FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1987421070> ?p ?o ?g. }

• W1987421070 endingPage "30191" @default.
• W1987421070 startingPage "30181" @default.
• W1987421070 abstract "P-type ATPases are a large family of enzymes that actively transport ions across biological membranes by interconverting between high (E1) and low (E2) ion-affinity states; these transmembrane transporters carry out critical processes in nearly all forms of life. In striated muscle, the archetype P-type ATPase, SERCA (sarco(endo)plasmic reticulum Ca2+-ATPase), pumps contractile-dependent Ca2+ ions into the lumen of sarcoplasmic reticulum, which initiates myocyte relaxation and refills the sarcoplasmic reticulum in preparation for the next contraction. In cardiac muscle, SERCA is regulated by phospholamban (PLB), a small inhibitory phosphoprotein that decreases the Ca2+ affinity of SERCA and attenuates contractile strength. cAMP-dependent phosphorylation of PLB reverses Ca2+-ATPase inhibition with powerful contractile effects. Here we present the long sought crystal structure of the PLB-SERCA complex at 2.8-Å resolution. The structure was solved in the absence of Ca2+ in a novel detergent system employing alkyl mannosides. The structure shows PLB bound to a previously undescribed conformation of SERCA in which the Ca2+ binding sites are collapsed and devoid of divalent cations (E2-PLB). This new structure represents one of the key unsolved conformational states of SERCA and provides a structural explanation for how dephosphorylated PLB decreases Ca2+ affinity and depresses cardiac contractility.Background: Phospholamban (PLB) regulates sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity and is thus a key regulator of cardiac contractility.Results: We present the crystal structure of SERCA in complex with PLB at 2.8-Å resolution.Conclusion: PLB stabilizes a divalent cation-free conformation of SERCA with collapsed Ca2+ binding sites. We call the structure E2-PLB.Significance: The E2-PLB structure explains how PLB decreases Ca2+ affinity and depresses cardiac contractility. P-type ATPases are a large family of enzymes that actively transport ions across biological membranes by interconverting between high (E1) and low (E2) ion-affinity states; these transmembrane transporters carry out critical processes in nearly all forms of life. In striated muscle, the archetype P-type ATPase, SERCA (sarco(endo)plasmic reticulum Ca2+-ATPase), pumps contractile-dependent Ca2+ ions into the lumen of sarcoplasmic reticulum, which initiates myocyte relaxation and refills the sarcoplasmic reticulum in preparation for the next contraction. In cardiac muscle, SERCA is regulated by phospholamban (PLB), a small inhibitory phosphoprotein that decreases the Ca2+ affinity of SERCA and attenuates contractile strength. cAMP-dependent phosphorylation of PLB reverses Ca2+-ATPase inhibition with powerful contractile effects. Here we present the long sought crystal structure of the PLB-SERCA complex at 2.8-Å resolution. The structure was solved in the absence of Ca2+ in a novel detergent system employing alkyl mannosides. The structure shows PLB bound to a previously undescribed conformation of SERCA in which the Ca2+ binding sites are collapsed and devoid of divalent cations (E2-PLB). This new structure represents one of the key unsolved conformational states of SERCA and provides a structural explanation for how dephosphorylated PLB decreases Ca2+ affinity and depresses cardiac contractility. Background: Phospholamban (PLB) regulates sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity and is thus a key regulator of cardiac contractility. Results: We present the crystal structure of SERCA in complex with PLB at 2.8-Å resolution. Conclusion: PLB stabilizes a divalent cation-free conformation of SERCA with collapsed Ca2+ binding sites. We call the structure E2-PLB. Significance: The E2-PLB structure explains how PLB decreases Ca2+ affinity and depresses cardiac contractility. Phospholamban (PLB), 3The abbreviations used are: PLBphospholambanSRsarcoplasmic reticulumSERCAsarco(endo)plasmic reticulum Ca2+-ATPaseSERCA1afast skeletal muscle isoform of SERCASERCA2acardiac isoform of SERCAWT-PLBwild-type PLBPLB4N27A, N30C, L37A, V49G-PLB2D12anti-PLB monoclonal antibodyMtransmembrane domainE1high Ca2+-affinity conformation of Ca2+-ATPaseE2low Ca2+ affinity conformation of Ca2+-ATPasedecyl maltosiden-decyl-β-d-maltopyranosidenonyl maltosiden-nonyl-β-d-maltopyranosidedodecyl maltosiden-dodecyl-β-d-maltopyranosideC12E8octaethylene glycol monododecyl etheroctyl glucosideoctyl β-d-glucopyranosideKCaCa2+ concentration required for half-maximal effectKMUSN-κ-maleimidoundecanoyl-oxysulfosuccinimide esterSLNsarcolipinTGthapsigargin. a single-span membrane protein of only 52 amino acids, is the principal membrane protein in the heart phosphorylated in response to β-adrenergic stimulation and a critical regulator of cardiac contractile strength (1Lindemann J.P. Jones L.R. Hathaway D.R. Henry B.G. Watanabe A.M. β-Adrenergic stimulation of phospholamban phosphorylation and Ca2+-ATPase activity in guinea pig ventricles.J. Biol. Chem. 1983; 258: 464-471Abstract Full Text PDF PubMed Google Scholar). In the dephosphorylated state, PLB decreases cardiac contractility by inhibiting the activity of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), an 110-kDa membrane protein with 10 membrane-spanning segments. PLB inhibits SERCA activity by decreasing Ca2+ affinity at its two Ca2+ binding sites (I and II) located in the SR membrane, thereby attenuating SR Ca2+ filling and contractile force development. Phosphorylation of PLB at Ser16 and Thr17 partially reverses PLB induced alterations in Ca2+ affinity, substantially augmenting contractility (2Simmerman H.K. Jones L.R. Phospholamban. Protein structure, mechanism of action, and role in cardiac function.Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (466) Google Scholar, 3Young H.S. Stokes D.L. The mechanics of calcium transport.J. Membr. Biol. 2004; 198: 55-63Crossref PubMed Scopus (28) Google Scholar, 4Wegener A.D. Simmerman H.K. Lindemann J.P. Jones L.R. Phospholamban phosphorylation in intact ventricles. Phosphorylation of serine 16 and threonine 17 in response to β-adrenergic stimulation.J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar). phospholamban sarcoplasmic reticulum sarco(endo)plasmic reticulum Ca2+-ATPase fast skeletal muscle isoform of SERCA cardiac isoform of SERCA wild-type PLB N27A, N30C, L37A, V49G-PLB anti-PLB monoclonal antibody transmembrane domain high Ca2+-affinity conformation of Ca2+-ATPase low Ca2+ affinity conformation of Ca2+-ATPase n-decyl-β-d-maltopyranoside n-nonyl-β-d-maltopyranoside n-dodecyl-β-d-maltopyranoside octaethylene glycol monododecyl ether octyl β-d-glucopyranoside Ca2+ concentration required for half-maximal effect N-κ-maleimidoundecanoyl-oxysulfosuccinimide ester sarcolipin thapsigargin. Chemical cross-linking was used previously to localize the PLB binding site on SERCA to a groove formed between transmembrane helices M2, M4, M6, and M9 of the Ca2+ pump (5Jones L.R. Cornea R.L. Chen Z. Close proximity between residue 30 of phospholamban and cysteine 318 of the cardiac Ca2+ pump revealed by intermolecular thiol cross-linking.J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 6Toyoshima C. Asahi M. Sugita Y. Khanna R. Tsuda T. MacLennan D.H. Modeling of the inhibitory interaction of phospholamban with the Ca2+ ATPase.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 467-472Crossref PubMed Scopus (171) Google Scholar, 7Chen Z. Stokes D.L. Rice W.J. Jones L.R. Spatial and dynamic interactions between phospholamban and the canine cardiac Ca2+ pump revealed with use of heterobifunctional cross-linking agents.J. Biol. Chem. 2003; 278: 48348-48356Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 8Chen Z. Stokes D.L. Jones L.R. Role of leucine 31 of phospholamban in structural and functional interactions with the Ca2+ pump of cardiac sarcoplasmic reticulum.J. Biol. Chem. 2005; 280: 10530-10539Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 9Chen Z. Akin B.L. Stokes D.L. Jones L.R. Cross-linking of C-terminal residues of phospholamban to the Ca2+ pump of cardiac sarcoplasmic reticulum to probe spatial and functional interactions within the transmembrane domain.J. Biol. Chem. 2006; 281: 14163-14172Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In conjunction with functional assays, these cross-linking studies led us to postulate that PLB binds to a unique Ca2+-free (E2) state of SERCA and decreases the Ca2+ affinity of the enzyme through direct effects on the Ca2+ binding sites (5Jones L.R. Cornea R.L. Chen Z. Close proximity between residue 30 of phospholamban and cysteine 318 of the cardiac Ca2+ pump revealed by intermolecular thiol cross-linking.J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Recently, a gain-of-function, cross-linkable PLB mutant, PLB4 (N27A, N30C, L37A, V49G-PLB) (Fig. 1), was developed (10Akin B.L. Chen Z. Jones L.R. Superinhibitory phospholamban mutants compete with Ca2+ for binding to SERCA2a by stabilizing a unique nucleotide-dependent conformational state.J. Biol. Chem. 2010; 285: 28540-28552Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) and shown to bind to the Ca2+-ATPase with exceedingly high affinity, comparable with or even greater than that of thapsigargin (TG), the well known, irreversible Ca2+-ATPase inhibitor (11Lytton J. Westlin M. Hanley M.R. Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps.J. Biol. Chem. 1991; 266: 17067-17071Abstract Full Text PDF PubMed Google Scholar, 12Sagara Y. Fernandez-Belda F. de Meis L. Inesi G. Characterization of the inhibition of intracellular Ca2+ transport ATPases by thapsigargin.J. Biol. Chem. 1992; 267: 12606-12613Abstract Full Text PDF PubMed Google Scholar). Here, we took advantage of PLB4 to stabilize the Ca2+-free state of SERCA and to crystallize it in complex with PLB. The 2.8-Å resolution structure, crystallized in nonyl maltoside, reveals a previously unresolved conformational state of the Ca2+ pump captured by PLB, which we define as E2-PLB. Importantly, the structure of E2-PLB is incompatible with Ca2+ (or Mg2+) binding to the enzyme, and confirms the hypothesis that mutually exclusive binding of PLB and Ca2+ to SERCA is the mechanism by which PLB decreases apparent Ca2+ affinity (5Jones L.R. Cornea R.L. Chen Z. Close proximity between residue 30 of phospholamban and cysteine 318 of the cardiac Ca2+ pump revealed by intermolecular thiol cross-linking.J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). SR vesicles sedimenting at 45,000 × g were isolated from rabbit back and hind leg muscles as described previously for cardiac SR vesicles (13Jones L.R. Besch Jr., H.R. Watanabe A.M. Regulation of the calcium pump of cardiac sarcoplasmic reticulum. Interactive roles of potassium and ATP on the phosphoprotein intermediate of the (K+,Ca2+)-ATPase.J. Biol. Chem. 1978; 253: 1643-1653Abstract Full Text PDF PubMed Google Scholar). SR vesicle pellets were extracted with 0.6 m KCl, 20 mm MOPS (pH 7.0), resuspended in 0.25 m sucrose, 20 mm MOPS (pH 7.2), and stored frozen in small aliquots at −40 °C at a protein concentration of 30 mg/ml. Recombinant WT-PLB and PLB4 were expressed and purified from Sf21 insect cells using anti-PLB monoclonal antibody (2D12) affinity chromatography as previously described (14Reddy L.G. Jones L.R. Cala S.E. O'Brian J.J. Tatulian S.A. Stokes D.L. Functional reconstitution of recombinant phospholamban with rabbit skeletal Ca2+-ATPase.J. Biol. Chem. 1995; 270: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Purified PLB was eluted from the column in 0.1% decyl maltoside or 0.01% dodecyl maltoside (Anatrace), which in control experiments were determined to be optimal detergents for co-crystallization of PLB with the solubilized Ca2+ pump. The purified, eluted PLB proteins were concentrated 100-fold with an Amicon concentrator, and then exhaustively dialyzed against 20 mm MOPS (pH 7.2), 20% glycerol, and 0.1% decyl maltoside or 0.01% dodecyl maltoside. The final working concentrations of PLB were 8–10 mg of protein/ml. PLB was stored frozen at −40 °C. Protein concentrations were determined by the Lowry method. The Ca2+ pump suitable for crystallization was solubilized directly from SR vesicles without prior purification or extraction of SR vesicles with low concentrations of deoxycholate (15Sørensen T.L. Olesen C. Jensen A.M. Møller J.V. Nissen P. Crystals of sarcoplasmic reticulum Ca2+-ATPase.J. Biotechnol. 2006; 124: 704-716Crossref PubMed Scopus (32) Google Scholar). Thawed SR vesicles were diluted 1:1 to a protein concentration of 15 mg/ml in buffer containing 2% nonyl maltoside (Anatrace), 20% glycerol, 100 mm MOPS (pH 7.0), 0.12 m sucrose, 80 mm KCl, 3 mm MgCl2, and 2.8 mm EGTA (final concentrations). The samples were allowed to stand for 7 min at room temperature, then ultracentrifuged at 4 °C at 100,000 × g for 15 min in a Beckman TLA 100.1 rotor. The supernatant was collected and PLB was added from the concentrated working solutions at a ratio of 0.14 mg of PLB/1.0 mg of solubilized SR vesicle protein, determined in control experiments to be a saturating concentration of PLB for inhibition of Ca2+-ATPase activity by lowering the apparent Ca2+ affinity. This amount of added PLB gave a molar ratio of PLB to SERCA of 2.9:1, as determined by quantitative immunoblotting (16Akin B.L. Jones L.R. Characterizing phospholamban to sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) protein binding interactions in human cardiac sarcoplasmic reticulum vesicles using chemical cross-linking.J. Biol. Chem. 2012; 287: 7582-7593Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Final volumes of mother liquors were adjusted by addition of 20% glycerol to make the final EGTA concentration 2 mm and samples were stored at 4 °C. Ca2+-ATPase prepared by this method (in 2 mm EGTA) retained 95–100% of the initially solubilized activity for at least 3 weeks at 4 °C in the presence and absence of PLB (Fig. 2B). In pilot studies, the Ca2+ pump was solubilized from SR vesicles using other detergents, including C12E8 and octyl glucoside. Solubilization conditions were identical to that described above, using 2% detergent concentrations (Fig. 2B). One day after the initial Ca2+-ATPase solubilization and addition of PLB, mother liquors were sedimented a second time by ultracentrifugation as described above. Hanging drops were made by mixing 1 μl of the sedimented mother liquors with 1 μl of reservoir solution (15% glycerol, 17% (w/v) PEG-2000, 200 mm NaOAc, and 5 mm β-mercaptoethanol) and crystals were grown by vapor diffusion at 4 °C. Single crystals appeared within 2 weeks and grew to a final size of 150 × 100 × 50 μm within 1 month. Crystals were mounted using nylon fiber loops and flash cooled in liquid nitrogen with no additional cryo-protectant. The x-ray diffraction data were collected at Beamline 19-ID operated by the Structural Biology Center at the Advanced Photon Source within Argonne National Laboratory. All diffraction data were collected at a wavelength of 0.979 Å from a single crystal at 100 K. The crystal was formed from PLB4 added in decyl maltoside. The diffraction data were integrated and scaled using the program package HKL3000 (17Otwinowski Z. Minor W. Processing of x-ray diffraction data collected in oscillation mode.Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38526) Google Scholar). The structure was solved by molecular replacement using the individual protein domains of SERCA (PDB code 2C8L) (18Jensen A.M. Sørensen T.L. Olesen C. Møller J.V. Nissen P. Modulatory and catalytic modes of ATP binding by the calcium pump.EMBO J. 2006; 25: 2305-2314Crossref PubMed Scopus (165) Google Scholar) as the search models. Solutions were found for the three cytoplasmic domains using Phaser (19McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. Phaser crystallographic software.J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (14440) Google Scholar), but no solution for the transmembrane region was obtained. The initial model was constructed from the three cytoplasmic domains and used to calculate initial electron density maps into which the individual transmembrane helices were manually fit using the program Coot (version 0.6.1 (20Emsley P. Lohkamp B. Scott W.G. Cowtan K. Features and development of Coot.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 486-501Crossref PubMed Scopus (17079) Google Scholar)) and the connectivity of the M4 and M5 helices to one of the cytoplasmic domains and the C-terminal transmembrane helix as points of reference. Helix M4 required fitting as two distinct sections and the connecting polypeptide was manually fit to the electron density in Coot. The structure was subjected to interative rounds of model building and refinement using the program Refmac5 (21Murshudov G.N. Vagin A.A. Dodson E.J. Refinement of macromolecular structures by the maximum-likelihood method.Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13854) Google Scholar) and included the use of TLS tensors (22Painter J. Merritt E.A. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion.Acta Crystallogr. D Biol. Crystallogr. 2006; 62: 439-450Crossref PubMed Scopus (1102) Google Scholar) to model the anisotropy of the individual domains and PLB. In addition to SERCA and PLB, the final model includes one potassium ion and 2 non-covalently associated maltose molecules, for which the acyl chains were not visible in the electron density and hence have been modeled simply as maltose residues. Initial attempts to include a magnesium ion bound to site I in the transmembrane domain gave rise to a negative difference peak at 4.2 σ in the Fo − Fc electron density map at the position of the modeled magnesium ion. Based on this information, combined with the lack of a positive peak in the Fo − Fc electron density map greater than 2.7 σ when the model is refined in the absence of magnesium and the fact that magnesium ions are not required for PLB cross-linking, we conclude that the PLB-SERCA complex lacks metal ions bound to the transmembrane metal sites. Ca2+-ATPase activity was determined colorimetrically by measuring inorganic phosphate release from ATP (16Akin B.L. Jones L.R. Characterizing phospholamban to sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) protein binding interactions in human cardiac sarcoplasmic reticulum vesicles using chemical cross-linking.J. Biol. Chem. 2012; 287: 7582-7593Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). 12 μg of solubilized Ca2+-ATPase protein (taken from mother liquors used for protein crystallization) were added to 1 ml of ATPase buffer containing 50 mm MOPS (pH 7.0), 3 mm MgCl2, 3 mm ATP, 100 mm KCl, 1 mm EGTA, 5 mm NaN3, and 3 μg/ml of A23187. Ionized Ca2+ concentrations were set by addition of CaCl2. Some reaction tubes in addition contained 60–80 μg of anti-PLB 2D12 antibody to reverse PLB inhibition of Ca2+-ATPase activity (23Chen Z. Akin B.L. Jones L.R. Mechanism of reversal of phospholamban inhibition of the cardiac Ca2+-ATPase by protein kinase A and by anti-phospholamban monoclonal antibody 2D12.J. Biol. Chem. 2007; 282: 20968-20976Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Reactions were conducted at 37 °C and Ca2+-dependent activities are reported. KCa values designate Ca2+ concentrations at which Ca2+-ATPase is half-maximally activated. Cross-linking assays were conducted with solubilized Ca2+-ATPase protein reconstituted with PLB4 taken from mother liquors used for protein crystallization. The Ca2+-ATPase protein (12 μg) was added to 500 μl of buffer identical to that indicated above for determination of Ca2+-ATPase activity with omission of NaN3. Cross-linking was conducted with 1 mm KMUS for 2 min at room temperature. Reactions were stopped with 7.5 μl of gel loading buffer containing 15% SDS and 100 mm dithiothreitol. Samples were subjected to SDS-PAGE and immunoblotting with the anti-PLB antibody, 2D12, for detection of PLB cross-linked to SERCA1a (skeletal muscle isoform) (Fig. 3C). Cross-linking of the canine cardiac isoform of the Ca2+ pump (SERCA2a) to N30C-PLB or PLB4 co-expressed in insect cells was conducted identically as previously described (10Akin B.L. Chen Z. Jones L.R. Superinhibitory phospholamban mutants compete with Ca2+ for binding to SERCA2a by stabilizing a unique nucleotide-dependent conformational state.J. Biol. Chem. 2010; 285: 28540-28552Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) in 50 mm MOPS (pH 7.0), 100 mm KCl, and 1 mm Ca2+, EGTA buffer in the presence or absence of 3 mm MgCl2 (Fig. 6). Cross-linking was conducted with 1 mm KMUS for 1 min at room temperature. The results shown in Fig. 6 were conducted in the absence of ATP, however, similar results were obtained with inclusion of 3 mm ATP. Ki values indicate Ca2+ concentrations at which cross-linking is inhibited by 50%.FIGURE 6Lack of effect of Mg2+ on PLB cross-linking. A, autoradiograph showing cross-linking of N30C-PLB or PLB4 to SERCA2a expressed in insect cell membranes. Cross-linking was conducted with KMUS over a range of Ca2+ concentrations in the presence and absence of 3 mm MgCl2. B, quantified results plotted as percentage of maximal cross-linking determined at zero Ca2+ concentration. Mg2+ had no effect on maximal cross-linking. Ki values (μm) for Ca2+ inhibition of cross-linking of N30C-PLB to SERCA were 0.37 ± 0.05 and 0.46 ± 0.07 in the absence and presence of Mg2+, respectively. Ki values for Ca2+ inhibition of PLB4 cross-linking were 1.52 ± 0.06 and 1.70 ± 0.10, respectively. Mean ± S.E. from three to five determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Crystallizing the novel Ca2+-free state of SERCA that binds PLB required that the solubilized enzyme be reconstituted with PLB in the absence of Ca2+ (presence of EGTA). However, the Ca2+-ATPase is extremely labile and prone to denaturation when solubilized without Ca2+ (24Toyoshima C. Nomura H. Structural changes in the calcium pump accompanying the dissociation of calcium.Nature. 2002; 418: 605-611Crossref PubMed Scopus (807) Google Scholar, 25Montigny C. Arnou B. Champeil P. Glycyl betaine is effective in slowing down the irreversible denaturation of a detergent-solubilized membrane protein, sarcoplasmic reticulum Ca2+-ATPase (SERCA1a).Biochem. Biophys. Res. Commun. 2010; 391: 1067-1069Crossref PubMed Scopus (3) Google Scholar), and the detergents typically used for SERCA solubilization/crystallization (C12E8) (15Sørensen T.L. Olesen C. Jensen A.M. Møller J.V. Nissen P. Crystals of sarcoplasmic reticulum Ca2+-ATPase.J. Biotechnol. 2006; 124: 704-716Crossref PubMed Scopus (32) Google Scholar, 18Jensen A.M. Sørensen T.L. Olesen C. Møller J.V. Nissen P. Modulatory and catalytic modes of ATP binding by the calcium pump.EMBO J. 2006; 25: 2305-2314Crossref PubMed Scopus (165) Google Scholar, 26Toyoshima C. Structural aspects of ion pumping by Ca2+-ATPase of sarcoplasmic reticulum.Arch. Biochem. Biophys. 2008; 476: 3-11Crossref PubMed Scopus (179) Google Scholar) and for PLB reconstitution with SERCA (C12E8 or octyl glucoside) (14Reddy L.G. Jones L.R. Cala S.E. O'Brian J.J. Tatulian S.A. Stokes D.L. Functional reconstitution of recombinant phospholamban with rabbit skeletal Ca2+-ATPase.J. Biol. Chem. 1995; 270: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 27Gorski P.A. Glaves J.P. Vangheluwe P. Young H.S. Sarco(endo)plasmic reticulum calcium ATPase (SERCA) inhibition by sarcolipin is encoded in its luminal tail.J. Biol. Chem. 2013; 288: 8456-8467Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) had strong detrimental effects on Ca2+ affinity (Fig. 2A) and/or stability of the enzyme over time (Fig. 2B). For example, solubilizing SERCA in the absence of Ca2+ in C12E8 caused a 3-fold decrease in Ca2+ affinity and loss of enzyme activity with a t½ of ∼3 days at 4 °C. Octyl glucoside, on the other hand, did not affect Ca2+ affinity, but caused even more rapid denaturation of the enzyme, with a t½ of less than 1 day at 4 °C. Consequently, a major challenge was to develop a new detergent system that would facilitate co-crystallization of SERCA reconstituted with PLB without denaturing the enzyme. The nonionic detergent nonyl maltoside was ultimately selected for solubilization of SERCA as it had little effect on the Ca2+ affinity of the enzyme (KCa = 0.25 versus 0.21 μm in the absence of detergent) (Fig. 2A), and preserved 90–100% of the solubilized Ca2+-ATPase activity for several weeks at 4 °C (Fig. 2B). SERCA solubilized in nonyl maltoside was then reconstituted with WT-PLB or PLB4 solubilized in decyl maltoside or dodecyl maltoside (shown with PLB4 solubilized in decyl maltoside in Fig. 2B), detergents that also preserved SERCA catalytic activity for weeks when stored at 4 °C. Ca2+-ATPase assays in the newly developed detergent system show that WT-PLB and PLB4 decreased the Ca2+ affinity of the solubilized enzyme by 2- and 5-fold, respectively, but had no significant effect on maximal ATPase activity measured at saturating Ca2+ concentrations (Fig. 3B). This alteration in the apparent Ca2+ affinity of SERCA is the hallmark of PLB inhibition (2Simmerman H.K. Jones L.R. Phospholamban. Protein structure, mechanism of action, and role in cardiac function.Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (466) Google Scholar, 3Young H.S. Stokes D.L. The mechanics of calcium transport.J. Membr. Biol. 2004; 198: 55-63Crossref PubMed Scopus (28) Google Scholar). As expected, addition of the 2D12 anti-PLB antibody (which mimics the effect of Ser16/Thr17 phosphorylation of PLB (23Chen Z. Akin B.L. Jones L.R. Mechanism of reversal of phospholamban inhibition of the cardiac Ca2+-ATPase by protein kinase A and by anti-phospholamban monoclonal antibody 2D12.J. Biol. Chem. 2007; 282: 20968-20976Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar)) largely reversed SERCA inhibition by WT-PLB and PLB4 (Fig. 3B, shaded dotted lines). Ca2+-dependent binding interactions between solubilized SERCA and PLB4 were also confirmed using chemical cross-linking. Cross-linking of residue N30C of PLB4 to Lys328 of SERCA with the heterobifunctional cross-linker KMUS (7Chen Z. Stokes D.L. Rice W.J. Jones L.R. Spatial and dynamic interactions between phospholamban and the canine cardiac Ca2+ pump revealed with use of heterobifunctional cross-linking agents.J. Biol. Chem. 2003; 278: 48348-48356Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 10Akin B.L. Chen Z. Jones L.R. Superinhibitory phospholamban mutants compete with Ca2+ for binding to SERCA2a by stabilizing a unique nucleotide-dependent conformational state.J. Biol. Chem. 2010; 285: 28540-28552Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) was inhibited over the same Ca2+ concentration range (Ki = 1.6 μm) (Fig. 3C) as enzyme activation occurred (KCa = 1.4 μm) (Fig. 3B). This strongly suggests that PLB decreases the Ca2+ affinity of SERCA by disrupting Ca2+ binding (10Akin B.L. Chen Z. Jones L.R. Superinhibitory phospholamban mutants compete with Ca2+ for binding to SERCA2a by stabilizing a unique nucleotide-dependent conformational state.J. Biol. Chem. 2010; 285: 28540-28552Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) to the enzyme. Fig. 3 further confirms that normal protein-binding interactions between PLB and SERCA were maintained in the newly developed detergent system, recapitulating results previously observed with intact SR vesicles (16Akin B.L. Jones L.R. Characterizing phospholamban to sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) protein binding interactions in human cardiac sarcoplasmic reticulum vesicles using chemical cross-linking.J. Biol. Chem. 2012; 287: 7582-7593Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) or with ER membranes co-expressing the recombinant proteins (10Akin B.L. Chen Z. Jones L.R. Superinhibitory phospholamban mutants compete with Ca2+ for binding to SERCA2a by stabilizing a unique nucleotide-dependent conformational state.J. Biol. Chem. 2010; 285: 28540-28552Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The remarkable stability of Ca2+-free SERCA solubilized in nonyl maltoside is reported here for the first time. It should be mentioned that rabbit skeletal muscle SR vesicles were used as the source of the Ca2+ pumps in this study due to the high content of the ATPase protein in the SR membrane, making enzyme purification unnecessary (15Sørensen T.L. Olesen C. Jensen A.M. Møller J.V. Nissen P. Crystals of sarcoplasmic reticulum Ca2+-ATPase.J. Biotechnol. 2006; 124: 704-716Crossref PubMed Scopus (32) Google Scholar) (Fig. 3D). (SERCA1a is highly homologous to the cardiac isoform, SERCA2a, and is regulated identically by PLB (6Toyoshima C. Asahi M. Sugita Y. Khanna R. Tsuda T. MacLennan D.H. Modeling of the inhibitory interaction of phospholamban with the Ca2+ ATPase.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 467-472Crossref PubMed Scopus (171) Google Scholar).) In addition, both WT-PLB and PLB4 prepared in alkyl mannosides were predominantly multimeric on SDS gels; WT-PLB was mostly pentameric, and PLB4 ran mostly as pentamers and tetramers. Boiling in 6% SDS prior to PAGE was required to destabilize these hig" @default.
• W1987421070 created "2016-06-24" @default.
• W1987421070 creator A5004272413 @default.
• W1987421070 creator A5018874505 @default.
• W1987421070 creator A5027046296 @default.
• W1987421070 creator A5031403355 @default.
• W1987421070 date "2013-10-01" @default.
• W1987421070 modified "2023-10-14" @default.
• W1987421070 title "The Structural Basis for Phospholamban Inhibition of the Calcium Pump in Sarcoplasmic Reticulum" @default.
• W1987421070 cites W1499775870 @default.
• W1987421070 cites W1518120013 @default.
• W1987421070 cites W1534028861 @default.
• W1987421070 cites W1539796472 @default.
• W1987421070 cites W1567439646 @default.
• W1987421070 cites W1577308337 @default.
• W1987421070 cites W1590474929 @default.
• W1987421070 cites W1593385535 @default.
• W1987421070 cites W1597612174 @default.
• W1987421070 cites W1604513294 @default.
• W1987421070 cites W1850997011 @default.
• W1987421070 cites W1966049529 @default.
• W1987421070 cites W1970953058 @default.
• W1987421070 cites W1973047758 @default.
• W1987421070 cites W1980664012 @default.
• W1987421070 cites W1998164234 @default.
• W1987421070 cites W2006505293 @default.
• W1987421070 cites W2007848144 @default.
• W1987421070 cites W2010576268 @default.
• W1987421070 cites W2016410233 @default.
• W1987421070 cites W2017699042 @default.
• W1987421070 cites W2025518120 @default.
• W1987421070 cites W2030756636 @default.
• W1987421070 cites W2033546351 @default.
• W1987421070 cites W2038840577 @default.
• W1987421070 cites W2046771956 @default.
• W1987421070 cites W2049445457 @default.
• W1987421070 cites W2056368880 @default.
• W1987421070 cites W2060075866 @default.
• W1987421070 cites W2068250491 @default.
• W1987421070 cites W2071160031 @default.
• W1987421070 cites W2071861370 @default.
• W1987421070 cites W2074404998 @default.
• W1987421070 cites W2075843198 @default.
• W1987421070 cites W2076089802 @default.
• W1987421070 cites W2090875155 @default.
• W1987421070 cites W2097761188 @default.
• W1987421070 cites W2098550162 @default.
• W1987421070 cites W2109503333 @default.
• W1987421070 cites W2109675385 @default.
• W1987421070 cites W2111464978 @default.
• W1987421070 cites W2124026197 @default.
• W1987421070 cites W2145537234 @default.
• W1987421070 cites W2162244749 @default.
• W1987421070 cites W2163341755 @default.
• W1987421070 cites W2165716854 @default.
• W1987421070 cites W2167019758 @default.
• W1987421070 cites W58055601 @default.
• W1987421070 doi "https://doi.org/10.1074/jbc.m113.501585" @default.
• W1987421070 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3798486" @default.
• W1987421070 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23996003" @default.
• W1987421070 hasPublicationYear "2013" @default.
• W1987421070 type Work @default.
• W1987421070 sameAs 1987421070 @default.
• W1987421070 citedByCount "111" @default.
• W1987421070 countsByYear W19874210702013 @default.
• W1987421070 countsByYear W19874210702014 @default.
• W1987421070 countsByYear W19874210702015 @default.
• W1987421070 countsByYear W19874210702016 @default.
• W1987421070 countsByYear W19874210702017 @default.
• W1987421070 countsByYear W19874210702018 @default.
• W1987421070 countsByYear W19874210702019 @default.
• W1987421070 countsByYear W19874210702020 @default.
• W1987421070 countsByYear W19874210702021 @default.
• W1987421070 countsByYear W19874210702022 @default.
• W1987421070 countsByYear W19874210702023 @default.
• W1987421070 crossrefType "journal-article" @default.
• W1987421070 hasAuthorship W1987421070A5004272413 @default.
• W1987421070 hasAuthorship W1987421070A5018874505 @default.
• W1987421070 hasAuthorship W1987421070A5027046296 @default.
• W1987421070 hasAuthorship W1987421070A5031403355 @default.
• W1987421070 hasBestOaLocation W19874210701 @default.
• W1987421070 hasConcept C101571972 @default.
• W1987421070 hasConcept C12554922 @default.
• W1987421070 hasConcept C134018914 @default.
• W1987421070 hasConcept C158617107 @default.
• W1987421070 hasConcept C17137333 @default.
• W1987421070 hasConcept C178790620 @default.
• W1987421070 hasConcept C181199279 @default.
• W1987421070 hasConcept C185592680 @default.
• W1987421070 hasConcept C23265538 @default.
• W1987421070 hasConcept C2778996579 @default.
• W1987421070 hasConcept C2781414619 @default.
• W1987421070 hasConcept C519063684 @default.
• W1987421070 hasConcept C55493867 @default.
• W1987421070 hasConcept C74884574 @default.
• W1987421070 hasConcept C86803240 @default.
• W1987421070 hasConcept C935647 @default.
• W1987421070 hasConceptScore W1987421070C101571972 @default.

• next