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

Abstract

Phospholamban (PLN) is an essential regulator of cardiac muscle contractility. The homopentameric assembly of PLN is the reservoir for active monomers that, upon deoligomerization form 1:1 complexes with the sarco(endo)plasmic reticulum Ca 2؉ -ATPase (SERCA), thus modulating the rate of calcium uptake. In lipid bilayers and micelles, monomeric PLN exists in equilibrium between a bent (or resting) T state and a more dynamic (or active) R state. Here, we report the high-resolution structure and topology of the T state of a monomeric PLN mutant in lipid bilayers, using a hybrid of solution and solid-state NMR restraints together with molecular dynamics simulations in explicit lipid environments. Unlike the previous structural ensemble determined in micelles, this approach gives a complete picture of the PLN monomer structure in a lipid bilayer. This hybrid ensemble exemplifies the tilt, rotation, and depth of membrane insertion, revealing the interaction with the lipids for all protein domains. The N-terminal amphipathic helical domain Ia (residues 1-16) rests on the surface of the lipid membrane with the hydrophobic face of domain Ia embedded in the membrane bilayer interior. The helix comprised of domain Ib (residues 23-30) and transmembrane domain II (residues 31-52) traverses the bilayer with a tilt angle of Ϸ24°. The specific interactions between PLN and lipid membranes may represent an additional regulatory element of its inhibitory function. We propose this hybrid method for the simultaneous determination of structure and topology for membrane proteins with compact folds or proteins whose spatial arrangement is dictated by their specific interactions with lipid bilayers.hybrid method ͉ membrane proteins ͉ oriented solid-state NMR ͉ molecular modeling ͉ PISEMA S tructure and topology are central to membrane protein function (1). Recently determined high-resolution structures reveal the compact folds for several membrane proteins, such as electron and proton-conducting proteins involved in photosynthesis and respiration (http://blanco.biomol.uci.edu/ Membrane Proteins xtal.html). However, a significant population of membrane proteins does not possess a compact tertiary fold, but has its fold space defined through interactions of secondary structure elements (helices, turns, and loops) with the lipid membrane, i.e., the topology (1). This is the case for phospholamban (PLN), a mammalian protein that is essential in the regulation of cardiac muscle contractility (2), and that has recently become a major target for gene therapy to ameliorate cardiomyopathies (3, 4). PLN is located in the sarco(endo)plasmic reticulum (SR) of cardiac myocytes, inhibiting the SR Ca 2ϩ -ATPase (SERCA) by shifting its relative Ca 2ϩ affinity (5). In vitro and in vivo experiments have shown PLN to exist as a homopentamer that deoligomerizes into active monomers that bind SERCA in a 1:1 molar ratio (6). The monomeric form of PLN exists in equilibrium between a dynamically disordered R state and a more restricted T state (7,8) and has 3 ...

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

“…Overall, the structure of each protomer within the pentameric assembly of PLN is similar to that of the monomeric PLN (AFA-PLN, 2KB7) (16). However, the two forms differ noticeably in the membrane topology (Fig.…”

“…1E shows the overlay of several oriented solid-state NMR spectra located in two distinct regions: (i) resonances clustered at 200 ppm that correspond to the TM domains and (ii) resonances at 65-100 ppm, assigned to the cytoplasmic domain Ia. We measured a total of 29 DC and 31 CSA values (Table S2) and converted them into restraints for the hybrid structure calculations, where both solution and solid-state NMR restraints are implemented into the target energy function (15,16).…”

“…The overall strategy for the hybrid NMR method consists of combining secondary structure information from solution NMR with orientational restraints obtained from solid-state NMR in lipid membranes (15,16). Here, we selected micelle and lipid conditions where PLN predominantly adopts a T-state conformation as probed by EPR and NMR (17)(18)(19) (Fig.…”

“…In this article, we present the high-resolution structure and topology of the PLN pentamer in its T state (14) as determined by a hybrid solution and solid-state NMR method (15,16). In its T state, PLN adopts a pinwheel topology with the cytoplasmic helices making extensive contacts with the lipid bilayer surface.…”

Proton evolved local field solid-state nuclear magnetic resonance using Hadamard encoding: Theory and application to membrane proteins

“…10 SLF experiments have been widely used by chemists and biophysicists to characterize the structures of liquid crystals as well as membrane proteins in mechanically or magnetically aligned lipid bilayers. [11][12][13][14][15][16][17][18] Since the introduction of the SLF experiment, several variants have emerged, such as PISEMA, [19][20][21] SAMPI4, 22 HIMSELF, 23 and more recently the sensitivity-enhanced and constant time versions. [24][25][26][27] These experiments are also known as rotating frame SLF (R-SLF) experiments and take advantage of the heteronuclear DC evolution under Hartmann-Hahn matching of RF fields for I and S spins and homonuclear decoupling for the abundant I spins.…”