Sites on the cytoplasmic region of phospholamban involved in interaction with the calcium‐activated ATPase of the sarcoplasmic reticulum

Abstract

Proton NMR studies have shown that when a peptide corresponding to the N-terminal region of phospholamban, PLB(1±20), interacts with the Ca 2+ ATPase of the sarcoplasmic reticulum, SERCA1a, docking involves the whole length of the peptide. Phosphorylation of Ser16 reduced the affinity of the peptide for the pump by predominantly affecting the interaction with the C-terminal residues of PLB(1±20). In the phosphorylated peptide weakened interaction occurs with residues at the N-terminus of PLB(1±20). PLB(1±20) is shown to interact with a peptide corresponding to residues 378±405 located in the cytoplasmic region of SERCA2a and related isoforms. This interaction involves the C-terminal regions of both peptides and corresponds to that affected by phosphorylation. The data provide direct structural evidence for complex formation involving residues 1±20 of PLB. They also suggest that phospholamban residues 1±20 straddle separate segments of the cytoplasmic domain of SERCA with the N-terminus of PLB associated with a region other than that corresponding to SERCA2a(378±405).Keywords: phospholamban; calcium-activated ATPase; sarcoplasmic reticulum; phosphorylation; NMR.The Ca 2+ ATPase of the sarcoplasmic reticulum (SERCA) is a calcium pump which functions in association with the Ca 2+ release channels, and is responsible for maintaining the calcium transients upon which the regulation of contractile activity in all types of muscle depends. The SERCA enzyme family consists of a number of isoforms encoded by three genes that are expressed in a tissue-specific manner. In cardiac muscle the isoform characteristic of that tissue, SERCA2a, is associated with phospholamban (PLB), a 52-residue polypeptide whose reversible phosphorylation enables the calcium pump to be modulated [1,2]. Interestingly, the same two proteins are expressed in slow-twitch skeletal muscle [3,4], whereas different isoforms, SERCA1a and 1b [5] but no PLB [6] are present in fast-twitch skeletal muscle. Nevertheless, SERCA isolated from fast skeletal muscle can be regulated by PLB in vitro [7±9], although it has been suggested that it is regulated in situ by a related protein, sarcolipin [10].The presence of PLB enables the calcium pumping rate of the sarcoplasmic reticulum to be increased in cardiac, and to a lesser extent in slow-twitch skeletal muscles, in response to b-adrenergic stimulation. Although the detailed mechanism of this process is not understood, a current view is that in the unphosphorylated form PLB interacts with SERCA and inhibits pumping activity, possibly by reducing its affinity for calcium ions (compare [11] with [12,13]), and reducing the maximum turnover rate at saturating calcium ion concentration, which is reflected in the Ca/MgATPase activity rate (reviewed in [14,15]). Phosphorylation of Ser16 of PLB by cAMP or cGMP dependent protein kinases and/or phosphorylation of Thr17 by the multifunctional calmodulin dependent protein kinase [16,17] results in increased activity of the pump [18,19].The detailed mechanism by which PLB m...

Phospholamban Interacts with HAX-1, a Mitochondrial Protein with Anti-apoptotic Function

“…8 Thus, binding of HAX-1 to this region may represent an important regulatory mechanism to any of the above reactions, as it may provide a mechanism controlling the conformation and activity of PLN. Indeed, phosphorylation of PLN or elevation of the concentration of Ca 2+ led to dissociation of HAX-1 from PLN, similar to findings on the PLN/SERCA2a interaction, 12,25,38,39 implicating the physiological/pathophysiological significance of this association in cardiac muscle.…”

A Structural Model of the Complex Formed by Phospholamban and the Calcium Pump of Sarcoplasmic Reticulum Obtained by Molecular Mechanics

“…This structure is in agreement with earlier studies that used either circular dichroism spectroscopy [19] or NMR spectroscopy on portions of PLN. [20,21] These studies provided evidence that PLN has both a-helical and b-sheet (or random coil) components and is not exclusively a helical, as recently suggested by Smith et al [22] The flexibility of the b turn would enable PLN to interact simultaneously with helix M6 of the SERCA pump and with a critical cytoplasmic portion of the pump (Lys397 ± Val402). [7] The detailed mechanism by which PLN interacts with the SERCA pump to modulate its activity is not yet understood.…”

NMR Solution Structure of Phospholamban

“…It was thus decided to determine the structure of monomeric PLN, specifically, that of the [F 41 ]PLN monomer (replacement of Cys 41 by Phe 41 ), which has no propensity to form pentamers and the biological activity of which is similar to that of wild-type PLN [11]. Structural studies on portions of PLN have provided evidence for a high helical content of the protein [12] [13], in agreement with preliminary NMR experiments on the PLN mutant [F 41 ]PLN (also called C41F) [14]. The work presented here describes the first determination of the three-dimensional structure of [F 41 ]PLN, obtained by two-dimensional homonuclear NMR spectroscopy.…”