Importance of Conserved N-domain Residues Thr441, Glu442, Lys515, Arg560, and Leu562 of Sarcoplasmic Reticulum Ca2+-ATPase for MgATP Binding and Subsequent Catalytic Steps

Clausen, Johannes; McIntosh, David B.; Vilsen, Bente; Woolley, David G.; Andersen, Jens Peter

doi:10.1074/jbc.m301122200

Cited by 50 publications

(66 citation statements)

References 65 publications

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“…Presence of a salt link between Glu-442 and Lys-515 in the case of SERCA (Fig. 1, B and C) has been suggested (42). In the case of Na/K-ATPase, the following observations suggest that a salt link, if present, is not required for Na/K-ATPase activity.…”

Section: Effect Of Substitution On the Apparent Affinity For Na ϩ Amentioning

confidence: 75%

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The Amino Acid Sequence 442GDASE446 in Na/K-ATPase Is an Important Motif in Forming the High and Low Affinity ATP Binding Pockets

Imagawa

Kaya

Taniguchi

2003

Journal of Biological Chemistry

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.5 ) and the apparent K m for Mg 2؉ , Na ؉ , K ؉ , and vanadate in Na/K-ATPase were also estimated. For all the mutants, the value for ATP was ϳ2-10-fold larger than that of the wild type. While the turnover number for Na/K-ATPase activity were unaffected or reduced by 20ϳ50% in mutants G442(A/P) and D443A. Although both affinities for ATP effects were reduced as a result of the mutations, the ratio, K m l /K m h , for each mutant was 1.3ϳ3.7, indicating that these mutations had a greater impact on the low affinity ATP effect than on the high affinity effect. Each K m P value with the turnover number suggests that these mutations favor the binding of pNPP over that of ATP. These data and others indicate that the sequence 442 GDASE 446 in the ATP binding pocket is an important motif that it is involved in both the high and low affinity ATP effects rather than in free Mg 2؉ , Na ؉ , and K ؉ effects.

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Section: Effect Of Substitution On the Apparent Affinity For Na ϩ Amentioning

confidence: 75%

“…1B) was reported, which corresponds to Ser-445 and Glu-446 (Fig. 4C) in Na/K-ATPase (42). The apparent acceleration by free Mg 2ϩ was saturated at around 0.2 mM.…”

Section: Larger Apparent Affinity Decrease For Atp Effects Of Each Mumentioning

confidence: 99%

The Amino Acid Sequence 442GDASE446 in Na/K-ATPase Is an Important Motif in Forming the High and Low Affinity ATP Binding Pockets

Imagawa

Kaya

Taniguchi

2003

Journal of Biological Chemistry

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“…20). A block or slowing of the E1P 3 E2P transition has previously been described for mutants Asp 601 3 Asn, Lys 684 3 Arg, Asp 703 3 Asn, and Asp 627 3 Glu, on the basis of measurements similar to those shown in Fig.…”

Section: (Closed Triangles)mentioning

confidence: 91%

Roles of Conserved P Domain Residues and Mg2+ in ATP Binding in the Ground and Ca2+-activated States of Sarcoplasmic Reticulum Ca2+-ATPase

McIntosh

Clausen

Woolley

et al. 2004

Journal of Biological Chemistry

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Residues in conserved motifs 625 The sarcoplasmic reticulum Ca 2ϩ -ATPase 1 from rabbit muscle couples the hydrolysis of ATP to the transport of Ca 2ϩ to the reticular lumen, and the lowering of the sarcoplasmic Ca 2ϩ concentration leads to muscle relaxation. This ion pump is a well studied example of P-type ATPases, the catalytic cycle of which includes a phosphorylated aspartyl intermediate and progression through E1 and E2 states. ATP-dependent phosphorylation takes place upon Ca 2ϩ binding in the E1 form, and hydrolysis of the aspartyl phosphoryl bond occurs in the E2P phosphoenzyme conformation following Ca 2ϩ translocation across the membrane. Two atomic structures of the protein have been elucidated by x-ray crystallography, one in the presence of Ca 2ϩ (designated E1(Ca 2 )) and another in the absence of Ca 2ϩ and presence of the specific inhibitor thapsigargin (E2(TG)) (1, 2). They reveal 10 membrane-spanning helices, connected through a stalk section to the phosphorylation (P) domain in association with nucleotide (N) and actuator (A) domains. The three head domains are loosely attached in E1(Ca 2 ) and closer together, forming a more compact entity, in E2(TG). For ATP-dependent phosphorylation of Asp 351 to occur in the Ca 2ϩ bound E1 state, domain N must close over the phosphorylation site, such that the nucleotide site is brought close to Asp 351 . ATP likely straddles both domains, the nucleoside portion anchored in domain N and the phosphates stretching into domain P (cf. Fig. 1A). Domain P contains the highly conserved segments 625 TGD, 676 FARXXPXXK, and 701 TGDGVND that surround the phosphorylation loop 351 DKTGTLT. Many of these residues have been implicated in ATP binding and catalysis on the basis of chemical labeling and cross-linking experiments, and mutagenesis of Ca 2ϩ -ATPase and related pumps (for review, see Ref.3). Most of them recur at the active site of a large class of soluble phosphohydrolases and phosphotransferases believed to have a similar reaction mechanism (4).

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“…Lys 515 lies in the depth of the ATP-binding pocket in SERCA1a (48) and is critical for nucleotide binding and SERCA1a function (8,31). A causal relationship between modification of Lys 515 and thermal inactivation of SERCA1a was suggested by our previous study showing that Hsp70 prevented both heat-induced reductions in FITC-binding capacity of SERCA1a and loss of function (50).…”

Section: Effects Of Heat Stress On Serca2a Structure and Functionmentioning

confidence: 92%

Protective effects of Hsp70 on the structure and function of SERCA2a expressed in HEK-293 cells during heat stress

Fu¹,

Tupling²

2009

American Journal of Physiology-Heart and Circulatory Physiology

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Fu MH, Tupling AR. Protective effects of Hsp70 on the structure and function of SERCA2a expressed in HEK-293 cells during heat stress. Am J Physiol Heart Circ Physiol 296: H1175-H1183, 2009. First published February 27, 2009 doi:10.1152/ajpheart.01276.2008.-Heat shock protein 70 (Hsp70) can physically interact with and prevent thermal inactivation of sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA) 1a, the SERCA isoform expressed in adult fasttwitch skeletal muscle. This study examined whether Hsp70 could physically interact with and prevent thermal inactivation of SERCA2a, the SERCA isoform expressed in heart. HEK-293 cells were cotransfected with cDNAs encoding human Hsp70 and rabbit SERCA2a (S2a/Hsp70). Cells cotransfected with SERCA2a cDNA and pMT2 (S2a/pMT2) were used as control. One-half of the cells was heat shocked at 40°C for 1 h (HS), and one-half was maintained at 37°C before harvesting the cells and isolating microsomes. Western blot analysis showed that Hsp70 and SERCA2a were colocalized in the microsomal fraction. The levels of Hsp70 were approximately fivefold higher (P Ͻ 0.05) in S2a/Hsp70 compared with S2a/pMT2 and approximately twofold higher (P Ͻ 0.05) following HS in all cells. Coimmunoprecipitation demonstrated that Hsp70 directly binds to SERCA2a. Following HS, maximal SERCA2a activity was reduced (ϳ52%, P Ͻ 0.05) in S2a/pMT2 but was increased (ϳ33%, P Ͻ 0.05) in S2a/Hsp70. Thermal inactivation of SERCA2a in S2a/pMT2 was associated with decreased (ϳ49%, P Ͻ 0.05) binding capacity for fluorescein isothiocyanate (FITC) and increased carbonyl (ϳ42%, P Ͻ 0.05) and nitrotyrosine (ϳ40%, P Ͻ 0.05) levels in SERCA2a. By contrast, the HS-induced increase in maximal SERCA2a activity observed in S2a/Hsp70 corresponded with no change (P Ͼ 0.05) in FITC-binding capacity and reductions in carbonyl (ϳ40%, P Ͻ 0.05) and nitrotyrosine (ϳ23%, P Ͻ 0.05) levels in SERCA2a compared with S2a/pMT2. These results show that Hsp70 forms a protective interaction with SERCA2a during HS actually reducing oxidation and nitrosylation of SERCA2a thus increasing its maximal activity. coimmunoprecipitatoin; carbonyl; nitrotyrosine; nucleotide binding site SARCO(ENDO)PLASMIC reticulum Ca 2ϩ -ATPases (SERCAs) are typical of the class of P-type ATPases that bind specific ions to be transported, hydrolyze ATP to form a phosphoprotein intermediate, and undergo conformational changes resulting in ion translocation (29). SERCA pumps are 110-kDa integral membrane proteins that consist of 10 transmembrane helexes (M1-M10), three cytoplasmic domains (A, actuator; N, nucleotide-binding; P, phosphorylation), and small luminal loops (48). In humans, 3 genes (ATP2A1-3) generate multiple isoforms (SERCAla,b, SERCA2a-c, SECA3a-f) by developmental or tissue-specific alternative splicing (14). Two SERCA isoforms predominate in adult striated muscle, namely SERCA1a and SERCA2a. The SERCA1a isoform consists of 994 amino acids and accounts for Ͼ99% of the SERCA isoforms expressed in adult fast-twitch skeletal muscle, whereas SERCA2a consists...

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Importance of Conserved N-domain Residues Thr441, Glu442, Lys515, Arg560, and Leu562 of Sarcoplasmic Reticulum Ca2+-ATPase for MgATP Binding and Subsequent Catalytic Steps

Cited by 50 publications

References 65 publications

The Amino Acid Sequence 442GDASE446 in Na/K-ATPase Is an Important Motif in Forming the High and Low Affinity ATP Binding Pockets

The Amino Acid Sequence 442GDASE446 in Na/K-ATPase Is an Important Motif in Forming the High and Low Affinity ATP Binding Pockets

Roles of Conserved P Domain Residues and Mg2+ in ATP Binding in the Ground and Ca2+-activated States of Sarcoplasmic Reticulum Ca2+-ATPase

Protective effects of Hsp70 on the structure and function of SERCA2a expressed in HEK-293 cells during heat stress

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