• Characterization of sarcoplasmic reticulum calcium(2+) ATPase by membrane reconstitution with preformed liposomes

      Yu, Xiang; Inesi, Giuseppe (1993)
      The Ca{dollar}\sp{lcub}2+{rcub}{dollar} transport ATPase of sarcoplasmic reticulum (SR) is an enzyme that catalyzes the transport of cytoplasmic Ca{dollar}\sp{lcub}2+{rcub}{dollar} into the SR lumen upon ATP hydrolysis. The most commonly used experimental system is the SR vesicles prepared from the muscle homogenate. The native SR vesicles have high protein content and electrolyte leakage. These properties have made it difficult to study some aspects of the enzyme mechanism, namely counter-ion transport, electrogenicity and the coupling ratio of Ca{dollar}\sp{lcub}2+{rcub}{dollar}/ATP. The SR Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase was reconstituted in unilamellar liposomes prepared by reverse phase evaporation. The size of the resulting proteoliposomes was similar to that of native SR vesicles, but with much lower protein/lipid ratio. The proteoliposomes sustained ATP dependent Ca{dollar}\sp{lcub}2+{rcub}{dollar} uptake to an asymptotic level of 2-3 {dollar}\mu{dollar}mole/mg protein without Ca{dollar}\sp{lcub}2+{rcub}{dollar} precipitating agents. The low permeability of the proteoliposomal membrane permitted direct demonstration of Ca{dollar}\sp{lcub}2+{rcub}{dollar}/H{dollar}\sp+{dollar} countertransport and electrogenicity by parallel measurements in the same experimental system. Countertransport of 1 H{dollar}\sp+{dollar} per 1 Ca{dollar}\sp{lcub}2+{rcub}{dollar} was demonstrated. Consistent with the countertransport stoichiometry, net positive charge displacement produced by Ca{dollar}\sp{lcub}2+{rcub}{dollar} transport was observed. The steady-state electrical potential observed under optimal conditions was approximately 50 mV. The estimated charge transfer associated with Ca{dollar}\sp{lcub}2+{rcub}{dollar} and H{dollar}\sp+{dollar} countertransport were well accounted for by the initial rate and steady-state values of membrane potentials. The coupling ratio of Ca{dollar}\sp{lcub}2+{rcub}{dollar}/ATP was determined by parallel measurements of both Ca{dollar}\sp{lcub}2+{rcub}{dollar} uptake and the ATP hydrolysis. Under optimal condition a 2:1 ratio was observed at early stage of filling of proteoliposomes. However, when maximal loading was achieved, the significant rate of ATP hydrolysis still remained with negligible Ca{dollar}\sp{lcub}2+{rcub}{dollar} uptake. The effects of different anions on Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase functions as well as membrane permeability were studied using the reconstitution system. The pH dependence of Ca{dollar}\sp{lcub}2+{rcub}{dollar}/H{dollar}\sp+{dollar} countertransport indicates the direct exchange of H{dollar}\sp+{dollar} for Ca{dollar}\sp{lcub}2+{rcub}{dollar} on ATPase acidic residues which are involved in cation translocation. The observation of futile cycle of ATP hydrolysis shows that as the concentration of lumenal Ca{dollar}\sp{lcub}2+{rcub}{dollar} rises above its dissociation constant, the phosphoenzyme still undergoes significant rates of hydrolytic cleavage even though retaining bound Ca{dollar}\sp{lcub}2+{rcub}{dollar}.
    • Detailed characterization of the cooperative mechanism of calcium(2+) binding and catalytic activation in the sarcoplasmic reticulum calcium(2+) transport (SERCA) ATPase

      Zhang, Zhongsen; Inesi, Giuseppe (2001)
      Occupation of two calcium-binding sites is required for catalytic activation of the sarcoplasmic reticulum Ca2+ ATPase (SERCA). The residues in the transmembrane domain, the cytoplasmic phosphorylation domain, and the L67 and L89 loops were subjected to mutational analysis. Direct measurements of Ca2+ binding and measurements of various enzymatic functions clarified the cooperative mechanism of calcium binding and catalytic activation of SERCA. The functional characterization and high-resolution structure of ATPase suggested cooperative and sequential calcium binding in which side chains of Glu771, Thr799, Asp800 and Glu908 contribute to site I, while Glu309, Asn796 and Asp800 contribute to site II. Mutational analysis of the L67 loop indicated its importance in protein folding and stabilization of the Ca 2+ ATPase. Single mutation of Pro820 to Ala resulted in negligible protein recovery while transcription occurred at normal levels. Single mutations of Lys819 or Arg822 interfered significantly with the formation of the phosphoenzyme intermediates. A triple conservative mutation of Asp813, 815 and 818 to Asn interfered mainly with the Ca2+-dependent activation of the ATPase but not Ca2+-independent phosphorylation by Pi. The effect of the triple mutation could be reproduced by a single mutation of Asp813 (but not of Asp815 or Asp818) to Asn. Functional and structural analysis of the experimental data demonstrates that the L67 loop plays an important role in protein folding and stabilization by linking the cytosolic catalytic domain and the transmembrane Ca2+ binding domain through a network of hydrogen bonds.
    • Mutational study on the coupling mechanism of catalysis and transport functions in the sarcoplasmic reticulum Ca(2+)-ATPase

      Zhang, Ziyu; Inesi, Giuseppe (1995)
      ATP utilization by P-type cation transport ATPases includes a phosphorylated intermediate which is formed by transfer of the ATP terminal phosphate onto an aspartyl residue at the catalytic site. The phosphorylation site and cation binding site of the ATPase molecule are separated by a fairly long distance of about 50 A. The coupling mechanism of these two functional sites is not yet fully understood and is currently under active investigation. Within the family of cation transport ATPases, the Ca2+-ATPase of sarcoplasmic reticulum (SR) provides an advantageous experimental system due to its abundance in the native membrane, and the availability of cDNA for expression of functional protein.;The sarcoplasmic reticulum Ca2+-ATPase segment extending from the phosphorylation site (Asp351) to the preceding transmembrane helix M4 (which is involved in Ca2+ binding in conjunction with transmembrane helices M5, M6 and M8), shares a marked sequence homology with the corresponding segments of other cation ATPases. We generated twenty six point mutations in this segment and expressed those mutant enzymes in COS-1 cells. We found that non-conservative mutations of residues which are homologous in various cation ATPases result in strong inhibition of catalytic and transport functions. Mutations of non-homologous residues to match the corresponding residues of other cation ATPases are not inhibitory, and in some cases produce higher activity. The inhibitory mutations specifically affect the phosphorylated intermediate turnover, which is associated with the vectorial translocation of bound Ca2+. The same mutations do not affect the kinetics of ATPase activation by Ca2+, which is required for enzyme phosphorylation by ATP. This indicates that activation of the phosphoryl transfer reaction by Ca2+ binding, and vectorial displacement of bound Ca2+ by enzyme phosphorylation, do not occur simply as the forward and reverse directions of the same process, but are linked to distinct structural features of the enzyme. The peptide segment extending from the phosphorylation site in the enzyme extramembranous headpiece, through the M4 helix in the membrane bound region, sustains a prominent role in transmission of the phosphorylation signal for displacement of bound Ca2+. A critical structural role of this segment is also demonstrated by the interference of specific mutations with membrane assembly of the expressed protein.
    • Thapsigargin: A potent inhibitor of the calcium ATPase in sarcoplasmic reticulum

      Sagara, Yutaka; Inesi, Giuseppe (1992)
      A Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase, a membranous protein in sarcoplasmic reticulum (SR) of skeletal muscle, is an enzyme that catalyzes transport of cytoplasmic Ca{dollar}\sp{lcub}2+{rcub}{dollar} into the SR lumen upon ATP hydrolysis. The endoplasmic reticulum (ER) Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase from rat liver cells, but not SR Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPases from skeletal or cardiac cells, was inhibited by thapsigargin, a sesquiterpene lactone from an umbelliferous plant Thapsia garganica. In this thesis project, thapsigargin (TG) was shown to be a potent inhibitor of all the SR/ER-type Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPases, but not the plasma membrane Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase in erythrocytes or the ryanodine receptor in skeletal muscle. TG inhibition is stoichiometric and potent: the affinity of TG to the Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase is high with the dissociation constant in subnanomolar. TG binding to the ATPase is apparently irreversible, even though the interaction is non-covalent. TG inhibits the steady state activities, Ca{dollar}\sp{lcub}2+{rcub}{dollar} transport and ATP hydrolysis of the SR Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase, as well as partial activities of the ATPase: Ca{dollar}\sp{lcub}2+{rcub}{dollar} and ATP binding at equilibrium and phosphoenzyme formation from inorganic phosphate are equally affected. Kinetic studies revealed that TG tightly binds an ATPase intermediate, which is produced during the ATPase catalytic cycle. The ATPase conformation (intermediate) sensitive to TG interaction can be also produced by the removal of Ca{dollar}\sp{lcub}2+{rcub}{dollar} by EGTA. The complex of TG and the ATPase intermediate does not react with any ATPase ligand and, therefore, is a dead-end complex. Spectroscopic and biochemical studies showed that this dead-end complex is in a different conformational state from the original intermediate that TG is initially binding. The changes in the ATPase conformation upon TG binding may be responsible for the global inhibition of ATPase partial activities. Since these partial activity sites (the nucleotide binding site, Ca{dollar}\sp{lcub}2+{rcub}{dollar} binding site, and the phosphorylation site) affected by TG are distantly located in the ATPase, the potent and stoichiometric inhibition of the ER/SR-type Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase by TG may occur at either a location that is critical in the function of all the partial activities of the ATPase or a location essential for transduction of the signals between the sites. Determination of the TG interaction site showed that TG is not binding to the phosphorylation site, the Ca{dollar}\sp{lcub}2+{rcub}{dollar} binding site, or the nucleotide binding site. (Abstract shortened with permission of author.)