Mapping the interaction surface of a membrane protein: Unveiling the conformational switch of phospholamban in calcium pump regulation

Abstract

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...

Phospholamban and Its Phosphorylated Form Interact Differently with Lipid Bilayers: A31P,2H, and13C Solid-State NMR Spectroscopic Study

“…In the second model, an allosteric interaction between the PLB monomer and SERCA takes place (65). When PLB is in its free form (not interacting with SERCA), the monomer interconverts between the bent form (T-state, inactive, highly ordered L-shape, cytoplasmic domain facing the lipids, predominant) and the extended form (R-state, active, more dynamically disordered with higher affinity to SERCA) (65).…”

“…When PLB is in its free form (not interacting with SERCA), the monomer interconverts between the bent form (T-state, inactive, highly ordered L-shape, cytoplasmic domain facing the lipids, predominant) and the extended form (R-state, active, more dynamically disordered with higher affinity to SERCA) (65). In the presence of SERCA, the preexisting equilibrium between the two forms is disturbed.…”

“…In the presence of SERCA, the preexisting equilibrium between the two forms is disturbed. The bent form inhibits SERCA because of protein-protein interactions between the transmembrane regions of PLB and SERCA (65). Only in the extended form, in which both the cytoplasmic domain and the transmembrane domain of PLB binds to SERCA, the SERCA-PLB complex is capable of reversing SERCA inhibition, because of phosphorylation or mutation (65,66).…”

“…Additionally, EPR spectroscopic studies from the Veglia and Thomas groups indicate that a two-state dynamic equilibrium exists for the cytosolic segment of AFA-PLB in a lipid bilayer (58,65,66). Similarly, the Chou group suggested that their structure of PLB in micelles sets the stage for a series of future experiments to characterize the effect of phosphorylation on the orientation and dynamics of its cytoplasmic helix (61).…”

Spectroscopic validation of the pentameric structure of phospholamban

“…In contrast, the conformational ensemble of the PLN monomer has much lower precision (1) due to psec-msec conformational disorder supported by EPR and solution and solid-state NMR spectroscopies (11)(12)(13)(14)(15). Although the PLN structure is dynamic, extensive data in lipids and micelles show that the predominant Structural models of wt-PLN.…”