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• W2071514381 abstract "The functional importance of the length of the A/M1 linker (Glu40-Ser48) connecting the actuator domain and the first transmembrane helix of sarcoplasmic reticulum Ca2+-ATPase was explored by its elongation with glycine insertion at Pro42/Ala43 and Gly46/Lys47. Two or more glycine insertions at each site completely abolished ATPase activity. The isomerization of phosphoenzyme (EP) intermediate from the ADP-sensitive form (E1P) to the ADP-insensitive form (E2P) was markedly accelerated, but the decay of EP was completely blocked in these mutants. The E2P accumulated was therefore demonstrated to be E2PCa2 possessing two occluded Ca2+ ions at the transport sites, and the Ca2+ deocclusion and release into lumen were blocked in the mutants. By contrast, the hydrolysis of the Ca2+-free form of E2P produced from Pi without Ca2+ was as rapid in the mutants as in the wild type. Analysis of resistance against trypsin and proteinase K revealed that the structure of E2PCa2 accumulated is an intermediate state between E1PCa2 and the Ca2+-released E2P state. Namely in E2PCa2, the actuator domain is already largely rotated from its position in E1PCa2 and associated with the phosphorylation domain as in the Ca2+-released E2P state; however, in E2PCa2, the hydrophobic interactions among these domains and Leu119/Tyr122 on the top of second transmembrane helix are not yet formed properly. This is consistent with our previous finding that these interactions at Tyr122 are critical for formation of the Ca2+-released E2P structure. Results showed that the EP isomerization/Ca2+-release process consists of the following two steps: E1PCa2 → E2PCa2 → E2P + 2Ca2+; and the intermediate state E2PCa2 was identified for the first time. Results further indicated that the A/M1 linker with its appropriately short length, probably because of the strain imposed in E2PCa2, is critical for the correct positioning and interactions of the actuator and phosphorylation domains to cause structural changes for the Ca2+ deocclusion and release. The functional importance of the length of the A/M1 linker (Glu40-Ser48) connecting the actuator domain and the first transmembrane helix of sarcoplasmic reticulum Ca2+-ATPase was explored by its elongation with glycine insertion at Pro42/Ala43 and Gly46/Lys47. Two or more glycine insertions at each site completely abolished ATPase activity. The isomerization of phosphoenzyme (EP) intermediate from the ADP-sensitive form (E1P) to the ADP-insensitive form (E2P) was markedly accelerated, but the decay of EP was completely blocked in these mutants. The E2P accumulated was therefore demonstrated to be E2PCa2 possessing two occluded Ca2+ ions at the transport sites, and the Ca2+ deocclusion and release into lumen were blocked in the mutants. By contrast, the hydrolysis of the Ca2+-free form of E2P produced from Pi without Ca2+ was as rapid in the mutants as in the wild type. Analysis of resistance against trypsin and proteinase K revealed that the structure of E2PCa2 accumulated is an intermediate state between E1PCa2 and the Ca2+-released E2P state. Namely in E2PCa2, the actuator domain is already largely rotated from its position in E1PCa2 and associated with the phosphorylation domain as in the Ca2+-released E2P state; however, in E2PCa2, the hydrophobic interactions among these domains and Leu119/Tyr122 on the top of second transmembrane helix are not yet formed properly. This is consistent with our previous finding that these interactions at Tyr122 are critical for formation of the Ca2+-released E2P structure. Results showed that the EP isomerization/Ca2+-release process consists of the following two steps: E1PCa2 → E2PCa2 → E2P + 2Ca2+; and the intermediate state E2PCa2 was identified for the first time. Results further indicated that the A/M1 linker with its appropriately short length, probably because of the strain imposed in E2PCa2, is critical for the correct positioning and interactions of the actuator and phosphorylation domains to cause structural changes for the Ca2+ deocclusion and release. Sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) 2The abbreviations used are: SERCA1a, adult fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase; SR, sarcoplasmic reticulum; EP, phosphoenzyme; E1P, ADP-sensitive phosphoenzyme; E2P, ADP-insensitive phosphoenzyme; TG, thapsigargin; MOPS, 3-(N-morpholino)propanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; Vi, orthovanadate; PrtK, proteinase K. is a representative member of P-type ion transporting ATPases and catalyzes Ca2+ transport coupled with ATP hydrolysis (Fig. 1) (Refs. 1Hasselbach W. Makinose M. Biochem. Z. 1961; 333: 518-528PubMed Google Scholar, 2Ebashi S. Lipmann F. J. Cell Biol. 1962; 14: 389-400Crossref PubMed Scopus (360) Google Scholar and for recent reviews see Refs. 3Inesi G. Sumbilla C. Kirtley M.E. Physiol. Rev. 1990; 70: 749-776Crossref PubMed Scopus (152) Google Scholar, 4Møller J.V. Juul B. le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (659) Google Scholar, 5MacLennan D.H. Rice W.J. Green N.M. J. Biol. Chem. 1997; 272: 28815-28818Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 6McIntosh D.B. Adv. Mol. Cell Biol. 1998; 23: 33-99Crossref Scopus (40) Google Scholar, 7Toyoshima C. Inesi G. Annu. Rev. Biochem. 2004; 73: 268-292Crossref Scopus (297) Google Scholar). In the catalytic cycle, the enzyme is activated by binding of two Ca2+ ions to the transport sites (E2to E1Ca2, steps 1 and 2) and then autophosphorylated at Asp351 with MgATP to form the ADP-sensitive phosphoenzyme (E1P, step 3), which can react with ADP to regenerate ATP in the reverse reaction. Upon formation of this EP, the bound Ca2+ ions are occluded in the transport sites (E1PCa2). The subsequent isomeric transition to the ADP-insensitive form (E2P), i.e. the loss of the ADP sensitivity at the catalytic site, results in rearrangements of the Ca2+-binding sites to deocclude Ca2+, reduce the affinity, open the luminal gate, and thus release Ca2+ into the lumen (steps 4 and 5). As an intermediate state in the EP isomerization/Ca2+-release process, E2PCa2 has been postulated (e.g. see Ref. 8Inao S. Kanazawa T. Biochim. Biophys. Acta. 1986; 857: 28-37Crossref PubMed Scopus (10) Google Scholar), although this state has never been identified. Finally, the E2P hydrolysis takes place and returns the enzyme into an unphosphorylated and Ca2+-unbound form (E2, steps 6 and 7). The transport cycle is totally reversible, e.g. E2P can be formed from Pi in the presence of Mg2+ and the absence of Ca2+ by reversal of its hydrolysis, and the subsequent addition of high concentrations of Ca2+ to E2P reverse the Ca2+-releasing step and the E1P to E2P isomerization. The enzyme has three cytoplasmic domains as follows: the nucleotide binding (N), phosphorylation (P), and actuator (A) domains, and 10 transmembrane helices M1-M10 (Fig. 2). During the EP isomerization/Ca2+-release E1PCa2 → E2P + 2Ca2+, the A domain largely rotates (by ∼110°) parallel to the membrane and associates with the P domain (see Refs. 9Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1613) Google Scholar, 10Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (808) Google Scholar, 11Sørensen T. L.-M. Møller J.V. Nissen P. Science. 2004; 304: 1672-1675Crossref PubMed Scopus (372) Google Scholar, 12Toyoshima C. Mizutani T. Nature. 2004; 430: 529-535Crossref PubMed Scopus (382) Google Scholar, 13Toyoshima C. Nomura H. Tsuda T. Nature. 2004; 432: 361-368Crossref PubMed Scopus (382) Google Scholar, 14Olesen C. Sørensen T. L.-M. Nielsen R.C. Møller J.V. Nissen P. Science. 2004; 306: 2251-2255Crossref PubMed Scopus (235) Google Scholar, 15Danko S. Daiho T. Yamasaki K. Kamidochi M. Suzuki H. Toyoshima C. FEBS Lett. 2001; 489: 277-282Crossref PubMed Scopus (60) Google Scholar, 16Danko S. Yamasaki K. Daiho T. Suzuki H. Toyoshima C. FEBS Lett. 2001; 505: 129-135Crossref PubMed Scopus (92) Google Scholar, 17Danko S. Yamasaki K. Daiho T. Suzuki H. J. Biol. Chem. 2004; 279: 14991-14998Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) (see E1·AlFx·ADP (the E1PCa2·ADP analog) → E2·MgF−4 (the E2·Pi analog) in Fig. 2). The interactions of the A domain with the P domain in the E2P state occur at three regions (Fig. 2, semitransparent purple, blue, and orange on E2·MgF−4): i.e. at the T181GES loop with the residues of the P domain around Asp351; at the Val200 loop (Asp196-Asp203) with the polar residues of the P domain (Arg678/Glu680/Arg656/Asp660); and at the Tyr122-hydrophobic cluster formed by seven hydrophobic residues gathered from the A domain (Ile179/Leu180/Ile232), the P domain (Val705/Val726), and the top part of M2 (the A/M2 linker region, Leu119/Tyr122). The formation of the A-P domain interaction at the T181GES loop has been predicted to be critical for the loss of ADP sensitivity at the catalytic site, i.e. the E1P to E2P isomerization, by the structural and mutation studies (18Clarke D.M. Loo T.W. MacLennan D.H. J. Biol. Chem. 1990; 265: 14088-14092Abstract Full Text PDF PubMed Google Scholar, 19Wang G. Yamasaki K. Daiho T. Suzuki H. J. Biol. Chem. 2005; 280: 26508-26516Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 20Anthonisen N. Clausen J.D. Andersen J.P. J. Biol. Chem. 2006; 281: 31572-31582Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The mutations at the latter two interaction regions were shown not to inhibit the E1P to E2P isomerization but to markedly retard the subsequent EP decay (19Wang G. Yamasaki K. Daiho T. Suzuki H. J. Biol. Chem. 2005; 280: 26508-26516Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 21Kato S. Kamidochi M. Daiho T. Yamasaki K. Wang G. Suzuki H. J. Biol. Chem. 2003; 278: 9624-9629Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 22Yamasaki K. Daiho T. Danko S. Suzuki H. J. Biol. Chem. 2004; 279: 2202-2210Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Its kinetics were consistent with the view that there is a Ca2+-releasing step from E2PCa2(E2PCa2 → E2P + 2Ca2+) before the E2P hydrolysis and that this Ca2+-releasing step is blocked and became the kinetic limit for the EP decay by the disruption of the A-P domain interactions at each of the latter two regions (19Wang G. Yamasaki K. Daiho T. Suzuki H. J. Biol. Chem. 2005; 280: 26508-26516Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 21Kato S. Kamidochi M. Daiho T. Yamasaki K. Wang G. Suzuki H. J. Biol. Chem. 2003; 278: 9624-9629Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 22Yamasaki K. Daiho T. Danko S. Suzuki H. J. Biol. Chem. 2004; 279: 2202-2210Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). It is therefore very interesting to know how the motions and interactions of the A and P domains progress during the postulated successive steps E1PCa2 → E2PCa2 → E2P + 2Ca2+ as the key structural events in the energy coupling between the cytoplasmic and transmembrane domains. In this respect, it is also critical to clarify and distinguish the structural roles of the three linkers connecting the A domain with M1′/M1, M2, and M3 (A/M1, A/M2, and A/M3 linkers). Tyr122/Leu119 involved in the aforementioned Tyr122-hydrophobic cluster is at the A/M2 linker region. The A/M3 linker, because of its strain, has been predicted to be important for the large rotation of the A domain in the EP isomerization (12Toyoshima C. Mizutani T. Nature. 2004; 430: 529-535Crossref PubMed Scopus (382) Google Scholar, 13Toyoshima C. Nomura H. Tsuda T. Nature. 2004; 432: 361-368Crossref PubMed Scopus (382) Google Scholar). Regarding the A/M1 linker, we recently found (23Daiho T. Yamasaki K. Wang G. Danko S. Iizuka H. Suzuki H. J. Biol. Chem. 2003; 278: 39197-39204Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) that its shortening by deletions of any single residues within this linker (Glu40-Ser48) blocks the E1P to E2P isomerization and the hydrolysis of the Ca2+-free form of E2P, whereas substitutions of any residues in this linker do not inhibit the function. Our results indicated that the A/M1 linker with its correct length critically contributes to the EP isomerization/Ca2+ release and to the E2P hydrolysis, and we pointed out the possible importance of this linker in the proper positioning of the A and P domains for their motions and association during these processes. Therefore, in this study, we further explored the structural roles of this linker and the structural events occurring in the processes by elongating this linker with insertion of glycines (see Fig. 2). Results demonstrated that the elongation of the linker markedly accelerates the E1PCa2 to E2PCa2 isomerization, strongly stabilizes E2PCa2 that possesses two occluded Ca2+ ions at the transport sites, and blocks the Ca2+ deocclusion and release from E2PCa2. Thus, for the first time, the intermediate state E2PCa2 was identified and trapped in this study. We were then able to characterize the structure of this state. Results revealed that the correct length of the A/M1 linker is critical for structural events in each of successive steps in E1PCa2 → E2PCa2 → E2P + 2Ca2+ and E2P + H2O → E2 + Pi, and they further suggested how the motions and interactions of the properly positioned A and P domains progress with the critical contribution of the linker to accomplish the successive structural events in these steps. Our study also revealed the importance of M1′ directly connected with the A/M1 linker likely for forming the base of this linker. Mutagenesis and Expression—The QuikChange™ site-directed mutagenesis method (Stratagene, La Jolla, CA) was utilized for the insertions and substitutions of residues in the rabbit SERCA1a cDNA. The ApaI-KpnI restriction fragments with the desired mutation were excised from the plasmid and ligated back into the corresponding region in the full-length SERCA1a cDNA in the pMT2 expression vector (24Kaufman R.J. Davies M.V. Pathak V.K. Hershey J.W.B. Mol. Cell. Biol. 1989; 9: 946-958Crossref PubMed Scopus (333) Google Scholar). The pMT2 DNA was transfected into COS-1 cells by the liposome-mediated transfection method. Microsomes were prepared from the cells as described previously (25Maruyama K. MacLennan D.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3314-3318Crossref PubMed Scopus (263) Google Scholar). The “control microsomes” were prepared from COS-1 cells transfected with the pMT2 vector containing no SERCA1a cDNA. The amount of expressed SERCA1a was quantified by a sandwich enzyme-linked immunosorbent assay as described previously (26Daiho T. Yamasaki K. Suzuki H. Saino T. Kanazawa T. J. Biol. Chem. 1999; 274: 23910-23915Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Expression levels of the wild-type SERCA1a and mutants examined in this study were 2-3% of total microsomal proteins, except a mutant 4950525354S for M1′ with serine substitutions of Leu49/Trp50/Leu52/Val53/Ile54, which showed markedly reduced expression (only ∼15-20% of the wild type). Ca2+-ATPase Activity—The rate of ATP hydrolysis was determined at 25 °C in a mixture containing 1 μg of microsomal protein, 0.1 mm [γ-32P]ATP, 1 μm A23187, 0.1 m KCl, 7 mm MgCl2, 0.55 mm CaCl2, 0.5 mm EGTA, and 50 mm MOPS/Tris (pH 7.0). The Ca2+-ATPase activity was obtained by subtracting the Ca2+-independent ATPase activity, which was determined in the presence of 5 mm EGTA without added CaCl2, otherwise as above. The ATPase activity/mg of expressed SERCA1a protein was calculated from the amount of expressed SERCA1a and the Ca2+-ATPase activity of expressed SERCA1a, which was obtained by subtracting the Ca2+-ATPase activity of the control microsomes from that of the microsomes expressing SERCA1a. This background level with the control microsomes was as low as 3% of the activity of microsomes expressing the wild-type SECRA1a. Formation and Hydrolysis of EP—Phosphorylation of SERCA1a in microsomes with [γ-32P]ATP or 32Pi and dephosphorylation of 32P-labeled SERCA1a were performed under conditions described in the figure legends. The reactions were quenched with ice-cold trichloroacetic acid containing Pi. Rapid kinetics measurements of phosphorylation and dephosphorylation were performed with a handmade rapid mixing apparatus (27Kanazawa T. Saito M. Tonomura Y. J. Biochem. (Tokyo). 1970; 67: 693-711Crossref PubMed Scopus (143) Google Scholar), otherwise as above. The precipitated proteins were separated by 5% SDS-PAGE at pH 6.0 according to Weber and Osborn (28Weber K. Osborn M. J. Biol. Chem. 1969; 244: 4406-4412Abstract Full Text PDF PubMed Google Scholar). The radioactivity associated with the separated Ca2+-ATPase was quantitated by digital autoradiography as described (29Daiho T. Suzuki H. Yamasaki K. Saino T. Kanazawa T. FEBS Lett. 1999; 444: 54-58Crossref PubMed Scopus (27) Google Scholar). The amount of EP formed with the expressed SERCA1a was obtained by subtracting the background radioactivity with the control microsomes. This background was less than 5% of the radioactivity of EP formed with the expressed wild-type SERCA1a. The amount of EP/mg of SERCA1a protein was calculated from the amount of EP thus obtained and the amount of expressed SERCA1a. Ca2+ Occlusion in EP—As described in Fig. 8 legend, the expressed mutant SERCA1a in microsomes was phosphorylated with ATP and 45CaCl2, and then the mixture was diluted by a “washing solution” containing excess EGTA and immediately filtered through a 0.45-μm nitrocellulose membrane filter (Millipore). The filter was washed four times with 2 ml of the washing solution, and 45Ca2+ remaining on the filter was quantitated. The amount of Ca2+ specifically bound to the transport sites of EP in the expressed SERCA1a was obtained by subtracting the amount of nonspecific Ca2+ binding, which was determined by including 1 μm thapsigargin (TG) in the phosphorylation mixture, otherwise as above. This background subtraction is ensured by the fact that TG inhibits the Ca2+ binding at the transport sites and the EP formation (30Sagara Y. Wade J.B. Inesi G. J. Biol. Chem. 1992; 267: 1286-1292Abstract Full Text PDF PubMed Google Scholar). The background level thus determined was ∼60% of the total amount of 45Ca2+ remaining on the filter when the maximum amount of EP was present (i.e. at the zero time of EP decay in Fig. 8). It should be noted that the specifically bound Ca2+ in EP thus determined represents the occluded one because it is not released even after the extensive washing by EGTA. The Ca2+ occluded/mg of expressed SERCA1a protein was calculated from the amount of expressed SERCA1a and the amount of occluded Ca2+. The Ca2+ occlusion resulted from Ca2+ binding to E2P in the reverse reaction of the Ca2+-release process was also determined. In this case, E2P was first formed from Pi in the absence of Ca2+, and 45Ca2+ was then added to E2P otherwise as described in Fig. 10 legend, and the amount of occluded 45Ca2+ was determined as above.FIGURE 10Accessibility of luminal Ca2+ in E2P formed from Pi without Ca2+. Microsomes expressing the wild type (A) or the mutant 2Gi-46/47 (B) were phosphorylated with 32Pi in 2.5 μl of a mixture as described in Fig. 9. The mixture was then cooled and diluted 100-fold at 0 °C with 247.5 μl of a solution containing various concentrations of CaCl2 in 1 μm A23187, 101 mm KCl, 1 mm EGTA, 7 mm MgCl2, and 50 mm MOPS/Tris (pH 7.0) to give the final Ca2+ concentrations as indicated with different symbols. The amount of EP remaining at the indicated time after this Ca2+ addition was determined and shown as percentage of the amount of EP at zero time, which was determined immediately before the Ca2+ addition. The EP decay occurred in two phases. The first and rapid phase completed within a few seconds corresponding to the hydrolysis of E2P without bound Ca2+ (see Fig. 7). Essentially the same results were observed in the mutants 3Gi-46/47 and 4Gi-46/47 (data not shown) as in 2Gi-46/47. C, content of EP in the slow and second phase at each Ca2+ concentration was obtained by extrapolating to the zero time and plotted versus the Ca2+ concentration. K0.5 values for the Ca2+ activation and Hill coefficients obtained by fitting to the Hill equation (solid lines) were 1.4 mm and 1.5 (wild type), 1.3 mm and 1.5 (1Gi-46/47), 1.2 mm and 1.4 (2Gi-46/47), 1.0 mm and 1.2 (3Gi-46/47), 0.9 mm and 1.4 (4Gi-46/47), as summarized in Table 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Limited Proteolysis of Major Intermediates and Western Blot Analysis—Major intermediates and its stable analogs of the Ca2+-ATPase were produced and subjected to the structural analysis by limited proteolysis with trypsin and proteinase K (PrtK) as described in Fig. 12 legend. The digests were separated by 10.5 or 7.5% SDS-PAGE, according to Laemmli (31Laemmli U.K. Nature. 1970; 277: 680-685Crossref Scopus (207233) Google Scholar), and blotted onto a polyvinylidene fluoride membrane and then incubated with IIH11 monoclonal antibody to the rabbit SERCA1a (Affinity Bioreagents), which recognizes an epitope between Ala199-Arg505. After incubation with secondary antibody (goat anti-mouse IgG-horseradish peroxidase-conjugated), the bound proteins were probed using an enhanced chemiluminescence-linked detection system (ECL Plus, GE Healthcare). Miscellaneous—Protein concentrations were determined by the method of Lowry et al. (32Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with bovine serum albumin as a standard. Data were analyzed by nonlinear regression using the program Origin (Microcal Software, Inc., Northampton, MA). Three-dimensional models of the enzyme were reproduced by the program VMD (33Humphrey W. Dalke A. Schulten K. J. Mol. Graphics. 1996; 14: 33-38Crossref PubMed Scopus (39018) Google Scholar). Ca2+-ATPase Activity—The specific Ca2+-ATPase activity of the expressed SERCA1a mutants was determined at saturating 50 μm Ca2+ and 25 °C and compared with that of the wild type (Fig. 3). Insertion of one glycine between Gly46 and Lys47 (1Gi-46/47) and between Pro42 and Ala43 (1Gi-42/43) within the A/M1 linker slowed the ATPase activity by ∼50%. Insertion of two or more glycines at each of these sites abolished the activity almost completely (2Gi-46/47 to 6Gi-46/47 and 4Gi-42/43). Thus, the elongation of the linker by the glycine insertion at the two different positions within the A/M1 linker exhibited the same inhibitory effects on the ATPase, indicating the importance of the correct length of this loop in the function. 3In this regard, we should describe the reasons why we chose the positions 42/43 and 46/47 for the insertions. First of all, to explore the importance of the length of the linker and to assign the effects of the insertions straightforwardly as those of the elongation of the linker, it is necessary or better to have insertions at two (at least) different positions. Then for the choice of the two, the positions approximately at one-third and at two-thirds of the length of the linker would be reasonable because the A/M1 linker, Glu40-Ser48 loop, is an extended loop without helical structure (see Fig. 2) and has no extensive interactions with other parts of the ATPase molecule (actually the substitutions of any single residues in this loop did not impair the function (23Daiho T. Yamasaki K. Wang G. Danko S. Iizuka H. Suzuki H. J. Biol. Chem. 2003; 278: 39197-39204Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar)). Also, the two positions should be not too close to the junctions of the A/M1 linker with the A domain and with M1′ (and to the membrane lipids). This is to avoid possible disruption of the structures at the junctions. With these reasons, we chose the two insertion positions at 42/43 and 46/47, and we therefore distributed the two on the linker but did not position them very close to each other at the middle part or at either side of the linker. As a reason for the choice of glycine(s) as the amino acid for the insertions, we intended to minimize possible disruption of the extended loop structure of the linker and to avoid possible formation of side-chain interactions. The results in Fig. 3 of the mutants with the insertions at the two different positions therefore agree with the view that the observed effects are because of the elongation of the A/M1 linker. The kinetic analysis shown in this study further revealed that the properties of the mutants with the insertions at the two different positions are essentially the same. We then examined the possible effects of the 4-amino acidinsertion in the C- and N-terminal regions of the A/M1 linker: between Val53 and Ile54 on the helix M1′ and between Thr22 and Gly23 in the immediate vicinity of the Thr25-Tyr36 helix (see Fig. 2b). In the mutants 4Ai-53/54 and 4Ai-22/23, alanines were inserted (for M1′, we intended to minimize possible disruption of the helical structure). These mutants exhibited the high ATPase activity (Fig. 3) and the Ca2+ transport coupled with the ATP hydrolysis (data not shown). Thus the insertions at the adjacent regions of the A/M1 linker did not inhibit the activity. We inserted amino acids also in the immediate N-terminal region of the A/M1 linker, for example at His32-Leu33 and at Gly37-His38; however, the protein expression levels of these mutants were extremely low (less than ∼10% of the wild type); therefore, their functional analysis was not possible. We also investigated the possible importance of amphipathic property of the helix M1′ (Trp50-Glu58 in E1·AlFx·ADP or Leu49-Gln56 in E2·MgF−4), which is directly connected with the A/M1 linker and formed by kinking of M1. M1′ lies on the membrane surface, having hydrophobic residues aligned on the membrane side (Leu49/Trp50/Val53/Ile54) and the polar residues (Glu51/Glu55/Gln56/Glu58) on the cytoplasmic side (Fig. 2b). Therefore, the hydrophobic interactions of M1′ with the membrane core and/or its hydrophilic interactions at the membrane surface may possibly be important for function (13Toyoshima C. Nomura H. Tsuda T. Nature. 2004; 432: 361-368Crossref PubMed Scopus (382) Google Scholar). Previously the mutations of the single residues on the M1′ region were found to have almost no or only a slight effect on the activity (23Daiho T. Yamasaki K. Wang G. Danko S. Iizuka H. Suzuki H. J. Biol. Chem. 2003; 278: 39197-39204Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 34Einholm A.P. Vilsen B. Andersen J.P. J. Biol. Chem. 2004; 279: 15888-15896Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). In this study, we therefore introduced the extensive nonconservative substitutions as follows: the serine substitution of all Leu49/Trp50/Leu52/Val53/Ile54 (4950525354S) and the alanine substitution of all Glu51/Glu55/Gln56/Glu58/Asp59 (5155565859A). The mutant 4950525354S exhibited the markedly reduced ATPase activity (17% that of the wild type), whereas the mutant 5155565859A exhibited a fairly high activity (70% that of the wild type). The results indicate that the hydrophobic interaction of M1′ with the membrane core may be important. EP Formation from ATP and the E2-E1Ca2 Transition—The amount of EP formed from ATP at saturating 50 μm Ca2+ was determined at steady state and 0 °C with 10 μm ATP under the conditions otherwise the same as those for the ATPase assay (open bars in Fig. 4). All the mutants formed EP with the amounts comparable with that of the wild type (3.31 ± 0.14 nmol/mg of the SERCA1a protein (n = 4)) except that the mutant 4950525354S exhibited a somewhat reduced amount. The Ca2+ affinity of the mutants in the E2to E1Ca2 transition was estimated by the Ca2+ dependence of the EP formation from ATP and was found to be nearly the same as that of the wild type (see the Ca2+ affinity and the Hill coefficient in Table 1). The mutant 4Ai-53/54 among those examined showed a slightly reduced affinity. We further found that the first-order rate constants of the E2to E1Ca2 transition (steps 1 and 2) in the examined mutants were nearly the same as in the wild type (Table 1). In this experiment, the enzyme was first preincubated in the absence of Ca2+ at pH 6, where the equilibrium between E1 and E2 is most shifted to E2 (35Pick U. Karlish S.J.D. J. Biol. Chem. 1982; 257: 6120-6126Abstract Full Text PDF PubMed Google Scholar), and then the phosphorylation was initiated by the simultaneous addition of saturating Ca2+ and ATP. When ATP was added to the enzyme preincubated with Ca2+, otherwise as above, the EP formation was much faster in the mutants as well as in the wild type, and therefore the rates obtained above actually reflect the rate-limiting E2to E1Ca2 transition.TABLE 1Kinetic parameters determined for partial reaction stepsE2 to E1Ca2E1Ca2 to E1PCa2aThe rate of the E1PCa2 formation from E1Ca2 in the presence of K+ was very similar to that in the absence of K+ (see Fig. 5, A and C) and therefore not shown for simplicity.Loss of ADP sensitivityDecay of EPATPHydrolysis of E2PPiE2P to E2PCa2/E1PCa2Ca2+ affinityRateCa2+ affinityK0.5n(−)K+(+)K+(−)K+K0.5nμms−1%s−1%s−1s−1%s−1%s−1%mmWild type0.161.870.213(100)1.37(100)bNot determined because the accumulation of ADP-insensitive EP was low.0.34(100)0.0390(100)0.64(100)1.41.54Ai-22/230.181.650.292(137)1.30(95)bNot determined because the accumulation of ADP-insensitive EP was low.0.63(188)0.0613(157)1.07(169)2.21.11Gi-42/430.161.880.157(74)1.91(140)bNot determined because the accumulation of ADP-insensitive EP was low.0.53(156)0.0247(63)1.07(169)2.31.54Gi-42/430.131.730.1" @default.
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