Roles of Tyr122-hydrophobic Cluster and K+ Binding in Ca2+-releasing Process of ADP-insensitive Phosphoenzyme of Sarcoplasmic Reticulum Ca2+-ATPase*

Tyr122-hydrophobic cluster (Y122-HC) is an interaction network formed by the top part of the second transmembrane helix and the cytoplasmic actuator and phosphorylation domains of sarcoplasmic reticulum Ca2+-ATPase. We have previously found that Y122-HC plays critical roles in the processing of ADP-insensitive phosphoenzyme (E2P) after its formation by the isomerization from ADP-sensitive phosphoenzyme (E1PCa2) (Wang, G., Yamasaki, K., Daiho, T., and Suzuki, H. (2005) J. Biol. Chem. 280, 26508–26516). Here, we further explored kinetic properties of the alanine-substitution mutants of Y122-HC to examine roles of Y122-HC for Ca2+ release process in E2P. In the steady state, the amount of E2P decreased so that of E1PCa2 increased with increasing lumenal Ca2+ concentration in the mutants with K0.5 110–320 μm at pH 7.3. These lumenal Ca2+ affinities in E2P agreed with those estimated from the forward and lumenal Ca2+-induced reverse kinetics of the E1PCa2-E2P isomerization. K0.5 of the wild type in the kinetics was estimated to be 1.5 mm. Thus, E2P of the mutants possesses significantly higher affinities for lumenal Ca2+ than that of the wild type. The kinetics further indicated that the rates of lumenal Ca2+ access and binding to the transport sites of E2P were substantially slowed by the mutations. Therefore, the proper formation of Y122-HC and resulting compactly organized structure are critical for both decreasing Ca2+ affinity and opening the lumenal gate, thus for Ca2+ release from E2PCa2. Interestingly, when K+ was omitted from the medium of the wild type, the properties of the wild type became similar to those of Y122-HC mutants. K+ binding likely functions via producing the compactly organized structure, in this sense, similarly to Y122-HC.

port coupled with ATP hydrolysis from the cytoplasm to lumen against a concentration gradient of ϳ10,000-fold (1)(2)(3)(4)(5)(6)(7)(8). In the initial steps (steps 1 and 2 in Scheme 1), the enzyme is activated by binding of two cytoplasmic Ca 2ϩ ions at the transport sites with a submicromolar high affinity (E2 to E1Ca 2 ). The activated enzyme is then auto-phosphorylated at Asp 351 by ATP and forms a phosphoenzyme intermediate (EP) (step 3), thereby the bound Ca 2ϩ ions are occluded in the transport sites. This EP is rapidly dephosphorylated by ADP in the reverse reaction reproducing ATP, therefore "ADP-sensitive EP" (E1P). In the next step (step 4), E1PCa 2 is isomerized to the ADP-insensitive form, E2PCa 2 . Upon this change at the catalytic site, the Ca 2ϩ sites are deoccluded and opened to the lumenal side, and the Ca 2ϩ affinity is largely reduced, releasing the bound Ca 2ϩ ions into the lumen (step 5). The Ca 2ϩ release process is thought to be very rapid with the wild-type Ca 2ϩ -ATPase, and the accumulation of E2PCa 2 intermediate had actually never been found until we recently identified and trapped successfully this intermediate by a mutation study (9). In the final step, the Asp 351 -acylphosphate of E2P is hydrolyzed to reproduce the dephosphorylated and inactive E2 form (step 6). The transport cycle is totally reversible, e.g. E2P can be formed from E2 by P i in the absence of Ca 2ϩ , and the subsequent lumenal Ca 2ϩ binding to E2P produces E1PCa 2 .
Three-dimensional structures in several intermediate states and their analogs have been solved (10 -18). The Ca 2ϩ -ATPase has three cytoplasmic domains, P (phosphorylation), N (nucleotide binding), and A (actuator or anchor), and ten transmembrane helices (M1-M10). The two Ca 2ϩ binding sites consist of residues on M4, M5, M6, and M8 (10). The P domain possesses the phosphorylation site (Asp 351 ) and is directly linked to the long helices M4 and M5. The ATP binding site is on the N domain connected to the P domain. The A domain is linked to M1, M2, and M3 via the A/M1-, A/M2-, and A/M3-linkers. The cytoplasmic three domains largely move and change their organization states during the Ca 2ϩ -transport cycle (19 -21), and these changes are linked with the rearrangements in the transmembrane helices for the Ca 2ϩ transport. As a most remarkable change, in the EP isomerization (loss of ADP sensitivity) and Ca 2ϩ release, the A domain largely rotates and the P domain largely inclines toward the A domain, and these domains produce their tight association (see Fig. 1 for the change E1Ca 2 ⅐AlF 4 Ϫ ⅐ADP 3 E2⅐MgF 4 2Ϫ as the model for the overall process E1ϳPCa 2 ⅐ADP 3 E2⅐P i , including the EP isomerization and Ca 2ϩ release). These structural changes therefore involve distinct events in distinct regions, yet they are coordinated; namely 1) the loss of ADP sensitivity at the cytoplasmic region, 2) the decrease in the Ca 2ϩ affinity at the transmembrane region, and 3) the opening of the Ca 2ϩ -releasing pathway (lumenal gating).
Recently, we found that mutations in a specific hydrophobic interaction network, "Tyr 122 -hydrophobic cluster" (Y122-HC), at the A-P domain interface disrupt markedly the processing of ADP-insensitive EP formed from ATP with Ca 2ϩ and also the hydrolysis of E2P formed from P i without Ca 2ϩ , thus causing nearly complete inhibition of the Ca 2ϩ -ATPase activity (22,23). In these Y122-HC mutants, the high affinity binding of cytoplasmic Ca 2ϩ , the resulting E1PCa 2 formation, and the loss of the ADP sensitivity were all found to occur normally as in the wild type (22,23). Y122-HC is formed by gathering of the seven residues of the three regions upon their motions; i.e. the largely rotated A domain (Ile 179 , Leu 180 , and Ile 232 ), the inclined P domain (Val 705 and Val 726 ), and the top part of the largely inclined M2 (or the A/M2-linker) (Leu 119 and Tyr 122 ). Thus Y122-HC produces the compactly organized structure of E2P. Our previous analyses indicate that, in the Y122-HC mutants, there is a kinetic limit after the loss of ADP sensitivity and before the hydrolysis of the Ca 2ϩ -free E2P, therefore the Ca 2ϩ release from E2PCa 2 is likely retarded (22,23). Almost the same kinetic results were found with the mutations in another A-P domain interaction network at the Val 200 loop of the A domain (24). Notably, E2PCa 2 , the ADP-insensitive EP with two Ca 2ϩ ions occluded at the transport sites was recently identified and trapped successfully by the elongation of the A/M1-linker with two or more amino acid insertions (9). In the elongation mutants, Y122-HC is not formed properly yet in E2PCa 2 trapped, but it is properly formed in the Ca 2ϩ -released form of E2P produced by P i without Ca 2ϩ . Thus the observation is consistent with the involvement of Y122-HC in the Ca 2ϩ release process from E2PCa 2 .
In the present study, to further clarify roles of Y122-HC in the Ca 2ϩ deocclusion/release processes and thus in the long range communication between the cytoplasmic and transmembrane regions, we explored kinetic features of the alanine-substitution mutants of Y122-HC. The results revealed that the mutations cause a marked increase in the apparent affinity of E2P for lumenal Ca 2ϩ and also a substantial retardation of the lumenal Ca 2ϩ access to E2P. Therefore, the formation of Y122-HC is critical for decreasing the affinity for Ca 2ϩ , for lumenal gating (opening of the release pathway), and thus for Ca 2ϩ release into lumen. Importantly, the assembling manner of the seven residues in Y122-HC in the very recently revealed crystal structure E2⅐BeF 3 Ϫ (17, 18) somewhat differs from that in E2⅐AlF 4 Ϫ and E2⅐MgF 4 2Ϫ . Therefore, we discussed the significance of this difference in terms of the possible sequential gathering of the seven residues into Y122-HC on the basis of the observed difference in the extents of their mutational effects. In addition, we found with the wild type that its kinetic behavior became similar to that of Y122-HC mutants when K ϩ was omitted from the medium of the wild type. Results revealed for the first time the critical role of K ϩ binding in the wild type for Ca 2ϩ deocclusion/release from E2PCa 2 .

EXPERIMENTAL PROCEDURES
Mutagenesis and Expression-Mutations were created by the QuikChange TM site-directed mutagenesis kit (Stratagene) and plasmid pGEM7-Zf(ϩ) or pGEM3-Zf(ϩ) (Promega, Madison, WI) containing ApaI-KpnI or KpnI-SalI fragments of rabbit SERCA1a cDNA as a template. The ApaI-KpnI or KpnI-SalI fragments were then excised from the products and used to replace the corresponding region in the full-length SERCA1a cDNA in the pMT2 expression vector (25). The pMT2 DNA was transfected into COS-1 cells by the liposome-mediated transfection method. Microsomes were prepared from the cells as described previously (26). The "control microsomes" were prepared from COS-1 cells transfected with the pMT2 vector containing no SERCA1a cDNA.
ATPase Activity-The rate of ATP hydrolysis was determined at 25°C in a mixture containing 20 g/ml microsomal Ϫ ⅐ADP (the analog for the transition state of the phosphoryl transfer E1ϳPCa 2 ⅐ADP, left panel) and E2⅐MgF 4 2Ϫ (E2⅐P i analog (21), right panel) of Ca 2ϩ -ATPase were obtained from the Protein Data Bank (PDB accession codes 1T5T and 1WPG, respectively (12,14) Formation and Hydrolysis of EP-Phosphorylation of SERCA1a in microsomes with [␥-32 P]ATP or 32 P i and dephosphorylation of 32 P-labeled SERCA1a were performed under conditions described in the figure legends. The reactions were quenched with ice-cold trichloroacetic acid containing P i . Rapid kinetics measurements of phosphorylation and dephosphorylation were performed with a handmade rapid mixing apparatus (27), otherwise the method was as above. The precipitated proteins were separated at pH 6.0 by 5% SDS-PAGE, according to Weber and Osborn (28). The radioactivity associated with the separated Ca 2ϩ -ATPase was quantitated by digital autoradiography as described previously (29). The amount of EP formed with the expressed SERCA1a was obtained by subtracting the background radioactivity with the control microsomes. This background was Ͻ1% of the radioactivity of EP formed with the expressed wild-type SERCA1a.
Miscellaneous-Protein concentrations were determined by the method of Lowry et al. (30) with bovine serum albumin as the standard. Free Ca 2ϩ concentrations were calculated by the Calcon program. Data were analyzed by nonlinear regression using the program Origin (MicroCal Software, Inc., Northampton, MA). Three-dimensional models of the enzyme were reproduced by using the program VMD (31).

RESULTS
Ca 2ϩ -induced Change in Accumulation of ADP-insensitive EP in the Presence of 0.1 M K ϩ at Steady State-We first determined the steady state Ca 2ϩ -ATPase activity in the presence of increasing Ca 2ϩ and ionophore A23187 with the alanine-substitution mutants of the seven residues of Y122-HC and the wild type. The Ca 2ϩ -ATPase activity was nearly completely inhibited in all the mutants in agreement with our previous observation (22,23), and the complete inhibition was found at all the Ca 2ϩ concentrations examined (see supplemental Fig. S1 for the representative mutant Y122A). Thus the possible lumenal Ca 2ϩ effect was not revealed by this type of measurements. Therefore in Fig. 2, to assess the affinity of the lumenally oriented Ca 2ϩ transport site of E2P (known as the low affinity sites with the mM over ϳ10 mM K d value), the amounts of ADPinsensitive EP were determined with the representative mutant Y122A at steady state at various Ca 2ϩ concentrations and pH values in the presence of A23187 and KCl. The total amounts of EP (ADP-sensitive EP plus ADP-insensitive EP) were nearly the same under all the sets of conditions.
In the mutant Y122A, the fraction of the ADP-insensitive EP was very high at the low Ca 2ϩ concentrations at all pHs (Fig. 2). This agrees with the property of this mutant (22,23) that the hydrolysis of E2P is nearly completely inhibited thus causing its accumulation. The fraction of ADP-insensitive EP in the mutant markedly decreased, and it was converted to the ADPsensitive EP with increasing Ca 2ϩ concentration at several tens of micromolar to the sub-millimolar range. The apparent Ca 2ϩ affinity in this Ca 2ϩ -induced change increased with increasing pH, and the Hill coefficients were found to be 2 in all pH values (see the legend to Fig. 2). In the wild type, the fraction of ADP-insensitive EP was low at pH 7.3 and 7.8 being ϳ10% or less, and was a significant level, 35% at pH 6.8 (supplemental Fig. S2). These levels were not changed at 1 M to 3 mM Ca 2ϩ . Consistently, the lumenal Ca 2ϩ affinity of E2P of the wild type is known to be in the millimolar to 10 mM range (see Ref. [32][33][34]. The results suggested that the lumenal Ca 2ϩ affinity of transport sites of E2P in the mutant may be significantly higher than that in the wild type. Time Courses of Forward and Ca 2ϩ -induced Reverse Conversions between E1PCa 2 and E2P-In Fig. 3 with Y122A, the ADP-insensitive EP, and the ADP-sensitive EP was first accumulated at steady state at 10 M Ca 2ϩ and 1 mM Ca 2ϩ , respectively, at pH 7.3. Then the Ca 2ϩ concentration jump was made from 10 M to 1 mM or from 1 mM to 80 nM, and the change in the fraction of the ADP-insensitive EP was followed. Because the hydrolysis of E2P was nearly completely blocked in Y122A (with the rate Ͻ Ͻ 0.01 s Ϫ1 ) (22,23), the time courses represent the forward and reverse isomerization between E1PCa 2 and E2P. When Ca 2ϩ was increased from 10 M to 1 mM, the fraction of ADP-insensitive EP rapidly decreased from 80% to 10% (i.e. it was converted to the ADP-sensitive EP) with a rate 0.4 s Ϫ1 . On the other hand, when the Ca 2ϩ concentration was decreased from 1 mM to 80 nM, i.e. virtually Ca 2ϩ was removed, the ADP-sensitive EP was converted to the ADP-insensitive EP with a rate 0.022 s Ϫ1 .
Effect of Ca 2ϩ Ionophore A23187 in Accumulation of ADPinsensitive EP-To ascertain that the Ca 2ϩ -dependent changes in the fraction of ADP-insensitive EP (Figs. 2 and 3) are caused by lumenal Ca 2ϩ , we examined also in the absence of A23187 the Ca 2ϩ dependence of the steady-state fraction of ADP-insensitive EP (Fig. 4). The Ca 2ϩ -dependent change was rather small in the absence of A23187, in contrast to the very large change in its presence. Therefore, the observed Ca 2ϩ -induced conversion from the ADP-insensitive EP to ADP-sensitive one in Y122A is due to the Ca 2ϩ binding to the lumenally oriented transport sites.
Ca 2ϩ -induced Change in Accumulation of ADP-insensitive EP of Seven Y122-HC Mutants in the Presence of 0.1 M K ϩ -In Fig. 5, each of the other six residues involved in the Y122-HC (Leu 119 /Ile 179 /Leu 180 /Ile 232 /Val 705 /Val 726 ) was substituted with alanine, and its effect on the ADP-insensitive EP level was examined as in Fig. 2 in the presence of A23187 and K ϩ . All the Y122-HC mutants exhibited the marked Ca 2ϩ -dependent change in the ADP-insensitive EP fraction. 3 The apparent affinities for lumenal Ca 2ϩ in the Y122-HC mutants were found to be between 110 and 320 M with a Hill coefficient of ϳ2 ( Table  1). The results showed that all these Y122-HC mutants possess the lumenally oriented transport sites with the affinities as high as that of Y122A.
Ca 2ϩ -induced Change in Accumulation of ADP-insensitive EP of Mutants and Wild Type in the Absence of K ϩ -In Fig. 6, the same sets of steady-state analysis as in Fig. 2 were done with the wild type and Y122A but here in the absence of K ϩ . It is well known (35,36) that, in the absence of K ϩ , the E2P hydrolysis of  After 10 s of this Ca 2ϩ jump, the total amount of EP and the fraction of the ADP-insensitive EP were determined as described in Fig. 2. The inclusion of Ruthenium Red in the presence of A23187 caused a slight decrease in the ADP-insensitive EP fraction at the Ca 2ϩ concentrations below ϳ100 M (cf. Fig. 2) for unknown reasons.  Table 1.
the wild type is markedly slowed, and therefore the ADP-insensitive EP significantly accumulates. The fraction of ADP-insensitive EP in the wild type in the absence of K ϩ decreased with increasing Ca 2ϩ concentration as in Y122A with the Hill coefficient ϳ2. The apparent affinity for lumenal Ca 2ϩ increased with increasing pH in the wild type as in Y122A. The pH-dependent changes are consistent with the fact that the residues for Ca 2ϩ ligation at the transport sites are also involved in the proton binding (and its counter transport); the observed Ca 2ϩinduced changes reflect the Ca 2ϩ binding to the lumenally oriented transport sites of E2P. At each pH, the affinity of the wild type was similar to or slightly lower than that of Y122A. Thus in the absence of 0.1 M K ϩ , the property of the wild type became similar to that of Y122A. In Y122A, elimination of K ϩ exhibited no significant effect on the apparent affinity for lumenal Ca 2ϩ (cf. Fig. 2).
The observed effect of K ϩ on the wild type is probably due to its binding in the cytoplasmic region. In crystallographic as well as mutational studies (12,37), the K ϩ binding site of the Ca 2ϩ -ATPase was identified to be in the cytoplasmic region but not in the lumenal or transmembrane regions (see Fig. 11). Actually, we found experimentally that, when K ϩ at 0.1 M was added without any K ϩ -ionophore to the Ca 2ϩ -ATPase in SR vesicles phosphorylated in the absence of K ϩ , the Ca 2ϩ -dependence of the ADP-insensitive EP fraction observed as in Fig. 6A became immediately (within 10 s after the K ϩ addition) that in the presence of 0.1 M K ϩ as in supplemental Fig. S2 (data not shown).
Kinetics of Lumenal Ca 2ϩ -induced E2P to E1PCa 2 Reverse Transition Followed by Its ADP-induced Rapid Decay to E1Ca 2 in the Presence of 0.1 M K ϩ -Then with the representative mutant Y122A, we explored kinetically the lumenal Ca 2ϩ accessibility to the lumenally oriented transport sites of E2P formed from P i without Ca 2ϩ and the resulting lumenal Ca 2ϩinduced E2P to E1PCa 2 reverse transition. In Fig. 7, we included ADP and thereby followed the Ca 2ϩ -and ADP-induced decay of E2P to E1Ca 2 via E1PCa 2 in the reverse reaction. The E2P hydrolysis in the absence of Ca 2ϩ was extremely slow (as previously demonstrated with the Y122-HC mutants (22,23)), and the E2P decay was dramatically accelerated by the addition of Ca 2ϩ and ADP (Fig. 7A). For example, the rate in the presence of 1 mM Ca 2ϩ was 200-times faster than that of the forward E2P hydrolysis in the absence of Ca 2ϩ . ADP alone without Ca 2ϩ or Ca 2ϩ alone without ADP did not accelerate the EP decay (data not shown). As shown in Fig. 3, the increase of Ca 2ϩ to 1 mM converted the ADP-insensitive EP (E2P) to the ADP-sensitive one (E1PCa 2 ), and E1PCa 2 thus formed was not decomposed in the absence of ADP. Therefore the Ca 2ϩ -and ADP-induced decay of E2P in Fig. 7A obviously occurred in the reverse reaction by the lumenal Ca 2ϩ binding; E2P ϩ 2Ca 2ϩ 3 E1PCa 2 , then E1PCa 2 ϩ ADP 3 E1Ca 2 ϩ ATP (Scheme 1). This view agrees with the previous demonstration with SR Ca 2ϩ -ATPase (34). The rate of the EP decay in the presence of Ca 2ϩ and ADP increased almost linearly with increasing Ca 2ϩ concentrations and was not saturated even at 3 mM (Fig. 7B). 4 Here, note that

TABLE 1 Parameters obtained for Ca 2؉ dependence of accumulation of ADPinsensitive EP in Y122-HC mutants
As shown in Fig. 5, the lumenal Ca 2ϩ -induced change in the steady-state accumulation of ADP-insensitive EP of the seven Y122-HC mutants in the presence of 0.1 M K ϩ were fitted to the Hill equation. The parameters thus obtained by the least squares fit are listed here. K 0.5 is the Ca 2ϩ concentration giving the half-maximum change in the fraction of ADP-insensitive EP among the total amount of EP, therefore the apparent affinity for lumenal Ca 2ϩ . The highest fraction of the ADP-insensitive EP at the low Ca 2ϩ concentration range (0 -10 M) and its lowest fraction at the high Ca 2ϩ concentration range (over ϳmM) are also listed as the obtained parameters in the fitting (see Fig. 5). The value n H is the Hill coefficient. The Ca 2ϩ -and ADP-dependent acceleration of the reverse E2P decay was assayed also with all the other Y122-HC mutants (supplemental Fig. S3). The rates of the reverse E2P decay increased almost linearly with increasing Ca 2ϩ concentrations even at 3 mM, except those of I232A and V705A 5 over ϳ1 mM Ca 2ϩ . Nevertheless, the slope of the Ca 2ϩ dependence below 1 mM Ca 2ϩ was estimated to be ϳ0.2 s Ϫ1 mM Ϫ1 in all the mutants as in Y122A. Therefore, the rate of the lumenal Ca 2ϩ access and binding to the transport sites is similar in all the mutants of Y122-HC.

Mutant
Kinetics of Lumenal Ca 2ϩ Access to E2P of Wild Type and Y122A with and without K ϩ -Then in Fig. 8, with the wild type and the representative mutant Y122A in the presence and absence of 0.1 M K ϩ , we analyzed the Ca 2ϩ -and ADP-dependent acceleration of the reverse decay of E2P formed from P i without Ca 2ϩ . As the well characterized property of the wild type, the forward hydrolysis of E2P without bound Ca 2ϩ is very slow in the absence of K ϩ , but markedly accelerated and thus very rapid in the presence of 0.1 M K ϩ (35, 36) (see the rates without Ca 2ϩ in Fig. 8A). Nevertheless, even with the wild type in the presence of K ϩ , we observed an apparently single exponential decay of E2P after the addition of Ca 2ϩ and ADP at all the Ca 2ϩ concentrations examined (time courses are not shown for simplicity). This is consistent with the kinetics described in the textbook by Fersht (39) that, in the parallel reactions in which a compound undergoes two or more single-step reactions simultaneously, its disappearance rate is described by a single exponential decay. In our case, the two reactions are the forward E2P hydrolysis and the Ca 2ϩ -/ADP-induced reverse E2P decay. The single decay rates thus obtained are plotted in Fig. 8A.
In the wild type in the presence of 0.1 M K ϩ , the Ca 2ϩ dependence of the EP decay rate was complicated because of the rapid E2P hydrolysis without Ca 2ϩ (ϳ0.4 s Ϫ1 ), no change in the rate at 0 -0.6 mM Ca 2ϩ , and the gradual increase above 0.6 mM. On the other hand, in the wild type in the absence of K ϩ in which the E2P hydrolysis without bound Ca 2ϩ is markedly slowed, the nearly linear increase in the rate of Ca 2ϩ -/ADPinduced reverse E2P decay was observed at least up to ϳ3 mM Ca 2ϩ as in the Y122-HC mutants in the presence of K ϩ . The slope of the wild type without K ϩ was actually close to that of Y122A with K ϩ . Therefore, the rate of lumenal Ca 2ϩ access and binding to the transport sites of E2P of the wild type in the absence of K ϩ is similar to that of Y122A. With the wild type in the presence of K ϩ , evaluation of the lumenal Ca 2ϩ access rate by this approach was not possible because of the complicated Ca 2ϩ -dependence curve. In Y122A, little effect was seen when K ϩ was omitted at 0 -1 mM Ca 2ϩ , although the slope became gradually less steep at the higher Ca 2ϩ concentration in the absence of K ϩ .
In Fig. 8B, by using the rates of the E2P decay in the presence of added Ca 2ϩ and ADP (determined in Fig. 8A) and the rates of 0.2 s Ϫ1 mM Ϫ1 , gave the apparent affinity for lumenal Ca 2ϩ in E2P of Y122A as 100 M. This value agreed very well with that (160 M) obtained in Fig. 2 under the same conditions (pH 7.3) at the steady state. 5 As a possible reason for the less steep Ca 2ϩ -dependent curve observed with I232A and V705A over ϳ1 mM, it might be possible that the rate in the transition from E2PCa 2 to E1PCa 2 is slower in these mutants than in the other mutants, and this step became rate-limiting at the high Ca 2ϩ concentrations where the lumenal Ca 2ϩ -induced change to E2PCa 2 became fast. the forward E1PCa 2 to E2P transition (as determined in Fig. 3), we simulated the fraction of the steady-state level of the ADPinsensitive EP (E2P) in the total amount of EP at each Ca 2ϩ concentration. Note that this simulation was made possible by the fact that nearly all the phosphorylation sites are phosphorylated at steady state (in either E1P or E2P form) under the conditions used for the steady-state and kinetic analyses at all the Ca 2ϩ concentrations in this study. Namely, the E2 to E1Ca 2 transition and the E1PCa 2 formation from E1Ca 2 with ATP are rapid enough to be ignored from the simulation. Therefore, the fraction of ADPinsensitive EP (E2P) in the steady state will be determined by the rate of its formation in the forward E1PCa 2 to E2P transition, v 1 , and by the rate of its decay, v 2 that includes both the forward hydrolysis of Ca 2ϩ -unbound E2P to E2 and the Ca 2ϩ -induced reverse transition to E1PCa 2 with the subsequent ADP-induced decay. This means that the simulation can be made even with the wild type in the presence of K ϩ (as v 2 can include the forward E2P hydrolysis). In the steady-state conditions, the decay rate (v 2 ) and formation rate (v 1 ) should be equal, therefore the fraction of ADPinsensitive EP (F E2P ) in the total amount of EP will be estimated by an equation: Here, the E2P decay rate (v 2 ) was obtained in Fig. 8A at each Ca 2ϩ concentration. The E1PCa 2 to E2P transition rate (v 1 ) was estimated from the Ca 2ϩ jump experiments from high (1 mM) to low (80 nM) for Y122A with and without 0.1 M K ϩ and the wild type without K ϩ , as described in Fig. 3.
With the wild type in the presence of 0.1 M K ϩ , the forward decay rate of E1PCa 2 formed by ATP was used as v 1 , because the E1PCa 2 to E2P transition (the loss of ADP sensitivity) is rate-limiting for the E1PCa 2 decay via E2P and its hydrolysis. The Ca 2ϩ -dependent curves thus obtained by the simulation for the steady-state level of ADP-insensitive EP for Y122A with and without K ϩ and the wild type without K ϩ agreed very well with the respective ones determined at the steady state (cf. Figs. 2 (with K ϩ ) and 6 (without K ϩ ) at pH 7.3). The affinities for lumenal Ca 2ϩ estimated from the simulated curves are in fact almost the same as those actually determined at steady state ( Table 2). The agreements assure the validity of the simulation and further allow us to estimate the lumenal Ca 2ϩ affinity of E2P of the wild type in the presence of K ϩ . In the simulation for the wild type in the presence of K ϩ (open circles and inset in Fig.  8B), the fraction of ADP-insensitive EP was very low, and the extent of its change was extremely small as expected from the FIGURE 8. Lumenal Ca 2؉ -and ADP-induced reverse E2P decay of wild type and Y122A in the absence and presence of K ؉ . A, microsomes (100 g/ml) expressing wild type (WT) or Y122A were phosphorylated with 32 P i in the presence of A23187 and absence of Ca 2ϩ , and then chilled on ice, as described in Fig. 7. Subsequently, the phosphorylated sample was diluted at 0°C with a 20-fold volume of a chase solution containing non-radioactive P i , various concentrations of CaCl 2 , and ADP in the presence of 105 mM KCl (open symbols) or 105 mM LiCl in place of KCl (closed symbols), otherwise as described in Fig. 7. The time courses of EP decay were fitted to single exponential (data not shown, see Fig. 7A as an example). The rates thus obtained were plotted versus the Ca 2ϩ concentrations. B, the fraction ADP-insensitive EP (F E2P ) in the total amount of EP at steady state was simulated by using the rate of E2P decay (v 2 ) and the rate of the E1PCa 2 to E2P isomerization (loss of ADP sensitivity, v 1 ) with an equation F E2P ϭ v 1 /(v 1 ϩ v 2 ). Here, v 2 is the E2P decay rate obtained above in panel A at each Ca 2ϩ concentrations. The v 1 was obtained as described in Fig. 3 by the Ca 2ϩ jump experiments from high (1 mM) to low (80 nM, virtually Ca 2ϩ removal) for Y122A with 0.1 M K ϩ or Li ϩ (without K ϩ ) and for the wild type with 0.1 M Li ϩ (without K ϩ ). For the wild type with 0.1 M K ϩ , the forward decay rate of E1PCa 2 formed from ATP was used as the v 1 value, because the E1PCa 2 to E2P transition (the loss of ADP sensitivity) is rate-limiting for the E1PCa 2 decay via E2P and its subsequent rapid hydrolysis. The v 1 values actually used for the calculation were 0.049 s Ϫ1 (wild type with K ϩ ), 0.071 s Ϫ1 (wild type with Li ϩ ), 0.021 s Ϫ1 (Y122A with K ϩ ), and 0.034 s Ϫ1 (Y122A with Li ϩ ). The fraction of ADP-insensitive EP thus calculated was plotted versus the Ca 2ϩ concentrations. The solid lines show the least squares fit to the Hill equation. In the inset, the ordinate is in a magnified scale for wild type with K ϩ . In Table 2, the affinities and the Hill coefficients of the wild type and the Y122-HC mutants thus "estimated by kinetic analyses" in the absence and presence of K ϩ at pH 7.3 are summarized together with those actually "determined by the steady-state analyses" of the lumenal Ca 2ϩ -induced change of the fraction of ADP-insensitive EP otherwise under the same conditions in Figs. 2 and 6.

TABLE 2 Affinities of E2P for lumenal Ca 2؉ estimated by kinetic analyses and those determined at steady-state analyses
As described in Fig. 8B, the lumenal Ca 2ϩ affinities (K 0.5 ) of E2P of the wild type and the mutant Y122A were estimated by the kinetic analyses of the lumenal Ca 2ϩinduced E2P to E1PCa 2 reverse transition and of the forward E1PCa 2 to E2P transition. The K 0.5 values and the Hill coefficients (n H ) thus estimated kinetically in Fig.  8 in the absence and presence of 0.1 M K ϩ at pH 7.3 are summarized here. Listed together are those determined by the steady-state analyses of the lumenal Ca 2ϩinduced change in the accumulated fraction of the ADP-insensitive EP (E2P) under otherwise the same conditions in Figs. 2 and 6 for the mutant Y122A with and without K ϩ and the wild type without K ϩ . steady-state measurements (cf. supplemental Fig. S2 (pH 7.3)). The apparent affinity of wild type for lumenal Ca 2ϩ in the presence of K ϩ was thus estimated by the small change to be 1.5 mM (see Table 2). This affinity was ϳ3.5-times lower than that of wild type without K ϩ and 10-times lower than that of Y122A with and without K ϩ . Thus, by omitting K ϩ , the lumenal Ca 2ϩ affinity of E2P in the wild type became higher and similar to that in Y122A.
Kinetics of Lumenal Ca 2ϩ -induced E2P to E1PCa 2 Reverse Transition of E2P of Wild Type in the Presence of 0.1 M K ϩ Was Revealed by the Absence of ADP-Unfortunately, in the above experimental design and approach of Fig. 8A, we were not able to estimate the lumenal Ca 2ϩ access rate in E2P of the wild type in the presence of K ϩ because of the observed complexity of the Ca 2ϩdependent curve. In Fig. 9, we therefore employed a modified and thus different approach to examine the lumenal Ca 2ϩ -induced reverse conversion from E2P to E1PCa 2 . Namely, E2P was formed with P i , and then a medium containing various concentrations of Ca 2ϩ but without ADP (in contrast to its presence in Fig. 8) was added to E2P, and the subsequent EP decay was followed (Fig. 9A). In the absence of Ca 2ϩ , E2P was all hydrolyzed rapidly to E2 in a single exponential function. The E2P hydrolysis was inhibited gradually with increasing Ca 2ϩ concentrations (over 0.1 mM), and the decay time course became biphasic as typically seen with 1 mM Ca 2ϩ . With increasing Ca 2ϩ concentration, the fraction of the first and rapid phase decreased, that of the second phase increased, and the rate of the second phase became slower. The observation agrees with the previous kinetics analysis (9,40). The first phase corresponds to the rapid and forward hydrolysis of the Ca 2ϩunbound E2P to E2. The EP species in the second phase was all ADP-sensitive (data not shown), therefore E1PCa 2 formed from E2P by the lumenal Ca 2ϩ binding. E1PCa 2 decayed very slowly in the absence of ADP, because the E1PCa 2 to E2P transition is much slower than the E2P hydrolysis, and this transition is retarded by the Ca 2ϩ replacement of Mg 2ϩ at the catalytic site of E1PCa 2 at the approximately millimolar high Ca 2ϩ concentrations (41,42).
The fraction of the second and slow phase of the EP decay was obtained by extrapolating to the zero time and plotted versus the Ca 2ϩ concentration (Fig. 9B). The plot showed saturation at 5-10 mM Ca 2ϩ . Here it is critical to note that, as previously discussed in detail (9), the fraction of EP of the second phase (the fraction remaining after the first phase) is dependent on the ratio between the rates of the forward E2P hydrolysis and of the reverse E2P to E1PCa 2 conversion upon the lumenal Ca 2ϩ binding to E2P. Namely, the plot in Fig. 9B reflects the relation between these forward and reverse rates of E2P rather than the lumenal Ca 2ϩ affinity of E2P. For example, at the 50% saturation of the curve, the rate of the Ca 2ϩ -induced E1PCa 2 formation from E2P is equal to that of the E2P hydrolysis to E2. Then in Fig. 9C for the wild type in the presence of K ϩ , the rate of the E1PCa 2 formation from E2P by the lumenal Ca 2ϩ binding to E2P was calculated (k rev , open circles) at each Ca 2ϩ concentration by using the fraction of the slow and second phase (F s ) and the E2P hydrolysis rate (k h ) with the equation, k rev ϭ k h F s /(1 Ϫ F s ). The rate increased largely with increasing Ca 2ϩ concentration.
In this kinetics, we eliminated the contribution of forward E2P hydrolysis on the overall E2P decay kinetics, and thereby revealed the rate of reverse E2P transition to E1PCa 2 induced by the lumenal Ca 2ϩ binding of the wild type in the presence of K ϩ . For comparison in Fig. 9C, the rates of the lumenal Ca 2ϩinduced reverse E2P decay estimated for the wild type without K ϩ and Y122A with K ϩ in Fig. 8A were replotted. Note again that, in these cases, the hydrolysis of Ca 2ϩ -unbound E2P was very slow and retarded; therefore, the observed Ca 2ϩ -/ADPinduced decay rates in their linear regions up to 3 mM Ca 2ϩ reflect mostly the rates of the lumenal Ca 2ϩ access and binding to E2P in the reverse E2P decay. Note also that the experimental design in Fig. 9A employed for the wild type with K ϩ was not applicable to the wild type without K ϩ and Y122A, because the E2P hydrolysis is very slow and almost completely retarded in these cases, and therefore the E2P decay upon the Ca 2ϩ addition cannot be described as the biphasic decay. Conversely, the experimental design employed in Fig. 8A to estimate the rates of the lumenal Ca 2ϩ access was not applicable to the wild type in the presence of K ϩ because of the complexity of the Ca 2ϩdependent curve as described above in Fig. 8A.
Thus in Fig. 9C, employing the inevitably different but most suitable experimental designs depending on the different kinetic properties, we were able to compare the rates of the E2P to E1PCa 2 reverse transition induced by the lumenal Ca 2ϩ binding to the transport sites of E2P at the limited Ca 2ϩ concentration range up to 3 mM. In the wild type in the presence of K ϩ , the rate was Ca 2ϩ -dependent and not saturated even at 3 mM, thus reflecting at least the Ca 2ϩ -dependent and rate-limiting process; i.e. the lumenal Ca 2ϩ -induced change from E2P to E2PCa 2 . This reverse transition rate in the wild type in the presence of K ϩ was significantly faster than those in the wild type in the absence of K ϩ and in Y122A (as well as in the other Y122-HC mutants (supplemental Fig. S3)) especially at the high Ca 2ϩ concentration over 1 mM.
Here it is also interesting to note that the affinity of E2P for the lumenal Ca 2ϩ in the wild type without K ϩ and the Y122-HC mutants is significantly higher than in the wild type with K ϩ (see Fig. 8B). If the rate of lumenal Ca 2ϩ access and binding to E2P is solely slowed in the wild type without K ϩ and Y122-HC mutants, a decrease in the affinity is rather the consequence, which is in contrast to the observed increase. Therefore the rates of the Ca 2ϩ release from E2PCa 2 in the wild type without K ϩ and in the Y122-HC mutants are also presumably retarded significantly as compared with that in the wild type in the presence of K ϩ . Namely, the mutations of Y122-HC and the lack of K ϩ binding affect the energy levels of Ca 2ϩ -free and -bound E2P states, as well as that of the transition state for lumenal gating (opening), and favor the Ca 2ϩ -bound state E2PCa 2 and the closed lumenal gate.

Roles of Y122-HC in Ca 2ϩ Release from E2PCa 2 and in E2P
Hydrolysis-In this study, we found that the mutations of any of the seven residues in Y122-HC increase the lumenal Ca 2ϩ affinity and retard the lumenal Ca 2ϩ access to the transport sites in E2P. These mutations also retard markedly the hydrolysis of the Ca 2ϩreleased form of E2P (22,23). Thus, the proper formation of Y122-HC from the seven residues is critical for both Ca 2ϩ release Tyr 122 -hydrophobic Cluster of SERCA1a for Ca 2؉ Release OCTOBER 24, 2008 • VOLUME 283 • NUMBER 43 into lumen from E2PCa 2 (reducing the Ca 2ϩ affinity and opening the lumenal gate), and formation of the E2P catalytic site for the subsequent Asp 351 -acylphosphate hydrolysis. The formation of Y122-HC therefore functions critically for realizing and stabilizing the compactly organized and thus distorted structure of the Ca 2ϩreleased form of E2P. The stabilization of this state is certainly important for making the time period long enough for Ca 2ϩ release into lumen and likely for proton bindings to the empty Ca 2ϩ sites, and for the fine rearrangement of the catalytic site for the subsequent Asp 351 -acylphosphate hydrolysis.
As shown in Fig. 10 and supplemental Fig. S4, the extents of the mutational effects on the lumenal Ca 2ϩ affinities and on the FIGURE 9. Kinetics of lumenal Ca 2؉ -induced change of E2P to E1PCa 2 in the wild type in the presence of K ؉ without ADP. A, the microsomes expressing the wild type were phosphorylated with 32 P i in the presence of A23187 and absence of Ca 2ϩ and chilled on ice, as described in Fig. 7. Subsequently, the phosphorylated sample was mixed at 0°C with a 20-fold volume of chase solution containing 105 mM KCl and various concentrations of CaCl 2 without ADP, otherwise as described in Fig. 7. The final free Ca 2ϩ concentrations were indicated in the figure. At the indicated time periods after this addition, the chase reaction was terminated by trichloroacetic acid, and the amount of EP was determined. Solid lines show the least squares fit to a double exponential decay. The EP remaining in the second and slow phase was all in the ADP-sensitive form (thus E1PCa 2 , data not shown), and the first and rapid phase is the forward hydrolysis of E2P without bound Ca 2ϩ . B, the fraction of EP in the second phase in the total amount of EP was obtained by extrapolating to the zero time in the double exponential decay fitting, and plotted versus the Ca 2ϩ concentration. The data were fitted well with the Hill equation (solid line), and the Ca 2ϩ concentration giving the 50% saturation and the Hill coefficient were found to be 750 M and 1.5. Here note that the EP amount in the second phase is dependent on the ratio between the rate of the forward E2P hydrolysis and that of the reverse E2P to E1PCa 2 conversion upon the lumenal Ca 2ϩ binding to E2P: the plot reflects the relative values between these forward and reverse rates of E2P rather than the lumenal Ca 2ϩ affinity of E2P. C, by using the data obtained in A and B with the wild type in the presence of K ϩ , the rate of the lumenal Ca 2ϩ -induced E1PCa 2 formation from E2P (k rev (E)) was calculated at each Ca 2ϩ concentration by the equation, k rev ϭ k h F s /(1 Ϫ F s ). Here, F s is the fraction of EP in the second phase, and k h is the forward hydrolysis rate of E2P without Ca 2ϩ . For comparison with the wild type in the absence of K ϩ (F) and Y122A in the presence of K ϩ (‚), their lumenal Ca 2ϩ access rates (the rates of the lumenal Ca 2ϩ -induced reverse E2P decay via E1PCa 2 in the presence of ADP) obtained in Fig. 8A are plotted. Ϫ bound at the phosphorylation site Asp 351 , and the bound potassium ion are shown by van der Waals spheres. The seven residues in Y122-HC are colored differently based on the strength of the retardation of the E2P hydrolysis rate (lower panel) and that of the increase in the lumenal Ca 2ϩ affinity (upper panel). The color changes gradually from red for the strongest effects to blue for weakening.
E2P hydrolysis rates varied significantly among the seven Y122-HC mutants and depended on their positions. The residues of which mutation exhibited the strongest effects on increasing lumenal Ca 2ϩ affinity were Leu 119 , Tyr 122 , and Leu 180 . This agrees with the critical role of M2 for rearrangement of the transmembrane helices for the Ca 2ϩ release; i.e. the tight association of the top part of M2 with the largely rotating A domain in Y122-HC functions for the lever-like inclination of M2 to push the lumenal part of M4 to open the lumenal gate (14). In fact, in E2PCa 2 trapped by the elongation of the A/M1linker, the Leu 119 /Tyr 122 region on the top part of M2 is not involved fully in Y122-HC (9).
The residues of which mutations exhibited the strongest retardation of the E2P hydrolysis were Ile 232 at the top part of the A/M3-linker and, again, Leu 119 and Tyr 122 on the A/M2linker (top part of M2). Thus these residues on the linkers seem to contribute most critically to produce the proper configuration of the catalytic site. Consistently, the proteolytic cleavage at Leu 119 on the A/M2-linker causes a marked inhibition of the E2P hydrolysis (43). The structural changes producing the Ca 2ϩ release may be transmitted to the catalytic site via these residues of Y122-HC on the linkers, thereby ensuring the E2P hydrolysis to occur after the Ca 2ϩ release. In any case, the different degree of the contributions of the seven residues of Y122-HC to the Ca 2ϩ release and subsequent formation of the E2P catalytic site may suggest a possible sequential gathering of the seven residues. This possibility will be discussed more in the last section of "Discussion" in relation to the crystal structure E2⅐BeF 3 Ϫ (17,18).

Structural Mechanism Involving Y122-HC and Other Critical Elements-In E1Ca 2 ⅐AlF 4
Ϫ ⅐ADP 3 E2⅐MgF 4 2Ϫ as an overall structural change, including the EP isomerization and Ca 2ϩ release (supplemental Fig. S5A), the A domain largely rotates and M2 largely inclines. Also the P domain markedly inclines toward the lower side of the A domain and rotates by ϳ20°a round the phosphorylation site (Asp 351 ) parallel to the membrane and in the opposite direction of the A-domain rotation. These motions involve (can be dissected into) the horizontal and vertical factors, parallel and perpendicular to the membrane plane. As a consequence of the motions, the A and P domains and M2 will come to their appropriate positions producing their tight association at Y122-HC. At the A-P domain interface in the E2P analog structures, there is another interaction network between these domains at the Val 200 loop, Asp 196 -Asp 203 of the A domain (Fig. 1, and (24) that this A-P domain interaction is critical for Ca 2ϩ release from E2PCa 2 and for formation of the E2P catalytic site, thus very similarly to Y122-HC. Then note that, in the E2P analog structures (see supplemental Fig. S5A for E2⅐MgF 4 2Ϫ ), the two networks at Y122-HC and at Val 200 are located at each side of the A-P domain interface on its top view and at the bottom and upper parts of the interface, respectively on its side view. Thus the two are situated horizontally and vertically with the specific relative positioning. It is very likely that this positioning of the two is most efficiently functioning to realize and stabilize the compactly organized and distorted structure of the Ca 2ϩ -re-leased E2P: i.e. the interactions at the two positions are most appropriate to produce the horizontal and vertical motions of the P and A domains and M2 required for Ca 2ϩ release from E2PCa 2 and to stabilize the Ca 2ϩ -released E2P state. Certainly these motions cause the rearrangements in the transmembrane helices for Ca 2ϩ release: e.g. the P-domain inclination with slight rotation is directly associated with the bending and slight rotation of connected M4/M5 and downward movement of M4, thus their twisting-like motion. The largely inclining M2 pushes the lumenal part of M4 (supplemental Fig. S5B). Hence the Ca 2ϩ sites are destroyed, and the lumenal gate is opened.
It should be noted that, for the loss of the ADP sensitivity E1PCa 2 3 E2PCa 2 , the large rotation of the A domain and its docking onto the P domain should occur so as to bring the T 181 GES loop above Asp 351 -acylphosphate to block the ADP access from the N domain. As the motive force of this large A-domain rotation approximately parallel to membrane plane, the strain imposed on the A/M3-linker in E1PCa 2 was predicted to be critical (13,14,20,44). Also, the sufficiently long length of the A/M1-linker was revealed to be critical for this EP isomerization, in this case, probably for realizing the E2PCa 2 structure, in which the A domain is positioned above the P domain (9,45). For the subsequent Ca 2ϩ release in E2PCa 2 3 E2P ϩ 2Ca 2ϩ , the A/M1-linker with its appropriately short length (therefore its strain) is critical (9). Actually, the elongation of this linker blocks completely Ca 2ϩ deocclusion/release from E2PCa 2 , thus trapping this E2PCa 2 state in which Y122-HC is not properly formed yet in contrast to its proper formation in the Ca 2ϩ -released form of E2P with the lumenally opened normal Ca 2ϩ release pathway (9). The results clearly demonstrated that the native and appropriately short length of A/M1-linker functions critically in inducing the motions from the E2PCa 2 state, especially inclination of the A and P domains and M2, to accomplish the Y122-HC formation and the Ca 2ϩ deocclusion/release from E2PCa 2 . During the Y122-HC formation, the interaction force being produced in Y122-HC will likely function to induce the final process of the vertical and horizontal motions of the P and A domain and M2 to realize and stabilize the Ca 2ϩ -released E2P structure (supplemental Fig. S6). Importantly also, the E2P catalytic site is produced by these rearrangements. In this mechanism, a possible hydrolysis of Asp 351 -acylphosphate without releasing Ca 2ϩ will be avoided; thereby the ordered reaction sequence of the Ca 2ϩ release from E2PCa 2 and the subsequent E2P hydrolysis will be accomplished for the energy coupling.
Possible Structural Role of K ϩ for Reducing Ca 2ϩ Affinity and Lumenal Gating-K ϩ is known to markedly accelerate the E2P hydrolysis (35,36) and also to modulate the E2 to E1Ca 2 transition in the non-phosphorylated Ca 2ϩ -ATPase (46,47). In the present study, we further found that the K ϩ binding is important for reducing the affinity for Ca 2ϩ and lumenal gating thus for Ca 2ϩ release from E2PCa 2 . In the crystal structure E1Ca 2 ⅐AlF 4 Ϫ ⅐ADP, K ϩ is situated at the bottom part of the P domain and coordinated by the backbone carbonyls of the loop Leu 711 -Glu 715 and by the Glu 732 side chain (Fig. 11). The K ϩ binding at this site was indeed previously found by the mutations to be critical for the stimulation of the E2P hydrolysis (37). In the structures E2P analogs and E2(TG), this K ϩ site of the P domain comes very close to the A/M3-linker, and actually K ϩ at this site is further coordinated by the Gln 244 side chain on the A/M3-linker (see E2⅐AlF 4 Ϫ in Fig. 11). Because the alanine substitution of Gln 244 and those of Glu-Gln-Asp 245 gave virtually no effect on Ca 2ϩ transport activity (48), K ϩ at this region may be coordinated by their neighboring residues or backbone carbonyls on the A/M3-linker and thereby perform a structural function. In the present study, we found that the lack of K ϩ binding has the consequences very similar to those of the mutations at Y122-HC. It is therefore possible that the K ϩ binding functions with similar structural effects as Y122-HC to produce the proper structure of the Ca 2ϩ -released form of E2P.
Then note that the K ϩ site of the P domain in E1Ca 2 ⅐AlF 4 Ϫ ⅐ADP is situated at much higher position from the membrane plane than the Gln 244 region on the A/M3-linker (Fig. 11) and that, in the change E1Ca 2 ⅐AlF 4 Ϫ ⅐ADP 3 E2⅐AlF 4 Ϫ (or E2⅐BeF 3 Ϫ and E2⅐MgF 4 2Ϫ ), the P domain with connected M4/M5 largely inclines toward the A domain, hence the K ϩ site with bound K ϩ on the P domain moves down to the Gln 244 region on the A/M3-linker to make contact. The interactions between the bottom part of the P domain and the A/M3-linker via bound K ϩ thus produced would likely cross-link them and hence contribute to formation and stabilization of this compactly organized Ca 2ϩ -released structure of E2P with the reduced Ca 2ϩ affinity and lumenally opened gate. Alternatively, it is also possible that the appropriate P-domain structure produced by K ϩ binding on this domain solely contributes to the formation of the Ca 2ϩ -released E2P structure.

Y122-HC in Crystal Structure of E2⅐BeF 3
Ϫ -The crystal structures of E2⅐BeF 3 Ϫ , the analog for the E2P ground state (21), were solved at the atomic level very recently with and without bound thapsigargin, TG (E2BeF 3 Ϫ and E2BeF 3 Ϫ (TG) (17,18)). Surprisingly, in this crystallized E2⅐BeF 3 Ϫ , the side chains of Ile 119 and Tyr 122 are somewhat pointing away from the clustered other five residues on the A and P domains (Ile 179 /Leu 180 /Ile 232 and Val 705 /Val 726 ), although all these seven residues are closely located in the E2⅐BeF 3 Ϫ structures of both 2ZBE (17) and 3B9B (18). On the other hand, Y122-HC is formed fully from all these seven residues in E2BeF 3 Ϫ (TG) as well as in the other E2P anal-ogous structures, E2⅐AlF 4 Ϫ and E2⅐MgF 4 2Ϫ . Thus, the assembling manner of the seven residues in the crystal structure E2⅐BeF 3 Ϫ seemingly conflicts with our results that the gathering of all the seven residues, including Tyr 122 /Ile 119 in Y122-HC, is required for producing the Ca 2ϩreleased E2P. Furthermore, Tyr 122 and Ile 119 on the top part of M2 (A/M2-linker) are likely most critical in Y122-HC and play central roles (Fig. 10). Our previous biochemical structural analysis of SR Ca 2ϩ -ATPase in solution by the proteolysis and the lumenal Ca 2ϩ accessibility demonstrated (21) that, in E2⅐BeF 3 Ϫ without TG, Leu 119 /Tyr 122 are surely gathered and involved in Y122-HC, and thereby the lumenal gate is opened and the lumenal Ca 2ϩ is accessible to the transport sites. Thus the crystal structure E2⅐BeF 3 Ϫ seems to conflict also with these biochemical results obtained in solution.
Nevertheless, as a comprehensive idea, the crystal structure of E2⅐BeF 3 Ϫ may be consistent with (or indicative of) the view that the gathering of the seven residues to form Y122-HC upon motions of the A and P domains and M2 (A/M2-linker) occurs in some ordered sequence but not necessarily at once (see Fig.  10 and under "Discussion"). Most peculiar to us is that Tyr 122 and Leu 119 , of which mutations exhibited the most inhibitory effects, are not involved yet in the hydrophobic cluster in the crystal structure E2⅐BeF 3 Ϫ . Here note that, in the structure E2⅐BeF 3 Ϫ , a Mg 2ϩ ion is bound near the Ca 2ϩ binding sites in the transmembrane domain because an extremely high Mg 2ϩ concentration employed for crystallization (18), or protonation on the residues of transmembrane helices, including Ca 2ϩ ligands, must have occurred as in low pH for crystallization (17). Thus, these ligations are probably involved critically in the stabilization of the transmembrane helices for the crystallization. This might mean that the transmembrane structure thus stabilized differs from that without any ligations, i.e. the state immediate after the Ca 2ϩ release (the empty Ca 2ϩ sites) that is realized by the contribution of Y122-HC. Therefore, Y122-HC is, in return, disrupted or not properly produced yet in the crystal structure E2⅐BeF 3 Ϫ as if it occurs with the lumenal Ca 2ϩ binding in the E2PCa 2 state as postulated in this study. Therefore the following sequential gathering of the seven residues to produce Y122-HC can be speculated: The five hydrophobic residues on the A domain (Ile 179 /Leu 180 /Ile 232 ) and P domain (Val 705 / Val 726 ) are first gathered through the motions of the A and P domains and top part of M2 (A/M2-linker), and subsequently, the top part of M2, including Ile 119 and Tyr 122 , makes further motions during the final process of the M2 inclination to join them and produce the fully assembled Y122-HC, thereby to realize and stabilize fully the gathered state of the A and P domains and top part of M2 (A/M2-linker) as in the Ca 2ϩreleased form of E2P. Being in agreement with this view, in E2PCa 2 trapped by the elongation of the A/M1-linker, Leu 119 / Ϫ ⅐ADP (E1ϳPCa 2 ⅐ADP analog, left) and E2⅐AlF 4 Ϫ (E2ϳP analog, right) around Y122-HC and the bound K ϩ ion are shown in schematic models (PDB codes: 1T5T and 1XP5 (12,15)). The two structures were manually aligned with M8 -M10 helices, which do not move virtually in the two. K ϩ bound in these structures is shown by a yellow van der Waals sphere. Gln 244 on the A/M3-linker at the immediate vicinity of the bound K ϩ in E2⅐AlF 4 Ϫ is indicated by ball and stick model.
Tyr 122 on the top part of M2 is not fully involved yet in Y122-HC (9). The Ca 2ϩ -released and empty Ca 2ϩ sites (without any protonation and stabilization immediately after the Ca 2ϩ release) will be subsequently protonated producing the E2P ground state for its hydrolysis.
Alternatively, if a possible contribution of such ligation in the transmembrane domain (Mg 2ϩ or protonation) should not be concerned in the crystallization of E2⅐BeF 3 Ϫ , the followings might be possible: first of all, the arrangements of helices of the Ca 2ϩ -released empty transport sites must be unstable, for example, due to possible repulsions between the negative charges of the Ca 2ϩ ligands. Then to relieve the instability, the most effective gathering of Tyr 122 /Leu 119 in Y122-HC, which produces and stabilizes the Ca 2ϩ -released empty state, might possibly be disrupted; thereby the helices may be rearranged so as to form the more stabilized arrangements that can be crystallized.