Spectroscopic validation of the pentameric structure of phospholamban

Abstract

Phospholamban (PLN) regulates calcium translocation within cardiac myocytes by shifting sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) affinity for calcium. Although the monomeric form of PLN (6 kDa) is the principal inhibitory species, recent evidence suggests that the PLN pentamer (30 kDa) also is able to bind SERCA. To date, several membrane architectures of the pentamer have been proposed, with different topological orientations for the cytoplasmic domain: (i) extended from the bilayer normal by 50-60 degrees; (ii) continuous alpha-helix tilted 28 degrees relative to the bilayer normal; (iii) pinwheel geometry, with the cytoplasmic helix perpendicular to the bilayer normal and in contact with the surface of the bilayer; and (iv) bellflower structure, in which the cytoplasmic domain helix makes approximately 20 degrees angle with respect to the membrane bilayer normal. Using a variety of cell membrane mimicking systems (i.e., lipid vesicles, oriented lipid bilayers, and detergent micelles) and a combination of multidimensional solution/solid-state NMR and EPR spectroscopies, we tested the different structural models. We conclude that the pinwheel topology is the predominant conformation of pentameric PLN, with the cytoplasmic domain interacting with the membrane surface. We propose that the interaction with the bilayer precedes SERCA binding and may mediate the interactions with other proteins such as protein kinase A and protein phosphatase 1.

Fig. 1.

Structural models of wt-PLN. The extended helix/sheet and continuous helix models shown were reconstructed from the original papers (7, 8) to give the reader a graphical illustration of the models. Graphics were prepared by using Pymol software (www.pymol.org). The pinwheel [1XNU (10)] and bellflower [1ZLL (2)] pentamers were taken directly from PDB coordinates.

Fig. 2.

Solid-state NMR spectra of PLN pentamer in lipid bilayers. (A) 1D cross-polarization spectrum of [U-¹⁵N] wt-PLN in DOPC/DOPE oriented lipid bilayers. (B and C) An overlay of selectively labeled PISEMA spectra for the transmembrane and cytoplasmic helices, respectively. The resonances are color-coded: [¹⁵N-Ala], green; [¹⁵N-Cys], purple; [¹⁵N-Leu], orange; [¹⁵N-Ile], red; [¹⁵N-Asn], gray; and [¹⁵N-Thr], blue. (D and E) Simulated PISA wheels (dashed lines) for both transmembrane (θ = 15°) and cytoplasmic (θ = 92°) domains. The PISEMA simulations assumed a regular α-helical geometry for both helical domains.

Fig. 3.

Simulated PISEMA spectra obtained for the pinwheel and bellflower models. The simulations for the cytoplasmic domain (A and B) assumed ideal α-helices with helix tilt angles of 92° (A) and 20° (B) with a rotation angle (defined in ref. 18) of 15°. PISEMA spectra for the transmembrane domains (tm) are calculated directly from the PDB coordinates (D and E). Unlike the pinwheel model, the bellflower model does not show any high-field resonances. Experimental PISEMA spectra show the remarkable agreement with the pinwheel model (C and F).

Fig. 4.

Solution NMR studies of PLN pentamer in DPC micelles. (A) Overlay of [¹H/¹⁵N]-TROSY-HSQC spectra of AFA-PLN monomer (black) and wt-PLN pentamer (red). (B) Difference plot of the combined ¹H and ¹⁵N chemical-shift variations between the monomeric and pentameric species (Δδ = [(Δδ_H² + Δδ_N²/25)/2)]^1/2). The three mutations in AFA-PLN (C36A, C41F, and C46A) are indicated with asterisks.

Fig. 5.

Paramagnetic mapping of PLN topology in DPC micelles. Intensity retention upon addition of Gd³⁺ (A), 5′-doxyl stearic acid (B), and 16′-doxyl stearic acid (C). The red bars in B and C highlight those residues quenched that face the micelle interior (Val-4, Leu-7, and Ala-11 in B and Leu-7 in C). The asterisks indicate overlapped resonances.

Fig. 6.

Structural models (A, pinwheel; B, bellflower) showing the sites of spin-label attachment (Lys-3) and the predicted interprotomer distances, measured from β-carbons. Pulsed EPR (DEER) decays (black squares) observed for wt-PLN, spin-labeled at Lys-3, in DPC micelles (C) and lipid bilayers (D). Solid curves in C and D show simulated decays predicted by the bellflower model (red), pinwheel model (blue), and the best-fit Gaussian distribution of distances (green).

Fig. 7.

EPR dynamics (A and B) and Ni²⁺ accessibility (C–E) data comparing AFA-PLN (data shown in blue) with wt-PLN (data shown in red). EPR spectra were obtained from membrane-reconstituted PLN with TEMPO-succinimide (SUCSL) attached to Lys-3 (A and C) or with TOAC substituted for Ala-11 (B and D). In B, the two low-field peaks correspond to the resolved T and R states. C and D are progressive saturation curves, showing the enhanced relaxation caused by collisions with membrane-surface-bound Ni²⁺. Relative accessibilities derived from C and D are shown in E.