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Abstract
Calcium transport across the sarcoplasmic reticulum of cardiac myocytes is regulated by a reversible inhibitory interaction between the Ca2+-ATPase and the small transmembrane protein phospholamban (PLB). A nullcysteine analogue of PLB, containing isotope labels in the transmembrane domain or cytoplasmic domain, was reconstituted into membranes in the absence and presence of the SERCA1 isoform of Ca2+-ATPase for structural investigation by cross-polarization magic-angle spinning (CP-MAS) NMR. PLB lowered the maximal hydrolytic activity of SERCA1 and its affinity for calcium in membrane preparations suitable for structural analysis by NMR. Novel backbone amide proton-deuterium exchange CP-MAS NMR experiments on the two PLB analogues co-reconstituted with SERCA1 indicated that labeled residues Leu42 and Leu44 were situated well within the membrane interior, whereas Pro21 and Ala24 lie exposed outside the membrane. Internuclear distance measurements on PLB using rotational resonance NMR indicated that the sequences Pro21-Ala24 and Leu42-Leu44 adopt an alpha-helical structure in pure lipid bilayers, which is unchanged in the presence of Ca2+-ATPase. By contrast, rotational echo double resonance (REDOR) NMR experiments revealed that the sequence Ala24-Gln26 switches from an alpha-helix in pure lipid membranes to a more extended structure in the presence of SERCA1, which may reflect local structural distortions which change the orientations of the transmembrane and cytoplasmic domains. These results suggest that Ca2+-ATPase has a long-range effect on the structure of PLB around residue 25, which promotes the functional association of the two proteins.
Highlights
Phospholamban (PLB)1 is a 52-amino acid membrane-spanning protein, which is expressed predominantly in the sarcoplasmic reticulum of cardiac myocytes [1]
Novel backbone amide proton-deuterium exchange cross-polarization magic-angle spinning (CP-MAS) NMR experiments on the two PLB analogues co-reconstituted with SERCA1 indicated that labeled residues Leu42 and Leu44 were situated well within the membrane interior, whereas Pro21 and Ala24 lie exposed outside the membrane
Previous studies have shown that the functional properties of SERCA1 and its inhibition by PLB are similar to those of SERCA2a [33], and replacement of SERCA2a by SERCA1 is valid here
Summary
Phospholamban (PLB) is a 52-amino acid membrane-spanning protein, which is expressed predominantly in the sarcoplasmic reticulum of cardiac myocytes [1]. The primary physiological function of PLB is to regulate the active transport of calcium ions into the sarcoplasmic reticulum lumen via an inhibitory association with SERCA2a, the cardiac isoform of. Co-crystallization studies have led to the proposal that a single PLB monomer interacts asymmetrically with two SERCA molecules such that the transmembrane domain of PLB lies close to the M3 helix of SERCA [16]. In support of the latter argument, thiol cross-linking analysis demonstrated that PLB residue 30 is in close proximity to Cys318 in helix M4 of Ca2ϩATPase [17].
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