TOAC Spin Label Conformation Resolved by MD Simulation of Membrane-Bound Phospholamban

Abstract

We have used all-atom molecular dynamics (MD) simulation to determine the conformational occupancy of the nitroxide spin label TOAC attached to membrane-bound phospholamban (PLB), as needed for interpretation of EPR spectra. EPR spectroscopy is sensitive primarily to the dynamic angular distribution of the probe principal axis relative to the magnetic field, θHP. MD simulations determine directly the orientation of the probe principal axis relative to the α-helix axis, θMP. Previously, θMP has been determined from X-ray crystallography of TOAC on α-helical peptides, which have indicated sole occupancy of a twistboat conformation, with θMP = 13.3° (Marsh 2006 JMagRes). We have employed all-atom MD simulations to determine the distribution of θMP values for TOAC bound to PLB. The PLB structure (PDB ID: 2KB7) was mutated with TOAC at residues 11, 24, 36, or 46, and placed in Monte-Carlo-generated lipid bilayers composed of either DOPC or DMPC:POPC. MD simulations were performed using NAMD with the CHARMM36 force field. Trajectories were computed to 100ns for chair, boat, and twistboat starting conformations of TOAC. Time-averaged geometry of PLB α-helices in the lipid bilayer membrane were calculated, and results agreed with published NMR data, validating the MD results. Furthermore, angle distributions in the probe frame - (θHP, ϕHP) and (θMP, ϕMP) - were calculated, indicating a bimodal TOAC occupancy model with sensitivity to label location. Coupling MD-derived conformational models with EPR data fitting has improved determination of the conformational distribution of PLB in a lipid bilayer and will allow us to determine PLB orientations in other conditions such as when bound to SERCA.