Structurele inzichten in SERCA2a regulatie

Ca2+ homeostasis is highly important for many fundamental physiological processes, including muscle contraction. Being responsible for the removal of Ca2+ from the cytoplasm back into the sarco(endo)plasmic reticulum, the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is one of the key proteins of muscle excitation-contraction coupling. Careful regulation of the activity, stability and expression levels of SERCA is essential and is accomplished by a variety of mechanisms. These include use of transcription factors, post-translational modifications and SERCA-interacting proteins, such as the small transmembrane regulators phospholamban, sarcolipin, myoregulin, DWORF as well as others that are described in this work in more detail. The regulation of SERCA as well as its functional properties such as Ca2+-affinity and turnover rate are different between SERCA isoforms. Disruption or alteration of proper SERCA function underlies severe disturbances in the contraction-relaxation cycle of muscle cells and leads to pathological conditions, including heart failure. A better understanding of SERCA function, regulation, dynamics and structure would help to explain the differences between SERCA isoforms, with this particular work focusing on skeletal muscle SERCA1a and cardiac SERCA2a, and may assist in developing highly specific therapies to treat diseases caused by malfunction of these proteins. This thesis describes the structural and functional analysis of cardiac SERCA2a, with crystal structures determined to 4.0 Å and 3.3 Å in two conformational states ([Ca2]E1-AMPPCP and [H2-3]E2-AlF- 4 - CPA, respectively). All domains as well as expected ligands (AMPPCP and CPA) can be clearly identified in the structure. The SERCA2a structures resemble those of skeletal muscle SERCA1a in the corresponding conformational states, suggesting a conserved Ca2+ transport mechanism. However, the structures alone could not fully explain the functional differences between the two isoforms, therefore additional experiments were performed. Mass spectrometry analysis identified unique post-translational modifications associated with each isoform, suggesting a distinct regulation pattern. Furthermore, molecular dynamics (MD) simulations of SERCA2a and the corresponding SERCA1a structure were performed to investigate whether each isoform may possess different intramolecular interactions. The MD analysis shows that 15.6% / 12.5% of salt bridge interactions and 36.9% / 53.1% of hydrogen