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Reaction cycle of Ca2{­ATPase of sarcoplasmic reticulum. A
simplied version. Boxed states show that the corresponding
crystal structures have been published. [Ca2{] indicates oc­
clusion of Ca2{.

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Á½B]ÁÄ 1 t[Ìf[^û2  úËõ Jan. 2011 Vol.24 No.1
(C) 2011 The Japanese Society for Synchrotron Radiation Researchðà ¡ CI|viP ^ ATPasejÌ\¢¶¨w»f·éÀèCÆË¹ÍâèÉÈçÈ¢CÆ¢¤_ÉÈÁ½B
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é½ßCÂÉ½sÉ X üªüËµ½ê

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Ca2{ ñÌ Ca2{­ATPase Ì»Ìæ¤ÉCê²ic²jªÉ[É·¢i`600 ÅjêCâèÍ[ÅÁ½B±Ìæ¤ÈêCSjI[^[wbhÉ90
A[ðµCc ²ªñ]²ÆÙÚêv·éæ¤ÉµÄf[^ûWðs¤ªC¿âpÌ½ßÌfKXªA[É½é±Fig. 2

Data collection in the gradual mode and variation of statis­
tics provided by Scalepack of a plate­like crystal of Na{, K{­ATPase. Inset shows the mounted crystal. The red squareindicates the start position of data collection and the beam
size (50 mm square). The arrow shows the direction of trans­
lation of the specimen. The spindle axis is parallel to thehorizontal axis of the gure. The angle of 90means that theincident X­ray beam is perpendicular to the plate­like crystal.
The wave length used was 0.9 Å, camera distance300 mm,
oscillation angle0.5
, exposure time0.7 sec (no attenua­
tor), total number of frames440. The crystal belongs to
the space group C2, and has unit cell dimensions of a
225.8, b51.0, c164.3 Å and b105.4
. The resolution
was 2.6 Å, and the mosaic spread 0.9_1.3
. The specimen
was cooled to 100 K by cold nitrogen gas. The detector used
was Rayonix MX225HE. The data were collected on June25, 2008.

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qÅâ³·éàÌÆl¦çêéBj·Èí¿C·xöqÅ@\ª¶Ý·éB»êÅÍ gating @\Í×E¿¤C×EúËõ Jan. 2011 Vol.24 No.1  3

Fig. 4

Fig. 3

Ribbon representation of the crystal structure of Ca2{­AT­
Pase in a Ca2{ bound form (E1¥2Ca2{ state). Broken ar­rows indicate movements that occur in transition into E1P.

Details of the transmembrane Ca2{ binding sites. Viewed
from the cytoplasmic side approximately perpendicular to
the membrane. Small spheres represent bound Ca2{ (cyan),and crystallographic water molecules (red). Cylinders showa­helices.

ATPase ÌÑÊwbNXÉÍWI a wbNXÍ­È­CM4, M6, M10ÍrÅÙÇ¯Ä¢éBM1 ÍóÔÉæÁÄ N ¤i×E¿¤j¼e}«wbNXiM1jO¤i¬EÌào¤jÇ¿çÅà¯¶©Æ¢¤Æ»¤ÅÍÆ C ¤iào¤jÉª©êÄÜêÈªèCM2 ÍóµÉ

È ¢ B Ca2{ |  v Í ³ m É Í Ca2{, H{ |  v ÅèæÁÄª«ûªÏ»·éiãqÌ M2 XCb`jBM4C

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µ½ H{ Í¨»ç­Í H{ `lðÊÁÄC³ÉßÁÄbNXÅµÜ¤B]ÁÄ H{ ZxùzÍ¶¶¸C{¿IÉÍ Ca2{

C g Asp351Ì ß T É Ê u · é B Na{, K{­ATPase Å ÍÌ|vÅéBÜ½CNa{, K{ |vÉÌãíèÉ Na{ ¾¯ªéBP hCÌêÆÈÁÄ¨èCÓ_»T

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Na{ Æ K{ ÌÉË¶µÄå«­zuðÏ¦éÂ\«èCéBào¤[vÍ M7_M8 ÔÈOÍ²­Z¢BM6©çC»ÌJjYÍ{¿IÉá¤±Æªí©éB»êªððµÄá¤½ßÉCÜ¸ÍCSÌ\¢©çà¾µæ¤B_M7 Ô[viL6/7jÍ P hCÌºðÁÄ¨èCØ¬EÌ Ca2{­ATPaseiSERCA1ajÍªqÊ11C

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4. Ca2{ ÌÆ×E¿¤Q[gÌì®@\èCÛ¶«ÌñíÉ¢ÌæÅéBNinucleotidej³ÄCE1¥2Ca2{ óÔÉ¨¢ÄC2 ÂÌ Ca2{ ÍàÌ×E¿¤\ÊÉärIß¢Æ±ëÉi©ç 5 Å ÙhCÍ P hCÆ b XghlÌqWÅÎÇ¸ê½ÊuÉj¡ÉÀñÅµÄ¢éiFig. 3, 4jBÆêCATP ÌAfjÂªÌÊðL·éBP ^àÉ 7 ÂÌ_f´qªzÊµÄ¢éªC»Ì«¿Íå«

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éB2 ÂÌ Ca2{ ÌéBÉÍÔªèCTCgÛÉÅÉ·éBÀÛCÊÁÙIÏÙÉæÁ
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Fig. 5

A cartoon illustrating the mechanism for closing thecytoplasmic gate by phosphorylation. The P­domain consistsof 2 parts and is bent by binding of Mg2{ and phosphate.
The A­domain sits on one end of the P­domain. The V­
shaped structure consisting of the M1 and M2 transmem­
brane helices is attached to the other end of the A­domain.
The change in inclination of the P­domain is amplied by theA­domain and pulls the V­shaped structure towards thecytoplasm to close the ion pathway.

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Fig. 6

A cartoon illustrating two_step rotation in gating of the ion
pathway during hydrolysis of aspartylphosphate. Small ar­rows indicate the movements of the transmembrane helices.

­ÍÉßÄÍ©Èá¢ÉæÁÄQ[gÌJÂª§ä³ê¾ªð½ª as­éÌ©BÀÛCf}[NO[vÍÁsociative Åé±Æª{¿Å

Mechanism for converting A­domain rotation into P­
domain inclination. The A­domain sits on the P­domain(wedge shaped) at one end and climbs up the P­domain
when it rotates by `90
. If the P­domain were spatially x­
ed, the M1/M2 helices would come out from the membrane(a). This is energetically very unfavourable as the transmem­
brane parts of the M1/M2 helices are hydrophobic. In reali­ty, the P­domain comes closer (actually inclines) to themembrane to keep the M1/M2 helices in the membrane (b).

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Details in the phosphorylation site in the E2P ground and E2`P transition states. The lumenal gates is opened in the sta­
ble analogue of the ground state (E2ÉBeF|
3 , a) and closed inthat of the transition state (E2ÉAlF|
4 , b). Note that watermolecules (small red spheres) are totally removed aroundBeF|
3 because Gly182 in the TGES loop (green) blocks ac­
cess of water (a). In the transition state (b), a further rota­tion of the A­domain moves the TGES loop to accommodate
only one water molecule (red circle), which is activated by
Glu183 and attacks (white arrow) the aspartylphosphate.

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10)

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8  úËõ Jan. 2011 Vol.24 No.1

 ÒÐî Lßåwªq×E¶¨w¤ ³ö
E­mail: ctiam.u­tokyo.ac.jpêåF\¢¶¨wmªðn
1983Nåwåw@wn¤È¨wêUmÛöC¹CwmBåwwèCÄX^tH[håw×E¶¨w³ºm¤õCp
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7 æèïx`¿§Ì\¢ðÍZ
^[EZ^[·Bðà ¡ CI|viP ^ ATPasejÌ\¢¶¨w

Structural Biology of Ion Pumps (P­type ATPases)
Chikashi TOYOSHIMA

Institute of Molecular and Cellular Biosciences The University of Tokyo
1_1_1, Yayoi, Bunkyo­ku, Tokyo, 113_0032, Japan

Abstract Ion pump proteins are membrane proteins that are integrated into biological membranes (lipid
bilayers). They are powered by ATP and are enzymes that hydrolyse ATP (ATPase). We have been working
on crystal structure determination and molecular dynamics simulations of Ca2{­ATPase from skeletal muscle
sarcoplasmic reticulum (SERCA1a). At present crystal structures are determined for 9 intermediates that ap­
proximately cover the entire reaction cycle. Thus, we can say that the mechanism of ion pumping is roughly
understood and answer fundamental questions, for instance, what ATP and phosphorylation do, why nearly100÷ eciency of energy conversion is possible, why the structure of ion pump has to be so. The focus of
this article is on the gating mechanism of ion pathway.

úËõ Jan. 2011 Vol.24 No.1  9