Phospholamban structural dynamics in lipid bilayers probed by a spin label rigidly coupled to the peptide backbone

Abstract

We have used chemical synthesis and electron paramagnetic resonance to probe the structural dynamics of phospholamban (PLB) in lipid bilayers. Derivatives of monomeric PLB were synthesized, each of which contained a single spin-labeled 2,2,6,6,-Tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid amino acid, with the nitroxide-containing ring covalently and rigidly attached to the alpha-carbon, providing direct insight into the conformational dynamics of the peptide backbone. 2,2,6,6,-tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid was attached at positions 0, 11, and 24 in the cytoplasmic domain or at position 46 in the transmembrane domain. The electron paramagnetic resonance spectrum of the transmembrane domain site (position 46) indicates a single spectral component corresponding to strong immobilization of the probe, consistent with the presence of a stable and highly ordered transmembrane helix. In contrast, each of the three cytoplasmic domain probes has two clearly resolved spectral components (conformational states), one of which indicates nearly isotropic nanosecond dynamic disorder. For the probe at position 11, an N-terminal lipid anchor shifts the equilibrium toward the restricted component, whereas Mg(2+) shifts it in the opposite direction. Relaxation enhancement, due to Ni(2+) ions chelated to lipid head-groups, provides further information about the membrane topology of PLB, allowing us to confirm and refine a structural model based on previous NMR data. We conclude that the cytoplasmic domain of PLB is in a dynamic equilibrium between an ordered conformation, which is in direct contact with the membrane surface, and a dynamically disordered form, which is detached from the membrane and poised to interact with its regulatory target.

Fig. 1.

Structural model of AFA-PLB, showing sites of TOAC spin labeling. Average structure was determined from NMR of AFA-PLB in detergent micelles (22). Dashed lines show approximate membrane surfaces.

Fig. 2.

Amino acid sequence of AFA-PLB (A), 0-TOAC-AFA-PLB (B), 11-TOAC-AFA-PLB (C), 24-TOAC-AFA-PLB (D), and 46-TOAC-AFA-PLB (E). The four proposed domains are indicated by shading. τ, TOAC.

Fig. 3.

CD spectra of AFA-PLB (—) and its TOAC derivatives in 10 mM Tris/0.2% octaethylene glycol monododecyl ether (C12E8), pH 7.0. CD spectra were recorded on a Jasco (Easton, MD) J-710 spectrophotometer at 25°C and analyzed as previously reported (21). Spectra are plotted as mean residue ellipticity, [θ].

Fig. 4.

Calcium-dependent ATPase activity at 25°C. The Ca-ATPase was reconstituted in the absence (○) and in the presence (•) of AFA-PLB alone and in the presence of AFA-PLB containing TOAC at positions 0 (★) (A), 11 (▪) (B), 24 (□)(C), and 46 (▿)(D). Data sets were fitbyEq. 1 and plotted as V/V_max. Each point represents the mean (n ≥ 6), and SEM was smaller than the plotted symbol.

Fig. 5.

EPR spectra of TOAC-labeled PLB, labeled at the indicated position (0, 11, 24, and 46) on AFA-PLB, incorporated into lipid bilayers. Spectra, obtained with a 120-G scan width at 4°C, were normalized to unit concentration by dividing by the double integral.

Fig. 6.

EPR spectra of membranes containing 11-TOAC-AFA-PLB (middle trace), as affected by an N-terminal lipid anchor (top trace) or by 20 mM Mg²⁺ (bottom trace). Spectra were acquired and plotted as in Fig. 5, except that the vertical scale was expanded by a factor of 2.

Fig. 7.

Accessibility of TOAC, at the indicated position (0, 11, 24, and 46) in AFA-PLB, to the membrane surface. ΔP_1/2 is the increase in P_1/2, determined from Eq. 3, due to surface-bound Ni²⁺ ions.

Fig. 8.

EPR spectra in lipid bilayers of AFA-PLB labeled at position 11 with Cys-directed methanethiosulfonate spin label (MTSSL) (Upper) or TOAC (Lower). Spectra were acquired and plotted as in Fig. 5.

Fig. 9.

Model of PLB topology and structural dynamics in the membrane, based on the NMR structure of AFA-PLB in detergent micelles (22) and on EPR of the four TOAC derivatives in the present study. Membrane head-group layers (10 Å thick) and the hydrocarbon layer (30 Å thick) are indicated schematically. (A) NMR solution structure of the ordered conformation, colored spectrally to indicate backbone dynamics, increasing from blue to green to yellow to red. (B) Two-state model supported by the EPR spectra (see Figs. 5 and 6), in which the ordered conformer (Left) is in dynamic equilibrium with a more dynamic conformer (Right).