Structural role of countertransport revealed in Ca2�

pump crystal structure in the absence of Ca2�

Koji Obara*†, Naoyuki Miyashita*‡, Cheng Xu§, Itaru Toyoshima†, Yuji Sugita*, Giuseppe Inesi§,and Chikashi Toyoshima*¶

*Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; †Department of InternalMedicine, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan; and §Department of Biochemistry, University of MarylandSchool of Medicine, Baltimore, MD 21201

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on May 3, 2005.

Contributed by Chikashi Toyoshima, July 21, 2005

Ca2�-ATPase of sarcoplasmic reticulum is an ATP-powered Ca2�

pump but also a H� pump in the opposite direction with nodemonstrated functional role. Here, we report a 2.4-Å-resolutioncrystal structure of the Ca2�-ATPase in the absence of Ca2� stabi-lized by two inhibitors, dibutyldihydroxybenzene, which bridgestwo transmembrane helices, and thapsigargin, also bound in themembrane region. Now visualized are water and several phospho-lipid molecules, one of which occupies a cleft between two trans-membrane helices. Atomic models of the Ca2� binding sites withexplicit hydrogens derived by continuum electrostatic calcula-tions show how water and protons fill the space and compensatecharge imbalance created by Ca2�-release. They suggest that H�

countertransport is a consequence of a requirement for maintain-ing structural integrity of the empty Ca2�-binding sites. For thisreason, cation countertransport is probably mandatory for allP-type ATPases and possibly accompanies transport of water aswell.

ion pump � SERCA � protonation � Ca2�-ATPase � continuum electrostaticcalculation

Ca2�-ATPase of skeletal muscle sarcoplasmic reticulum(SERCA1a), an integral membrane protein consisting of 994

aa (1), transfers two Ca2� from the cytoplasm into the lumen ofsarcoplasmic reticulum per ATP hydrolyzed and thereby estab-lishes a �104 concentration gradient across the membrane (2).At the same time, Ca2�-ATPase pumps two or three H� in theopposite direction (3–6) during the reaction cycle. According tothe classical E1�E2 theory (7–9), transmembrane ion-bindingsites have high affinity for Ca2� and face the cytoplasm in E1,whereas they have low affinity and face the lumen of sarcoplas-mic reticulum in E2. The opposite applies to H�, which binds tothe ATPase in E2 and dissociates in E1, presumably in exchangewith Ca2�. As Na�K�-ATPase and gastric H�K�-ATPase coun-tertransport K�, instead of H�, countertransport of monovalentcations may be a common feature of the P-type ATPase super-family (2, 10), of which SERCA1a is the best-studied member(11, 12). However, the sarcoplasmic reticulum membrane isleaky to monovalent cations including H� (13). As has beenpointed out before (14), the membrane potential and pH gra-dient must be minimized to achieve such a large concentrationgradient of Ca2�. Therefore, the physiological role of H�

countertransport has been a puzzle. Although protonation ofcarboxyls in the Ca2�-binding site has been suggested frombiochemical (15, 16) and mutagenesis studies (17, 18), crystalstructures of SERCA1a in various states (19–24) did not clarifythe role of countertransport or identify protonation sites.

These questions, however, can be addressed computationallyby calculating stabilization energy provided by H� binding(25–27). Protonation probability of a particular residue is relateddirectly to the free energy difference between protonated andunprotonated forms. Such calculations, therefore, should be ableto identify residues likely to be protonated in crystal structures

and were indeed successful (28) for a Ca2�-bound form[E1�2Ca2� (19); PDB ID code 1SU4]. As confirmed by all-atommolecular dynamics simulation (28), protonation of Ca2�-coordinating residues has an important structural role even inE1�2Ca2�. Such a computational approach requires accurateatomic models that include crystallographic water at least. Theresolution (3.1 Å) of the published model for a Ca2�-unboundform [E2(TG); Protein Data Bank (PDB) ID code 1IWO (20)]was not good enough for this purpose, although the structure wasstabilized by thapsigargin (TG), a very potent inhibitor (29). Wenow overcome this problem by stabilizing the transmembraneregion with 2,5-di-tert-butyl-1,4-dihydroxybenzene (BHQ), inaddition to TG. BHQ, commonly used as an antioxidant, is ahigh-affinity inhibitor (Kd � 20 nM) of Ca2�-ATPase and has abinding site distinct from TG (30). We show that BHQ bridgestwo transmembrane helices and sterically blocks the conforma-tion changes of Glu-309 that works as the cytoplasmic gate of theion pathway (18, 21, 24, 31), thereby fixing the pump in aH�-occluded state.

In the new 2.4-Å resolution structure, water and portions ofseveral phospholipid molecules are resolved. Hence, electro-static calculations could be applied reliably to yield atomicmodels with explicit hydrogens. These models show that phos-pholipids, water, and protons fill in space and compensate for thecharge imbalance created by Ca2� release. The models thusexplain why H� countertransport is required, although H�-gradient is dissipated by other means, and further suggest thatwater molecules might be transported together with H�.

Materials and MethodsCrystallization. Affinity-purified enzyme (32, 33) (20 �M) in octa-ethyleneglycol mono-n-dodecylether (C12E8) and 1 mM Ca2� wasmixed with 30 �M TG, 0.12 mM BHQ, and 3 mM EGTA, thendialyzed against a buffer consisting of 2.75 M glycerol, 4% poly-ethylene glycol 400, 3 mM MgCl2, 0.04 mM BHQ, 2.5 mM NaN3,2 �g�ml butylhydroxytoluene, 0.2 mM DTT, 1 mM EGTA, and 20mM Mes (pH 6.1) for �1 month. Crystals were grown to 300 �300 � 50 �m and were flash-frozen in cold nitrogen gas.

Data Collection. Diffraction data were collected at BL41XU ofSPring-8 (Hyogo, Japan) by using an R-Axis V imaging platedetector (Rigaku, Tokyo) and a Q315 charge-coupled devicedetector (Area Detector Systems Corporation, Poway, CA).Diffraction intensities from two best crystals were merged

Abbreviations: SERCA, sarco(endo)plasmic reticulum Ca2�-ATPase; TG, thapsigargin; BHQ,2,5-di-tert-butyl-1,4-dihydroxybenzene; PDB, Protein Data Bank.

Data deposition: The atomic coordinates for the E2(TG�BHQ) form of Ca2�-ATPase havebeen deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2AGV).

‡Present address: Department of Chemistry, Boston University, Boston, MA 02215.

¶To whom correspondence should be addressed. E-mail: ct@iam.u-tokyo.ac.jp.

© 2005 by The National Academy of Sciences of the USA

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(Rmerge � 7.5%; I�� � 29.3; redundancy � 10.6; 27.1%, 4.5, 5.7,respectively, for the highest-resolution bin, 2.5–2.4 Å) by usingDENZO and SCALEPACK (34). The crystals had a P41 symmetrywith the unit cell parameters of a � b � 71.39 Å, and c � 591.02Å. One asymmetric unit contained two protein molecules.

Modeling. Because the unit cell parameters were identical tothose of E2(TG) crystals (20), molecular replacement (35) wasperformed with the atomic model for the E2(TG) crystals (PDBID code 1IWO) (20). Then, TG, BHQ, phospholipids modeledas PE, and water molecules were added. Many omit maps werecalculated to examine the presence of water and phospholipidmolecules. Several errors in the original model (1IWO), inparticular the orientations of side chains, were corrected.

The final model included two protomers, virtually identical,each of which contains Ca2�-ATPase, TG, BHQ, Na�, threephospholipids, and 124 water molecules. It was refined againstthe diffraction data consisting of 113,814 reflections (99.9%completeness) to Rfree of 0.263 and Rcryst of 0.239 at 2.4-Åresolution; rms deviation of the bond length and angle were0.008 Å and 1.3°, respectively.

Recombinant ATPase Expression and Mutagenesis. The chicken fastmuscle SERCA-1 cDNA (36) was subcloned into the SV40-pAdlox vector and transfected into COS-1 cells for overexpres-sion of protein under control of the SV40 promoter. COS-1 cellcultures and transfection methods were described by Sumbillaet al. (37). For site-directed mutagenesis, primers of 20–30 bp inlength were synthesized for each individual mutation. Theseprimers were used to hybridize DNA sequences internal to theflanking primers and were used for PCR mutagenesis by theoverlap extension method (38). Briefly, two overlapping frag-ments containing the mismatched bases of the targeted sequencewere amplified in separate PCRs. The reaction products werethen mixed and amplified by PCR using both flanking primers.The mutant cassette then was exchanged with the correspondingcassette of wild-type cDNA in SV40-pAdlox, which was used fortransfection into COS-1 cells and expression of the mutantprotein.

Functional Studies. The microsomal fraction of transfected COS-1cells was obtained by differential centrifugation of homogenizedcells (37). Immunodetection of expressed ATPase in the micro-somal fraction was obtained by Western blotting. SERCA AT-

Pase activity was assayed in a reaction mixture containing 20 mMMops (pH 7.0), 80 mM KCl, 3 mM MgCl2, 10 �M free Ca2�, 5mM sodium azide, 50 nM Ca2�-ATPase, 3 �M ionophoreA23187, 3 mM ATP, and 0–10 �M TG. Ca2�-independentATPase activity was assayed in the presence of 2 mM EGTAwithout added Ca2�. The reaction was started at 37°C by addingATP, and samples were taken at serial times for determinationof Pi (39). The Ca2�-dependent activity was calculated bysubtracting the Ca2�-independent ATPase activity from thetotal ATPase activity and was corrected to account for the levelof expressed protein in each microsomal preparation as revealedby immunoactivity and with reference to microsomes obtainedfrom COS-1 cells transfected with wild-type SERCA1 cDNA.

Continuum Electrostatic Calculation. In continuum electrostaticcalculations, protonation probability of a residue is obtainedfrom the difference between �Gpro and �Gsol, which are freeenergy differences (�G) between protonated and unprotonatedstates of the residue in protein and in solution, respectively. �Gis related to the equilibrium between protonated (AH) andunprotonated (A�) forms of the residue (A) as

�G � �RT lnKa � �RT ln�AH�A�H�� .

The MEAD program suite (40) used here solves a finite-difference Poisson equation, assuming continuum dielectric forthe interior and exterior of a protein, to evaluate the protonationprobability. The program allows, however, only two regions ofdifferent dielectric constants (�) for a whole system. For the bulksolvent, � of 80 was used. This assignment leaves only onedielectric constant to be assigned to the rest, including lipidbilayer modeled as a slab of 30-Å thickness. Because the residuesof concern are all within the bilayer, � of 4 appeared mostappropriate. However, to examine the robustness, all of thecalculations were done also with � � 20.

At first, the probabilities for all 30 titratable residues aroundthe transmembrane Ca2�-binding sites (Fig. 1) were calculatedto find the residues that exhibited large differences between theE1�2Ca2� and the E2 states. At this stage, atomic charges of thetitratable residues were adjusted rather than explicitly addingprotons, because positions of protons strongly affected proto-nation probabilities, and the positions of protons refined byenergy minimization were highly dependent on the initial (in-evitably incorrect) positions. These calculations identified four

Fig. 1. Structural model and the locations of 30 titratable residues around the Ca2�-binding sites in E1�2Ca2� (Left) and E2 (Right), as used in continuumelectrostatic calculations. Ca2�-ATPase and the lipid bilayer (modeled as a slab of 30-Å thickness) were treated as a single low dielectric object (� � 4 or 20; yellow),and the solvent was treated as a high dielectric object (� � 80; light blue). Two purple spheres show bound Ca2�, and cyan and red dots represent ionizable andprotonatable residues, respectively. Nine residues (E55, E58, D59, D254, E258, E309, E771, D800, and E908) examined with explicit protons are circled. Each imageis an enlarged view of the boxed area in the Inset, in which the entire model is shown.

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such residues (Glu-58, Glu-309, Glu-771, and Asp-800) andGlu-908, protonated in both states, as target residues for moreaccurate calculations with explicit protons. For this purpose, theresidues that could affect the protonation states of the targetresidues were identified by calculating protonation probabilitiesby using only the target residues. A significant difference fromthat obtained with all 30 residues was found only with Glu-58 inE2. Conversely, calculations with nine residues that includedfour acidic residues surrounding Glu-58 (Glu-55, Asp-59, Asp-254, and Glu-258; see Fig. 1) yielded identical results with thoseincluding all 30. Hence, the final calculations were done by usingthese nine residues with explicit protons added.

In all of the calculations, a two-step focusing procedureimplemented in MEAD was used for minimizing calculation costs.First calculations were done on a grid of 341 � 341 � 341 pointsfor evaluating �Gpro and on a grid of 121 � 121 � 121 points for�Gsol. In either case, a grid size of 1.0 Å was used. Secondcalculations for �Gpro and �Gsol were done both on a grid of 71 �71 � 71 points at 0.25-Å interval, which was centered at thetarget residue. All hydrogen atoms were added by using MOLX,a utility program in the MARBLE software package (41). Theirpositions were refined by energy minimization and moleculardynamics simulations with MARBLE (41), keeping protein heavyatoms fixed.

Continuum electrostatic calculations were tried also withMCCE (42), which allows multiconformers for titratable residuesbut treats lipid bilayer as a part of bulk solvent (i.e., � � 80).Because multiconformer calculations were not essential as dem-onstrated by molecular dynamics simulations (see below) andbecause several critical residues are located near the protein–lipid interface, we did not use this program primarily. The resultswere, however, virtually the same as those obtained with MEADand are not described here.

ResultsStructure Determination and Phospholipids. In this study, we gen-erated crystals of Ca2�-ATPase in the absence of Ca2� stabilizedwith BHQ alone [E2(BHQ)], TG alone [E2(TG)], and BHQ plusTG [E2(TG�BHQ)]. To generate the crystals, affinity-purifiedCa2�-ATPase in the presence of Ca2� was supplemented withexogenous phosphatidylcholine, mixed with EGTA and theinhibitors, and then dialyzed against a crystallization buffer (20).Inclusion of two inhibitors in crystallization improved the res-

olution to 2.4 Å, with no changes in unit cell parameters fromE2(TG). Therefore, the atomic model was built by molecularreplacement starting from the model for E2(TG) [PDB ID code1IWO (20)] and refined to Rfree of 26.3% at 2.4-Å resolution.BHQ alone yielded crystals of the same symmetry but withslightly different unit cell parameters. The resolution of theE2(BHQ) crystals was limited to 3.0 Å, and the structure ofthe ATPase was virtually the same as those of E2(TG�BHQ)and E2(TG), including the paths of the M3 and M7 helicesthat constitute the main part of the TG-binding pocket (Fig.2). Therefore, we describe here the crystal structure ofE2(TG�BHQ) only. Although side-chain conformations ofsome residues were corrected for, paths of the main chain were

Fig. 2. Superimposition of two E2 structures viewed in stereo. Yellow,E2(BHQ); light green, E2(TG) (PDB ID code 1IWO). Atomic models of TG andBHQ are shown in ball-and-stick. Transmembrane helices and three cytoplas-mic domains are labeled.

Fig. 3. Locations of two transmembrane inhibitors and a phospholipid in thecrystal structure of Ca2�-ATPase in E2(TG�BHQ). (a) Side view. (b) Top view. (c)Details of BHQ interaction. Violet nets represent an Fo � Fc map, showing extraelectron densities that cannot be explained by Ca2�-ATPase. The map wascalculated at 2.4-Å resolution and contoured at 4� in a or 3.5 � in b and c,before introducing BHQ and lipid into the model. The map of TG is shown forindicating the expected level of electron density. Atomic models of a BHQ anda phospholipid [modeled as phosphatidylethanolamine (PE)] are shown inball-and-stick; C� traces of Ca2�-ATPase, together with a few side chains,appear in b as yellow [E2(TG�BHQ)] and violet (E1�2Ca2�) sticks. In c, atomicmodel of E2(TG�BHQ) (atom color) is shown. Cylinders represent transmem-brane helices (numbered). Dotted line in a indicates approximate position ofthe cytoplasmic boundary of the hydrophobic core of the lipid bilayer. Arrowsin b indicate the movements of transmembrane helices in the transitionE2(TG�BHQ)3 E1�2Ca2�.

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virtually identical to the published model of E2(TG) (rmsdeviation � 0.57 Å for C� atoms). With the E2(TG�BHQ)crystals, temperature factors of the residues in the transmem-brane region (average � 55) were even smaller than those(average � 80) in the E2�MgF4

2� crystals (22) that diffracted to2.3-Å resolution. The ATPase in the E2�MgF4

2� crystals isthought to be in a state analogous to E2�Pi, stabilized withMgF4

2�, a stable phosphate analog, as well as TG. Positions ofwater molecules observed in the Ca2�-binding sites were verysimilar to those in E2�MgF4

2� (PDB ID code 1WPG), but onemore water molecule with a large temperature factor wasidentified around Glu-908.

A difference Fourier map (Fig. 3) calculated after refining theatomic model without BHQ and phospholipids showed two clearextra densities present near Glu-309. The one closer to themiddle of the membrane had a tri-partite structure, exactlymatching BHQ (Fig. 3). Slightly above this density, there wasanother peak with a shape characteristic of the head group of aphospholipid, which was tentatively modeled as phosphatidyleth-anolamine. Electron density was clear to the carbonyl groups(Fig. 3). Because only phosphatidylcholine was added exog-enously, phosphatidylethanolamine probably remained from thenative membrane (43). Its location in the V-shaped cavitybetween the M2 and M4 helices (Figs. 3b and 4d) suggests thatit acts as a wedge to keep those helices apart in E2. In fact,although somewhat weaker, clear electron densities were presentat the same position in the maps of E2(TG) (Fig. 4b) andE2(BHQ). However, no corresponding electron density waspresent in the map of E2�MgF4

2� (Fig. 4c), apparently reflectinga narrower cleft (Fig. 4d). There is no such a cavity in E1�2Ca2�

(Fig. 3b) (19). Thus, this lipid molecule is likely to move into thisposition during the E2P3 E2 transition to stabilize the groundE2 state and move out in E23 E1�2Ca2� in the normal reactioncycle. As the acyl chains of this lipid molecule would collide withM2 in E1�2Ca2� (Fig. 3b), such movements are expected.

Presumably to facilitate such movements, the phosphate moi-ety of this lipid molecule is not stabilized by any hydrogen bonds;Gln-108 and Thr-316 are the closest polar residues but are �3.5Å away (Fig. 3b). There are no nearby Arg or Lys residues thatcould make salt bridges to stabilize the phosphate moiety, asobserved with many phospholipids found in crystals of othermembrane proteins [e.g., cytochrome bc1 complex (44)]. Theelectron density map showed hints of phospholipid molecules atother places, but only two more were identified with certainty.They were located between neighboring protein molecules in thecrystal lattice and were common to the maps of E2(TG),E2(BHQ), and E2(TG�BHQ).

BHQ Binding Site and Mode of Binding. BHQ binds to the ATPaseby bridging the M1 and M4 helices with hydrogen bondsinvolving the two hydroxyl groups of BHQ, one with the carboxylof Asp-59 on M1 and the other with the carbonyl of Pro-308 onM4 (Fig. 3c). The positions of M1 in E2(TG�BHQ), E2(BHQ),and E2(TG) are identical (Figs. 2 and 4), showing that BHQstabilizes an E2 conformation the same as E2(TG). A cleardifference from the published model of E2(TG), however, is thatthe Glu-309 side chain points toward the Ca2�-binding sites (seebelow). BHQ fixes this side-chain conformation of Glu-309 byoccupying the space that would be necessary for the side chainto point outwards (Fig. 3c). Because Glu-309 works as the gatingresidue for cytoplasmic Ca2� to reach the binding cavity (18, 21,24, 31), Ca2� cannot pass if BHQ is present. Thus, this situationis reminiscent of those in E1�AMPPCP and E1�AlFx�ADP (21,24), in which Leu-65 on M1 blocks the conformation changes ofthe Glu-309 side chain to occlude Ca2�. Here BHQ presumablyoccludes H� by the same mechanism.

Hydroxyl groups of BHQ thus are expected to have vital rolesin the compound’s inhibitory action, as demonstrated by aweaker inhibition with the Asp-593Ala mutant (Fig. 5; see alsoTable 1, which is published as supporting information on the

Fig. 4. Fo � Fc maps for three different E2 structures around the cleft between M4 and M1–M2 helices. The maps are calculated at 2.4-Å resolution and contouredat 3.5� [a and d, E2(TG�BHQ)], 3.1 Å and at 2.5� [b, E2(TG) (20)], and 2.3 Å and at 2.5� [c, E2�MgF4

2� (22)] before introducing phospholipids into the model.Structures are viewed along the membrane plane. Atomic models for BHQ and phosphatidylethanolamine (up to carbonyl groups) at the expected positions aresuperimposed. In d, models of E2(TG�BHQ) and E2�MgF4

2� are superimposed and viewed from the right in a; Asn-101 side chain in E2�MgF42� would collide with

the phospholipid located at the position found in E2(TG�BHQ) or E2(TG).

Fig. 5. Inhibition by BHQ of various mutants. Effects of various mutations onthe BHQ concentration dependence of ATPase inhibition are shown. Squares,wild type; diamonds, F256V; triangles, V62E; inverted triangles, D59A; circles,L311A. Mutations of Pro-308 and Pro-312 also were tried but interfered withprotein expression. See Table 1 for the numbers deduced from this figure.

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PNAS web site). However, its butyl groups appear more criticalbecause our mutation study shows a much stronger effect withthe Leu-311 3 Ala mutant (Fig. 5). Fig. 3c shows that BHQmakes extensive van der Waals contacts with M1 and M4 helices:the butyl group on the M4 side fits in the space between Pro-308and Pro-312, and contacts Leu-61 and Leu-311; the other butylgroup on the M1 side contacts Leu-61, Val-62, and Leu-65.These results are consistent with a previous study (30) thatexamined derivatives of BHQ, which showed that hydroxylgroups are important, but the affinity is changed nearly 10,000-fold by replacing the butyl groups with amyl or propyl groups.The binding site of BHQ is thus distinctly different from that ofTG, which binds to the groove surrounded by M3, M5, and M7(20). In fact, Phe-256 on M3 is critical for binding of TG (45, 46)but has only small effects for BHQ (Fig. 5 and Table 1).Nevertheless, the effects of BHQ, such as inhibition of E2Pformation by Pi, are very similar to those of TG (30, 47). Atpresent, we do not understand why these two inhibitors have suchsimilar effects.

Conformation of Glu-309. It is now well established that Glu-309,which caps site II Ca2� (19), works as a cytoplasmic gate bychanging the side-chain conformation. With BHQ present, the

Glu-309 side chain points inwards and forms hydrogen bondswith the Asn-796 side chain and the Val-304 carbonyl (Fig. 6b).The latter requires protonation of Glu-309 (see below). Asn-796provides the two protons of the side-chain amide to Glu-771 andGlu-309 (Fig. 6b). Therefore, this Asn is critical and is changedto Asp in Na�K�-ATPase and H�K�-ATPase that counter-transport (and coordinate) K� instead of H�.

At 2.4-Å resolution, the main-chain conformation of Glu-309 is unambiguous. Its peptide bond is f lipped (the differencein � angle of Glu-309 is 168°) from that in the E1�2Ca2� state.In E2(TG�BHQ), Glu-309 takes a helix conformation,whereas in E1�2Ca2� it is in a �-strand conformation. To allowthis conformation change, the carbonyl group of Glu-309 is notfixed by hydrogen bonds with protein atoms in either E1�2Ca2�

or E2(TG�BHQ); only the amide group makes a hydrogenbond with the Ile-307 carbonyl in E2(TG�BHQ) but withnonoptimal geometry (Fig. 6b). However, the Gly-310 amidemakes a hydrogen bond with the Asn-796 carbonyl (Fig. 6b),which, in E1�2Ca2�, makes a hydrogen bond with Asp-800 onthe same M6 helix (Fig. 6a). As a result, the distance betweenPro-308 and Pro-312 is 2.5 Å shorter in E2(TG�BHQ), andthe orientation of the luminal half of M4 is changed by �15°with respect to the cytoplasmic half. These changes are

Fig. 6. Atomic models of the Ca2�-binding sites with explicit hydrogens viewed approximately perpendicular to the membrane from the cytoplasmic side.Identical region is shown in stereo. (a) E1�2Ca2� (PDB ID code 1SU4). (b) E2(TG�BHQ). Side chains are shown only for residues that involved in hydrogen bondsor Ca2�-coordination. Hydrogen atoms are shown only for water, Asn, Thr, and protonated carboxyls. Small white spheres represent bound protons, and redones represent water. Cyan spheres (marked I and II) in a represent bound Ca2�. Broken lines show hydrogen bonds and Ca2� coordination. Hydrogen bondsbetween main-chain atoms are in green. Interhelix hydrogen bonds are marked with arrows. The figure was prepared with MOLSCRIPT (50).

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probably needed to allow the movements of M4 perpendicularto the membrane plane (20) (Fig. 7). Similar changes takeplace at Asp-703 (G703DG), which binds Mg2� and undergoeslarge conformation changes between phosphorylated (or�-phosphate bound) and unphosphorylated forms (21, 24).This kind of peptide bond f lipping is not uncommon (48) andis observed frequently in molecular dynamics simulations [e.g.,for Val-76 preceding Gly-77 in a K�-channel (49)].

Water Molecules in the Ca2�-Binding Sites. This higher-resolutionstructure allows deeper understanding of the E2 structure andE1–E2 transition: movements of water molecules and changesin protonation of carboxyl groups play important roles here.For example, compared with E1�2Ca2�, the part of M6 be-tween Asn-796 and Asp-800 is rotated nearly 90°, so thatAsn-796 approaches Glu-771 on M5 (Figs. 7 and 8) (20). At thesame time, the Asn-796 carbonyl changes its hydrogen-bondingpartner from the Asp-800 amide to that of Gly-310 (on M4)and a water molecule (Fig. 6). The short distance (2.7 Å)between the two juxtaposed oxygen atoms of Asn-796 andGlu-771 side chains clearly requires protonation of the Glu-771carboxyl, and the formation of a hydrogen bond (Fig. 6b). Therotation of M6, stabilized by the interhelix hydrogen bondswith residues in M4, M5, and M9 (Fig. 6b, arrows), creates alarger space around the Trp-794 side chain near the luminalsurface (Fig. 7). Now a water molecule (6 in Figs. 6–8)occupies that space, forming hydrogen bonds with Trp-794,Thr-799, and His-944 side chains (Figs. 7 and 8). Thus, thiswater molecule may work as a lock of the rotated configurationof the M6 helix.

In the E1�2Ca2� crystal structure (PDB ID code 1SU4), onlytwo water molecules (a–b, in Figs. 6–8) are present in theCa2�-binding cavity and coordinate site I Ca2�. InE2(TG�BHQ), five water molecules (1–5 in Figs. 6–8) areidentified, filling the space created by Ca2� release. Threewater molecules (1–3) are located near the positions taken bytwo Ca2� (cyan spheres in Figs. 6 and 7) in E1�2Ca2�. They arelikely to be hydrogen bonded to one another and to linktransmembrane helices. A water molecule (1 in Figs. 6–8) nearGly-310 amide (M4) is connected with Asn-796 carbonyl (M6)

and another water molecule (2) hydrogen bonded to Asn-768(M5); this latter water, in turn, is linked to another (3) thatforms hydrogen bonds with Asn-796, Asp-800 (M6), andSer-767 (M5) (Fig. 6b). Water molecules 4 and 5 (Figs. 6–8)are detached from others and located near Glu-908 carboxyl,filling the space between M5 and M8 created by the bendingof M5 above Gly-770 during Ca2� release (20) (Fig. 7). Thesetwo are likely to be hydrogen bonded to Glu-908 and Ser-766(Fig. 6b).

Protonation in the Empty Ca2�-Binding Sites. Ca2� release createsnot only empty space, it also causes severe charge imbalance.The four carboxyl groups clustered in the Ca2�-binding sitesare likely to be deleterious to structural integrity if left ionized.If no metal binding is allowed, such imbalance must becompensated for by the formation of hydrogen bonds withother residues following conformation changes, by the intro-duction of water molecules, or, more directly, by protonation.To understand how Ca2�-ATPase handles this problem, wegenerated atomic models with explicit hydrogens by applyingcontinuum electrostatic calculations (25). These calculationsestimate protonation probability by evaluating free energydifference between protonated and unprotonated forms ofselected residues for all possible combinations of protonationstates. Of the 30 protonatable�ionizable residues examined(Fig. 1), the calculations show that only 4 transmembraneresidues, Glu-771, Asp-800, Glu-309, and Glu-908 will beprotonated (in descending order of probability; see Table 2,which is published as supporting information on the PNAS website) in E2(TG�BHQ). If we assume � � 4, for all four of them,protonation probabilities are �99% (gain in free energy byprotonation is �11 kcal�mol; see Table 2) for pH 6–8. If weassume � � 20, the protonation probabilities for three of themdo not change much, but that of Glu-908 decreases and ispH-dependent; at pH 8, it is reduced to �20% (see Table 3,which is published as supporting information on the PNAS website).

In most cases, the requirement for the protonation of theseresidues can be inferred from the atomic model, because oxygenatoms are juxtaposed within a hydrogen-bond distance (e.g., 2.6

Fig. 7. Superimposition of the C�-traces of E2(TG�BHQ) (green) and E1�2Ca2� (violet) around the Ca2�-binding sites, viewed in stereo. Water molecules arerepresented by small spheres in red (E2(TG�BHQ); 1–6) and violet (E1�2Ca2�; a–e). Ca2� in E1�2Ca2� appear as cyan spheres (marked I and II). Side chains [atomcolor for E2(TG�BHQ) and violet for E1�2Ca2�] are shown for some of the residues that may be involved in moving water or binding Ca2�. Arrows shows themovements of the transmembrane helices in the transition E1�2Ca2� 3 E2(TG�BHQ). Orange broken lines represent hydrogen bonds, and cyan lines showCa2�-coordination.

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Å between Glu-309O�2 and Val-305 carbonyl). Such a closedistance will be allowed only when a hydrogen bond is formedbetween them. In fact, the locations of hydrogens and protonsshown in Fig. 6 are uniquely determined by this requirement,making multiconformer calculations for titratable residues (e.g.,ref. 42) unnecessary. To further examine the stability of theproposed atomic model, molecular dynamics simulations wereperformed for E2, as was already done for E1�2Ca2� (28). Thesystem was stable at least for 2.6 nsec with the arrangements ofwater and hydrogens shown in Fig. 6b.

As listed in Table 4, which is published as supportinginformation on the PNAS web site, the 12 protein oxygenatoms used for Ca2� coordination in E1�2Ca2� are nowinvolved in 15 hydrogen bonds, 5 with protein amide, 5 withwater, and 5 with bound protons (Fig. 6b). As a result, thereare more interhelix hydrogen bonds in E2(TG�BHQ) than inE1�2Ca2� (arrows in Fig. 6). In fact, almost all carboxyl andcarbonyl oxygen atoms within the transmembrane region ofE2(TG�BHQ) are involved in hydrogen bonds or are proton-ated. One of the exceptions is the carbonyl of Leu-797, locatedin the part of M6 that undergoes a large rotation (20) (Fig. 7).The unprotonated carboxyl oxygen of Asp-800 appears to forma weak hydrogen bond with the hydrogen of Val-795 C�.Protonation could occur on this oxygen, if water 3 (Fig. 6b)were H3O�, but there is no oxygen atom that could form ahydrogen bond with that proton.

Proton Countertransport. From this explicit model of the hydro-gen-bonding network for E2(TG�BHQ), we can now addressquestions on proton countertransport by Ca2�-ATPase. InE1�2Ca2�, as described in ref. 28, only Glu-58 and Glu-908 areexpected to be protonated in the transmembrane region (Table2). Thus, four residues are likely to be protonated in E2, andtwo are likely to be protonated in E1�2Ca2�. Nevertheless, wecannot derive the number of H� countertransported during thereaction cycle because it depends on the side (i.e., cytoplasmicor luminal) from which protons are introduced into the bindingsites.

Because all of the residues protonated in E2(TG�BHQ)coordinate Ca2� in E1�2Ca2�, it is most likely that all of themare introduced from the luminal side during Ca2�-release.However, ambiguity remains with Glu-58, which is hydrogenbonded to Glu-309 in E1�2Ca2� but is located in the cytoplasmin E2. If the proton that appears on Glu-58 in E1�2Ca2� bindsfrom the cytoplasmic side (model 1), it will not affect thenumber of protons countertransported, which is three in thiscase. However, if that proton comes from the luminal side, forexample, via Glu-309 by an exchange reaction for H� on Ca2�

binding (model 2), the protonation state of Glu-58 negativelyaffects the number of protons countertransported, which willbe 2.6 at pH 6.0 and 2.2 at pH 8.0 (Table 2).

As mentioned earlier, we also carried out all of the calcu-lations for � � 20. In this case, the situation is more complexbecause the protonation probability of Glu-908 is decreasedand pH dependent (Table 3). The expected number of protonscountertransported is �2.5 for model 1 and 1.7 for model 2.We further examined the case in which the Glu-309 side chainorients to the outside as in the published model of E2(TG)(20). Its protonation probability remains high because manyacidic residues surround this residue on the cytoplasmic side(Fig. 1).

Are Water Molecules Countertransported? Because the number ofwater molecules in the Ca2�-binding sites is two in E1�2Ca2�

and five in E2(TG�BHQ) (Figs. 6–8), these atomic modelswill mean that three water molecules are countertransportedwith protons, if they come from the luminal side and arereleased into the cytoplasm. However, it is also conceivablethat they are introduced from cavities within the protein onCa2� release and returned to the same places in Ca2� binding.In this case, no net f low of water across the membrane results.For the candidates, we can propose two crystallographic watermolecules (d and e in Fig. 7) located below the Arg-762 sidechain and those just beyond Glu-309 on the opposite side of theCa2�-binding cavity. Water d and e may be introduced byrearrangements of the long side chains of Glu-759, Arg-762,and Glu-918 (Fig. 7). Those on the Glu-309 side may enter thebinding cavity possibly past Gly-310 or, for molecule c (Figs.6 and 7), by the rotation of Leu-797 in M6. In any event, threewater molecules must be excluded from the Ca2�-binding siteswhen two Ca2� bind to the ATPase. Whether this exclusionresults in the transport of water across the membrane remainsto be seen.

DiscussionAs described, inclusion of BHQ, in addition to TG, stabilizedthe transmembrane region of Ca2�-ATPase in the E2 state toa level similar to that in E1�2Ca2� and resolved severalphospholipid and water molecules. This result has allowed usto propose atomic models of the Ca2�-binding sites withexplicit hydrogens, and to show how empty spaces and chargeimbalance created by Ca2�-release are compensated for byevents that could result in countertransport, as summarized inFig. 8. A phospholipid moves to fill a large lipid-facing space,and water molecules do the same for the protein interior.

Fig. 8. Schematic representation of the Ca2�-binding sites in E1�2Ca2� andE2(TG�BHQ). Cyan spheres represent Ca2�; red ones represent water. Boundprotons appear as red circles. Arrows indicate the movements of transmem-brane helices in the transition E1�2Ca2�3 E2(TG�BHQ). Dotted arrows indi-cate potential movements of water molecules. Dotted pink lines indicatehydrogen bonds, and those in light green indicate Ca2� coordination. Label-ing of water molecules follows Figs. 6 and 7.

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Charge imbalance is solved predominantly by protonation,which is certainly the most direct means. However, protona-tion alone is not enough, particularly when both oxygen atomsof a carboxyl group are used for coordination. Conformationalchanges and incorporation of water molecules are needed(Table 4). Conversely, water alone cannot substitute for metalions either, because a water molecule is larger (atomic radius:1.4 Å) than a Ca2� (ionic radius: 0.99 Å) and can accommodateless oxygen atoms (four vs. six to eight). Ionization of a clusterof carboxyls within the hydrophobic core of the membranewould incur a huge energetic cost (�100 kcal�mol in this case;Table 2). These considerations make the countertransport ofeither metal ions or H�, and possibly water, mandatory for allP-type ATPases, although for many of them the ions coun-tertransported are unknown as yet. It could be difficult toidentify them, because ion gradients generated by counter-transport might be dissipated by other means.

In the Ca2�-ATPase, all of the residues protonated areinvolved in Ca2�-coordination, indicating that Ca2�-binding is,in fact, an exchange reaction with H�. This finding provides at

least a partial explanation for the well-known pH dependenceof Ca2�-binding affinity (10 times weaker at pH 6.0 than at pH7.0) (15). The presence of one titratable residue per eachCa2�-binding site (15, 16) agrees with the electrostatic calcu-lation described here, but strong pH dependence of thoseresidues in the neutral range (5, 15, 16) does not. Thisinconsistency is understandable because substantial confor-mation changes take place during Ca2�-binding, whereas theelectrostatic calculations assume rigid structures.

We thank M. Kawamoto, H. Sakai, N. Shimizu, and T. Tsuda for datacollection at BL41XU of SPring-8 and Y. Ohuchi for computerprograms. We thank D. Bashford (St. Jude Children’s ResearchHospital, Memphis, TN) for providing us with his MEAD program; M.Ikeguchi (Yokohama City University, Yokohama, Japan) for theMARBLE program; and D. B. McIntosh and D. H. MacLennan for theirhelp in improving the manuscript. This work was supported in part bya Creative Science Project Grant from the Ministry of Education,Culture, Sports, Science and Technology; the Japan New Energy andIndustry Technology Development Organization; and the HumanFrontier Science Program.

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