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Phospholamban and Sarcolipin Oligomers Directly Modulate Ca2+-ATPase Activity
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- Author / Creator
- Primeau, Joseph
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Oscillating cytosolic calcium concentrations dictate the contraction-relaxation cycles of muscle cells. Calcium release from the sarcoplasmic reticulum (SR) to the cytosol stimulates muscle contraction, while active transport of calcium back into the SR triggers muscle relaxation. Calcium reuptake involves the transport of calcium against its concentration gradient, which is facilitated by the sarcoendoplasmic reticulum calcium ATPase, or SERCA. SERCA has been traditionally described as an E1-E2 enzyme that follows the Post-Albers scheme for metal ion transport. SERCA undergoes substantial structural changes from a high-affinity state (E1) to a low-affinity state (E2) to facilitate calcium transport. SERCA’s calcium transport properties are regulated by small, tissue-specific, transmembrane protein subunits; the most notable include phospholamban (PLN) and sarcolipin (SLN). PLN and SLN alter SERCA’s calcium transport properties by reversibly interacting with the transmembrane domain of SERCA, allowing for dynamic control of muscle contractility. Crystal structures have revealed that PLN and SLN were both found to stabilize a calcium-free E1-like intermediate by interacting with an inhibitory groove formed by M2, M6, and M9 of SERCA. This interaction is involved in altering SERCA’s calcium affinity and was canonically thought to be the primary mode of SERCA inhibition. We have identified through two-dimensional electron crystallography (2D crystals) of SERCA and PLN/SLN in a lipid membrane that PLN/SLN interacts with SERCA at a site distinct from the inhibitory groove, transmembrane segment M3. Both PLN and SLN 2D crystals reveal that this interaction is mediated by an oligomer consistent with a homopentamer.
Here, we functionally characterize the oligomeric interaction with SERCA for wild-type PLN, wild-type SLN and determined that this interaction alters SERCA’s maximum turnover rate, with PLN eliciting a stimulatory effect and SLN eliciting an inhibitory effect. Protein docking and molecular dynamics simulations provided a model for this novel interaction, revealing a potential mechanism for the distinct regulatory roles of the PLN and SLN interaction with M3. In addition, we identified that SLN’s regulatory function is dependent on the E1-E2 conformation of SERCA, suggesting that there are multiple modes of SERCA-SLN interaction that can persist throughout SERCA’s calcium transport pathway and multiple turnover events. Based on our results, we have concluded that the PLN pentamer naturally associates with SERCA, the SLN pentamer naturally and persistently associates with SERCA, and the regulatory nature of these interactions with M3 is dependent on local micro-environment perturbations. -
- Graduation date
- Spring 2020
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.