Stefania Danko

Organization of cytoplasmic domains of sarcoplasmic reticulum Ca2+‐ATPase in E1P and E1ATP states: a limited proteolysis study

In order to characterize the domain organization of sarcoplasmic reticulum Ca2+‐ATPase in different physiological states, limited proteolysis using three proteases (proteinase K (prtK), V8 and trypsin) was conducted systematically and quantitatively. The differences between E2 and E2P were examined in our previous study and E2P 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 E1ATP, E1PADP and E1P 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 E1P. All these states were characterized by strong (E1ATP) to complete (E1PADP and E1P) 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 (Arg198) 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 E1P and E2P. 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.

ADP-insensitive phosphoenzyme intermediate of sarcoplasmic reticulum Ca2+-ATPase has a compact conformation resistant to proteinase K, V8 protease and trypsin

Sarcoplasmic reticulum Ca2+‐ATPase was digested with proteinase K, V8 protease and trypsin in the absence of Ca2+. Unphosphorylated enzyme was rapidly degraded. In contrast, ADP‐insensitive phosphoenzyme formed with Pi and phosphorylated state analogues produced by the binding of F− or orthovanadate, were almost completely resistant to the proteolysis except for tryptic cleavage at the T1 site (Arg505). 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.

Comprehensive Analysis of Expression and Function of 51 Sarco(endo)plasmic Reticulum Ca2+-ATPase Mutants Associated with Darier Disease

We examined possible defects of sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b) associated with its 51 mutations found in Darier disease (DD) pedigrees, i.e. most of the substitution and deletion mutations of residues reported so far. COS-1 cells were transfected with each of the mutant cDNAs, and the expression and function of the SERCA2b protein was analyzed with microsomes prepared from the cells and compared with those of the wild type. Fifteen mutants showed markedly reduced expression. Among the other 36, 29 mutants exhibited completely abolished or strongly inhibited Ca2+-ATPase activity, whereas the other seven possessed fairly high or normal ATPase activity. In four of the aforementioned seven mutants, Ca2+ transport activity was significantly reduced or almost completely lost, therefore uncoupled from ATP hydrolysis. The other three were exceptional cases as they were seemingly normal in protein expression and Ca2+ transport function, but were found to have abnormalities in the kinetic properties altered by the three mutations, which happened to be in the three DD pedigrees found by us previously (Sato, K., Yamasaki, K., Daiho, T., Miyauchi, Y., Takahashi, H., Ishida-Yamamoto, A., Nakamura, S., Iizuka, H., and Suzuki, H. (2004) J. Biol. Chem. 279, 35595-35603). Collectively, our results indicated that in most cases (48 of 51) DD mutations cause severe disruption of Ca2+ homeostasis by the defects in protein expression and/or transport function and hence DD, but even a slight disturbance of the homeostasis will result in the disease. Our results also provided further insight into the structure-function relationship of SERCAs and revealed critical regions and residues of the enzyme.

Critical Role of Glu40-Ser48 Loop Linking Actuator Domain and First Transmembrane Helix of Ca2+-ATPase in Ca2+ Deocclusion and Release from ADP-insensitive Phosphoenzyme

The functional importance of the length of the A/M1 linker (Glu40-Ser48) connecting the actuator domain and the first transmembrane helix of sarcoplasmic reticulum Ca2+-ATPase was explored by its elongation with glycine insertion at Pro42/Ala43 and Gly46/Lys47. Two or more glycine insertions at each site completely abolished ATPase activity. The isomerization of phosphoenzyme (EP) intermediate from the ADP-sensitive form (E1P) to the ADP-insensitive form (E2P) was markedly accelerated, but the decay of EP was completely blocked in these mutants. The E2P accumulated was therefore demonstrated to be E2PCa2 possessing two occluded Ca2+ ions at the transport sites, and the Ca2+ deocclusion and release into lumen were blocked in the mutants. By contrast, the hydrolysis of the Ca2+-free form of E2P produced from Pi without Ca2+ was as rapid in the mutants as in the wild type. Analysis of resistance against trypsin and proteinase K revealed that the structure of E2PCa2 accumulated is an intermediate state between E1PCa2 and the Ca2+-released E2P state. Namely in E2PCa2, the actuator domain is already largely rotated from its position in E1PCa2 and associated with the phosphorylation domain as in the Ca2+-released E2P state; however, in E2PCa2, the hydrophobic interactions among these domains and Leu119/Tyr122 on the top of second transmembrane helix are not yet formed properly. This is consistent with our previous finding that these interactions at Tyr122 are critical for formation of the Ca2+-released E2P structure. Results showed that the EP isomerization/Ca2+-release process consists of the following two steps: E1PCa2 → E2PCa2 → E2P + 2Ca2+; and the intermediate state E2PCa2 was identified for the first time. Results further indicated that the A/M1 linker with its appropriately short length, probably because of the strain imposed in E2PCa2, is critical for the correct positioning and interactions of the actuator and phosphorylation domains to cause structural changes for the Ca2+ deocclusion and release.

Deletions of Any Single Residues in Glu40-Ser48 Loop Connecting A Domain and the First Transmembrane Helix of Sarcoplasmic Reticulum Ca2+-ATPase Result in Almost Complete Inhibition of Conformational Transition and Hydrolysis of Phosphoenzyme Intermediate

Possible roles of the Glu40-Ser48 loop connecting A domain and the first transmembrane helix (M1) in sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) were explored by mutagenesis. Deletions of any single residues in this loop caused almost complete loss of Ca2+-ATPase activity, while their substitutions had no or only slight effects. Single deletions or substitutions in the adjacent N- and C-terminal regions of the loop (His32-Asn39 and Leu49-Ile54) had no or only slight effects except two specific substitutions of Asn39 found in SERCA2b in Dariers disease pedigrees. All the single deletion mutants for the Glu40-Ser48 loop and the specific Asn39 mutants formed phosphoenzyme intermediate (EP) from ATP, but their isomeric transition from ADP-sensitive EP (E1P) to ADP-insensitive EP (E2P) was almost completely or strongly inhibited. Hydrolysis of E2P formed from Pi was also dramatically slowed in these deletion mutants. On the other hand, the rates of the Ca2+-induced enzyme activation and subsequent E1P formation from ATP were not altered by the deletions and substitutions. The results indicate that the Glu40-Ser48 loop, with its appropriate length (but not with specific residues) and with its appropriate junction to A domain, is a critical element for the E1P to E2P transition and formation of the proper structure of E2P, therefore, most likely for the large rotational movement of A domain and resulting in its association with P and N domains. Results further suggest that the loop functions to coordinate this movement of A domain and the unique motion of M1 during the E1P to E2P transition.

Multiple and distinct effects of mutations of Tyr122, Glu123, Arg324, and Arg334 involved in interactions between the top part of second and fourth transmembrane helices in sarcoplasmic reticulum Ca2+-ATPase: Changes in cytoplasmic domain organization during isometric transition of phosphoenzyme intermediate and subsequent Ca2+ release

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 Ca2+-bound structure of sarcoplasmic reticulum Ca2+-ATPase, Tyr122-Arg324, and Glu123-Arg334 at the top part of second transmembrane helix (M2) connected to the A domain and fourth transmembrane helix (M4) in the P domain. The observed substitution effects indicated that Glu123, Arg334, and Tyr122 contributed to the rapid transition between the Ca2+-unbound and bound states of the unphosphorylated enzyme. Results further showed the more profound inhibitory effects of the substitutions in the M4/P domain (Arg324 and Arg334) upon the isomeric transition of phosphorylated intermediate (EP) (loss of ADP sensitivity) and those in M2/A domain (Tyr122 and Glu123) upon the subsequent processing and hydrolysis of EP. The observed distinct effects suggest that the interactions seen in the Ca2+-bound structure are not functionally important but indicate that Arg334 with its positive charge and Tyr122 with its aromatic ring are critically important for the above distinct steps. On the basis of the available structural information, the results strongly suggest that Arg334 moves downward and forms new interactions with M2 (likely Asn111); 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, Tyr122 most likely functions in the subsequent Ca2+-releasing step to produce hydrophobic interactions at the A-P domain interface formed upon their gathering and thus to produce the Ca2+-released form of EP. During the Ca2+-transport cycle, the four residues seem to change interaction partners and thus contribute to the coordinated movements of the cytoplasmic and transmembrane domains.

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.

Formation of the Stable Structural Analog of ADP-sensitive Phosphoenzyme of Ca2+-ATPase with Occluded Ca2+ by Beryllium Fluoride STRUCTURAL CHANGES DURING PHOSPHORYLATION AND ISOMERIZATION

As a stable analog for ADP-sensitive phosphorylated intermediate of sarcoplasmic reticulum Ca2+-ATPase E1PCa2·Mg, a complex of E1Ca2·BeFx, was successfully developed by addition of beryllium fluoride and Mg2+ to the Ca2+-bound state, E1Ca2. In E1Ca2·BeFx, most probably E1Ca2·BeF3−, two Ca2+ are occluded at high affinity transport sites, its formation required Mg2+ binding at the catalytic site, and ADP decomposed it to E1Ca2, as in E1PCa2·Mg. Organization of cytoplasmic domains in E1Ca2·BeFx was revealed to be intermediate between those in E1Ca2·AlF4− ADP (transition state of E1PCa2 formation) and E2·BeF3−·(ADP-insensitive phosphorylated intermediate E2P·Mg). Trinitrophenyl-AMP (TNP-AMP) formed a very fluorescent (superfluorescent) complex with E1Ca2·BeFx in contrast to no superfluorescence of TNP-AMP bound to E1Ca2·AlFx. E1Ca2·BeFx with bound TNP-AMP slowly decayed to E1Ca2, being distinct from the superfluorescent complex of TNP-AMP with E2·BeF3−, which was stable. Tryptophan fluorescence revealed that the transmembrane structure of E1Ca2·BeFx mimics E1PCa2·Mg, and between those of E1Ca2·AlF4−·ADP and E2·BeF3−. E1Ca2·BeFx at low 50–100 μm Ca2+ was converted slowly to E2·BeF3− releasing Ca2+, mimicking E1PCa2·Mg → E2P·Mg + 2Ca2+. Ca2+ replacement of Mg2+ at the catalytic site at approximately millimolar high Ca2+ decomposed E1Ca2·BeFx to E1Ca2. Notably, E1Ca2·BeFx was perfectly stabilized for at least 12 days by 0.7 mm lumenal Ca2+ with 15 mm Mg2+. Also, stable E1Ca2·BeFx was produced from E2·BeF3− at 0.7 mm lumenal Ca2+ by binding two Ca2+ to lumenally oriented low affinity transport sites, as mimicking the reverse conversion E2P· Mg + 2Ca2+ → E1PCa2·Mg.

Stable Structural Analog of Ca2+-ATPase ADP-insensitive Phosphoenzyme with Occluded Ca2+ Formed by Elongation of A-domain/M1′-linker and Beryllium Fluoride Binding

We have developed a stable analog for the ADP-insensitive phosphoenzyme intermediate with two occluded Ca2+ at the transport sites (E2PCa2) of sarcoplasmic reticulum Ca2+-ATPase. This is normally a transient intermediate state during phosphoenzyme isomerization from the ADP-sensitive to ADP-insensitive form and Ca2+ deocclusion/release to the lumen; E1PCa2 → E2PCa2 → E2P + 2Ca2+. Stabilization was achieved by elongation of the Glu40-Ser48 loop linking the Actuator domain and M1 (1st transmembrane helix) with four glycine insertions at Gly46/Lys47 and by binding of beryllium fluoride (BeFx) to the phosphorylation site of the Ca2+-bound ATPase (E1Ca2). The complex E2Ca2·BeF3− was also produced by lumenal Ca2+ binding to E2·BeF3− (E2P ground state analog) of the elongated linker mutant. The complex was stable for at least 1 week at 25 °C. Only BeFx, but not AlFx or MgFx, produced the E2PCa2 structural analog. Complex formation required binding of Mg2+, Mn2+, or Ca2+ at the catalytic Mg2+ site. Results reveal that the phosphorylation product E1PCa2 and the E2P ground state (but not the transition states) become competent to produce the E2PCa2 transient state during forward and reverse phosphoenzyme isomerization. Thus, isomerization and lumenal Ca2+ release processes are strictly coupled with the formation of the acylphosphate covalent bond at the catalytic site. Results also demonstrate the critical structural roles of the Glu40-Ser48 linker and of Mg2+ at the catalytic site in these processes.

Roles of Interaction between Actuator and Nucleotide Binding Domains of Sarco(endo)plasmic Reticulum Ca2+-ATPase as Revealed by Single and Swap Mutational Analyses of Serine 186 and Glutamate 439

Roles of hydrogen bonding interaction between Ser186 of the actuator (A) domain and Glu439 of nucleotide binding (N) domain seen in the structures of ADP-insensitive phosphorylated intermediate (E2P) of sarco(endo)plasmic reticulum Ca2+-ATPase were explored by their double alanine substitution S186A/E439A, swap substitution S186E/E439S, and each of these single substitutions. All the mutants except the swap mutant S186E/E439S showed markedly reduced Ca2+-ATPase activity, and S186E/E439S restored completely the wild-type activity. In all the mutants except S186E/E439S, the isomerization of ADP-sensitive phosphorylated intermediate (E1P) to E2P was markedly retarded, and the E2P hydrolysis was largely accelerated, whereas S186E/E439S restored almost the wild-type rates. Results showed that the Ser186-Glu439 hydrogen bond stabilizes the E2P ground state structure. The modulatory ATP binding at sub-mm∼mm range largely accelerated the EP isomerization in all the alanine mutants and E439S. In S186E, this acceleration as well as the acceleration of the ATPase activity was almost completely abolished, whereas the swap mutation S186E/E439S restored the modulatory ATP acceleration with a much higher ATP affinity than the wild type. Results indicated that Ser186 and Glu439 are closely located to the modulatory ATP binding site for the EP isomerization, and that their hydrogen bond fixes their side chain configurations thereby adjusts properly the modulatory ATP affinity to respond to the cellular ATP level.