ABCMdb

ABCC8 p.Asp818Ala

Predicted by SNAP2:	A: D (75%), C: D (75%), E: D (75%), F: D (71%), G: D (85%), H: D (80%), I: D (80%), K: D (85%), L: D (75%), M: D (80%), N: D (75%), P: D (91%), Q: D (75%), R: D (85%), S: D (75%), T: D (80%), V: D (75%), W: D (85%), Y: D (75%),
Predicted by PROVEAN:	A: D, C: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Involvement of the cytoplasmic loop L6-7 in the en... J Biol Chem. 2002 Apr 12;277(15):13016-28. Epub 2002 Jan 18. Menguy T, Corre F, Juul B, Bouneau L, Lafitte D, Derrick PJ, Sharma PS, Falson P, Levine BA, Moller JV, le Maire M
Involvement of the cytoplasmic loop L6-7 in the entry mechanism for transport of Ca2+ through the sarcoplasmic reticulum Ca2+-ATPase.
J Biol Chem. 2002 Apr 12;277(15):13016-28. Epub 2002 Jan 18., [PMID:11801592]

Abstract [show]
We previously found that mutants of conserved aspartate residues of sarcoplasmic reticulum Ca(2+)-ATPase in the cytosolic loop, connecting transmembrane segments M6 and M7 (L6-7 loop), exhibit a strongly reduced sensitivity toward Ca(2+) activation of the transport process. In this study, yeast membranes, expressing wild type and mutant Ca(2+)-ATPases, were reacted with Cr small middle dotATP and tested for their ability to occlude (45)Ca(2+) by HPLC analysis, after cation resin and C(12)E(8) treatment. We found that the D813A/D818A mutant that displays markedly low calcium affinity was capable of occluding Ca(2+) to the same extent as wild type ATPase. Using NMR and mass spectrometry we have analyzed the conformational properties of the synthetic L6-7 loop and demonstrated the formation of specific 1:1 cation complexes of the peptide with calcium and lanthanum. All three aspartate Asp(813)/Asp(815)/Asp(818) were required to coordinate the trivalent lanthanide ion. Overall these observations suggest a dual function of the loop: in addition to mediating contact between the intramembranous Ca(2+)-binding sites and the cytosolic phosphorylation site (Zhang, Z., Lewis, D., Sumbilla, C., Inesi G., and Toyoshima, C. (2001) J. Biol. Chem. 276, 15232-15239), the L6-7 loop, in a preceding step, participates in the formation of an entrance port, before subsequent high affinity binding of Ca(2+) inside the membrane.

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2 We found that the D813A/D818A mutant that displays markedly low calcium affinity was capable of occluding Ca2؉ to the same extent as wild type ATPase. Login to comment
39 This part of the loop contains three conserved aspartic residues (Asp813 , Asp815 , and Asp818 ) whose cluster mutation generated the D813A/D818A and D813A/ D815A/D818A mutants that we found to display a marked reduction in the apparent affinity with which Ca2ϩ controls ATPase phosphorylation and turn-over (12). Login to comment
49 EXPERIMENTAL PROCEDURES Mutation and Expression of Ca2ϩ -ATPase in Yeast-The single mutants E309Q and E771Q and the cluster mutants D813A/D818A and D813A/D815A/D818A (referred to as ADA and AAA mutants, respectively) were obtained as previously described in Refs. 12 and 25. Login to comment
150 In Fig. 2B, it is seen that with the D813A/D818A mutant in the L6-7 loop, the radioactivity profile is slightly lower than that observed for the wild type ATPase, but the Ca2ϩ -ATPase content is also lower (the reason for this difference probably is unrelated to differences in the level of expression, see below). Login to comment
180 Comparison of the Ca2؉ -occlusion reaction in presence of Cr⅐ATP for the wild type, L6-7 loop ADA mutated Ca2؉ -ATPase (D813A/ D818A), E309Q mutated Ca2؉ -ATPase and SR ؉ control membrane using gel filtration coupled to Western blot. Login to comment
181 The calcium occlusion induced by Cr⅐ATP is tested on light membrane fractions containing wild type Ca2ϩ -ATPase (chromatogram A), the ADA mutant (D813A/ D818A) in the L6-7 loop (chromatogram B) and the E309Q mutant (chromatogram D) Data for control membranes alone (Control Mb; chromatogram A, B, and C) or in the presence of SR Ca2ϩ -ATPase (SR ϩ Control Mb; chromatogram C) are also shown. Login to comment
212 Nanomole of occluded Ca2ϩ per mg of Ca2ϩ -ATPase Aggregation state of the Ca2ϩ -ATPaseLower estimates Upper estimates SR ϩ control membrane 5.6 Ϯ 0.2 11 Ϯ 0.18 Monomeric Wild type 5.4 Ϯ 0.9 9.8 Ϯ 0.9 Monomeric D813A-D818A (ADA) 4.2 Ϯ 0.9 9.6 Ϯ 0.9 Monomeric E309Q 0 0 Aggregated or oligomeric E771Q Not measurable Not measurable Not solubilized D813A-D815A-D818A (AAA) Not measurable Not measurable Not solubilized helical turn involving residues 816-819 (4). Login to comment

[hide] Involvement of the cytoplasmic loop L6-7 in the en... Ann N Y Acad Sci. 2003 Apr;986:90-5. Corre F, Jaxel C, Fuentes J, Menguy T, Falson P, Levine BA, Moller JV, le Maire M
Involvement of the cytoplasmic loop L6-7 in the entry mechanism for transport of Ca2+ through the sarcoplasmic reticulum Ca2+-ATPase.
Ann N Y Acad Sci. 2003 Apr;986:90-5., [PMID:12763779]

Abstract [show]
We have found that despite a markedly low calcium affinity the D813A/D818A mutant is capable, after complexation with Cr.ATP, of occluding Ca(2+) to the same extent (1-2 Ca(2+) per ATPase monomer) as wild- type ATPase. The inherent ability of the synthetic L6-7 loop peptide to bind Ca(2+) was demonstrated with murexide and mass spectrometry. NMR analysis indicated the formation of specific 1:1 cation complexes of the peptide with calcium and lanthanum with coordination by all three aspartate residues D813/D815/D818 that resulted in an altered conformation of the peptide chain. Overall our observations suggest that, in addition to mediating contact between the intramembranous Ca(2+) binding sites and the cytosolic phosphorylation site as previously suggested, the L6-7 loop, in a preceding step, participates in the formation of an entrance port important for lodging Ca(2+) at a high-affinity binding site inside the membrane.

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6 Bioquímica y Biología Molecular y Genética, E.U. Enfermería y T.O., Universidad de Extremadura, Cáceres, Spain cSchool of Biosciences, University of Birmingham, B15 2TT United Kingdom dDepartment of Biophysics, University of Aarhus, DK-8000 Aarhus C, Denmark ABSTRACT: We have found that despite a markedly low calcium affinity the D813A/D818A mutant is capable, after complexation with Cr.ATP, of occluding Ca2+ to the same extent (1-2 Ca2+ per ATPase monomer) as wild-type ATPase. Login to comment
13 lemairem@dsvidf.cea.fr part of the L6-7 loop (p808-818) interacted with Ca2+.1 Cluster mutation Ca2+- ATPase constructs, D813A-D818A (later called ADA) and D813A-D815A-D818A, displayed a marked reduction in the apparent affinity with which Ca2+ controls ATPase phosphorylation and turnover.2,3 We have rationalized our findings in terms of involvement of the conserved aspartic residues of the L6-7 loop in the interaction with calcium ions during the initial steps of the Ca2+ binding process.4 The crystal structures of SERCA 1a,5,6 however, show the acidic residues of the L6-7 loop in a conformation unsuitable for multiple coordination by Ca2+. Login to comment
20 Solubilization by C12E8 of Various Cr.ATP-reacted Ca2+-ATPase Species as a Function of Calcium In similar experiments we found that, as previously reported,7 the E309Q mutant does not occlude Ca2+, but several attempts to measure Cr.ATP-induced Ca2+ occlusion by the E771Q (previously also reported to display no calcium occlusion7) and the D813A-D815A-D818A mutants failed, because we were unable to recover any Ca2+-ATPase after elution from the HPLC column. Login to comment

[hide] The cytoplasmic loop located between transmembrane... J Biol Chem. 1998 Aug 7;273(32):20134-43. Menguy T, Corre F, Bouneau L, Deschamps S, Moller JV, Champeil P, le Maire M, Falson P
The cytoplasmic loop located between transmembrane segments 6 and 7 controls activation by Ca2+ of sarcoplasmic reticulum Ca2+-ATPase.
J Biol Chem. 1998 Aug 7;273(32):20134-43., [PMID:9685357]

Abstract [show]
During active cation transport, sarcoplasmic reticulum Ca2+-ATPase, like other P-type ATPases, undergoes major conformational changes, some of which are dependent on Ca2+ binding to high affinity transport sites. We here report that, in addition to previously described residues of the transmembrane region (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478), the region located in the cytosolic L6-7 loop connecting transmembrane segments M6 and M7 has a definite influence on the sensitivity of the Ca2+-ATPase to Ca2+, i.e. on the affinity of the ATPase for Ca2+. Cluster mutation of aspartic residues in this loop results in a strong reduction of the affinity for Ca2+, as shown by the Ca2+ dependence of ATPase phosphorylation from either ATP or Pi. The reduction in Ca2+ affinity for phosphorylation from Pi is observed both at acidic and neutral pH, suggesting that these mutations interfere with binding of the first Ca2+, as proposed for some of the intramembranous residues essential for Ca2+ binding (Andersen, J. P. (1995) Biosci. Rep. 15, 243-261). Treatment of the mutated Ca2+-ATPase with proteinase K, in the absence or presence of various Ca2+ concentrations, leads to Ca2+-dependent changes in the proteolytic degradation pattern similar to those in the wild type but observed only at higher Ca2+ concentrations. This implies that these effects are not due to changes in the conformational state of Ca2+-free ATPase but that changes affecting the proteolytic digestion pattern require higher Ca2+ concentrations. We conclude that aspartic residues in the L6-7 loop might interact with Ca2+ during the initial steps of Ca2+ binding.

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27 1 The abbreviations used are: SR, sarcoplasmic reticulum; SERCA, sarco(endo)plasmic reticulum Ca2ϩ -ATPase; p95, C-terminal proteolytic fragment of SR Ca2ϩ -ATPase starting at residue Lys-120; p83C, C-terminal proteolytic fragment of SR Ca2ϩ -ATPase starting at residue Glu-243; p28N, N-terminal proteolytic fragment of SR Ca2ϩ -ATPase ending at residue Thr-242; p20C or p20, C-terminal proteolytic fragment of SR Ca2ϩ -ATPase starting at residue Gly-808; p19C or p19, C-terminal proteolytic fragment of SR Ca2ϩ -ATPase starting at residue Asp-818; C12E8, octaethylene glycol monododecyl ether; Tes, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; Ab, Antibody; WT, wild type; ADA, aspartate mutant D813A/D818A; AAA aspartate mutant D813A/ D815A/D818A; PVDF, polyvinylidine difluoride; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid; E1, conformational state of Ca2ϩ -ATPase of high affinity for calcium; E2, conformational state of Ca2ϩ -ATPase of low affinity for calcium. Login to comment
43 The N-terminal part of the loop contains three aspartic residues (Asp-813, -815, and -818), which were mutated in a cluster, D813A/D818A and D813A/D815A/D818A. Login to comment
47 The experiments were designed to characterize the functional consequences of mutations of aspartic residues, D813A/D818A and D813A/D815A/D818A, as well as those of mutations of the proline residues in the same loop, Pro-811, -812, -820, and -821. Login to comment
52 MATERIALS AND METHODS Mutation and Expression of Ca2ϩ -ATPase in Yeast-The single mutants E309Q and E771Q and the cluster mutants D813A/D818A (referred to later as ADA) and D813A/D815A/D818A (referred to later as AAA) were obtained as in Ref. 32. Login to comment
92 Therefore, these residues were mutated to alanine residues to give the following mutants: D813A/D818A (referred to as ADA), D813A/ D815A/D818A (referred to as AAA), P811A/P812A, P812A, P820A/P821A, and P821A. Login to comment
211 An important outcome of our investigation is that cluster mutations of the aspartic residues shifted the Ca2ϩ -ATPase affinity for Ca2ϩ to much higher concentrations than those required to activate wild type ATPase; the K0.5 for Ca2ϩ activation of phosphorylation from ATP of the D813A/D818A mutated ATPase was shifted from the micromolar to the millimolar range, while the maximal level of phos- FIG. 6. Login to comment
219 In agreement with this, the double and triple mutants D813A/D818A and D813A/D815A/D818A only displayed a very low ATPase activity when they were tested at pCa 4, (Fig. 2). Login to comment

[hide] The cytoplasmic loop between putative transmembran... J Biol Chem. 1997 Jul 11;272(28):17258-62. Falson P, Menguy T, Corre F, Bouneau L, de Gracia AG, Soulie S, Centeno F, Moller JV, Champeil P, le Maire M
The cytoplasmic loop between putative transmembrane segments 6 and 7 in sarcoplasmic reticulum Ca2+-ATPase binds Ca2+ and is functionally important.
J Biol Chem. 1997 Jul 11;272(28):17258-62., [PMID:9211861]

Abstract [show]
Limited proteolysis by proteinase K of rabbit SERCA1 Ca2+-ATPase generates a number of fragments which have been identified recently. Here, we have focused on two proteolytic C-terminal fragments, p20C and p19C, starting at Gly-808 and Asp-818, respectively. The longer peptide p20C binds Ca2+, as deduced from changes in migration rate by SDS-polyacrylamide gel electrophoresis performed in the presence of Ca2+ as well as from labeling with 45Ca2+ in overlay experiments. In contrast, the shorter peptide p19C, a proteolysis fragment identical to p20C but for 10 amino acids missing at the N-terminal side, did not bind Ca2+ when submitted to the same experiments. Two cluster mutants of Ca2+-ATPase, D813A/D818A and D813A/D815A/D818A, expressed in the yeast Saccharomyces cerevisiae, were found to have a very low Ca2+-ATPase activity. Region 808-818 is thus essential for both Ca2+ binding and enzyme activity, in agreement with similar results recently reported for the homologous gastric H+, K+-ATPase (Swarts, H. G. P., Klaassen, C. H. W., de Boer, M., Fransen, J. A. M. , and De Pont, J. J. H. H. M. (1996) J. Biol. Chem. 271, 29764-29772). However, the accessibility of proteinase K to the peptidyl link between Leu-807 and Gly-808 clearly shows that the transmembrane segment M6 ends before region 808-818. It is remarkable that critical residues for enzyme activity are located in a cytoplasmic loop starting at Gly-808.

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4 Two cluster mutants of Ca2؉ -ATPase, D813A/D818A and D813A/D815A/D818A, expressed in the yeast Saccharomyces cerevisiae, were found to have a very low Ca2؉ -ATPase activity. Login to comment
46 One single mutation E309Q, one double mutation D813A/D818A (referred to later as ADA), and one triple mutation D813A/D815A/D818A (AAA) were introduced. Login to comment
121 Preliminary attempts with molecular modeling of the Ca2ϩ -ATPase 808-818 sequence by energy minimization according to the ␣-lactalbumin Ca2ϩ binding site suggested that the side chains of Asp-813 and Asp-818, together with the peptide carbonyl group between Asp-815 and Ile-816 could be involved in Ca2ϩ binding to the L6-7 loop.2 We then took advantage of the functional expression of Ca2ϩ -ATPase in yeast (53) to test the activity of two cluster mutants in this region, i.e. D813A/D818A (ADA) and D813A/D815A/D818A (AAA). Login to comment
142 The reaction medium contained 50 ␮g/ml yeast membranes with wild type or mutant Ca2ϩ -ATPases: D813A/D818A (ADA), D813A/D815A/D818A (AAA), or E309Q. Login to comment