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

Abstract

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 (BeF⅐E2), bound aluminum fluoride (AlF⅐E2), and bound magnesium fluoride (MgF⅐E2), 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 BeF⅐E2 as in E2P but hydrophilic in MgF⅐E2 and AlF⅐E2; 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 BeF⅐E2 as in E2P, but only very slightly (hence, the release pathway is likely closed without thapsigargin) in MgF⅐E2 and AlF⅐E2 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. BeF⅐E2 thus has the features very similar to those characteristic of the E2P ground state, although AlF⅐E2 and MgF⅐E2 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؉ .Sarcoplasmic reticulum (SR) 1 Ca 2ϩ -ATPase is a representative member of P-type ion-transporting ATPases and catalyzes Ca 2ϩ transport coupled with ATP hydrolysis (Fig. 1) (Refs. 1 and 2 and, for recent reviews, see Refs. 3-6). In the catalytic cycle, the enzyme is activated by the binding of two Ca 2ϩ ions (E2 to Ca 2 E1, steps 1-2) and then autophosphorylated at Asp 351 by MgATP to form ADP-sensitive phosphoenzyme (E1P, steps 3-4). The subsequent isomeric transition to the ADPinsensitive form (E2P) will result in a reduction in affinity, a change in orientation of the Ca 2ϩ binding sites, and Ca 2ϩ release into lumen (steps 5-6). Finally, hydrolysis takes place and returns the enzyme into an unphosphorylated and Ca 2ϩ -unbound form (E2, steps 7-8). E2P can also be formed from P i in the presence of Mg 2ϩ and the absence of Ca 2ϩ by reversal of its hydrolysis, and the subsequent addition of high concentrations of Ca 2ϩ (from the lumenal side) can readily reverse the Ca 2ϩ ...

Interdomain communication in calcium pump as revealed in the crystal structures with transmembrane inhibitors

“…6). Intrinsic Trp fluorescence levels substantiate this finding (28). The fluorescence of E2⅐MgF 4 2Ϫ is low and similar to that of E2(TG), and the difference between E2 and E2P is likely to reflect the environment of Trp-288 at the bottom of M4, because this is the most prominent change among the transmembrane helices between these two structures.…”

Trinitrophenyl derivatives bind differently from parent adenine nucleotides to Ca 2+ -ATPase in the absence of Ca 2+

“…There is particular interest in the origin of the superfluorescence of TNP-AMP in the E2P ground state. E2 · BeF 3 − is a stable analogue of the E2P ground state, and superfluorescence is observed even in the presence of TG (25). This is in marked contrast to E2 · AlF 4 − , a stable transition state analogue, in which TNP-AMP exhibits little fluorescence (25).…”

“…E2 · BeF 3 − is a stable analogue of the E2P ground state, and superfluorescence is observed even in the presence of TG (25). This is in marked contrast to E2 · AlF 4 − , a stable transition state analogue, in which TNP-AMP exhibits little fluorescence (25). Hence we soaked TNP-AMP into E2 · BeF 3 − ðTGÞ and E2 · AlF 4 − ðTGÞ crystals.…”

“…Superfluorescence is characteristic of the E2P ground state and not observed with the transition state analogue (25). Hence TNP-AMP bound structures were also determined for E2 · BeF 3 − (E2P ground state analogue) and E2 · AlF 4 − (E2 ∼ P transition state analogue), to explore the origin of the superfluorescence.…”

Analysis of the Gastric H,K ATPase for Ion Pathways and Inhibitor Binding Sites

“…For the K + counter-transport pumps E 2 P must allow for both the escape of the outward moving ion (sodium or hydronium) and for the passage of luminal K + to the ion occlusion site. There is no open path in the PDB 1wpg structure from the ion site to the lumen, however, and it has been suggested that this structure represents the product-bound state, E 2 ·P, in which Ca 2+ has been released to the lumen, the ion sites have incorporated protons for counter transport, and the luminal gate has been closed (34). Nevertheless, the PDB 1wpg form has bound MgADP which is associated with an expanded luminal vestibule compared to the previously published E 2 conformation (PDB code 1iwo).…”