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Cryo-electron microscopy of SERCA interacting with oligomeric phospholamban and oligomeric sarcolipin

  • Author / Creator
    Glaves, John Paul J
  • In 2007, heart disease was the second leading cause of death in Canadians. In heart muscle cells, calcium (Ca2+) is released from the sarcoplasmic reticulum (SR) during contraction and must be replenished for relaxation to occur. Three proteins restore Ca2+ to the SR following contraction: the sarco(endo)plasmic reticulum ATPase (SERCA) and its regulatory binding partners, phospholamban (PLB) and sarcolipin (SLN). The importance and sensitivity of SERCA regulation has been highlighted by identified human mutations in PLB and SLN that lead to heart disease and heart failure. This thesis aims to provide upstream biochemical insights into regulatory complexes formed between SERCA and PLB and between SERCA and SLN for the development of heart disease therapies. The structural information suggests novel interactions between SERCA and its regulatory binding partners. We studied SERCA-PLB and SERCA-SLN complexes using electron microscopy (EM) of two-dimensional co-crystals. Cryo-EM of SERCA and PLB co-crystals revealed a novel mode of binding between the PLB pentamer and SERCA. The binding site involved the third transmembrane helix of SERCA and represented a site distinct from the SERCA binding site for monomeric PLB. Cryo-EM of SERCA and SLN co-crystals suggested that SLN can also bind the third transmembrane helix of SERCA. The functional mutation of PLB affected co-crystal frequency and the structure of PLB in the projection maps. The co-crystal frequency was directly related to the functional state of PLB. The structure of PLB was also influenced by phosphorylation, but the interaction was maintained between the phosphorylated PLB pentamer and SERCA. Combined, the results support an active, functional interaction between pentamers of PLB and SERCA. The function of the pentameric interaction is proposed to deliver monomeric PLB to its inhibitory interaction with SERCA at a different binding site.

  • Subjects / Keywords
  • Graduation date
    2011-11
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3D700
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Biochemistry
  • Supervisor / co-supervisor and their department(s)
    • Young, Howard S (Biochemistry)
  • Examining committee members and their departments
    • Young, James (Physiology)
    • Fliegel, Larry (Biochemistry)
    • Glover, JN Mark (Biochemistry)
    • Marassi, Francesca (Sanford Burnham Medical Research Institute)