Probing the interaction of Arg9Cys mutated phospholamban with phospholipid bilayers by solid-state NMR spectroscopy

Abstract

Phospholamban (PLB) is a 52 amino acid integral membrane protein that interacts with the sarcoplasmic reticulum Ca2+ ATPase (SERCA) and helps to regulate Ca2+ flow. PLB inhibits SERCA impairing Ca2+ translocation. The inhibition can be relieved upon phosphorylation of PLB. The Arg9 to Cys (R9C) mutation is a loss of function mutation with reduced inhibitory potency. The effect R9C PLB has on the membrane surface and the hydrophobic region dynamics was investigated by 31P and 2H solid-state NMR spectroscopy in multilamellar vesicles (MLVs). The 31P NMR spectra indicate that, like the phosphorylated PLB (P-PLB), the mutated R9C-PLB protein has significantly less interaction with the lipid bilayer headgroup when compared to wild-type PLB (WT-PLB). Similar to P-PLB, R9C-PLB slightly decreases 31P T1 values in the lipid headgroup region. 2H SCD order parameters of 2H nuclei along the lipid acyl chain decrease less dramatically for R9C-PLB and P-PLB when compared to WT-PLB. The results suggest that R9C-PLB interacts less with the membrane surface and hydrophobic region than WT-PLB. Detachment of the cytoplasmic domain of R9C-PLB from the membrane surface could be related to its loss of function.

Secondary Structure, Backbone Dynamics, and Structural Topology of Phospholamban and Its Phosphorylated and Arg9Cys-Mutated Forms in Phospholipid Bilayers Utilizing 13C and 15N Solid-State NMR Spectroscopy

“… 49 Both P-PLB and R9C-PLB have significantly less interaction with the membrane. 26 , 49 Therefore, interaction with lipids may be involved in regulating PLB function. WT-PLB is dominated by the pentamer form.…”

“…However, 2 H NMR spectra investigating lipid acyl chain dynamics do not show any effect upon R9C-PLB or P-PLB addition. 26 More likely, the secondary structure change occurs upon R9C mutation or phosphorylation. Unwinding is observed at Ala24 in domain Ib, suggesting a population shift from the dominant folded state toward an unfolded state.…”

“…It is believed that the membrane is not only acting as an environment to stabilize or localize SERCA and PLB but is also involved actively in regulating function. 13 , 23 , 26 …”

Acute Inotropic and Lusitropic Effects of Cardiomyopathic R9C Mutation of Phospholamban

“…A, schematic representation of primary sequence of WT-PLB and the mutant PLB constructs. The amino acid sequence of the PLB protomer shows the N-terminal cytosolic domain Ia (residues 1-16), flexible linker (residues 17-22), domain Ib (residues [23][24][25][26][27][28][29][30], and the C-terminal transmembrane domain II (residues 31-52). Cer or YFP was fused to the N terminus.…”

“…Proposed mechanisms include trapping of PKA (8), disruption of PLB phosphorylation (8,9,20,21), and loss of PLB inhibitory function (8, 9, 20 -22). Other studies have suggested that the R9C mutation mimics PLB phosphorylation by partial unfolding of the cytoplasmic helix resulting in decreased helical conformation (23) or detachment of PLB cytoplasmic domain from the membrane surface (24,25). Moreover, we have previ-ously proposed that the R9C mutation induces oxidation-dependent cross-linking of adjacent R9C-PLB protomers (20).…”