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 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.

Keywords: calcium ATPase; calcium imaging; calcium intracellular release; calcium transport; conformational change; endoplasmic reticulum (ER); fluorescence resonance energy transfer (FRET); membrane biophysics; membrane transport; sarcoplasmic reticulum (SR).

Figure 1.

MD simulations of SERCA structural dynamics. For C–E, WT–SERCA (black), AAA (red), D426A (blue), E429A (green), E435A (pink) are shown. Data represent average of 6 MD run productions. A, SERCA starting X-ray structure of PDB 1SU4 for simulations showing the actuator (A), nucleotide-binding (N), phosphorylation (P), and TM domains. The Nβ5–β6 loop is highlighted in orange, and three negatively charged residues Asp-426, Glu-429, and Glu-435 are labeled in the magnified inset. B, covariance matrices Cα atoms for WT–SERCA (upper left) and AAA–SERCA (lower right). Covariance analysis of WT–SERCA residue dynamics as measured from Cα revealed positively (red) and negatively (blue) correlated motions. Dotted boxes highlight regions of covariance of the N- and A-domains. For AAA–SERCA, covariance analysis indicated similar global dynamics yet reduced anti-correlated N- and A-domain motions compared with WT–SERCA. Data show representative of 6 MD runs. C, number of contacts between the N- and A-domains during MD trajectories. D, quantification of results in C. E, AAA–SERCA shows an increase in N- to A-domains separation distance compared with WT-SERCA. F, negative correlation of separation distance on domain–domain contacts, from results in D and E. G, first and second principal components of SERCA domain motions. H, relative sampling of the top two principal components by WT–SERCA (black) and AAA–SERCA (red) trajectories. Each point represents a conformation extracted from the MD trajectories at an interval of 0.1 ns. For comparison, gray dots represent X-ray structures with open (1SU4), closed (1VFP), and intermediate (3W5B) headpiece conformations.

Figure 2.

N-domain β5–β6 loop triple mutation decreases SERCA function. A, calcium-dependent ATPase activity of cells expressing WT– or AAA–SERCA, or nontransfected cells (Ctrl). B, Ca²⁺ sensitivity of ATPase activity (K_Ca), as in A, p = 0.61. C, maximal Ca²⁺-dependent ATPase rate (V_max), as in A, *, p = 0.015. D, triple-transfected cells: SERCA, RyR, and R-CEPIA1er. ER Ca²⁺ was depleted by caffeine (Caf) addition, followed by ER Ca²⁺ store recovery in the presence of RyR blocker ruthenium red (RR). E, maximal Ca²⁺ uptake rate, as in D. *, p = 0.016 for AAA versus WT and p = 0.002 for Ctrl versus WT. F, maximal ER Ca²⁺ load, as in D. *, p = 4 × 10⁻⁵ versus WT.

Figure 3.

Ligand-induced control of SERCA structure in ER microsomes. A, WT 2-color SERCA shows increased FRET with increasing Ca²⁺, as detected by confocal fluorescence microscopy. Black error bars represent S.E., whereas gray bars represent S.D. (n = 6 experiments). B, FRET of WT 2-color SERCA stabilized in key enzymatic states. *, p ≤ 0.008 compared with H⁺. C, calculated FRET distances compared with distances between the fluorescent protein fusion sites measured from X-ray crystal structures. Select structures are labeled for comparison, other data are identified in Table 1.

Figure 4.

Ca²⁺-dependent redistribution of SERCA conformers in live cells. A, FRET of 2-color SERCA constructs expressed in basal HEK-293 cells, as detected by acceptor-sensitized fluorescence microscopy, *, p ≤ 0.005. B, TG-induced E2 conformers of WT 2-color SERCA and the four mutant constructs show reduced FRET in HEK-293 cells, whereas DMSO vehicle-only control (Ctrl, dark blue) had no significant effect. C, addition of the Ca²⁺ ionophore Iono resulted in a biphasic FRET response (1 = quick decrease, 2 = slow recovery) for WT and single-point mutants, as detected using epifluorescence microscopy. AAA–SERCA (red) showed only the first phase: a quick decrease in FRET. Data represent the average of n = 6–25 cells for each condition.

Figure 5.

SERCA structural dynamics measured by FRET in HEK-293 live cells. A, anti-correlated changes in Cer and YFP fluorescence intensity indicate rhythmic FRET fluctuations in intact cells. B, the ratio of YFP/Cer, as in A, was used as an index of FRET (gray). FRET (bottom panel, gray) was inversely correlated to cytosolic Ca²⁺, as measured by X-Rhod fluorescence (black, top panel). WT–SERCA FRET decreased during cytosolic Ca²⁺ elevations due to spontaneous ER Ca²⁺ release through RyR. C, a decrease in 2-color SERCA intramolecular FRET (bottom trace) occurs simultaneously with depletion of ER Ca²⁺ stores (top trace). D, quantification of intermolecular FRET between Cer–SERCA and YFP–SERCA using progressive acceptor photobleaching (started at black arrow). Data presented are the mean F/F₀ of Cer and YFP fluorescence measured in 6 cells. E, SERCA–SERCA intermolecular FRET did not change in response to ionomycin addition. F, SERCA–SERCA intermolecular FRET did not change with spontaneous Ca²⁺ release events (top trace). The data indicate that changes in 2-color SERCA FRET are due to changes in intramolecular FRET rather than changes in intermolecular FRET. G, after addition of ionomycin, both cytosolic Ca²⁺ and WT–SERCA FRET increased, as detected by confocal fluorescence microscopy. H, addition of caffeine (Caf) transiently increased cytosolic Ca²⁺ and decreased WT–SERCA FRET. I, AAA–SERCA FRET decreased during spontaneous cytosolic Ca²⁺ elevations. J, in contrast to WT–SERCA FRET, AAA–SERCA FRET decreased with addition of ionomycin. K, AAA–SERCA FRET increased in response to caffeine, similar to WT–SERCA FRET (H). L, addition of ionomycin causes an increase in ER Ca²⁺ content, but this increase occurs more slowly than the second phase of the observed FRET response of 2-color SERCA. We conclude that the phase 2 FRET increase of 2-color SERCA is not due to saturation of SERCA luminal Ca²⁺-binding sites (i.e. low affinity E2 orientation).

Figure 6.

Redistribution of SERCA conformers during Ca²⁺ signaling. A, a simplified Post-Albers cycle. Blue boxes enclose states with similar intramolecular FRET efficiency. B, schematic diagram of changes in cytosolic Ca²⁺ (red) and changes in FRET (gray) as the population of SERCA redistributes among structural states, with the predominant state shown in blue. The WT FRET response to ionomycin shows two phases (phases 1 and 2), whereas AAA FRET shows only a decrease in FRET (dotted line).