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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We examined possible defects of sarco(endo)plasmic reticulum Ca catalyze Ca 2ϩ transport coupled with ATP hydrolysis (Fig. 1) and play an essential role in maintaining Ca 2ϩ homeostasis in the cytoplasm and endoplasmic reticulum lumen of cells (1-7). SERCAs have three cytoplasmic domains: phosphorylation (P), nucleotide binding (N), and actuator (A) and 10 transmembrane helices (M1-M10 or 11 in the SERCA2b isoform, M11). In the Ca 2ϩ transport cycle, the ATPase is activated by the binding of two Ca 2ϩ ions from the cytoplasm to the transport sites composed of M4, M5, M6, and M8 (E2 3 E1Ca 2 , step 1). Asp 351 in the P domain is then phosphorylated with MgATP to form the phosphorylated intermediate (EP) (step 2). During dephosphorylation of EP, the Ca 2ϩ ions are released into the lumen. In the detailed mechanism, the dephosphorylation process includes the conformational transition of EP associated with Ca 2ϩ release (step 3) and the subsequent hydrolysis of the acylphosphate bond (step 4).The three human SERCA genes encode SERCA isoforms (8 -10). Mutations in the SERCA2 gene (ATP2A2) and the resulting defects in the SERCA2b housekeeping isoform cause an autosomal dominant genetic skin disease, Darier disease (DD) (11,12). Over 100 mutations have been found with the DD pedigrees (11-24). They include many nonsense mutations, and also substitution and deletion mutations of amino acid residues. The mutations are located throughout the SERCA2b molecule and show no "hot spots" on the primary sequence. To understand how each of the substitution and deletion mutations affects SERCA2b protein, a limited number of mutations had been explored (9 by Ahn et al. (25), 10 by Dode et al. (23), 3 by Sato et al. (26) (a total of 20 because of overlap in Refs. 23 and 25)). To provide a comprehensive insight into the molecular basis of DD, as well as to understand the basis for each case of the DD pedigrees, it is necessary to analyze further the many unexplored substitution and deletion mutations. We therefore carried out in this study a comprehensive analysis of the expression and function of most of the DD causing substitution and deletion mutations reported, i.e. the 51 mutations shown in Fig. 2. Our results showed that most of the mutations (48 of the 51) cause severe defects in protein expression and/or Ca 2ϩ transport function. The loss of the transport function was ascribed to markedly reduced ATP hydrolysis or uncoupling from ATP hydrolysis. The remaining three mutations were exceptional in that they exhibited seemingly normal protein expression and

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We explored, by mutational substitutions and kinetic analysis, possible roles of the four residues involved in the hydrogen-bonding or ionic interactions found in the Ca 2؉ -bound structure of sarcoplasmic reticulum Ca 2؉ ; it thus contributes to the inclination of the M4/P domain toward the M2/A domain, which is crucial for the appropriate gathering between the P domain and the largely rotated A domain to cause the loss of ADP sensitivity. On the other hand, Tyr 122 most likely functions in the subsequent Ca 2؉ -releasing step to produce hydrophobic interactions at the A-P domain interface formed upon their gathering and thus to produce the Ca 2؉ -released form of EP. During the Ca 2؉ -transport cycle, the four residues seem to change interaction partners and thus contribute to the coordinated movements of the cytoplasmic and transmembrane domains.Sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA1a) 1 is a representative member of P-type ion-transporting ATPases; it catalyzes Ca 2ϩ transport coupled with ATP hydrolysis ( Fig. 1; Refs. 1 and 2, and for recent reviews, see Refs. 3 and 4). In the catalytic cycle, the enzyme is activated by the binding of two Ca 2ϩ ions (E2 to E1Ca 2 , steps 1 and 2) and then autophosphorylated at Asp 351 by MgATP to form ADP-sensitive phosphoenzyme (E1P, step 3). Upon formation of this EP, the bound Ca 2ϩ ions are occluded in the transport sites. The subsequent isomeric transition to the ADP-insensitive form (E2P) will result in a reduction in affinity and a change in orientation of the Ca 2ϩ -binding sites and, thus, the Ca 2ϩ release into lumen (steps 4 -5). Finally, hydrolysis takes place and returns the enzyme into an unphosphorylated and Ca 2ϩ -unbound form (E2, step 6). 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. The enzyme has three cytoplasmic domains (N, P, and A) which are widely separated in the Ca 2ϩ -bound form (E1Ca 2 ) but are associated in the Ca 2ϩ -unbound and thapsigarginbound form E2(TG) (Refs. 5 and 6; Fig. 2). In E2(TG), the A domain has largely rotated, and the P domain has significantly inclined together with the transmembrane helices M4 and M5 toward A domain to associate with A domain. We showed previously in the proteolysis experiments (7,8) that the large rotation of A domain and its gathering with P and N domains most likely occur during the E1P to E2P transition and Ca 2ϩ release (steps 4 -5) to form the most compactly organized single headpiece in the Ca 2ϩ -released form of E2P; we further suggested that the stabilization energy provided by the intimate contacts between three cytoplasmic domains in E2P will provide energy for moving transmembrane helices and release the bound Ca 2ϩ into lumen. To gain further insight into the energy coupling between cytoplasmic and transmembrane domains, it is crucial to find out the structural elements essential for the changes in the cytoplasmic domain organization and the coordinated movements of transmembrane helices and reveal the actual roles of ...