Superinhibitory phospholamban mutants compete with Ca2+ for binding to SERCA2a by stabilizing a unique nucleotide-dependent conformational state

Brandy L. Akin, Zhenhui Chen, Larry R. Jones

Research output: Contribution to journalArticle

20 Scopus citations


Three cross-linkable phospholamban (PLB) mutants of increasing inhibitory strength (N30C-PLB < N27A,N30C,L37A-PLB (PLB3) < N27A,N30C,L37A,V49G-PLB (PLB4)) were used to determine whether PLB decreases the Ca2+ affinity of SERCA2a by competing for Ca2+ binding. The functional effects of N30C-PLB, PLB3, and PLB4 on Ca2+-ATPase activity and E1∼P formation were correlated with their binding interactions with SERCA2a measured by chemical cross-linking. Successively higher Ca2+ concentrations were required to both activate the enzyme co-expressed with N30C-PLB, PLB3, and PLB4 and to dissociate N30CPLB, PLB3, and PLB4 from SERCA2a, suggesting competition between PLB and Ca2+ for binding to SERCA2a. This was confirmed with the Ca2+ pump mutant, D351A, which is catalytically inactive but retains strong Ca2+ binding. Increasingly higher Ca2+ concentrations were also required to dissociate N30C-PLB, PLB3, and PLB4 from D351A, demonstrating directly that PLB antagonizes Ca 2+ binding. Finally, the specific conformation of E2 (Ca 2+-free state of SERCA2a) that binds PLB was investigated using the Ca2+-pump inhibitors thapsigargin and vanadate. Cross-linking assays conducted in the absence of Ca2+ showed that PLB bound preferentially to E2 with bound nucleotide, forming a remarkably stable complex that is highly resistant to both thapsigargin and vanadate. In the presence of ATP, N30C-PLB had an affinity for SERCA2a approaching that of vanadate (micromolar), whereas PLB3 and PLB4 had much higher affinities, severalfold greater than even thapsigargin (nanomolar or higher). We conclude that PLB decreases Ca 2+ binding to SERCA2a by stabilizing a unique E2·ATP state that is unable to bind thapsigargin or vanadate.

Original languageEnglish (US)
Pages (from-to)28540-28552
Number of pages13
JournalJournal of Biological Chemistry
Issue number37
StatePublished - Sep 10 2010

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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