Structure of the Transmembrane Cysteine Residues in Phospholamban

Arkin, Isaiah T.; Adams, Paul D.; Brünger, A.T.; Aimoto, Saburo; Engelman, Donald M.; Smith, Steven O.

doi:10.1007/s002329900172

Cited by 27 publications

(20 citation statements)

References 41 publications

(47 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This chimeric construct facilitates expression and purification of these TM domains in E. coli and analogous SN-TM fusion proteins have been used to study the self-association of a wide variety of transmembrane helices, including glycophorin A (GpA), colon carcinoma kinase 4 (CCK4), as well as BNIP-3, erbB1-4, Vpr1, Vpx2, Gag and phospholamban. [12][13][14][15][16][17][18] Because SN shows no detectable propensity to self-associate in the absence of a dimerizing C-terminal region, 19 this expression strategy has the advantage of providing a method of untangling the thermodynamic influences of the TM from that of the remaining domains in the full-length receptor protein. Most notably, this fusion protein architecture has been especially instrumental in dissecting the sequence dependence of the stability of glycophorin A dimerization.…”

Section: Resultsmentioning

confidence: 99%

Dimerization of the Erythropoietin Receptor Transmembrane Domain in Micelles

Ebie

Fleming

2007

Journal of Molecular Biology

View full text Add to dashboard Cite

Section: Resultsmentioning

confidence: 99%

Dimerization of the Erythropoietin Receptor Transmembrane Domain in Micelles

Ebie

Fleming

2007

Journal of Molecular Biology

View full text Add to dashboard Cite

“…Peptide elution was achieved with a linear gradient to a final solvent composition of 5% water, 38% acetonitrile, and 57% isopropanol. 39 Fractions containing peptides were lyophilized to yield full-length PLB (15.4% yield based on starting resin) and TM-AFA-PLB (22% yield based on starting resin).…”

Section: Synthesis Of Full-length Plb and Tm-plb Domainmentioning

confidence: 99%

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

Lockwood

Zhang

et al. 2003

Biopolymers

View full text Add to dashboard Cite

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 (PLB(1-52)) and its cytoplasmic (PLB(1-25)K: phospholamban residues 1-25 plus a C-terminal lysine), and transmembrane (PLB(26-52)) domains, and a 38-residue model alpha-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 alpha-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 PLB(1-25)K 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 PLB(1-25) amphiphile to that of PLB and its isolated transmembrane and cytoplasmic domains. Our results indicate that the cytoplasmic domain PLB(1-25)K 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.

show abstract

“…Thus while Ser 16 provides activation of calcium transport triggered by increasing cAMP levels from β-adrenergic stimulation, Thr 17 phosphorylation requires the sustained elevated calcium concentrations that activate CaM kinase and thus is likely to be important in protection of the cell against conditions of calcium overload [18]. (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), IB (21)(22)(23)(24)(25)(26)(27)(28)(29)(30), and II , as originally defined [24].…”

Section: Plb Regulation Of the Ca-atpasementioning

confidence: 98%

“…In particular, the membrane-spanning peptide is a highly conserved and hydrophobic sequence composed almost entirely of Ile and Leu residues with the notable exception of 3 polar cysteines (Cys 36, Cys 41 , Cys 46 ). These cysteines are not involved in disulfide bonds, and their exact role in PLB structure is presently unclear [26]. Commonly, these cysteines are mutated to a non-reactive amino acid for use in structural studies in order to avoid disulfide crosslinking.…”

Section: Structure Of Plbmentioning

confidence: 99%

“…Subsequent structural measurements have revised the initial secondary structure predictions, but the domains as defined by Fujii and coworkers remain in common use as a means to identify locations within PLB. From the N-terminus, the cytoplasmic portion of the enzyme is divided into two sequences; IA, corresponding to the first twenty amino acids that contain an amphipathic helix and the phosphorylation sites (Ser 16 and Thr 17 ) and 1B, (21)(22)(23)(24)(25)(26)(27)(28)(29)(30), which is part of a continuous helix that contains both cytosolic (i.e., sequence 1B) and membrane-spanning (sequence II) elements (Fig. 2).…”

Section: Structure Of Plbmentioning

confidence: 99%

See 1 more Smart Citation

Coil-to-Helix Transition within Phospholamban Underlies Release of Ca- ATPase Inhibition in Response to β-Adrenergic Signaling

Bigelow¹,

Squier²

2006

CEI

View full text Add to dashboard Cite

Phospholamban (PLB) is a major target of β-adrenergic signaling, whose phosphorylation results in enhanced rates of relaxation in the heart. Prior to phosphorylation, PLB functions to reduce the calcium sensitivity of the Ca-ATPase, resulting in slower rates of calcium resequestration into the sarcoplasmic reticulum after each contractile event. Recent structures indicate that the inhibitory interaction between PLB and the Ca-ATPase requires PLB to assume an extended structure, where the transmembrane and cytosolic portions of PLB undergo specific binding interactions with distant sites on the Ca-ATPase. In the extended conformation, PLB binding to the Ca-ATPase functions to inhibit the Ca-ATPase through a reduction in the rates of catalytically important motions involving the nucleotide binding domain. Phosphorylation of PLB at either Ser 16 or Thr 17 releases the inhibitory interaction between PLB and the Ca-ATPase. These sites of phosphorylation are within a hinge region in PLB that separates the highly structured transmembrane and cytosolic portions that associate with the Ca-ATPase. The helical content of the hinge region increases following the phosphorylation of PLB, which induces a shortening of the maximal dimensions of PLB and a release of the inhibitory interaction with the Ca-ATPase. Following phosphorylation, PLB remains associated with the Ca-ATPase in a more compact form that has no inhibitory capability. Thus, the conformational switch involving PLB regulation of the Ca-ATPase relies upon a physical mechanism, whereby the phosphorylationdependent stabilization of the structure of PLB functions to destabilize the inhibitory interaction between PLB and the Ca-ATPase. Upon hydrolysis of the phosphoester linkages by endogenous phosphatases, PLB is poised to reassume the inhibited state through re-association with inhibitory sites on the nucleotide binding domain of the Ca-ATPase. PHYSIOLOGICAL ROLE OF PLBPhospholamban (PLB) is a 52 amino acid integral membrane protein that constitutes a key element in the responsiveness of muscle contractility to β-adrenergic stimulation through its regulation of the sarcoplasmic reticulum (SR) Ca-ATPase [1]. The SR Ca-ATPase is the primary mediator of muscle relaxation in its role of active transport of cytosolic calcium ions into the SR lumen after each contractile event which is induced, in turn, by rapid release of calcium into the cytosol. As the limiting step in contractility, the resequestration of cytosolic calcium provides a major control point for both contractile function and calcium signaling events within the cell [2]. PLB is coexpressed with the SERCA2a isoform of the Ca-ATPase in cardiac, smooth, and slow-twitch skeletal muscle where the nonphosphorylated form of PLB acts an inhibitor of the CaATPase. Upon PLB phosphorylation at Ser 16 or Thr 17 , mediated in vivo by PKA or CaM-kinase II, respectively, relief of inhibition is manifest in increased rates of calcium transport with concomitant increased contractile rates.Alterations in PLB regulation of ca...

show abstract

Structure of the Transmembrane Cysteine Residues in Phospholamban

Cited by 27 publications

References 41 publications

Dimerization of the Erythropoietin Receptor Transmembrane Domain in Micelles

Dimerization of the Erythropoietin Receptor Transmembrane Domain in Micelles

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

Coil-to-Helix Transition within Phospholamban Underlies Release of Ca- ATPase Inhibition in Response to β-Adrenergic Signaling

Contact Info

Product

Resources

About

Structure of the Transmembrane Cysteine Residues in Phospholamban

Cited by 27 publications

References 41 publications

Dimerization of the Erythropoietin Receptor Transmembrane Domain in Micelles

Dimerization of the Erythropoietin Receptor Transmembrane Domain in Micelles

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

Coil-to-Helix Transition within Phospholamban Underlies Release of Ca- ATPase Inhibition in Response to &#946;-Adrenergic Signaling

Contact Info

Product

Resources

About

Coil-to-Helix Transition within Phospholamban Underlies Release of Ca- ATPase Inhibition in Response to β-Adrenergic Signaling