Allosteric regulation of SERCA by phosphorylation-mediated conformational shift of phospholamban

Abstract

The membrane protein complex between the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and phospholamban (PLN) controls Ca(2+) transport in cardiomyocytes, thereby modulating cardiac contractility. β-Adrenergic-stimulated phosphorylation of PLN at Ser-16 enhances SERCA activity via an unknown mechanism. Using solid-state nuclear magnetic resonance spectroscopy, we mapped the physical interactions between SERCA and both unphosphorylated and phosphorylated PLN in membrane bilayers. We found that the allosteric regulation of SERCA depends on the conformational equilibrium of PLN, whose cytoplasmic regulatory domain interconverts between three different states: a ground T state (helical and membrane associated), an excited R state (unfolded and membrane detached), and a B state (extended and enzyme-bound), which is noninhibitory. Phosphorylation at Ser-16 of PLN shifts the populations toward the B state, increasing SERCA activity. We conclude that PLN's conformational equilibrium is central to maintain SERCA's apparent Ca(2+) affinity within a physiological window. This model represents a paradigm shift in our understanding of SERCA regulation by posttranslational phosphorylation and suggests strategies for designing innovative therapeutic approaches to enhance cardiac muscle contractility.

Keywords: magic angle spinning; paramagnetic relaxation enhancement; protein-protein interactions; solid-state NMR.

Fig. 1.

Structures of PLN and SERCA. (A) Primary sequence and domains of PLN^AFA. 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], PLN^AFA 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 SERCA^AEDANS (donor) and PLN^Dab-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.

Fig. 2.

NMR mapping of PLN^AFA changes on SERCA binding. (A) CSI (δC_α-δC_β) of PLN^AFA in complex with SERCA obtained from [¹³C,¹³C]-DARR and single quantum-double quantum (SQ-DQ) correlation experiments at −20 °C, 4 °C, and 20 °C in 8:1:1 PC/PE/PA bilayers . The CSI was calculated as δ_13Cα−δ_13Cβ−(δ_13Cα,RC−δ_13Cβ,RC), where RC stands for the random coil chemical shifts. (B) Cα chemical shift changes on SERCA binding. Chemical shift perturbations (CSPs) are calculated as the difference in the chemical shifts with and without SERCA (δ_T,+SERCA - δ_T-SERCA for the T state; δ_B,+SERCA - δ_R,-SERCA for the B state). The peaks corresponding to E2, S16, T17, I18, and E19 were not resolved. (C) Portion of the [¹³C,¹³C]-DARR spectrum showing C_α-C_δ correlations of Leu-7 in the absence (Upper) and presence (Lower) of SERCA. In the absence of SERCA, the T state is in slow exchange with the R state. SERCA binding shifts the populations toward the B state. Shaded areas indicate α-helical (green) and coil (gray) chemical shift ranges. (D) CSP of the TM methyl groups. (E) TM region of the molecular model by Toyoshima et al. (9), with PLN residues with large CSP highlighted.

Fig. 3.

Mapping protein–protein interactions using PREs effects from SERCA^C674-sl to PLN^AFA. (A) Selected region of the [¹³C,¹³C]-DARR spectra (4 °C) of ¹³C,¹⁵N-labeled PLN^AFA at Val-4, Leu-7, Ala-11, Ile-12, Ala-15, and Ile-18 (PLN^AFA6cyt) in complex with SERCA^C674-sl before and after addition of DTT. (B) Portion of the [¹³C,¹³C]-DARR spectra showing C_α-C_β and C_α-C_δ correlations for Ala-11 and Leu-7 corresponding to the T and B states. (C) Relative peak intensities (I_B/I_T) for T- and B-state resonances for V4, L7, and A11/A15 peaks in the presence of reduced (black bars) and oxidized (red bars) SERCA^C674-sl, respectively.

Fig. 4.

Mapping the interactions between domain Ia of PLN and SERCA. (A) Methyl region of rINEPT spectra in the presence and absence of SERCA^C674-sl. Lipid peaks are marked with asterisks. (B) Intensity retention plot for residues in domain Ia, as probed by rINEPT experiments. I and I_o are the peak intensities before and after addition of DTT. Error bars reflect the signal-to-noise ratio in the spectra. Resonances broadened beyond detection are marked with asterisks. (C) PRE effects mapped onto PLN structure: affected residues are in red, and unaffected are in cyan. Dashed box highlights resonances broadened beyond detection. The model was obtained by manual unfolding of domain Ia from the Toyoshima complex (9). (D) [¹H,¹³C] heteronuclear single quantum correlation (HSQC) spectrum of ¹³C-MTC-SERCA in dodecylphosphocholine (DPC) micelles. (E) Water-edited CP spectra of ¹³C-MTC-SERCA in the presence of PLN^AFA-sl before and after addition of ascorbate to reduce the spin label. The additional peaks correspond to the natural abundance ¹³C resonances of lipids and proteins.

Fig. 5.

Lipid association for the T state of domain Ia mapped by PREs. (A, Upper) Chemical structure and nomenclature of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); (Lower) PRE effects of SERCA/PLN^AFA-sl complex on the PC/PE/PA lipid resonances. I and I₀ correspond to the peak intensities before and after addition of 10 mM ascorbate, respectively. (B) Representative regions of [¹³C,¹³C]-DARR spectra of V4, L7, and I12 of PLN^AFA in complex with SERCA in the presence of PE^sl lipids before and after addition of 10 mM DTT, showing that the T state of domain Ia is quenched by the spin label on the membrane surface. Experiments were acquired at 4 °C.

Fig. 6.

Effects of Ser-16 phosphorylation of PLN on the conformational equilibrium. (A) Representative region of [¹³C,¹³C]-DARR spectra showing Ala resonances of PLN^AFA and PLN^pS16AFA in complex with SERCA. The DARR spectra were acquired at 20 °C with 200 ms mixing time. (B) Intensity retention of phosphorylated and unphosphorylated PLN in the presence of SERCA^C674-sl. Undetectable peaks in the presence of spin label are marked with asterisks.

Fig. 7.

Allosteric regulatory model of SERCA by PLN. Conformational equilibrium of PLN bound to SERCA with inhibitory T and R states and noninhibitory B state. Phosphorylation shifts the equilibrium toward the noninhibitory B state, whereas deletion of cytoplasmic domain Ia results in an inhibitory effect greater than full-length PLN, which is outside the physiological window of inhibition.