Structural and dynamics analysis of pathogenic modifications in cardiac sarcoplasmic reticulum proteins involved in Ca2+ transport

Abstract

Calcium signaling pathways are essential for the coordination of contraction and relaxation in cardiac muscle. Disruption of cardiac calcium cycling by pathogenic modifications in calcium transport proteins leads to a variety of cardiomyopathies including dilated cardiomyopathy and arrhythmias. The following thesis summarizes the structural and dynamic characterization of key regulatory proteins involved in calcium release and reuptake in the sarcoplasmic reticulum (SR). Calmodulin (CaM), a calcium-sensing protein that regulates its cellular targets based on the level of calcium in the cell, mediates calcium release from the SR via the homotetrameric calcium release channel, ryanodine receptor (RyR). The CaM-RyR complex has been a challenging structural target due to the size and complexity of the RyR. By applying a combination of solution and solid-state NMR techniques we have begun to develop a molecular model for CaM’s regulation of the RyR and how this regulation is disrupted by pathogenic modifications such as oxidation and mutation. Disruptions in calcium reuptake to the SR due to mutations in the small transmembrane protein, phospholamban (PLN), result from dysregulation of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). These PLN mutations are primarily associated with the development of dilated cardiomyopathy and by applying solution and solid-state NMR techniques we have begun to develop a model for how changes in PLN’s structure and dynamics correlate to the dysregulation of SERCA. Together, the structural and dynamic studies outlined in this thesis provide further insights into the correlations between protein structure and function and the crucial roles CaM and PLN play in cardiac function.