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Structure and transport mechanism of the human calcium

pump SPCA1
2,4 ✉ 1✉
Mengqi Wu1,5, Cang Wu 1,5
, Tiefeng Song2,5, Kewu Pan3, Yong Wang and Zhongmin Liu

© The Author(s) 2023

Secretory-pathway Ca2+-ATPases (SPCAs) play critical roles in maintaining Ca2+ homeostasis, but the exact mechanism of SPCAs-
mediated Ca2+ transport remains unclear. Here, we determined six cryo-electron microscopy (cryo-EM) structures of human SPCA1
(hSPCA1) in a series of intermediate states, revealing a near-complete conformational cycle. With the aid of molecular dynamics
simulations, these structures offer a clear structural basis for Ca2+ entry and release in hSPCA1. We found that hSPCA1 undergoes
unique conformational changes during ATP binding and phosphorylation compared to other well-studied P-type II ATPases. In
addition, we observed a conformational distortion of the Ca2+-binding site induced by the separation of transmembrane helices 4L
and 6, unveiling a distinct Ca2+ release mechanism. Particularly, we determined a structure of the long-sought CaE2P state of P-type
IIA ATPases, providing valuable insights into the Ca2+ transport cycle. Together, these findings enhance our understanding of Ca2+
transport by hSPCA1 and broaden our knowledge of P-type ATPases.
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Cell Research (2023) 33:533–545; https://doi.org/10.1038/s41422-023-00827-x

INTRODUCTION epidermal keratinocytes, SPCA1 contributes ~67% of Ca2+ uptake

Calcium ion (Ca2+) is a vital signaling molecule in cells that into the GA, making keratinocytes highly sensitive to changes in
regulates numerous cellular processes, such as proliferation, functional SPCA1 level.3,17 Haploinsufficiency of SPCA1 leads to
differentiation, apoptosis, secretion, contraction, and fertilization.1 the development of Hailey-Hailey disease (HHD), a heritable
The Golgi apparatus (GA), along with the endoplasmic reticulum autosomal dominant skin disease.18 Deficiency of SPCA1-mediated
(ER) and mitochondria, serve as critical Ca2+ storage organelles Ca2+ transport appears to be the causative factor of HHD.19,20
that regulate Ca2+ homeostasis and subsequent Ca2+ signaling in Additionally, SPCA1 has been implicated in basal-type breast
cells.2 Maintaining an appropriate Ca2+ concentration in the GA cancer,21 and its upregulation is associated with microcalcifica-
lumen is critical for normal protein synthesis, processing, and tions during breast cancer development,22,23 a process dependent
sorting.3 Several proteins, including sarcoplasmic/endoplasmic on SPCA1’s Ca2+-pumping activity.22 Therefore, a thorough
reticulum Ca2+-ATPases (SERCAs), inositol-1,4,5-trisphosphate characterization of SPCA1-mediated Ca2+ transport may provide
receptors (IP3Rs), ryanodine receptors (RYRs), and secretory novel insight into understanding diseases associated with SPCA1.
pathway Ca2+-ATPases (SPCAs), play essential roles in balancing SPCA1 is a member of the P-type IIA ATPases24 and transports
the Ca2+ homeostasis of GA.2,4 While the transport mechanisms of substrates through a series of intermediate states in a process
SERCAs,5–7 IP3Rs,8,9 and RYRs10–13 have been well structurally known as the Post-Albers reaction cycle.25 This cycle includes
characterized using X-ray crystallography and single-particle cryo- several intermediate states such as CaE1, CaE1-ATP, CaE1P-ADP,
electron microscopy (cryo-EM), the transport mechanism of SPCAs CaE1P, CaE2P, E2P, E2~P, E2Pi, E2, and E1 (where P indicates
remains elusive due to a lack of crucial structural information. protein phosphorylation and Pi indicates non-covalently bound
Moreover, unlike other Ca2+ transporters distributed on multiple phosphate).26–28 It is worth noting that CaE1P is an ADP-sensitive
organelles,4 SPCAs are mainly localized to the GA3,14 and may play state in which the phosphorylated Asp is far away from the TGE
unique roles in regulating the functions of GA. motif, allowing the binding of ADP to regenerate ATP in a reverse
The SPCA family in mammals consists of at least two subtypes, reaction.29,30 Due to the loss of ADP binding affinity at the
SPCA1 and SPCA2, which are encoded by the ATP2C1 and ATP2C2 catalytic site, CaE2P is believed to be an ADP-insensitive state with
genes, respectively.3,4 SPCA1 was identified as a Ca2+ pump earlier occluded Ca2+ at the transport site.29,31 E2P is an intermediate
than SPCA2,15 and as a result, its physiological function and state after Ca2+ release.28 SERCAs, also members of P-type IIA
properties are better understood. SPCA1 is crucial for maintaining ATPases, have been extensively studied and serve as a model
the proper structure and function of the GA.14,16 Silencing of system for understanding the Ca2+ transport mechanism of P-type
SPCA1 induces fragmentation of the GA ribbon and slows down Ca2+-ATPases.32,33 However, there are some key differences
protein transport in the Golgi compartments.14 In human between SERCAs and SPCA1. For example, SERCAs contain two

1
Department of Immunology and Microbiology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, Guangdong, China. 2College of Life Sciences,
Zhejiang University, Hangzhou, Zhejiang, China. 3School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China. 4The Provincial International Science
and Technology Cooperation Base on Engineering Biology, International Campus of Zhejiang University, Haining, Zhejiang, China. 5These authors contributed equally: Mengqi
Wu, Cang Wu, Tiefeng Song. ✉email: yongwang_isb@zju.edu.cn; liuzm@sustech.edu.cn

Received: 14 February 2023 Accepted: 8 May 2023

Published online: 31 May 2023
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high-affinity Ca2+-binding sites, namely site I and site II, and S6a). Three characteristic cytosolic amino acid motifs in P-type
transport two Ca2+ per ATP hydrolysis cycle,5,6 whereas SPCA1 ATPase (480KGA in the N domain for ATP binding, 350DKT in the P
may transport only one divalent cation per Post-Albers cycle.34 domain for phosphorylation, and 189TGE in the A domain for
Additionally, antagonists that effectively inhibit SERCAs, such as dephosphorylation)42 were well solved in the EM structure
thapsigargin (TG) and cyclopiazonic acid (CPA), show weaker (Supplementary information, Fig. S6b). An AMP-PCP molecule
inhibitory effects on SPCA1,35,36 suggesting a distinct inhibitory was observed near the conserved 480KGA and 350DKT motifs,
mechanism. Notably, there is still no structure in the CaE2P state interacting with an Mg2+ ion and several residues, including
available even for the most extensively studied SERCAs, limiting Thr352, Lys424, Glu427, Phe454, Lys459, Lys480, Gly481, Arg523,
our understanding of the complete Ca2+ transport cycle of P-type Leu525, Thr570, Gly571, and Arg619 (Fig. 3b), similar to the
IIA ATPases. Recently, the structures of hSPCA1 in the CaE1-ATP reported CaE1-ATP structure.37 Additionally, a well-defined Ca2+
and E2P states were reported, providing detailed insights into ATP density was observed in the TM domain (TMD) (Fig. 2a, b; see the
and Ca2+ binding as well as some information on the structural next section for more details). These structural features allowed us
basis for Ca2+ release.37 However, the current structural informa- to assign the AMP-PCP-bound structure of hSPCA1 to the CaE1-
tion is insufficient to fully explain the complete Ca2+ transport ATP state.
mechanism of hSPCA1.
In this study, we determined the cryo-EM structures of hSPCA1 Structural basis for Ca2+ binding
in six intermediate states, which allowed us to characterize the To gain insight into how hSPCA1 recognizes Ca2+, we successfully
Ca2+-binding pocket and capture the conformational rearrange- obtained the structures of hSPCA1 bound to Ca2+ in three
ments of hSPCA1 throughout a near-complete Post-Albers cycle. different states: CaE1, CaE1-ATP, and CaE1P-ADP. These structures
Further all-atom molecular dynamics (MD) simulations with had resolutions of 3.59 Å, 3.52 Å, and 3.71 Å, respectively
explicit solvent enabled us to identify functional states and (Supplementary information, Figs. S2, S3). We found that the
capture substrate entry and exit pathways. Our results provide TMDs were nearly identical in all three structures (Fig. 3c).
unprecedented insight into the Ca2+ transport mechanism of Additionally, we observed an ion-like EM density between TM4
hSPCA1, expand our understanding of the working mode of and TM6 (Fig. 2a, b; Supplementary information, Fig. S6d),
P-type II ATPases, and advance our knowledge of P-type ATPases. surrounded by highly conserved residues across the SPCA family
(Supplementary information, Fig. S6c). Based on the biochemical
assay43 and 1-µs explicit solvent all-atom MD simulations of the
RESULTS CaE1-ATP structure (Fig. 2c), we identified this density as a Ca2+
Structure determination and overall architecture of hSPCA1 ion that is stably bound to the protein.
To study the structure of hSPCA1, we isolated and purified the Structural analysis revealed that the Ca2+ coordinates with the
hSPCA1 proteins (see more details in Materials and Methods). side-chain oxygen atoms of several residues, including Glu308,
Analysis using size-exclusion chromatography (SEC) and sodium Asn738, and Asp742, and the main-chain carbonyl oxygen atoms
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of other residues, including Val303, Ala304, and Ile306 (Fig. 2b),
showed that hSPCA1 was highly pure and eluted as a single and consistent with the results reported by Chen et al.37 We also found
symmetric peak (Supplementary information, Fig. S1a, b). Western that the Ca2+-binding site in hSPCA1 corresponds to site II in
blot assay confirmed the identity of the protein (Supplementary hSERCA2 (Fig. 2d, e). However, hSPCA1 does not have a
information, Fig. S1c), and an ATPase activity assay demonstrated corresponding site I because some related residues (e.g., Asn767
that it has ATP hydrolysis activity (Supplementary information, Fig. and Glu770 in hSERCA2) are replaced by smaller ones (Ser710 and
S1d). These results indicated that the purified hSPCA1 was Ala713 in hSPCA1) and the potential Ca2+-binding site is further
qualified for cryo-EM analysis. occupied by a bulky residue Met741 in hSPCA1 (Fig. 2d–f). Asp799
To gain a detailed understanding of the transport cycle of in TM6 and Glu907 in TM8 coordinate with the Ca2+ in the site I of
hSPCA1, we incubated the protein with different ATP or hSERCA2 with a distance of ~4.4 Å between the two residues
phosphate analogs before preparing cryo-EM samples using (Fig. 2d, e), but the counterpart distance (Asp742 and Asp819) in
methods described in previous studies (see more details in hSPCA1 is ~8.8 Å (Fig. 2e), too large to coordinate with a Ca2+ ion.
Materials and Methods).6,29,38,39 We recorded and processed This disrupts the coordination geometry of the potential Ca2+-
movies of hSPCA1 in different states using cryoSPARC40 and binding site I in hSPCA1, while site II retains its ability to bind Ca2+
Relion41 softwares (Supplementary information, Figs. S2–S5), and (Fig. 2e). This may explain why hSPCA1 only transports one Ca2+
obtained cryo-EM structures representing six intermediate states ion per cycle.
(Fig. 1c) at overall resolutions ranging from 3.2 Å to 3.7 Å
according to the gold-standard Fourier shell correlation (FSC) A unique movement of cytosolic domains and TMD during
0.143 criterion (Supplementary information, Figs. S2–S5 and Table phosphorylation
S1). This allowed us to build accurate atomic models of most Phosphorylation is a crucial step in the transport reaction of P-type
regions of hSPCA1 (except the two flexible terminals, Met1 to ATPases. However, hSPCA1 exhibits a distinct movement during
Glu15, and Gln906 to Val919) and to assign the structures to this process compared to SERCAs and sodium-potassium pumps
specific intermediate states in the transport cycle (Fig. 1c). We first (Na+/K+-ATPases, NKAs).
focused on the AMP-PCP-bound structure in the CaE1-ATP state to Like SERCAs5 and NKAs,44 the cytosolic domains of hSPCA1
characterize the overall structure of hSPCA1. were loosely arranged in the CaE1 state to allow ATP binding
The overall structure of hSPCA1 is typical of a P-type ATPase, (Supplementary information, Fig. S6e, f). In hSPCA1, the N domain
with ten transmembrane (TM) helices (TM1–TM10) and three interacted with the P domain through several residues, including
conserved cytosolic domains: the actuator (A), phosphorylation Gly518, Ser519, Ala520, Gly521, Ile545, Glu575, Thr576, Ala579, and
(P), and nucleotide-binding (N) domains (Fig. 1a–c). The A domain Ile580, with a distance between the 480KGA motif and the 350DKT
is connected to TM1–TM3, while the P domain is linked to TM4 motif at ~25.0 Å (Fig. 3a). Meanwhile, the A domain interacted
and TM5. The N domain is connected to the P domain through with the P domain through multiple residues, including Val165,
two linkers (Fig. 1a–c). Notably, TM1, TM4, and TM6 are divided Gly166, Asp167, Arg168, and Leu222 in the A domain, and Val646,
into two sub-helices (TM1C and TM1L, TM4C and TM4L, TM6C and Asp667, Val668, and Glu671 in the P domain, with a distance
TM6L, C indicated the cytosol and L represented the GA lumen) of 33.2 Å from the 350DKT motif to the 189TGE motif (Fig. 3a). As
(Fig. 1a; Supplementary information, Fig. S6a). TM2–TM5 have long expected, there was no interaction observed between the A and N
cytoplasmic extensions (Fig. 1c; Supplementary information, Fig. domains (Fig. 3a).

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Fig. 1 Overall architecture of hSPCA1. a Topology diagram of hSPCA1. Conserved cytoplasmic domains and TM helices are schematically
illustrated. The domains are colored as follows: A domain, cornflower blue; N domain, forest green; P domain, peru (orange-brown); N-terminal
region, red; C-terminal region, deep blue; TM1–TM2, cyan; TM3–TM4, hot pink; TM5–TM10, medium purple. b Cryo-EM map (contoured at 5.3
σ) of hSPCA1 in the CaE1-ATP state, shown with the same color scheme as in a. c Atomic models of the six resolved structures of hSPCA1.

The binding of AMP-PCP caused the N domain to rotate ~57.0° and ~8.3°, respectively, towards the P domain (Fig. 3d). Moreover,
towards the P domain, bringing the 480KGA motif and the 350DKT TM1 moved upward by ~13.0 Å towards TM4 (Fig. 3d). Interest-
motif closer together ( ~17.8 Å) (Fig. 3b). No significant conforma- ingly, the cytosolic domains of hSPCA1 in the CaE1-ATP state are
tional changes were observed in the TMD and the A and P highly similar to those of NKAs in the NaE1-ATP state,44 with no
domains during the transition of CaE1 → CaE1-ATP, with the root contact between the A and N domains (Fig. 3e).
mean square deviation (RMSD) being 0.83 Å (Fig. 3c). In contrast, The hydrolysis of ATP and transfer of a phosphate group to
SERCA1 underwent notable movement in these domains upon Asp350 in hSPCA1 resulted in the CaE1P-ADP state. This
ATP binding (Supplementary information, Fig. S6g), resulting in a phosphorylation did not cause significant changes in the overall
compact headpiece with the A and N domains in extensive structure of hSPCA1 (RMSD is 0.87 Å) (Fig. 3c), similar to what was
contact and an upward movement of TM1 (Supplementary observed in SERCA1.6 In contrast, the Na+-bound NaE1P-ADP state
information, Fig. S6g).5,6 Compared to the CaE1-ATP state in of NKAs39 displayed a movement of the A and N domains towards
hSPCA1, the A domain and N domain of SERCA1 rotated ~18.6° each other compared to the NaE1-ATP state (Supplementary

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Fig. 2 Structural basis for Ca2+ binding. a Side view of hSPCA1 in the CaE1-ATP state. N and P domains are colored forest green and peru,
respectively. A domain and TM1–TM2 are hidden for a clearer view. TM3 is colored hot pink. TM4 and TM6 are colored orange-red. TM5 and
TM7–TM10 are medium purple. Ca2+ is shown as the lime sphere. The Ca2+ density (contoured at 15 σ) is shown as black mesh. b Atomic
models and cryo-EM densities of Ca2+ and Ca2+-binding site of hSPCA1 in the CaE1-ATP state. Ca2+-binding residues are shown as the cyan
sticks and Ca2+ is shown as the lime sphere. The densities are shown as black mesh, contoured at 15 σ. The interactions between residues and
Ca2+ are shown by magenta dashed lines. c The stability of the Ca2+ substrate in the binding site observed in a 1-µs explicit solvent all-atom
MD simulation. It shows the trajectory of the distance between the Ca2+ ion and the center of the lipid bilayer. d The two Ca2+-binding sites of
hSERCA2 in the CaE1-ATP state (PDB: 6LLE). Ca2+ ions are shown as dark gray spheres. Ca2+-binding residues are shown as sticks. The magenta
dashed lines represent the metal coordination bonds. e Comparison of the Ca2+-binding sites between hSPCA1 and hSERCA2 in the CaE1-ATP
state. Site I and site II counterpart residues in hSPCA1 are shown as cyan sticks. Dark gray sticks are the Ca2+-binding site I residues in
hSERCA2. The black dashed lines show the distance between the Ca2+-binding oxygen atoms from Asp799 and Glu907 in hSERCA2 and the
distance between the oxygen atoms from Asp742 and Asp819 in hSPCA1, respectively. f Sequence alignment of hSPCA1 and hSERCA2. Black
asterisks indicate residues contributing to Ca2+-binding site I. Blue asterisks indicate residues contributing to Ca2+-binding site II. The red
asterisk indicates the residue contributing to both sites I and II.

information, Fig. S6h), yielding ~15.8° and ~6.3° rotation, 1 mM CaCl2 to capture the phosphorylated states of hSPCA1. This
respectively, in comparison to the CaE1P-ADP state of hSPCA1 resulted in two cryo-EM maps with different conformations, BeF3–-
(Fig. 3f). This leads to a more compact headpiece and upward Class I and BeF3–-Class II, with resolutions of 3.42 Å and 3.25 Å,
movement of TM1 towards TM4 ( ~11.2 Å compared to hSPCA1, respectively (Fig. 4a; Supplementary information, Fig. S4). The
Fig. 3f). near-atomic resolutions of these two maps allowed us to trace the
In summary, the transport process in P-type ATPases can vary BeF3– densities which were clearly visible near Asp350 (Fig. 4a).
within the same subfamily. The fact that the cytosolic headpiece In the BeF3–-Class I state, compared to the CaE1P-ADP state, the
remains open and that the TMD remains almost stationary in N domain rotated ~46.9° against the P domain, the P domain
SPCAs during the phosphorylation reaction CaE1 → CaE1- tilted ~27.2° towards the A domain, and the A domain rotated
ATP → CaE1P-ADP may represent a key feature that distinguishes ~64.4° towards the P domain (Fig. 4b). This resulted in the
them from other P-type II ATPases, such as SERCAs and NKAs. movements of the conserved 189TGE and 350DKT motifs by ~19.8 Å
and ~12.1 Å, respectively (Fig. 4c), shortening the distance
A compact headpiece in the Ca2+-occluded CaE2P state between them ( ~11.5 Å) (Fig. 4c). The A domain extensively
To understand the movement of Ca2+ in the following state after contacted both the P and N domains. When aligned based on the
the CaE1P-ADP state, we incubated the proteins with BeF3– and P domain, the ADP-binding site was mainly occupied by the

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Fig. 3 A unique movement of cytosolic domains and TMD during phosphorylation. a Cytoplasmic domains of hSPCA1 in the CaE1 state. A,
N, and P domains are colored cornflower blue, forest green, and peru, respectively. Conserved 189TGE, 480KGA, and 350DKT motifs are colored
hot pink, orange-red, and cyan, respectively. The dashed lines in the left inset show the distances from the Cα of Gly481 to the Cα of Lys351
and from the Cα of Glu191 to the Cα of Asp350. The right upper inset and right lower inset show the interaction surface between domains N
and P, and that between domains A and P, respectively. The residues are shown as sticks. b Cytoplasmic domains of hSPCA1 in the CaE1-ATP
state. AMP-PCP (purple ribbon), Mg2+ (dark red sphere), and the corresponding cryo-EM densities (black mesh, contoured at 16.8 σ) are
shown. The dashed lines in the left inset show the distances from the Cα of Gly481 to the Cα of Lys351, and from the Cα of Glu191 to the Cα of
Asp350. c Structural comparison of states CaE1 (tan), CaE1-ATP (light sky blue) and CaE1P-ADP (plum) of hSPCA1 by global alignment.
d Structural comparison by P domain alignment (left) and TM3–TM4 alignment (right) of hSPCA1 (light sky blue) and SERCA1 (dark gray, PDB:
1T5S) in the CaE1-ATP state. The red arrow (right) shows the distance from the Cα of Pro82 in hSPCA1 to the Cα of Leu60 in SERCA1.
e Structural comparison by global alignment (left) and TM3–TM4 alignment (right) of hSPCA1 (light sky blue) and NKA (dark gray, PDB: 7E21)
in the substrate-bound E1-ATP state. f Structural comparison by P domain alignment (left) and TM3–TM4 alignment (right) of hSPCA1 (plum)
and NKA (dark gray, PDB: 8D3U) in the substrate-bound E1P-ADP state. The red arrow (right) shows the distance from the Cα of Pro82 in
hSPCA1 to the Cα of Gly89 in NKA.

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Fig. 4 Structural details of hSPCA1 in the CaE2P and early E2P states. a Atomic models (color ribbon) and cryo-EM densities (dark gray
mesh, contoured at 7.0 σ) of hSPCA1 in the CaE2P and early E2P states. The cryo-EM densities of BeF3–, Mg2+, Asp350, and Asp644 of hSPCA1
in the CaE2P and early E2P states are shown (black mesh, contoured at 10.0 σ). b Structural comparison of hSPCA1 cytoplasmic domains in the
CaE2P (color) and CaE1P-ADP (dark gray) states by global alignment. c The movement of the 189TGE and 350DKT motifs from the CaE1P-ADP
state to the CaE2P state. The black dashed lines show the distance between Cα of Asp350 in the CaE1P-ADP state and that in the CaE2P state,
and the distance between Cα of Glu191 in the CaE1P-ADP state and that in the CaE2P state. The deep red dashed line shows the distance
between Cα of Glu191 and Cα of Asp350 in the CaE2P state. d The conserved 189TGE motif (hot pink) in the CaE2P state and the ADP
molecule (dark gray) in the CaE1P-ADP state. e Structural comparison of hSPCA1 TMD in the CaE2P (color) and CaE1P-ADP (dark gray) states.
The red text shows the distance from the Cα of Leu96 in the CaE1P-ADP state to the Cα of Leu96 in the CaE2P state. f Structural comparison of
hSPCA1 in the early E2P (color) and CaE2P (dark gray) states by global alignment. g Structural comparison of the 189TGE and 350DKT motifs in
the early E2P (color) and CaE2P states (dark gray). h Structural comparison of hSPCA1 TMD in the early E2P (color) and CaE2P (dark gray) states.
i Structural comparison of hSPCA1 TM4 and TM6 in the early E2P (orange-red) and CaE2P (dark gray) states by alignment of TM4 and TM6. The
black dashed lines (right) show the distances from the Ca2+ ion (dark gray sphere) in the CaE2P state to the main-chain carbonyl oxygen
atoms of Val303, Ala304, and Ile306, and the side-chain oxygen atoms of Glu308, Asn738, and Asp742.

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189
TGE motif from the A domain (Fig. 4d), ruling out the possibility Based on the structural features and comparisons of BeF3–-Class
of assigning the BeF3–-Class I to the ADP-sensitive CaE1P state. II to the adjacent CaE2P and E2P states, we confidently assigned
Moreover, significant rotation of the cytosolic domains caused BeF3–-Class II to an early E2P state of hSPCA1 along the transport
notable conformational rearrangements of TMD compared to that cycle. The structural rearrangement during the CaE2P → early
in CaE1P-ADP, with TM1–TM2 moving ~9.2 Å towards the cytosolic E2P → E2P transition provides clear information on Ca2+ release
leaflet as a whole rigid body and the cytoplasmic parts of TM4 and from hSPCA1.
TM5 (TM4C and TM5C) inclining towards TM1–TM2 (Fig. 4e).
However, the remaining part of the TMD, including the Ca2+- Conformational changes during dephosphorylation
binding residues, had a similar arrangement in both structures To understand the conformational changes during hSPCA1
(RMSD is 0.74 Å; Fig. 4e; Supplementary information, Fig. S6d). dephosphorylation, we trapped hSPCA1 in the E2~P state by
Interestingly, a Ca2+-like density was resolved in the Ca2+-binding incubating the protein with AlF4– (Fig. 5d). The EM densities
site (Supplementary information, Fig. S6d), indicating that Ca2+ derived from AlF4– were clearly visible near Asp350 (Fig. 5e).
can be stably trapped in the BeF3–-Class I state of hSPCA1; this was Superimposition of the reported E2P37 and our E2~P structures
confirmed by MD simulation (Supplementary information, Video showed that the headpiece of hSPCA1 underwent significant
S1). rearrangements during the E2P to E2~P transition (Fig. 5d, e). Both
The structural features of an ADP-insensitive headpiece and the A and N domains moved towards the P domain, rotating
Ca2+ occluded at the binding site strongly support that BeF3–- ~28.8° and ~17.0°, respectively (Fig. 5e), consistent with what was
Class I should be assigned to the CaE2P state rather than the observed in SERCA2b.30 This changed the interface between the
189
CaE1P state. Since no substrate-bound E2P structure of P-type TGE motif and the P domain during the E2P to E2~P transition
Ca2+-ATPases has been reported before, our hSPCA1 structure in (Fig. 5f, g). In the E2P state, Asp572, Arg619, and Asn647 were
the CaE2P state reveals the unprecedented structural changes that essential for the interaction of the 189TGE motif with the P domain
occur during the CaE1P-ADP → CaE2P reaction. (Fig. 5g). However, in the E2~P structure, the 189TGE motif
interacted with Thr352, Gly571, Asp572, and Asn647 in the P
A lumen-facing cavity in the early E2P state domain, moving Glu191 closer to the phosphorylated Asp350
In the BeF3–-Class II structure, compared to the CaE2P structure, (Fig. 5f, g), which is important for initiating dephosphorylation.45,46
the movement of the headpiece mainly occurred in the A domain As domain A rotated, TMD experienced a slight shift, with the
which rotated ~10.2° towards the P domain (Fig. 4f). However, the lumen-facing cavity remaining closed (Fig. 5d), suggesting that the
position of the 189TGE motif was similar in both structures (Fig. 4g), dephosphorylation process has minimal effect on the gating of
indicating that BeF3–-Class II was also trapped in an ADP- the Ca2+ release pathway.
insensitive state. Interestingly, the conformations of TMDs were
noticeably rearranged in BeF3–-Class II compared to CaE2P. A full Ca2+ transport pathway
The rotation of the A domain in BeF3–-Class II caused TM1–TM2 To better understand the complete Ca2+ transport pathway, we
to move further upwards by ~4.3 Å and TM3 by ~4.2 Å (Fig. 4f, h). used explicit solvent all-atom MD simulations to study the four key
The luminal part of TM4 (TM4L) swung away from TM6, whereas states (CaE1, CaE2P, early E2P, and E2~P). These simulations
TM4C rotated towards it (Fig. 4h). TM5L moved towards TM6, allowed us to study the hydration of the substrate-binding site
while TM5C remained fixed. TM6–TM10 rotated away from TM4 and solvent-accessible pathways in detail, which are challenging
(Fig. 4h). The separation of TM4L and TM6 created a cavity facing to capture in the cryo-EM structures.
the GA lumen formed by TM2 and TM4–TM6. Interestingly, a large MD simulations of the CaE1 state showed a solvent-accessible
and extended EM density was observed in the lumen-facing cavity facing the cytosol formed by TM1, TM2, and TM4 (Fig. 6a).
cavity, surrounded by TM2, TM4–TM6, and TM8 (Supplementary This cavity provided access to the Ca2+-binding site, suggesting a
information, Fig. S7a). possible entry pathway for Ca2+. It remained open during the
Aligning TM4 and TM6 of both BeF3–-bound structures showed transition of CaE1 → CaE1-ATP → CaE1P-ADP. In the CaE2P state,
no evident movement in TM4C. However, TM4L of BeF3–-Class II the Ca2+-binding site was still intact, but the kink in TM1 moved
swung ~34.5° away from TM6L, while TM6 rotated ~12.5° away towards the cytoplasmic side. As a result, the cavity was blocked
from TM4 compared to the CaE2P structure (Fig. 4i). As a result, by several amino acids, including Gln78, Asn81, Val114, Ala117,
several residues involved in Ca2+ binding, including Val303, Pro311, and Ile312, making the cavity dehydrated and sealing the
Ala304, Ile306, Glu308, and Asn738, moved away from Ca2+ (with Ca2+ within the TMD (Fig. 6b; Supplementary information, Fig. S6d
distances of ~6.3 Å, ~3.9 Å, ~5.9 Å, ~6.7 Å, and ~5.2 Å, respectively, and Video S1).
Fig. 4i). This disrupted the conformation of the Ca2+-binding site In the following early E2P state, TM4L and TM6 moved apart
and lowered its affinity to Ca2+, consistent with the absence of (Fig. 4h, i), creating a highly hydrated cavity facing the lumen
Ca2+-like EM density in the Ca2+-binding site of the BeF3–-Class II (Fig. 6c; Supplementary information, Video S2). This suggests a
EM map (Supplementary information, Figs. S6d, S7a). potential pathway for releasing Ca2+. The lumen-facing cavity was
Structural comparison showed a similar headpiece (RMSD is blocked in the middle of the membrane by two amino acids,
0.63 Å) but a different TM region in the BeF3–-Class II structure of Gly309 and Gln747, while the cytosol-facing cavity was closed at
hSPCA1 compared to the recently reported E2P structure37 the membrane surface. These separate cavities prevented Ca2+
(Fig. 5a). In the E2P structure, TM1–TM2 moved ~6.2 Å back backflow and allowed unidirectionally transport from the cytosol
towards the luminal leaflet of the lipid bilayer (Fig. 5a). TM3 and to the GA lumen (Fig. 6c).
TM4 moved towards TM1, while TM5 bent towards TM3. Finally, In the subsequent E2~P state, MD simulations showed that the
the lumen-facing cavity closed due to the movement of lumen-facing cavity became dehydrated, indicating closure of the
TM6–TM10 close to TM4 (Fig. 5a, b), indicating that Ca2+ had Ca2+ release pathway (Fig. 6d). At the same time, movement of
been completely released from hSPCA1 in the E2P state. TM1–TM2 towards the luminal side partially opened the cytosol-
It is worth noting that TM6 of the E2P state twisted ~27.0° facing cavity (Fig. 6d). This allowed solvent from the cytosol to
compared to the BeF3–-Class II structure (Fig. 5c). As a result, the reach the surface above the Ca2+-binding site (Fig. 6d). Since the
coordination geometry of the Ca2+-binding site became unsui- TMD conformation of the recently reported E2P state is almost
table for trapping Ca2+ (Fig. 5c). The twist of TM6 distorted the identical to our E2~P structure (Fig. 5d), it is likely that the partially
conformation of the Ca2+-binding site and closed the lumen- opened cytosol-facing cavity is already formed in the previous E2P
facing cavity, preventing the re-trapping of Ca2+. state.

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Fig. 5 Structural comparison of hSPCA1 in three E2 states. a Global alignment of the early E2P (color) and E2P (dark gray, PDB: 7YAM) states
of hSPCA1. The red text shows the distance from the Cα of Leu96 in the early E2P state to the Cα of Leu96 in the E2P state. b Structural
comparison of hSPCA1 TMD in the early E2P (color) and E2P (dark gray) states. c Comparison of TM4 and TM6, and the Ca2+-binding site
between the early E2P state (orange-red) and the E2P state (dark gray) in hSPCA1. d Global alignment of the E2~P (color) and E2P (dark gray,
PDB: 7YAM) states of hSPCA1. e Structural comparison of hSPCA1 cytoplasmic domains in the E2~P (color) and E2P (dark gray, PDB: 7YAM)
states. The upper right panel shows the cryo-EM densities of AlF4–, Mg2+, Asp350, and Asp644 (black mesh, contoured at 10.0 σ). f The
movement of the 189TGE motif from the E2P state (dark gray, PDB: 7YAM) to the E2~P (color) state. The black dashed line show the distance
between Cα of Glu191 in the E2P state and that in the E2~P state. g The interfaces between 189TGE motif and P domain in the E2~P (color) and
E2P states (dark gray, PDB: 7YAM). The EM densities of the 189TGE motif and Thr352, Gly571, Asp572, and Asn647 in the E2~P state are shown
(black mesh, contoured at 10.0 σ).

By cryo-EM analysis of hSPCA1 and all-atom MD simulations, we A proposed model of Ca2+ transport by hSPCA1
were able to deduce a complete Ca2+ transport pathway for Based on all our solved cryo-EM structures and MD simulation
hSPCA1 based on changes in hydration and openness of the results of hSPCA1, as well as the reported E2P structure,37 we
cytosol-facing and lumen-facing cavities. analyzed the structural basis and rearrangement for Ca2+ binding,

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541

Fig. 6 The Ca2+ transport pathway of hSPCA1. a The open cytosol-facing cavity of hSPCA1 in the CaE1 state. TM1, TM2, TM4, and TM6 are
colored medium blue, saddle brown, forest green, and magenta, respectively. Water molecules and Ca2+ are shown as small red balls and the
lime sphere, respectively. The black arrow indicates the possible Ca2+ entry pathway. b The closed cytosol-facing cavity of hSPCA1 in the
CaE2P state. The red arrow (bottom) shows the distance from the Cα of Pro82 in the CaE1 state to the Cα of Pro82 in the CaE2P state. c The
closed cytosol-facing cavity and open lumen-facing cavity of hSPCA1 in the early E2P state. The black arrow indicates the Ca2+ release
pathway. d The semi-open cytosol-facing cavity and closed lumen-facing cavity of hSPCA1 in the E2~P state. The Ca2+-binding residues are
colored forest green, magenta, or black. The surfaces are shown with colors ranging from dark cyan (most hydrophilic) to white (intermediate)
to dark goldenrod (most lipophilic).

translocation and release, as well as the Ca2+ entry and releasing dephosphorylation, the A and N domains move towards the P
pathway of hSPCA1. This allows us to propose a model of how domain, whereas the P domain and TMD remain unchanged.
hSPCA1 transports Ca2+ across the GA membrane (Fig. 7; Finally, dephosphorylation of Asp350 loosens the compact head-
Supplementary information, Video S3). piece, and makes TM1–TM2 move back towards the luminal
During the Ca2+ transport cycle, hSPCA1 starts with a loosely leaflet. A new Ca2+ from the cytosol enters the binding site to
arranged headpiece of cytosolic domains, with the N domain begin a new transport cycle.
ready to bind ATP. Ca2+ from the cytosol can then enter the
binding site through the cytosol-facing cavity. The N domain
binds an ATP molecule and rotates ~57.0°, followed by ATP DISCUSSION
hydrolysis and phosphorylation of Asp350. This leads to the In our study, we determined six cryo-EM structures of hSPCA1
phosphorylated E1 state of hSPCA1 (the CaE1P-ADP state) with representing the CaE1, CaE1-ATP, CaE1P-ADP, CaE2P, early E2P,
little changes in the overall structure. After ADP release, but prior and E2~P states, respectively. Together with the previously
to Ca2+ release, the A and N domains rotate towards and against reported E2P state, these results provide new insights into the
the P domain, respectively, forming a compact headpiece. The dynamics of hSPCA1 during the phosphorylation and depho-
conserved 189TGE motif moves close to the phosphorylation site sphorylation process involved in Ca2+ transport.
Asp350 (the CaE2P state; Figs. 4c, 7a). As a result, the integrated
movement of TM1–TM2 closes the cytosol-facing cavity and Structures of hSPCA1 in the CaE1-ATP state bearing different
dehydrates the Ca2+-binding site, resulting in a Ca2+-occluded conformations
CaE2P state. During the transition from the CaE2P state to the Recently, Chen et al.37 reported a CaE1-ATP structure that showed
early E2P state, TM4L and TM6 move apart to disrupt the Ca2+- a compact headpiece and upward movement of TM1 and TM2
binding site and create a large lumen-facing cavity formed by towards the cytoplasm compared to our CaE1-ATP structure
TM2, TM4–TM6 for Ca2+ release. Further movement of TM4 and (Supplementary information, Fig. S7c). The conformational differ-
TM6 in the E2P state closes the lumen-facing cavity, ensuring the ences between the reported and our structures may be due to
complete release of Ca2+. The movement of TM1–TM2 towards different sample preparation methods. In the reported CaE1-ATP
the luminal leaflet partially opens the cytosol-facing cavity. After structure, a megabody 14 (Mb14) with a molecular weight of
entering the E2~P state, the headpiece rearranges for ~56 kDa was used to stabilize the conformation of hSPCA1 while

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542

Fig. 7 A proposed model of Ca2+ transport by hSPCA1. Schematic models of the stages of the transport cycle in hSPCA1. A, N, and P
domains are shown as light cornflower blue, light forest green, and peru cylinders, respectively. TM1–TM2, TM3–TM4, and TM5–TM10 are
shown as cyan, light pink, and light-medium purple rectangles. The red arrows indicate movements of the cytosolic domains (with respect to
the previous state in the forward reaction).

biochemically abolishing its Ca2+-dependent ATPase activity.37 from the CaE1P state to the CaE2P state, and that Ca2+ release
The binding of Mb14, with its relatively large volume, to the may be triggered only in the subsequent transition.
headpiece would limit hSPCA1’s conformational rearrangements.
In contrast, we prepared our cryo-EM sample of the CaE1-ATP- Ca2+ release triggered by a ~10.2° rotation of the A domain
state hSPCA1 by directly incubating the ATP analog with hSPCA1 It is generally accepted that the rotation of the A domain is
protein, a method which was widely used to trap the E1-ATP state coupled with Ca2+ release,33,49–51 but the detailed structural
of the representative P-type ATPases.6,39,47,48 mechanism remains unclear. Our structures revealed that during
the transition from the CaE2P to early E2P states, the cytosolic
TGE motif moves towards the phosphorylation site before domains maintain almost identical conformations except for
Ca2+ release a ~10.2° rotation of the A domain (Fig. 4f). This rotation induces
The TGE motif, which is believed to be closely related to protein a series of conformational changes in the TMD (Fig. 4h), including
dephosphorylation,33,42,45,46 undergoes a large movement during the upward movement of TM1–TM2 towards the cytosolic leaflet,
substrate transport. However, the coupling of this movement with TM3 moving away from TM1, and TM4L moving away from TM6.
substrate transport is not well understood. A recent study showed Additionally, TM6–TM10 move away from TM4, resulting in the
that in the CaE1P state of hSERCA2, the TGE motif was located in a destruction of the Ca2+-binding site and the formation of the
similar position to that in the CaE1-ATP structure, far from the lumen-facing cavity that allows Ca2+ to be released into the GA
phosphorylated aspartic acid ( ~27.0 Å; Supplementary informa- lumen. Therefore, the small rotation of the A domain during the
tion, Fig. S7d).30 In our study, we determined the CaE2P structure, transition from CaE2P to early E2P state may play a crucial role in
providing new insights into the movement of the TGE motif. inducing Ca2+ release.
Compared to the reported CaE1P structure of hSERCA2 (Supple-
mentary information, Fig. S7d), our CaE2P structure of A putative regulator of hSPCA1
hSPCA1 shows that the TGE motif is positioned near the Previous studies have shown that small-molecule drugs targeting
phosphorylation site prior to Ca2+ release. We speculate that the the lumen-facing cavity formed by TM2 and TM4–TM6 in NKAs are
movement of the TGE motif likely occurs during the transition effective in treating heart diseases.52 In the early E2P structure of

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543
hSPCA1, we also observed a blob of EM density in the lumen- different intermediate states along the transport cycle using the following
facing cavity, surrounded by Asp102 in TM2, Ala304 and Glu308 in conditions: the CaE1-ATP state was captured with 1 mM ATP analog β,γ-
TM4, Ile717 in TM5, Asn738, Asp742, and Gln747 in TM6, and methyleneadenosine 5’-triphosphate (AMP-PCP); the CaE1P-ADP state was
Thr811 in TM8 (Supplementary information, Fig. S7a, b). Our MD captured with 5 mM ADP, 5 mM NaF, and 1 mM AlCl3; the CaE2P/early E2P
simulations suggest that this cavity is highly hydrated (Fig. 6c). states were captured with 10 mM NaF and 2 mM BeSO4; and the E2~P state
was captured with 10 mM NaF and 2 mM AlCl3. After incubating with
Therefore, we hypothesize that the unassigned EM density in the substrates for 1 h on ice, the protein solutions were applied to a freshly
cavity may be derived from certain cations and/or hydrophilic glow-discharged Quantifoil holey carbon grid (PELCO easiGlow, 0.39 mBar,
molecules that could potentially regulate the transport activity of air, 15 mA, 40 s, R1.2/1.3, Au, 300 mesh). The cryo-EM samples were
hSPCA1. Further study is needed to confirm this hypothesis. prepared using a Vitrobot Mark IV (Thermo Fischer Scientific) at 4 °C with a
In summary, by determining the structures of six consecutive blotting time of 3–4 s under 99% humidity conditions, and then the grids
intermediate states along the Post-Albers reaction cycle, we were were plunge-frozen in liquid ethane. All grids were then transferred to
able to define a near-complete Ca2+ transport pathway for liquid nitrogen and stored there for data collection. For the CaE1 state,
hSPCA1. The specific Ca2+ transport mechanism of hSPCA1 differs hSPCA1 samples were freshly purified and concentrated in SEC buffer, then
used immediately.
from that of SERCAs and other P-type II ATPases in many ways.
This information not only expands our understanding of P-type
ATPases, but also holds great potential for the development of Cryo-EM data collection
specific agonists and inhibitors targeting hSPCA1, which could be Cryo-EM data were collected by EPU2 software package (Thermo Fisher
beneficial in treating diseases related to hSPCA1. Scientific) on a 300 kV Titan Krios G3i transmission electron microscope
(Thermo Fischer Scientific). Prior to detection, inelastically scattered
electrons were filtered out with a GIF Quantum energy filter (Gatan) using
a slit width of 20 eV. Images were acquired in counting mode (super
MATERIALS AND METHODS resolution) on a K3 Summit detector (Gatan) at a nominal magnification of
DNA constructs 105,000×, resulting in a pixel size of 0.84 Å/pixel. Images were exposed for
The full-length hSPCA1a-coding sequence (NM_001199179.1) was a total of 1.83 s with a dose rate of 20 e–/pixel/s resulting in a total dose of
obtained by PCR amplification from HEK293T cDNA. The coding sequence 50 e–/Å2, which was fractionated into 32 frames.
was cloned into the pCAG vector and fused with a C-terminal 2× Strep tag.
Cryo-EM data processing
Protein expression and purification Movie frames were aligned using the Patch motion correction (multi)
HEK293F cells were cultured in SMM 293-TII medium (Sino Biological Inc.) integrated in cryoSPARC (v3.3). The contrast transfer function (CTF)
supplemented with 1× penicillin/streptomycin at 37 °C with 5% CO2. Each parameters were estimated from the aligned micrographs using Patch
liter of cells was transiently transfected with 1.8 mg DNA using 4.5 mg CTF estimation (multi) in cryoSPARC. One thousand images were used to
polyethyleneimine (Polysciences, Inc.) at a density of 2.0 × 106 to 2.5 × 106 generate an initial particle-set by blob picker in cryoSPARC; particle
cells/mL. The transfected cells were cultured for 48 h before being extraction was carried out with a box size of 360 pixels; and two-
harvested. About 4 L of transfected cells were used for one batch of dimensional (2D) classification was performed in cryoSPARC. High-quality
protein purification. All procedures below were carried out at 4 °C or on ice. 2D class averages representing projections in different orientations were
The cell pellet was resuspended and solubilized in lysis buffer containing selected as templates for Topaz training of the entire dataset. The particles
100 mM Tris (pH 8.0), 100 mM KCl, 5 mM MgCl2, 1 mM CaCl2, 20% (v/v) were then subjected to 2D classification in cryoSPARC or 3D classification
glycerol, 1% (w/v) DDM (Anatrace) & 0.2% (w/v) CHS (Sigma), 1 mM DTT, in Relion. After ab-initio model building and heterogeneous refinement in
1 mM PMSF, and 1× EDTA-free protease inhibitor cocktail (Roche), and cryoSPARC, most bad particles were removed, and the best class was used
stirred gently for 2 h. After centrifugation at 20,000× g for 1 h, the to generate the final map by using Non-uniform refinement in cryoSPARC.
supernatant was collected and incubated with Strep-Tactin® Sepharose® Subtraction of detergent density and local refinement yielded an improved
(IBA, LifeSciences) for 1.5 h. The resin was washed with 50 column volumes map with better details using cryoSPARC (v3.3). The resolution was
of wash buffer containing 100 mM Tris (pH 8.0), 100 mM KCl, 5 mM MgCl2, estimated by using the gold-standard FSC 0.143 criterion.
1 mM CaCl2, 10% (v/v) glycerol, 0.05% (w/v) DDM & 0.01% (w/v) CHS, 1 mM
DTT, and 1 mM PMSF. Subsequently, the protein was eluted with 50 mM
Tris (pH 8.0), 100 mM KCl, 5 mM MgCl2, 1 mM CaCl2, 0.025% (w/v) DDM & Model building and refinement
0.005% (w/v) CHS and 50 mM biotin (VETEC). The protein was concentrated The model of the hSPCA1 was built by fitting the model predicted by
using a 50-kDa MWCO Amicon Ultra centrifugal filter. The protein was AlphaFold254 into the density map using UCSF ChimeraX,55 followed by
further purified by SEC using a Superose 6 Increase 5/150 GL column (GE manual model construction in COOT56 and real-space refinement with
Healthcare) equilibrated with 25 mM Tris (pH 8.0), 100 mM KCl, 5 mM secondary structure restraints in PHENIX.57 The model statistics are
MgCl2, 1 mM CaCl2, and 0.06% (w/v) digitonin (Sigma). 1 mM CaCl2 was presented in Supplementary information, Table S1.
absent in the SEC buffer for the E2~P state. Peak fractions were collected
for Cryo-EM sample preparation and ATPase activity assay. MD simulations
The models of hSPCA1 in four different states (CaE1, CaE2P, early E2P, and
ATPase activity assay E2~P) were constructed using the corresponding cryo-EM structures. These
The peak fractions after SEC were diluted to a suitable concentration and models were then embedded into a flat, mixed lipid bilayer consisting of 1-
used in an ATPase activity assay. The assay was performed using a palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and solvated in a
commercially available kit by measuring the release of inorganic cubic water box containing 0.10 M KCl and 0.1 M CaCl2. The size of the box
phosphate (Pi) from ATP according to the kit protocol (Nanjing Jiancheng was 12.6 nm, 12.6 nm, and 12.9 nm in the x, y, and z dimensions,
Bioengineering Institute, China). The absorbance was measured at 636 nm respectively, resulting in ∼210,000 atoms in total for each model. The
using a microplate reader (Tecan, Switzerland). The protein concentration Orientations of Proteins in Membranes (OPM) webserver was used to align
was determined by Implen P330 ultra-micro spectrophotometer. the TMD of hSPCA1 in the lipid bilayer. The systems were built with the
CHARMM-GUI webserver58 and underwent an energy minimzation step
using the steepest descent algorithm followed by a six-step equilibration
Preparation of Cryo-EM samples during which position constraints in the systems were gradually removed.
Biochemical experiments and structural data have proven that the ligation Finally, production runs in semi-iso-thermal-isobaric (NPT) conditions were
of BeF3– at the aspartic acid phosphorylation site would simulate the performed. The CHARMM36m force field59 was used for proteins,
CaE1P, CaE2P, and E2P states.29–31,38 AlF4– possesses planar geometry, CHARMM36 for lipids, and TIP3P for water. Force field parameters used
which coordinates the Asp-oxygen and the hydrolytic water at apical for the phosphorylated Asp350 were the same as previously described.60
positions, producing the bipyramidal structure superimposable to the In the MD simulations, the temperature was kept constant at 310 K using
penta-coordinated phosphorus in the transition state (E2~P) during the a Nose–Hoover thermostat with a 1 ps coupling constant, and the pressure
aspartylphosphate hydrolysis.29,38,53 Thus, the purified hSPCA1 (concen- was kept at 1.0 bar using the Parrinello–Rahman barostat with a 5 ps
trated to ~10 mg/mL) was incubated with different substrates to trap coupling constant. A cut-off of 1.2 nm was applied for the van der Waals

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interactions using a switch function starting at 1.0 nm. The cut-off for the 25. Dyla, M., Kjaergaard, M., Poulsen, H. & Nissen, P. Structure and mechanism of
short-range electrostatic interactions was also set at 1.2 nm and the long- P-Type ATPase ion pumps. Annu. Rev. Biochem. 89, 583–603 (2020).
range electrostatic interactions were calculated by means of the particle 26. Wuytack, F., Raeymaekers, L. & Missiaen, L. Molecular physiology of the SERCA
mesh Ewald decomposition algorithm with a mesh spacing of 0.12 nm. A and SPCA pumps. Cell Calcium 32, 279–305 (2002).
reciprocal grid of 108 × 108 × 108 cells was used with fourth-order B-spline 27. He, W. & Hu, Z. The role of the Golgi-resident SPCA Ca(2)(+)/Mn(2)(+) pump in
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version of Gromacs 2021.5.61 500 ns or 1000 ns simulation was performed 28. Danko, S., Yamasaki, K., Daiho, T. & Suzuki, H. Membrane perturbation of ADP-
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DATA AVAILABILITY structural analog of ADP-sensitive phosphoenzyme of Ca2+-ATPase with occlu-
The cryo-EM structures of hSPCA1 in CaE1, CaE1-ATP, CaE1P-ADP, CaE2P, early E2P, ded Ca2+ by beryllium fluoride: structural changes during phosphorylation and
and E2~P states have been deposited at the Protein Data Bank (PDB) with the isomerization. J. Biol. Chem. 284, 22722–22735 (2009).
accession codes 8IWP, 8IWR, 8IWW, 8IWS, 8IWT and 8IWU, respectively. The cryo-EM 30. Zhang, Y. et al. Multiple sub-state structures of SERCA2b reveal conformational
density maps of these structures have been deposited at the Electron Microscopy overlap at transition steps during the catalytic cycle. Cell Rep. 41, 111760 (2022).
Data Bank (EMDB) with the codes EMD-35776, EMD-35777, EMD-35781, EMD-35778, 31. Daiho, T., Danko, S., Yamasaki, K. & Suzuki, H. Stable structural analog of Ca2+-
EMD-35779, and EMD-35780, respectively. ATPase ADP-insensitive phosphoenzyme with occluded Ca2+ formed by elon-
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32. Bublitz, M., Poulsen, H., Morth, J. P. & Nissen, P. In and out of the cation pumps:
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We thank all staff members of the Cryo-EM Centre, Southern University of Science appropriate credit to the original author(s) and the source, provide a link to the Creative
and Technology, and Chunlong Guo, Zhenqian Guo, Fanhao Meng, Li Li and other Commons license, and indicate if changes were made. The images or other third party
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thank Ruiqian Bu, Jianqiang Mu, Ying Huang, and other members of Zhongmin Liu’s indicated otherwise in a credit line to the material. If material is not included in the
laboratory for their discussion on this project. This work was supported by funds from article’s Creative Commons license and your intended use is not permitted by statutory
the National Natural Science Foundation of China (32000850 to Z.L.), Shenzhen regulation or exceeds the permitted use, you will need to obtain permission directly
Municipal Basic Research projects (JCYJ20210324105007020 to Z.L.), the start-up from the copyright holder. To view a copy of this license, visit http://
funds of Southern University of Science and Technology to Z.L. Y.W. acknowledges creativecommons.org/licenses/by/4.0/.
the financial support from the National Key R&D Program of China (2021YFF1200404),
the Fundamental Research Funds for the Central Universities of China (K20220228)
and the start-up funds of Zhejiang University, as well as the access to computational © The Author(s) 2023

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