Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biology

Abstract

The sarco/endoplasmic reticulum calcium ATPase (SERCA) is the major regulator of Ca2+ levels in the cell. Deficient calcium handling in the heart has been linked to heart failure, a leading cause of death in developed countries. As of today, targeting SERCA to enhance cardiac function has not been successful due to lack of details about SERCA structural dynamics during Ca2+ transport.

In my research, I utilized MD simulations and variety of physical assays to determine the role of Nβ5-β6 loop in regulation of SERCA structural dynamics during Ca2+ transport. Previous MD simulations by our lab predicted that the Nβ5-β6 loop regulates a SERCA structural transition from an open to a closed conformation. Here, I showed that rationally designed mutations of three acidic residues of the Nβ5-β6 loop decrease SERCA headpiece closed conformation in silico and in live cells, proving that the Nβ5-β6 loop facilitates SERCA structural dynamics. I provided evidence that the mutated transporter is able to hydrolyze ATP without a change in Ca2+ sensitivity, but with significantly reduced maximal activity. I propose that the decreased transporter activity is the direct result of the deficit in structural dynamics regulated by the Nβ5-β6 loop.

FRET measurements of the fluorescently labeled SERCA during Ca2+ transport in vitro and ex vivo revealed an open SERCA conformation during transient Ca2+ elevations, which characterizes steady-state population of SERCA in the rate-limiting step of the Ca2+ cycle. Sustained Ca2+ elevation resulted in the accumulation of wild-type SERCA in a non-physiological high-Ca2+ affinity closed conformation. In contrast, the mutated transporter accumulated in an open conformation due to a deficit in the kinetics of the cytosolic headpiece closure.

This study provided insights into SERCA structural rearrangements during the Ca2+ transport and demonstrated the importance of the Nβ5-β6 loop in SERCA structural dynamics and transporter function. These insights could be relevant in rational design of novel therapeutics aimed to improve cardiac function by targeting specific SERCA conformations during rate-limiting steps of Ca2+ transport cycle.

Download

Share

COinS