Simmerman, Heather K. B., and Larry R. Jones. Phospholamban: Protein Structure, Mechanism of Action, and Role in Cardiac Function. Physiol. Rev. 78: 921–947, 1998. — A comprehensive discussion is p...

http://agrino.org/skalifourta/Phospholamban-%20Protein%20Structure,%20Mechanism%20of%20Action,%20and%20Role%20in%20Cardiac%20Function.pdf

Phospholamban: Protein Structure, Mechanism of Action, and Role in Cardiac Function

Phospholamban is a 52-amino-acid protein that assembles into a pentamer in sarcoplasmic reticulum membranes. The protein has a role in the regulation of the resident calcium ATPase through an inhibitory association that can be reversed by phosphorylation. The phosphorylation of phospholamban is initiated by -adrenergic stimulation, identifying phospholamban as an important component in the stimulation of cardiac activity by -agonists. It is this role of phospholamban that has motivated studies in recent decades. There is evidence that phospholamban may also function as a Ca 2C -selective ion channel. The structural

Structural perspectives of phospholamban, a helical transmembrane pentamer

Structure of the 1-36 Amino-Terminal Fragment of Human Phospholamban by Nuclear Magnetic Resonance and Modeling of the Phospholamban Pentamer

We have used synthetic lipidated peptides (“peptide-amphiphiles”) to study the structure and function of isolated domains of integral transmembrane proteins. We used 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis to prepare full-length phospholamban (PLB1–52) and its cytoplasmic (PLB1–25K: phospholamban residues 1–25 plus a C-terminal lysine), and transmembrane (PLB26–52) domains, and a 38-residue model α-helical sequence as a control. We created peptide-amphiphiles by linking the C-terminus of either the isolated cytoplasmic domain or the model peptide to a membrane-anchoring, lipid-like hydrocarbon tail. Circular dichroism measurements showed that the model peptide-amphiphile, either in aqueous suspension or in lipid bilayers, had a higher degree of α-helical secondary structure than the unlipidated model peptide. We hypothesized that the peptide-amphiphile system would allow us to study the function and structure of the PLB1–25K cytoplasmic domain in a native-like configuration. We compared the function (inhibition of the Ca-ATPase in reconstituted membranes) and structure (via CD) of the PLB1–25 amphiphile to that of PLB and its isolated transmembrane and cytoplasmic domains. Our results indicate that the cytoplasmic domain PLB1–25K has no effect on Ca-ATPase (calcium pump) activity, even when tethered to the membrane in a manner mimicking its native configuration, and that the transmembrane domain of PLB is sufficient for inhibition of the Ca-ATPase. © 2003 Wiley Periodicals, Inc. Biopolymers 69: 283–292, 2003

Structure and function of integral membrane protein domains resolved by peptide-amphiphiles: Application to phospholamban

Phospholamban (PLB), the regulator of the cardiac sarcoplasmic reticulum (SR) Ca2+ pump is specifically phosphorylated at Ser16 andThr17 by cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase (CaMK), respectively. The regulation of this dual-site phosphorylation of amino acid residues in direct proximity is only poorly understood. In order to study the site-specific phosphorylation of PLB, we used a synthetic peptide (PLB-24) corresponding to the cytosolic part of the PLB monomer with the phosphorylation sites as a model substrate. PLB-24 possesses substrate properties as the native PLB as demonstrated by phosphorylation with exogenous, purified PKA, cGMP-dependent protein kinase (PKG) and a type II CaMK (CaMKII). In isolated vesicles of cardiac SR there was a rapid phosphorylation of the peptide by the endogenous PKA (SR-PKA) and CaMK (SR-CaMK), but not under conditions that activate PKG. Both SR-PKA and SR- CaMK incorporated the same amount of 32P into PLB-24, 0.60 ± 0.01 nmol 32P/mg SR protein and 0.61 ± 0.03 nmol 32P/mg SR protein, respectively. Phosphorylation by SR-PKA was abolished by the specific PKA inhibitor (IC50 = 0.2 μM), whereas SR-CaMK phosphorylation was inhibited by calmidazolium (IC50 =1.6 μM) and a CaMKII-specific inhibitor peptide (IC50 = 2.5 μM). Phosphorylation by SR-PKA was exclusively at Ser, whereas SR-CaMK phosphorylated only Thr. After simultaneous activation of both SR-kinases 32P incorporation into PLB-24 was additive and occurred at Ser as well as at Thr. Sequential activation of SR-PKA and SR-CaMK also caused the additive phosphorylation of PLB-24 independently of which kinase was activated first. Thus, at the monomeric level of PLB the respective phosphorylation site appears to be accessible to its related SR protein kinase in vitro even when the adjacent site is phosphorylated.

Site-specific phosphorylation of a phospholamban peptide by cyclic nucleotide- and Ca2+/calmodulin-dependent protein kinases of cardiac sarcoplasmic reticulum.

Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy have been used to characterize the conformation of the putative cytoplasmic domain of phospholamban (PLB), an oligomeric membrane-bound protein which regulates the activity of the cardiac sarcoplasmic reticulum Ca(2+)-dependent ATPase. In aqueous solution the 25-residue peptide adopts a number of rapidly interconverting conformers with no secondary structural type obviously predominating. However, in trifluoroethanol (TFE) the conformation, while still highly dynamic, is characterized by a high proportion of helical structures. Evidence for this is provided by alpha CH chemical shifts and low NH chemical shift temperature coefficients, small NH-alpha CH intraresidue scalar coupling constants, a substantial number of distinctive interresidue nuclear Overhauser effects (NOEs) [dNN(i, i + 1), d alpha N(i, i + 3), d alpha beta(i, i + 3) and d alpha N(i, i + 4)] and characteristic CD bands at 190 (positive), 206 (negative) and 222 nm (negative). The helicity is interrupted around Pro-21. The activity of PLB is regulated by phosphorylation at either Ser-16 or Thr-17. CD shows that phosphorylation at Ser-16 by the cAMP-activated protein kinase causes about an 11% decrease in alpha-helical content in TFE.

Conformation of the cytoplasmic domain of phospholamban by NMR

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Conformation of the cytoplasmic domain of phospholamban by NMR