Cross-linking of C-terminal residues of phospholamban to the Ca 2+ pump of cardiac sarcoplasmic reticulum to probe spatial and functional interactions within the transmembrane domain

Zhenhui Chen, Brandy L. Akin, David L. Stokes, Larry Jones

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Abstract

Interactions between the transmembrane domains of phospholamban (PLB) and the cardiac Ca2+ pump (SERCA2a) have been investigated by chemical cross-linking. Specifically, C-terminal, transmembrane residues 45-52 of PLB were individually mutated to Cys, then cross-linked to V89C in the M2 helix of SERCA2a with the thiol-specific cross-linking reagents Cu2+- phenanthroline, dibromobimane, and bismaleimidohexane. V49C-, M50C-, and L52C-PLB all crosslinked strongly to V89C-SERCA2a, coupling to 70-100% of SERCA2a molecules. Residues 45-48 and 51 of PLB also cross-linked to V89C of SERCA2a, but more weakly. Evidence for the mechanism of PLB regulation of SERCA2a was provided by the conformational dependence of cross-linking. In particular, the required absence of Ca2+ for cross-linking implicated the E2 conformation of SERCA2a, and its enhancement by ATP confirmed E2·ATP as the conformation with the highest affinity for PLB. In contrast, E2 phosphorylated with inorganic phosphate (E2P) and E2 inhibited by thapsigargin (E2·TG) both failed to cross-link to PLB. These results with transmembrane PLB residues are completely consistent with cytoplasmic PLB residues studied previously, suggesting that the dissociation of PLB from the Ca2+ pump is complete, not partial, when the pump binds Ca 2+ (E1·Ca2) or adopts the E2P or E2·TG conformations. V49C of PLB cross-linked to 100% of SERCA2a molecules, suggesting that this residue might have functional importance for regulation. Indeed, we found that mutation of Val49 to smaller side-chained residues V49A or V49G augmented PLB inhibition, whereas mutation to the larger hydrophobic residue, V49L, prevented PLB inhibition. A model for the interaction of PLB with SERCA2ais presented, showing that Val49 fits into a constriction at the lumenal end of the M2 helix of SERCA, possibly controlling access of PLB to its binding site on SERCA.

Original languageEnglish
Pages (from-to)14163-14172
Number of pages10
JournalJournal of Biological Chemistry
Volume281
Issue number20
DOIs
StatePublished - May 19 2006

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Sarcoplasmic Reticulum
Pumps
Conformations
phospholamban
Cross-Linking Reagents
Mutation
Molecules
Thapsigargin
Phenanthrolines
Sulfhydryl Compounds
Constriction

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{c661f3629dbb43129f8cbffa102f4ac9,
title = "Cross-linking of C-terminal residues of phospholamban to the Ca 2+ pump of cardiac sarcoplasmic reticulum to probe spatial and functional interactions within the transmembrane domain",
abstract = "Interactions between the transmembrane domains of phospholamban (PLB) and the cardiac Ca2+ pump (SERCA2a) have been investigated by chemical cross-linking. Specifically, C-terminal, transmembrane residues 45-52 of PLB were individually mutated to Cys, then cross-linked to V89C in the M2 helix of SERCA2a with the thiol-specific cross-linking reagents Cu2+- phenanthroline, dibromobimane, and bismaleimidohexane. V49C-, M50C-, and L52C-PLB all crosslinked strongly to V89C-SERCA2a, coupling to 70-100{\%} of SERCA2a molecules. Residues 45-48 and 51 of PLB also cross-linked to V89C of SERCA2a, but more weakly. Evidence for the mechanism of PLB regulation of SERCA2a was provided by the conformational dependence of cross-linking. In particular, the required absence of Ca2+ for cross-linking implicated the E2 conformation of SERCA2a, and its enhancement by ATP confirmed E2·ATP as the conformation with the highest affinity for PLB. In contrast, E2 phosphorylated with inorganic phosphate (E2P) and E2 inhibited by thapsigargin (E2·TG) both failed to cross-link to PLB. These results with transmembrane PLB residues are completely consistent with cytoplasmic PLB residues studied previously, suggesting that the dissociation of PLB from the Ca2+ pump is complete, not partial, when the pump binds Ca 2+ (E1·Ca2) or adopts the E2P or E2·TG conformations. V49C of PLB cross-linked to 100{\%} of SERCA2a molecules, suggesting that this residue might have functional importance for regulation. Indeed, we found that mutation of Val49 to smaller side-chained residues V49A or V49G augmented PLB inhibition, whereas mutation to the larger hydrophobic residue, V49L, prevented PLB inhibition. A model for the interaction of PLB with SERCA2ais presented, showing that Val49 fits into a constriction at the lumenal end of the M2 helix of SERCA, possibly controlling access of PLB to its binding site on SERCA.",
author = "Zhenhui Chen and Akin, {Brandy L.} and Stokes, {David L.} and Larry Jones",
year = "2006",
month = "5",
day = "19",
doi = "10.1074/jbc.M601338200",
language = "English",
volume = "281",
pages = "14163--14172",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
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TY - JOUR

T1 - Cross-linking of C-terminal residues of phospholamban to the Ca 2+ pump of cardiac sarcoplasmic reticulum to probe spatial and functional interactions within the transmembrane domain

AU - Chen, Zhenhui

AU - Akin, Brandy L.

AU - Stokes, David L.

AU - Jones, Larry

PY - 2006/5/19

Y1 - 2006/5/19

N2 - Interactions between the transmembrane domains of phospholamban (PLB) and the cardiac Ca2+ pump (SERCA2a) have been investigated by chemical cross-linking. Specifically, C-terminal, transmembrane residues 45-52 of PLB were individually mutated to Cys, then cross-linked to V89C in the M2 helix of SERCA2a with the thiol-specific cross-linking reagents Cu2+- phenanthroline, dibromobimane, and bismaleimidohexane. V49C-, M50C-, and L52C-PLB all crosslinked strongly to V89C-SERCA2a, coupling to 70-100% of SERCA2a molecules. Residues 45-48 and 51 of PLB also cross-linked to V89C of SERCA2a, but more weakly. Evidence for the mechanism of PLB regulation of SERCA2a was provided by the conformational dependence of cross-linking. In particular, the required absence of Ca2+ for cross-linking implicated the E2 conformation of SERCA2a, and its enhancement by ATP confirmed E2·ATP as the conformation with the highest affinity for PLB. In contrast, E2 phosphorylated with inorganic phosphate (E2P) and E2 inhibited by thapsigargin (E2·TG) both failed to cross-link to PLB. These results with transmembrane PLB residues are completely consistent with cytoplasmic PLB residues studied previously, suggesting that the dissociation of PLB from the Ca2+ pump is complete, not partial, when the pump binds Ca 2+ (E1·Ca2) or adopts the E2P or E2·TG conformations. V49C of PLB cross-linked to 100% of SERCA2a molecules, suggesting that this residue might have functional importance for regulation. Indeed, we found that mutation of Val49 to smaller side-chained residues V49A or V49G augmented PLB inhibition, whereas mutation to the larger hydrophobic residue, V49L, prevented PLB inhibition. A model for the interaction of PLB with SERCA2ais presented, showing that Val49 fits into a constriction at the lumenal end of the M2 helix of SERCA, possibly controlling access of PLB to its binding site on SERCA.

AB - Interactions between the transmembrane domains of phospholamban (PLB) and the cardiac Ca2+ pump (SERCA2a) have been investigated by chemical cross-linking. Specifically, C-terminal, transmembrane residues 45-52 of PLB were individually mutated to Cys, then cross-linked to V89C in the M2 helix of SERCA2a with the thiol-specific cross-linking reagents Cu2+- phenanthroline, dibromobimane, and bismaleimidohexane. V49C-, M50C-, and L52C-PLB all crosslinked strongly to V89C-SERCA2a, coupling to 70-100% of SERCA2a molecules. Residues 45-48 and 51 of PLB also cross-linked to V89C of SERCA2a, but more weakly. Evidence for the mechanism of PLB regulation of SERCA2a was provided by the conformational dependence of cross-linking. In particular, the required absence of Ca2+ for cross-linking implicated the E2 conformation of SERCA2a, and its enhancement by ATP confirmed E2·ATP as the conformation with the highest affinity for PLB. In contrast, E2 phosphorylated with inorganic phosphate (E2P) and E2 inhibited by thapsigargin (E2·TG) both failed to cross-link to PLB. These results with transmembrane PLB residues are completely consistent with cytoplasmic PLB residues studied previously, suggesting that the dissociation of PLB from the Ca2+ pump is complete, not partial, when the pump binds Ca 2+ (E1·Ca2) or adopts the E2P or E2·TG conformations. V49C of PLB cross-linked to 100% of SERCA2a molecules, suggesting that this residue might have functional importance for regulation. Indeed, we found that mutation of Val49 to smaller side-chained residues V49A or V49G augmented PLB inhibition, whereas mutation to the larger hydrophobic residue, V49L, prevented PLB inhibition. A model for the interaction of PLB with SERCA2ais presented, showing that Val49 fits into a constriction at the lumenal end of the M2 helix of SERCA, possibly controlling access of PLB to its binding site on SERCA.

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DO - 10.1074/jbc.M601338200

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