FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2008477360> ?p ?o ?g. }

• W2008477360 endingPage "39204" @default.
• W2008477360 startingPage "39197" @default.
• W2008477360 abstract "Possible roles of the Glu40-Ser48 loop connecting A domain and the first transmembrane helix (M1) in sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) were explored by mutagenesis. Deletions of any single residues in this loop caused almost complete loss of Ca2+-ATPase activity, while their substitutions had no or only slight effects. Single deletions or substitutions in the adjacent N- and C-terminal regions of the loop (His32-Asn39 and Leu49-Ile54) had no or only slight effects except two specific substitutions of Asn39 found in SERCA2b in Darier's disease pedigrees. All the single deletion mutants for the Glu40-Ser48 loop and the specific Asn39 mutants formed phosphoenzyme intermediate (EP) from ATP, but their isomeric transition from ADP-sensitive EP (E1P) to ADP-insensitive EP (E2P) was almost completely or strongly inhibited. Hydrolysis of E2P formed from Pi was also dramatically slowed in these deletion mutants. On the other hand, the rates of the Ca2+-induced enzyme activation and subsequent E1P formation from ATP were not altered by the deletions and substitutions. The results indicate that the Glu40-Ser48 loop, with its appropriate length (but not with specific residues) and with its appropriate junction to A domain, is a critical element for the E1P to E2P transition and formation of the proper structure of E2P, therefore, most likely for the large rotational movement of A domain and resulting in its association with P and N domains. Results further suggest that the loop functions to coordinate this movement of A domain and the unique motion of M1 during the E1P to E2P transition. Possible roles of the Glu40-Ser48 loop connecting A domain and the first transmembrane helix (M1) in sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) were explored by mutagenesis. Deletions of any single residues in this loop caused almost complete loss of Ca2+-ATPase activity, while their substitutions had no or only slight effects. Single deletions or substitutions in the adjacent N- and C-terminal regions of the loop (His32-Asn39 and Leu49-Ile54) had no or only slight effects except two specific substitutions of Asn39 found in SERCA2b in Darier's disease pedigrees. All the single deletion mutants for the Glu40-Ser48 loop and the specific Asn39 mutants formed phosphoenzyme intermediate (EP) from ATP, but their isomeric transition from ADP-sensitive EP (E1P) to ADP-insensitive EP (E2P) was almost completely or strongly inhibited. Hydrolysis of E2P formed from Pi was also dramatically slowed in these deletion mutants. On the other hand, the rates of the Ca2+-induced enzyme activation and subsequent E1P formation from ATP were not altered by the deletions and substitutions. The results indicate that the Glu40-Ser48 loop, with its appropriate length (but not with specific residues) and with its appropriate junction to A domain, is a critical element for the E1P to E2P transition and formation of the proper structure of E2P, therefore, most likely for the large rotational movement of A domain and resulting in its association with P and N domains. Results further suggest that the loop functions to coordinate this movement of A domain and the unique motion of M1 during the E1P to E2P transition. Sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) 1The abbreviations used are: SERCA1a, adult fast twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase; EP, phosphoenzyme; E1P, ADP-sensitive phosphoenzyme; E2P, ADP-insensitive phosphoenzyme; TG, thapsigargin; E2V, decavanadate-bound crystal structure; MOPS, 3-(N-morpholino)propanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid. 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 and 2Ebashi S. Lipmann F. J. Cell Biol. 1962; 14: 389-400Crossref PubMed Scopus (360) Google Scholar, and for recent reviews, see Refs. 3M⊘ller J.V. Juul B. le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (660) Google Scholar and 4MacLennan 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). In the catalytic cycle, the enzyme is activated by binding of two Ca2+ ions (E2 to E1Ca2, steps 1 and 2) and then autophosphorylated by MgATP to form ADP-sensitive phosphoenzyme (E1P, step 3). Upon formation of this EP, the bound Ca2+ ions are occluded in the transport sites. The subsequent isomeric transition to ADP-insensitive form (E2P, step 4) will result in a reduction in affinity and a change in orientation of the Ca2+ binding sites, and thus a Ca2+ release into lumen (step 5). Finally, hydrolysis takes place and returns the enzyme into an unphosphorylated and Ca2+-unbound form (E2, step 6). E2P can also be formed from Pi in the presence of Mg2+ and absence of Ca2+ by reversal of its hydrolysis. The enzyme has three cytoplasmic domains (N, P, and A), which are widely separated in the Ca2+ bound form (E1Ca2) and associated in the Ca2+-unbound and thapsigargin-bound form (E2(TG)) (5Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1619) Google Scholar, 6Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar) (Fig. 2). The modeling of tubular crystals formed with decavanadate (E2V) revealed (5Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1619) Google Scholar) that three cytoplasmic domains gather to form a most compactly organized single headpiece in E2V. With the limited proteolysis experiments, we previously showed (7Danko S. Daiho T. Yamasaki K. Kamidochi M. Suzuki H. Toyoshima C. FEBS Lett. 2001; 489: 277-282Crossref PubMed Scopus (60) Google Scholar, 8Danko S. Yamasaki K. Daiho T. Suzuki H. Toyoshima C. FEBS Lett. 2001; 505: 129-135Crossref PubMed Scopus (92) Google Scholar) 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 ∼90° (5Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1619) Google Scholar)) 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 Ca2+ ions. It is thus crucial to find out structural elements essential for the A domain movement and resulting domain organization and for transmitting these changes to transmembrane helices. We have recently identified the Lys189-Lys205 outermost loop of A domain as to make intimate contact with P domain for formation of the proper structure of Ca2+-released form of E2P in step 5 (9Kato 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). P domain was actually documented with the crystal structures E1Ca2 and E2(TG) to function as a coordinator for transmitting the movements of the transmembrane helices to the cytoplasmic domains (6Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar). In addition, it is also possible that the loops connecting A domain with the transmembrane helices (M1-M3) may play roles in the movement of A domain and in transmitting the movement to transmembrane helices. The likely importance of interactions of M2- and M3-connecting loops with P domain was previously pointed out (6Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar, 10M⊘ller J.V. Lenoir G. Marchand C. Montigny C. le Maire M. Toyoshima C. Juul B.S. Champeil P. J. Biol. Chem. 2002; 277: 38647-38659Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), and in fact the proteolytic cut or mutation of the M3-connecting loop at or near the interaction sites was shown to cause almost complete loss of the Ca2+-ATPase activity due to blockage of the E1P to E2P transition (10M⊘ller J.V. Lenoir G. Marchand C. Montigny C. le Maire M. Toyoshima C. Juul B.S. Champeil P. J. Biol. Chem. 2002; 277: 38647-38659Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 11Andersen J.P. Vilsen B. Leberer E. MacLennan D.H. J. Biol. Chem. 1989; 264: 21018-21023Abstract Full Text PDF PubMed Google Scholar, 12Imamura Y. Kawakita M. J. Biochem. (Tokyo). 1989; 105: 775-781Crossref PubMed Scopus (26) Google Scholar, 13le Maire M. Lund S. Viel A. Champeil P. M⊘ller J.V. J. Biol. Chem. 1990; 265: 1111-1123Abstract Full Text PDF PubMed Google Scholar, 14Juul B. Møller J.V. Taniguchi K., and Kaya, S. Na/K-ATPase and Related ATPases. Elsevier Science Publishers B. V., Amsterdam2000: 233-236Google Scholar). On the other hand, the Glu40-Ser48 loop with an extended structure, the major part of the M1-connecting loop, is well separated from and not having significant interactions with other parts of the molecule in E2V as well as in E1Ca2 and E2(TG) (Fig. 2), and possible roles of this loop remain unknown. Interestingly, not only A domain, but also M1 connected to this loop, seems to undergo very large and unique structural changes during Ca2+ transport cycle, i.e. up-and-down and horizontal movements and bending near the membrane surface (6Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar). In the present study, we focused on and explored possible roles of the Glu40-Ser48 loop by site-specific mutagenesis and found that deletions of any single residues in this loop result in almost complete loss of the Ca2+-ATPase activity, while their substitutions have no or only slight effects. Results further showed that both the E1P to E2P transition and the E2P hydrolysis are almost completely inhibited in all these single deletion mutants. The results indicate that the Glu40-Ser48 loop is critical for formation of the proper structure of E2P and further suggest that the loop may coordinate the motions of A domain and M1 during the E1P to E2P transition and thus possibly contribute to the rearrangement of the transmembrane helices. Mutagenesis and Expression—Mutations were created by PCR using the QuikChange™ site-directed mutagenesis kit (Stratagene) and plasmid pGEM7-Zf(+) (Promega) containing the ApaI-KpnI fragment of the rabbit SERCA1a cDNA as a template. The ApaI-KpnI fragments were then excised from the PCR products and used to replace the corresponding region in the full-length SERCA1a cDNA in the pMT2 expression vector (15Kaufman 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 (16Maruyama K. MacLennan D.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3314-3318Crossref PubMed Scopus (264) 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 (17Daiho 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). The expression levels of all the mutants in the microsomes were comparable with that of the wild type. ATPase Activity—The rate of ATP hydrolysis was determined at 25 °C in a mixture containing 20 μg/ml microsomal protein, 0.1 mm [γ-32P]ATP, 1 μm A23187, 7 mm MgCl2, 0.1 m KCl, 50 mm MOPS/Tris (pH 7.0), 0.55 mm CaCl2, and 0.5 mm EGTA. 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 specific 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 legends to figures. 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 (18Kanazawa T. Saito M. Tonomura Y. J. Biochem. (Tokyo). 1970; 67: 693-711Crossref PubMed Scopus (143) Google Scholar) or otherwise as above. The precipitated proteins were separated at pH 6.0 by 5% SDS-polyacrylamide gel electrophoresis according to Weber and Osborn (19Weber 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 previously (20Daiho 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 4% 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. Miscellaneous—Protein concentrations were determined by the method of Lowry et al. (21Lowry 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 (Theoretical Biophysics Group, University of Illinois at Urbana-Champaign). Effects of Deletions and Substitutions on ATP Hydrolysis—The specific Ca2+-ATPase activities of the expressed mutant and wild-type SERCA1a were determined at 25 °C (Fig. 3). Deletions of any single residues in the Glu40-Ser48 loop resulted in almost complete loss of the activity, while their nonconservative substitutions caused only partial decrease or rather slight increase in the activity. Single deletions or substitutions of the residues in the adjacent N-terminal (His32-Asn39) and C-terminal (Leu49-Ile54) regions of the Glu40-Ser48 loop had only slight or moderate effect on the activity, except that the specific substitutions of Asn39 (N39D and N39T, but not N39A) had a significantly reduced activity. Quadruple alanine substitutions of Asn39, Glu40, Glu44, and Glu45 were previously shown to cause no loss of function (22Clarke D.M. Maruyama K. Loo T.W. Leberer E. Inesi G. MacLennan D.H. J. Biol. Chem. 1989; 264: 11246-11251Abstract Full Text PDF PubMed Google Scholar), and the present results are in essential agreement. Formation of EP from ATP—We then performed detailed kinetic analysis with the mutants. EP was formed from ATP at 0 °C under conditions otherwise similar to those for the ATPase assay. All the mutants possessed the ability to form EP, and the amount formed was comparable with that of wild type (Fig. 4). In Fig. 5, the fraction of E2P accumulated was determined at steady state (15 s after addition of ATP). In the presence of K+, which strongly accelerates decay of E2P and thus suppresses its accumulation in the wild type (23Shigekawa M. Dougherty J.P. J. Biol. Chem. 1978; 253: 1451-1457Abstract Full Text PDF PubMed Google Scholar), the amount of E2P accumulated in all the mutants was very low as in the wild type. In the absence of K+, the fraction of E2P largely increased in the wild type (to 73% of the total amount of EP (E1P plus E2P)), but it remained very low in the single deletion mutants for the Glu40-Ser48 loop and in the mutants N39D and N39T, indicating that almost all EP accumulated was E1P in these mutants. On the other hand, as in the wild type, the fraction of E2P largely increased in the other mutants, in which the residues in the loop were substituted or those in the adjacent N- and C-terminal regions of the loop were deleted or substituted (with the exception of N39D and N39T). The results strongly suggest that the E1P to E2P transition in step 4 is inhibited in the single deletion mutants for the Glu40-Ser48 loop and in the substitution mutants N39D and N39T. We, therefore, examined the decay of E1P accumulated in the presence of K+.Fig. 5Accumulation of E2P from ATP. Microsomes were phosphorylated with [γ-32P]ATP in the presence of 0.1 m KCl (black bars) or 0.1 m LiCl without added KCl (gray bars) or otherwise as described in the legend to Fig. 4. The reaction was quenched with trichloroacetic acid at 15 s after the start of phosphorylation, and the total amount of EP was determined. For determination of E2P, an equal volume (50 μl) of a mixture containing 5 mm ADP, 3 mm EGTA, 50 mm MOPS/Tris (pH 7.0), and 0.1 m KCl (black bars) or 0.1 m LiCl without added KCl (gray bars) was added to the above phosphorylation mixture at 15 s after the start of phosphorylation. At 1 s after this addition, the reaction was quenched with trichloroacetic acid. E1P disappeared entirely within 1 s after the addition of ADP. The amount of E2P is shown as percentage of the total amount of EP. The values presented are the mean ± S.D. (n = 3-4).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Decay of EP Formed from ATP—Decay of EP formed from ATP in the presence of K+ was determined at 0 °C by first phosphorylating with [γ-32P]ATP in the presence of K+ and Ca2+ for 15 s under the conditions in which almost all EP formed was E1P (see Fig. 5) and then terminating phosphorylation by adding excess EGTA to prevent further phosphorylation and thus allow decay of 32P-labeled EP. The EP decay was well fitted with a single exponential as shown in Fig. 6 with the representative mutants and wild type. The decay rates were thus obtained with all the mutants and summarized in Table I. The decay was almost completely blocked by any single deletions in the Glu40-Ser48 loop, and the rates were only 1-3% of that in the wild type. On the other hand, the rates in the substitution mutants for the Glu40-Ser48 loop and in all the deletion and substitution mutants for the adjacent regions of the Glu40-Ser48 loop were comparable with or only slightly lower than that in the wild type, except the mutants N39D and N39T (but not N39A) that had the significantly reduced rate (dashed lines in Fig. 6).Table IRate constants for partial reaction steps of all the mutantsDecay of E1P formed from ATPHydrolysis of E2P formed from PiTransition from E2 to E1Ca2 (formation of E1P from E2)s −1%s −1%s −1%WT0.0546(100.0)0.837(100.0)0.327(100.0)Δ320.0306(56.1)0.906(108.3)0.120(36.6)Δ330.0455(83.4)0.786(93.9)0.194(59.6)Δ340.0562(103.0)0.626(74.9)0.159(48.7)Δ360.0335(61.3)0.581(69.5)0.313(95.7)Δ370.0371(67.9)1.317(157.4)0.243(74.2)Δ380.0527(96.5)0.574(68.6)0.247(75.5)Δ390.0457(83.7)0.271(32.4)0.242(74.1)Δ400.0004(0.7)0.003(0.4)0.172(52.7)Δ410.0006(1.1)0.003(0.4)0.201(61.4)Δ420.0006(1.2)0.003(0.3)0.217(66.2)Δ430.0006(1.1)0.005(0.6)0.165(50.4)Δ440.0007(1.3)0.013(1.6)0.241(73.8)Δ450.0007(1.4)0.011(1.3)0.162(49.5)Δ460.0011(2.0)0.019(2.2)0.245(75.1)Δ470.0014(2.6)0.023(2.8)0.219(67.1)Δ480.0016(2.9)0.017(2.0)0.275(84.0)Δ490.0203(37.2)0.669(80.0)0.228(69.8)Δ500.0150(27.5)0.581(69.5)0.222(67.9)Δ510.0263(48.2)0.811(97.0)0.297(90.8)Δ520.0571(104.6)0.712(85.1)0.256(78.4)Δ530.0426(78.1)0.313(37.4)0.173(52.9)Δ540.0457(83.7)0.341(40.7)0.131(40.3)G37P0.0363(66.5)0.972(116.2)0.373(114.2)H38A0.0570(104.4)1.856(221.9)0.450(137.7)N39A0.0655(120.0)1.192(142.5)0.279(85.3)N39D0.0117(21.4)1.023(122.3)0.148(45.4)N39T0.0147(26.9)0.789(94.4)0.148(45.1)E40A0.0602(110.3)2.123(253.8)0.229(69.9)L41G0.0310(56.7)0.211(25.2)0.273(83.4)P42G0.0427(78.2)1.209(144.6)0.378(115.8)P42A0.0330(60.4)0.403(48.2)0.248(75.8)A43G0.0469(85.8)1.713(204.8)0.280(85.5)E44A0.0372(68.1)1.263(150.9)0.461(141.0)E45A0.0419(76.6)1.546(184.8)0.288(88.0)G46P0.0490(89.8)1.041(124.4)0.307(94.0)K47A0.0695(127.2)2.279(272.4)0.411(125.6)S48A0.0591(108.3)1.233(147.4)0.279(85.2)L49A0.0651(119.2)1.173(140.3)0.428(131.1)I54V0.0448(82.0)0.475(56.7)0.302(92.5) Open table in a new tab Hydrolysis of E2P Formed from P i —E2P was formed by Pi in the absence of Ca2+ and K+ and presence of 35% (v/v) Me2SO, which extremely favors E2P formation (24de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar). In some of the single deletion mutants, including those for the Glu40-Ser48 loop, the amount of EP formed was somewhat reduced (Fig. 7A). Nevertheless the hydrolysis of 32P-labeled E2P was examined with all the mutants at 0 °C by diluting the above phosphorylated samples with a large volume of a solution containing K+ and non-radioactive Pi. The conditions were thus made otherwise identical to those used for the decay of EP formed from ATP in Fig. 6. The hydrolysis of E2P proceeded with first-order kinetics as shown in Fig. 7, B and C, with the representative mutants and wild type. The hydrolysis rates were thus obtained with all the mutants and summarized in Table I. The hydrolysis was markedly slowed or blocked by any single deletions in the Glu40-Ser48 loop, and the rates were only 0.3-3% of that in the wild type. In contrast, the hydrolysis in the substitution mutants for the Glu40-Ser48 loop and in the deletion and substitution mutants for the adjacent regions of the loop including N39D and N39T (dashed lines in Fig. 7C) was as rapid as, or only slightly slower than, that in the wild type. Transition from E2 to E1Ca 2 —The mutants and wild type were preincubated in the absence of Ca2+ at pH 6 where equilibrium between E1 and E2 is most shifted to E2 (25Pick U. Karlish S.J.D. J. Biol. Chem. 1982; 257: 6120-6126Abstract Full Text PDF PubMed Google Scholar) and then phosphorylated at 0 °C by simultaneous addition of saturating concentrations of Ca2+ and ATP under conditions otherwise similar to those for the ATPase assay. The time course of EP formation was well described by the first-order kinetics as shown in Fig. 8 with the representative mutants and wild type. The rates were thus obtained with all the mutants and summarized in Table I. The rates of all the mutants, including the single deletion mutants for the Glu40-Ser48 loop, were comparable with that of the wild type and not significantly reduced. When ATP was added to the enzyme preincubated with Ca2+ otherwise as above, the EP formation proceeded at much faster rate (5 s-1 in the wild type and the comparable rate (4-7 s-1) in all the mutants). The results show that the Ca2+-induced E2 to E1Ca2 transition, which is rate-limiting for the EP formation from E2, is essentially not inhibited in all the mutants. Roles of Glu40-Ser48 Loop—In the present study, we explored possible roles of the Glu40-Ser48 loop connecting A domain and M1 helix by mutagenesis and found that deletions of any single residues within this loop (but not their substitutions) almost completely block or strongly inhibit both the E1P to E2P transition and the E2P hydrolysis. Results indicate that the loop with its appropriate length (but not with specific residues) is critical for the rapid isomeric transition and hydrolysis of EP. During the E1P to E2P transition and Ca2+ release into lumen, a large rotation of A domain by ∼90° and its intimate contact with P and N domains occur to form the most compactly organized cytoplasmic domains in E2P without bound Ca2+ (7Danko S. Daiho T. Yamasaki K. Kamidochi M. Suzuki H. Toyoshima C. FEBS Lett. 2001; 489: 277-282Crossref PubMed Scopus (60) Google Scholar, 8Danko S. Yamasaki K. Daiho T. Suzuki H. Toyoshima C. FEBS Lett. 2001; 505: 129-135Crossref PubMed Scopus (92) Google Scholar). In this E2P, the hydrophobic atmosphere (24de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar, 26de Meis L. Inesi G. J. Biol. Chem. 1982; 257: 1289-1294Abstract Full Text PDF PubMed Google Scholar, 27Dupont Y. Pougeois R. FEBS Lett. 1983; 156: 93-98Crossref PubMed Scopus (72) Google Scholar, 28de Meis L. Biochim. Biophys. Acta. 1989; 973: 333-349Crossref PubMed Scopus (140) Google Scholar) is thus realized around the phosphorylation site and a specific water molecule can now attack the acylphosphate bond to hydrolyze. It is therefore likely that the formation of the proper structure of E2P, i.e. the movement of A domain and resulting domain organization, was impaired by the deletions of even single residues (∼3.5 Å shortening) in the Glu40-Ser48 loop. The facts found in the structural model E2V, an E2P analogue (7Danko S. Daiho T. Yamasaki K. Kamidochi M. Suzuki H. Toyoshima C. FEBS Lett. 2001; 489: 277-282Crossref PubMed Scopus (60) Google Scholar, 8Danko S. Yamasaki K. Daiho T. Suzuki H. Toyoshima C. FEBS Lett. 2001; 505: 129-135Crossref PubMed Scopus (92) Google Scholar), that the Glu40-Ser48 loop is extended and not interacting with other parts of the molecule (Fig. 2), is consistent with the view that no specific residues are involved in the role of this loop but that its length being crucial. In the detailed and well accepted reaction mechanism for the E1P to E2P transition and Ca2+ release, the process consists of two steps (steps 4 and 5, Fig. 1). The single deletions in the Glu40-Ser48 loop obviously caused blocking of the loss of ADP sensitivity in step 4 (i.e. the E1P to E2P transition). We have recently found (9Kato 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) that the mutations in the Lys189-Lys205 outermost loop on A domain do not inhibit the loss of ADP sensitivity but strongly inhibit the subsequent processing of E2P in step 5 and indicated that the final process of gathering of A and P domains is accomplished in step 5 to release the bound Ca2+ by the intimate contact of the Lys189-Lys205 loop with P domain. Together with this study, we thus could identify two structural elements crucial for the successive, but distinct, two steps; the large rotation of A domain and its association with P and N domains (to some extent) in step 4 and the final process for intimate contact of A and P domains in step 5. Importantly, it was found in comparison of the structures E1Ca2 and E2(TG) (6Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar) that upon Ca2+ dissociation, M1 connected to the Glu40-Ser48 loop undergoes horizontal and upward movements and a large structural change, i.e. bending at Asp59, forming an amphipathic helix M1′ that has hydrophobic residues on one side and charged residues on the other (Fig. 2). M1′ is thus likely to be situated at the membrane surface by interactions with lipids and possibly with Arg63 in M1. This large motion is likely caused by the steric collision of M1 with M3 that inclines together with M4-M6 and P domain toward M1 and A domain (6Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar). The unique structure of M1′ and M1 are also found in E2V, an analogue of E2P. Then it is possible that the shortening of the Glu40-Ser48 loop by the deletions may inhibit such motion of M1 and thus block the appropriate movements of M3-M6, P domain, and A domain for the E1P to E2P transition. Alternatively, it may also be possible that the appropriate movement of A domain would be inhibited if M1′ first formed is not flexible enough to move upward from the position at the membrane surface and to compensate the shortening of the Glu40-Ser48 loop by the deletions. In any case, it is likely that, not only the crucial large rotation of A domain and gathering of the cytoplasmic domains, but also the unique movement and structural change of M1, likely contribute to formation of the proper structure of E2P. It is possible that the Glu40-Ser48 loop functions in coordinating these motions of A domain and M1 during the E1P to E2P transition and thus in rendering A domain to accept the inclined P domain at an appropriate position and contributing to cross-talk between the cytoplasmic and transmembrane domains. The observation showing essentially no effects of the deletions in the Glu40-Ser48 loop on the rate of the E2 to E1Ca2 transition (and subsequent E1P formation) (steps 1-3, Fig. 8) indicates that the structural importance of the Glu40-Ser48 loop may be already lost in E2. This view is consistent with our previous observation (7Danko S. Daiho T. Yamasaki K. Kamidochi M. Suzuki H. Toyoshima C. FEBS Lett. 2001; 489: 277-282Crossref PubMed Scopus (60) Google Scholar, 8Danko S. Yamasaki K. Daiho T. Suzuki H. Toyoshima C. FEBS Lett. 2001; 505: 129-135Crossref PubMed Scopus (92) Google Scholar) that the cytoplasmic domain organization in the more relaxed E2 state is significantly different from that in the most compactly organized E2P state. The view may also be compatible with the notion (6Toyoshima C. Nomura H. N" @default.
• W2008477360 created "2016-06-24" @default.
• W2008477360 creator A5002113575 @default.
• W2008477360 creator A5019219270 @default.
• W2008477360 creator A5026465948 @default.
• W2008477360 creator A5047085626 @default.
• W2008477360 creator A5055906267 @default.
• W2008477360 creator A5083428632 @default.
• W2008477360 date "2003-10-01" @default.
• W2008477360 modified "2023-10-02" @default.
• W2008477360 title "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" @default.
• W2008477360 cites W1481361731 @default.
• W2008477360 cites W1486586853 @default.
• W2008477360 cites W1493803422 @default.
• W2008477360 cites W1500554454 @default.
• W2008477360 cites W1515311330 @default.
• W2008477360 cites W1519486794 @default.
• W2008477360 cites W1533641603 @default.
• W2008477360 cites W1543280300 @default.
• W2008477360 cites W1561646380 @default.
• W2008477360 cites W1565119049 @default.
• W2008477360 cites W1579731099 @default.
• W2008477360 cites W1590194063 @default.
• W2008477360 cites W1591793880 @default.
• W2008477360 cites W1625660792 @default.
• W2008477360 cites W1641590304 @default.
• W2008477360 cites W1775749144 @default.
• W2008477360 cites W1968266886 @default.
• W2008477360 cites W1969512778 @default.
• W2008477360 cites W1997605861 @default.
• W2008477360 cites W1999722457 @default.
• W2008477360 cites W2007979534 @default.
• W2008477360 cites W2012803276 @default.
• W2008477360 cites W2013578935 @default.
• W2008477360 cites W2024927200 @default.
• W2008477360 cites W2036162307 @default.
• W2008477360 cites W2039899658 @default.
• W2008477360 cites W2046630229 @default.
• W2008477360 cites W2049291517 @default.
• W2008477360 cites W2050185615 @default.
• W2008477360 cites W2063404335 @default.
• W2008477360 cites W2076691657 @default.
• W2008477360 cites W2079538130 @default.
• W2008477360 cites W2081009955 @default.
• W2008477360 cites W2087726424 @default.
• W2008477360 cites W2088839366 @default.
• W2008477360 cites W2092175923 @default.
• W2008477360 cites W2097383319 @default.
• W2008477360 cites W2098550162 @default.
• W2008477360 cites W2130139433 @default.
• W2008477360 cites W2151825841 @default.
• W2008477360 cites W2153331570 @default.
• W2008477360 cites W2161549748 @default.
• W2008477360 cites W2416384825 @default.
• W2008477360 cites W997323785 @default.
• W2008477360 doi "https://doi.org/10.1074/jbc.m305200200" @default.
• W2008477360 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12857730" @default.
• W2008477360 hasPublicationYear "2003" @default.
• W2008477360 type Work @default.
• W2008477360 sameAs 2008477360 @default.
• W2008477360 citedByCount "43" @default.
• W2008477360 countsByYear W20084773602012 @default.
• W2008477360 countsByYear W20084773602013 @default.
• W2008477360 countsByYear W20084773602014 @default.
• W2008477360 countsByYear W20084773602016 @default.
• W2008477360 countsByYear W20084773602018 @default.
• W2008477360 countsByYear W20084773602019 @default.
• W2008477360 countsByYear W20084773602020 @default.
• W2008477360 countsByYear W20084773602021 @default.
• W2008477360 countsByYear W20084773602022 @default.
• W2008477360 crossrefType "journal-article" @default.
• W2008477360 hasAuthorship W2008477360A5002113575 @default.
• W2008477360 hasAuthorship W2008477360A5019219270 @default.
• W2008477360 hasAuthorship W2008477360A5026465948 @default.
• W2008477360 hasAuthorship W2008477360A5047085626 @default.
• W2008477360 hasAuthorship W2008477360A5055906267 @default.
• W2008477360 hasAuthorship W2008477360A5083428632 @default.
• W2008477360 hasBestOaLocation W20084773601 @default.
• W2008477360 hasConcept C104317684 @default.
• W2008477360 hasConcept C114614502 @default.
• W2008477360 hasConcept C118892022 @default.
• W2008477360 hasConcept C12554922 @default.
• W2008477360 hasConcept C134306372 @default.
• W2008477360 hasConcept C141315368 @default.
• W2008477360 hasConcept C158617107 @default.
• W2008477360 hasConcept C170493617 @default.
• W2008477360 hasConcept C181199279 @default.
• W2008477360 hasConcept C184670325 @default.
• W2008477360 hasConcept C185592680 @default.
• W2008477360 hasConcept C18903297 @default.
• W2008477360 hasConcept C194232998 @default.
• W2008477360 hasConcept C23265538 @default.
• W2008477360 hasConcept C24530287 @default.
• W2008477360 hasConcept C2778530040 @default.
• W2008477360 hasConcept C2779965526 @default.
• W2008477360 hasConcept C33923547 @default.
• W2008477360 hasConcept C36503486 @default.
• W2008477360 hasConcept C41625074 @default.

• next