The structural topology of wild‐type phospholamban in oriented lipid bilayers using ¹⁵N solid‐state NMR spectroscopy

Abstract

For the first time, 15 N solid-state NMR experiments were conducted on wild-type phospholamban (WT-PLB) embedded inside mechanically oriented phospholipid bilayers to investigate the topology of its cytoplasmic and transmembrane domains.15 N solid-state NMR spectra of site-specific 15 N-labeled WT-PLB indicate that the transmembrane domain has a tilt angle of 13°6 6°with respect to the POPC (1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine) bilayer normal and that the cytoplasmic domain of WT-PLB lies on the surface of the phospholipid bilayers. Comparable results were obtained from sitespecific 15 N-labeled WT-PLB embedded inside DOPC/DOPE (1,2-dioleoyl-sn-glycero-3-phosphocholine/ 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) mechanically oriented phospholipids' bilayers. The new NMR data support a pinwheel geometry of WT-PLB, but disagree with a bellflower structure in micelles, and indicate that the orientation of the cytoplasmic domain of the WT-PLB is similar to that reported for the monomeric AFA-PLB mutant.

Phospholamban Oligomerization, Quaternary Structure, and Sarco(endo)plasmic Reticulum Calcium ATPase Binding Measured by Fluorescence Resonance Energy Transfer in Living Cells

“…6). Several new NMR studies of PLB in phospholipid bilayers provide evidence that PLB cytoplasmic domains are membrane-parallel (10,14) and in contact with the bilayer surface (15,16). Those studies are compatible with the pinwheel model of PLB pentamer structure, which was developed from the NMR solution structure of monomeric PLB in DPC micelles (7) in combination with FRET-based distance constraints from in-gel anisotropy of PLB in SDS detergent (13).…”

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach

“…Ser-16 and Thr-17 (the 2 phosphorylatable sites) are exposed to the bulk solvent, prone to interact with their respective kinases (i.e., protein kinase A and CamKII kinase, respectively). The amphipathic interactions of domain Ia with the lipid bilayer are also present in the oligomeric state of PLN, as measured by solution NMR in detergent micelles (14,50), solid-state NMR in lipid bilayers (13,52), EPR saturation transfer experiments in lipid bilayers and detergent micelles (8,12,13), and f luorescence resonance energy transfer in intact cells (46) and detergent reconstituted systems (53).…”

Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method

“…Although micelle studies give useful insights into the topology of PLN, they may not reproduce the architecture of this protein in cell membranes (25). A more rigorous approach is to supplement micelle studies with solidstate NMR techniques in lipid bilayers (26)(27)(28)(29)(30). Therefore, we carried out solid-state NMR experiments in mechanically aligned lipid bilayers (31,53,54).…”

“…PLN is a 52-residue transmembrane (TM) protein highly conserved across mammals (2). Its helix-loop-helix secondary structure is further subdivided into four dynamic domains: domain Ia (1-16), loop (17)(18)(19)(20)(21)(22), domain Ib (23)(24)(25)(26)(27)(28)(29)(30), and domain II (3,4). The hydrophobic TM domain II is the most conserved and responsible for SERCA inhibition, whereas the cytoplasmic domain harbors two phosphorylation sites that reverse PLN inhibitory function (2).…”

“…In its T state, PLN adopts a pinwheel topology with the cytoplasmic helices making extensive contacts with the lipid bilayer surface. The TM helices traverse the membrane at an angle of approximately 11°with respect to the bilayer normal and have a narrow hydrophobic pore (average radius approximately 2 Å), which tapers to approximately 1 Å at the juxtamembrane region (residues [23][24][25][26][27][28][29][30].…”