Takashi Daiho

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.

Quasi-irreversible inactivation of the sarcoplasmic reticulum Ca2+-ATPase by simultaneous tight binding of magnesium and fluoride to the catalytic site

The sarcoplasmic reticulum Ca(2+)-ATPase was inactivated quasi-irreversibly by the treatment with KF in the presence of Mg2+ and absence of Ca2+. This inactivation was Mg(2+)-dependent, and prevented by high-affinity Ca2+ binding. The enzyme was completely protected by ATP against the inactivation with an affinity consistent with that of the catalytic site for ATP. The affinity for Mg2+ in this inactivation was in agreement with that for Mg2+ in phosphorylation of the enzyme with Pi. Mg.ATP did not bind to the inactivated enzyme, whereas metal-free ATP did bind to it with a high affinity. These findings suggest that the Mg2+ binding sub-site in the catalytic site of the inactivated enzyme is occupied by tightly-bound Mg2+. The enzyme was completely protected by Pi against the inactivation with an affinity consistent with that of the catalytic site for Pi. The inactivated enzyme showed neither phosphorylation with Pi nor high-affinity vanadate binding. These findings suggest that the phosphorylation site of the inactivated enzyme is occupied by tightly-bound F-. The contents of tightly-bound Mg2+ and F- in the inactivated enzyme were determined after unbound Mg2+ and F- were removed by gel filtration. 2.3 mol of Mg2+ and 3.7 mol of F- per mol of phosphorylation sites were tightly bound to the enzyme. The tight binding of these ligands depended on the presence of each other, and was completely prevented by high-affinity Ca2+ binding. Linear relationships were found between the contents of the tightly-bound ligands and the extent of the enzyme inactivation. The tightly-bound Mg2+ and F- were entirely released by low-affinity Ca2+ binding, and correspondingly the ATPase activity was restored. It is concluded that the observed enzyme inactivation is caused by simultaneous tight binding of Mg2+ and F- to the catalytic site.

Distinct Types of Abnormality in Kinetic Properties of Three Darier Disease-causing Sarco(endo)plasmic Reticulum Ca2+-ATPase Mutants That Exhibit Normal Expression and High Ca2+ Transport Activity

The possible functional abnormalities in three different Darier disease-causing Ca2+-ATPase (SERCA2b) mutants, Ile274 → Val at the lumenal end of M3, Leu321 → Phe on the cytoplasmic part of M4, and Met719 → Ile in P domain, were explored, because they exhibited nearly normal expression and localization in COS-1 cells and the high ATPase and coupled Ca2+ transport activities that were essentially identical (L321F) or slightly lower (I274V by ∼35% and M719I by ∼30%) as compared with those of the wild type. These mutations happened to be in Japanese patients found previously by us. Kinetic analyses revealed that each of the mutants possesses distinct types of abnormalities; M719I and L321F possess the 2–3-fold reduced affinity for cytoplasmic Ca2+, whereas I274V possesses the normal high affinity. L321F exhibited also the remarkably reduced sensitivity to the feedback inhibition of the transport cycle by accumulated lumenal Ca2+, as demonstrated with the effect of Ca2+ ionophore on ATPase activity and more specifically with the effects of Ca2+ (up to 50 mm) on the decay of phosphoenzyme intermediates. The results on I274V and M719I suggest that the physiological requirement for Ca2+ homeostasis in keratinocytes to avoid haploinsufficiency is very strict, probably much more than considered previously. The insensitivity to lumenal Ca2+ in L321F likely brings the lumenal Ca2+ to an abnormally elevated level. The three mutants with their distinctively altered kinetic properties will thus likely cause different types of perturbation of intracellular Ca2+ homeostasis, but nevertheless all types of perturbation result in Darier disease. It might be possible that the observed unique feature of L321F could possibly be associated with the specific symptoms in the pedigree with this mutation, neuropsychiatric disorder, and behavior problems. The results also provided further insight into the global nature of conformational changes of SERCAs for ATP-driven Ca2+ transport.

Mutations of Arg198 in sarcoplasmic reticulum Ca2+‐ATPase cause inhibition of hydrolysis of the phosphoenzyme intermediate formed from inorganic phosphate

Arg198 of sarcoplasmic reticulum Ca2+‐ATPase was substituted with lysine, glutamine, glutamic acid, alanine, and isoleucine by site‐directed mutagenesis. Kinetic analysis was performed with microsomal membranes isolated from COS‐1 cells which were transfected with the mutated cDNAs. The rate of dephosphorylation of the ADP‐insensitive phosphoenzyme was determined by first phosphorylating the Ca2+‐ATPase with 32Pi and then diluting the sample with non‐radioactive Pi. This rate was reduced substantially in the mutant R198Q, more strongly in the mutants R198A and R198I, and most strongly in the mutant R198E, but to a much lesser extent in R198K. The reduction in the rate of dephosphorylation was consistent with the observed decrease in the turnover rate of the Ca2+‐ATPase accompanied by the steady‐state accumulation of the ADP‐insensitive phosphoenzyme formed from ATP. These results indicate that the positive charge and high hydrophilicity of Arg198 are critical for rapid hydrolysis of the ADP‐insensitive phosphoenzyme.

Deletions or Specific Substitutions of a Few Residues in the NH2-terminal Region (Ala3 to Thr9) of Sarcoplasmic Reticulum Ca2+-ATPase Cause Inactivation and Rapid Degradation of the Enzyme Expressed in COS-1 Cells

Amino acid residues in the NH2-terminal region (Glu2 – Ala14) of adult fast twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) were deleted or substituted, and the mutants were expressed in COS-1 cells. Deletion of any single residue in the Ala3–Ser6 region or deletion of two or more consecutive residues in the Ala3–Thr9 region caused strongly reduced expression. Substitution mutants A4K, A4D, and H5K also showed very low expression levels. Deletion of any single residue in the Ala3–Ser6 region caused only a small decrease in the specific Ca2+ transport rate/mg of SERCA1a protein. In contrast, other mutants showing low expression levels had greatly reduced specific Ca2+ transport rates. In vitroexpression experiments indicated that translation, transcription, and integration into the microsomal membranes were not impaired in the mutants that showed very low expression levels in COS-1 cells. Pulse-chase experiments using [35S]methionine/cysteine labeling of transfected COS-1 cells demonstrated that degradation of the mutants showing low expression levels was substantially faster than that of the wild type. Lactacystin, a specific inhibitor of proteasome, inhibited the degradation accelerated by single-residue deletion of Ala3. These results suggest that the NH2-terminal region (Ala3 –Thr9) of SERCA1a is sensitive to the endoplasmic reticulum-mediated quality control and is thus critical for either correct folding of the SERCA1a protein or stabilization of the correctly folded SERCA1a protein or both.