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-Å 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.
authors: From the Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois 60153 and.
authors: Neha
authors: N
authors: Abrol
authors: From the Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois 60153 and.
authors: Nikolai
authors: N
authors: Smolin
authors: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
authors: Gareth
authors: G
authors: Armanious
authors: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
authors: Delaine K
authors: DK
authors: Ceholski
authors: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
authors: Catharine A
authors: CA
authors: Trieber
authors: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
authors: Howard S
authors: HS
authors: Young
authors: From the Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois 60153 and srobia@luc.edu.
authors: Seth L
authors: SL
authors: Robia
mesh_terms: D000818:Animals; D055592:Biophysical Phenomena; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006333:Heart Failure / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D046912:Multiprotein Complexes; D011485:Protein Binding; D020836:Protein Structure, Quaternary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D049452:Green Fluorescent Proteins; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban
keywords: Calcium ATPase; Confocal Microscopy; Fluorescence Resonance Energy Transfer (FRET); Fret; Heart Failure; Membrane Biophysics; Membrane Protein; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum (SR)