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 approximately 24 degrees . 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.

Fig. 1.

Solid-state NMR spectra of AFA-PLN in lipid bilayers. (A) Primary sequence of AFA-PLN showing assigned residues in gray (CSA and DC assignments in Table S1). (B) Assigned PISEMA (Leu, Ala) and SAMPI4 spectra (Ser, Arg) of domain Ia. (C) [U-¹⁵N] PISEMA spectrum of PLN domain Ib and II displaying a uniform intensity across the PISA wheel. (D) Selectively labeled PLN PISEMA spectra of domain Ib and II. Overlay shown in Fig. S3.

Fig. 2.

Experimental CSA (A) and DC (C) values from Fig. 1 plotted vs. residue number. (B and D) show correlation plots of calculated vs. experimental CSA and DC for the hybrid ensemble, respectively. The parallel lines in B and D indicate the experimental errors used in the structural calculations.

Fig. 3.

Comparison between the solution NMR ensemble and the conformational ensemble generated with the hybrid protocol. (A) Angles used to describe the topology of monomeric AFA-PLN. (B) Rotation (ρ) versus tilt angle (θ) for the 20 structures in the deposited ensemble. The Insets better allow for variations to be seen in the angles describing the topology with respect to the membrane normal for domains Ib and II (Left, circles) and domain Ia (Right, triangles). (C) Comparison of the ensemble of structures generated from solution NMR restraints alone (squares) with the hybrid solution and solid-state NMR method (circles).

Fig. 4.

Structural topology of monomeric PLN in a DOPC lipid bilayer. (A) Probability distribution profile for the chemical groups of DOPC in the molecular dynamics simulation of PLN within the bilayer. (B) Structure of monomeric AFA-PLN in a DOPC lipid bilayer. (C) Detailed images of the residues within the amphipathic domain Ia helix show that the hydrophobic residues (Val-4, Leu-7, Ala-11, Ile-12, Ala-15) face the interior of the bilayer. (D) Structure highlighting the face of PLN within domains Ib and II that have been shown by mutagenesis and cross-linking data to interact with SERCA (56). Residues in D shown with side chains in sticks and labeled on the structure reside on the same helix of PLN, i.e., they are positioned for binding SERCA. In both B and D, the colors reflect the hydrophobicity: light gray, hydrophobic; blue/red/purple, hydrophilic; green, aromatic (Phe); specifically Ser and Thr residues are shown in purple, Glu is shown in red, and Asn and Gln are shown in blue.