Article

Structural Basis of Sarco/Endoplasmic Reticulum
Ca2+-ATPase 2b Regulation via Transmembrane
Helix Interplay
Graphical Abstract

Authors
Michio Inoue, Nanami Sakuta,
Satoshi Watanabe, ..., Tomoya Tsukazaki,
Kazuhiro Nagata, Kenji Inaba

Correspondence
kinaba@tagen.tohoku.ac.jp

In Brief
Inoue et al. present a crystal structure ofhuman SERCA2b, a ubiquitously
expressed Ca2+-ATPase that facilitates
Ca2+ uptake into the endoplasmic
reticulum and thereby plays central roles
in intracellular calcium homeostasis. The
structural and biochemical analyses
clarify molecular mechanisms of
SERCA2b regulation via TM helix
interplay.

Highlightsd

Crystal structure was solved for human SERCA2b and
SERCA2a in E1-2Ca2+-AMPPCP stated

The location and orientation of TM11 helix characteristic of
SERCA2b were revealeddAmino acid residues involved in interaction with TM11 wereidentifieddMolecular mechanisms of TM11-mediated SERCA2b
regulation are proposed

Inoue et al., 2019, Cell Reports 27, 1221–1230
April 23, 2019 ª 2019 The Author(s).
https://doi.org/10.1016/j.celrep.2019.03.106

Cell Reports

ArticleStructural Basis of Sarco/Endoplasmic Reticulum
Ca2+-ATPase 2b Regulation
via Transmembrane Helix Interplay
Michio Inoue,1,6 Nanami Sakuta,1,6 Satoshi Watanabe,1,6 Yuxia Zhang,1,6 Kunihito Yoshikaie,2 Yoshiki Tanaka,2
Ryo Ushioda,2,6 Yukinari Kato,4 Junichi Takagi,5 Tomoya Tsukazaki,2 Kazuhiro Nagata,3,6 and Kenji Inaba1,6,7,*
1Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan
2Graduate School of Biological Sciences, NARA Institute of Science and Technology, Ikoma 630-0192, Japan
3Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, Japan
4Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan
5Institute for Protein Research, Osaka University, Suita 565-0871, Japan
6Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Saitama, Kawaguchi, Japan
7Lead Contact*Correspondence: kinaba@tagen.tohoku.ac.jp
https://doi.org/10.1016/j.celrep.2019.03.106

SUMMARY

Sarco/endoplasmic reticulum (ER) Ca2+-ATPase 2b(SERCA2b) is a ubiquitously expressed membraneprotein that facilitates Ca2+ uptake from the cytosol
to the ER. SERCA2b includes a characteristic 11th
transmembrane helix (TM11) followed by a luminal
tail, but the structural basis of SERCA regulation by
these C-terminal segments remains unclear. Here,
we determined the crystal structures of SERCA2b
and its C-terminal splicing variant SERCA2a, bothin the E1-2Ca2+-adenylyl methylenediphosphonate(AMPPCP) state. Despite discrepancies with thepreviously reported structural model of SERCA2b,
TM11 was found to be located adjacent to TM10
and to interact weakly with a part of the L8/9 loop
and the N-terminal end of TM10, thereby inhibiting
the SERCA2b catalytic cycle. Accordingly, mutational disruption of the interactions between TM11
and its neighboring residues caused SERCA2b to
display SERCA2a-like ATPase activity. We proposethat TM11 serves as a key modulator of SERCA2b
activity by fine-tuning the intramolecular interactions
with other transmembrane regions.
INTRODUCTION
The endoplasmic reticulum (ER) is an essential organelle
responsible for Ca2+ storage, in which sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) imports Ca2+ against a huge(>1,000-fold) Ca2+ concentration gradient between the ER
and the cytosol. Human cells contain 3 different SERCA isoforms divided into 11 splice variants (SERCA1a–1b, -2a–2c,
and -3a–3f) (Altshuler et al., 2012). SERCA1a and SERCA2a
are expressed mainly in skeletal and cardiac muscle, respectively, while SERCA2b is a ubiquitously expressed housekeeping membrane protein responsible for maintaining Ca2+

homeostasis in cells. Residues 1–993 of SERCA2b share an
identical amino acid sequence with its splice variant SERCA2a,but the former also possesses a 49-residue C-terminal extension (Campbell et al., 1991) (Figure 1A) that is predicted tocontain the 11th transmembrane helix (TM11) and the 12-residue luminal extension (LE) (Campbell et al., 1992). SERCA2aand SERCA1a isoforms, both of which lack TM11 and the
C-terminal tail, exhibit higher ATPase activity and lower affinity
for Ca2+ than SERCA2b (Dode et al., 2003; Verboomen et al.,
1992, 1994), indicating a regulatory role for the C-terminal
extension in SERCA2b. Accordingly, truncation of the C-terminal 49 residues resulted in a 2-fold higher turnover rate thanwild-type (WT) SERCA2b (Vandecaetsbeek et al., 2009; Verboomen et al., 1994).
A docking model of SERCA2b predicted that TM11 interacts
with TM7 and TM10 and that the LE segment binds thegroove formed between loops L5/6 and L7/8 (Vandecaetsbeek
et al., 2009). Exogenous addition of a synthetic LE peptide(Met1039–Ser1042) to SERCA2a inhibits the ATPase activity
and increases the apparent Ca2+ affinity, possibly by stabilizing
the Ca2+-bound E1-2Ca2+ state, suggesting that the LE
segment serves as a functional modulator of SERCA2b (Vandecaetsbeek et al., 2009). Moreover, co-reconstitution of asynthetic TM11 peptide (Gly1013–Tyr1030) with SERCA1a has
a similar functional effect (lower Vmax and higher Ca2+ affinity)
to that of LE (Gorski et al., 2012). However, the molecular mechanisms underpinning TM11-mediated SERCA2b regulation
remain poorly understood due to a lack of structural information
on SERCA2b.
Herein, we present crystal structures of human SERCA2b and
SERCA2a, both in the E1-2Ca2+-adenylyl methylenediphosphonate (AMPPCP) state (Figure 1B). While crystal structures wererecently released for E2-AlF4- and E1-2Ca2+-AMPPCP formsof SERCA2a from pig ventricular muscle (Sitsel et al., 2019),
we report on the crystal structure of SERCA2b. The crystal structure, in combination with anomalous signal analysis using thesingle-wavelength anomalous dispersion (SAD) dataset of selenomethionine (SeMet)-labeled SERCA2b, revealed that despite
discrepancies with the previously predicted model, TM11 is

Cell Reports 27, 1221–1230, April 23, 2019 ª 2019 The Author(s). 1221
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Figure 1. Alignment of the Amino Acid Sequences of Human SERCA Family Members and Catalytic Cycle Exerted by SERCA Family
Enzymes(A) Transmembrane helices TM1–TM10 and TM11 are shown in green and red, respectively. Cytosolic A, N, and P domains are shown in cyan, salmon pink, andorange, respectively. Residues Phe1018 and Val1029 are blue.
(B) Key states generated along the ATP-driven Ca2+ pump cycle of SERCA. Note that AMPPCP, a non-hydrolyzable ATP analog, traps the Ca2+-bound state of
SERCA to generate the E1-2Ca2+-AMPPCP state.

located adjacent to TM10 and does not interact with TM7;
instead, the N-terminal and C-terminal ends of TM11 interact
with a part of the L8/9 loop and TM10, respectively. Structurebased biochemical studies demonstrated that the functional
properties of SERCA2b were significantly affected by the mutations of TM11 residues interacting with the L8/9 loop or TM10.
We propose that TM11 serves to fine-tune the turnover rate
and Ca2+ affinity of SERCA2b through the interplay with otherTM regions.

1222 Cell Reports 27, 1221–1230, April 23, 2019

RESULTS AND DISCUSSION
Overall Structures of SERCA2b and SERCA2a
We developed human stable cell lines expressing recombinanthuman SERCA2b and SERCA2a with an N-terminal dodecapeptide PA-tag (GVAMPGAEDDVV) using the PiggyBac Cumate Switch Inducible system. For stable expression of
SERCA2b or SERCA2a, HEK293T cells harboring either one of
the SERCA2 genes and the promoter sequence transposedTable 1. X-Ray Diffraction Data Collection and Refinement Statistics
SERCA2b

SERCA2aSeMet-Labeled SERCA2b

Data Collection
Beamline

SPring-8 BL32XU

SPring-8 BL32XU

SPring-8 BL32XU

Wavelength

1.000

1.000

0.9780Space group

P21212

P21212

P21212Cell Dimensions
a, b, c (Å)

165.08, 84.64, 118.83

165.58, 84.36, 122.19

165.31, 84.87, 121.22

Oscillation 3 No. crystals

5 3 21, 10 3 25180 3 1

10 3 42

Resolution (Å)a48.2–3.45 (3.57–3.45)

49.48–3.20 (3.31–3.20)

49.48–5.5 (5.70–5.50)

Completeness (%)a93.7 (77.0)

100 (99.9)

99.1 (99.7)

Redundancya

8.9 (5.1)

6.6 (6.6)

14.0 (14.6)

I/s (I)a4.7 (1.8)

4.7 (1.3)

4.2 (1.3)

Rmeas (%)a,b

33.8 (91.7)

26.4 (147)

44.6 (304)

CC1/2a0.963 (0.511)

0.999 (0.524)

0.660 (0.404)

Refinement Statistics
Resolution (Å)

48.2–3.45

49.48–3.20–

Rwork/Rfree (%)c,d

24.9/30.8

21.1/25.6–

RMSDs
Bond lengths (Å)

0.007

0.010

–

Bond angles ( )

0.726

0.736

–

Ramachandran Plot
Favored (%)

94.0

94.0

–

Allowed (%)

6.0

6.0

–

Outliers (%)

0.0

0.0

–

a

Values in parentheses are for the highest-resolution shell.
bRmeas = Shkl {n/[n1]}1/2 SijIi(hkl)  < I(hkl) > j/ShklSIi(hkl), where Ii(hkl) is the ith observed intensity and < I(hkl) > is the average intensity from multiple
observations.
c
Rwork = SjFo-Fcj/SjFoj, where Fo and Fc are observed and calculated structure factors, respectively.
d
Rfree is R factor for a selected subset (5%) of randomly chosen reflections that were omitted in prior refinement calculations.

onto the chromosomes were established through selection byrepeated passage in the presence of 10 mg/mL puromycin.
Recombinant SERCA2b and SERCA2a were overexpressedusing cumate induction (Ushioda et al., 2016) and purified by
the established methodology for rapid high-yield purification
using the high-affinity antibody NZ-1 (Fujii et al., 2016), followedby size-exclusion chromatography (Figures S1A and S1B).
SERCA2a and SERCA2b fully purified in the presence of
1 mM DTT were obtained with a yield of 0.6–1 mg/L culture medium. SERCA2b and SERCA2a were crystallized in complex
with Ca2+ and AMPPCP, a non-hydrolyzable ATP analog, using
the lipidic cubic phase (LCP) method (Figure S1C). X-ray diffraction images of SERCA2b (Figure S1D) were collected from >10
microcrystals, and images of SERCA2a were collected from asingle crystal, at SPring-8 beamline BL32XU, and refined to3.45 and 3.2 Å resolution, respectively (Table 1). Phases were
determined by molecular replacement using a previously published crystal structure of AMPPCP-bound SERCA1a (PDB:
1T5S) as a search model.
In both SERCA2b and SERCA2a, clear electron density wasobserved for the cytosolic A, N, and P domains, and the 10 TM helices (Figure 2A; Videos S1 and S2), allowing model building of

their overall structures (Figure 2B). The locations of the 3 cytosolic
domains and the arrangement of TM1–TM10 are almost identicalto those previously reported for AMPPCP-bound SERCA1a (Sørensen et al., 2004; Toyoshima and Mizutani, 2004). The modes of
ATP binding and calcium coordination in SERCA2b and SERCA2a
are also similar to those in SERCA1a, suggesting common mechanisms of ATP hydrolysis and Ca2+ transport among the SERCA
family members (Figures 1B, S2, and S3). In agreement with theuse of reduced forms of SERCA2b and SERCA2a in crystallization, the electron density map of the L7/8 loop indicated thatboth Cys875 and Cys887 were in reduced form (Figure S4). Enzymatic assays showed that the ATPase activity of SERCA2b reconstituted into liposome was 2-fold lower than that of SERCA2a(Figure S1E), as reported previously (Vandecaetsbeek et al.,2009). Thus, our SERCA2 preparations retained their intrinsic
activities.
Modeling and Physical Location of TM11
Significant electron density was visible adjacent to TM10 in
SERCA2b, but not in SERCA2a (Figures 2A and 2B; Videos S1and S2), indicating that it is derived from TM11 and is characteristic of SERCA2b. To further corroborate our assignment

Cell Reports 27, 1221–1230, April 23, 2019 1223

Figure 2. Overall Structures of SERCA2b and
SERCA2a in the E1-2Ca2+-AMPPCP State(A) Electron density maps of SERCA2b (left) and
SERCA2a (right). 2Fo-Fc maps at the 1.0 s contour
level are shown as blue mesh. The insets show
enlarged views of the electron density map in thevicinity of TM10 in SERCA2b (left) and SERCA2a(right). Green and red meshes display positive andnegative Fo-Fc maps, respectively, at the 3.0 s
contour level. The Ca traces of SERCA2b and
SERCA2a modeled based on the 2Fo-Fc maps areshown in ribbon representation.
(B) Crystal structures of SERCA2b and SERCA2a in
the E1-2Ca2+-AMPPCP state shown in ribbon representation. Cytosolic A, N, and P domains areshown in cyan, salmon pink, and orange, respectively. TM11 (numbered M11) and the transmembrane (TM) helix bundle domain composed of
TM1–TM10 (numbered M1–M10) are red and green,
respectively. Green sticks in the cytosolic domains
denote bound AMPPCP, while yellow and grayspheres represent bound Ca2+ and Mg2+ ions,
respectively. The lower ribbon diagrams show the
top view of TM helices from the cytosol.

of TM11, we crystallized SeMet-substituted recombinant
SERCA2b and collected a diffraction dataset at the Se K-edge
wavelength (0.9780 Å). An anomalous difference Fourier map
contoured at 3 s identified 20 of the 32 selenium sites (Figure 3A).
Of particular note, a significant anomalous peak (4.0 s) was
observed close to the putative TM11 backbone, likely derived
from Met1023 located in TM11 (Figure 3B). Based on thisanomalous peak and the 2Fo-Fc map at 3.45 Å, we modeled
the F1018–Y1030 segment of the TM11 helix (Figures 3B
and 3C), as previously performed on disulfide bond formation
protein B (DsbB), a membranous sulfhydryl oxidase from
Escherichia coli (Inaba et al., 2006, 2009). Consequently, the1224 Cell Reports 27, 1221–1230, April 23, 2019

N- and C-terminal ends of TM11 were
found to interact with a part of the L8/9
loop and the ER luminal end of
TM10, respectively; Trp927 (L8/9) forms
van der Waals contacts with Phe1018(TM11), while Val1029 (TM11) is surrounded by Leu967, Leu970, and Lys971(TM10; Figure 3C).
The TM11 region has high B-factor
values (135–186 Å2), suggesting its fluctuating property and/or partially disorderedhelical conformation (Figure 3D). In supportof this, the electron density map of
SERCA2b at 1.5–2.0 s counter levels (Figure S5) showed that similar to the L7/8
and L9/10 loops, the TM11 region had fragmented electron density with lower intensity compared to the other TM helices.
Accordingly, TM11 is significantly isolated
from the TM helix bundle domain, generating a cavity with a volume of >35 Å3 between TM10 and TM11 and a distance of
13.7 Å between the Ca atoms of Ile977 and Met1023 (Figure 3E).
No electron density higher than the 0.4 s contour level wasobserved in this cavity, suggesting that there are few, if any,
tightly bound phospholipids that intervene between these 2 TM
helices. Electron density is missing also for the L10/11 loop
and the ER luminal tail following TM11 (Figure 1A). It is likelythat these 2 regions are even more fluctuating and/or disorderedthan TM11. Thus, TM11 and the subsequent tail appear toengage in only limited interactions with other TM regions of
SERCA2b. The present finding tempts us to speculate that
TM11 may be readily released from the TM helix bundle domain
in other intermediate states. It will be interesting to exploreFigure 3. Structural Modeling and Locationof TM11 and Its Interactions with Neighboring Residues in SERCA2b(A) Anomalous difference Fourier map at 6 Å resolution, contoured at 3 s (magenta mesh). The map
was calculated using the dataset from SeMetlabeled SERCA2b crystals. Methionine residues in
SERCA2b are shown in stick representation.
(B) Close-up view of the vicinity of TM11 in
SERCA2b (black box in 2A). The Ca trace of TM11
is superimposed on a 2Fo-Fc map contoured at
1.0 s (gray). The anomalous difference Fourier
map contoured at 3 s is also shown in magenta,
indicating an anomalous peak from SeMet1023 on
TM11.
(C) Close-up view of the intramolecular interactions of TM11 with loops L8/9 and TM10 in
SERCA2b. The side chains of TM11 and the residues involved in interactions with TM11 areshown as a stick model. Yellow dotted lines indicate pairs of carbon atoms (within 4 Å distance)
between hydrophobic side chains present in L8/9,
TM10, and TM11.
(D) B-factor distribution of SERCA2b. Residues
with B factors between 50 and 200 Å2 are indicated by rainbow coloring.
(E) Cavities formed in the TM region of SERCA2b.
TM helices of SERCA2b are shown in ribbon
representation. Cavities >35 Å3 in the TM region of
SERCA2b are visualized as a set of intersecting
yellow spheres.

whether TM11 serves to fine-tune the SERCA2b activity throughthe association with or release from the TM helix bundle domain
to maintain the Ca2+ homeostasis in the ER (see also thefollowing sections).
Roles of TM11 in SERCA2b Regulation
To quantify the specific activities of SERCA2b and SERCA2a in
the membrane-embedded state, we first prepared microsomesof HEK293T cells overexpressing WT SERCA2b and SERCA2a.
In agreement with previous results obtained using microsomesof COS cells overexpressing SERCA2a or SERCA2b (Vandecaetsbeek et al., 2009), the Vmax value of SERCA2b (1.8 3 102
min1) was nearly 2.3-fold lower than that of SERCA2a (4.2 3
102 min1), while the Kd values for Ca2+ binding to SERCA2b
and SERCA2a were 0.21 and 0.35 mM, respectively (Figure 4;
Table S1). Thus, SERCA2b displayed compromised ATPase
activity and increased apparent affinity for Ca2+ comparedwith SERCA2a, implying that TM11 and the subsequent luminal

tail, which are characteristic of SERCA2b,
serve as an intramolecular uncompetitive
inhibitor.
To explore the functional roles of the intramolecular interactions between TM11
and its neighboring residues, and thereby
verify the actual occurrence of the interactions, Phe1018 and Val1029 on TM11
were simultaneously mutated to glycine,and the ATPase activity of the resultant
mutant (F1018G/V1029G) was measured at various Ca2+ concentrations. Microsomal SERCA2b F1018G/V1029G displayeda 1.7-fold higher Vmax value and 1.5-fold lower affinity forCa2+ than microsomal SERCA2b WT (Figure 4). Thus, disruptionof the interactions between TM11 and its neighboring TM regions
caused the catalytic turnover rate and Ca2+-binding affinity of
SERCA2b to resemble those of SERCA2a. Meanwhile, neitherof the single mutations F1018G or V1029G greatly affected theATPase activity of SERCA2b, suggesting that formation of either
one of the interactions is sufficient for the TM11-mediated
SERCA2b regulation. Collectively, the TM11-involved interactions identified in this crystallographic study play a critical role
in regulating the SERCA2b activity.
In this context, Mikkelsen et al. (2018) demonstrated thateither the mutation of Glu917, a residue located in the L8/9
loop, or of Arg835, a TM7 residue hydrogen bonded to Glu917,
abrogated the inhibitory effect of TM11. It was inferred that the
specific interactions between the L8/9 loop and TM7 may be

Cell Reports 27, 1221–1230, April 23, 2019 1225

the Ca2+ pump activity of SERCA2b (Anderson et al., 2016). The
competition assay for binding to SERCA2b suggested that ALN interacts with the same region of the Ca2+-ATPase as PLB and SLN.
The present study revealed that TM11 is located at acompletely different site from those micropeptides, suggestingthat TM11 regulates SERCA activity in a different manner (formore details, see the following 2 sections). PLB and SLN are reported to bind and preferentially stabilize the Ca2+-free E2 state
and/or the E1-Mg2+ state before Ca2+ binding, thereby inhibiting
the transition to the E1-2Ca2+ state (MacLennan and Kranias,
2003; Toyoshima et al., 2003, 2013; Winther et al., 2013). In
this context, SLN contributes to muscle-based thermogenesis
in mammalian cells by uncoupling SERCA-mediated ATP hydrolysis from Ca2+ transport (Bal et al., 2012; Ikeda et al., 2017). Previous studies showed that TM11 is critical for low rates of the E2P
dephosphorylation step, E2P to E2, and the conformational transition of the dephosphoenzyme, E2 to E1 (Gorski et al., 2012),
whereas the subsequent LE region prevents the E1P to E2P transition, independent of TM11 (Clausen et al., 2012; Dode et al.,2003). Thus, TM11 and the LE region likely inhibit the catalytic
cycle of SERCA2b in different ways, while their possible rolesin the exothermic activity of SERCA2b remains to be explored.

Figure 4. Regulatory Roles of TM11 and Its Interactions with
Neighboring Residues in the SERCA2b ATPase Cycle
ATPase activities of microsomal SERCA2b and SERCA2a. The specific activities of SERCA2b WT (black), F1018G/V1029G (blue), F1018G (green), V1029G(orange), and SERCA2a WT (red) are plotted as a function of Ca2+ concentration. Bar graphs indicate the maximal turnover rate of the ATPase activity (Vmax;
left) and the Ca2+-binding affinity (Kd for Ca2+; right) of SERCA2b WT, its mutants, and SERCA2a WT. Results presented in graphs are means ± SD of 3
independent experiments. ns, not significant; *p < 0.05 and **p < 0.005.

involved in regulating the position of TM11 and its interactions
with the surrounding regions. However, the present crystallographic study reveals that TM11 is located on the opposite
side to TM7, close to TM10, and makes no direct contact with
TM7 (Figure 1B). We surmise that mutational disruption of the
Arg835 (TM7)-Glu917 (L8/9) interaction could greatly alter theL8/9 conformation, thereby diminishing interactions between
L8/9 and TM11 and hence reducing inhibition by TM11.
Different Binding Sites for TM11 and Phospholamban
and Sarcolipin Proteins in SERCA Family Members
It is well known that SERCA2a and SERCA1a are regulated by thesmall single-spanning membrane protein phospholamban (PLB)
and its homologous protein sarcolipin (SLN) (Hughes et al.,
1994; Odermatt et al., 1998). Similar to SERCA2a and SERCA1a,
PLB and SLN are abundant in cardiac and skeletal muscle,
respectively, whereas SERCA2b is expressed ubiquitously (Burket al., 1989). The crystal structure of SERCA1a in complex withSLN demonstrated that SLN binds to the TM groove formed by
TM2, TM4, TM6, and TM9 (Figure 5A) (Toyoshima et al., 2013;
Winther et al., 2013). More recently, a transmembrane micropeptide, another-regulin (ALN), which is expressed ubiquitously andshares key amino acids with PLB and SLN, was identified to inhibit

1226 Cell Reports 27, 1221–1230, April 23, 2019

Similar Binding Sites of TM11 in SERCA2b and the TM
Helix of Neuroplastin in PII-type ATPase-1
Neuroplastin (NPTN) and Basigin, members of the immunoglobulin (Ig) superfamily with a single TM helix, have recently been
identified as obligatory auxiliary subunits of the plasma membrane Ca2+ ATPase (PMCA) (Schmidt et al., 2017). PMCA proteins belong to the PII-type ATPase family, like SERCA proteins,and endogenously form a heterotetramer involving NPTN or
Basigin to fulfill the Ca2+ transport activity. The single TM helixof NPTN or Basigin plays particular roles in stabilizing and trafficking the PMCA complexes (Schmidt et al., 2017). The latest
study revealed the cryo-EM structure of PMCA1 in complex
with NPTN (Gong et al., 2018), in which PMCA1 assumes aconformation similar to an E1-Mg2+ state, a transient statefrom E2 to E1-2Ca2+ states. The TM helix and the Ig domain of
NPTN were found to interact with TM10 and the L7/8 loop of
PMCA1, respectively. The superposition of SERCA2b with the
PMCA1-NPTN complex indicates that TM11 in SERCA2b islocated at a similar position to the NPTN TM helix in the
PMCA1-NPTN complex, even though these 2 TM helices arearranged in different angles with respect to the TM helix bundle
domains (Figure 5B). The NPTN TM helix (21 residues), which islonger than SERCA2b TM11 (13 residues), interacts tightly with
TM10 of PMCA1 (Figure S6A). The contact area between the
NPTN TM helix and PMCA1 TM10 is 259.1 Å2, while that between
TM11 and TM10 in SERCA2b is 80.5 Å2. Accordingly, the NPTN
TM helix region has low B-factor values (50–100 Å2), suggesting
its rigid conformation (Figure S6B). Unlike TM11 in SERCA2b,
NPTN may serve as an essential partner rather than a weakly
or transiently interacting modulator of PMCA1.
Possible Mechanisms Underlying TM11-Mediated
SERCA2b Regulation
The membranous domain of SERCA can be subdivided into 3 subdomains consisting of TM1–TM2, TM3–TM4, and TM5–TM10

Figure 5. Positional Comparisons of the TM
Helices of the Auxiliary Subunits of Ca2+
ATPases and TM11 in SERCA2b(A) Crystal structures of SERCA2b in the E12Ca2+-AMPPCP state and sarcolipin (SLN)-bound
SERCA1a in the E1-Mg2+ form (PBD: 3W5A) aresuperimposed such that the RMSD of the Caatoms in TM5–TM10 is minimized. SERCA2b,
SERCA1a, TM11 of SERCA2b, and SLN are
shown in blue, yellow, red, and green, respectively. The side view of the superimposition (left)
and the top view from the cytosol (right) are shown.
(B) Crystal structures of SERCA2b in the E12Ca2+-AMPPCP state and plasma membrane
Ca2+ ATPase-1 (PMCA1) in complex with neuroplastin (NPTN) (PBD: 6A69) are superimposedsuch that the RMSD of the Ca atoms in TM5–TM10
is minimized. SERCA2b, TM11 of SERCA2b,
PMCA1, and NPTN are shown in blue, red, orange,
and cyan, respectively. The side view of thesuperimposition (left) and the top view from thecytosol (right) are shown.

helices, respectively. During the transition between Ca2+-boundE1 and Ca2+-released E2 states, the association and separationof these 3 subdomains take place to control the closing and opening of the luminal Ca2+ pathway (Olesen et al., 2007). Concomitantly, the TM1–TM2 subdomain undergoes striking upward anddownward shifts, and TM4 becomes kinked at a conserved proline residue, influencing the orientation and intra-subdomain
arrangement of TM5–TM10 (Møller et al., 2010). Although no information is yet available for the structures of SERCA2b in intermediate states other than the E1-2Ca2+-AMPPCP state, it is possiblethat the extra TM helix weakly interacting with other TM regions
serves to control such rearrangement of the TM helix bundle
domain during the SERCA catalytic cycle.
In this context, when the cytosolic A, N, and P domainsof SERCA2b and SERCA2a are superimposed such that the
root-mean-square deviation (RMSD) of their Ca atoms is minimized, the TM1–TM2, TM3–TM4, and TM5–TM10 subdomainsof SERCA2b are shifted by 2.6 –3.4 at the position of Ala327

with respect to those in SERCA2a, andend up further apart from TM11 as aconsequence (Figure 6A). Accordingly,
W1028 and V1029 of TM11 in SERCA2b
would undergo steric clashes with L967,
L970, and K971 of TM10 in SERCA2a(Figure 6A). To avoid these stericclashes, the movement of the TM helix
bundle domain appears to be more
restricted in SERCA2b than in SERCA2a(Figure 6B).
It has been established that SERCA
family enzymes undergo significant dynamics in both the cytosolic and TM domains during the catalytic cycle (Møller
et al., 2010). In this regard, we note thatwhen any of the TM helix subdomains issuperimposed between SERCA2a and
SERCA2b, a slight but significant orientation shift is seen in theircytosolic domains, while there is no striking difference in theother TM helix subdomains (Figure S7). Thus, it is also interpretable that the presence of TM11 alters the tilt angle of the cytosolic domains relative to the TM helix bundle domain in the
E1-2Ca2+-ATP state. Although structural information for other reaction intermediates is required to completely delineate how
TM11 affects the location and dynamics of each TM helix andcytosolic domain during the reaction cycle, we propose apossible role for TM11 in controlling the relative arrangementof the TM helix bundle and cytosolic domains in SERCA2b forthe modulation of its activity (Figure 6B).
Further Perspectives
Functional regulation by accessory TM regions has been reported previously for other P-type ATPases. In P1B-type
ATPases, 2 additional N-terminal TMs modulate the entry andexit of Zn2+ and Cu2+ ions (Gourdon et al., 2011; Wang et al.,Cell Reports 27, 1221–1230, April 23, 2019 1227

Figure 6. SERCA2b Regulation via TM11
Interplay with Other TM Helices(A) Superposition of crystal structures of SERCA2b(cyan) and SERCA2a (magenta) highlights different
tilt angles of the TM helix clusters TM1–TM2, TM3–
TM4, and TM5–TM10, between the 2 proteins, inwhich Ala327 serves as the cardinal point. Arrows
indicate that TM helices in SERCA2b are shifted by
2.6 –3.4 at the position of Ala327 with respect tothose in SERCA2a, most likely due to the presenceof TM11. The inset shows a close-up view of thevicinity of TM11. TM11 residues that would experience steric clashes with TM10 of SERCA2a areshown in stick representation. Yellow dotted lines in
the inset indicate pairs of atoms located within 2.4 Åbetween TM11 of SERCA2b and TM10 of SERCA2a.
(B) Proposed model of TM11-dependent regulationof the relative domain orientation in SERCA2b. The
presence of TM11 restricts the movement of the TM
helix bundle domain relative to the cytosolic domains due to the steric hindrance shown in (A),leading to lower ATPase activity for SERCA2b. The
length of double-headed arrows reflects the mobilityof the TM helices with respect to the cytosolic domains in SERCA2b (left) and SERCA2a (right).

2014). Na+/K+-ATPase forms a heterotrimer composed of an
a-subunit containing A, N, and P domains, 10 TMs, and single
TM b- and g-subunits (Morth et al., 2007). Association of theb- and g-subunits with the a-subunit modulates the affinity for
K+ and/or Na+ (Geering, 2005; Hasler et al., 2001). Moreover, inthe structure of H+-ATPase in the AMPPCP-bound state, theC-terminal regulatory domain (R domain) lacks electron density,
presumably due to high flexibility (Pedersen et al., 2007). The R
domain is believed to function as an auto-inhibitor that restricts
TM helix movement and/or access of ions to the membranetransport core during the reaction cycle (Morth et al., 2011).
Thus, regulating the interactions between additional TM moieties
and the main helix bundle domain likely represents an effective
and common strategy for the functional regulation of P-type
ATPases.
Several recent reports suggest that ER-resident redox enzymes such as ERdj5, ERp57, TMX1, and SEPN1 are involved

1228 Cell Reports 27, 1221–1230, April 23, 2019

in regulating SERCA2b activity (Li and Camacho, 2004; Marino et al., 2015; Raturi
et al., 2016; Ushioda et al., 2016). While
SEPN1 helps to maintain SERCA2b activity
by protecting the hyperoxidation of the ER
luminal Cys875 (Marino et al., 2015), theER-resident disulfide reductase ERdj5
stimulates SERCA2b activity by reducing
the Cys875-Cys887 disulfide in the L7/8
loop (Ushioda et al., 2016). ER chaperones
calnexin and calreticulin and the CREC
(Ca2+-binding protein of 45 kDa [Cab45],
reticulocalbin, ER Ca2+-binding protein of
55 kDa [ERC-55], and calumenin) family
protein Cab45S also bind SERCA2b,
thereby inhibiting its Ca2+ pump activity(Chen et al., 2016; John et al., 1998; Roderick et al., 2000).
TMTC1 and TMTC2, which consist of multiple transmembranesegments and tetratricopeptide repeat (TPR)-containing adaptor
domains, associate with SERCA2b to influence intracellular
calcium levels (Sunryd et al., 2014). Thus, several ER luminal or
membrane proteins are involved in the regulation of SERCA2b
activity, although detailed mechanisms have yet to be uncovered. However, based on the findings of the present study, we
surmise that such regulatory partner proteins may affect thelocation of TM11 and/or its interaction with other SERCA2b regions, thereby modulating SERCA2b activity but not SERCA2aactivity. In line with this, the ER chaperone calnexin physicallyinteracts with the C terminus of SERCA2b to control the Ca2+
store in the ER (Roderick et al., 2000). The structural insightgained in this study provides a framework for understanding
the mechanism underlying TM11-mediated SERCA2b regulation
for maintaining Ca2+ homeostasis in the ER.

STAR+METHODSREFERENCES

Detailed methods are provided in the online version of this paper
and include the following:

Adams, P.D., Afonine, P.V., Bunkóczi, G., Chen, V.B., Davis, I.W., Echols, N.,
Headd, J.J., Hung, L.-W., Kapral, G.J., Grosse-Kunstleve, R.W., et al. (2010).
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d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURSE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILSd METHOD DETAILSB Construction of plasmids and cell lines
B Crystallization of SERCA2b and SERCA2a
B Data collection and structure determination
B Preparation and analysis of SeMet-labeled SERCA2b
B Preparation of microsomal SERCA2b and SERCA2a
B ATPase activity assay
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND SOFTWARE AVAILABILITY

SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.
celrep.2019.03.106.
ACKNOWLEDGMENTS
We thank the beamline scientists of SPring-8 and Photon Factory for theirassistance with X-ray diffraction data collection. This work was supported
by CREST, JST (to K.I. and K.N., grant number JPMJCR13M6), Grants-inAid for Scientific Research on Innovative Areas from MEXT (15H04335 and26116005 to K.I. and 26119007 to T.T.), the Takeda Science Foundation (toK.I.), and the Platform Project for Supporting Drug Discovery and Life Science
Research/Platform for Drug Discovery, Informatics, and Structural Life Science (PDIS) from the Japan Agency for Medical Research and Development(AMED) (to K.I.). This work was also supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED
(JP18am0101075 and JP18am0101078 to J.T. and Y.K.).
AUTHOR CONTRIBUTIONS
M.I. constructed a large-scale expression system for SERCA2b and
SERCA2a and established the purification methods. M.I. and J.T. prepared
the vector for the overexpression of PA-tagged SERCA2b. N.S. purified
SERCA2a on a large scale. M.I., N.S., K.Y., Y.T., and T.T. crystallized
SERCA2a and SERCA2b using the LCP method. M.I., N.S., and S.W.
collected and analyzed X-ray diffraction data and determined the structuresof SERCA2a and SERCA2b. S.W. merged the diffraction data and refined thestructures. M.I. and Y.Z. constructed plasmids for expressing SERCA2b
mutants; prepared microsomal SERCA2b WT, F1019/V1028 mutants, and
SERCA2a WT; and measured their ATPase activities. Y.K. prepared antiPA antibody and conjugated Sepharose beads. K.I., R.U., and K.N. designed
the study. K.I. supervised the work. M.I. and S.W. prepared the figures. K.I.,
M.I., and S.W. wrote the manuscript. All of the authors discussed the results
and approved the manuscript.
DECLARATION OF INTERESTS

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Received: December 3, 2018
Revised: February 28, 2019
Accepted: March 27, 2019
Published: April 23, 2019

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STAR+METHODSKEY RESOURCES TABLEREAGENT or RESOURCESOURCEIDENTIFIER

Antibodies
SERCA2 Antibody (N-19)

Santa Cruz Biotechnology

sc-8095

Donkey F(ab’)2 Anti-Goat IgG (HRP)

Abcam

Ab98519

Cumate (p-Isopropylbenzoic Acid)

FUJIFILM Wako Pure Chemical Corporation

037-06412

Phorbol 12-myristate 13-acetate

FUJIFILM Wako Pure Chemical Corporation

168-23593

Monoolein

Nu-Chek-Prep, Inc.

M-239

Chemicals, Peptides, and Recombinant Proteinsb, g -Methyleneadenosine 50 -triphosphate disodium salt

Sigma

M7510

Polyethylenimine

Sigma

408727

Seleno-L-methionine

NACALAI TESQUE

02286-75

PA-SERCA2b

Ushioda et al., 2016

N/A

PA-SERCA2aThis study

N/A

Thermo Fisher Scientific

E6646

SERCA2b E1-AMPPCP form

This paper

PDB: 5ZTF

SERCA2a E1-AMPPCP form

This paper

PDB: 6JJU

ATCC

CRL-3216

Critical Commercial Assays
EnzCheckTM Phosphate Assay Kit
Deposited Data

Experimental Models: Cell Lines
Human: HEK293T cells
Oligonucleotides
Primer: SERCA2a forward: taatacgactcactataggg

This paper

N/A

Primer: SERCA2a reverse: tgcggccgcttactccagta
ttgcaggttccaggtagttgcgggcc

This paper

N/A

Primer: SERCA2bF1018G forward: taccctaaagga
ccggcccacacgacgagtattac

This paper

N/A

Primer: SERCA2bF1018G reverse: forward:catta
tgagcagcacacccggccaggaaatcccat

This paper

N/A

Primer: SERCA2bV1029G forward: cggggaccact
agaccccaatatcgtgtctgtgat

This paper

N/A

Primer: SERCA2bV1029G reverse: tagtgtctgtgct
ataaccccagatcaccaggggc

This paper

N/A

Plasmid: pcDNA3.1/PA-SERCA2b

Ushioda et al., 2016

N/A

PiggyBac Cumate Switch Inducible Vector

System Biosciences, LLC, CA, USA#PBQM812A-1

Super PiggyBac Transposase Expression Vector

System Biosciences, LLC, CA, USA#PB200PA-1

Recombinant DNA

Software and Algorithms
Prism 7GraphPad software

N/A

XDS

Kabsch, 2010

http://xds.mpimf-heidelberg.
mpg.deKAMO

Yamashita et al., 2018

N/A

DIALS

Waterman et al., 2016

N/A

MOLREP

Vagin and Teplyakov, 2010

http://www.ccp4.ac.uk/html/
molrep.html(Continued on next page)

Cell Reports 27, 1221–1230.e1–e3, April 23, 2019 e1

Continued
REAGENT or RESOURCESOURCEIDENTIFIER

AIMLESS

Evans and Murshudov, 2013

N/A

Image Lab

Bio-Rad Laboratories, Inc., CA, USA

N/A

COOT

Emsley and Cowtan, 2004

https://www2.mrc-lmb.cam.
ac.uk/personal/pemsley/coot/

PHENIX

Adams et al., 2010https://www.phenix-online.org

Pymol

De Lano, 2006

https://pymol.org/2/

CONTACT FOR REAGENT AND RESOURSE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Kenji
Inaba (kinaba@tagen.tohoku.ac.jp).
EXPERIMENTAL MODEL AND SUBJECT DETAILSHEK293T cells were used for overexpression of recombinant human SERCA2b and SERCA2a.
METHOD DETAILS
Construction of plasmids and cell lines
In the present crystallographic studies, we engineered PA-tagged SERCA2a (PA-SERCA2a) using plasmid pcDNA3.1/PA-SERCA2b
constructed in our previous study (Ushioda et al., 2016) as a template. The open reading frame (ORF) of SERCA2a was amplified by
PCR using primers 50 -taatacgactcactataggg-30 and 50 -tgcggccgcttactccagtattgcaggttccaggtagttgcgggcc-3 0 and subcloned into
pcDNA3.1. The ORF of PA-SERCA2b or PA-SERCA2a was isolated from pcDNA3.1 by digestion with NheI and NotI, and inserted
into the NheI and NotI sites of the PiggyBac Cumate Switch Inducible Vector (System Biosciences, LLC, CA, USA) to establish acell line that inducibly expresses PA-SERCA2b or PA-SERCA2a, respectively. For functional assays, we also engineered theF1018G, V1029G and F1018G/V1029G mutants of PA-SERCA2b using the plasmid pcDNA3.1/PA-SERCA2b as a template. The
SERCA2b mutants were prepared by site-directed mutagenesis with appropriate primers as follows: 50 -taccctaaaggaccggcccacac
gacgagtattac-30 and 50 -cattatgagcagcacacccggccaggaaatcccat-30 for F1018G, 50 -cggggaccactagaccccaatatcgtgtctgtgat-30 and
50 -tagtgtctgtgctataaccccagatcaccaggggc-30 for V1029G and the all four primers for F1018G/V1029G.
Crystallization of SERCA2b and SERCA2aExpression and purification of PA-SERCA2b and PA-SERCA2a were performed as described previously (Ushioda et al., 2016),
except that 1 mM sodium butyrate was not included in induction reagents. Briefly, HEK293T cells were co-transfected with the
PA-SERCA2b- or PA-SERCA2a-inserted PiggyBac Cumate Switch Inducible vector and Super PiggyBac Transposase Expression
Vector (System Biosciences, LLC, CA, USA) using polyethylenimine (Sigma) to establish a cell line stably expressing each of theproteins. High expressions of PA-SERCA2b and HA-SERCA2a were induced with cumate and 50 ng/ml phorbol 12-myristate 13-acetate (PMA). The cells were cultured in DMEM supplemented with 4% inactivated fetal calf serum at 37 C for 48 hr after induction.
Harvested cells were solubilized with 1% (w/v) DDM, and PA-SERCA2b and PA-SERCA2a were purified by anti-PA Sepharose
beads. During the final step of purification, PA-SERCA2b and PA-SERCA2a were passed through a Superose 6 column (GE Healthcare UK Ltd., Buckinghamshire, UK) equilibrated with buffer containing 100 mM MOPS (pH 6.8), 80 mM KCl, 20% glycerol, 1 mM
CaCl2, 1 mM MgCl2, 1 mM DTT and 0.1% n-dodecyl-b-D-maltoside (DDM). PA-SERCA2b and PA-SERCA2a thus purified wereconcentrated to 1018 mg ml-1, mixed with 1 mM AMPPCP and incubated overnight at 4 C. After centrifugation at 22,300 g for
30 min, proteins were reconstituted into monoolein at a protein-to-lipid ratio of 2:3 (v/v) (Caffrey and Cherezov, 2009). Crystals of
PA-SERCA2b were grown at 20 C in reservoir solution (2834% polyethylene glycol 250 dimethylethanol, 100 mM Tris-HCl (pH
7.17.3), 100 mM KSCN) for 23 days. Similarly, crystals of PA-SERCA2a were grown at 16 C in 28.529.5% polyethylene glycol
250 dimethylethanol, 100 mM Tris-HCl (pH 7.7) and 50 mM NH4NO3 for 23 days.
Data collection and structure determination
X-ray diffraction data were collected at SPring-8 beamline BL32XU. Small wedges of data were collected from a number of microcrystals and scaled together. For reduced SERCA2a, a complete dataset was collected from a single crystal. Diffraction data were
integrated with XDS (Kabsch, 2010) using the KAMO system (Yamashita et al., 2018) or DIALS (Waterman et al., 2016). Multiple datasets were analyzed with BLEND, followed by scaling and merging with AIMLESS (Evans and Murshudov, 2013) based on CC1/2
values. Initial phases were calculated by molecular replacement with MOLREP using the rabbit SERCA1a E1-AMPPCP structure

e2 Cell Reports 27, 1221–1230.e1–e3, April 23, 2019

(PBD ID: 1T5S). Model building and refinement were carried out with COOT (Emsley et al., 2010) and PHENIX (Adams et al., 2010),
respectively. Final models were refined to reasonable Rfree values with good geometry, compared with all published crystal structuresof comparable resolution.
Preparation and analysis of SeMet-labeled SERCA2b
Cells inducibly expressing PA-SERCA2b were cultured in 4% FCS and Dulbecco’s modified Eagles’ medium (DMEM) without methionine. Expression of SeMet-labeled PA-SERCA2b was induced with 30 mg/ml Seleno-L-methionine, 50 nM phorbol 12-myristate
13-acetate and 150 mg/ml cumate. Cells were harvested after 2 days of incubation at 37 C. Purification, crystallization and X-ray
diffraction data collection for SeMet-labeled SERCA2b were performed following the same procedures as described above, exceptthat radiation with a wavelength of 0.978 Å was employed for data collection and calculation of an anomalous difference Fourier map
to identify SeMet residues in SERCA2b.
Preparation of microsomal SERCA2b and SERCA2aHEK293T cells transfected with WT, F1018G, V1029G or F1018G/V1029G mutants of PA-SERCA2b or WT PA-SERCA2a wereincubated in DMEM at 37 C for 2 days. Harvested cells were repeatedly homogenized in 250 mM sucrose, 150 mM KCl, 10 mM
Tris-HCl (pH 7.5), 100 nM CaCl2 and 0.2% NaN3 with a Dounce Tissue Grinder (DWK Life Sciences GmbH, Wertheim/Main,
Germany), and subsequently centrifuged at 10,000 g for 20 min at 4 C. Homogenates were ultracentrifuged at 100,000 g for
1 hour at 4 C, and pellets were resuspended in 50 mM HEPES (pH 7.0), 100 mM NaCl and 20% glycerol, and homogenized by repeatedly passing the suspension through a 1 mL syringe with a 25 G needle. The concentrations of SERCA2b and SERCA2a in
microsomes were estimated by western blotting with anti-SERCA2 antibody, based on a calibration curve calculated from theband intensity of purified SERCA2b of known concentrations. Band intensities were quantified and analyzed by Image Lab (Bio-Rad
Laboratories, Inc., CA, USA).
ATPase activity assay
The ATPase activities of SERCA2b and SERCA2a in microsomes were measured as described previously (Ushioda et al., 2016).
Briefly, the microsomal SERCA2b or SERCA2a was incubated in buffer containing 50 mM HEPES (pH 7.0), 100 mM NaCl, 20% glycerol, 50 mM NaN3, 5 mM MgCl2, 1 mM EGTA and various concentrations of CaCl2. 1 mM ATP was added to initiate the SERCA
ATPase cycle. Each calcium concentration in the reaction buffer was determined using the chelator program (Schoenmakers
et al., 1992). The reaction was stopped by 5 mM EGTA, and the resultant solution was mixed with EnzCheck phosphate assay kit(Thermo Fisher Scientific). The mixture was incubated at 22 C for 30 min, and the absorbance at 360 nm was measured by
U-3900 Spectrophotometer (HITACHI). Curve fitting of the SERCA2b and SERCA2a activities at different calcium concentrations
was performed using Prism7 (Graphpad software) to determine Kd for Ca2+, Vmax and Hill slope values (Table S1).
QUANTIFICATION AND STATISTICAL ANALYSIS
Biochemical assays shown in Figure 4 were performed in three independent replicates. Statistical analysis was performed inGraphPad Prism7 using one-way ANOVA analysis followed by Tukey’s multiple comparison test. Values are expressed asmeans ± SD. Differences with a p-value of < 0.05 are considered to be significant.
DATA AND SOFTWARE AVAILABILITY
Atomic coordinates and structure factors of human SERCA2b and SERCA2a have been deposited in the Protein Data Bank underaccession codes 5ZTF and 6JJU, respectively.

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