Close Proximity between Residue 30 of Phospholamban and Cysteine 318 of the Cardiac Ca2+ Pump Revealed by Intermolecular Thiol Cross-linking
2002; Elsevier BV; Volume: 277; Issue: 31 Linguagem: Inglês
10.1074/jbc.m204085200
ISSN1083-351X
AutoresLarry R. Jones, Răzvan L. Cornea, Zhenhui Chen,
Tópico(s)Ion Transport and Channel Regulation
ResumoPhospholamban (PLB) is a 52-amino acid inhibitor of the Ca2+-ATPase in cardiac sarcoplasmic reticulum (SERCA2a), which acts by decreasing the apparent affinity of the enzyme for Ca2+. To localize binding sites of SERCA2a for PLB, we performed Cys-scanning mutagenesis of PLB, co-expressed the PLB mutants with SERCA2a in insect cell microsomes, and tested for cross-linking of the mutated PLB molecules to SERCA2a using 1,6-bismaleimidohexane, a 10-Å-long, homobifunctional thiol cross-linking agent. Of several mutants tested, only PLB with a Cys replacement at position 30 (N30C-PLB) cross-linked to SERCA2a. Cross-linking occurred specifically and with high efficiency. The process was abolished by micromolar Ca2+ or by an anti-PLB monoclonal antibody and was inhibited 50% by phosphorylation of PLB by cAMP-dependent protein kinase. The SERCA2a inhibitors thapsigargin and cyclopiazonic acid also completely prevented cross-linking. The two essential requirements for cross-linking of N30C-PLB to SERCA2a were a Ca2+-free enzyme and, unexpectedly, a micromolar concentration of ATP or ADP, demonstrating that N30C-PLB cross-links preferentially to the nucleotide-bound, E2 state of SERCA2a. Sequencing of a purified proteolytic fragment in combination with SERCA2a mutagenesis identified Cys318 of SERCA2a as the sole amino acid cross-linked to N30C-PLB. The proximity of residue 30 of PLB to Cys318 of SERCA2a suggests that PLB may interfere with Ca2+ activation of SERCA2a by a protein interaction occurring near transmembrane helix M4. Phospholamban (PLB) is a 52-amino acid inhibitor of the Ca2+-ATPase in cardiac sarcoplasmic reticulum (SERCA2a), which acts by decreasing the apparent affinity of the enzyme for Ca2+. To localize binding sites of SERCA2a for PLB, we performed Cys-scanning mutagenesis of PLB, co-expressed the PLB mutants with SERCA2a in insect cell microsomes, and tested for cross-linking of the mutated PLB molecules to SERCA2a using 1,6-bismaleimidohexane, a 10-Å-long, homobifunctional thiol cross-linking agent. Of several mutants tested, only PLB with a Cys replacement at position 30 (N30C-PLB) cross-linked to SERCA2a. Cross-linking occurred specifically and with high efficiency. The process was abolished by micromolar Ca2+ or by an anti-PLB monoclonal antibody and was inhibited 50% by phosphorylation of PLB by cAMP-dependent protein kinase. The SERCA2a inhibitors thapsigargin and cyclopiazonic acid also completely prevented cross-linking. The two essential requirements for cross-linking of N30C-PLB to SERCA2a were a Ca2+-free enzyme and, unexpectedly, a micromolar concentration of ATP or ADP, demonstrating that N30C-PLB cross-links preferentially to the nucleotide-bound, E2 state of SERCA2a. Sequencing of a purified proteolytic fragment in combination with SERCA2a mutagenesis identified Cys318 of SERCA2a as the sole amino acid cross-linked to N30C-PLB. The proximity of residue 30 of PLB to Cys318 of SERCA2a suggests that PLB may interfere with Ca2+ activation of SERCA2a by a protein interaction occurring near transmembrane helix M4. sarco(endo)plasmic reticulum Ca2+-ATPase 1,6-bismaleimidohexane 3-(N-morpholino)propanesulfonic acid canine PLB with Asn30 replaced by Cys and Cys residues 36, 41, and 46 replaced by Ala phospholamban catalytic subunit of cyclic AMP-dependent protein kinase fast skeletal muscle isoform of SERCA cardiac isoform of SERCA sarcoplasmic reticulum adenosine 5′-(β,γ-methylenetriphosphate) 5′-adenylyl-β, γ-imidodiphosphate Ca2+-ATPase activities or SERCA1 enzymes are 100-kDa integral membrane proteins responsible for the active transport of Ca2+ into the sarco(endo)plasmic reticulum (1MacLennan D.H. Rice W.J. Green N.M. J. Biol. Chem. 1997; 272: 28815-28818Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar). In heart, the predominant Ca2+-ATPase expressed is SERCA2a (1MacLennan D.H. Rice W.J. Green N.M. J. Biol. Chem. 1997; 272: 28815-28818Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar), which pumps Ca2+ into the SR lumen, causing cardiac muscle relaxation (2Bers D.M. Nature. 2002; 415: 198-205Crossref PubMed Scopus (3207) Google Scholar). A unique property of cardiac muscle is the regulation of SERCA2a by PLB, a small, single span membrane protein (3Simmerman H.K.B. Collins J.H. Theibert J.L. Wegener A.D. Jones L.R. J. Biol. Chem. 1986; 261: 13333-13341Abstract Full Text PDF PubMed Google Scholar, 4Fujii J.K. Ueno A. Kitano K. Tanaka S. Kadoma M. Tada M. J. Clin. Invest. 1987; 79: 301-304Crossref PubMed Scopus (135) Google Scholar), which inhibits the Ca2+-ATPase by decreasing its apparent affinity for Ca2+ (5Cantilina T. Sagara Y. Inesi G. Jones L.R. J. Biol. Chem. 1993; 268: 17018-17025Abstract Full Text PDF PubMed Google Scholar). Phosphorylation of PLB at Ser16 by PKA and at Thr17 by Ca2+/calmodulin-dependent protein kinase relieves the inhibitory action of PLB on SERCA2a, giving an increase in the rate of cardiac muscle relaxation as well as a positive inotropic effect (6Simmerman H.K.B. Jones L.R. Physiol. Rev. 1997; 78: 921-947Crossref Scopus (458) Google Scholar, 7Schmidt A.G. Edes I. Kranias E.G. Cardiovasc. Drugs Ther. 2001; 15: 387-396Crossref PubMed Scopus (59) Google Scholar). Currently, there is much interest in understanding the molecular mechanism of SERCA2a inhibition by PLB, both as a paradigm for understanding membrane protein interactions and for the potential of targeting drugs to this system to treat heart failure (6Simmerman H.K.B. Jones L.R. Physiol. Rev. 1997; 78: 921-947Crossref Scopus (458) Google Scholar, 7Schmidt A.G. Edes I. Kranias E.G. Cardiovasc. Drugs Ther. 2001; 15: 387-396Crossref PubMed Scopus (59) Google Scholar). Phospholamban has an interesting structure (6Simmerman H.K.B. Jones L.R. Physiol. Rev. 1997; 78: 921-947Crossref Scopus (458) Google Scholar). Containing only 52 amino acids, the protein is a homopentamer in the membrane held together by Leu/Ile zipper interactions occurring in the transmembrane region (residues 32–52) (8Simmerman H.K.B. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). The cytoplasmic domain (residues 1–31) is highly charged and basic and is postulated to interact with SERCA2a by both electrostatic and hydrophobic interactions (9James P. Inui M. Tada M. Chiesi M. Carafoli E. Nature. 1989; 342: 90-92Crossref PubMed Scopus (370) Google Scholar, 10Toyofuku T. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1994; 269: 3088-3094Abstract Full Text PDF PubMed Google Scholar). PLB mutagenesis studies suggest that the PLB monomer is the active inhibitory species, which dissociates from the pentamer, binds to SERCA2a, and inhibits the Ca2+-ATPase by direct protein interactions (11Kimura Y. Kurzydlowski K Tada M. MacLennan D.H. J. Biol. Chem. 1997; 272: 15061-15064Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 12Cornea R.L. Jones L.R. Autry J.M. Thomas D.D. Biochemistry. 1997; 36: 2960-2967Crossref PubMed Scopus (158) Google Scholar, 13Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 14Asahi M. Kimura Y. Kurzydlowski K Tada M. MacLennan D.H. J. Biol. Chem. 1999; 274: 32855-32862Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 15Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 16Reddy L.G. Autry J.M. Jones L.R. Thomas D.D. J. Biol. Chem. 1999; 274: 7649-7655Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Considerable attention has been given to delineating the three-dimensional structural interactions between PLB and SERCA2a, especially in light of the recent crystal structure determination of SERCA1a (the skeletal muscle isoform) to 2.6-Å resolution (17Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1594) Google Scholar). An indirect approach for studying protein structure is to use mutagenesis and, by producing loss of function or gain of function PLB mutants, infer sites in both the cytoplasmic (10Toyofuku T. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1994; 269: 3088-3094Abstract Full Text PDF PubMed Google Scholar, 18Kimura Y. Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1998; 273: 14238-14241Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) and membrane (11Kimura Y. Kurzydlowski K Tada M. MacLennan D.H. J. Biol. Chem. 1997; 272: 15061-15064Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 13Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 15Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) regions of PLB that may be important for regulatory interactions with the Ca2+ pump. Another approach is to use chemical cross-linking to directly identify physical contact points. In an earlier reconstitution study by James et al. (9James P. Inui M. Tada M. Chiesi M. Carafoli E. Nature. 1989; 342: 90-92Crossref PubMed Scopus (370) Google Scholar), Lys3 of PLB was shown to photoaffinity-label to residues 397–400 of SERCA2a. A functional requirement for these SERCA2a residues in transducing PLB inhibition was subsequently demonstrated by Toyofuku et al. (19Toyofuku T. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1994; 269: 22929-22932Abstract Full Text PDF PubMed Google Scholar) using replacement mutagenesis. However, this strategy of purification and reconstitution followed by photoaffinity labeling (9James P. Inui M. Tada M. Chiesi M. Carafoli E. Nature. 1989; 342: 90-92Crossref PubMed Scopus (370) Google Scholar) is difficult to execute with PLB and SERCA2a and has not been duplicated in other laboratories. More recent approaches to address physical interactions between PLB and SERCA2a are cryoelectron microscopy of PLB/SERCA2a co-crystals (20Young H.S. Jones L.R. Stokes D.L. Biophys. J. 2001; 81: 884-894Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and assessment of fusion protein interactions (21Kimura Y. Inui M. Mol. Pharmacol. 2002; 61: 667-673Crossref PubMed Scopus (10) Google Scholar). Here we describe direct chemical cross-linking of PLB to SERCA2a in microsomes in situ with no reconstitution required. A Cys-substituted mutant of PLB (N30C-PLB) and native SERCA2a were co-expressed in Sf21 insect cell microsomes (13Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 15Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and cross-linking of the two molecules was achieved with the homobifunctional thiol probe, BMH (22Green N.S. Reisler E. Houk K.N. Protein Sci. 2001; 10: 1293-1304Crossref PubMed Scopus (197) Google Scholar). N30C-PLB is shown to label SERCA2a specifically and with high efficiency. Coupling occurs at only one of the 26 (13) endogenous Cys residues of SERCA2a, Cys318. Characterization of factors modulating the cross-linking signal gives several new insights into the mechanism of Ca2+ pump inhibition by PLB. BMH was obtained from Pierce. Thapsigargin and cyclopiazonic acid were purchased from Sigma. Mouse recombinant PKA was from Calbiochem, and endo-Asp-N and endo-Lys-C were obtained from Roche Molecular Biochemicals. Point mutations were introduced into the cDNA encoding the canine isoform of PLB, using the Altered Sites II Mutagenesis SystemTM (13Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 15Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). All single Cys replacements in PLB described presently were made on the Cys-less PLB background, which is canine PLB with Cys residues 36, 41, and 46 changed to Ala. The Cys-less PLB mutant retains full functional activity (23Autry J.M. Kobayashi Y. Jones L.R. Biophys. J. 1998; 74 (abstr.): 337Google Scholar, 24Karim C.B. Paterlini M.G. Reddy L.G. Hunter G.W. Barany G. Thomas D.D. J. Biol. Chem. 2001; 276: 38814-38819Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Mutated PLB inserts were subcloned into the BglII site of the pVL1393 transfection vector and co-transfected into Sf21 insect cells using BaculoGoldTM (Pharmingen) linearized baculovirus DNA. Baculoviruses were plaque-purified and amplified as described (13Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 15Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Wild-type canine SERCA2a cDNA (13Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) was mutated directly in the transfection vector pVL1393 using the QuikChangeTM XL-Gold system (Stratagene). All mutations were confirmed by DNA sequencing. Canine SERCA2a and PLB were co-expressed in Sf21 insect cells as described (13Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 15Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Most co-expressions were with wild-type SERCA2a and N30C-PLB (on the Cys-less PLB background). Microsomes were harvested 60 h after initiating baculovirus infections and stored frozen in small aliquots at −40 °C at a protein concentration of 6–10 mg/ml (25Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Cross-linking between N30C-PLB and SERCA2a remained intact after several freeze-thaw cycles of microsomal membranes. Sulfhydryl cross-linking was performed at room temperature using the homobifunctional cross-linking agent BMH (Pierce). The reactions were conducted with 11 μg of microsomal protein in 12 μl of buffer A, consisting of 40 mm MOPS (pH 7.0), 3.2 mm MgCl2, 75 mm KCl, 3 mm ATP, and 1 mm EGTA. Incubations were routinely conducted for 1 h in the presence of 0.1 mmBMH. Reactions were started by adding 0.75 μl of BMH from a 1.6 mm stock solution in dimethyl sulfoxide and stopped by adding 7.5 μl of SDS-PAGE sample-loading buffer (13Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) containing 15% SDS plus 100 mm dithiothreitol. After terminating the reactions, samples were subjected to SDS-PAGE and immunoblotting. To assess Ca2+ effects on cross-linking, ionized Ca2+ was varied by adding CaCl2 to buffer A (13Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 15Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). When PKA effects on cross-linking were tested, 1 μg of the catalytic subunit was added to 11 μg of microsomes in buffer A, and phosphorylation was conducted for 10 min at room temperature before initiating cross-linking by the addition of BMH. For PKA experiments, the final concentration of BMH was increased to 2.0 mm to compensate for the ∼1.5 mm final concentration of mercaptoethanol contributed by the PKA preparation. In some experiments, ATP in buffer A was replaced by other nucleotides, as indicated. SDS-PAGE using 8% polyacrylamide (26Porzio M.A. Pearson A.M. Biochim. Biophys. Acta. 1977; 490: 27-34Crossref PubMed Scopus (504) Google Scholar) and immunoblotting were performed as described previously (13Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 15Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). For detection of PLB, blots were probed with anti-PLB monoclonal antibody, 2D12, which recognizes residues 7–13 of canine PLB. For detection of SERCA2a, blots were probed with anti-SERCA2a monoclonal antibody, 2A7-A1, which recognizes residues 386–396 of canine SERCA2a. Epitopes were mapped (27Koybayashi Y.M. Jones L.R. J. Biol. Chem. 1999; 274: 28660-28668Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) using PepSpotsTM (Jerini Bio Tools). Antibody-binding protein bands were visualized by 125I-protein A and autoradiography and quantified using a Bio-Rad Molecular Imager Fx. In two experiments, the procedure was modified slightly. In the experiment in which the anti-PLB monoclonal antibody effect on BMH cross-linking was examined (Fig. 4 A), the125I-protein A step was omitted, and the immunoblot was probed with 125I-2D12 directly, iodinated using IODO-GEN (Bio-Rad). This was done to eliminate interference from125I-protein A binding to the 2D12 antibody carried over from the cross-linking samples. In the experiment in which the PKA effect on cross-linking was examined (Fig. 4 B), the immunoblot was probed with our anti-PLB monoclonal antibody, 1F1, raised to residues 1–10 of canine PLB, instead of 2D12. In control experiments, we observed that phosphorylation of PLB at Ser16 by PKA partially inhibited 2D12 binding to PLB, apparently due to steric or conformational effects. Phosphorylation of PLB by PKA had no effect on 1F1 binding to PLB. Ca2+-ATPase activity of microsomes co-expressing SERCA2a and N30C-PLB was measured at 37 °C in the presence and absence of the anti-PLB antibody, 2D12, in buffer containing 50 mm MOPS (pH 7.0), 3 mmMgCl2, 100 mm KCl, 5 mmNaN3, 3 μg/ml A23187, 3 mm ATP, and 1 mm EGTA (13Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 15Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Ionized Ca2+ concentration was varied by adding CaCl2 (13Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 15Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In order to identify which Cys residue of SERCA2a was cross-linked to N30C-PLB, we scaled up the cross-linking reaction 15,000-fold to allow purification of the two cross-linked proteins to homogeneity. Proteolysis was then performed, and the SERCA2a peptide covalently attached to PLB was isolated and sequenced. For maximal cross-linking, 166 mg of Sf21 microsomes co-expressing N30C-PLB and SERCA2a were cross-linked with 0.1 mm BMH for 1 h at room temperature in 180 ml of buffer A. 10 mmdithiothreitol was added to terminate the cross-linking reaction, and the sample was sedimented to yield the cross-linked microsomal pellet. Three rounds of sequential monoclonal antibody-affinity chromatographies were then used to obtain a purified SERCA2a peptide covalently attached to N30C-PLB, as follows. In round 1, SERCA2a was isolated from the cross-linked microsomal pellet using anti-SERCA (2A7-A1) monoclonal antibody affinity chromatography essentially as described previously (28Reddy L.G. Jones L.R. Cala S.E. O'Brian J.J. Tatulian S.A. Stokes D.L. J. Biol. Chem. 1995; 270: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The microsomal pellet was dissolved in 1% SDS followed by 3.5% Triton X-100, and 64 ml of solubilized microsomal proteins were loaded over 6.6 ml of 2A7-A1 affinity beads. A flow-through fraction was collected, and the beads were then washed with four consecutive 6.6-ml rinses of 20 mm MOPS (pH 7.2), 0.5 m NaCl, and 0.2% Triton X-100 (fractions 1–4), followed by three additional 6.6-ml rinses with 20 mm MOPS (pH 7.2) and 0.1% Triton X-100 (fractions 5–7). Purified SERCA2a was then eluted in fractions 8–12 with consecutive 6.6-ml rinses of 20 mm glycine (pH 2.4), 0.1% Triton X-100. Fractions 8–12 were eluted into 110 volume of concentrated MOPS buffer to return the pH to 7.2. Column fractions were analyzed by SDS-PAGE, followed by Coomassie Blue staining and immunoblotting. For economy, only results from peak column fractions are displayed in the figures. In round 2, fractions 8–12 from round 1 containing purified SERCA2a were pooled and subjected to anti-PLB (2D12) monoclonal antibody affinity chromatography (28Reddy L.G. Jones L.R. Cala S.E. O'Brian J.J. Tatulian S.A. Stokes D.L. J. Biol. Chem. 1995; 270: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Pooled fractions 8–12 from round 1 were re-equilibrated in 1% SDS, 3.5% Triton X-100 and then loaded over 2.8 ml of 2D12 affinity beads. The column was processed identically to that described for round 1, employing 2.8-ml rinses for each fraction. The Ca2+-ATPase molecules not cross-linked to N30C-PLB passed freely through the column and were recovered in the flow-through fraction and fraction 1. Only SERCA2a molecules cross-linked to N30C-PLB were retained by the column and recovered in the acidic pH elution fractions (fractions 8–12). Fractions 8–12 were eluted into concentrated MOPS to return the pH to 7.2 as described above. In round 3, the purified, cross-linked product from round 2 was proteolyzed, and the cross-linked peptide was isolated. Fractions 8–12 from round 2 in 90 mm MOPS, 18 mm glycine, and 0.1% Triton X-100 (pH 7.2) were pooled and Amicon-concentrated to 1 ml. 8 μg of endo-Asp-N was then added, and proteolysis was conducted for 4 h at 37 °C. Digestion was terminated by adding 10 mm EDTA, and then 20 μg of endo-Lys-C was added, and the proteolysis continued overnight at 37 °C. The next morning, endo-Lys-C digestion was terminated by the addition of 0.3 mm 1-chloro-3-tosylamido-7-amino-l-2-heptanone, followed by the addition of 1% SDS and placement of the sample in a boiling water bath for 10 min. 3.5% Triton X-100 was then added, and the SERCA2a peptide cross-linked to N30C-PLB was isolated using anti-PLB monoclonal antibody affinity chromatography as described in round 2 above. Peptides were subjected to SDS-PAGE and transferred to Immobilon-PSQ (Millipore Corp.) for sequencing. Sequencing was with an Applied Biosystems 492 Protein Sequencer at the Laboratory for Macromolecular Structure (Purdue University, W. Lafayette, IN). To screen for residues of PLB interacting with SERCA2a, we performed Cys-scanning mutagenesis of PLB and co-expressed the PLB mutants with wild-type SERCA2a in Sf21 microsomes (13Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 15Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Cross-linking of the mutated PLB molecules to SERCA2a was then tested using the homobifunctional thiol cross-linking agent, BMH, a 10-Å-long probe (22Green N.S. Reisler E. Houk K.N. Protein Sci. 2001; 10: 1293-1304Crossref PubMed Scopus (197) Google Scholar). Taking advantage of the fact that PLB devoid of Cys residues is fully functional (23Autry J.M. Kobayashi Y. Jones L.R. Biophys. J. 1998; 74 (abstr.): 337Google Scholar, 24Karim C.B. Paterlini M.G. Reddy L.G. Hunter G.W. Barany G. Thomas D.D. J. Biol. Chem. 2001; 276: 38814-38819Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), we made the Cys substitutions on the Cys-less PLB background, which is native PLB with its three endogenous cysteines (residues 36, 41, and 46) changed to alanine. Fig. 1 shows cross-linking results obtained when single Cys replacements at residues 30–41 of PLB were scanned for cross-linking to SERCA2a. (Upstream and downstream PLB mutations have subsequently been scanned, which will be reported on in a later work.) Sf21 microsomes from each co-expression were incubated with 0.1 mm BMH for 60 min in buffer A and then subjected to SDS-PAGE and immunoblotting. The blots were probed with anti-PLB monoclonal antibody 2D12 (Fig. 1 A) and anti-SERCA2a monoclonal antibody 2A7-A1 (Fig. 1 B). PLB with the single Cys replacement at residue 30 (N30C-PLB) cross-linked strongly to SERCA2a, as indicated by the very intense anti-PLB signal obtained at a molecular mass just greater than 100 kDa (Fig.1 A, PLB/SER). This PLB-positive band corresponded to the 6-kDa PLB monomer (4Fujii J.K. Ueno A. Kitano K. Tanaka S. Kadoma M. Tada M. J. Clin. Invest. 1987; 79: 301-304Crossref PubMed Scopus (135) Google Scholar) cross-linked to the 109-kDa Ca2+ pump protein (13Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). None of the other PLB mutants tested cross-linked to SERCA2a (Fig. 1 A); nor did wild-type PLB with all three Cys residues left intact (data not shown). All mutated PLB molecules were efficiently expressed and are seen in different mobility forms ranging from pentamers (PLB 5 ) through monomers (PLB 1 ) at the bottom of the immunoblot (Fig. 1 A). SERCA2a was also expressed well in all of the microsomal preparations, and a slight mobility shift was evident in the Ca2+ pump protein after it was cross-linked to N30C-PLB (Fig. 1 B). Maximal cross-linking of N30C-PLB to SERCA2a by 0.1 mm BMH in buffer A was achieved at an incubation time of 60 min; half-maximal cross-linking occurred at 15 min (Fig.2 A). The mobility of the SERCA2a band decreased gradually with increasing cross-linking (Fig.2 B), but due to the broadness of the Ca2+ pump band and the absence of clear doublet formation, the mobility shift could not be used to accurately assess the percentage of Ca2+-ATPase molecules cross-linked, which is considerable (see Fig. 8). When cross-linking was conducted for 60 min in buffer A, half-maximal cross-linking of N30C-PLB to SERCA2a occurred at a BMH concentration of 20–30 μm; maximal cross-linking was achieved at 100 μm BMH (Fig. 2 C). Twenty-fold higher concentrations of BMH (2 mm) gave no additional cross-linking (data not shown).Figure 8Purification of SERCA2a cross-linked to N30C-PLB using anti-PLB monoclonal antibody affinity chromatography (round 2). Fractions 8–12 from round 1 containing the purified Ca2+-ATPase were pooled (pool 8–12), and the Ca2+-ATPase was repurified by anti-PLB monoclonal antibody affinity chromatography using 2D12. Processing and nomenclature is the same as described for Fig. 7. Std., protein standards.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Under the conditions of SDS-PAGE utilized, N30C-PLB (expressed on the Cys-less PLB background) migrated primarily as a monomer (Fig. 2,A and C, lane 1). BMH induced the rapid dimerization of PLB monomers, a process that was already complete at an incubation time of 5 min in the presence of 0.1 mmBMH (Fig. 2 A, lane 2, PLB 2 ), when only 14% of the maximal level of PLB·SERCA2a heterodimers had formed, or at an incubation time of 60 min in the presence at 10 μm BMH (Fig. 2 C, lane 2,PLB 2 ), when only 6% of the maximal level of PLB·SERCA2a heterodimers had formed. We found that even without cross-linking agents, N30C-PLB could retain pentamers on SDS-PAGE but that these pentamers were unstable and dissociated readily at low concentrations of SDS (Fig. 2 D). With only 0.2% SDS in the sample loading buffer prior to electrophoresis, N30C-PLB was mostly pentameric on SDS-PAGE; with 1.4% or higher concentrations of SDS in the sample loading buffer, the protein was mostly monomeric, with some dimers (Fig. 2 D). These results suggest that in intact microsomal membranes, N30C-PLB is predominantly a pentamer. Cross-linking between PLB monomers preassembled as pentamers is expected to be a very rapid process (29Guan L. Murphy F.D. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3475-3480Crossref PubMed Scopus (47) Google Scholar). Cross-linking of PLB monomers to SERCA2a, in contrast, is a slower process. It should be pointed out that even after rapid homodimer formation by PLB monomers within pentamers, there should always be at least one uncross-linked monomer per pentamer that is free to dissociate from the complex and interact with SERCA2a. Using purified PLB and SERCA2a as standards, we calculated a molar ratio of 4:1 for N30C-PLB to SERCA2a in Sf21 microsomes. A molar ratio of 2:1 for the naturally occurring proteins in dog cardiac SR vesicles was found. PLB inhibits the Ca2+-ATPase at low ionized Ca2+ concentration by decreasing the apparent affinity of the enzyme for Ca2+(5Cantilina T. Sagara Y. Inesi G. Jones L.R. J. Biol. Chem. 1993; 268: 17018-17025Abstract Full Text PDF PubMed Google Scholar). No inhibition is typically observed at high ionized Ca2+ concentration (6Simmerman H.K.B. Jones L.R. Physiol. Rev. 1997; 78: 921-947Crossref Scopus (458) Google Scholar). This suggests that Ca2+may disrupt the physical interaction between PLB and SERCA2a (9James P. Inui M. Tada M. Chiesi M. Carafoli E. Nature. 1989; 342: 90-92Crossref PubMed Scopus (370) Google Scholar, 30Asahi M. McKenna E. Kurzydlowski K. Tada M. MacLennan D.L. J. Biol. Chem. 2000; 275: 15034-15038Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Fig. 3 A demonstrates
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