Electrostatic interactions between single arginine and phospholipids modulate physiological properties of sarcoplasmic reticulum Ca2+-ATPase.

Arg324 of sarcoplasmic reticulum Ca2+-ATPase forms electrostatic interactions with the phosphate moiety of phospholipids in most reaction states, and a hydrogen bond with Tyr122 in other states. Using site-directed mutagenesis, we explored the functional roles of Arg324 interactions, especially those with lipids, which at first glance might seem too weak to modulate the function of such a large membrane protein. The hydrogen bond forms transiently and facilitates Ca2+ binding from the cytoplasmic side. The contributions of the electrostatic interactions to the reaction steps were quantified using a rate vs activity coefficient plot. We found that the interaction between Arg324 and lipids decreases the affinity for luminal Ca2+. The transformation rate of the phosphoenzyme intermediate is facilitated by the electrostatic interactions, and the function of these interactions depends not only on the type but also on the composition of the phospholipids. The properties observed in microsomes could not be reproduced with any single phospholipid, but with a mixture of phospholipids that mimics the native membrane. These results suggest the importance of swapping of the lipid partners of different headgroups in the reaction step. This study shows that Arg324 plays a role in the reaction cycle via complex intra-protein and protein-lipid interactions.

Cryo-EM reveals mechanistic insights into lipid-facilitated polyamine export by human ATP13A2.

The cytoplasmic polyamine maintains cellular homeostasis by chelating toxic metal cations, regulating transcriptional activity, and protecting DNA. ATP13A2 was identified as a lysosomal polyamine exporter responsible for polyamine release into the cytosol, and its dysfunction is associated with Alzheimer's disease and other neural degradation diseases. ATP13A2 belongs to the P5 subfamily of the P-type ATPase family, but its mechanisms remain unknown. Here, we report the cryoelectron microscopy (cryo-EM) structures of human ATP13A2 under four different conditions, revealing the structural coupling between the polyamine binding and the dephosphorylation. Polyamine is bound at the luminal tunnel and recognized through numerous electrostatic and π-cation interactions, explaining its broad specificity. The unique N-terminal domain is anchored to the lipid membrane to stabilize the E2P conformation, thereby accelerating the E1P-to-E2P transition. These findings reveal the distinct mechanism of P5B ATPases, thereby paving the way for neuroprotective therapy by activating ATP13A2.

Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging.

The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) transports Ca2+ ions across the membrane coupled with ATP hydrolysis. Crystal structures of ligand-stabilized molecules indicate that the movement of actuator (A) domain plays a crucial role in Ca2+ translocation. However, the actual structural movements during the transitions between intermediates remain uncertain, in particular, the structure of E2PCa2 has not been solved. Here, the angle of the A-domain was measured by defocused orientation imaging using isotropic total internal reflection fluorescence microscopy. A single SERCA1a molecule, labeled with fluorophore ReAsH on the A-domain in fixed orientation, was embedded in a nanodisc, and stabilized on Ni-NTA glass. Activation with ATP and Ca2+ caused angle changes of the fluorophore and therefore the A-domain, motions lost by inhibitor, thapsigargin. Our high-speed set-up captured the motion during EP isomerization, and suggests that the A-domain rapidly rotates back and forth from an E1PCa2 position to a position close to the E2P state. This is the first report of the detection in the movement of the A-domain as an angle change. Our method provides a powerful tool to investigate the conformational change of a membrane protein in real-time.

Nanodisc-based kinetic assays reveal distinct effects of phospholipid headgroups on the phosphoenzyme transition of sarcoplasmic reticulum Ca2+-ATPase.

Sarco(endo)plasmic reticulum Ca2+-ATPase catalyzes ATP-driven Ca2+ transport from the cytoplasm to the lumen and is critical for a range of cell functions, including muscle relaxation. Here, we investigated the effects of the headgroups of the 1-palmitoyl-2-oleoyl glycerophospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylglycerol (PG) on sarcoplasmic reticulum (SR) Ca2+-ATPase embedded into a nanodisc, a lipid-bilayer construct harboring the specific lipid. We found that Ca2+-ATPase activity in a PC bilayer is comparable with that of SR vesicles and is suppressed in the other phospholipids, especially in PS. Ca2+ affinity at the high-affinity transport sites in PC was similar to that of SR vesicles, but 2-3-fold reduced in PE and PS. Ca2+ on- and off-rates in the non-phosphorylated ATPase were markedly reduced in PS. Rate-limiting phosphoenzyme (EP) conformational transition in 0.1 m KCl was as rapid in PC as in SR vesicles, but slowed in other phospholipids, especially in PS. Using kinetic plots of the logarithm of rate versus the square of mean activity coefficient of solutes in 0.1-1 m KCl, we noted that PC is optimal for the EP transition, but PG and especially PS had markedly unfavorable electrostatic effects, and PE exhibited a strong non-electrostatic restriction. Thus, the major SR membrane lipid PC is optimal for all steps and, unlike the other headgroups, contributes favorable electrostatics and non-electrostatic elements during the EP transition. Our analyses further revealed that the surface charge of the lipid bilayer directly modulates the transition rate.

Membrane Perturbation of ADP-insensitive Phosphoenzyme of Ca2+-ATPase Modifies Gathering of Transmembrane Helix M2 with Cytoplasmic Domains and Luminal Gating.

Ca2+ transport by sarcoplasmic reticulum Ca2+-ATPase involves ATP-dependent phosphorylation of a catalytic aspartic acid residue. The key process, luminal Ca2+ release occurs upon phosphoenzyme isomerization, abbreviated as E1PCa2 (reactive to ADP regenerating ATP and with two occluded Ca2+ at transport sites) → E2P (insensitive to ADP and after Ca2+ release). The isomerization involves gathering of cytoplasmic actuator and phosphorylation domains with second transmembrane helix (M2), and is epitomized by protection of a Leu119-proteinase K (prtK) cleavage site on M2. Ca2+ binding to the luminal transport sites of E2P, producing E2PCa2 before Ca2+-release exposes the prtK-site. Here we explore E2P structure to further elucidate luminal gating mechanism and effect of membrane perturbation. We find that ground state E2P becomes cleavable at Leu119 in a non-solubilizing concentration of detergent C12E8 at pH 7.4, indicating a shift towards a more E2PCa2-like state. Cleavage is accelerated by Mg2+ binding to luminal transport sites and blocked by their protonation at pH 6.0. Results indicate that possible disruption of phospholipid-protein interactions strongly favors an E2P species with looser head domain interactions at M2 and responsive to specific ligand binding at the transport sites, likely an early flexible intermediate in the development towards ground state E2P.

Glycine 105 as Pivot for a Critical Knee-like Joint between Cytoplasmic and Transmembrane Segments of the Second Transmembrane Helix in Ca2+-ATPase.

The cytoplasmic actuator domain of the sarco(endo)plasmic reticulum Ca2+-ATPase undergoes large rotational movements that influence the distant transmembrane transport sites, and a long second transmembrane helix (M2) connected with this domain plays critical roles in transmitting motions between the cytoplasmic catalytic domains and transport sites. Here we explore possible structural roles of Gly105 between the cytoplasmic (M2c) and transmembrane (M2m) segments of M2 by introducing mutations that limit/increase conformational freedom. Alanine substitution G105A markedly retards isomerization of the phosphoenzyme intermediate (E1PCa2 → E2PCa2 → E2P + 2Ca2+), and disrupts Ca2+ occlusion in E1PCa2 and E2PCa2 at the transport sites uncoupling ATP hydrolysis and Ca2+ transport. In contrast, this substitution accelerates the ATPase activation (E2 → E1Ca2). Introducing a glycine by substituting another residue on M2 in the G105A mutant (i.e. "G-shift substitution") identifies the glycine positions required for proper Ca2+ handling and kinetics in each step. All wild-type kinetic properties, including coupled transport, are fully restored in the G-shift substitution at position 112 (G105A/A112G) located on the same side of the M2c helix as Gly105 facing M4/phosphorylation domain. Results demonstrate that Gly105 functions as a flexible knee-like joint during the Ca2+ transport cycle, so that cytoplasmic domain motions can bend and strain M2 in the correct direction or straighten the helix for proper gating and coupling of Ca2+ transport and ATP hydrolysis.

Assembly of a Tyr122 Hydrophobic Cluster in Sarcoplasmic Reticulum Ca2+-ATPase Synchronizes Ca2+ Affinity Reduction and Release with Phosphoenzyme Isomerization.

The mechanism whereby events in and around the catalytic site/head of Ca(2+)-ATPase effect Ca(2+) release to the lumen from the transmembrane helices remains elusive. We developed a method to determine deoccluded bound Ca(2+) by taking advantage of its rapid occlusion upon formation of E1PCa2 and of stabilization afforded by a high concentration of Ca(2+). The assay is applicable to minute amounts of Ca(2+)-ATPase expressed in COS-1 cells. It was validated by measuring the Ca(2+) binding properties of unphosphorylated Ca(2+)-ATPase. The method was then applied to the isomerization of the phosphorylated intermediate associated with the Ca(2+) release process E1PCa2 → E2PCa2 → E2P + 2Ca(2+). In the wild type, Ca(2+) release occurs concomitantly with EP isomerization fitting with rate-limiting isomerization (E1PCa2 → E2PCa2) followed by very rapid Ca(2+) release. In contrast, with alanine mutants of Leu(119) and Tyr(122) on the cytoplasmic part of the second transmembrane helix (M2) and Ile(179) on the A domain, Ca(2+) release in 10 µm Ca(2+) lags EP isomerization, indicating the presence of a transient E2P state with bound Ca(2+). The results suggest that these residues function in Ca(2+) affinity reduction in E2P, likely via a structural rearrangement at the cytoplasmic part of M2 and a resulting association with the A and P domains, therefore leading to Ca(2+) release.

Second transmembrane helix (M2) and long range coupling in Ca²âº-ATPase.

The actuator (A) domain of sarco(endo)plasmic reticulum Ca(2+)-ATPase not only plays a catalytic role but also undergoes large rotational movements that influence the distant transport sites through connections with transmembrane helices M1 and M2. Here we explore the importance of long helix M2 and its junction with the A domain by disrupting the helix structure and elongating with insertions of five glycine residues. Insertions into the membrane region of M2 and the top junctional segment impair Ca(2+) transport despite reasonable ATPase activity, indicating that they are uncoupled. These mutants fail to occlude Ca(2+). Those at the top segment also exhibited accelerated phosphoenzyme isomerization E1P → E2P. Insertions into the middle of M2 markedly accelerate E2P hydrolysis and cause strong resistance to inhibition by luminal Ca(2+). Insertions along almost the entire M2 region inhibit the dephosphorylated enzyme transition E2 → E1. The results pinpoint which parts of M2 control cytoplasm gating and which are critical for luminal gating at each stage in the transport cycle and suggest that proper gate function requires appropriate interactions, tension, and/or rigidity in the M2 region at appropriate times for coupling with A domain movements and catalysis.

Roles of long-range electrostatic domain interactions and K+ in phosphoenzyme transition of Ca2+-ATPase.

Sarcoplasmic reticulum Ca(2+)-ATPase couples the motions and rearrangements of three cytoplasmic domains (A, P, and N) with Ca(2+) transport. We explored the role of electrostatic force in the domain dynamics in a rate-limiting phosphoenzyme (EP) transition by a systematic approach combining electrostatic screening with salts, computer analysis of electric fields in crystal structures, and mutations. Low KCl concentration activated and increasing salt above 0.1 m inhibited the EP transition. A plot of the logarithm of the transition rate versus the square of the mean activity coefficient of the protein gave a linear relationship allowing division of the activation energy into an electrostatic component and a non-electrostatic component in which the screenable electrostatic forces are shielded by salt. Results show that the structural change in the transition is sterically restricted, but that strong electrostatic forces, when K(+) is specifically bound at the P domain, come into play to accelerate the reaction. Electric field analysis revealed long-range electrostatic interactions between the N and P domains around their hinge. Mutations of the residues directly involved and other charged residues at the hinge disrupted in parallel the electric field and the structural transition. Favorable electrostatics evidently provides a low energy path for the critical N domain motion toward the P domain, overcoming steric restriction. The systematic approach employed here is, in general, a powerful tool for understanding the structural mechanisms of enzymes.

Ca2+ release to lumen from ADP-sensitive phosphoenzyme E1PCa2 without bound K+ of sarcoplasmic reticulum Ca2+-ATPase.

During Ca(2+) transport by sarcoplasmic reticulum Ca(2+)-ATPase, the conformation change of ADP-sensitive phosphoenzyme (E1PCa(2)) to ADP-insensitive phosphoenzyme (E2PCa(2)) is followed by rapid Ca(2+) release into the lumen. Here, we find that in the absence of K(+), Ca(2+) release occurs considerably faster than E1PCa(2) to E2PCa(2) conformation change. Therefore, the lumenal Ca(2+) release pathway is open to some extent in the K(+)-free E1PCa(2) structure. The Ca(2+) affinity of this E1P is as high as that of the unphosphorylated ATPase (E1), indicating the Ca(2+) binding sites are not disrupted. Thus, bound K(+) stabilizes the E1PCa(2) structure with occluded Ca(2+), keeping the Ca(2+) pathway to the lumen closed. We found previously (Yamasaki, K., Wang, G., Daiho, T., Danko, S., and Suzuki, H. (2008) J. Biol. Chem. 283, 29144-29155) that the K(+) bound in E2P reduces the Ca(2+) affinity essential for achieving the high physiological Ca(2+) gradient and to fully open the lumenal Ca(2+) gate for rapid Ca(2+) release (E2PCa(2) → E2P + 2Ca(2+)). These findings show that bound K(+) is critical for stabilizing both E1PCa(2) and E2P structures, thereby contributing to the structural changes that efficiently couple phosphoenzyme processing and Ca(2+) handling.

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 Ca(2+) at the transport sites (E2PCa(2)) of sarcoplasmic reticulum Ca(2+)-ATPase. This is normally a transient intermediate state during phosphoenzyme isomerization from the ADP-sensitive to ADP-insensitive form and Ca(2+) deocclusion/release to the lumen; E1PCa(2) --> E2PCa(2) --> E2P + 2Ca(2+). Stabilization was achieved by elongation of the Glu(40)-Ser(48) loop linking the Actuator domain and M1 (1st transmembrane helix) with four glycine insertions at Gly(46)/Lys(47) and by binding of beryllium fluoride (BeF(x)) to the phosphorylation site of the Ca(2+)-bound ATPase (E1Ca(2)). The complex E2Ca(2)xBeF(3)(-) was also produced by lumenal Ca(2+) binding to E2xBeF(3)(-) (E2P ground state analog) of the elongated linker mutant. The complex was stable for at least 1 week at 25 degrees C. Only BeF(x), but not AlF(x) or MgF(x), produced the E2PCa(2) structural analog. Complex formation required binding of Mg(2+), Mn(2+), or Ca(2+) at the catalytic Mg(2+) site. Results reveal that the phosphorylation product E1PCa(2) and the E2P ground state (but not the transition states) become competent to produce the E2PCa(2) transient state during forward and reverse phosphoenzyme isomerization. Thus, isomerization and lumenal Ca(2+) 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 Glu(40)-Ser(48) linker and of Mg(2+) at the catalytic site in these processes.

[Mechanism of ca(2+) pump as revealed by mutations, development of stable analogs of phosphorylated intermediates, and their structural analyses].

Sarco(endo)plasmic reticulum Ca(2+)-ATPase is a representative member of P-type cation transporting ATPases and catalyzes Ca(2+) transport coupled with ATP hydrolysis. The ATPase possesses three cytoplasmic domains (N, P, and A) and ten transmembrane helices (M1-M10). Ca(2+) binding at the transport sites in the transmembrane domain activates the ATPase and then the catalytic aspartate is auto-phosphorylated to form the phosphorylated intermediate (EP). Structural and functional studies have shown that, during the isomerization of EP in the Ca(2+) transport cycle, large motions of the three cytoplasmic domains take place, which then rearranges the transmembrane helices thereby destroying the Ca(2+) binding sites, opening the lumenal gate, and thus releasing the Ca(2+) into lumen. Stable structural analogues for the Ca(2+)-occluded and -released states of phosphorylated intermediates and for the transition and product states of the phosphorylation and dephosphorylation reactions were developed for biochemical and atomic-level structural studies to reveal the coupled changes in the catalytic and transport sites. Mutation studies identified the residues and structural regions essential for the structural changes and Ca(2+) transport function. Genetic dysfunction of Ca(2+)-ATPase causes various isoform-specific diseases. In this manuscript, recent understanding of the Ca-ATPase will be reviewed.

Roles of interaction between actuator and nucleotide binding domains of sarco(endo)plasmic reticulum Ca(2+)-ATPase as revealed by single and swap mutational analyses of serine 186 and glutamate 439.

Roles of hydrogen bonding interaction between Ser(186) of the actuator (A) domain and Glu(439) of nucleotide binding (N) domain seen in the structures of ADP-insensitive phosphorylated intermediate (E2P) of sarco(endo)plasmic reticulum Ca(2+)-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 Ca(2+)-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 Ser(186)-Glu(439) hydrogen bond stabilizes the E2P ground state structure. The modulatory ATP binding at sub-mm approximately 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 Ser(186) and Glu(439) 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.

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 Ca(2+)-ATPase E1PCa(2).Mg, a complex of E1Ca(2).BeF(x), was successfully developed by addition of beryllium fluoride and Mg(2+) to the Ca(2+)-bound state, E1Ca(2). In E1Ca(2).BeF(x), most probably E1Ca(2).BeF(3)(-), two Ca(2+) are occluded at high affinity transport sites, its formation required Mg(2+) binding at the catalytic site, and ADP decomposed it to E1Ca(2), as in E1PCa(2).Mg. Organization of cytoplasmic domains in E1Ca(2).BeF(x) was revealed to be intermediate between those in E1Ca(2).AlF(4)(-) ADP (transition state of E1PCa(2) formation) and E2.BeF(3)(-).(ADP-insensitive phosphorylated intermediate E2P.Mg). Trinitrophenyl-AMP (TNP-AMP) formed a very fluorescent (superfluorescent) complex with E1Ca(2).BeF(x) in contrast to no superfluorescence of TNP-AMP bound to E1Ca(2).AlF(x). E1Ca(2).BeF(x) with bound TNP-AMP slowly decayed to E1Ca(2), being distinct from the superfluorescent complex of TNP-AMP with E2.BeF(3)(-), which was stable. Tryptophan fluorescence revealed that the transmembrane structure of E1Ca(2).BeF(x) mimics E1PCa(2).Mg, and between those of E1Ca(2).AlF(4)(-).ADP and E2.BeF(3)(-). E1Ca(2).BeF(x) at low 50-100 microm Ca(2+) was converted slowly to E2.BeF(3)(-) releasing Ca(2+), mimicking E1PCa(2).Mg --> E2P.Mg + 2Ca(2+). Ca(2+) replacement of Mg(2+) at the catalytic site at approximately millimolar high Ca(2+) decomposed E1Ca(2).BeF(x) to E1Ca(2). Notably, E1Ca(2).BeF(x) was perfectly stabilized for at least 12 days by 0.7 mm lumenal Ca(2+) with 15 mm Mg(2+). Also, stable E1Ca(2).BeF(x) was produced from E2.BeF(3)(-) at 0.7 mm lumenal Ca(2+) by binding two Ca(2+) to lumenally oriented low affinity transport sites, as mimicking the reverse conversion E2P. Mg + 2Ca(2+) --> E1PCa(2).Mg.

Roles of Tyr122-hydrophobic cluster and K+ binding in Ca2+ -releasing process of ADP-insensitive phosphoenzyme of sarcoplasmic reticulum Ca2+ -ATPase.

Tyr(122)-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 Ca(2+)-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 (E1PCa(2)) (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 Ca(2+) release process in E2P. In the steady state, the amount of E2P decreased so that of E1PCa(2) increased with increasing lumenal Ca(2+) concentration in the mutants with K(0.5) 110-320 microm at pH 7.3. These lumenal Ca(2+) affinities in E2P agreed with those estimated from the forward and lumenal Ca(2+)-induced reverse kinetics of the E1PCa(2)-E2P isomerization. K(0.5) 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 Ca(2+) than that of the wild type. The kinetics further indicated that the rates of lumenal Ca(2+) 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 Ca(2+) affinity and opening the lumenal gate, thus for Ca(2+) release from E2PCa(2). 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.

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 (Glu(40)-Ser(48)) connecting the actuator domain and the first transmembrane helix of sarcoplasmic reticulum Ca(2+)-ATPase was explored by its elongation with glycine insertion at Pro(42)/Ala(43) and Gly(46)/Lys(47). 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 E2PCa(2) possessing two occluded Ca(2+) ions at the transport sites, and the Ca(2+) deocclusion and release into lumen were blocked in the mutants. By contrast, the hydrolysis of the Ca(2+)-free form of E2P produced from P(i) without Ca(2+) was as rapid in the mutants as in the wild type. Analysis of resistance against trypsin and proteinase K revealed that the structure of E2PCa(2) accumulated is an intermediate state between E1PCa(2) and the Ca(2+)-released E2P state. Namely in E2PCa(2), the actuator domain is already largely rotated from its position in E1PCa(2) and associated with the phosphorylation domain as in the Ca(2+)-released E2P state; however, in E2PCa(2), the hydrophobic interactions among these domains and Leu(119)/Tyr(122) on the top of second transmembrane helix are not yet formed properly. This is consistent with our previous finding that these interactions at Tyr(122) are critical for formation of the Ca(2+)-released E2P structure. Results showed that the EP isomerization/Ca(2+)-release process consists of the following two steps: E1PCa(2) --> E2PCa(2) --> E2P + 2Ca(2+); and the intermediate state E2PCa(2) 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 E2PCa(2), is critical for the correct positioning and interactions of the actuator and phosphorylation domains to cause structural changes for the Ca(2+) deocclusion and release.

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.

ATP2C1 is specifically localized in the basal layer of normal epidermis and its depletion triggers keratinocyte differentiation.

BACKGROUND: ATP2C1 is a calcium/manganese-ATPase localized in the Golgi apparatus and known as responsible gene for Hailey-Hailey disease. But its localization and roles in the epidermis are not fully elucidated. OBJECTIVE: To explore the localization and biological role of ATP2C1 in normal epidermis in terms of differentiation states. METHODS: We examined the immunohistochemical distribution of ATP2C1 in normal epidermis and measured the expression of ATP2C1 in cultured keratinocytes following forced detachment from culture dish or following treatment with high concentrations of calcium. Furthermore, we knockdown ATP2C1 expression in cultured keratinocytes by using RNA interference procedure to abrogate cation accumulation in cell organelles. RESULTS: ATP2C1 is specifically localized at the basal cell layer in normal epidermis. Neither detachment of keratinocyte from culture dish nor treatment with high concentrations of calcium suppressed ATP2C1 expression, while both procedures induced differentiation markers, K10 keratin and involucrin. In contrast, knockdown of ATP2C1 induced these differentiation markers of cultured keratinocytes. Furthermore, treatment of keratinocytes with a calcium ionophore, A23187, did not up-regulate differentiation markers of keratinocytes, while a more manganese selective ionophore Br-A23187 up-regulated these differentiation markers. CONCLUSION: Our results suggest that ATP2C1 plays an essential role for basal keratinocytes to keep in the undifferentiated state and that its reduction evokes differentiation and up-localization to suprabasal layers most likely via the manganese starvation in the Golgi apparatus of keratinocytes.

Critical hydrophobic interactions between phosphorylation and actuator domains of Ca2+-ATPase for hydrolysis of phosphorylated intermediate.

Functional roles of seven hydrophobic residues on the interface between the actuator (A) and phosphorylation (P) domains of sarcoplasmic reticulum Ca2+-ATPase were explored by alanine and serine substitutions. The residues examined were Ile179/Leu180/Ile232 on the A domain, Val705/Val726 on the P domain, and Leu119/Tyr122 on the loop linking the A domain and M2 (the second transmembrane helix). These residues gather to form a hydrophobic cluster around Tyr122 in the crystal structures of Ca2+-ATPase in Ca2+-unbound E2 (unphosphorylated) and E2P (phosphorylated) states but are far apart in those of Ca2+-bound E1 (unphosphorylated) and E1P (phosphorylated) states. The substitution-effects were also compared with those of Ile235 on the A domain/M3 linker and those of T181GE of the A domain, since they are in the immediate vicinity of the Tyr122-cluster. All these substitutions almost completely inhibited ATPase activity without inhibiting Ca2+-activated E1P formation from ATP. Substitutions of Ile235 and T181GE blocked the E1P to E2P transition, whereas those in the Tyr122-cluster blocked the subsequent E2P hydrolysis. Substitutions of Ile235 and Glu183 also blocked EP hydrolysis. Results indicate that the Tyr122-cluster is formed during the E1P to E2P transition to configure the catalytic site and position Glu183 properly for hydrolyzing the acylphosphate. Ile235 on the A domain/M3 linker likely forms hydrophobic interactions with the A domain and thereby allowing the strain of this linker to be utilized for large motions of the A domain during these processes. The Tyr122-cluster, Ile235, and T181GE thus seem to have different roles and are critical in the successive events in processing phosphorylated intermediates to transport Ca2+.

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 Ca(2+)-ATPase (SERCA2b) mutants, Ile(274) --> Val at the lumenal end of M3, Leu(321) --> Phe on the cytoplasmic part of M4, and Met(719) --> Ile in P domain, were explored, because they exhibited nearly normal expression and localization in COS-1 cells and the high ATPase and coupled Ca(2+) transport activities that were essentially identical (L321F) or slightly lower (I274V by approximately 35% and M719I by approximately 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 Ca(2+), 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 Ca(2+), as demonstrated with the effect of Ca(2+) ionophore on ATPase activity and more specifically with the effects of Ca(2+) (up to 50 mm) on the decay of phosphoenzyme intermediates. The results on I274V and M719I suggest that the physiological requirement for Ca(2+) homeostasis in keratinocytes to avoid haploinsufficiency is very strict, probably much more than considered previously. The insensitivity to lumenal Ca(2+) in L321F likely brings the lumenal Ca(2+) to an abnormally elevated level. The three mutants with their distinctively altered kinetic properties will thus likely cause different types of perturbation of intracellular Ca(2+) 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 Ca(2+) transport.