The structural natures of stable analogues for the ADP-insensitive phosphoenzyme (E2P) of Ca 2؉ -ATPase formed in sarcoplasmic reticulum vesicles, i.e. the enzymes with bound beryllium fluoride (BeF⅐E2), bound aluminum fluoride (AlF⅐E2), and bound magnesium fluoride (MgF⅐E2), were explored and compared with those of actual E2P formed from P i without Ca 2؉ . Changes in trinitrophenyl-AMP fluorescence revealed that the catalytic site is strongly hydrophobic in BeF⅐E2 as in E2P but hydrophilic in MgF⅐E2 and AlF⅐E2; yet, the three cytoplasmic domains are compactly organized in these states. Thapsigargin, which was shown in the crystal structure to fix the transmembrane helices and, thus, the postulated Ca 2؉ release pathway to lumen in a closed state, largely reduced the tryptophan fluorescence in BeF⅐E2 as in E2P, but only very slightly (hence, the release pathway is likely closed without thapsigargin) in MgF⅐E2 and AlF⅐E2 as in dephosphorylated enzyme. Consistently, the completely suppressed Ca 2؉ -ATPase activity in BeF-treated vesicles was rapidly restored in the presence of ionophore A23187 but not in its absence by incubation with Ca 2؉ (over several millimolar concentrations) at pH 6, and, therefore, lumenal Ca 2؉ is accessible to reactivate the enzyme. In contrast, no or only very slow restoration was observed with vesicles treated with MgF and AlF even with A23187. BeF⅐E2 thus has the features very similar to those characteristic of the E2P ground state, although AlF⅐E2 and MgF⅐E2 most likely mimic the transition or product state for the E2P hydrolysis, during which the hydrophobic nature around the phosphorylation site is lost and the Ca 2؉ release pathway is closed. The change in hydrophobic nature is probably associated with the change in phosphate geometry from the covalently bound tetrahedral ground state (BeF 3 ؊ ) to trigonal bipyramidal transition state (AlF 3 or AlF 4 ؊ ) and further to tetrahedral product state (MgF 4 2؊ ), and such change likely rearranges transmembrane helices to prevent access and leakage of lumenal Ca 2؉ .Sarcoplasmic reticulum (SR) 1 Ca 2ϩ -ATPase is a representative member of P-type ion-transporting ATPases and catalyzes Ca 2ϩ transport coupled with ATP hydrolysis (Fig. 1) (Refs. 1 and 2 and, for recent reviews, see Refs. 3-6). In the catalytic cycle, the enzyme is activated by the binding of two Ca 2ϩ ions (E2 to Ca 2 E1, steps 1-2) and then autophosphorylated at Asp 351 by MgATP to form ADP-sensitive phosphoenzyme (E1P, steps 3-4). The subsequent isomeric transition to the ADPinsensitive form (E2P) will result in a reduction in affinity, a change in orientation of the Ca 2ϩ binding sites, and Ca 2ϩ release into lumen (steps 5-6). Finally, hydrolysis takes place and returns the enzyme into an unphosphorylated and Ca 2ϩ -unbound form (E2, steps 7-8). E2P can also be formed from P i in the presence of Mg 2ϩ and the absence of Ca 2ϩ by reversal of its hydrolysis, and the subsequent addition of high concentrations of Ca 2ϩ (from the lumenal side) can readily reverse the Ca 2ϩ ...

In order to characterize the domain organization of sarcoplasmic reticulum Ca 2+ -ATPase in different physiological states, limited proteolysis using three proteases (proteinase K (prtK), V8 and trypsin) was conducted systematically and quantitatively. The differences between E 2 and E 2 P were examined in our previous study and E 2 P was characterized by the complete resistance to all three proteases (except for trypsin attack at the very top of the molecule (T1 site)). The same strategies were employed in this study for E 1 ATP, E 1 PADP and E 1 P states. Because of the transient nature of these states, they were either stabilized by non-hydrolyzable analogues or made predominant by adjusting buffer conditions. Aluminum fluoride (without ADP) was found to stabilize E 1 P. All these states were characterized by strong (E 1 ATP) to complete (E 1 PADP and E 1 P) resistance to prtK and to V8 but only weak resistance to trypsin at the T2 site. Because prtK and V8 primarily attack the loops connecting the A domain to the transmembrane helices whereas the trypsin T2 site (Arg 198 ) is located on the outermost loop in the A domain, these results lead us to propose that the A domain undergoes a large amount of rotation between E 1 P and E 2 P. Combined with previous results, we demonstrated that four states can be clearly distinguished by the susceptibility to three proteases, which will be very useful for establishing the conditions for structural studies. ß

2ϩ -release process, E2PCa 2 has been postulated (e.g. see Ref. 8), although this state has never been identified. Finally, the E2P hydrolysis takes place and returns the enzyme into an unphosphorylated and Ca 2ϩ -unbound form (E2, steps 6 and 7). The transport cycle is totally reversible, e.g. E2P can be formed from P i in the presence of Mg 2ϩ and the absence of Ca 2ϩ by reversal of its hydrolysis, and the subsequent addition of high concentrations of Ca 2ϩ to E2P reverse the Ca 2ϩ -releasing step and the E1P to E2P isomerization.The enzyme has three cytoplasmic domains as follows: the nucleotide binding (N), phosphorylation (P), and actuator (A) domains, and 10 transmembrane helices M1-M10 (Fig. 2). During the EP isomerization/Ca 2ϩ -release E1PCa 2 3 E2P ϩ 2Ca 2ϩ , the A domain largely rotates (by ϳ110°) parallel to the membrane and associates with the P domain (see Refs. 9 -17) (see E1⅐AlF x ⅐ADP (the E1PCa 2 ⅐ADP analog) 3 E2⅐MgF 4 2Ϫ (the E2⅐P i analog) in Fig. 2). The interactions of the A domain with the P domain in the E2P state occur at three regions (Fig. 2, semitransparent

Sarcoplasmic reticulum Ca 2+ -ATPase was digested with proteinase K, V8 protease and trypsin in the absence of Ca 2+ . Unphosphorylated enzyme was rapidly degraded. In contrast, ADP-insensitive phosphoenzyme formed with P i and phosphorylated state analogues produced by the binding of F 3 or orthovanadate, were almost completely resistant to the proteolysis except for tryptic cleavage at the T1 site (Arg 505 ). The results indicate that the phosphoenzyme and its analogues have a very compact form in the cytoplasmic region, being consistent with large domain motions (gathering of three cytoplasmic domains). Results further show that the structure of the enzyme with bound decavanadate is very similar to ADP-insensitive phosphoenzyme. Thapsigargin did not affect the changes in digestion time course induced by the formation of the phosphorylated state analogues. ß

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