Structural Basis of Dilated Cardiomyopathy

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DESCRIPTION (provided by applicant): The overall goal of this research is to elucidate the structural basis of dilated cardiomyopathy (DCM). Cardiac muscle function depends critically on Ca-ATPase, a membrane-embedded Ca transport enzyme inhibited by phospholamban (PLB). Upon adrenergic stimulation, this inhibition is reversed by cAMP-dependent protein kinase (PKA), phosphorylating PLB at S16. Disruptions to this system result in heart disease. A specific PLB mutation (R9C-PLB) has been linked directly to hereditary DCM. R9C-PLB irreversibly inhibits PKA, hampering the phosphorylation of wt-PLB, interfering with Ca transport regulation, and causing heart failure. This finding represents a breakthrough that has been widely reported in the national media. We will determine the structural basis of DCM using molecular biology, peptide synthesis, NMR, and EPR methods to accomplish the following AIMs: AIM 1: Characterizing the phosphorylation kinetics of both PLB and its lethal R9C mutant. AIM 2: Probing the binding surfaces and the structures of PKA/PLB and PKA/R9C-PLB complexes. AIM 3: Mapping the structural dynamics of PKA and PKA complexes with PLB and its R9C-mutant. AIM 4: Elucidating the membrane architecture of the PKA/PLB complex in lipids: influence of N-myristoylation of PKA. The immediate outcome of this research will be to characterize the high-resolution structure of the R9C-PLB/PKA complex and to elucidate the structural links between R9C mutation and DCM. The long-term goal is to develop a molecular framework for new therapeutic approaches involving calcium regulation and heart muscle contractility. To the best of our knowledge, these are the first biophysical studies of protein-protein interactions between integral membrane proteins and soluble proteins ever carried out using NMR spectroscopy.