Role of leucine 31 of phospholamban in structural and functional interactions with the ca2+ pump of cardiac sarcoplasmic reticulum

Zhenhui Chen, David L. Stokes, Larry R. Jones

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30 Scopus citations

Abstract

The ability of two loss-of-function mutants, L31A and L31C, of phospholamban (PLB) to bind to and inhibit the Ca2+ pump of cardiac sarcoplasmic reticulum (SERCA2a) was investigated using a molecular cross-linking approach. Leu31 of PLB, located at the cytoplasmic membrane boundary, is a critical amino acid shown previously to be essential for Ca2+-ATPase inhibition. We observed that L31A or L31C mutations of PLB prevented the inhibition of Ca2+-ATPase activity and disabled the cross-linking of N27C and N30C of PLB to Lys328 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 Leu31 of PLB within 10 Å of Thr317 of SERCA2a in the M4 helix. Thus, contrary to previous suggestions, PLB with loss-of-function mutations at Leu31 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 Ca2+ 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 Ca2+ pump is in the Ca2+-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.

Original languageEnglish (US)
Pages (from-to)10530-10539
Number of pages10
JournalJournal of Biological Chemistry
Volume280
Issue number11
DOIs
StatePublished - Mar 18 2005

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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