Resolving Structural Dynamics of the SERCA-PLB Complex by Merging MD Simulation and Orientation-Sensitive EPR Measurement

Abstract

We are using molecular dynamics (MD) simulation to resolve the structural dynamics of the membrane protein phospholamban (PLB), a binding inhibitor to the protein pump sarco(endo)plasmic reticulum calcium-ATPase (SERCA). Increase of cytosolic calcium concentration drives cell contraction via the actin-myosin mechanism, while subsequent uptake of calcium into the SR by SERCA allows cell relaxation. Over-inhibition of SERCA by PLB has been correlated with dysfunctional contractility and heart disease, but the structural basis of this mechanism is not fully understood. We combine MD simulation with electron paramagnetic resonance (EPR) spectroscopy to elucidate the structural mechanism of the PLB-SERCA complex. Previously, we have used MD simulation to determine the dynamic model of the spin label, TOAC, rigidly attached to PLB at residues 24, 36, and 46. Based on these results, which provided a bimodal structural model for TOAC at each attachment position, we have refined analysis of EPR measurement to determine PLB's orientation relative to the bilayer normal. We now extend this combined MD and EPR approach to study the structural changes of PLB in the presence and absence of SERCA. Furthermore, we have used EPR measurements on the SERCA-PLB complex to constrain MD simulation of the system, thus extending spatial and temporal resolution of membrane-bound PLB in the presence and absence of SERCA. Computations were carried out at the Minnesota Supercomputing Institute and experiments were performed in the Biophysical Technology Center. This work was funded by grants from NIH to DDT (R01 GM27906 and T32 AR007612).