The structural basis for phospholamban inhibition of the calcium pump in sarcoplasmic reticulum

Abstract

P-type ATPases are a large family of enzymes that actively transport ions across biological membranes by interconverting between high (E1) and low (E2) ion-affinity states; these transmembrane transporters carry out critical processes in nearly all forms of life. In striated muscle, the archetype P-type ATPase, SERCA (sarco(endo)plasmic reticulum Ca(2+)-ATPase), pumps contractile-dependent Ca(2+) ions into the lumen of sarcoplasmic reticulum, which initiates myocyte relaxation and refills the sarcoplasmic reticulum in preparation for the next contraction. In cardiac muscle, SERCA is regulated by phospholamban (PLB), a small inhibitory phosphoprotein that decreases the Ca(2+) affinity of SERCA and attenuates contractile strength. cAMP-dependent phosphorylation of PLB reverses Ca(2+)-ATPase inhibition with powerful contractile effects. Here we present the long sought crystal structure of the PLB-SERCA complex at 2.8-Å resolution. The structure was solved in the absence of Ca(2+) in a novel detergent system employing alkyl mannosides. The structure shows PLB bound to a previously undescribed conformation of SERCA in which the Ca(2+) binding sites are collapsed and devoid of divalent cations (E2-PLB). This new structure represents one of the key unsolved conformational states of SERCA and provides a structural explanation for how dephosphorylated PLB decreases Ca(2+) affinity and depresses cardiac contractility.

Keywords: Calcium ATPase; Calcium Transport; Crystal Structure; Phospholamban; Protein Cross-linking; Protein Crystallization; SERCA; Sarcolipin; Sarcoplasmic Reticulum (SR).

FIGURE 1.

Amino acid sequences of PLB4 and WT-PLB (canine isoforms), and rabbit SLN. Cytoplasmic and transmembrane domains are shown as well as phosphorylated residues Ser¹⁶ and Thr¹⁷ of PLB. Mutations in PLB4 are shaded in blue. Amino acid residues common to both PLB and SLN are shaded in yellow.

FIGURE 2.

Effect of detergent solubilization of SERCA on apparent Ca²⁺ affinity (A) and Ca²⁺-ATPase stability over time (B). Mother liquors were prepared from rabbit skeletal SR vesicles in buffer containing 2% detergent concentrations and 2 mm EGTA as described under “Experimental Procedures.” A, apparent Ca²⁺ affinity (K_Ca) of the solubilized Ca²⁺-ATPase was determined immediately after membrane solubilization by measuring Ca²⁺ activation of ATP hydrolysis. Results are expressed as percentage of the maximal Ca²⁺-ATPase activity obtained for each detergent tested: NM (nonyl glucoside), OG (octyl glucoside), DM (decyl maltoside), and DDM (dodecyl maltoside). Control membranes (Con) were not treated with detergent. K_Ca values (μm) were 0.21 ± 0.005 (Con), 0.25 ± 0.02 (NM), 0.23 ± 0.02 (OG), 0.63 ± 0.02 (C₁₂E₈), 0.29 ± 0.01 (DM), and 0.40 ± 0.02 (DDM). Mean ± S.E. from four to eight determinations are shown. B, enzyme stability of SERCA was monitored after storage of the mother liquors in different detergents at 4 °C for the times indicated. At the designated times of storage, aliquots were taken for assay of Ca²⁺-ATPase activity (micromole of P_i/mg of protein/h) at saturating Ca²⁺ concentration (50 μm Ca²⁺). NM + PLB4 designates SERCA solubilized in NM reconstituted with PLB in DM, an optimal condition for crystal formation. Shown is one representative experiment, which was repeated at least three times for all the different detergents with similar results.

FIGURE 3.

Crystal structure and functional characterization of the PLB4-SERCA complex. A, a ribbon model of the complex between SERCA and PLB4. In SERCA the cytoplasmic headpiece consists of actuator (A) (gray), phosphorylation (P) (gold), and nucleotide-binding (N) (green) domains. The transmembrane domain helices of SERCA are colored cyan, except for M4, which is colored blue. The bound PLB4 molecules are colored magenta (chain B) and yellow (chain C) (Table 1). The figure was generated using PyMOL (The PyMOL Molecular Graphics System, version 1.5.0.4, Schrödinger, LLC). B, Ca²⁺-ATPase activity of nonyl maltoside-solubilized SERCA alone and after reconstitution with purified, solubilized WT-PLB or PLB4. Activity was measured in the presence and absence of the anti-PLB monoclonal antibody, 2D12. K_Ca values (μm) for Ca²⁺ activation of ATP hydrolysis were: no PLB, 0.26 ± 0.01; WT-PLB, 0.62 ± 0.03 and 0.35 ± 0.02 (+ 2D12); PLB4, 1.38 ± 0.07 and 0.53 ± 0.03 (+ 2D12). Mean ± S.E. from three to six determinations are shown. C, anti-PLB immunoblot showing the Ca²⁺ effect on cross-linking of PLB4 to SERCA with KMUS under the same conditions used for determination of Ca²⁺-ATPase activity. K_i value (μm) for Ca²⁺ inhibition of cross-linking was 1.60 ± 0.16. Mean ± S.E. from 6 determinations are shown. D, Coomassie Blue-stained gel showing SERCA solubilized from rabbit skeletal muscle SR membranes and WT-PLB and PLB4 purified in decyl maltoside (± boil). 10 μg of membrane protein were electrophoresed per gel lane.

FIGURE 4.

Binding of PLB4 to the transmembrane domain of SERCA. A, a view of PLB4 bound to SERCA roughly orthogonal to that in Fig. 3A including the side chains of PLB4 interacting with the transmembrane domain; the color scheme is identical to Fig. 3A. B, a horizontal section of the SERCA transmembrane domain showing the interaction area of PLB4 in proximity of the Ca²⁺-binding sites, the color scheme is identical to Fig. 3A. Critical side chains are labeled and displayed, as are the nearby transmembrane helices. The dotted lines and numerical values indicate the distances between potential hydrogen bond donors and acceptors. The figure was generated using PyMOL.

FIGURE 5.

Critical interactions at the interface between SERCA and PLB4. A, a horizontal section of the SERCA transmembrane domain centered near the key inhibitory residue Asn³⁴ of PLB4. The color scheme is identical to Fig. 4A. Key residues at the interaction surface are labeled, as are the individual transmembrane helices in this region. The dotted line indicates inferred hydrogen bonding between donor and acceptor atoms and the numerical value indicates the distance between atoms potentially sharing hydrogens. B, a “peeled” surface view of the interaction surface between PLB4 and SERCA. For this figure, PLB4 was rotated 180 degrees in the vertical plane and translated to the right-hand side of this figure to expose the interacting surfaces and their chemical character. The color scheme utilizes “atom type” coloring (blue for nitrogen, red for oxygen, yellow for sulfur, and green for carbon atoms not involved in the contact surface between PLB4 and SERCA). Carbon atoms in amino acids residing either in PLB4 or SERCA that have at least one atom at the contact surface are colored gray. Amino acid residues that form the contact surface are labeled. C, the original figure-of-merit σA-weighted 2F_o − F_c (blue) and F_o − F_c (green) electron density maps for PLB4 bound to the transmembrane domain of SERCA were generated prior to their inclusion in the model, superimposed on the final refined coordinates for the complex. D, structural super-positioning of the PLB4-SERCA complex (cyan and magenta ribbons for SERCA and PLB4) with that of the Ca²⁺-free E2 complex stabilized by TG (green ribbons, RCSB code 1IWO (24)). The structures were super-imposed utilizing the Cα atoms of residues 750–990. Key residues involved in Ca²⁺ binding are labeled, as is Ile³⁸ of PLB4.

FIGURE 6.

Lack of effect of Mg²⁺ on PLB cross-linking. A, autoradiograph showing cross-linking of N30C-PLB or PLB4 to SERCA2a expressed in insect cell membranes. Cross-linking was conducted with KMUS over a range of Ca²⁺ concentrations in the presence and absence of 3 mm MgCl₂. B, quantified results plotted as percentage of maximal cross-linking determined at zero Ca²⁺ concentration. Mg²⁺ had no effect on maximal cross-linking. K_i values (μm) for Ca²⁺ inhibition of cross-linking of N30C-PLB to SERCA were 0.37 ± 0.05 and 0.46 ± 0.07 in the absence and presence of Mg²⁺, respectively. K_i values for Ca²⁺ inhibition of PLB4 cross-linking were 1.52 ± 0.06 and 1.70 ± 0.10, respectively. Mean ± S.E. from three to five determinations.