Simulation and FRET Analyses of SERCA, Phospohlamban, and Sarcolipin Complexes

Abstract

We have used molecular modeling and experimental FRET constraints of fluorescent protein fusion constructs to study molecular interactions and small-molecule effects on SERCA and its transmembrane regulatory peptide phospholamban (PLB) and sarcolipin (SLN). The sarcoplasmic reticulum calcium transport ATPase (SERCA) is reversibly inhibited in heart and muscle by PLB and SLN, with phosphorylation-induced relief of inhibition. We previously utilized fluorescent fusion-protein biosensors and FRET assays to screen for small-molecule modulators of SERCA structural dynamics and activation (Schaaf et al., Biosensors 2018). In order to interpret FRET results (dynamic population distributions) in a three-dimensional, structural context, we performed molecular modeling and conformational sampling simulations. Starting points for molecular simulations were SERCA x-ray crystal structures in the following biochemical states: E1-2Ca (1SU4), E1P-2Ca-ADP (2ZBD), E2P-ADP (1WPG), E2 (1IWO), E1-2Ca+SLN-bound (3W5A), and E1-2Ca+PLB-bound (4KYT). Simulations generated an ensemble of conformations from which the inter-probe distance (R) and the orientation factor (κ2) were calculated. These two simulation-based parameters were then used to calculate donor lifetime change due to FRET from GFP to RFP tags. Simulation results were correlated to distances measured experimentally using subnanosecond-resolved fluorescence. Spectroscopy studies were performed in the Biophysical Technology Center, University of Minnesota Department of Biochemistry, Molecular Biology, and Biophysics. Simulations were run at the University of Minnesota Supercomputing Institute. This work is supported by NIH grants to DDT (GM27906, HL129814, AG026160).