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

Abstract

Phospholamban (PLB) is a membrane protein that regulates heart muscle relaxation rates via interactions with the sarcoplasmic reticulum Ca2+ ATPase (SERCA). When PLB is phosphorylated or Arg9Cys (R9C) is mutated, inhibition of SERCA is relieved. 13C and 15N solid-state NMR spectroscopy is utilized to investigate conformational changes of PLB upon phosphorylation and R9C mutation. 13C=O NMR spectra of the cytoplasmic domain reveal two α-helical structural components with population changes upon phosphorylation and R9C mutation. The appearance of an unstructured component is observed on domain Ib. 15N NMR spectra indicate an increase in backbone dynamics of the cytoplasmic domain. Wild-type PLB (WT-PLB), Ser16-phosphorylated PLB (P-PLB), and R9C-mutated PLB (R9C-PLB) all have a very dynamic domain Ib, and the transmembrane domain has an immobile component. 15N NMR spectra indicate that the cytoplasmic domain of R9C-PLB adopts an orientation similar to P-PLB and shifts away from the membrane surface. Domain Ib (Leu28) of P-PLB and R9C-PLB loses the alignment. The R9C-PLB adopts a conformation similar to P-PLB with a population shift to a more extended and disordered state. The NMR data suggest the more extended and disordered forms of PLB may relate to inhibition relief.

Acute Inotropic and Lusitropic Effects of Cardiomyopathic R9C Mutation of Phospholamban

“…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).…”

“…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.…”

Phospholamban phosphorylation, mutation, and structural dynamics: a biophysical approach to understanding and treating cardiomyopathy

“…13 C solid-state NMR studies have shown that it is a hydrophobic α-helix that is aligned with domain II (Yu and Lorigan 2014). Upon S16 phosphorylation, domain Ib changes its structure from an α-helix to an uncoiled coil and loses its alignment with domain II (Yu and Lorigan 2014).…”

“…13 C solid-state NMR studies have shown that it is a hydrophobic α-helix that is aligned with domain II (Yu and Lorigan 2014). Upon S16 phosphorylation, domain Ib changes its structure from an α-helix to an uncoiled coil and loses its alignment with domain II (Yu and Lorigan 2014). Alanine scanning of a peptide containing PLB residues 21-30 revealed that domain Ib residues N27 (which is K27 in humans), and N30 were also important for SERCA2a binding (Asahi et al 2001).…”

“…However, glycine mutations along domain Ib may be able to mimic the phosphorylation-induced unwinding of its α-helix and misalignment with domain II (Yu and Lorigan 2014). …”