Allosteric regulation of SERCA by phosphorylation- mediated conformational shift of phospholamban Martin Gustavssona, Raffaello Verardia, Daniel G. Mullena, Kaustubh R. Moteb, Nathaniel J. Traasetha,1, T. Gopinatha, and Gianluigi Vegliaa,b,2 aDepartment of Biochemistry, Molecular Biology, and Biophysics and bDepartment of Chemistry, University of Minnesota, Minneapolis, MN 55455 Edited by Chikashi Toyoshima, University of Tokyo, Tokyo, Japan, and approved September 13, 2013 (received for review May 26, 2013) The membrane protein complex between the sarcoplasmic re- cytoplasmic domain binds SERCA’s N domain in an α-helical + ticulum Ca2 -ATPase (SERCA) and phospholamban (PLN) controls conformation, suggesting that the inhibitory effect may be eli- + Ca2 transport in cardiomyocytes, thereby modulating cardiac con- cited via an induced fit mechanism. tractility. β-Adrenergic-stimulated phosphorylation of PLN at Ser- Interestingly, PLN’s cytoplasmic domain in the recent X-ray 16 enhances SERCA activity via an unknown mechanism. Using structure of the complex is completely unresolved (8), leaving solid-state nuclear magnetic resonance spectroscopy, we mapped many questions regarding its regulatory function unanswered. the physical interactions between SERCA and both unphosphory- Moreover, there have been few structural data on the complex lated and phosphorylated PLN in membrane bilayers. We found between SERCA and phosphorylated PLN. On the basis of cross- that the allosteric regulation of SERCA depends on the conforma- linking and sparse spectroscopic data, two different mechanistic tional equilibrium of PLN, whose cytoplasmic regulatory domain models have been proposed: a dissociative model, in which phos- interconverts between three different states: a ground T state phorylation causes PLN detachment from SERCA, reestablishing + (helical and membrane associated), an excited R state (unfolded Ca2 flux (14), and the subunit model, in which phosphorylation and membrane detached), and a B state (extended and enzyme- induces conformational rearrangements of PLN’s cytoplasmic bound), which is noninhibitory. Phosphorylation at Ser-16 of PLN domain without dissociation (15–17). Nevertheless, the two models shifts the populations toward the B state, increasing SERCA activ- fall short in the interpretation of the recent structure–function ity. We conclude that PLN’s conformational equilibrium is central – correlations (15, 18 20). BIOPHYSICS AND 2+ to maintain SERCA’s apparent Ca affinity within a physiological In lipid bilayers, PLN adopts a helix-turn-helix structure ar- window. This model represents a paradigm shift in our under- ranged in an L-shaped configuration (Fig. 1) (21), with the helical COMPUTATIONAL BIOLOGY standing of SERCA regulation by posttranslational phosphoryla- membrane-spanning region (inhibitory) divided into hydrophilic tion and suggests strategies for designing innovative therapeutic domain Ib and hydrophobic domain II and an amphipathic region approaches to enhance cardiac muscle contractility. (regulatory) domain Ia, which harbors the recognition sequence for cAMP-dependent protein kinase A. A prominent feature of solid-state NMR | magic angle spinning | protein-protein interactions | PLN is its conformational dynamics. Although domain II is rel- paramagnetic relaxation enhancement atively rigid and membrane-embedded, the cytoplasmic domain Ia, domain Ib, and the intervening loop are more dynamic. Im- + he sarcoplasmic reticulum Ca2 -ATPase (SERCA)/phos- portantly, domain Ia is metamorphic, adopting a helical structure + Tpholamban (PLN) complex regulates Ca2 translocation into when in contact with membrane (T or ground state) and becoming the sarcoplasmic reticulum (SR) of cardiomyocytes and con- unfolded when detached (R or high energy, conformationally ex- stitutes the main mechanism of cardiac relaxation (diastole) (1–3). cited state) (11, 15, 18, 22). On average, the L-shaped T state is + SERCA is a P-type ATPase that translocates two Ca2 ions per the most populated in either monomeric or pentameric PLN + ATP molecule hydrolyzed in exchange for three H (Fig. 1) (4, 5). (Fig. 1) (21, 23), with the R state accounting for 16–20% of the PLN binds and allosterically inhibits SERCA function, decreas- conformational ensemble (24). Remarkably, PLN’s conformational + ing its apparent affinity for Ca2 ions (3, 6). On β-adrenergic stimulation, cAMP-dependent protein kinase A phosphorylates Significance PLN at Ser-16, reversing the inhibition and augmenting cardiac output (3). Disruptions in this regulatory mechanism degenerate 2+ 2+ The sarcoplasmic reticulum Ca -ATPase (SERCA)/phospholamban into Ca mishandling and heart failure (3). complex regulates cardiac muscle contractility by controlling + Several X-ray structures of SERCA have been determined Ca2 transport from the cytosol to the lumen of the sarco- along its enzymatic coordinates, providing atomic details on the fi plasmic reticulum. By mapping the interactions between these structural transitions in the absence of PLN (4, 5). The rst two membrane proteins, we found that SERCA function depends image of the SERCA/PLN complex resulted from cryo-EM on the equilibria between transient conformational states of studies (7), but the low-resolution data prevent an atomic view of phospholamban. Phosphorylation of phospholamban shifts the PLN structure and architecture within the complex. In addition, equilibria, enhancing SERCA function. This mechanism explains mutagenesis and cross-linking data were used to model the why tuning phospholamban’s structural dynamics can modu- complex, suggesting that the inhibitory transmembrane (TM) late SERCA function and may aid in designing innovative region of PLN is positioned into a binding groove far from the + therapeutic approaches to heart failure. putative Ca2 entry, as well as the ATP binding site, and located between TM helices M2, M4, M6, and M9 of SERCA. The lo- Author contributions: M.G., R.V., D.G.M., K.R.M., N.J.T., T.G., and G.V. designed research; cation of PLN’s TM domain agrees with a recent crystal structure M.G., R.V., D.G.M., K.R.M., N.J.T., and T.G. performed research; M.G., R.V., K.R.M., N.J.T., of the SERCA/PLN complex (8) and is remarkably similar to the and G.V. analyzed data; and M.G., R.V., and G.V. wrote the paper. one recently identified for a PLN homolog, sarcolipin, in com- The authors declare no conflict of interest. plex with SERCA (9, 10) (Fig. 1). This article is a PNAS Direct Submission. In the SERCA/PLN model, which was further refined using 1Present address: Chemistry Department, New York University, New York, NY 10003. NMR constraints (11), the loop bridging the TM and cytoplas- 2To whom correspondence should be addressed. E-mail: [email protected]. fi mic domain of PLN adopts an unfolded con guration, stretch- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ing toward the N domain of the enzyme (12, 13). Also, PLN’s 1073/pnas.1303006110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1303006110 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Fig. 1. Structures of PLN and SERCA. (A) Primary sequence and domains of PLNAFA. The S16 phosphorylation site is marked with an arrow, and mutation sites (C36A, C41F, C46A) are indicated with asterisks. (B) 3D structures of PLN pentamer [Protein Data Bank (PDB) ID code 2KYV], PLNAFA monomer in the T (PDB ID code 2KB7) and R states (PDB ID code 2LPF), and SERCA in complex with sarcolipin (PDB ID code 3W5A). SERCA includes 10 TM helices and three cytoplasmic domains: nucleotide binding (N domain), actuator (A domain), and phosphorylation (P domain). (C) Fluorescence energy transfer (FRET) between SERCAAEDANS (donor) and PLNDab-AFA (acceptor), showing the formation of the complex. (D)Ca2+-dependent ATPase activity of the SERCA/PLN complex in PC/PE/PA lipid vesicles at lipid:PLN:SERCA molar ratios of 700:1:1 and 700:5:1. equilibrium has been detected in both synthetic and natural SR T and R states simultaneously but requires selective 13C labeling lipid membranes (22) and persists in the presence of SERCA to eliminate the intense signals arising from residues of domains (15, 25). Phosphorylation of PLN at Ser-16 shifts the equilib- Ib and II (SI Appendix, Fig. S1) (22). rium, inducing an order-to-disorder transition and increasing To map the conformational equilibrium in lipid membranes, the R state population (15, 18, 26, 27). To date, however, PLN’s we coreconstituted SERCA with 13C labeled PLN. To eliminate conformational equilibrium and its different structural states have the pentamer/monomer equilibrium, we used a functional mo- never been correlated to SERCA’sregulation. nomeric variant of PLN, PLNAFA,carryingC36A,C41F,and Here, we used magic angle spinning (MAS) solid-state NMR C46A mutations (29). The SERCA/PLNAFA complex was then co- spectroscopy in combination with different isotopic and spin-la- reconstituted in phosphatidylcholine/phosphatidylethanolamine/ beling schemes to elucidate the role of PLN’s conformational phosphatidic acid (PC/PE/PA) lipid vesicles (8:1:1 molar ratio) equilibrium and phosphorylation in the context of the allosteric at a 150:1:1 lipid:SERCA:PLNAFA molar ratio to mimic the SR regulation of SERCA. We found that in the presence of SERCA, membrane composition (30). Under these experimental con- the TM domain of PLN remains anchored to the ATPase, whereas ditions, SERCA and PLNAFA form a fully functional complex the cytoplasmic domain Ia populates three conformational states: (Fig. 1 C and D), with their cytoplasmic domains facing mostly T, R, and SERCA-bound (B). The inhibitory T state is in equi- the outside of the lipid vesicles (31, 32). On binding SERCA, librium with both the excited R state and a sparsely populated B the Cα chemical shifts of the residues of PLNAFA domains II + state, which is noninhibitory and maintains the Ca2 flux within and Ib are virtually superimposable on those of the free state, a physiological window. Phosphorylation at Ser-16 shifts the with a chemical shift index (CSI) indicative of a helical confor- equilibrium toward the B state, reversing PLN inhibitory action.