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

Abstract

Phospholamban (PLN) is a type II membrane protein that inhibits the sarcoplasmic reticulum Ca 2þ -ATPase (SERCA), thereby regulating calcium homeostasis in cardiac muscle. In membranes, PLN forms pentamers that have been proposed to function either as a storage for active monomers or as ion channels. Here, we report the T-state structure of pentameric PLN solved by a hybrid solution and solid-state NMR method. In lipid bilayers, PLN adopts a pinwheel topology with a narrow hydrophobic pore, which excludes ion transport. In the T state, the cytoplasmic amphipathic helices (domains Ia) are absorbed into the lipid bilayer with the transmembrane domains arranged in a left-handed coiled-coil configuration, crossing the bilayer with a tilt angle of approximately 11°with respect to the membrane normal. The tilt angle difference between the monomer and pentamer is approximately 13°, showing that intramembrane helix-helix association forces dominate over the hydrophobic mismatch, driving the overall topology of the transmembrane assembly. Our data reveal that both topology and function of PLN are shaped by the interactions with lipids, which fine-tune the regulation of SERCA.hybrid NMR method | PISEMA | calcium regulation | oligomeric protein | dipolar assisted rotational resonance recoupling T he membrane protein complex formed by Ca 2þ -ATPase (SERCA) and phospholamban (PLN) regulates Ca 2þ concentration within the sarcoplasmic reticulum (SR), thereby controlling muscle excitation-contraction coupling (1, 2). 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), and domain II (31-52) (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). PLN has a direct role in the pathophysiology of the heart muscle, with three lethal mutations linked to dilated cardiomyopathy in humans (R9C-PLN, R14del, and L39-truncated-PLN) (5). In both synthetic and cell membranes, PLN forms pentamers that dissociate into monomers upon interacting with SERCA (1, 6). Although the stoichiometry of the SERCA/PLN complex has been assessed (1, 6), both the role and the structure of the PLN pentamer remain a matter of active debate. Because PLN expression in both atria and ventricles is higher than SERCA, it is likely that oligomerization may participate in SERCA regulation (7). Insights into PLN organization in the membrane have come from biochemical and biophysical data (2,6,8). Initial electrophysiological measurements indicated that PLN formed Ca 2þ channels (9). However, more recent electrochemical studies concluded that PLN does not conduct Cl − or Ca 2þ ions (10).Divergent structural models for the PLN pentamer have been proposed in the literature (8). Although very similar in the secondary structure content, these models differ in t...

Structure of CrgA, a cell division structural and regulatory protein fromMycobacterium tuberculosis, in lipid bilayers

“…The use of such bilayer preparations for supporting the native-like conformation of the M2 protein from Influenza A has been validated with the comparison of spectra from synthetic bilayers and from cellular membranes where the protein has been inserted by the cellular machinery and never removed from this environment or exposed to a detergent environment (28). Multiple recent membrane protein structures have now been determined by OS ssNMR (29)(30)(31)(32)(33)(34), and the first membrane protein structure has been obtained from MAS ssNMR (35). OS ssNMR generates information on the orientations of peptide planes with respect to the bilayer normal, and for a TM helix it yields the tilt angle of the helix relative to the lipid bilayer normal and rotational orientation about the helical axis along the entire length of helix.…”

Allosteric regulation of SERCA by phosphorylation-mediated conformational shift of phospholamban

“…On average, the L-shaped T state is the most populated in either monomeric or pentameric PLN ( Fig. 1) (21,23), with the R state accounting for 16-20% of the conformational ensemble (24). Remarkably, PLN's conformational…”

Phospholamban C-terminal Residues Are Critical Determinants of the Structure and Function of the Calcium ATPase Regulatory Complex

“…PLB is a homopentameric, integral sarcoplasmic reticulum membrane protein that upon deoligomerization into active monomers reversibly inhibits sarco/ endoplasmic reticulum calcium ATPase (SERCA) (13)(14)(15), thereby directly regulating cardiac Ca 2ϩ kinetics and contractility (16 -21). PLB has a "helix-loop-helix" tertiary structure consisting of the N-terminal cytosolic domain IA (residues 1-16), flexible linker (residues [17][18][19][20][21][22], domain IB (residues [23][24][25][26][27][28][29][30], and C-terminal transmembrane (TM) domain II (residues 31-52) (22,23). The C-terminal TM domain is highly conserved among species (13) and may be important for PLB oligomerization (24,25) and SERCA regulation (26).…”