Molecular mechanisms and regulation of the calcium pump in the heart

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Project Summary The long-term goal of this project is to elucidate the molecular mechanisms and regulation of the calcium pump (sarcoplasmic reticulum Ca2+-ATPase, SERCA) in the heart. SERCA clears cytosolic Ca2+ in cardiomyocytes, thus playing a central role in Ca2+ regulation in the heart. SERCA is regulated by phospholamban (PLB), a 52-residue phosphorylation-regulated membrane protein that inhibits the activity of the pump. A key molecular dysfunction in heart failure (HF) involves impaired Ca2+ transport during diastole, usually associated with insufficient SERCA expression and unaltered PLB levels, thus yielding lower SERCA activity due to PLB inhibition. Therefore, there is an urgent need for time-resolved, atomistic characterization of SERCA activation and SERCA-PLB regulation to understand the molecular basis of Ca2+ dysregulation, and to design appropriate approaches to HF. These mechanisms are complex, requiring structural changes and interdomain allosteric communication pathways that are difficult to determine experimentally. Since complete experimental characterization of these changes is likely to remain an intractable problem, we propose to use molecular simulations as a complementary approach. The central hypothesis of this project is that molecular simulations at appropriate spatiotemporal scales are uniquely suited to provide a time-resolved detection of SERCA mechanisms and regulation at a level of resolution currently inaccessible through experiments alone. The high-resolution mechanistic information from these studies can be directly used for computer-aided discovery of hits that activate SERCA through specifically targeting the SERCA-PLB interaction. To verify and consolidate these hypotheses, we have developed a robust battery of computational biophysics and virtual high-throughput screening approaches to SERCA and SERCA-PLB. Three Specific Aims will be pursued: (1) Map ligand-induced structural changes associated with SERCA activation. (2) Determine the molecular mechanisms for PLB regulation of SERCA. (3) Perform a structure-based search of hits that activate SERCA. For this project, we focus on skeletal SERCA1a because crystal structures have been obtained only for this isoform, but the structural results from our simulations are directly applicable to cardiac SERCA2a because there are no significant differences in the kinetics and function of both isoforms, including regulation by PLB. The simulation work will be closely coupled to experimental studies through collaborations; the combination of structural and functional data will provide the experimental tests necessary to verify our simulations and refine our structural models. Activation of SERCA is a widely pursued therapeutic goal in heart failure, and this project has great potential for pushing important frontiers in our understanding of SERCA function and regulation, ultimately enabling a more rational approach to address a critical problem in human health.