Close proximity between residue 30 of phospholamban and cysteine 318 of the cardiac Ca2+ pump revealed by intermolecular thiol cross-linking.
Phospholamban (PLB) is a 52-amino acid inhibitor of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SERCA2a), which acts by decreasing the apparent affinity of the enzyme for Ca(2+). 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-A-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 Ca(2+) 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 Ca(2+)-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 Cys(318) of SERCA2a as the sole amino acid cross-linked to N30C-PLB. The proximity of residue 30 of PLB to Cys(318) of SERCA2a suggests that PLB may interfere with Ca(2+) activation of SERCA2a by a protein interaction occurring near transmembrane helix M4.
Jones
Larry R
LR
Krannert Institute of Cardiology and the Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA. lrjones@iupui.edu
Cornea
Razvan L
RL
Chen
Zhenhui
Z
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D003432:Cross-Linking Reagents; D003545:Cysteine; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D013438:Sulfhydryl Compounds; D014162:Transfection; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D003432:Cross-Linking Reagents; D003545:Cysteine; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D000099211:Phospholamban