Phospholamban and its phosphorylated form interact differently with lipid bilayers: a 31P, 2H, and 13C solid-state NMR spectroscopic study

Abstract

Phospholamban (PLB) is a 52-amino acid integral membrane protein that helps to regulate the flow of Ca(2+) ions in cardiac muscle cells. Recent structural studies on the PLB pentamer and the functionally active monomer (AFA-PLB) debate whether its cytoplasmic domain, in either the phosphorylated or dephosphorylated states, is alpha-helical in structure as well as whether it associates with the lipid head groups (Oxenoid, K. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 10870-10875; Karim, C. B. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 14437-14442; Andronesi, C.A. (2005) J. Am. Chem. Soc. 127, 12965-12974; Li, J. (2003) Biochemistry 42, 10674-10682; Metcalfe, E. E. (2005) Biochemistry 44, 4386-4396: Clayton, J. C. (2005) Biochemistry 44, 17016-17026). Comparing the secondary structure of the PLB pentamer and its phosphorylated form (P-PLB) as well as their interaction with the lipid bilayer is crucial in order to understand its regulatory function. Therefore, in this study, the full-length wild-type (WT) PLB and P-PLB were incorporated into 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) phospholipid bilayers and studied utilizing solid-state NMR spectroscopy. The analysis of the (2)H and (31)P solid-state NMR data of PLB and P-PLB in POPC multilamellar vesicles (MLVs) indicates that a direct interaction takes place between both proteins and the phospholipid head groups. However, the interaction of P-PLB with POPC bilayers was less significant compared that with PLB. Moreover, the secondary structure using (13)C=O site-specific isotopically labeled Ala15-PLB and Ala15-P-PLB in POPC bilayers suggests that this residue, located in the cytoplasmic domain, is a part of an alpha-helical structure for both PLB and P-PLB.

Figure 1

Proposed structural models of pentameric and monomeric phospholamban in a lipid bilayer. (A). The “L” shape monomeric NMR solution structure of AFA-PLB by the Veglia group (60). (B) The solid-state NMR monomeric structure of AFA-PLB by the Baldus group (57). (C) The NMR solution structure of pentameric PLB by the Chou group (61). This figure was generated using the MOLMOL software and a G5 Apple Mac computer (94). The three-dimensional structures in ((A), (B) and (C)) were obtained from the published reports and the protein data bank.

Figure 2

²H NMR powder-pattern spectra of the POPC bilayers in the absence (A, solid line) and in the presence of 2 mol% (B) and 4 mol% (C) PLB (dashed lines) and P-PLB (dotted lines) with respect to the lipids at 25 °C.

Figure 3

The smoothed acyl chains orientational order S_CD profiles calculated from the dePaked ²H NMR spectra of (Figure 2). The open squares, circles, and triangles (A) represent POPC bilayers with 0 mol%, 2 mol% and 4 mol% P-PLB, respectively. The closed squares, circles and triangles (B) represent POPC bilayers with 0 mol%, 2 mol% and 4 mol% PLB, respectively.

Figure 4

Static ³¹P solid-state NMR spectrum of P-PLB without POPC lipids investigated at 25°C ((A), solid line). Static ³¹P solid-state NMR spectra of POPC phospholipid in the absence ((B), solid line) and in the presence of 2 mol% (C) and 4 mol% (D) P-PLB (dotted lines) and PLB (dashed lines) with respect to the POPC lipids investigated at 25°C.

Figure 5

³¹P spin-lattice relaxation T₁ as a function of temperature (25°C–55°C) of POPC phospholipid bilayers with and without 4 mol% protein with respect to the lipids. The open circles represent a POPC control (without protein) sample. The open triangles and squares represent POPC bilayers with 4 mol% P-PLB and PLB, respectively. The error bars were obtained by averaging ³¹P T₁ values from two different sample sets.

Figure 6

¹³C-CPMAS solid-state NMR spectra of ¹³C=O labeled Leu39-PLB (A), Ala15-PLB (B), and Ala15-P-PLB (C), upon insertion into the POPC bilayers at −25 °C. At the same temperature (−25 °C), the ¹³C-CPMAS solid-state NMR spectrum of the natural abundance ¹³C carbonyl of POPC bilayers (without protein) is shown in (D).