Redistribution of SERCA calcium pump conformers during intracellular calcium signaling

Abstract

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 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.

Dynamics-driven allostery underlies pre-activation of the regulatory Ca2+-ATPase/phospholamban complex

“…Interestingly, the Ca 2+ -bound complex populates a relatively small conformation where Arg139 and Asp426 come close together similar to that found in the crystal structure of SERCA that precedes full activation (Fig 4C, right panel, green trace) [37]. This observation is significant because the interaction between the loop Nβ5-β6 and residues 133-139 of the A-domain has been proposed to be an important step that initiates SERCA activation [25,40]. These specific contacts and concerted motions are suggestive of activation-precursor events, and also suggest that activation of the complex occurs as a series of sequential structural steps controlled primarily by Ca 2+ binding [22,41].…”

“…2B). The headpiece motions captured by the MD simulations are consistent with the structural transitions of SERCA in the absence of PLB, where apo SERCA populates a partially open cytosolic headpiece prior to Ca 2+ -induced activation [25,34], with SERCA undergoing a structural shift toward a closed conformation during Ca 2+ signaling and activation [25,35].…”

“…While the application of a variety of simulation and experimental approaches alone has led to a greater insight into the mechanisms for SERCA-PLB regulation, the structural changes induced by Ca 2+ and how those are communicated to couple enzymatic activity with active transport remain poorly defined. More specifically, it remains unknown whether binding of a single Ca 2+ ion initiates SERCA-PLB activation by stabilizing the transport sites [17,22,23] and producing a SERCA structure that is compatible with Ca 2+ binding [24], or by inducing large-scale domain arrangements that produce catalytically competent conformations of SERCA [25,26]. Answering these questions requires knowledge of the structural changes and motions at high spatiotemporal resolution.…”

“…The differences in structural landscape and domain motions captured by our µs-long simulations suggest that Ca 2+ binding to SERCA-PLB induces a disorder-toorder transition in the headpiece of SERCA. We tested this hypothesis by quantifying intramolecular FRET between fluorescent proteins fused to the A-and N-domains of SERCA (2-color SERCA) as an index of the structural changes of the SERCA cytoplasmic headpiece [25,35,38,39]. We explored the Ca 2+ -dependent change in FRET of microsomal preparations of 2-color SERCA co-expressed with the non-phosphorylatable PLBS16A mutant to mimic the MD conditions, and 2-color SERCA alone as a control (see Methods, Supporting Information).…”

“…Arg139 (A domain) and Asp426 (loop Nβ5-β6), which play a key role in initiating the open-to-closed conformational change of SERCA [25,40], and Thr171 (A domain) and…”