thapsigargin and beryllium-fluoride

The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding, Ca2+ translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively. The structures complete the cycle of nucleotide binding and cation transport of Ca2+-ATPase. Phosphorylation of the enzyme triggers the onset of a conformational change that leads to the opening of a luminal exit pathway defined by the transmembrane segments M1 through M6, which represent the canonical membrane domain of P-type pumps. Ca2+ release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen. The mechanism explains how P-type ATPases are able to form the steep electrochemical gradients required for key functions in eukaryotic cells.

Topics: Adenosine Triphosphate; Animals; Beryllium; Calcium; Crystallography, X-Ray; Fluorides; Ion Transport; Mass Spectrometry; Models, Molecular; Muscle, Skeletal; Phosphorylation; Protein Conformation; Rabbits; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Structure-Activity Relationship; Thapsigargin

We document here the intrinsic fluorescence and 45Ca2+ binding properties of putative "E2P-related" complexes of Ca2+-free ATPase with fluoride, formed in the presence of magnesium, aluminum, or beryllium. Intrinsic fluorescence measurements suggest that in the absence of inhibitors, the ATPase complex with beryllium fluoride (but not those with magnesium or aluminum fluoride) does constitute an appropriate analog of the "ADP-insensitive" phosphorylated form of Ca2+-ATPase, the so-called "E2P" state. 45Ca2+ binding measurements, performed in the presence of 100 mm KCl, 5 mm Mg2+, and 20% Me2SO at pH 8, demonstrate that this ATPase complex with beryllium fluoride (but again not those with magnesium or aluminum fluoride) has its Ca2+ binding sites accessible for rapid, low affinity (submillimolar) binding of Ca2+ from the luminal side of SR. In addition, we specifically demonstrate that in this E2P-like form of ATPase, the presence of thapsigargin, 2,5-di-tert-butyl-1,4-dihydroxybenzene, or cyclopiazonic acid prevents 45Ca2+ binding (i.e. presumably prevents opening of the 45Ca2+ binding sites on the SR luminal side). Since crystals of E2P-related forms of ATPase have up to now been described in the presence of thapsigargin only, these results suggest that crystallizing an inhibitor-free E2P-like form of ATPase (like its complex with beryllium fluoride) would be highly desirable, to unambiguously confirm previous predictions about the exit pathway from the ATPase transmembrane Ca2+ binding sites to the SR luminal medium.

Topics: Adenosine Triphosphatases; Aluminum; Aluminum Compounds; Animals; Beryllium; Binding Sites; Biochemistry; Biological Transport; Calcium; Calcium-Transporting ATPases; Cell Membrane; Enzyme Inhibitors; Fluorides; Hydrogen-Ion Concentration; Indoles; Ions; Magnesium; Microscopy, Fluorescence; Phosphorylation; Protein Binding; Protein Conformation; Rabbits; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Time Factors; Tryptophan

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+).

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Aluminum Compounds; Animals; Beryllium; Biochemical Phenomena; Biochemistry; Calcimycin; Calcium; Calcium-Transporting ATPases; Catalytic Domain; Cell Membrane; Chelating Agents; Crystallography, X-Ray; Cytoplasm; Dose-Response Relationship, Drug; Fluorides; Hydrogen-Ion Concentration; Hydrolysis; Magnesium Compounds; Models, Chemical; Muscle, Skeletal; Phosphorylation; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Rabbits; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Spectrometry, Fluorescence; Thapsigargin; Time Factors; Tryptophan; Vanadates