ADP‐insensitive phosphoenzyme intermediate of sarcoplasmic reticulum Ca²⁺‐ATPase has a compact conformation resistant to proteinase K, V8 protease and trypsin

Abstract

Sarcoplasmic reticulum Ca 2+ -ATPase was digested with proteinase K, V8 protease and trypsin in the absence of Ca 2+ . Unphosphorylated enzyme was rapidly degraded. In contrast, ADP-insensitive phosphoenzyme formed with P i and phosphorylated state analogues produced by the binding of F 3 or orthovanadate, were almost completely resistant to the proteolysis except for tryptic cleavage at the T1 site (Arg 505 ). The results indicate that the phosphoenzyme and its analogues have a very compact form in the cytoplasmic region, being consistent with large domain motions (gathering of three cytoplasmic domains). Results further show that the structure of the enzyme with bound decavanadate is very similar to ADP-insensitive phosphoenzyme. Thapsigargin did not affect the changes in digestion time course induced by the formation of the phosphorylated state analogues. ß

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

“…Proteolysis-SR vesicles (0.45 mg/ml) were digested at 25°C with proteinase K (0.05 mg/ml) or trypsin (0.1 mg/ml) as described previously (10,11) in a buffer containing 50 mM LiCl, 2 mM EGTA, and 50 mM MES/Tris (pH 6). When possible effects of TG or K ϩ on the digestion rate were examined, 4 M TG or 100 mM KCl (in place of LiCl) was included in the above buffer.…”

“…When possible effects of TG or K ϩ on the digestion rate were examined, 4 M TG or 100 mM KCl (in place of LiCl) was included in the above buffer. The digested samples were subjected to Laemmli SDS-polyacrylamide gel electrophoresis (29), and the densitometric analyses of the gels stained with Coomassie Brilliant Blue R-250 were performed as described previously (10,11). The rate constant for the degradation of a 110-kDa ATPase chain by proteinase K and that for the cleavage at T2 site (Arg 198 ) by trypsin were obtained from the time courses (0 -60 min) by least squares fit as described (11).…”

“…It is likely that the interactions between the three cytoplasmic domains are somewhat rearranged (yet in a compactly organized state) upon the change from the tetrahedral ground state to the trigonal bipyramidal transition state and, thus, upon the associated possible rearrangements in coordination of the bound phosphate during the E2P hydrolysis and, further, it is likely that such changes rearrange the connected transmembrane helices so as to close the postulated Ca 2ϩ release pathway and gate. These conformational changes may be small but essential for the transport cycle and also could be the initial events in the process from the most compactly organized (E2P) to more relaxed (E2) state in the cytoplasmic domains (10,11). It is also possible that conformational changes of the P domain around Asp 351 during E2P hydrolysis may directly affect the arrangements in the transmembrane helices.…”

“…Our results further suggested that the stabilization energy provided by intimate contacts between the three domains in E2P will provide energy for moving transmembrane helices to open the Ca 2ϩ release pathway to lumen and thus release the bound Ca 2ϩ ions. During the subsequent E2P hydrolysis to E2, the compactly organized state of cytoplasmic domains becomes more relaxed (10,11), and the postulated Ca 2ϩ release pathway (the area surrounded by M3-M5 (8)) and lumenal gate (formed with contribution of lumenal loop connecting M7-M8 (L78) (18) and possibly by its interaction with L34 (9)) should be closed to prevent possible Ca 2ϩ leakage and prepare for the entry of new Ca 2ϩ from the cytoplasmic side to the transport sites (9). In the crystal structure of E2 fixed with TG, the postulated lumenal gate and pathway are, in fact, closed (9).…”

Deletions of Any Single Residues in Glu40-Ser48 Loop Connecting A Domain and the First Transmembrane Helix of Sarcoplasmic Reticulum Ca2+-ATPase Result in Almost Complete Inhibition of Conformational Transition and Hydrolysis of Phosphoenzyme Intermediate

“…The modeling of tubular crystals formed with decavanadate (E2V) revealed (5) that three cytoplasmic domains gather to form a most compactly organized single headpiece in E2V. With the limited proteolysis experiments, we previously showed (7,8) that E2V is very similar to E2P in domain organization and that E2P is the intermediate having the most compactly organized headpiece in the catalytic cycle. The results further indicated that a large rotation of A domain (by ϳ90° (5)) and its strong association with P and N domains most likely occur during the E1P to E2P transition and suggested that stabilization energy provided by intimate contacts between all three cytoplasmic domains in E2P will provide energy for moving transmembrane helices and release the bound Ca 2ϩ ions.…”