of the CDF superfamily.

Zinc-coordination structures in hZnT7
The cryo-EM map of Zn2+-bound hZnT7 demonstrated that the primary
Zn2+-binding site (hereafter, named STM) is located at around thehalfway point of the TMD (Fig. 4a). At the STM in the IF protomer of theZn2+ state 1, a tetrahedral Zn2+ complex is formed by His70 (TM2) andAsp74 (TM2), Asp244 (TM5), and His164 (His-loop) while His240 (TM5)moves away from the STM due to the bending of the N-terminal segment of TM5 (Fig. 6a and Supplementary Fig. 16a), as aforementioned.
In the IF protomer of Zn2+ state 2, His164 is replaced with His240 toform a similar tetrahedral complex to that in the Zn2+ state 1 (Fig. 6band Supplementary Fig. 16b). In the OF protomer of the Zn2+ state 2,
the side chain of His70 is moved away from Zn2+ (Fig. 6d and Supplementary Fig. 16d), concomitant with signiﬁcant increase of the distance between the Cα atoms of His70 and Asp244, seemingly
facilitating the release of Zn2+ to the Golgi luminal side. In support ofthis, His70 appears to further swing away from the STM in the Zn2+unbound OF protomer with formation of a hydrogen bond between
Asp74 and His240 (Fig. 6e and Supplementary Fig. 16e). In the OF
protomer of the Zn2+ state 3, the Nε atom of His70 is located beyond
the range of direct Zn2+ coordination (Fig. 6c and Supplementary
Fig. 16c), and some extra density is seen between His70 and Zn2+,
suggesting the presence of a water molecule mediating the interaction
between these two. Thus, the sidechain of His70 appears to be well
separated from Zn2+ and mobile in the Zn2+-bound OF protomer, possibly representing an initial stage in rehydration of the metal ion prior
to its release.
The previously published structures of the Zn2+-bound OF form of
EcYiiP, the Zn2+-bound IF form of SoYiiP, and the Zn2+-bound OF formof hZnT8 demonstrate the presence of additional Zn2+-binding sites(Supplementary Fig. 17a). A second Zn2+-binding site of hZnT8 islocated at the TMD-CTD interface (SIF) (Supplementary Fig. 17b),
although this site had a lower afﬁnity for Zn2+19. Similarly, Zn2+-bindingsites are present near the TMD-CTD interface of EcYiiP and SoYiiP(TM2-TM3 loop). hZnT7 lacks the second Zn2+-binding site around the
TMD-CTD interface, consistent with the absence of histidine residues
in this region (Supplementary Fig. 17b).
In contrast to hZnT7, hZnT8 and bacterial YiiP have two additional
Zn2+ binding sites (SCD1 and SCD2) in the CTD (Supplementary Fig. 13c).
hZnT8 contains two cysteines near the C-terminus, and a unique HCH(His52-Cys53-His54) motif in a non-conserved N-terminal extension of

Nature Communications | (2023)14:4770

Structural and functional roles of the histidine-rich loopof hZnT7
The His-loop of hZnT7, positioned between TM4 and TM5 on thecytosolic side, is exceptionally long compared with other zinc transporters (Fig. 1). The cryo-EM density map at 2.2 Å resolution allowed
for visualization of only the N-terminal (residues 229-232) andC-terminal (residues 159-163) segments of the His-loop near the ends of
TM4 and TM5, respectively (Fig. 2e), suggesting that the major portionof the His-loop is highly ﬂexible. To explore the structural and functional roles of the His-loop, its major portion (residues His176 to
Pro221) was deleted, and the resultant mutant protein (hZnT7ΔHisloop) was used for structural and functional analyses. Cryo-EM structures of the hZnT7ΔHis-loop-Fab complex in Zn2+-bound and -unboundstates were determined at nominal resolutions of 3.4 Å (Supplementary Fig. 19 and Supplementary Table 2). Since the major class (Class 4;
67.4%; 127,941 particles) showed unusually bent TM helices and a poor
density map, the second major-class density map (32.6%; 69,877 particles) was used for the model building of hZnT7ΔHis-loop (Supplementary Fig. 19).
Both Zn2+-bound and -unbound forms of hZnT7ΔHis-loop were
found to adopt an OF conformation with similar location and orientation of the TM helices to those in wild-type hZnT7 (hZnT7 WT),
indicating that deletion of the His-loop had little impact on the overall
TMD of hZnT7 (Supplementary Fig. 20a-c). Similarly, the CTD of
hZnT7ΔHis-loop is almost superimposable to that of hZnT7 WT, with
an RMSD for all Cα atoms in this domain of 0.3 Å (Supplementary
Fig. 20d). The structure of the CTD is almost insensitive to Zn2+, displaying an RMSD for all Cα atoms in this domain of 0.3 Å between the
Zn2+-unbound and -bound states (Supplementary Fig. 20e). Collectively, while the His-loop may possibly contribute to the stabilization of
the IF conformation via its incorporation into the widened cytosolic
cavity seen in the IF protomer of the Zn2+ state 1 (Fig. 5e), it does notappreciably determine the structure of the OF protomer.

Essentiality of histidine residues in the His-loop for efﬁcient Zn2+transport
The amino acid sequence (Fig. 1) indicates that the distribution ofhistidine residues in the His-loop can be classiﬁed into two sites: onenear the C-terminus of TM4 (site 1 with 7 histidine residues including
His164) and the other in the C-terminal half of the His-loop (site 2 with
14 histidine residues). To characterize functional roles of the sites-1
and 2 in Zn2+ binding and Zn2+ uptake activity, we prepared three sortsof ZnT7 mutants (HS1, HS2 and HS3). HS1 and HS2 have His-to-Ser
6Articlehttps://doi.org/10.1038/s41467-023-40521-5aZn2+ state 1

Zn2+ state 2

Zn2+ state 3

Golgi lumen
membrane
Zn2+
3’

5’

4’

6’

Zn2+

Zn2+
4
3

2

3’
5’

4’

5 1b

3

5

2’
6

Cytosoliccavity

α1
α2

2

6

1a

2’

1a’

1a

His-loopsegment

CTD

β3

IF

OF

IF

90o
6’

OF

3’

OF

90o

1a

5’
Bend
4’

6’

1b

Cytosoliccavity

β1b
β2
β1a

4
1b

3

Zn2+

His-loopsegment

1a

4’

TMD

5

6

3’

5’

4

2

6’

Zn2+

2
5’

6’

2

5’

4

1b’
2’

3

6

5

3

2’

1a’

2+

Zn2+ state 1

1b

6

4

1b’
3

5

6

2’

1a’

b Zn -unbound

2
3’

4’

4
1b’

6’

1b

4’

1b

1a

90o

1a

3’

OF

5

1a’

Zn2+ state 2

Zn2+ state 3cZn2+ state 1
TM4’

LuminalcavityCytosoliccavityCytosoliccavityCytosoliccavityTM5’
TM1b’

Cytosoliccavity (Open)CTD’

Fig. 4 | Cryo-EM structures of the Zn2+-bound hZnT7 dimers (Zn2+ state 1, Zn2+
state 2 and Zn2+ state 3). a Cartoon representations show the overall structures of
two types of Zn2+-bound hZnT7 heterodimers named Zn2+ state 1 (left) and Zn2+
state 2 (middle), and a Zn2+-bound hZnT7 homodimer named Zn2+ state 3 (right).
The side (upper) and cytosolic (lower) views are shown. The black dashed-line

square indicates the region that is highlighted in Fig. 5e. b Coronal sections of theelectrostatic potential surfaces of the hZnT7 dimers in Zn2+-unbound state, Zn2+
state 1, Zn2+ state 2 and Zn2+ state 3. c Electrostatic potential surface around thecytosolic cavity of the IF protomer in the Zn2+ state 1. Surface colors indicate
Coulombic potentials (red, negative; white, neutral; blue, positive).

mutations at site 1 and site 2, respectively, while all histidine residues inthe His-loop are mutated to Ser in HS3 (Fig. 7a). Zn2+ binding afﬁnity
and stoichiometry for hZnT7 WT and HS1-HS3 were analyzed by isothermal titration calorimetry (ITC) at 20 °C, pH 7.5. A set of ITC thermograms (Fig. 7b) revealed different Zn2+-binding properties of thesefour proteins. We found that each protomer of hZnT7 WT bound 3
molar equivalents of Zn2+ with an apparent Kd value of 11.3 µM (Fig. 7c).
A protomer of hZnT7 AAAA, in which the HDHD motif at the TM Zn2+binding site was all mutated to alanine, bound ~2 molar equivalents of
Zn2+ with an apparent Kd value of 12.2 µM (Fig. 7c), suggesting thepresence of one Zn2+-binding site in the TM domain and two in the Hisloop of hZnT7. Each protomer of HS1 and HS2 was found to bind 2.4

and 1.7 molar equivalents of Zn2+ with apparent Kd values of 8.8 and11.6 µM, respectively (Fig. 7c), Altogether, it is surmised that one Zn2+binding site is contained in the site 2 of the His-loop and that another
Zn2+-binding site is constituted by both sites-1 and 2 while the site-1 orsite-2 alone can bind the Zn2+ partially. HS3 could bind only one Zn2+
most likely in the TM domain.
The thermodynamic parameters, enthalpy (ΔH) and entropychanges (TΔS), obtained by the ITC analysis (Fig. 7c) suggest that Zn2+binding to hZnT7 is enthalpy-driven while Zn2+-binding to the His-loop
reduced the entropy due to the decrease of conformational freedom
associated with Zn2+ binding. Notably, the binding enthalpy for hZnT7
HS3 (–8.0 kcal/mol) greatly decreased compared to those of hZnT7Nature Communications | (2023)14:4770

7

Articlehttps://doi.org/10.1038/s41467-023-40521-5a

G o l g i l u m i n a l vi e w
1a’

3
1b’

4’

Cytosolic view

6

1a

5’

5

2

Bent helix
6’

2’

1b
4

Zn2+

Zn2+
4’

4
5’
6’

3’

2

3’
1b

1b’

2’

3

1a’
Bent helix

5
6

1a

IF

OF

IF

OF

IF-OF heterodimer (Zn2+-unbound): ◉TM1’ to TM6’ and ◉TM1 to TM6
IF/Zn-OF heterodimer (Zn2+ state 1): ◉TM1’-TM2’; ◉TM3’-TM6’; ◉TM4’-TM5’ (in IF) and ◉TM1 to TM6 (in OF)

b

c

2+
TM5’ Zn

d
TM5’

H240

TM5’

TM5’

e Cytosolic view
K160
4’

H240

F159

H161

5’

6’
H164

Zn2+
TM4’

T245

TM4’
T245

Bent helix

Zn2+
D244

3’

2’

1b’
H240
4’

L242

Bent helix

1b’

I241
67o

H240
Zn2+-unbound (IF)Zn2+ state 1 (IF)Zn2+ state 1 (IF)Zn2+ state 2 (IF)

6’
Zn2+

Zn2+

H240

H240
His-loop
Bent helix
segment

Bent
helix

2’

His-loopsegment

H164

H240
K160

Bent
helix

5’

Side viewFig. 5 | The His-rich loop incorporates into the cytosolic cavity of the IF protomer. a Superimposition of the TMDs of the hZnT7 heterodimers in IF-OF form
(gray) and Zn2+ state 1 (colored as indicated), viewed from the Golgi lumen (left) andcytosol (right). Black arrows indicate the movement of TM5 during the conversion
from the IF-OF form to Zn2+ state 1. b Bending of the N-terminal portion of TM5 in
the IF protomer of Zn2+ state 1. The density map of TM5 is shown by blue mesh at acontour level of 0.17σ. c Superimposition of TM5 in the IF protomers of the hZnT7
heterodimers in IF-OF form (gray) and Zn2+ state 1 (yellow). Black and red arrows
indicate the movements of TM5 and His240 in TM5 during the transition from theZn2+-unbound state to Zn2+ state 1, respectively. d Superimposition of TM4 and TM5

between the IF protomers in the Zn2+ state 1 (yellow) and Zn2+ state 2 (blue). The
yellow and blue spheres indicate an Zn2+ ion bound to the Zn2+ state 1 and Zn2+ state
2, respectively. The red arrow indicates the movement of His240 during the transition from the Zn2+ state 1 to state 2. The black dash-line square is highlighted in theright upper inset with the density map around His240 in the Zn2+ state 2.
e Highlighted representation of the His-loop segment viewed from the cytosol(upper) and side (lower). The His-loop segment is shown in magenta with its densitymap (blue) at a contour level of 0.17σ. TM4 and TM5 were shown in yellow. A red
sphere indicates a Zn2+ ion coordinated by His164.

WT, HS1 and HS2 (-15 to -16 kcal/mol), suggesting that histidine residues in the His-loop contribute to stabilizing the Zn2+-bound conformation of hZnT7. Consistently, the Kd for Zn2+ value of HS3 issigniﬁcantly higher (i.e. lower afﬁnity) than those of WT, HS1 andHS2 (Fig. 7c).
To explore the roles of the His-loop histidine residues for efﬁcient
Zn2+ transport by hZnT7, we measured the Zn2+ uptake activities of HS1HS3 by preparing the proteoliposome containing one of the mutants
and Fluozin-3, and compared them with that of WT. In line with thepresent observation that HS1 and HS2 retained Zn2+-binding afﬁnity
despite lower stoichiometry, their Zn2+ uptake rates were comparable
to or slightly lower than that of WT (Fig. 7d and Supplementary ﬁg. 21).
The Zn2+ uptake activity of HS3 was much lower than those of the otherthree. Given that the His-loop directly participates in Zn2+ recruitmentin the IF protomer (Fig. 5d, e), one likely interpretation is that Zn2+
binding to the HDHD motif is promoted by the His-loop. However, HS1,

which includes a mutation of H164S, still retained the Zn2+ transport
activity (Fig. 7d), tempting to speculate that one or several of the site-2
histidine residues can also serve as a substitute for His164. It is also
notable that the ITC proﬁle for Zn2+ binding to hZnT7 WT did not show
stepwise Zn2+ bindings to different sites with different afﬁnities. Thus,
we surmise that histidine residues in the His-loop cooperate with theZn2+-binding residues in the TMD for efﬁcient Zn2+ recruitment
by hZnT7.

Nature Communications | (2023)14:4770

Discussion
We herein present near-atomic resolution cryo-EM structures of hZnT7
in both Zn2+-unbound and -bound states, in complex with the Fabfragment that speciﬁcally binds the cytosolic end of hZnT7. The cryoEM structures illuminate multiple sub-state conformations of thehZnT7 dimer, in which two protomers adopt homodimers composedof two OF protomers and heterodimers composed of IF and OF
8

Articlehttps://doi.org/10.1038/s41467-023-40521-5

1
Zn2+-bound (IF)(Zn2+ state 1)

a

TM5

2
Zn2+-bound (IF)(Zn2+ state 2)

b

TM2

TM5

3
Zn2+-bound (OF)(Zn2+ state 3)

c
TM5

TM2

TM2
H70

H70

Bent
helix

H70

8.4 Å

9.9 Å
D244

8.3 Å
2.0 Å

Zn2+

2.0 Å
D244
H164

2.1 Å
D244
2.1 Å

D74

Zn2+

D74

Zn2+
D74
H240

H240
H240

His-rich loop
segmentd4
Zn2+-bound (OF)(Zn2+ state 2)
TM5

e
TM2

5
Zn2+-unbound (OF)(Zn2+ state 1)
TM5

TM2

H70

H70

10.1 Å

D244

10.6 Å

D74
D244
Zn2+
H240

D74
2.3 Å

H240

Fig. 6 | Zn2+-coordination structure of hZnT7. Close-up views of the Zn2+-bindingsites in the IF protomer in Zn2+ state 1 (a) and Zn2+ state 2 (b), in the OF protomer in
Zn2+ state 3 (c), Zn2+ state 2 (d) and Zn2+ state 1 (e). The red spheres indicate boundZn2+. The density map and Zn2+-binding residues are represented by blue mesh andyellow sticks, respectively. The main chains are represented by yellow ribbons. Red
dashed lines indicate the distance between the Cα atoms of His70 and Asp244.
Black dashed lines indicate the distances between the Nδ/Nε atom of His70, His164
or His240 and Zn2+.

protomers regardless of Zn2+ binding. In a Zn2+-bound IF protomer of
Zn2+ state 1, an N-terminal His-loop segment is inserted into the cytosolic cavity via the bending of the cytosolic half of TM5, allowing
His164 to directly coordinate to Zn2+. In another Zn2+-bound IF protomer of Zn2+ state 2, His240 engages in Zn2+ coordination at the TM Zn2+binding site in place of His164. Zn2+ state 1 was identiﬁed as the major
subclass conformation when using the sample puriﬁed throughout inthe presence of 10 µM Zn2+, whereas Zn2+ state 2 was observed after theexogenous addition of 200 µM or 300 µM Zn2+ to the hZnT7. Indeed,
our ITC analysis showed that the Kd for Zn2+ value of puriﬁed hZnT7 isaround 10 μM (Fig. 7). However, Outten & O’Halloran (2001) estimatedthe labile Zn2+ concentration in the cytosol to be in the picomolar
range, much below 10 μM20. More recently, Liu et at. (2022) reported
that the cytosolic labile Zn2+ concentration in Hela cells is ~0.13 nM21.
Thus, we need to carefully consider the discrepancy between the Zn2+binding afﬁnity of hZnT7 determined by the in vitro biophysicalapproach and the Zn2+ availability in the physiological environment asa central enigma in mechanisms of zinc transporters. Nonetheless, thedifferent His-loop conformations seen at the different Zn2+ concentrations may suggest that low concentrations of Zn2+ allow the Hisloop to efﬁciently transfer Zn2+ to the Zn2+-binding site in the TMD,
whereas the large excess of Zn2+ could limit the mobility of the His-loop
possibly via multiple Zn2+ binding, resulting in trapping the Zn2+ state 2

with the His-loop located away from the TM Zn2+-binding site. No
homogeneous IF-IF dimer was identiﬁed in either Zn2+-bound or
unbound form, implying that this state may be energetically unstableas suggested by the well-separated locations of TM2 of one protomer
and TM3 of another protomer in the modelled IF-IF homodimer(Supplementary Fig. 22).
Like hZnT7, an Arabidopsis thaliana vacuolar Zn2+/H+ antiporter(AtMTP1) also has a long cytosolic loop with 25 histidine residues,
which can be divided into the 1st (near the C-terminus of TM4) and 2nd(central part) histidine-rich segments22,23. The ITC analysis showed thata polypeptide chain of the AtMTP1 His-rich loop was able to bind four
Zn2+ ions20. Nonetheless, the deletion of the N-terminal 32 amino acidresidues in the His-rich loop led to much higher Zn2+ uptake activity
than the wild type23. Thus, the His-rich loop may rather serve to suppress the Zn2+ transport activity of AtMTP1, presumably to maintainintracellular Zn2+ homeostasis in accordance with the Zn2+ concentration. Molecular mechanisms underlying the opposite functional rolesof the His-loop between hZnT7 and AtMTP1 need to be further
explored.
The bending of TM5 in hZnT7 is caused by the kink at Thr245,
leading to the large movements of His240 (Fig. 5c). As a result, theconventional Zn2+-binding HDHD motif is partially affected by thetransient coordination of His164 to Zn2+. Similar TM helix bending canNature Communications | (2023)14:4770

9

Articlehttps://doi.org/10.1038/s41467-023-40521-5

ahZnT7 WT
His

1

2

3

hZnT7 HS1

hZnT7 HS2

hZnT7 HS3

hZnT7 AAAA
Ala

Asp

4

5

Site 1(7 His)61Site 2(14 His)

2

3

4

Ser

5

1

6

2

3

4

5

6

1

2

3

4

5

1

6

2

3

4

5

6

His

His-rich loopbhZnT7 WThZnT7 HS1

hZnT7 HS3

hZnT7 AAAAhZnT7 HS2

d

c
N

Kd
(μM)

∆H
(kcal/mol)T∆S(kcal/mol)∆G(kcal/mol)

hZnT7 WT

3.0

11.3

-15.3

-8.6

-6.6

hZnT7 HS1

2.4

8.8

-15.1

-8.3

-6.8

hZnT7 HS2

1.7

11.6

-15.9

-9.2

-6.6

hZnT7 HS3

1.2

13.2

-8.0

-1.5

-6.6

hZnT7 AAAA

2.3

12.2

-13.6

-7.0

-6.6

be seen in other transporters of known structures, including Pyrococcus furiosus MATE (PfMATE)24 and Arabidopsis thaliana DTX14(AtDTX14)25 that belong to the MATE family transporters responsible
for efﬂux of endogenous cationic, lipophilic substances, and xenobiotics. In PfMATE24, the kink at Pro26 is somehow remotely inducedby the protonation of Asp23 on the N-terminal lobe of TM1, leading to
the bending of this helix. A similar event has been observed in

Nature Communications | (2023)14:4770

AtDTX1425, where Glu265 (TM1) at the substrate-binding site is protonated, inducing the kink of TM1 at Pro257. Although such a criticalproline residue does not exist near Asp244 in TM5 of hZnT7, thedeprotonation/protonation of Asp244 may possibly control thebending of TM5. In any case, Zn2+ binding that induces the incorporation of the His-loop into the cytosolic cavity seems likely to beclosely coupled to the bending of TM5 in hZnT7.
10

Articlehttps://doi.org/10.1038/s41467-023-40521-5

Fig. 7 | Roles of the His-loop in Zn2+ binding afﬁnity and Zn2+ uptake activity of
hZnT7. a Cartoon representations show the distribution of histidine residues in the
His-loop, and the constructions of HS1, HS2, HS3 and AAAA mutants of hZnT7.
b ITC analyses for Zn2+ binding to hZnT7 WT and its His-to-Ser mutants (HS1, HS2
and HS3) and AAAA mutant with mutations of the HDHD motif to alanine. The
upper panels show the heat generation during the calorimetric titrations with 1 μL
injections of 500 μM ZnCl2 into hZnT7 WT (18 μM), HS1 (20 μM), HS2 (20 μM), HS3(30 μM) and AAAA (20 μM) in the reaction cell with a volume of 280 μL. The lower
panels display the integrated heat values calculated from the upper panels as afunction of the molar ratio of Zn2+ to hZnT7. The solid lines represent the best ﬁt to
the observed ITC curves. At least two independent replicates for each construct.
c The resulting ﬁtting parameters are summarized. d Initial Zn2+ uptake rates per μgof hZnT7 protein determined with the proteoliposomes containing the indicated
ZnT7 protein and Fluozin3 in buffer containing 100 μM Zn2+. Data are averages fromthree independent experiments. Error-bars indicate mean ± SEM (n = 3); numbers
indicate p values analyzed by one-way ANOVA. Source numerical data are provided
in the Source Data ﬁle. See also Supplementary Fig. 21.

The present cryo-EM analysis also demonstrates that the Zn2+bound OF form has structural features suitable for Zn2+ release to theGolgi lumen. The luminal cavity of the Zn2+ transport pore is open,
whereas the cytosolic cavity is fully closed to prevent further Zn2+ entry
from the cytosol. Presumably, H+ from the weakly acidic Golgi lumen
protonates residues of the HDHD motif, triggering the release ofbound Zn2+. The negatively charged surface of the luminal cavity(Fig. 2d) likely facilitates Zn2+ transfer to the luminal gate in the OF
form. Protonations at the HDHD motif may also induce the subsequentconversion from the OF to the IF form, followed by the release of H+
into the cytosol. This mechanism of action nicely explains the physiological function of hZnT7 as a Zn2+/H+ antiporter in the Golgi
membrane.
Taken all together, we propose a likely scenario of hZnT7mediated Zn2+ transport from the cytosol to the Golgi lumen (Fig. 8 and
Supplementary Movie 3). The Zn2+-unbound IF protomer possesses anegatively charged cytosolic cavity around the TMD-CTD interface,near which the exceptionally long and ﬂexible His-loop is located(stage 1). Upon Zn2+-binding, an N-terminal segment of the His-loop isincorporated into the widely opened cytosolic cavity via the bendingof the cytosolic half of TM5, where His164 in the His-loop directly andtransiently coordinates to Zn2+ to form a tetrahedral complex with
His70, Asp74 and Asp244 in the TMD (stage 2). This conformationalchange seems advantageous for the efﬁcient recruitment and shuttlingof Zn2+ to the metal transport pathway in the IF protomer. The next
step is the exchange of a Zn2+-coordinating residue from His164 to
His240 (TM5) via the return of the bent segment of TM5 (stage 3). This
state likely allows the conversion from the IF to OF conformations,concomitant with the movement of His70 away from the bound Zn2+
possibly via protonation to the Zn2+-binding residues (stages 4 and 5).
Finally, Zn2+ is released to the Golgi lumen (stage 6).
While the present study has signiﬁcantly advanced our understanding of the Zn2+ transport mechanism exerted by an hZnT7 protomer, it remains an important open question how two protomers
work cooperatively within the hZnT7 dimer during the Zn2+-transport
cycle. It is also possible that two protomers work independently. Further exploration of other intermediate states of the hZnT7 dimer byadvanced structural approaches including the time-resolved cryo-EM
single-particle analysis26 may reveal a whole picture of the hZnT7mediated Zn2+ transport. ZnT7 is likely to collaborate with other ZnTand ZiP family members for the maintenance of Zn2+ homeostasis in
the early secretory pathway. Detailed mechanisms of intracellular Zn2+
homeostasis resulting from the cooperation of multiple Zn2+ transporters is undoubtedly an important topics of the present-day molecular cell biology, in light of the physiological roles of Zn2+ in thefolding, maturation, and quality control of Zn2+-binding proteins, cellsignaling and regulation, and many other cellular events.

PiggyBac-hZnT7-expressing HEK293T cells were cultured in DMEM(High glucose; 4.5 g/L D-(+)-glucose with L-glutamine, phenol red andsodium pyruvate; Nacalai Tesque, Cat# 08458-16), supplemented with
4% fetal bovine serum (FBS, Nichirei Biosciences, Cat# 175012), 1%
Penicillin-Streptomycin mixed solution (PS, Nacalai Tesque, Cat#
26253-84) and 4 μg/mL Puromycin (InvivoGen, Cat# ant-pr-1), andgrown at 37 oC in an atmosphere containing 5% CO2. After 3 days, thecells were passaged into fresh DMEM (high glucose) supplemented
with 4% FBS and 1% PS. After growth for 24 h, expression was inducedby the addition of 4-Isopropylbenzoic acid (Cumate, Wako, Cat# 03906411) and Phorbol 12-Myristate 13-Acetate (PMA, Wako, Cat# 16223591) to ﬁnal concentrations of 10X (300 μg/mL) and 1X (50 nM),
respectively. Cells were continuously cultured at 37 oC for 3 h in 5% CO2
before being incubated at 30 oC in 5% CO2. After 48 h, the cells were
harvested, washed with cold-1X phosphate-buffered saline (PBS), andstored at -80 oC.
To prepare the hZnT7ΔHis-loop, the DNA segment encoding
residues His176 to Pro221 of hZnT7 was deleted using the primers asshown in Supplementary Data 1. PCR product was ampliﬁed with PrimeSTAR® Max DNA Polymerase (Takara, Cat# R045Q) and ligated by
In‑Fusion Cloning (In-Fusion HD kit, Takara, Cat# ST2317). All primer
and double-strand DNA in this study were synthesized by Euroﬁns. The
medium was changed to fresh serum-free DMEM containing 1% PS, andHEK293T cells (ATCC, Cat# CRL-3216) were transfected with
pcDNA3.1-PA-hZnT7ΔHis-loop plasmids using PEI reagent (SigmaAldrich, Cat# 408727-100 ML) at a ratio of 50 μg DNA to 150 μL PEI
reagent (1 mg/mL). Cells were continuously cultured for 24 h before
10 mM sodium butyrate (Nacalai Tesque, Cat# 12328-44) was added.
After 54 h of culturing at 30 oC and 5% CO2, the cells were harvested.
To prepare the hZnT7 HS1, HS2 and HS3 mutants, histidine residues in the His-loop were mutated to serine. For hZnT7 HS1, 7 histidine
residues near the C-terminus of TM4 (all histidine residue numbers,
e.g. His161, 164, 166, 168, 172, 174 and 176) were mutated to serine. For
hZnT7 HS2, 14 histidine residues near the N-terminus of TM5 (all histidine residue numbers, e.g. His187, 189, 192, 194, 196, 200, 204, 206,
208, 210, 212, 214, 216 and 218) were mutated to serine. In hZnT7 HS3,
all histidine residues raised above were mutated to serine. Primers anddouble-strand DNA were shown in Supplementary Data 1. For hZnT7
AAAA mutant, His70 and Asp74 (TM2), and His240 and Asp244 (TM5)were mutated to alanine.

Methods
Cloning and protein expression
A cDNA encoding human ZnT7 (hZnT7; UniPort ID: Q8NEW0) was
synthesized with codon optimization for expression in human cells.
The hZnT7 gene with an N-terminal PA-tag sequence (GVAMPGAEDDVV) was cloned into a PiggyBac Cumate Switch Inducible Vector(System Biosciences). For large-scale expression of hZnT7, stable

Nature Communications | (2023)14:4770

Protein puriﬁcation
Proteins were puriﬁed and treated at 4 oC. Cells were washed with icecold-1X PBS buffer and collected by centrifugation. Cells from 2 L of
medium (wet-weight, ~8 g) were lysed by resuspension in buffer A
containing 20 mM Tris-HCl (Nacalai Tesque, Cat# 35434-34), pH 7.5,
150 mM NaCl (Nacalai Tesque, Cat# 31320-05) and 10% glycerol(Nacalai Tesque, Cat# 17018-83), supplemented with 2 mM DTT(Nacalai Tesque, Cat# 14112-94), 0.05 mg/mL DNase I (Wako, Cat# 04326773), 5 mM MgCl2 (Nacalai Tesque, Cat# 20908-65), and 1× proteaseinhibitor cocktail (Nacalai Tesque, Cat# 25955-11). The cells weresonicated for 3 min in an ice-water cup, and the cell membrane fraction
was collected by ultracentrifugation at 200,000 × g (micro Ultracentrifuge CS100FNX, Hitachi) for 1 h at 4 oC. The pellet was dissolvedin a solution containing 1% (v/v) n-dodecyl-β-D-maltoside (DDM,
11

Articlehttps://doi.org/10.1038/s41467-023-40521-52
Zn2+ state 1 (IF)1
Zn2+-unbound (IF)
H+

Lumen
3
1b

H+

3

6 5
2

H+

3

6 5
2

1b

4

His70

3
Zn2+ state 2 (IF)

1b

4

6 5
2

4

Asp244
His240

Asp74

Bent helix

His161 Zn2+

His164

His240

His161

His164

His164

Cytosol

Flexible region

Histidine-rich loop

4
Zn2+ state 3 (OF)5
Zn2+-bound (OF)6
Zn2+-unbound (OF)

H+

Lumen
3
1b

NegativechargeStable region

6

3

5

2

1b

4
His70

6

2
His70

His1614

1b2

His161
His164

1a

3

5

5
4

His161
His164

1a

6

His164

1a

Cytosol
Fig. 8 | Proposed model of Zn2+ transport mediated by a protomer of Golgiresident Zn2+/H+ antiporter hZnT7. During its Zn2+ transport cycle, an hZnT7
protomer undergoes TM-helix rearrangement and striking His-loop movement,
leading to the facilitated Zn2+ recruitment from the cytosol to the negatively
charged cytosolic cavity. During this process, the exchange of a Zn2+-coordinating
residue from His164 to His240 takes place. Once Zn2+ is bound to the HDHD motif,
the IF form converts to the OF form, concomitant with moving away of His70. In themeantime, protons likely enter the luminal cavity to protonate His70 and possibly
His240. These conformational changes around the Zn2+-binding site likely serve tofacilitate the release of Zn2+ to the luminal side. The TM helices are presented as

tubes; TM1a, TM1b, TM3 and TM6 in gray, TM2 and TM5 in green, and TM4 inyellow. The visible His-loop segment around the C-terminus of TM4 and N-terminusof TM5 is represented by dark blue lines while dashed lines indicate the invisible
region of the His-loop. Zn2+ is represented by a red sphere. The negatively charged
surfaces of the cytosolic cavity in the IF conformation and the Zn2+ release gate in
the OF conformation are represented by thick red lines. A proton (H+) is represented by a green sphere. Note that the CTD, TM loops and another protomer of thehZnT7 homodimer are omitted for simpliﬁcation. How two protomers interplay
each other within the hZnT7 dimer during the Zn2+-transport cycle remains elusive.
See also Supplementary Fig. 23.

Nacalai Tesque, Cat# 14239-54), 0.5% (v/v) cholesteryl hemisuccinate(CHS, Sigma-Aldrich, Cat# C6512-5G), 20 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 10% glycerol and 2 mM DTT, and incubated for 2 h at 4 oC withconstant stirring. The supernatant containing PA-tagged hZnT7 wascollected by centrifugation at 15,000 × g for 30 min at 4 oC and incubated with 12 ml anti-PA-tag antibody beads (net weight, 6 ml; Wako,
Cat# 018-25843) overnight at 4 oC with gentle rotation. The resin wascollected in a glass column (Bio-Rad) and washed with 15 column
volume (CV) of Buffer B [Buffer A supplemented with 0.02% GDN(Anatrace, Cat# GDN101-1G)]. PA-tagged hZnT7 was eluted with Buffer
C [Buffer B supplemented with 0.2 mg/mL PA peptide (PH Japan Co.,
Ltd; Cat# 180688)]. The protein was concentrated using a 50-kDa
cutoff Amicon ﬁlter (Sigma-Aldrich) and puriﬁed on a SuperoseTM 6
Increase 10/300 GL gel ﬁltration column (GE Healthcare) preequilibrated with buffer B. The eluted fractions were concentratedusing a 50-kDa cutoff Amicon ﬁlter and mixed with an excess amountof the Fab#1 fragment. The hZnT7-Fab#1 mixture was further subjected to SuperdexTM 200 Increase 10/300 GL gel ﬁltration column (GEHealthcare) pre-equilibrated with glycerol-free buffer B to remove theresidual Fab#1 fragments and concentrated to ~10 mg/mL using a 100kDa cutoff Amicon ﬁlter (Sigma-Aldrich) for negative staining EM andcryo-EM measurements. To obtain higher resolution cryo-EM maps,
puriﬁcation of Zn2+-unbound hZnT7 was performed throughout in thepresence of EDTA.
hZnT7ΔHis-loop, HS1, HS2, HS3 and AAAA mutants were puriﬁed
as described for wild-type hZnT7 (hZnT7 WT). The elution fractions
from the afﬁnity chromatography column were concentrated using a
50-kDa cutoff Amicon ﬁlter and then incubated with 1 mM EDTA
(Nacalai Tesque, Cat# 15105-35) for 30 min on ice before being puriﬁed
on a SuperoseTM 6 Increase 10/300 GL gel ﬁltration column pre-Nature Communications | (2023)14:4770

12

Articleequilibrated with buffer B (20 mM Tris-HCl, pH 7.5, 150 mM NaCl and
0.02% GDN). The eluted fractions were concentrated using a 50-kDacutoff Amicon ﬁlter and used for the ITC analysis. For cryo-EM analysis,
hZnT7ΔHis-loop protein was eluted from the anti-PA-tag antibody
beads, concentrated using a 50-kDa cutoff Amicon ﬁlter, and puriﬁed
on a SuperoseTM 6 Increase 10/300 GL gel ﬁltration column preequilibrated with buffer B. The eluted fractions were again concentrated using a 50-kDa-cutoff Amicon ﬁlter and used for preparationof the hZnT7ΔHis-loop-Fab#1 complex in the same way as the hZnT7
WT-Fab#1 complex.

Preparation and screening of monoclonal antibody Fab fragments to hZnT7
All animal experiments conformed to the guidelines of the Guide for
the Care and Use of Laboratory Animals of Japan and were approved by
the Kyoto University Animal Experimentation Committee. Mouse
monoclonal antibodies against hZnT7 were generated essentially as
described27. Brieﬂy, a proteoliposome antigen was prepared byreconstituting puriﬁed hZnT7 at high density into phospholipid vesicles consisting of a 10:1 mixture of chicken egg yolk phosphatidylcholine (egg PC, Avanti Polar Lipids) and adjuvant lipid A (SigmaAldrich) to facilitate the immune response. 6-weeks old female MRL/lpr mice maintained at temperature and humidity ranges of 22 to 26 oC
and 40% to 60%, respectively, under a 12-h light and 12-h dark cycle
were injected three times at 2-week intervals with the proteoliposome
antigen. Antibody-producing hybridoma cell lines were generatedusing a conventional fusion protocol. Biotinylated proteoliposomes
were prepared by reconstituting hZnT7 with a mixture of egg PC and1,2-dipal-mitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl)(16:0 biotinyl Cap-PE; Avanti), and used as binding targets forconformation-speciﬁc antibody selection. The targets were immobilized onto streptavidin-coated microplates (Nunc). Hybridoma clones
producing antibodies recognizing conformational epitopes of hZnT7
were selected by enzyme-linked immunosorbent assay on immobilizedbiotinylated proteoliposomes (liposome ELISA), allowing positive
selection of the antibodies that recognized the native conformation ofhZnT7. Serum was diluted in the range of 3/10,000,000 to 1/1,000, and1-10 μg/mL of the hZnT7 antigen was used for ELISA experiments.
Clones were additionally screened for reduced antibody binding to
SDS-denatured hZnT7, resulting in negative selection of linear epitoperecognizing antibodies. The formation of stable complexes between
hZnT7 and each antibody clone was determined by ﬂuorescencedetection SEC. Five monoclonal antibodies were found to speciﬁcallybind to and stabilize conformational epitopes of hZnT7. The sequences of the Fabs were determined by standard 5’-RACE using total RNA
isolated from hybridoma cells (Supplementary Data 2).

Preparation of hZnT7-Fab complexes
Puriﬁed hZnT7 was mixed at a molar ratio of 1:3 with each of the ﬁveselected Fab fragments (Fab#1, YN7114-08; Fab#2, YN7117-01; Fab#3,
YN7114-03; Fab#4, YN-7148-12 and Fab#5, YN7179-03). Following
incubation for 30 min at 4 oC with gentle rotation, the mixtures were
centrifuged at 15,000 rpm for 10 min at 4 oC and injected into a
SuperdexTM 200 Increase 10/300 GL gel ﬁltration column. Fractions
containing both hZnT7 and Fab were collected and concentrated witha 100-kDa cutoff Amicon ﬁlter to around 10 mg/mL for cryo-EM
measurements.
To prepare Zn2+-bound hZnT7, hZnT7 puriﬁed in Zn2+-free buffer
was mixed with 10 μM Zn2+ and incubated on ice for 10 min. This
mixture was incubated with a 3-molar excess of Fab#1 for 0.5 h at 4 oC
with gentle rotation, centrifuged at high speed to remove precipitates,
and injected onto an SEC column, as described above, with the SEC
buffer containing 10 μM Zn2+. Fractions containing both hZnT7 andFab#1 were pooled and concentrated using a 100-kDa cutoff Amiconﬁlter to ~8-10 mg/mL for cryo-EM measurements.
Nature Communications | (2023)14:4770

https://doi.org/10.1038/s41467-023-40521-5

Negative stain EM
The hZnT7-Fab complexes were assessed by negative-stain EM, which
provided 2D reconstruction images useful for Fab selection. Grids fornegative-stain EM were prepared according to the standard protocol.
Speciﬁcally, 5 μL of puriﬁed hZnT7-Fab complex was applied to glowdischarged carbon-coated grids (ELC-C10), with the excess solution
removed with ﬁlter paper. The grid was immediately stained with 1%(w/v) uranyl acetate solution. The negatively stained EM grids wereimaged on a JEOL JEM-2010F electron microscope (JEOL) operated at
200 kV. Particles were extracted by phase-ﬂipping, followed byreference-free 2D class averaging using RELION v3.128,29.

EM data acquisition
For the initial data collection using a Talos Arctica TEM (ThermoFisher
Scientiﬁc), 3 μL of the hZnT7-Fab#1 or #3 complex samples wereapplied to glow-discharged QUANTIFOIL® (QUANTIFOIL® R 1.2/1.3,
300 mesh, Cu-Rh). The grids were blotted and immersed in liquidethane using Vitrobot Mark IV systems (FEI; ThermoFisher Scientiﬁc)
operated at 4 oC and 100% humidity. The blotting parameters for
hZnT7 WT were set at a wait time of 3 s, a blot time of 3.5-4 s, and a blot
force of 10; and the blotting parameters for hZnT7ΔHis-loop were set
at a wait time of 3 s, a blot time of 3 s, and a blotting force of -5. For theﬁnal data collection using a CRYO ARM™ 300II (JEOL), the hZnT7Fab#1 complex sample with or without Zn2+ was applied to glowdischarged QUANTIFOIL® (QUANTIFOIL® R 1.2/1.3, 200 mesh, Au). The
grids were blotted and immersed in liquid ethane using Vitrobot MarkIV systems (ThermoFisher Scientiﬁc) operated at 4 oC and 100%
humidity, with a wait time of 3 s, a blot time of 3 s, and a blot force of -5.
For preparation of the hZnT7-Fab#1 complex with exogenously added
Zn2+, the sample puriﬁed in the presence of 10 µM Zn2+ was mixed with
200 or 300 µM Zn2+ and then immediately blotted under the same
condition as above.
The grids were initially imaged using a Talos Arctica TEM (ThermoFisher Scientiﬁc) equipped with a Gatan K2 summit. The ﬁnal
datasets of the hZnT7-Fab#1 and hZnT7ΔHis-loop-Fab#1 complexes
were collected on a CRYO ARM™ 300II (JEOL) operated at 300 kV andequipped with a JEOL in-column Omega energy ﬁlter and a Gatan K3
BioQuantum detector. Data were automatically collected using SerialEM v3.730. The data collection parameters are summarized in Supplementary Tables 1 and 2.

EM data processing
Details of image processing are summarized in Supplementary Fig. 3,
13, 15 and 19. All movie stacks were subjected to motion correction
using the motion correction program implemented in RELION v3.128,29
or v4.031, and the contrast transfer function (CTF) parameters were
estimated with Patch CTF estimation in cryoSPARC v3.332 or CTFFIND4
v4.1.533. In the initial data collection by using Titan Krios G3i, we
determined a cryo-EM structure of the hZnT7-Fab#1 complex at 2.8 Å
resolution, which was used as 2D templates for template picking in theanalysis of the following datasets.
For the dataset of Zn2+-unbound hZnT7, particles were picked bytemplate picker in cryoSPARC from 6,280 micrographs with CTF
resolution of less than 6 Å resolution, and then subjected to 2D classiﬁcation. The best particles (403,333 particles) were subjected to abinitio modeling and heterogeneous reﬁnement. Particle belonging to
the best class (272,633 particles) were re-extracted at a pixel size of
0.788 Å, and then subjected to non-uniform (NU) reﬁnement. The
reﬁned particles were subjected to two arounds of Bayesian polishingand NU-reﬁnement with global CTF and defocus reﬁnement. The UCSF
pyem v0.534 was used to data conversion from cryoSPARC to RELION.
After an additional ab-initio modeling and heterogeneous reﬁnement,
particles belonging to the best two classes with high-resolution maps
(183,862 particles) were subjected to NU-reﬁnement in C2 symmetry,
resulting in an EM map reconstructed at 2.07 Å resolution (consensus
13Articlemap). To estimate the conformational heterogeneity of hZnT7, 3D
Variability Analysis (3DVA)35 and its clustering analysis in cryoSPARC
were performed by using a mask covering the hZnT7 portion. Particles
belonging to three clusters in which two protomers adopted an OF
conformation (OF-OF form), with clear density of the TM regions, wereselected and subjected to NU-reﬁnement. We found that in anothercluster (28,335 particles), one protomer adopted in an IF conformationand the other in an OF conformation (IF-OF form). The particles
belonging to this cluster were also selected and subjected to NUreﬁnement in the C1 symmetry. Finally, to improve the density of thehZnT7 portion, the reﬁned particles were subjected to local reﬁnement
using a mask covering the hZnT7 dimer, at rotation and shift standard
deviation of 3o and 2 Å, respectively. The ﬁnal maps of the OF-OF and
IF-OF forms of Zn2+-unbound hZnT7 were reconstructed at 2.21 and
2.79 Å-resolution, respectively (Supplementary Fig. 3).
For the dataset of Zn2+-bound hZnT7 prepared in the presence of
10 µM Zn2+ (named Zn2+ sample 1), particles were picked by template
picker from 6,775 micrographs, and then subjected to 2D classiﬁcation. The best classes (300,742 particles) were subjected to two rounds
of ab-initio modeling and heterogeneous reﬁnement. Particles
belonging to the best class (195,376 particles) were re-extracted at a
pixel size of 0.788 Å, and then subjected to NU-reﬁnement, followed by
two rounds of Bayesian polishing and NU-reﬁnement with global CTF
and defocus reﬁnement. After 3DVA, particles belonging to goodclusters with clear density of the TM regions were selected and subjected to NU reﬁnement and local reﬁnement. The ﬁnal map of the Zn2+
state 1 (IF/Zn-OF form) was reconstructed at 2.68 Å resolution, in which
one protomer adopted a Zn2+-bound IF conformation and the other ina Zn2+-unbound OF conformation (Supplementary Fig. 13).
For the datasets of the other Zn2+-bound hZnT7 sample preparedby addition of 200 µM or 300 µM Zn2+, particles were picked by template picker from 6,135 micrographs and then subjected to 2D classiﬁcation. The best classes (229,309 particles) were subjected to tworounds of ab-initio modeling and heterogeneous reﬁnement. Particles
belonging to the best class (137,123 particles) were re-extracted at a
pixel size of 0.788 Å, and then subjected to NU-reﬁnement, followed by
two rounds of Bayesian polishing and NU reﬁnement with global CTF
and defocus reﬁnement. Subsequent 3DVA focused on the IF protomerof the hZnT7 portion revealed that particles were classiﬁed into two
different forms, an OF/Zn-OF/Zn homodimer form (63,620 particles)
and an IF/Zn-OF heterodimer form (60,692 particles). Similar results
were obtained from the sample prepared by addition of 200 µM Zn2+.
Subsequently, the classes of these two forms were merged and subjected to NU-reﬁnement, resulting in the improvement of overall
density for each class. For the IF/Zn-OF/Zn form (Zn2+ state 2), particles
were further selected after additional heterogenous reﬁnement(98,881 particles), and then subjected to NU-reﬁnement and local
reﬁnement. The ﬁnal map of the Zn2+ state 2 was determined at 2.92 Å
resolution, in which one protomer existed in a Zn2+-bound IF conformation and the other in a Zn2+-bound OF conformation. For the OF/
Zn-OF/Zn form (Zn2+ state 3), additional ab-initio modeling, heterogenous reﬁnement, and 3DVA were performed. The best particles(56,678 particles) were subjected to NU-reﬁnement and local reﬁnement, resulting in an EM map of “Zn2+ state 3” at 3.12 Å resolution, in
which both protomer adopted a Zn2+-bound OF conformation (Supplementary Fig. 15).
For the dataset of hZnT7ΔHis-loop in Zn2+-free buffer, particles
were picked from 1,213 movies using LoG-based auto-picking in
RELION to create 2D references for template-based picking from thefull dataset of 4,850 movies. After two rounds of 2D classiﬁcation,
325,036 particles were selected and subjected to two rounds of 3Dclassiﬁcation using the 3D initial model generated by RELION. Particles
classiﬁed into the best class (188,918 particles) were re-extracted at a
pixel size of 1.182 Å and subjected to 3D reﬁnement in theC2 symmetry, CTF reﬁnement, and Bayesian polishing. SubsequentNature Communications | (2023)14:4770

https://doi.org/10.1038/s41467-023-40521-5

focused non-aligned 3D classiﬁcation in the C2 symmetry using anencompassing mask of the TM domain improved the local resolutionof the latter. The best particles with clear density for the TM domain(Class 2, 60,977 particles) were reﬁned at 3.4 Å resolution with nonuniform-reﬁnement in cryoSPARC (Supplementary Fig. 19).
For the dataset of the hZnT7ΔHis-loop in Zn2+-containing buffer,particles were picked from 500 movies by using LoG-based autopicking in RELION to create 2D references for template-based picking
from the full dataset of 6,150 movies. After two rounds of 2D classiﬁcation, a total of 424,349 particles were selected and subjected to tworounds of 3D classiﬁcation. Particles classiﬁed into the best class(142,746 particles) were re-extracted at a pixel size of 1.182 Å andsubjected to 3D reﬁnement in C2 symmetry, CTF reﬁnement, andBayesian polishing. Subsequent focused non-aligned 3D classiﬁcationin C2 symmetry using an encompassing mask of the TM domain
improved the local resolution of the latter. The best particles (142,624
particles) were reﬁned at 3.4 Å resolution with non-uniform reﬁnementin cryoSPARC (Supplementary Fig. 19).
The global resolution was estimated in RELION v3.1 or v4.0 andcryoSPARC v3.3 with a Fourier shell correlation (FCS) of 0.143. The EM
maps were sharpened and locally ﬁltered based on local resolution
with cryoSPARC v3.3.

Model building, reﬁnement and validation
An initial model of the Zn2+-free hZnT7-Fab#1 complex was automatically built with Buccaneer in CCP-EM v1.6.036. Further manualmodel building was performed with COOT v0.9.8.737,38. The model was
reﬁned against the locally ﬁltered map using phenix.real_space_reﬁnein PHENIX v1.20.139. The ﬁnal model included hZnT7 residues 22-376,
Fab light chain residues 1-218, and Fab heavy chain residues 1-234(Supplementary Data 2). The large part of the His-loop (residues 164 to228) was missing from the EM map. Residues 135-140 of hZnT7 were
removed from the model because of poor density. Structures of theother forms of hZnT7 were initially modeled using the Zn2+-unbound
OF-OF structure, followed by further manual model building with
COOT v0.9.8.7. The models were reﬁned against the locally ﬁlteredmaps using phenix.real_space_reﬁne in PHENIX v1.20.1.
The ﬁnal models were validated using MolProbity40. All the ﬁgures
were prepared in PyMOL v2.5.241, UCSF Chimera v1.1742 and UCSF
ChimeraX v1.543. Amino acid sequence alignments were analyzed by
the MAFFT version 7 online service with defaults44. Electrostatic
potential maps were calculated with APBS-PDB2PQR online service
with pKa Options setup at pH 7.545,46.

Isothermal titration calorimetry analysis
Isothermal titration calorimetry (ITC) experiments were carried out
using the MicroCal™ iTC200 calorimeter (GE Healthcare). Typically, a
0.4 µL initial injection and 1.0 µL of titrant solution [500 µM ZnCl2(Nacalai Tesque, Cat# 36920-24)] were injected into protein solution(300 μL of 20 µM ZnT7) at 150 s intervals with gentle stirring at
750 rpm at 20 °C. The buffer used was 20 mM Tris–HCl, pH 7.5,
150 mM NaCl and 0.02% GDN. The temperature was kept at 20 °C
throughout the measurement. The titration data were ﬁtted to the One
Set of Sites model provided by the Microcal Origin software (Origin 7
SR4 v7.0552; OriginLab Corporation). The binding enthalpy change(ΔH, kcal/mol), dissociation constant (Kd, μM), and the binding stoichiometry (N) were permitted to ﬂoat during the least-squares minimization and taken as the best-ﬁt values.

Zn2+ transport assay with proteoliposomes
The Zn2+ transport activity of hZnT7 was measured using FluoZin-3(ThermoFisher Scientiﬁc, Cat# F24194), a Zn2+-sensitive ﬂuorophore.
To avoid bleaching of the ﬂuorophore, the sample was shielded fromdirect light throughout the experiments. Brieﬂy, 1 mg of puriﬁed hZnT7
or hZnT7ΔHis-loop was added to 5 mg egg yolk phosphatidylcholine
14

Article(egg PC, Avanti Polar Lipids, Cat# 840051 P) in 1 mL of PBS containing
0.8% sodium cholate (Sigma-Aldrich, Cat# C6445-10G). The solution
was incubated with 100 mg of wet fresh Bio-Beads SM-2 (Bio-Rad, Cat#
152-8920) overnight at 4 °C, with gentle rotation. The beads were
removed, and the supernatant was centrifuged at 200,000 × g for
20 min at 4 oC. Each pellet was resuspended in 100 μL Buffer IN [20 mM
HEPES (Nacalai Tesque, Cat# 17546-05), pH 6.5 and 150 mM KCl(Nacalai Tesque, Cat# 28514-75)], and mixed with 50 μM Fluozin-3. The
mixtures were sonicated for 30 sec, frozen in liquid nitrogen, andthawed on ice. This procedure was repeated twice. The mixtures wereagain sonicated for 30 sec and applied to a PD-10 column (GE Healthcare, Cat# 17085101). The eluted fractions were centrifuged at 200,000
× g for 20 min at 4 oC. The pellets were resuspended in 100 μL Buffer IN
and kept on ice prior to the assay. As a negative control, empty liposomes were prepared using the same protocol. The proteoliposomesor liposomes thus prepared were mixed with 100 μM ZnSO4 (Hampton
Research, Cat# HR2-245) in a cuvette containing 500 μL buffer OUT(20 mM HEPES, pH 7.5 and 150 mM KCl). Fluorescence spectra wererecorded at an excitation wavelength of 490 nm using a Fluorescence
Spectrophotometer F-7000 (Hitachi). To normalize the timedependent ﬂuorescence signal from each proteoliposome preparation (FP), 1% octyl-β-D-glucloside (OG, Dojindo, Cat# 344-05034)
detergent solution in buffer OUT was mixed with the proteoliposome
for 1 h on ice in the dark, and the maximal ﬂuorescence signal (FPmax)
was measured. Background signal from the protein-free liposome (FL)
was also observed and normalized with the maximal ﬂuorescence signal from the protein-free liposome treated with 1% OG detergent(FLmax). Zn2+ transport activity was calculated as FP/FPmax - FL/FLmax
over time, with initial transport rates (ΔF s−1) determined by linear
regression of the data during the ﬁrst 2 s16. Data analysis was performed
using GraphPad Prism v9.3.1 (GraphPad Software, LLC).

Reporting summary
Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article.

Data availability
The data that support this study are available from the corresponding
authors upon request. Cryo-EM density maps of hZnT7 have been
deposited in the Electorn Microscopy Data Bank (EMDB) underaccession codes EMD-36048 (OF-OF), EMD-36049 (OF/Zn-OF/Zn form,
i.e. Zn2+ state 3), EMD-36050 (IF-OF form), EMD-36055 (IF/Zn-OF form,
i.e. Zn2+ state 1), EMD-36051 (IF/Zn-OF/Zn form, i.e. Zn2+ state 2), and
EMD-36053 (Zn2+-hZnT7ΔHis-loop in OF/Zn-OF/Zn form). The atomic
coordinates of human ZnT7 have been deposited in the Protein Data
Bank (PDB) under accession codes 8J7T (hZnT7-Fab complex comprising two Zn2+-unbound OF protomers; OF-OF form), 8J7U (hZnT7Fab complex comprising two Zn2+-bound OF protomers; OF/Zn-OF/Znform, i.e. Zn2+ state 3), 8J7V (hZnT7-Fab complex comprising Zn2+unbound IF and OF protomers; IF-OF form), 8J80 (hZnT7-Fab complex
comprising Zn2+-bound IF and Zn2+-unbound OF protomers; IF/Zn-OF
form, i.e. Zn2+ state 1), 8J7W (hZnT7-Fab complex comprising Zn2+bound IF and Zn2+-bound OF protomers; IF/Zn-OF/Zn form, i.e. Zn2+
state 2), 8J7X (Zn2+-unbound hZnT7ΔHis-loop-Fab complex in OF-OF
form; Apo-hZnT7ΔHis-loop) and 8J7Y (Zn2+-bound hZnT7ΔHis-loopFab complex in OF/Zn-OF/Zn form; Zn2+-hZnT7ΔHis-loop). The source
data underlying Fig. 7d and Supplementary Figure 1c are provided as a
Source Data ﬁle. Source data are provided with this paper.

References
1.

2.

Andreini, C., Banci, L., Bertini, I. & Rosato, A. Counting the zincproteins encoded in the human genome. J. Proteome Res. 5,
196–201 (2006).
Eide, D. J. Zinc transporters and the cellular trafﬁcking of zinc.
Biochim. Biophys. Acta 1763, 711–722 (2006).

Nature Communications | (2023)14:4770

https://doi.org/10.1038/s41467-023-40521-5
3.

Kambe, T. Molecular architecture and function of ZnT transporters.
Curr. Top. Membr. 69, 199–220 (2012).
4. Kambe, T., Taylor, K. M. & Fu, D. Zinc transporters and their functional integration in mammalian cells. J. Biol. Chem. 296,
100320 (2021).
5. Gao, H. L. et al. Golgi apparatus localization of ZNT7 in the mouse
cerebellum. Histol. Histopathol. 24, 567–572 (2009).
6. Chi, Z. H. et al. ZNT7 and Zn2+ are present in different cell
populations in the mouse testis. Histol. Histopathol. 24,
25–30 (2009).
7. Kirschke, C. P. & Huang, L. ZnT7, a novel mammalian zinc transporter, accumulates zinc in the Golgi apparatus. J. Biol. Chem. 278,
4096–4102 (2003).
8. Gao, H. L. et al. Expression of zinc transporter ZnT7 in mousesuperior cervical ganglion. Auton. Neurosci. 140, 59–65 (2008).
9. Kowada, T. et al. Quantitative imaging of labile Zn2+ in the Golgi
apparatus using a localizable small-molecule ﬂuorescent probe.
Cell Chem. Biol. 27, 1521–1531.e8 (2020).
10. Amagai, Y. et al. Zinc homeostasis governed by Golgi-resident ZnTfamily members regulates ERp44-mediated proteostasis at the ERGolgi interface. Nat. Commun. 14, 2683 (2023).
11. Huang, L. et al. ZnT7 (Slc30a7)-deﬁcient mice display reduced bodyzinc status and body fat accumulation. J. Biol. Chem. 282,
37053–37063 (2007).
12. Tepaamorndech, S., Huang, L. & Kirschke, C. P. A null-mutation in
the Znt7 gene accelerates prostate tumor formation in a transgenic
adenocarcinoma mouse prostate model. Cancer Lett. 308,
33–42 (2011).
13. Tepaamorndech, S. et al. Zinc transporter 7 deﬁciency affects lipid
synthesis in adipocytes by inhibiting insulin-dependent Akt activation and glucose uptake. FEBS J. 283, 378–394 (2016).
14. Lu, M. & Fu, D. Structure of the zinc transporter YiiP. Science 317,
1746–1748 (2007).
15. Lu, M., Chai, J. & Fu, D. Structural basis for autoregulation of the zinctransporter YiiP. Nat. Struct. Mol. Biol. 16, 1063–1067 (2009).
16. Coudray, N. et al. Inward-facing conformation of the zinc transporter YiiP revealed by cryoelectron microscopy. Proc. Natl Acad.
Sci. 110, 2140–2145 (2013).
17. Lopez-Redondo, M. et al. Structural basis for the alternating accessmechanism of the cation diffusion facilitator YiiP. Proc. Natl Acad.
Sci. 115, 3042–3047 (2018).
18. Lopez-Redondo, M. et al. Zinc binding alters the conformational
dynamics and drives the transport cycle of the cation diffusion
facilitator YiiP. J. Gen. Physiol. 153, e202112873 (2021).
19. Xue, J. et al. Cryo-EM structures of human ZnT8 in both outwardand inward-facing conformations. Elife 9, e58823 (2020).
20. Outten, C. E. & O’Halloran, T. V. Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis. Science 292,
2488–2492 (2001).
21. Liu, R. et al. Organelle-level labile Zn2+ mapping based on targetable
ﬂuorescent sensors. ACS Sens. 7, 748–757 (2022).
22. Tanaka, N., Kawachi, M., Fujiwara, T. & Maeshima, M. Zinc-binding
and structural properties of the histidine-rich loop of Arabidopsis
thaliana vacuolar membrane zinc transporter MTP1. FEBS Open Bio.
3, 218–224 (2013).
23. Kawachi, M., Kobae, Y., Mimura, T. & Maeshima, M. Deletion of ahistidine-rich loop of AtMTP1, a vacuolar Zn(2+)/H(+) antiporter of
Arabidopsis thaliana, stimulates the transport activity. J. Biol. Chem.
283, 8374–8383 (2008).
24. Tanaka, Y. et al. Structural basis for the drug extrusion mechanism
by a MATE multidrug transporter. Nature 496, 247–251 (2013).
25. Miyauchi, H. et al. Structural basis for xenobiotic extrusion by
eukaryotic MATE transporter. Nat. Commun. 8, 1633 (2017).
26. Dandey, V. P. et al. Time-resolved cryo-EM using Spotiton. Nat.
Methods 17, 897–900 (2020).
15

Article
27. Jaenecke, F. et al. Generation of conformation-speciﬁc antibody
fragments for crystallization of the multidrug resistance transporter
MdfA. Methods Mol. Biol. 1700, 97–109 (2018).
28. Scheres, S. H. W. RELION: Implementation of a Bayesian approachto cryo-EM structure determination. J. Struct. Biol. 180,
519–530 (2012).
29. Zivanov, J. et al. New tools for automated high-resolution cryo-EM
structure determination in RELION-3. Elife 7, e42166 (2018).
30. Mastronarde, D. N. Automated electron microscope tomographyusing robust prediction of specimen movements. J. Struct. Biol. 152,
36–51 (2005).
31. Kimanius, D., Dong, L., Sharov, G., Nakane, T. & Scheres, S. H. W.
New tools for automated cryo-EM single-particle analysis in
RELION-4.0. Biochem. J. 478, 4169–4185 (2021).
32. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
33. Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus
estimation from electron micrographs. J. Struct. Biol. 192,
216–221 (2015).
34. Asarnow, D., Palovcak, E. & Cheng, Y. Asarnow/pyem: UCSF pyem
v0.5 (v0.5). Zenodo https://doi.org/10.5281/zenodo.3576630 (2019).
35. Punjani, A. & Fleet, D. J. 3D variability analysis: Resolving continuous
ﬂexibility and discrete heterogeneity from single particle cryo-EM.
J. Struct. Biol. 213, 107702 (2021).
36. Burnley, T., Palmer, C. M. & Winn, M. Recent developments in the
CCP-EM software suite. Acta Crystallogr. D. Struct. Biol. 73,
469–477 (2017).
37. Emsley, P. & Cowtan, K. Coot: Model-building tools for moleculargraphics. Acta Crystallogr. D. Bio. Crystallogr. 60, 2126–2132 (2004).
38. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features anddevelopment of Coot. Acta Crystallogr. D. Bio. Crystallogr. 66,
486–501 (2010).
39. Adams, P. D. et al. PHENIX: a comprehensive Python-based system
for macromolecular structure solution. Acta Crystallogr. D. Bio.
Crystallogr. 66, 213–221 (2010).
40. Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35,
W375–W383 (2007).
41. DeLano, W. L. The PyMOL molecular graphics system. http://www.
pymol.org (2002).
42. Pettersen, E. F. et al. UCSF Chimera-a visualization system for
exploratory research and analysis. J. Comput. Chem. 25,
1605–1612 (2004).
43. Goddard, T. D. et al. UCSF ChimeraX: Meeting modern challenges
in visualization and analysis. Protein Sci. 27, 14–25 (2018).
44. Katoh, K., Rozewicki, J. & Yamada, K. D. MAFFT online service:
multiple sequence alignment, interactive sequence choice andvisualization. Brief. Bioinform. 20, 1160–1166 (2019).
45. Jurrus, E. et al. Improvements to the APBS biomolecular solvation
software suite. Protein Sci. 27, 112–128 (2018).
46. Unni, S. et al. Web servers and services for electrostatics calculations with APBS and PDB2PQR. J. Comput. Chem. 32,
1488–1491 (2011).

Acknowledgements
We thank T. Yokoyama, K. Nanatani, J. Inoue, S. Koshiba and M. Yamamoto for management of the cryo-EM facility at Tohoku University
Medical Megabank. This work was supported by funding from AMED-Nature Communications | (2023)14:4770

https://doi.org/10.1038/s41467-023-40521-5
CREST (21gm1410006h0001) to K.I., JSPS KAKENHI to K.I. (18H03978,
21H04758 and 21H05247), Canon Medical Systems Corporation to K.K.
and K.I., and the Basis for Supporting Innovative Drug Discovery and Life
Science Research (BINDS) from the Japan Agency for Medical Researchand Development (AMED) under grant numbers JP19am0101115 (support
number: 1025), JP19am0101078 (support No. 2293), JP21am0101079(support no. 2343) and JP22ama121038.

Author contributions
H.B.B. performed almost all experiments, structure modeling andstructure reﬁnement. S.W. performed acquisition of cryo-EM images
with CRYO-ARM300II, image processing, structure modeling andreﬁnement and assisted in sample preparation. A.T. and M.K. acquired
cryo-EM images with Talos Arctica and Titan Krios G3i. N.N., K.L., T.U. and
S.I. prepared the monoclonal antibody Fab fragment recognizing hZnT7.
Y.K. provided anti-PA-tag antibody beads for puriﬁcation of PA-tagged
hZnT7. M.I. prepared a stable expression cell line for hZnT7. H.F. and K.K.
provided many insights into structures and mechanisms of SLC transporters including zinc transporters to improve the manuscript. H.B.B.,
S.W. and K.I. prepared the ﬁgures and wrote the manuscript. All authors
discussed the results, critically read the manuscript, and approved themanuscript for submission. K.I. supervised this work.

Competing interests
The authors declare no competing interests.

Additional information
Supplementary information The online version contains
supplementary material available athttps://doi.org/10.1038/s41467-023-40521-5.
Correspondence and requests for materials should be addressed toKenji Inaba.
Peer review information Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A
peer review ﬁle is available.
Reprints and permissions information is available at
http://www.nature.com/reprints
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, aslong as you give appropriate credit to the original author(s) and thesource, provide a link to the Creative Commons license, and indicate ifchanges were made. The images or other third party material in thisarticle are included in the article’s Creative Commons license, unlessindicated otherwise in a credit line to the material. If material is notincluded in the article’s Creative Commons license and your intended
use is not permitted by statutory regulation or exceeds the permitteduse, you will need to obtain permission directly from the copyrightholder. To view a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/.
© The Author(s) 2023

16