Phospholamban (PLN) is an integral membrane protein that regulates calcium homeostasis by inhibiting sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) in cardiac muscle. PLN exists in two primary oligomeric forms: (1) a monomer that directly binds to and inhibits SERCA (1, 2) and (2) a pentamer that indirectly influences SERCA activity by regulating the pentamer-monomer equilibrium (1, 2). To completely describe the fold space and ultimately the biological function of PLN and other membrane proteins, it is necessary to determine the membrane protein’s structure and the specific interactions with the lipid bilayer (i.e., topology). In this work, we describe a new hybrid method to calculate the structures of the PLN monomer and pentamer using a combination of solution and solid-state NMR restraints in detergent micelles and lipid bilayers, respectively (3, 4). These high-resolution images of the PLN not only describe the structures, but also the topologies of all the protein domains with respect to the lipid bilayer.
We obtained short-range distance and angular restraints from solution NMR of PLN reconstituted into DPC detergent micelles and orientation restraints (anisotropic chemical shifts and dipolar couplings) from 2D separated local field experiments such as PISEMA (6, 7) in mechanically aligned lipid bilayers. The final stage of refinement was to incorporate explicit lipids around the protein structure and carry out minimization to reveal the interactions between the lipid and protein domains. In our structures, we find that the N-terminal helical domain Ia (residues 1-16) of monomer and pentamer rests on the surface of the lipid bilayer with the hydrophobic face of domain Ia embedded in the bilayer interior. The helix comprised of domain Ib (residues 23-30) and transmembrane domain II (residues 31-52) traverses the bilayer with tilt angles of ~11° (pentamer) and ~24° (monomer; structure in Figure 1). Hybrid methods such as the one presented in this work will be necessary to tackle challenging biophysical problems such as membrane protein structure determination.
Figure 1. Structure of PLN monomer from Traaseth et al. (Ref. 3).
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