Phospholamban C-terminal residues are critical determinants of the structure and function of the calcium ATPase regulatory complex Journal Article

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Authors: Abrol, N.; Smolin, N; Armanious, G.; Ceholski, D. K.; Trieber, C. A.; Young, H. S.; Robia, S. L.
Article Title: Phospholamban C-terminal residues are critical determinants of the structure and function of the calcium ATPase regulatory complex
Abstract: To determine the structural and regulatory role of the C-terminal residues of phospholamban (PLB) in the membranes of living cells, we fused fluorescent protein tags to PLB and sarco/endoplasmic reticulum calcium ATPase (SERCA). Alanine substitution of PLB C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB, suggesting a change in quaternary conformation of PLB pentamer and SERCA-PLB regulatory complex. Val to Ala substitution at position 49 (V49A) had particularly large effects on PLB pentamer structure and PLB-SERCA regulatory complex conformation, increasing and decreasing probe separation distance, respectively. We also quantified a decrease in oligomerization affinity, an increase in binding affinity of V49A-PLB for SERCA, and a gain of inhibitory function as quantified by calcium-dependent ATPase activity. Notably, deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. C-terminal truncations also resulted in progressive loss of PLB-PLB FRET due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the binding affinity of truncated PLB for SERCA and loss of inhibitory potency. However, despite decreased SERCA-PLB binding, intermolecular FRET for Val(49)-stop (V49X) truncation mutant was paradoxically increased as a result of an 11.3-A decrease in the distance between donor and acceptor fluorophores. We conclude that PLB C-terminal residues are critical for localization, oligomerization, and regulatory function. In particular, the PLB C terminus is an important determinant of the quaternary structure of the SERCA regulatory complex.
Journal Title: The Journal of biological chemistry
Volume: 289
Issue: 37
ISSN: 1083-351X; 0021-9258
Publisher: by The American Society for Biochemistry and Molecular Biology, Inc  
Journal Place: United States
Date Published: 2014
Start Page: 25855
End Page: 25866
Language: eng
DOI/URL:

10.1074/jbc.M114.562579
Notes: CI: (c) 2014; GR: R01 HL092321/HL/NHLBI NIH HHS/United States; GR: Canadian Institutes of Health Research/Canada; JID: 2985121R; 0 (Calcium-Binding Proteins); 0 (Multiprotein Complexes); 0 (Recombinant Fusion Proteins); 0 (phospholamban); 147336-22-9 (Green Fluorescent Proteins); EC 3.6.3.8 (Sarcoplasmic Reticulum Calcium-Transporting ATPases); SY7Q814VUP (Calcium); OID: NLM: PMC4162186 [Available on 09/12/15]; OTO: NOTNLM; PMCR: 2015/09/12 00:00; 2014/07/29 [aheadofprint]; ppublish