Supporting information
Hierarchical Domain-Motion Analysis of Conformational
Changes in Sarcoplasmic Reticulum Ca2+-ATPase
Chigusa Kobayashi1, Ryotaro Koike2, Motonori Ota2, and Yuji Sugita1,3,4,5
1
RIKEN Advanced Institute for Computational Science, 7-1-26 Minatojima-minamimachi, Chuo-ku,
Kobe, Hyogo Kobe 640-0047, Japan, 2Graduate School of Information Science, Nagoya University,
Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan, 3RIKEN Theoretical Molecular Science
Laboratory, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan, 4RIKEN Quantitative Biology
Center, 7-1-26 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo Kobe 640-0047, Japan, 5RIKEN
iTHES, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
Movie S1
Conformational changes during the whole pump cycle. Intermediate structures are calculated by
Climber1.
Figure S1. MT and rigid domains in the reaction from E1·Mg2+ to E1·2Ca2+, (reaction G)
The diagram and cartoons are depicted as in Fig. 3. The bottom cartoons show the crystal structure
in the E1·Mg2+ state, and is the top left one the crystal structure in the E1·2Ca2+ state.
Figure S2. MT and rigid domains in the reaction from E1·2Ca2+ to E1·ATP, (reaction A)
The diagram and cartoons are depicted as in Fig. 3. The bottom cartoons show the crystal structure
in the E1·2Ca2+ state, and is the top left one the crystal structure in the E1·ATP state
Figure S3. MT and rigid domains in the reaction from E1·ATP to E1~P·ADP, (reaction B)
The diagram and cartoons are depicted as in Fig. 3. The bottom cartoons show the crystal structure
in the E1·ATP state, and the left cartoon shows the crystal structure in the E1~P·ADP state.
Figure S4. MT and rigid domains in the reaction from E1~P·ADP to E2P, (reaction C)
The diagram and cartoons are depicted as in Fig. 3. The bottom cartoons show the crystal structure
in the E1~P·ADP state, and is the top left one the crystal structure in the E2P state.
Figure S5. MT and rigid domains in the reaction from E2P to E2·Pi. (reaction D)
The diagram and cartoons are depicted as in Fig. 3. The bottom cartoons show the crystal structure
in the E2P state, and is the top left one the crystal structure in the E2·Pi state.
Figure S6. Accumulated RMSD of intermediate structures on the morphing trajectory for each
reaction step. Boxes represent RMSDs of CRDs in the same colors as Fig. 6 and total RMSD in
black.
Table S1. Crystal structures of SR Ca2+-ATPase in PDB. “PCA” corresponds to reaction states in
Fig. 2. “N/A” means that the structure is not included in PCA analysis due to missing residues.
E1·Mg
E1·Mg2+
E1·2Mg2+
E1·2Ca2+
E1·2Ca2+
E1·2Ca2+ (E309Q)
E1·Mg
E1·Mg2+
N/A
E1·2Ca2+
E1·2Ca2+
N/A
TNPAMP, Mg
TNPAMP, Mg2+
AMPPCP, Mg2+
Mg
Mg2+
2Mg2+
2Ca2+
2Ca2+
2Ca2+
Resolution
(Å)
3.01
3.01
3.1
2.4
3
3.5
E1·ATP
E1·ATP
E1·ATP
E1·ATP
E1·ATP
E1~P·ADP
E1~P·ADP
E1P·ADP
E1·ATP
E1·ATP
E1·ATP
E1·ATP
N/A
E1~P·ADP
E1~P·ADP
N/A
AMPPCP, Ca2+
AMPPCP, Ca2+
AMPPCP, Mg2+
AMPPCP, Mg2+
AMPPCP, Mg2+
ADP, AlF4-, 2Mg2+
ADP, AlF4-, 2Mg2+
AMPPN, Ca2+
2Ca2+
2Ca2+
2Ca2+
2Ca2+
2Ca2+
2Ca2+
2Ca2+
2Ca2+
2.5
2.58
2.6
2.9
2.95
2.4
2.9
2.8
3AR27
3N8G8
1T5S9
1VFP 10
3TLM11
2ZBD12
1T5T9
3BA613
E2P
E2P
E2P
E2P
E2P
E2P
BeF3-, Mg2+
BeF3-, Mg2+
BeF3-, Mg2+
Mg2+
TG1
2.65
3.8
2.4
3B9B13
2ZBE14
2ZBF14
E2P·ATP
E2~P
E2~P
E2~P
E2P
E2~P
E2~P
E2~P
BeF3-, Mg2+, TNP-AMP
AlF4-, Mg2+
AlF4-, Mg2+
AlF4-, Mg2+
TG1
TG1
TG1
TG1
2.6
2.55
2.2
3
3AR97
2ZBG14
3N5K8
1XP515
E2~P·ATP
E2~P·TNP-AMP
E2~P·ATP
E2~P·ATP
N/A
AlF4-, Mg2+, AMPPCP
AlF4- ,Mg2, TNP-AMP
TG1
3
2.6
3B9R13
3AR87
1HT9
2.65
2.3
2.5
2.5
2.55
2.45
2.4
3.1
3.4
3.1
3.4
2.8
2.9
2.65
3.1
3.1
3.1
3.1
3.1
2.8
3.2
2O9J16
1WPG12
3FGO17
4BEW18
3FPB17
3W5D2
2AGV19
3NAN20
2EAS21
3W5C2
2OA016
2EAU21
2EAT21
3NAL20
3NAM20
1IWO22
2C8L5
2EAR21
2YFY23
4KYT24
4J2T 25
CPA2, Mg2+
TG1
Boc-12ADT10
TG1
TG1
TG1
TG1
TG1
TG1
TG1
3.2
2.3
3.3
2.15
2.5
2.8
3.1
2.2
2.2
2.15
3FPS17
3AR37
2BY426
3AR47
2DQS27
2C8K5
2C885
3AR67
3AR57
3AR77
State
2+
E2·Pi
E2·Pi
E2·Pi
E2·Pi
E2·Pi
E2·Pi
E2
E2
E2
E2
E2
E2
E2
E2
E2
E2
E2
E2
E2
E2
E2
E2·ADP
E2·ADP
E2·ATP
E2·ATP
E2·ATP
E2·ATP
E2·ATP
E2·ATP
E2·ATP
E2·ATP
1TG:
PCA
2+
E2·Pi
E2·Pi
E2·Pi
E2·Pi
E2·Pi
E2
E2
E2
E2
N/A
E2
E2
E2
E2
E2
E2
E2
E2
N/A
E2
E2·ADP
E2·ADP
E2·ATP
E2·ATP
E2·ATP
E2·ATP
E2·ATP
E2·ATP
E2·ATP
E2·ATP
P-domain ligands
2+
MgF42-, Mg2+
MgF42-, ADP, Mg2+
MgF42-, AMPPCP, Mg2+, Mn2+
MgF3-, AMPPCP, Mg2+, Mn2+
MgF42-, Mg2+,ATP
SO42Mg2+
ADP, Mg2+
Mg2+
Mg2+
Mg2+
ADP, Mg2+
ADP,2Mg2+
AMPPCP, 2Mg2+
ATP, 2Mg2+
AMPPCP, 2Mg2+
AMPPCP, Mg2+
AMPPCP, Mg2+
TNP-ATP, Mg2+
TNP-AMP
TNP-ATP
TM ligands
2+
CPA2
TG1
CPA2 , Mn2+
CPA2 , Mn2+
CPA2 , Mg2+
TG1, BHQ3
Boc-Tg4
CPA2
CPA2
CPA2, CC5
CPA2, TG1
DTB6
dOTg7
TG1
TG1
TG1
dBTG8
PDB ID
3W5A2
3W5B2
4H1W3
1SU44
2C9M5
4NAB6
thapsigargin,
-cyclopiazonic acid,
2,5-di-tert-butyl-1,4-dihydroxybenzene,
4Boc-Tg: [2-N-tert-butoxyl-carbonyl,4-hydroxy[-4-phenyl-butanoyl-8-O-debutanoyl]thapsigargin,
5CC: curcumin,
6DTB: 8-O-(dodecanoyl-8-O-debutanoyltrilobolide),
7dOTg: 2-deoctanoyl-4,5-dihydrothapsigargin,
8dBTG: debutanoyl thapsigargin,
91HT:(3S,3aR,4S,6S,6aR,7S,8S,9bS)-6-(acetyloxy)-3a,4-bis(butanoyloxy)-3-hydroxy-3,6,9-trimethyl-8-([(2E)-2-methylbut-2-enoyl]oxy)-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-decahydroaz
uleno[4,5-b]furan-7-yl octanoate
10Boc-12ADT: N-tert-butoxycarbonyl-12-aminododecanoyl-8-O-debutanoyl thapsigargin,
2CPA:
3BHQ:
Table S2. Residues of rigid domains in individual Motion Trees for the reaction steps.
Step
1
2
3
4
5
6
7
8
9
A
1-44
51-59
60-75
89-125
236-244
245-258
259-273
344-361
362-502
293-343
600-687
509-599
688-688
689-692
693-820
821-822
126-235
823-856
894-950
952-962
966-989
B
1-44
237-242
51-75
89-236
243-273
293-502
509-856
894-950
952-962
966-989
C
1-44
51-75
329-359
360-502
754-807
131-135
89-130
241-273
293-328
602-753
509-601
836-856
140-240
136-139
808-835
894-929
930
931-950
952-962
966-989
D
1-44
51-75
125-240
89-111
112-124
241-273
293-351
352-355
356-356
357-502
604-856
509-603
894-950
952-962
966-989
E
1-44
51-75
110-239
89-109
703-704
254-273
727-729
293-345
240-253
346-361
362-502
600-689
509-599
240-273
293-327
328-360
361-502
767-768
750-766
602-749
509-601
771-813
769-770
814-830
690-702
705-726
730-856
894-950
952-962
966-989
F
1-44
51-75
126-239
89-111
112-125
831-856
894-950
952-962
966-989
G
1-44
51-59
60-75
236-244
245-273
309-332
361-502
509-600
127-235
89-126
333-341
342-360
724
293-308
755-768
601-723
769-772
773-786
725-754
787-799
800-813
814-828
829-856
894-950
952-962
966-989
Table S3. Common rigid domains (CRDs) during the whole reaction cycle of SR Ca2+-ATPase.
Domain
residue ID (# of residues)
A
1-44,131-135,140-235 (145)
M1
60-75 (16)
M2L
89-109 (21)
M2C
112-124(13)
M3L
259-273 (15)
M4L
293-308 (16)
M4C
309-327 (19)
P1
346-351,604-687 (90)
N
362-502,509-599 (232)
P2
329-332,693-702,705-723,730-749,814-820,823-828 (66)
M5M
755-766 (12)
M5L-M10
773-786,800-807,836-856,894-929,931-950,952-962,966-989 (134)
M6L
787-799 (13)
M6C,M7C
808-813,831-835 (11)
References
1.
Weiss DR, Levitt M. Can Morphing Methods Predict Intermediate Structures? J Mol Biol 2009;385:665-674.
2.
Toyoshima C, Iwasawa S, Ogawa H, Hirata A, Tsueda J, Inesi G. Crystal structures of the calcium pump and
sarcolipin in the Mg2+-bound E1 state. Nature 2013;495:260-264.
3.
Winther AML, Bublitz M, Karlsen JL, Moller JV, Hansen JB, Nissen P, Buch-Pedersen MJ. The
sarcolipin-bound calcium pump stabilizes calcium sites exposed to the cytoplasm. Nature 2013;495:265-269.
4.
Toyoshima C, Nakasako M, Nomura H, Ogawa H. Crystal structure of the calcium pump of sarcoplasmic
reticulum at 2.6 angstrom resolution. Nature 2000;405:647-655.
5.
Jensen AML, Sorensen TLM, Olesen C, Moller JV, Nissen P. Modulatory and catalytic modes of ATP binding
by the calcium pump. EMBO J 2006;25:2305-2314.
6.
Clausen JD, Bublitz M, Arnou B, Montigny C, Jaxel C, Moller JV, Nissen P, Andersen JP, le Maire M. SERCA
mutant E309Q binds two Ca2+ ions but adopts a catalytically incompetent conformation. Embo J
2013;32:3231-3243.
7.
Toyoshima C, Yonekura SI, Tsueda J, Iwasawa S. Trinitrophenyl derivatives bind differently from parent
adenine nucleotides to Ca2+-ATPase in the absence of Ca2+. Proc Natl Acad Sci USA 2011;108:1833-1838.
8.
Bublitz M, Olesen C, Poulsen H, Morth JP, Moller J, Nissen P. To be Published.
9.
Sorensen TLM, Moller JV, Nissen P. Phosphoryl transfer and calcium ion occlusion in the calcium pump.
Science 2004;304:1672-1675.
10.
Toyoshima C, Mizutani T. Crystal structure of the calcium pump with a bound ATP analogue. Nature
2004;430:529-535.
11.
Sacchetto R, Bertipaglia I, Giannetti S, Cendron L, Mascarello F, Damiani E, Carafoli E, Zanotti G. Crystal
structure of sarcoplasmic reticulum Ca2+-ATPase (SERCA) from bovine muscle. J Struct Biol 2012;178:38-44.
12.
Toyoshima C, Nomura H, Tsuda T. Lumenal gating mechanism revealed in calcium pump crystal structures
with phosphate analogues. Nature 2004;432:361-368.
13.
Olesen C, Picard M, Winther AML, Gyrup C, Morth JP, Oxvig C, Moller JV, Nissen P. The structural basis of
calcium transport by the calcium pump. Nature 2007;450:1036-U1035.
14.
Toyoshima C, Norimatsu Y, Iwasawa S, Tsuda T, Ogawa H. How processing of aspartylphosphate is coupled to
lumenal gating of the ion pathway in the calcium pump. Proc Natl Acad Sci USA 2007;104:19831-19836.
15.
Olesen C, Sorensen TLM, Nielsen RC, Moller JV, Nissen P. Dephosphorylation of the calcium pump coupled
to counterion occlusion. Science 2004;306:2251-2255.
16.
Moncoq K, Trieber CA, Young HS. The molecular basis for cyclopiazonic acid inhibition of the sarcoplasmic
reticulum calcium pump. J Biol Chem 2007;282:9748-9757.
17.
Laursen M, Bublitz M, Moncoq K, Olesen C, Moller JV, Young HS, Nissen P, Morth JP. Cyclopiazonic Acid Is
Complexed to a Divalent Metal Ion When Bound to the Sarcoplasmic Reticulum Ca 2+-ATPase. J Biol Chem
2009;284:13513-13518.
18.
Mattle D, Drachmann ND, Liu XY, Gourdon P, Pedersen BP, Morth P, Wang J, Nissen P. To be published.
19.
Obara K, Miyashita N, Xu C, Toyoshima L, Sugita Y, Inesi G, Toyoshima C. Structural role of countertransport
revealed in Ca2+ pump crystal structure in the absence of Ca2+. Proc Natl Acad Sci USA
2005;102:14489-14496.
20.
Winther AML, Liu HZ, Sonntag Y, Olesen C, le Maire M, Soehoel H, Olsen CE, Christensen SB, Nissen P,
Moller JV. Critical Roles of Hydrophobicity and Orientation of Side Chains for Inactivation of Sarcoplasmic
Reticulum Ca2+-ATPase with Thapsigargin and Thapsigargin Analogs. J Biol Chem 2010;285:28883-28892.
21.
Takahashi M, Kondou Y, Toyoshima C. Interdomain communication in calcium pump as revealed in the crystal
structures with transmembrane inhibitors. Proc Natl Acad Sci USA 2007;104:5800-5805.
22.
Toyoshima C, Nomura H. Structural changes in the calcium pump accompanying the dissociation of calcium.
Nature 2002;418:605-611.
23.
Sonntag Y, Musgaard M, Olesen C, Schiott B, Moller JV, Nissen P, Thogersen L. Mutual adaptation of a
membrane protein and its lipid bilayer during conformational changes. Nature Comm 2011;2:304.
24.
Akin BL, Hurley TD, Chen Z, Jones LR. The Structural Basis for Phospholamban Inhibition of the Calcium
Pump in Sarcoplasmic Reticulum. J Biol Chem 2013;388:30181-30191.
25.
Paulsen ES, Villadsen J, Tenori E, Liu HZ, Bonde DF, Lie MA, Bublitz M, Olesen C, Autzen HE, Dach I,
Sehgal P, Nissen P, Moller JV, Schiott B, Christensen SB. Water-Mediated Interactions Influence the Binding
of Thapsigargin to Sarco/Endoplasmic Reticulum Calcium Adenosinetriphosphatase. J Med Chem
2013;56:3609-3619.
26.
Sohoel H, Jensen AML, Moller JV, Nissen P, Denmeade SR, Isaacs JT, Olsen CE, Christensen SB. Natural
products as starting materials for development of second-generation SERCA inhibitors targeted towards
prostate cancer cells. Biorg Med Chem 2006;14:2810-2815.
27.
Toyoshima C, Norimatsu Y, Tsueda J. To be published.