Phospholamban and Its Phosphorylated Form Interact Differently with Lipid Bilayers: A31P,2H, and13C Solid-State NMR Spectroscopic Study

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Abstract
Phospholamban (PLB) is a 52-amino acid integral membrane protein that helps to regulate the flow of Ca2+ 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 α-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) Biochemistry42, 10674−10682; Metcalfe, E. E. (2005) Biochemistry44, 4386−4396: Clayton, J. C. (2005) Biochemistry44, 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 2H and 31P 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 13CO 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 α-helical structure for both PLB and P-PLB.

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