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Phosphorylation and Mutation of Phospholamban Alter Physical Interactions with the Sarcoplasmic Reticulum Calcium Pump

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Phosphorylation and Mutation of Phospholamban Alter Physical Interactions with the Sarcoplasmic Reticulum Calcium Pump doi:10.1016/j.jmb.2010.11.014 J. Mol. Biol. (2011) 405, 707–723 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb Phosphorylation and Mutation of Phospholamban Alter Physical Interactions With the Sarcoplasmic Reticulum Calcium Pump John Paul Glaves1,2, Catharine A. Trieber1,2, Delaine K. Ceholski1, David L. Stokes3,4 and Howard S. Young1,2⁎ 1Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 2National Institute for Nanotechnology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 3Skirball Institute of Biomolecular Medicine, School of Medicine, New York University, New York, NY 10016, USA 4New York Structural Biology Center, New York, NY 10027, USA Received 19 July 2010; Phospholamban physically interacts with the sarcoplasmic reticulum received in revised form calcium pump (SERCA) and regulates contractility of the heart in response 2 November 2010; to adrenergic stimuli. We studied this interaction using electron microscopy accepted 8 November 2010 of 2D crystals of SERCA in complex with phospholamban. In earlier studies, Available online phospholamban oligomers were found interspersed between SERCA dimer 23 November 2010 ribbons and a 3D model was constructed to show interactions with SERCA. In this study, we examined the oligomeric state of phospholamban and the Edited by W. Baumeister effects of phosphorylation and mutation of phospholamban on the interaction with SERCA in the 2D crystals. On the basis of projection maps Keywords: from negatively stained and frozen-hydrated crystals, phosphorylation of SERCA; Ser16 selectively disordered the cytoplasmic domain of wild type phospho- phospholamban; lamban. This was not the case for a pentameric gain-of-function mutant phosphorylation; (Lys27Ala), which retained inhibitory activity and remained ordered in the electron crystallography; phosphorylated state. A partial loss-of-function mutation that altered the 2D crystals charge state of phospholamban (Arg14Ala) retained an ordered state, while a complete loss-of-function mutation (Asn34Ala) was also disordered. The functional state of phospholamban was correlated with an order-to-disorder transition of the phospholamban cytoplasmic domain in the 2D co-crystals. Furthermore, co-crystals of the gain-of-function mutant (Lys27Ala) facilitated data collection from frozen-hydrated crystals. An improved projection map was calculated to a resolution of 8 Å, which supports the pentamer as the oligomeric state of phospholamban in the crystals. The 2D co-crystals with SERCA require a functional pentameric form of phospholamban, which physically interacts with SERCA at an accessory site distinct from that used by the phospholamban monomer for the inhibitory association. © 2010 Elsevier Ltd. All rights reserved. Introduction *Corresponding author. E-mail address: Cation transport by the P-type ion pumps is an [email protected]. essential process in all eukaryotic cells, where Abbreviations used: SERCA, sarcoplasmic reticulum changes in intracellular cation concentrations are calcium ATPase; SR, sarcoplasmic reticulum; PLB, linked to precise physiological responses. The best phospholamban; PKA, cAMP-dependent protein kinase. understood members of this transport family include 0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved. 708 Co-crystals of SERCA and Phospholamban the sarcoplasmic reticulum calcium ATPase binds to and inhibits SERCA, and phosphorylation (SERCA) found in muscle cells and the plasma disrupts this inhibitory complex.24,25 However, membrane sodium-potassium ATPase (Na+-K+ there is contradictory evidence about whether PLB pump) found in all cell types. These two P-type ion is physically dissociated from SERCA following pumps are particularly important in cardiac con- phosphorylation. Fluorescence energy transfer tractility and are major drug targets for the clinical experiments suggest that PLB inhibits and aggre- improvement of heart disease. An extensive series of gates SERCA, and that phosphorylation reverses X-ray and electron crystallographic studies have this process and causes dissociation of PLB and resulted in structures of a variety of SERCA reaction SERCA.26 Similarly, cross-linking experiments in- intermediates, thus revealing how ATP hydrolysis is dicate that phosphorylation weakens the physical coupled to calcium transport across the sarcoplasmic association of PLB with SERCA and makes the recticulum (SR) membrane in order to achieve complex more susceptible to dissociation by – muscle relaxation.1 14 These studies show that sub-saturating concentrations of calcium.27,28 In SERCA is composed of a transmembrane domain contrast, studies using co-immunoprecipitation,29 that contains the calcium-binding sites and three fluorescence30,31 and EPR spectroscopy32 all sug- cytosolic domains that are responsible for nucleotide gest that PLB remains associated with SERCA binding, phosphorylation, and communication with following phosphorylation. Rather than dissocia- the transmembrane domain. It has been shown that tion of PLB from SERCA after phosphorylation, the intermediate states (calcium binding, phosphor- EPR and NMR studies point to a transition from ylation, calcium transport, dephosphorylation and order to disorder in the cytoplasmic domain of proton counter-transport) involve coupled domain PLB.32,33 Such a transition is consistent with the movements that link the cation-binding sites with results of a variety of biophysical studies showing the phosphorylation state of the enzyme. that phosphorylation causes a partial unwinding Despite this wealth of structural information, the and disordering of the PLB N-terminal α-helix – regulation of the calcium pump in cardiac muscle around the Ser16 phosphorylation site.34 38 Regu- remains an elusive target of study. In cardiac and lation by phosphorylation is thought to occur in the smooth muscle, SERCA is regulated by phospho- context of a complex between monomeric PLB and lamban (PLB), a 52 residue integral membrane SERCA yet it has been suggested that the PLB protein. PLB engages in an inhibitory interaction pentamer is necessary for regulation of cardiac with SERCA that reduces its apparent calcium contractility in a physiological context;39 and a affinity. This is a dynamic process that depends on direct interaction has been proposed between the – the cytosolic calcium concentration, as well as the PLB pentamer and SERCA.40 42 EPR measurements phosphorylation and oligomeric states of PLB, which of boundary lipids suggest that phosphorylation of is in dynamic equilibrium between monomeric and PLB shifts the population towards the oligomeric homo-oligomeric states, with pentameric forms being state.16 These results raise questions about the role dominant in SDS-PAGE.15,16 Mutation of key leucine of PLB oligomeric states in the regulation of SERCA. and isoleucine residues in the transmembrane do- In earlier work, we observed a direct interaction main of PLB destabilizes the pentameric structure between an oligomeric form of PLB and SERCA in 2D and has been shown to shift this equilibrium in favor crystals.42 Specifically, we characterized co-crystals of of the monomer. These pentamer-disrupting muta- SERCA and a super-inhibitory mutant of PLB tions are associated with increased inhibition of (Ile40Ala; I40A).42 While SDS-PAGE indicated that SERCA, leading to the speculation that the PLB this mutant of PLB was monomeric,17 our projection – monomer is the active inhibitory species,17 19 and map revealed that I40A formed an oligomer, which that the pentamer is an inactive storage form.20,21 was later supported by fluorescence resonance energy Unfortunately, it has not been possible to test this transfer experiments.43,44 These results reinforce the model directly with well defined PLB oligomeric conclusion reached by Jones and co-workers, that states within a lipid membrane. In any case, SERCA SDS-PAGE might indicate the relative stability of PLB inhibition by the PLB monomer can be reversed either oligomers but it is not a definitive means of assessing by elevated cytoplasmic calcium concentrations or by the oligomeric species adopted by PLB within the phosphorylation of PLB. The primary physiological lipid bilayer.18 A 3D model based on our projection mechanism for relieving SERCA inhibition is through map suggested that PLB pentamers interact with the phosphorylation of PLB at Ser16 by cAMP- SERCA at two potential sites; one near transmem- dependent protein kinase (PKA), and PLB can be brane segment M3 and another near the C-terminus. phosphorylated at Thr17 by either calcium/calmod- These contact sites are distinct from the inhibitory site ulin-dependent protein kinase II22 or by Akt.23 occupied by the PLB monomer, which is adjacent to The functional effect of PLB phosphorylation on M2, M4 and M6 of SERCA according to the results of – SERCA regulation is clear but the mechanism for mutagenesis and cross-linking studies.45 49 In this this effect is less certain. The original model for study, we investigated the effects of PLB phosphory- SERCA regulation suggested that monomeric PLB lation and mutation on the interaction between a PLB Co-crystals of SERCA and Phospholamban 709 oligomer and SERCA in the context of 2D crystals. Table 1. Apparent calcium affinity (KCa) determined for Our results show a correlation between PLB function SERCA in the absence and in the presence of wild type, and crystal formation, suggesting that
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