Spatial and Dynamic Interactions between Phospholamban and the Canine Cardiac Ca2+ Pump Revealed with Use of Heterobifunctional Cross-linking Agents
2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês
10.1074/jbc.m309545200
ISSN1083-351X
AutoresZhenhui Chen, David L. Stokes, William J. Rice, Larry R. Jones,
Tópico(s)Cardiac pacing and defibrillation studies
ResumoHeterobifunctional thiol to amine cross-linking agents were used to gain new insights on the dynamics and conformational factors governing the interaction between the cardiac Ca2+ pump (SERCA2a) and phospholamban (PLB). PLB is a small protein inhibitor of SERCA2a that reduces enzyme affinity for Ca2+ and thereby regulates cardiac contractility. We found that the PLB monomer with Asn27 or Asn30 changed to Cys (N27C-PLB or N30C-PLB) cross-linked to lysine of SERCA2a within seconds with ≥80% efficiency. Optimal cross-linking occurred at spacer chain lengths of 10 and 15 Å for N27C and N30C, respectively. The rapid time course of cross-linking indicated that neither dissociation of PLB pentamers nor binding of PLB monomers to SERCA2a was rate-limiting. Cross-linking occurred only to the E2 (Ca2+-free) conformation of SERCA2a, was strongly favored by nucleotide binding to this state, and was completely inhibited by thapsigargin. Protein sequencing in combination with mutagenesis identified of Lys328 of SERCA2a as the target of cross-linking. A three-dimensional map of interacting residues indicated that the cross-linking distances were entirely compatible with the 10-Å distance recently determined between N30C of PLB and Cys318 of SERCA2a. In contrast, Lys3 of PLB did not cross-link to any Lys (or Cys) of SERCA2a, suggesting that previous three-dimensional models that constrain Lys3 near residues 397–400 of thapsigargin-inhibited SERCA2a should be viewed with caution. Furthermore, although earlier models of PLB·SERCA2a are based on thapsigargin-bound SERCA, our results suggest that the nucleotide-bound, E2 conformation is substantially different and represents the key conformational state for interacting with PLB. Heterobifunctional thiol to amine cross-linking agents were used to gain new insights on the dynamics and conformational factors governing the interaction between the cardiac Ca2+ pump (SERCA2a) and phospholamban (PLB). PLB is a small protein inhibitor of SERCA2a that reduces enzyme affinity for Ca2+ and thereby regulates cardiac contractility. We found that the PLB monomer with Asn27 or Asn30 changed to Cys (N27C-PLB or N30C-PLB) cross-linked to lysine of SERCA2a within seconds with ≥80% efficiency. Optimal cross-linking occurred at spacer chain lengths of 10 and 15 Å for N27C and N30C, respectively. The rapid time course of cross-linking indicated that neither dissociation of PLB pentamers nor binding of PLB monomers to SERCA2a was rate-limiting. Cross-linking occurred only to the E2 (Ca2+-free) conformation of SERCA2a, was strongly favored by nucleotide binding to this state, and was completely inhibited by thapsigargin. Protein sequencing in combination with mutagenesis identified of Lys328 of SERCA2a as the target of cross-linking. A three-dimensional map of interacting residues indicated that the cross-linking distances were entirely compatible with the 10-Å distance recently determined between N30C of PLB and Cys318 of SERCA2a. In contrast, Lys3 of PLB did not cross-link to any Lys (or Cys) of SERCA2a, suggesting that previous three-dimensional models that constrain Lys3 near residues 397–400 of thapsigargin-inhibited SERCA2a should be viewed with caution. Furthermore, although earlier models of PLB·SERCA2a are based on thapsigargin-bound SERCA, our results suggest that the nucleotide-bound, E2 conformation is substantially different and represents the key conformational state for interacting with PLB. PLB 1The abbreviations used are: PLBphospholambanSERCAsarco(endo)plasmic reticulum Ca2+-ATPaseSERCA2acardiac isoform of SERCASERCA1afast skeletal muscle isoform of SERCASRsarcoplasmic reticulumN27C-PLB and N30C-PLBcanine PLB with Asn27 and Asn30, respectively, replaced by Cys, and Cys residues 36, 41, and 46 replaced by AlaMOPS3-(N-morpholino)propanesulfonic acidNHSN-hydroxysuccinimideWTwild-typeBMH1,6-bismaleimidohexane;EMCS, N-(ϵ-maleimidocaproyloxy)succinimide ester; KMUS, N-(κ-maleimidoundecanoyloxy)sulfosuccinimide ester; E1, high Ca2+ affinity conformation of Ca2+-ATPase; E2, low Ca2+ affinity conformation of Ca2+-ATPase. is a small phosphoprotein modulator of the Ca2+-pump (SERCA2a) in cardiac SR, which is critically involved in regulating the strength and duration of the heartbeat (1Simmerman H.K.B. Jones L.R. Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (467) Google Scholar, 2Schmidt A.G. Edes I. Kranias E.G. Cardiovasc. Drugs Ther. 2001; 15: 387-396Crossref PubMed Scopus (61) Google Scholar). In the dephosphorylated state, PLB inhibits the Ca2+-ATPase by decreasing its apparent affinity for Ca2+ (3Cantilina 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 and Thr17 during β-adrenergic stimulation of the heart reverses PLB inhibition and augments Ca2+ loading of the SR (4Wegener A.D. Simmerman H.K.B. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar), thus producing positive inotropic and lusitropic effects (2Schmidt A.G. Edes I. Kranias E.G. Cardiovasc. Drugs Ther. 2001; 15: 387-396Crossref PubMed Scopus (61) Google Scholar, 5Bers D.M. Nature. 2002; 415: 198-205Crossref PubMed Scopus (3391) Google Scholar). PLB is a single-span membrane protein composed of 52 amino acids, which forms a homopentamer within the SR membrane (1Simmerman H.K.B. Jones L.R. Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (467) Google Scholar, 4Wegener A.D. Simmerman H.K.B. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar). Recent work suggests that there is a dynamic equilibrium between PLB monomers and pentamers (6Cornea R.L. Jones L.R. Autry J.M. Thomas D.D. Biochemistry. 1997; 36: 2960-2967Crossref PubMed Scopus (160) Google Scholar), and that the PLB monomer is responsible for binding to SERCA2a (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and inhibiting it (8Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 9Kimura Y. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1997; 272: 15061-15064Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). In intact myocardium, β-adrenergic receptor stimulation disrupts the inhibitory interaction between PLB and SERCA2a rapidly; PLB phosphorylation, Ca2+ transport, and contractility all increase within seconds after β-receptor activation (4Wegener A.D. Simmerman H.K.B. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar, 10Lindemann J.P. Jones L.R. Hathaway D.R. Henry B.G. Watanabe A.M. J. Biol. Chem. 1983; 258: 464-471Abstract Full Text PDF PubMed Google Scholar). This suggests that PLB monomers must associate and dissociate quickly, over a time course of seconds or faster, to allow for dynamic regulation of SERCA2a and the strength of contractility. phospholamban sarco(endo)plasmic reticulum Ca2+-ATPase cardiac isoform of SERCA fast skeletal muscle isoform of SERCA sarcoplasmic reticulum canine PLB with Asn27 and Asn30, respectively, replaced by Cys, and Cys residues 36, 41, and 46 replaced by Ala 3-(N-morpholino)propanesulfonic acid N-hydroxysuccinimide wild-type 1,6-bismaleimidohexane; The emerging picture for the mechanism of SERCA2a inhibition by PLB suggests mutually exclusive binding of Ca2+ and PLB to SERCA2a. Chemical cross-linking (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 11James P. Inui M. Tada M. Chiesi M. Carafoli E. Nature. 1989; 342: 90-92Crossref PubMed Scopus (374) Google Scholar) and immunoprecipitation (12Asahi M. McKenna E. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 2000; 275: 15034-15038Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) indicate that PLB binds preferentially to the Ca2+-free conformation of SERCA2a, dubbed E2 (3Cantilina T. Sagara Y. Inesi G. Jones L.R. J. Biol. Chem. 1993; 268: 17018-17025Abstract Full Text PDF PubMed Google Scholar). The alternative conformation induced by Ca2+, E1, does not appear to be capable of binding PLB (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), but instead is primed for catalyzing the hydrolysis of ATP and subsequent steps leading to Ca2+ transport across the SR membrane (13Inesi G. Zhang Z. Lewis D. Biophys. J. 2002; 83: 2327-2332Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). By stabilizing SERCA2a in the E2 conformation, PLB slows the transition from E2 to E1 during the catalytic cycle (3Cantilina T. Sagara Y. Inesi G. Jones L.R. J. Biol. Chem. 1993; 268: 17018-17025Abstract Full Text PDF PubMed Google Scholar), thus impeding overall Ca2+ transport (Fig. 1). Thus, the apparent reduction of Ca2+ affinity is a kinetic effect of PLB on conformational changes, not necessarily a direct effect on Ca2+ binding sites (3Cantilina T. Sagara Y. Inesi G. Jones L.R. J. Biol. Chem. 1993; 268: 17018-17025Abstract Full Text PDF PubMed Google Scholar). Recently, the crystal structure of SERCA1a, the skeletal muscle isoform of the Ca2+ pump, was determined, both in the E1 state with bound Ca2+ (14Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1619) Google Scholar), and in the E2 state, bound with the irreversible inhibitor thapsigargin (15Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar). These structures reveal a relatively complex molecule composed of ∼1000 amino acids, ten transmembrane helices, and three distinct cytoplasmic domains (16MacLennan 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). Although no crystal structure of PLB has yet been reported, numerous biophysical methods have been used to study its structure (17Slovic A.M. Summa C.M. Lear J.D. DeGrado W.F. Protein Sci. 2003; 12: 337-348Crossref PubMed Scopus (41) Google Scholar). Because of technical limitations of the various techniques, no consensus has yet been reached for the complete structure of PLB within biological membranes or, more particularly, in association with SERCA2a (1Simmerman H.K.B. Jones L.R. Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (467) Google Scholar, 17Slovic A.M. Summa C.M. Lear J.D. DeGrado W.F. Protein Sci. 2003; 12: 337-348Crossref PubMed Scopus (41) Google Scholar). Nevertheless, there is great interest in understanding the physical basis for Ca2+ pump regulation (1Simmerman H.K.B. Jones L.R. Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (467) Google Scholar, 2Schmidt A.G. Edes I. Kranias E.G. Cardiovasc. Drugs Ther. 2001; 15: 387-396Crossref PubMed Scopus (61) Google Scholar, 5Bers D.M. Nature. 2002; 415: 198-205Crossref PubMed Scopus (3391) Google Scholar). As a result, several models of the three-dimensional interactions between different residues of PLB and SERCA2a have recently been proposed (18Young H.S. Jones L.R. Stokes D.L. Biophys. J. 2001; 81: 884-894Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 19Hutter M.C. Krebs J. Meiler J. Griesinger C. Carafoli E. Helms V. Chembiochem. 2002; 3: 1200-1208Crossref PubMed Scopus (37) Google Scholar, 20Toyoshima C. Asahi M. Sugita Y. Khanna R. Tsuda T. MacLennan D.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 467-472Crossref PubMed Scopus (172) Google Scholar), using the E2 conformation of SERCA determined by crystallography (15Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar) or by cryoelectronmicroscopy (21Stokes D.L. Green N.M. Annu. Rev. Biophys. Biomol. Struct. 2003; 32: 445-468Crossref PubMed Scopus (78) Google Scholar) as a structural template. Biochemical work has demonstrated that the PLB pentamer is stabilized by a network of interdigitating Leu/Ile residues along one face of the transmembrane helix between residues 37 and 51 of each monomer (17Slovic A.M. Summa C.M. Lear J.D. DeGrado W.F. Protein Sci. 2003; 12: 337-348Crossref PubMed Scopus (41) Google Scholar, 22Simmerman H.K. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Point mutations made at these Leu/Ile zipper residues destabilized the pentamer (22Simmerman H.K. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), and the concomitant enhancement of SERCA2a inhibition led to the idea that the PLB monomer is the species responsible for enzyme inhibition (8Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 9Kimura Y. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1997; 272: 15061-15064Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 24Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). This has now been verified directly by chemical cross-linking (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Point mutations along the opposite face of the transmembrane helix attenuated SERCA2a inhibition, and residues here were proposed to constitute an interaction site between the two molecules (9Kimura Y. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1997; 272: 15061-15064Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), although certain residues in the zipper domain now also appear to interact directly with SERCA2a (24Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Amino acid substitutions within the cytoplasmic region of PLB (residues 1–31) also affected SERCA2a activity (23Kimura 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, 25Toyofuku T. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1994; 269: 3088-3094Abstract Full Text PDF PubMed Google Scholar), suggesting that this region of PLB is equally important for direct physical and functional interactions with the Ca2+ pump. In particular, replacement of Asn27 or Asn30 of PLB with Ala had no major effect on the equilibrium between PLB pentamers and monomers, but significantly enhanced inhibition of SERCA2a, suggesting that these two PLB residues are directly involved in the interaction with SERCA2a (18Young H.S. Jones L.R. Stokes D.L. Biophys. J. 2001; 81: 884-894Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 23Kimura 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, 24Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 26Mahaney J. Barlow A. Honaker B. Huffman J. Muchnok T. Arch. Biochem. Biophys. 1999; 372: 408-413Crossref PubMed Scopus (8) Google Scholar). Finally, an interaction between the extreme N terminus of PLB and an exposed loop in the nucleotide domain of SERCA2a was suggested by chimeric constructs (27Toyofuku T. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1994; 269: 22929-22932Abstract Full Text PDF PubMed Google Scholar) and by early cross-linking studies (11James P. Inui M. Tada M. Chiesi M. Carafoli E. Nature. 1989; 342: 90-92Crossref PubMed Scopus (374) Google Scholar). To characterize further the interactions between the PLB monomer and SERCA2a at the molecular level, we initiated a series of studies using cross-linking agents as molecular rulers to measure distances between key amino acid residues of PLB and SERCA2a (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Using the homobifunctional, thiol cross-linking agent BMH, we demonstrated a highly specific cross-link between Asn30 of PLB changed to cysteine (N30C-PLB) and Cys318 of SERCA2a at the cytoplasmic boundary of M4. The length of the cross-linking spacer chain suggested that these two residues are 10 Å apart. Cross-linking of PLB to SERCA2a occurred only in the absence of Ca2+ and was strikingly potentiated by ATP or ADP, providing strong physical evidence that monomeric PLB binds preferentially to the nucleotide-stabilized, E2 conformation of SERCA2a (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Significantly, the irreversible SERCA inhibitor thapsigargin completely prevented cross-linking of PLB to SERCA2a (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), supporting previous evidence that the E2-like conformation stabilized by thapsigargin is conformationally distinct from the native E2 conformation (28DeJesus F. Girardet J.L. Dupont Y. FEBS Lett. 1993; 332: 229-232Crossref PubMed Scopus (28) Google Scholar, 29Inesi G. Sagara Y. Arch. Biochem. Biophys. 1992; 298: 313-317Crossref PubMed Scopus (161) Google Scholar) (Fig. 1). Here we report on the use of second generation cross-linking agents, which are heterobifunctional and provide additional constraints on the interaction and dynamics between PLB and SERCA2a. The new cross-linking agents contain a maleimide group at one end for coupling to Asn27 or Asn30 of PLB replaced with cysteine, and an NHS-ester group at the other end, for coupling to nearby lysine residues of WT-SERCA2a (30Hermanson G.T. Bioconjugate Techniques. Academic Press, New York1996Google Scholar). We show that these heterobifunctional cross-linking agents work faster and even more efficiently than the homobifunctional agent, BMH, reported on previously (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Nearly quantitative coupling between PLB and SERCA2a is achieved with heterobifunctional cross-linking agents. At the same time, the heterobifunctional cross-linkers remain highly specific, and react only with Lys328 of SERCA2a when attached to residues 27 and 30 of PLB, with optimal linker lengths of 10 and 15 Å, respectively. Materials—All of the cross-linking agents used were obtained from Pierce. The heterobifunctional cross-linkers were: N-(α-maleimidoacetoxy)succinimide ester (cross-linking distance 4.4 Å), N-(β-maleimidopropyloxy)succinimide ester (6.9 Å), N-(ϵ-maleimidocaproyloxy)succinimide ester (EMCS; 9.4 Å), m-maleimidobenzoyl-N-hydroxysuccinimide ester (9.9 Å), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (11.6 Å), succinimidyl-6-(β-maleimidopropionamido)hexanoate (14.3 Å), and N-(κ-maleimidoundecanoyloxy)sulfosuccinimide ester (KMUS; 15.7 Å). The homobifunctional cross-linking agent was 1,6-bismaleimidohexane (BMH; 10.2 Å) (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Thapsigargin was purchased from Sigma. Iodogen was from Pierce. Endo-Asp-N and endo-Lys-C were obtained from Roche Molecular Biochemicals. Mutagenesis and Baculovirus Production—Point mutations in the cDNA encoding canine PLB were made as previously described (8Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 22Simmerman H.K. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 24Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). N27C-PLB and N30C-PLB were expressed on Cys-less PLB background, which is canine PLB with Cys residues 36, 41, and 46 replaced by Ala (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Wild-type canine SERCA2a cDNA was mutated directly in the transfection vector pVL1393 using the QuikChange™ XL-Gold system (Stratagene) (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). All cDNA mutations were confirmed by DNA sequencing. Baculoviruses encoding mutated proteins were generated by co-transfecting into Sf21 insect cells mutated cDNAs in pVL1393 with BaculoGold™ (Pharmingen) linearized baculovirus DNA (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 8Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 24Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Protein Co-expression in Insect Cells and Isolation of Microsomes—Canine SERCA2a and N27C-PLB or N30C-PLB were co-expressed in Sf21 insect cells as described (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 8Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 24Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). 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. Protein assay and Ca2+-ATPase activities of microsomes were determined as previously described (8Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Cross-linking—Cross-linking between single Cys residues of N27C-PLB or N30C-PLB and endogenous Lys residue(s) of WT-SERCA2a was conducted using the heterobifunctional cross-linking agents of increasing lengths listed under "Materials." All heterobifunctional agents tested contained a maleimide group at one end for coupling to Cys residues and an NHS-ester group at the other end for coupling to Lys residues (30Hermanson G.T. Bioconjugate Techniques. Academic Press, New York1996Google Scholar). Cross-linking between PLB and SERCA2a was conducted essentially as recently described (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Reactions were conducted with 11 μg of microsomal protein in 12 μl of buffer A, which contained 40 mm MOPS (pH 7.0), 3.2 mm MgCl2, 75 mm KCl, 3 mm ATP, and 1 mm EGTA. Reactions were started by adding 0.75 μl of cross-linking agent from a 1.6 mm stock solution in dimethyl sulfoxide (0.1 mm final cross-linker concentration) and stopped by adding 7.5 μl of SDS-PAGE sample-loading buffer containing 15% SDS plus 100 mm dithiothreitol. Heterobifunctional cross-linking was conducted for 10 min at room temperature unless otherwise indicated. Homobifunctional cross-linking between Cys residues with 0.1 mm BMH was conducted for 1 h at room temperature (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). 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 (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In some experiments, ATP in buffer A was omitted or was replaced by other nucleotides, as indicated. To determine antibody effects on cross-linking, affinity-purified anti-PLB monoclonal antibody, 2D12, or anti-SERCA2a monoclonal antibody, 2A7-A1, dialyzed in 20 mm MOPS (pH 7) and 150 mm NaCl, were used (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). 5.5 μg of antibody were included with 11 μg of microsomal protein in buffer A. SDS-PAGE and Immunoblotting—SDS-PAGE was performed in 8% polyacrylamide, and immunoblots were probed with anti-PLB monoclonal antibody, 2D12, to detect free and cross-linked forms of PLB (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In the experiment of Fig. 9, the immunoblot was also probed with anti-SERCA2a monoclonal antibody, 2A7-A1, to detect Ca2+ pumps not cross-linked to PLB (8Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Antibody-binding protein bands were visualized with 125I-protein A, except for the experiment depicted in Fig. 7, in which 125I-2D12 was used directly for PLB visualization (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), to avoid interference from antibodies carried over from the cross-linking reactions. Antibody-binding bands were quantified with a Bio-Rad Personal Fx phosphorimager.Fig. 7Monoclonal antibody effects on N27C-PLB and N30C-PLB cross-linking to WT-SERCA2a. 11 μg of microsomes co-expressing WT-SERCA2a and N27C-PLB or N30C-PLB were cross-linked in the absence (CON) or presence of 5.5 μg of anti-PLB monoclonal antibody (2D12) or anti-SERCA monoclonal antibody (2A7-A1) (top) as described in Fig. 5 legend. The immunoblot was probed with 125I-2D12. Only PLB/SERCA2a cross-linked bands are shown on the autoradiograph.View Large Image Figure ViewerDownload (PPT) Identification of Cross-linked Lys Residue of SERCA2a—SERCA2a cross-linked to N27C-PLB with EMCS, or to N30C-PLB with KMUS, was purified to homogeneity from insect cell microsomes in separate runs by sequential anti-SERCA2a (2A7-A1) and anti-PLB (2D12) monoclonal affinity chromatographies, as recently described in detail (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Each type of purified, cross-linked Ca2+ pump was then digested with endo-Asp-N and endo-Lys-C, and the limit SERCA2a fragment cross-linked to PLB was isolated by a second round of 2D12 chromatography and sequenced, as described (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). By this analysis, the cross-linked amino acid of SERCA2a was restricted to just three potential Lys residues (see Fig. 8). Replacement-mutagenesis of Lys with Ala was then used to determine which of these three Lys residues was the one cross-linked to N27C-PLB and to N30C-PLB. Modeling—Coordinates for PLB residues 19–52 (42Lamberth S. Schmid H. Muenchback M. Vorherr T. Krebs E. Carafoli E. Griesinger C. Helv. Chim. Acta. 2000; 83: 2141-2152Crossref Scopus (79) Google Scholar) (Protein Data Bank accession code 1FJK) were manually docked to E2 coordinates of SERCA1a (15Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar) (Protein Data Bank accession code 1IWO) using cross-linking constraints as a guide. The model was then subjected to a round of energy minimization in XPLOR, with 1IWO held rigid, to eliminate any steric clashes. Phospholamban residues 27 and 30 were mutated to Cys using the DeepView Swiss-PdbViewer program. A search for optimal rotomers for PLB residues, Cys27 and Cys30, and SERCA residue, Lys328, was then performed using this program. The model was rendered using Pymol (W. L. DeLano, www.pymol.org). Specific Cross-linking of N27C-PLB and N30C-PLB to SERCA2a—We recently showed that N30C-PLB cross-links to Cys318 of native SERCA2a near M4 using the homobifunctional thiol cross-linking agent BMH (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In the work described here, we tested for cross-linking N27C-PLB and N30C-PLB to Lys residues of native SERCA2a using a series of heterobifunctional thiol to amine cross-linking agents of different lengths, spanning distances from 4 to 16 Å. N27C-PLB and N30C-PLB were expressed on the Cys-less PLB background, and were fully functional in their abilities to inhibit SERCA2a by lowering Ca2+ affinity (7Jones L.R. Cornea R.L. Chen Z. J. Biol. Chem. 2002; 277: 28319-28329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Fig. 2A shows that N27C-PLB and N30C-PLB cross-linked strongly to wild-type SERCA2a in insect cell microsomes with use of heterobifunctional cross-linking agents. When immunoblots were probed with the anti-PLB antibody, 2D12, intense cross-linking signals were obtained at molecular masses of just over 100 kDa (PLB/SER), corresponding to heterodimers formed between PLB and SERCA2a. Some weaker homodimerization of PLB monomers was also detected (PLB2). 2We should point out that Cys-less PLB is predominately a pentamer in insect cell microsomal membranes, even though it migrates primaril
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