Probing ground and excited states of phospholamban in model and native lipid membranes by magic angle spinning NMR spectroscopy

Abstract

In this paper, we analyzed the ground and excited states of phospholamban (PLN), a membrane protein that regulates sarcoplasmic reticulum calcium ATPase (SERCA), in different membrane mimetic environments. Previously, we proposed that the conformational equilibria of PLN are central to SERCA regulation. Here, we show that these equilibria detected in micelles and bicelles are also present in native sarcoplasmic reticulum lipid membranes as probed by MAS solid-state NMR. Importantly, we found that the kinetics of conformational exchange and the extent of ground and excited states in detergent micelles and lipid bilayers are different, revealing a possible role of the membrane composition on the allosteric regulation of SERCA. Since the extent of excited states is directly correlated to SERCA inhibition, these findings open up the exciting possibility that calcium transport in the heart can be controlled by the lipid bilayer composition. This article is part of a Special Issue entitled: Membrane protein structure and function.

Figure 1

Conformational equilibria and primary sequence. A.) Conformational equilibria of monomeric PLN. The T state is represented by its high resolution structure (PDB ID: 1N7L)[16]. B.) Primary sequence of AFA-PLN.

Figure 2

Assignment of domain Ib and II in lipids and comparison with DPC micelles. A.) SQ-DQ correlation experiments of AFA-PLN reconstituted into egg PC/PE/PA 8/1/1. B.) ¹³C_α-¹³C_β CSI for residues in domain Ib and II of AFA-PLN in DPC micelles and egg lipids. CSI was calculated as δ_13Cα – δ_13Cβ – (δ_13Cα,RC – δ_13Cβ,RC), where RC stands for the random coil chemical shifts [43]. C.) Correlation between CSI values in DPC micelles and egg lipids. Residues in domain Ib are shown as open circles and residues in domain II as filled circles.

Figure 3

PLN in isotropic bicelles. A.) [¹⁵N]-HSQC of AFA-PLN in DMPC/DHPC isotropic bicelles with q=0.33. Peak corresponding to residues S10, S16 and T17 (marked with x) were detected at a lower noise level as shown in the inset. B.) Helicity of domain Ia in different membrane mimics. ¹³C_α – ¹³C_β CSI for domain Ia of AFA-PLN in DPC micelles (black), DMPC/DHPC isotropic bicelles (red) and a peptide corresponding to residues 1–20 of PLN[48] (PLN_1-20, orange).

Figure 4

Negatively charged lipids shifts the equilibrium towards the T state. A–D.) ¹³C HSQC experiments of AFA-PLN in DPC micelles (black, T state), DMPC/DHPC, q=0.33, isotropic bicelles (grey) DMPC/DMPG/DHPC (4:1 DMPC:DMPG) isotropic bicelles (green) and PLN_1-20 (orange, R state). E.) CO CSI for AFA-PLN in different membrane mimics.

Figure 5

CP-transfers detect chemical shifts that are absent from INEPT-based experiments. Comparison of rINEPT (top) and DARR (bottom, 200ms mixing time) experiments of AFA-PLN_6cyt reconstituted into d₅₄-DMPC MLVs at 30°C. Dashed lines highlight examples of peaks that are detected in both experiments.

Figure 6

Conformational equilibrium of AFA-PLN in DMPC at different temperatures. A.) Cross-peaks from DARR spectra corresponding to a C_α-C_γ correlation for Val4 (left) and C_α-C_δ2 correlation for Leu7 (right) at 30°C (top) and −25°C (bottom). Blue and red dashed lines highlight the chemical shift regions of the folded T state and unfolded R state respectively. B) Cα-Cβ CSI of the folded T state (measured from experiments at −25°C, blue bars) and unfolded R state (measured from experiments at 30°C, red bars) compared to DPC micelles (black dotted lines, predominantly T state) and PLN_1-20 (orange dotted line, predominantly R state).

Figure 7

The conformational equilibrium of AFA-PLN is conserved in native SR lipids. A.) Overlay of DARR experiments of AFA-PLN_6cyt reconstituted into DMPC (black) or extracted SR lipids (purple). Experiments in SR lipids were acquired at −25°C and 20°C and experiments DMPC were acquired at −25°C and 30°C. All experiments utilized a 200ms mixing time. B.) ¹³C_α – ¹³C_β CSI of the R and T state components in DMPC (black) and SR lipids (purple).

Figure 8

The T state is inserted into the lipid bilayer. A.) Region from DARR spectra of AFA-PLN_6cyt in SR lipids acquired in the presence (turquoise) and absence (black) of 20 mM Gd³⁺. B.) Signal retention of peaks corresponding to the T and R states quantified from DARR spectra acquired in the presence and absence of 20 mM Gd³⁺. Signal retention was calculated as I_Gd3+/I₀. in DARR spectra quenching of T and R states of AFA-PLN. For Ala15 and Ile18 the T and R state peaks are overlapped and therefore the signal retention reports on a combination of T and R states.