Redistribution of SERCA calcium pump conformers during intracellular calcium signaling

Olga N. Raguimova, Nikolai Smolin, Elisa Bovo, Siddharth Bhayani, Joseph M. Autry, Aleksey V. Zima, Seth L. Robia

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The conformational changes of a calcium transport ATPase were investigated with molecular dynamics (MD) simulations as well as fluorescence resonance energy transfer (FRET) measurements to determine the significance of a discrete structural element for regulation of the conformational dynamics of the transport cycle. Previous MD simulations indicated that a loop in the cytosolic domain of the SERCA calcium transporter facilitates an open-to-closed structural transition. To investigate the significance of this structural element, we performed additional MD simulations and new biophysical measurements of SERCA structure and function. Rationally designed in silico mutations of three acidic residues of the loop decreased SERCA domain–domain contacts and increased domain– domain separation distances. Principal component analysis of MD simulations suggested decreased sampling of compact conformations upon N-loop mutagenesis. Deficits in headpiece structural dynamics were also detected by measuring intramolecular FRET of a Cer–YFP–SERCA construct (2-color SERCA). Compared with WT, the mutated 2-color SERCA shows a partial FRET response to calcium, whereas retaining full responsiveness to the inhibitor thapsigargin. Functional measurements showed that the mutated transporter still hydrolyzes ATP and transports calcium, but that maximal enzyme activity is reduced while maintaining similar calcium affinity. In live cells, calcium elevations resulted in concomitant FRET changes as the population of WT 2-color SERCA molecules redistributed among intermediates of the transport cycle. Our results provide novel insights on how the population of SERCA pumps responds to dynamic changes in intracellular calcium.

Original languageEnglish (US)
Pages (from-to)10843-10856
Number of pages14
JournalJournal of Biological Chemistry
Issue number28
StatePublished - Jul 13 2018

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    Raguimova, O. N., Smolin, N., Bovo, E., Bhayani, S., Autry, J. M., Zima, A. V., & Robia, S. L. (2018). Redistribution of SERCA calcium pump conformers during intracellular calcium signaling. Journal of Biological Chemistry, 293(28), 10843-10856.