title: Role of leucine 31 of phospholamban in structural and functional interactions with the Ca2+ pump of cardiac sarcoplasmic reticulum.
abstract: The ability of two loss-of-function mutants, L31A and L31C, of phospholamban (PLB) to bind to and inhibit the Ca(2+) pump of cardiac sarcoplasmic reticulum (SERCA2a) was investigated using a molecular cross-linking approach. Leu(31) of PLB, located at the cytoplasmic membrane boundary, is a critical amino acid shown previously to be essential for Ca(2+)-ATPase inhibition. We observed that L31A or L31C mutations of PLB prevented the inhibition of Ca(2+)-ATPase activity and disabled the cross-linking of N27C and N30C of PLB to Lys(328) and Cys(318) of SERCA2a. Although L31C-PLB failed to cross-link to any Cys or Lys residue of wild-type SERCA2a, L31C did cross-link with high efficiency to T317C of SERCA2a with use of the homobifunctional sulfhydryl cross-linking reagent, 1,6-bismaleimidohexane. This places Leu(31) of PLB within 10 angstroms of Thr(317) of SERCA2a in the M4 helix. Thus, contrary to previous suggestions, PLB with loss-of-function mutations at Leu(31) retains the ability to bind to SERCA2a, despite losing inhibitory activity. Cross-linking of L31C-PLB to T317C-SERCA2a occurred only in the absence of Ca(2+) and in the presence of nucleotide and was prevented by thapsigargin and by anti-PLB antibody, demonstrating for a fourth cross-linking pair that PLB interacts near M4 only when the Ca(2+) pump is in the Ca(2+)-free, nucleotide-bound E2 conformation, but not in the E2 state inhibited by thapsigargin. L31I-PLB retained full functional and cross-linking activity, suggesting that a bulky hydrophobic residue at position 31 of PLB is essential for productive interaction with SERCA2a. A model for the three-dimensional structure of the interaction site is proposed.
authors.affiliation: Krannert Institute of Cardiology and the Department of Medicine, Indiana University School of Medicine, 1800 N. Capitol Ave., Indianapolis, Indiana 46202, USA.
authors.forename: Zhenhui
authors.initials: Z
authors.lastname: Chen
authors.forename: David L
authors.initials: DL
authors.lastname: Stokes
authors.forename: Larry R
authors.initials: LR
authors.lastname: Jones
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D000495:Allosteric Site; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007313:Insecta; D007930:Leucine / Q000737:chemistry*; D008301:Maleimides / Q000737:chemistry; D008861:Microsomes / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013997:Time Factors; D000099211:Phospholamban
chemical_list: D000251:Adenosine Triphosphatases; D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D007930:Leucine; D008301:Maleimides; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban; C075594:1,6-bismaleimidohexane