A structural model for the catalytic cycle of Ca(2+)-ATPase

Ca(2+)-ATPase is responsible for active transport of calcium ions across the sarcoplasmic reticulum membrane. This coupling involves an ordered sequence of reversible reactions occurring alternately at the ATP site within the cytoplasmic domains, or at the calcium transport sites within the transmembrane domain. These two sites are separated by a large distance and conformational changes have long been postulated to play an important role in their coordination. To characterize the nature of these conformational changes, we have built atomic models for two reaction intermediates and postulated the mechanisms governing the large structural changes. One model is based on fitting the X-ray crystallographic structure of Ca(2+)-ATPase in the E1 state to a new 6 A structure by cryoelectron microscopy in the E2 state. This fit indicates that calcium binding induces enormous movements of all three cytoplasmic domains as well as significant changes in several transmembrane helices. We found that fluorescein isothiocyanate displaced a decavanadate molecule normally located at the intersection of the three cytoplasmic domains, but did not affect their juxtaposition; this result indicates that our model likely reflects a native E2 conformation and not an artifact of decavanadate binding. To explain the dramatic structural effect of calcium binding, we propose that M4 and M5 transmembrane helices are responsive to calcium binding and directly induce rotation of the phosphorylation domain. Furthermore, we hypothesize that both the nucleotide-binding and beta-sheet domains are highly mobile and driven by Brownian motion to elicit phosphoenzyme formation and calcium transport, respectively. If so, the reaction cycle of Ca(2+)-ATPase would have elements of a Brownian ratchet, where the chemical reactions of ATP hydrolysis are used to direct the random thermal oscillations of an innately flexible molecule.     

Locating the thapsigargin-binding site on Ca(2+)-ATPase by cryoelectron microscopy

Thapsigargin (TG) is a potent inhibitor of Ca(2+)-ATPase from sarcoplasmic and endoplasmic reticula. Previous enzymatic studies have concluded that Ca(2+)-ATPase is locked in a dead-end complex upon binding TG with an affinity of <1 nM and that this complex closely resembles the E(2) enzymatic state. We have studied the structural effects of TG binding by cryoelectron microscopy of tubular crystals, which have previously been shown to comprise Ca(2+)-ATPase molecules in the E(2) conformation. In particular, we have compared 3D reconstructions of Ca(2+)-ATPase in the absence and presence of either TG or its dansylated derivative. The overall molecular shape of Ca(2+)-ATPase in the reconstructions is very similar, demonstrating that the TG/Ca(2+)-ATPase complex does indeed physically resemble the E(2) conformation, in contrast to massive domain movements that appear to be induced by Ca(2+) binding. Difference maps reveal a consistent difference on the lumenal side of the membrane, which we conclude corresponds to the thapsigargin-binding site. Modeling the atomic structure for Ca(2+)-ATPase into our density maps reveals that this binding site is composed of the loops between transmembrane segments M3/M4 and M7/M8. Indirect effects are proposed to explain the effects of the S3 stalk segment on thapsigargin affinity as well as thapsigargin-induced changes in ATP affinity. Indeed, a second difference density was observed at the decavanadate-binding site within the three cytoplasmic domains, which we believe reflects an altered affinity as a result of the long-range conformational coupling that drives the reaction cycle of this family of ATP-dependent ion pumps.     

Ca2+ -ATPase structure in the E1 and E2 conformations: mechanism,helix-helix and helix-lipid interactions

The determination of the crystal structure of the Ca(2+)-ATPase of sarcoplasmic reticulum (SR) in its Ca(2+)-bound [Nature 405 (2000) 647] and Ca(2+)-free forms [Nature 418 (2002) 605] gives the opportunity for an analysis of conformational changes on the Ca(2+)-ATPase and of helix-helix and helix-lipid interactions in the transmembrane (TM) region of the ATPase. The locations of the ends of the TM alpha-helices on the cytoplasmic side of the membrane are reasonably well defined by the location of Trp residues and by the location of Lys-262 that snorkels up to the surface. The locations of the lumenal ends of the helices are less clear. The position of Lys-972 on the lumenal side of helix M9 suggests that the hydrophobic thickness of the protein is only about 21 A, rather than the normal 30 A. The experimentally determined TM alpha-helices do not agree well with those predicted theoretically. Charged headgroups are required for strong interaction of lipids with the ATPase, consistent with the large number of charged residues located close to the lipid-water interface. Helix packing appears to be rather irregular. Packing of helices M8 and M10 is of the 3-4 ridges-into-grooves or knobs-into-holes types. Packing of helices M5 and M7 involves two Gly residues in M7 and one Gly residue in M5. Packing of the other helices generally involves just one or two residues on each helix at the crossing point. The irregular packing of the TM alpha-helices in the Ca(2+)-ATPase, combined with the diffuse structure of the ATPase on the lumenal side of the membrane, is suggested to lead to a relative low activation energy for changing the packing of the TM alpha-helices, with changes in TM alpha-helical packing being important in the process of transfer of Ca(2+) ions across the membrane. The inhibitor thapsigargin binds in a cleft between TM alpha-helices M3, M5 and M7. It is suggested that this and other similar clefts provide binding sites for a variety of hydrophobic molecules affecting the activity of the Ca(2+)-ATPase.     

Calcium binding to the photosystem II subunit CP29

We have identified a Ca(2+)-binding site of the 29-kDa chlorophyll a/b-binding protein CP29, a light harvesting protein of photosystem II most likely involved in photoregulation. (45)Ca(2+) binding studies and dot blot analyses of CP29 demonstrate that CP29 is a Ca(2+)-binding protein. The primary sequence of CP29 does not exhibit an obvious Ca(2+)-binding site therefore we have used Yb(3+) replacement to analyze this site. Near-infrared Yb(3+) vibronic side band fluorescence spectroscopy (Roselli, C., Boussac, A., and Mattioli, T. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12897-12901) of Yb(3+)-reconstituted CP29 indicated a single population of Yb(3+)-binding sites rich in carboxylic acids, characteristic of Ca(2+)-binding sites. A structural model of CP29 presents two purported extra-membranar loops which are relatively rich in carboxylic acids, one on the stromae side and one on the lumenal side. The loop on the lumenal side is adjacent to glutamic acid 166 in helix C of CP29, which is known to be the binding site for dicyclohexylcarbodiimide (Pesaresi, P., Sandonà, D., Giuffra, E. , and Bassi, R. (1997) FEBS Lett. 402, 151-156). Dicyclohexylcarbodiimide binding prevented Ca(2+) binding, therefore we propose that the Ca(2+) in CP29 is bound in the domain including the lumenal loop between helices B and C.     

Modeling a dehalogenase fold into the 8-A density map for Ca(2+)-ATPase defines a new domain structure

Members of the large family of P-type pumps use active transport to maintain gradients of a wide variety of cations across cellular membranes. Recent structures of two P-type pumps at 8-A resolution have revealed the arrangement of transmembrane helices but were insufficient to reveal the architecture of the cytoplasmic domains. However, recent proposals of a structural homology with a superfamily of hydrolases offer a new basis for modeling these domains. In the current work, we have extended the sequence comparison for the superfamily and delineated domains in the 8-A density map of Ca(2+)-ATPase. The homology suggests a new domain structure for Ca(2+)-ATPase and, specifically, that the phosphorylation domain adopts a Rossman fold. Accordingly, the atomic structure of L-2 haloacid dehalogenase has been fitted into the relevant domain of Ca(2+)-ATPase. The resulting model suggests the existence of two ATP sites at the interface between two domains. Based on this new model, we are able to reconcile numerous results of mutagenesis and chemical cross-linking within the catalytic domains. Furthermore, we have used the model to predict the configuration of Mg.ATP at its binding site. Based on this prediction, we propose a mechanism, involving a change in Mg(2+) liganding, for initiating the domain movements that couple sites of ion transport to ATP hydrolysis.     

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.     

A structure-function study of the C2 domain of cytosolic phospholipase A2. Identification of essential calcium ligands and hydrophobic membrane binding residues

The C2 domain of cytosolic phospholipase A2 (cPLA2) is involved in the Ca2+-dependent membrane binding of this protein. To identify protein residues in the C2 domain of cPLA2 essential for its Ca2+ and membrane binding, we selectively mutated Ca2+ ligands and putative membrane-binding residues of cPLA2 and measured the effects of mutations on its enzyme activity, membrane binding affinity, and monolayer penetration. The mutations of five Ca2+ ligands (D40N, D43N, N65A, D93N, N95A) show differential effects on the membrane binding and activation of cPLA2, indicating that two calcium ions bound to the C2 domain have differential roles. The mutations of hydrophobic residues (F35A, M38A, L39A, Y96A, Y97A, M98A) in the calcium binding loops show that the membrane binding of cPLA2 is largely driven by hydrophobic interactions resulting from the penetration of these residues into the hydrophobic core of the membrane. Leu39 and Val97 are fully inserted into the membrane, whereas Phe35 and Tyr96 are partially inserted. Finally, the mutations of four cationic residues in a beta-strand (R57E/K58E/R59E/R61E) have modest and negligible effects on the binding of cPLA2 to zwitterionic and anionic membranes, respectively, indicating that they are not directly involved in membrane binding. In conjunction with our previous study on the C2 domain of protein kinase C-alpha (Medkova, M., and Cho, W. (1998) J. Biol. Chem. 273, 17544-17552), these results demonstrate that C2 domains are not only a membrane docking unit but also a module that triggers membrane penetration of protein and that individual Ca2+ ions bound to the calcium binding loops play differential roles in the membrane binding and activation of their parent proteins.     

Homology modeling identifies C-terminal residues that contribute to the Ca2+ sensitivity of a BKCa channel

Activation of BK(Ca) channels by direct Ca(2+) binding and membrane depolarization occur via independent and additive molecular processes. The "calcium bowl" domain is critically involved in Ca(2+)-dependent gating, and we have hypothesized that a sequence within this domain may resemble an EF hand motif. Using a homology modeling strategy, it was observed that a single Ca(2+) ion may be coordinated by the oxygen-containing side chains of residues within the calcium bowl (i.e., (912)ELVNDTNVQFLD(923)). To examine these predictions directly, alanine-substituted BK(Ca) channel mutants were expressed in HEK 293 cells and the voltage and Ca(2+) dependence of macroscopic currents were examined in inside-out membrane patches. Over the range of 1-10 microM free Ca(2+), single point mutations (i.e., E912A and D923A) produced rightward shifts in the steady-state conductance-voltage relations, whereas the mutants N918A or Q920A had no effect on Ca(2+)-dependent gating. The double mutant E912A/D923A displayed a synergistic shift in Ca(2+)-sensitive gating, as well as altered kinetics of current activation/deactivation. In the presence of 1, 10, and 80 mM cytosolic Mg(2+), this double mutation significantly reduced the Ca(2+)-induced free energy change associated with channel activation. Finally, mutations that altered sensitivity of the holo-channel to Ca(2+) also reduced direct (45)Ca binding to the calcium bowl domain expressed as a bacterial fusion protein. These findings, along with other recent data, are considered in the context of the calcium bowl's high affinity Ca(2+) sensor and the known properties of EF hands.     

Regulation of intra-Golgi membrane transport by calcium

Calcium cations play a critical role in regulating vesicular transport between different intracellular membrane-bound compartments. The role of calcium in transport between the Golgi cisternae, however, remains unclear. Using a well characterized cell-free intra-Golgi transport assay, we now show that changes in free Ca(2+) concentration in the physiological range regulate this transport process. The calcium-chelating agent 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocked transport with an IC(50) of approximately 0.8 mm. The effect of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid was reversible by addition of fresh cytosol and was irreversible when performed in the presence of a Ca(2+) ionophore that depletes calcium from lumenal stores. We demonstrate here that intra-Golgi transport is stimulated by low Ca(2+) concentrations (20-100 nm) but is inhibited by higher concentrations (above 100 nm). Further, we show that calmodulin antagonists specifically block intra-Golgi transport, implying a role for calmodulin in mediating the effect of calcium. Our results suggest that Ca(2+) efflux from intracellular pools may play an essential role in regulating intra-Golgi transport.     

Proton paths in the sarcoplasmic reticulum Ca(2+) -ATPase

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) pumps Ca(2+) and countertransport protons. Proton pathways in the Ca(2+) bound and Ca(2+)-free states are suggested based on an analysis of crystal structures to which water molecules were added. The pathways are indicated by chains of water molecules that interact favorably with the protein. In the Ca(2+) bound state Ca(2)E1, one of the proposed Ca(2+) entry paths is suggested to operate additionally or alternatively as proton pathway. In analogs of the ADP-insensitive phosphoenzyme E2P and in the Ca(2+)-free state E2, the proton path leads between transmembrane helices M5 to M8 from the lumenal side of the protein to the Ca(2+) binding residues Glu-771, Asp-800 and Glu-908. The proton path is different from suggested Ca(2+) dissociation pathways. We suggest that separate proton and Ca(2+) pathways enable rapid (partial) neutralization of the empty cation binding sites. For this reason, transient protonation of empty cation binding sites and separate pathways for different ions are advantageous for P-type ATPases in general.     

Interdomain fluorescence resonance energy transfer in SERCA probed by cyan-fluorescent protein fused to the actuator domain

We have used a biosynthetically incorporated fluorescent probe to monitor domain movements involved in ion transport by the sarcoendoplasmic reticulum Ca-ATPase (SERCA) from rabbit fast-twitch skeletal muscle. X-ray crystal structures suggest that the nucleotide-binding (N) and actuator (A) domains of SERCA move apart by several nanometers upon Ca binding. To test this hypothesis, cDNA constructs were created to fuse cyan-fluorescent protein (CFP) to the N terminus of SERCA (A domain). This CFP-SERCA fluorescent fusion protein retained activity when expressed in Sf21 insect cells using the baculovirus system. Fluorescence resonance energy transfer (FRET) was used to monitor the A-N interdomain distance for CFP-SERCA selectively labeled with fluorescein isothiocyanate (FITC) at Lys 515 in the N domain. At low [Ca (2+)] (E2 biochemical state), the measured FRET efficiency between CFP (donor in A domain) and FITC (acceptor in N domain) was 0.34 +/- 0.03, indicating a mean distance of 61.6 +/- 2.0 A between probes on the two domains. An increase of [Ca (2+)] to 0.1 mM (E1-Ca biochemical state) decreased the FRET efficiency by 0.06 +/- 0.03, indicating an increase in the mean distance by 3.0 +/- 1.2 A. Quantitative molecular modeling of dual-labeled SERCA, including an accurate calculation of the orientation factor, shows that the FRET data observed in the absence of Ca is consistent with the E2 crystal structure, but the increase in distance (decrease in FRET) induced by Ca is much less than predicted by the E1 crystal structure. We conclude that the E1 crystal structure does not reflect the predominant structure of SERCA under physiological conditions in a functional membrane bilayer.     

A high-affinity, calmodulin-sensitive (Ca2+ + Mg2+)-ATPase and associated calcium-transport pump in the Ehrlich ascites tumor cell plasma membrane

A unique cytoplast preparation from Ehrlich ascites tumor cells (G. V. Henius, P. C. Laris, and J. D. Woodburn (1979) Exp. Cell. Res. 121, 337-345), highly enriched in plasma membranes, was employed to characterize the high-affinity plasma membrane calcium-extrusion pump and its associated adenosine triphosphatase (ATPase). An ATP-dependent calcium-transport system which had a high affinity for free calcium (K0.5 = 0.040 +/- 0.005 microM) was identified. Two different calcium-stimulated ATPase activities were detected. One had a low (K0.5 = 136 +/- 10 microM) and the other a high (K0.5 = 0.103 +/- 0.077 microM) affinity for free calcium. The high-affinity enzyme appeared to represent the ubiquitous high-affinity plasma membrane (Ca2+ + Mg2+)-ATPase (calcium-stimulated, magnesium-dependent ATPase) seen in normal cells. Both calcium transport and the (Ca2+ + Mg2+)-ATPase were significantly stimulated by the calcium-dependent regulatory protein calmodulin, especially when endogenous activator was removed by treatment with the calcium chelator ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid. Other similarities between calcium transport and the (Ca2+ + Mg2+)-ATPase included an insensitivity to ouabain (0.5 mM), lack of activation by potassium (20 mM), and a requirement for magnesium. These similar properties suggested that the (Ca2+ + Mg2+)-ATPase represents the enzymatic basis of the high-affinity calcium pump. The calcium pump/enzyme system was inhibited by orthovanadate at comparatively high concentrations (calcium transport: K0.5 congruent to 100 microM; (Ca2+ + Mg2+)-ATPase: K0.5 greater than 100 microM). Upon Hill analysis, the tumor cell (Ca2+ + Mg2+)-ATPase failed to exhibit cooperative activation by calcium which is characteristic of the analogous enzyme in the plasma membrane of normal cells.     

Distinct natures of beryllium fluoride-bound, aluminum fluoride-bound, and magnesium fluoride-bound stable analogues of an ADP-insensitive phosphoenzyme intermediate of sarcoplasmic reticulum Ca2+-ATPase: changes in catalytic and transport sites during phosphoenzyme hydrolysis

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 (BeF.E2), bound aluminum fluoride (AlF.E2), and bound magnesium fluoride (MgF.E2), 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 BeF.E2 as in E2P but hydrophilic in MgF.E2 and AlF.E2; 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 BeF.E2 as in E2P, but only very slightly (hence, the release pathway is likely closed without thapsigargin) in MgF.E2 and AlF.E2 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. BeF.E2 thus has the features very similar to those characteristic of the E2P ground state, although AlF.E2 and MgF.E2 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+).     

Calcium transport by sarcoplasmic reticulum Ca(2+)-ATPase. Role of the A domain and its C-terminal link with the transmembrane region

After treatment of sarcoplasmic reticulum Ca(2+)-ATPase with proteinase K (PK) in the presence of Ca(2+) and a protecting non-phosphorylated ligand (e.g. adenosine 5'-(beta,gamma-methylenetriphosphate), we were able to prepare in high yield an ATPase species that only differs from intact ATPase because of excision of the MAATE(243) sequence from the loop linking the A domain with the third transmembrane segment. The PK-treated ATPase was unable to transport Ca(2+) and to catalyze ATP hydrolysis, but it could bind two calcium ions with high affinity and react with ATP to form a classical ADP-sensitive phosphoenzyme, Ca(2)E1P, with occluded Ca(2+). The ability of Ca(2)E1P to become converted to the Ca(2+)-free ADP-insensitive form, E2P, was strongly reduced, as was the ability of PK-treated ATPase to react with orthovanadate or to form an E2P intermediate from inorganic phosphate in the absence of Ca(2+). PK-treated ATPase also reacted with thapsigargin to form a complex with altered properties, and the tryptic cleavage "T2" site in the A domain was no longer protected in the absence of Ca(2+). It is probable that disrupting the C-terminal link of the A domain with the transmembrane region severely compromises reorientation of A and P domains and the functionally critical cross-talk of these domains with the membrane-bound Ca(2+) ions.     

The importance of carboxyl groups on the lumenal side of the membrane for the function of the Ca(2+)-ATPase of sarcoplasmic reticulum

The conventional model for transport of Ca(2+) by the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) involves a pair of binding sites for Ca(2+) that change upon phosphorylation of the ATPase from being high affinity and exposed to the cytoplasm to being low affinity and exposed to the lumen. However, a number of recent experiments suggest that in fact transport involves two separate pairs of binding sites for Ca(2+), one pair exposed to the cytoplasmic side and the other pair exposed to the lumenal side. Here we show that the carbodiimide 1-ethyl-3-[3-(dimethylamino)-propyl] carbodiimide (EDC) is membrane-impermeable, and we use EDC to distinguish between cytoplasmic and lumenal sites of reaction. Modification of the Ca(2+)-ATPase in sealed SR vesicles with EDC leads to loss of ATPase activity without modification of the pair of high affinity Ca(2+)-binding sites. Modification of the purified ATPase in unsealed membrane fragments was faster than modification in SR vesicles, suggesting the presence of more quickly reacting lumenal sites. This was confirmed in experiments measuring EDC modification of the ATPase reconstituted randomly into sealed lipid vesicles. Modification of sites on the lumenal face of the ATPase led to loss of the Ca(2+)-induced increase in phosphorylation by P(i). It is concluded that carboxyl groups on the lumenal side of the ATPase are involved in Ca(2+) binding to the lumenal side of the ATPase and that modification of these sites leads to loss of ATPase activity. The presence of MgATP or MgADP leads to faster inhibition of the ATPase by EDC in unsealed membrane fragments than in sealed vesicles, suggesting that binding of MgATP or MgADP to the ATPase leads to a conformational change on the lumenal side of the membrane.     

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.     

The N-terminal domain of 5-lipoxygenase binds calcium and mediates calcium stimulation of enzyme activity

Human 5-lipoxygenase (5-LO) is a key enzyme in the conversion of arachidonic acid into leukotrienes and lipoxins, mediators and modulators of inflammation. In this study, we localized a stimulatory Ca(2+)-binding site to the N-terminal region of the enzyme. Thus, in a (45)Ca(2+) overlay assay, the N-terminal 128 amino acids of recombinant human 5-LO (fused to glutathione S-transferase) bound radioactive calcium to about the same extent as intact 5-LO. The glutathione S-transferase fusion protein of the C-terminal part of 5-LO (amino acids 120-673) showed much weaker binding. A model of a putative 5-LO N-terminal domain was calculated based on the structure of rabbit reticulocyte 15-LO. This model resembles beta-sandwich C2 domains of other Ca(2+)-binding proteins. Comparison of our model with the C2 domain of cytosolic phospholipase A(2) suggested a number of amino acids, located in the loops that connect the beta-strands, as potential Ca(2+) ligands. Indeed, mutations particularly in loop 2 (N43A, D44A, and E46A) led to decreased Ca(2+) binding and a requirement for higher Ca(2+) concentrations to stimulate enzyme activity. Our data indicate that an N-terminal beta-sandwich of 5-LO functions as a C2 domain in the calcium regulation of enzyme activity.     

Ca2+ induces clustering of membrane proteins in the plasma membrane via electrostatic interactions

Membrane proteins and membrane lipids are frequently organized in submicron-sized domains within cellular membranes. Factors thought to be responsible for domain formation include lipid-lipid interactions, lipid-protein interactions and protein-protein interactions. However, it is unclear whether the domain structure is regulated by other factors such as divalent cations. Here, we have examined in native plasma membranes and intact cells the role of the second messenger Ca(2+) in membrane protein organization. We find that Ca(2+) at low micromolar concentrations directly redistributes a structurally diverse array of membrane proteins via electrostatic effects. Redistribution results in a more clustered pattern, can be rapid and triggered by Ca(2+) influx through voltage-gated calcium channels and is reversible. In summary, the data demonstrate that the second messenger Ca(2+) strongly influences the organization of membrane proteins, thus adding a novel and unexpected factor that may control the domain structure of biological membranes.     

Calcium transport across the sarcoplasmic reticulum: structure and function of Ca2+-ATPase and the ryanodine receptor.

Contraction of striated muscle results from a rise in cytoplasmic calcium concentration in a process termed excitation/contraction coupling. Most of this calcium moves back and forth across the sarcoplasmic-reticulum membrane in cycles of contraction and relaxation. The channel responsible for release from the sarcoplasmic reticulum is the ryanodine receptor, whereas Ca2+-ATPase effects reuptake in an ATP-dependent manner. The structures of these two molecules have been studied by cryoelectron microscopy, with helical crystals in the case of Ca2+-ATPase and as isolated tetramers in the case of ryanodine receptor. Structures of Ca2+-ATPase at 8-A resolution reveal the packing of transmembrane helices and have allowed fitting of a putative ATP-binding domain among the cytoplasmic densities. Comparison of ATPases in different conformations gives hints about the conformational changes that accompany the reaction cycle. Structures of ryanodine receptor at 30-A resolution reveal a multitude of isolated domains in the cytoplasmic portion, as well as a distinct transmembrane assembly. Binding sites for various protein ligands have been determined and conformational changes induced by ATP, calcium and ryanodine have been characterized. Both molecules appear to use large conformational changes to couple interactions in their cytoplasmic domains with calcium transport through their membrane domains, and future studies at higher resolution will focus on the mechanisms for this coupling.     

Cytosolic phosphorylation of calnexin controls intracellular Ca(2+) oscillations via an interaction with SERCA2b

Calreticulin (CRT) and calnexin (CLNX) are lectin chaperones that participate in protein folding in the endoplasmic reticulum (ER). CRT is a soluble ER lumenal protein, whereas CLNX is a transmembrane protein with a cytosolic domain that contains two consensus motifs for protein kinase (PK) C/proline- directed kinase (PDK) phosphorylation. Using confocal Ca(2+) imaging in Xenopus oocytes, we report here that coexpression of CLNX with sarco endoplasmic reticulum calcium ATPase (SERCA) 2b results in inhibition of intracellular Ca(2+) oscillations, suggesting a functional inhibition of the pump. By site-directed mutagenesis, we demonstrate that this interaction is regulated by a COOH-terminal serine residue (S562) in CLNX. Furthermore, inositol 1,4,5-trisphosphate- mediated Ca(2+) release results in a dephosphorylation of this residue. We also demonstrate by coimmunoprecipitation that CLNX physically interacts with the COOH terminus of SERCA2b and that after dephosphorylation treatment, this interaction is significantly reduced. Together, our results suggest that CRT is uniquely regulated by ER lumenal conditions, whereas CLNX is, in addition, regulated by the phosphorylation status of its cytosolic domain. The S562 residue in CLNX acts as a molecular switch that regulates the interaction of the chaperone with SERCA2b, thereby affecting Ca(2+) signaling and controlling Ca(2+)-sensitive chaperone functions in the ER.