No abstract

We have used chemical synthesis and electron paramagnetic resonance to probe the structural dynamics of phospholamban (PLB) in lipid bilayers. Derivatives of monomeric PLB were synthesized, each of which contained a single spin-labeled 2,2,6,6,-Tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid amino acid, with the nitroxide-containing ring covalently and rigidly attached to the ␣-carbon, providing direct insight into the conformational dynamics of the peptide backbone. 2,2,6,6,-tetramethyl-piperidine-Noxyl-4-amino-4-carboxylic acid was attached at positions 0, 11, and 24 in the cytoplasmic domain or at position 46 in the transmembrane domain. The electron paramagnetic resonance spectrum of the transmembrane domain site (position 46) indicates a single spectral component corresponding to strong immobilization of the probe, consistent with the presence of a stable and highly ordered transmembrane helix. In contrast, each of the three cytoplasmic domain probes has two clearly resolved spectral components (conformational states), one of which indicates nearly isotropic nanosecond dynamic disorder. For the probe at position 11, an N-terminal lipid anchor shifts the equilibrium toward the restricted component, whereas Mg 2؉ shifts it in the opposite direction. Relaxation enhancement, due to Ni 2؉ ions chelated to lipid headgroups, provides further information about the membrane topology of PLB, allowing us to confirm and refine a structural model based on previous NMR data. We conclude that the cytoplasmic domain of PLB is in a dynamic equilibrium between an ordered conformation, which is in direct contact with the membrane surface, and a dynamically disordered form, which is detached from the membrane and poised to interact with its regulatory target. Muscle function depends critically on the active transport of calcium into the lumen of the sarcoplasmic reticulum (SR) by the Ca-ATPase, the SERCA gene product (1). SERCA activity is modulated in the heart by interaction with phospholamban (PLB), a 52-residue integral membrane protein (2-4). PLB inhibits SERCA at submicromolar calcium, but ␤-adrenergic stimulation causes phosphorylation of PLB at Ser-16 and͞or Thr-17, relieving this inhibition (2, 5), activating calcium transport across the SR, and accelerating cardiac muscle relaxation (6-8). PLB is a major regulator of the dynamics of cardiac contractility (9, 10), and the SERCA-PLB calcium-regulatory system has been implicated in cardiovascular disease (11)(12)(13).PLB is predominantly a homopentamer, with a small fraction of monomers, but it has been shown by electron paramagnetic resonance (EPR) (14, 15), fluorescence (16), and mutagenesis (17-19) that the less predominant monomeric form of PLB is primarily responsible for inhibition of SERCA. Therefore, the present study focuses on a stable PLB monomer, designated AFA-PLB, obtained by mutating the three Cys residues 36, 41, and 46 to Ala, Phe, and Ala, respectively (20)(21)(22). The isolated transmembrane domain of PLB inhibits SERCA just as effectively as d...

We have used magnetic resonance to map the interaction surface of an integral membrane protein for its regulatory target, an integral membrane enzyme. Phospholamban (PLN) regulates cardiac contractility via its modulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity. Impairment of this regulatory process causes heart failure. To map the molecular details of the PLN͞SERCA interaction, we have functionally reconstituted SERCA with labeled PLN in dodecylphosphocholine micelles for highresolution NMR spectroscopy and in both micelles and lipid bilayers for EPR spectroscopy. Differential perturbations in NMR linewidths and chemical shifts, measured as a function of position in the PLN sequence, provide a vivid picture of extensive SERCA contacts in both cytoplasmic and transmembrane domains of PLN and provide structural insight into previously reported functional mutagenesis data. NMR and EPR data show clear and complementary evidence for a dynamic (s-to-ms) equilibrium between two conformational states in the cytoplasmic domain of PLN. These results support the hypothesis that SERCA attracts the cytoplasmic domain of PLN away from the lipid surface, shifting the preexisting equilibrium of PLN conformers toward a structure that is poised to interact with the regulatory target. EPR shows that this conformational switch behaves similarly in micelles and lipid membranes. Based on structural and dynamics data, we propose a model in which PLN undergoes allosteric activation upon encountering SERCA.NMR spectroscopy ͉ protein-protein interaction S arco(endo)plasmic reticulum calcium ATPase (SERCA) designates a family of P-type calcium pumps embedded in the sarco(endo)plasmic reticulum. In muscle, SERCA is a 110-kDa enzyme, containing 10 transmembrane helices and three distinct cytoplasmic domains (1), which restores cytosolic calcium to submicromolar levels via ATP hydrolysis, resulting in relaxation. In cardiac muscle, phospholamban (PLN) regulates SERCA, inhibiting the enzyme at submicromolar Ca 2ϩ (2). After ␤-adrenergic stimulation, PLN is phosphorylated by protein kinase A, reversing SERCA inhibition. SERCA1a, the fast-twitch skeletal muscle isoform, is functionally identical to SERCA2a, the cardiac isoform, both in the presence and absence of PLN (3). Because SERCA1a is a better-characterized enzyme and is readily purified in 100-mg quantities, the present study used this isoform.PLN is a 52-aa, single-pass transmembrane protein that undergoes a dynamic equilibrium between a monomeric inhibitory form and a pentameric storage form (4). PLN has three structural domains, as determined by NMR in dodecylphosphocholine (DPC) micelles (5). The N-terminal cytosolic helix (domain Ia: residues 2-16) is amphipathic, with the hydrophobic face pointing toward the lipid surface (5, 6). A flexible loop (residues 17-21) connects this helix to the C-terminal helix (residues 22-50), introducing an average angle of Ϸ80°between the two helices (5, 6). The C-terminal helix ends with a hydrophobic membrane-embedded seque...

The integral membrane protein complex between phospholamban (PLN) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) regulates cardiac contractility. In the unphosphorylated form, PLN binds SERCA and inhibits Ca2+ flux. Upon phosphorylation of PLN at Ser16, the inhibitory effect is reversed. Although structural details on both proteins are emerging from X-ray crystallography, cryo-electron microscopy, and NMR studies, the molecular mechanisms of their interactions and regulatory process are still lacking. It has been speculated that SERCA regulation depends on PLN structural transitions (order to disorder, i.e., folding/unfolding). Here, we investigated PLN conformational changes upon chemical unfolding by a combination of electron paramagnetic resonance and NMR spectroscopies, revealing that the conformational transitions involve mostly the cytoplasmic regions, with two concomitant phenomena: (1) membrane binding and folding of the amphipathic domain Ia and (2) folding/unfolding of the juxtamembrane domain Ib of PLN. Analysis of phosphorylated and unphosphorylated PLN with two phosphomimetic mutants of PLN (S16E and S16D) shows that the population of an unfolded state in domains Ia and Ib (T′ state) is linearly correlated to the extent of SERCA inhibition measured by activity assays. Inhibition of SERCA is carried out by the folded ground state (T state) of the protein (PLN), while the relief of inhibition involves promotion of PLN to excited conformational states (Ser16 phosphorylated PLN). We propose that PLN population shifts (folding/unfolding) are a key regulatory mechanism for SERCA.

The BH3-only protein, Noxa, is induced in response to apoptotic stimuli, such as DNA damage, hypoxia, and proteasome inhibition in most human cells. Noxa is constitutively expressed in proliferating cells of hematopoietic lineage and required for apoptosis in response to glucose stress. We show that Noxa is phosphorylated on a serine residue (S(13)) in the presence of glucose. Phosphorylation promotes its cytosolic sequestration and suppresses its apoptotic function. We identify Cdk5 as the Noxa kinase and show that Cdk5 knockdown or expression of a Noxa S(13) to A mutant increases sensitivity to glucose starvation, confirming that the phosphorylation is protective. Both glucose deprivation and Cdk5 inhibition promote apoptosis by dephosphorylating Noxa. Paradoxically, Noxa stimulates glucose consumption and may enhance glucose turnover via the pentose phosphate pathway rather than through glycolysis. We propose that Noxa plays both growth-promoting and proapoptotic roles in hematopoietic cancers with phospho-S(13) as the glucose-sensitive toggle switch controlling these opposing functions.