Concerted but Noncooperative Activation of Nucleotide and Actuator Domains of the Ca-ATPase upon Calcium Binding

Chen, Baowei; Mahaney, James E.; Mayer, Michael; Bigelow, Diana J.; Squier, Thomas C.

doi:10.1021/bi8014289

Cited by 20 publications

(16 citation statements)

References 52 publications

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“…This change was somewhat smaller in magnitude than the 30+ Å change predicted by the first X-ray crystallographic structures ( Fig. 1 A), but larger than that measured by other FRET studies [8], [9], [29], Most notably, the distance change observed here was opposite in direction compared to early crystal structure predictions [4]. The apparent decrease in FRET distance with Ca also contrasts with previous FRET studies that used reactive dyes as donors/acceptors.…”

Section: Discussioncontrasting

confidence: 89%

2-Color Calcium Pump Reveals Closure of the Cytoplasmic Headpiece with Calcium Binding

Hou

Blackwell

et al. 2012

PLoS ONE

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The sarco(endo)plasmic reticulum calcium ATPase (SERCA) undergoes conformational changes while transporting calcium, but the details of the domain motions are still unclear. The objective of the present study was to measure distances between the cytoplasmic domains of SERCA2a in order to reveal the magnitude and direction of conformational changes. Using fluorescence microscopy of live cells, we measured intramolecular fluorescence resonance energy transfer (FRET) from a donor fluorescent protein fused to the SERCA N-terminus to an acceptor fluorescent protein fused to either the N-, P-, or transmembrane domain. The “2-color” SERCA constructs were catalytically active as indicated by ATPase activity in vitro and Ca uptake in live cells. All constructs exhibited dynamic FRET changes in response to the pump ligands calcium and thapsigargin (Tg). These FRET changes were quantified as an index of SERCA conformational changes. Intramolecular FRET decreased with Tg for the two N-domain fusion sites (at residue 509 or 576), while the P- (residue 661) and TM-domain (C-terminus) fusions showed increased FRET with Tg. The magnitude of the Tg-dependent conformational change was not decreased by coexpression of phospholamban (PLB), nor did PLB slow the kinetics of Tg binding. FRET in ionophore-permeabilized cells was lower in EGTA than in saturating calcium for all constructs, indicating a decrease in domain separation distance with the structural transition from E2 (Ca-free) to E1 (Ca-bound). The data suggest closure of the cytoplasmic headpiece with Ca-binding. The present results provide insight into the structural dynamics of the Ca-ATPase. In addition, the 2-color SERCA constructs developed for this study may be useful for evaluating candidate small molecule regulators of Ca uptake activity.

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Section: Discussioncontrasting

confidence: 89%

2-Color Calcium Pump Reveals Closure of the Cytoplasmic Headpiece with Calcium Binding

Hou

Blackwell

et al. 2012

PLoS ONE

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“…All these three distance profiles within residues in the transmembrane region were exhibited as a result of conformational changes triggered by the binding of RA to SERCA1a during the calcium transport process. As described by Chen et al, 2008 [58], the salt bridge between Arg762 and Asp981 was formed after calcium binding to site I [58]. Thus, this salt bridge was hindered by RA binding, which was confirmed by the Arg762NH -Asp981OD1 distance staying at ~13 Å during the 200 ns MD simulation ( Figure 5B).…”

Section: Molecular Dynamics Simulation Of Serca1a-ra-popc Systemsupporting

confidence: 70%

“…Thus, this salt bridge was hindered by RA binding, which was confirmed by the Arg762NH -Asp981OD1 distance staying at ~13 Å during the 200 ns MD simulation ( Figure 5B). On the other hand, the pairwise distances of Glu80-Arg290 and Glu90-Lys297, which were formed after Ca 2+ binding to site II [58], alternated and complemented between two states allowing the formation and/or breaking of a salt bridge ( Figure 5B). According to our calculations, RA occupied the Ca 2+ -binding site I (Glu908, Glu771 and Asp800, Asn768, Thr799 as described in [12]) instead of Ca 2+ -binding site II (Ala305, Glu309, Asn796, Asp800 as described in [12]).…”

Section: Molecular Dynamics Simulation Of Serca1a-ra-popc Systemmentioning

confidence: 99%

“…The RA was also stabilized by a network of hydrophobic, π-π, and water interactions. It is worth mentioning that SERCA1a remained in the E2 intermediate state during the MD simulation avoiding the formation of key salt bridges between several residues side chains, including Arg762 and Asp981, that otherwise would enable the occupancy of Ca 2+ -binding site II of SERCA1a neutralizing the positive charge of Arg762 [58].…”

Section: Interactionmentioning

confidence: 99%

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Structural Changes of Sarco/Endoplasmic Reticulum Ca2+-ATPase Induced by Rutin Arachidonate: A Molecular Dynamics Study

Rodrı́guez

Májeková

2020

Biomolecules

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Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) maintains the level of calcium concentration in cells by pumping calcium ions from the cytoplasm to the lumen while undergoing substantial conformational changes, which can be stabilized or prevented by various compounds. Here we attempted to clarify the molecular mechanism of action of new inhibitor rutin arachidonate, one of the series of the acylated rutin derivatives. We performed molecular dynamics simulations of SERCA1a protein bound to rutin arachidonate positioned in a pure dipalmitoylphosphatidylcholine bilayer membrane. Our study predicted the molecular basis for the binding of rutin arachidonate towards SERCA1a in the vicinity of the binding site of calcium ions and near the location of the well-known inhibitor thapsigargin. The stable hydrogen bond between Glu771 and rutin arachidonate plays a key role in the binding. SERCA1a is kept in the E2 conformation preventing the formation of important salt bridges between the side chains of several residues, primarily Glu90 and Lys297. All in all, the structural changes induced by the binding of rutin arachidonate to SERCA1a may shift proton balance near the titrable residues Glu771 and Glu309 into neutral species, hence preventing the binding of calcium ions to the transmembrane binding sites and thus affecting calcium homeostasis. Our results could lead towards the design of new types of inhibitors, potential drug candidates for cancer treatment, which could be anchored to the transmembrane region of SERCA1a by a lipophilic fatty acid group.

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“…46 The region that is most frequently used for TC environment design is a protein loop: the motif is incorporated either into a protein‐chain turn, or into a domain‐connecting region, by insertion of a TC sequence or by replacement of the existing amino acids (Figure 2 C). 14, 47–61 This approach is often used when the protein structure is known, but it is possible to identify exposed loops by bioinformatic prediction and experimental research. It should be noted that the fluorescence of biarsenical TC loops varies significantly, even within a single protein, and it strongly depends on the surrounding amino acids 47.…”

Section: Placement Of Tc Motif In Proteinsmentioning

confidence: 99%

Exploration of Biarsenical Chemistry—Challenges in Protein Research

Pomorski

Krężel

2011

ChemBioChem

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The fluorescent modification of proteins (with genetically encoded low-molecular-mass fluorophores, affinity probes, or other chemically active species) is extraordinarily useful for monitoring and controlling protein functions in vitro, as well as in cell cultures and tissues. The large sizes of some fluorescent tags, such as fluorescent proteins, often perturb normal activity and localization of the protein of interest, as well as other effects. Of the many fluorescent-labeling strategies applied to in vitro and in vivo studies, one is very promising. This requires a very short (6- to 12-residue), appropriately spaced, tetracysteine sequence (-CCXXCC-); this is either placed at a protein terminus, within flexible loops, or incorporated into secondary structure elements. Proteins that contain the tetracysteine motif become highly fluorescent upon labeling with a nonluminescent biarsenical probe, and form very stable covalent complexes. We focus on the development, growth, and multiple applications of this protein research methodology, both in vitro and in vivo. Its application is not limited to intact-cell protein visualization; it has tremendous potential in other protein research disciplines, such as protein purification and activity control, electron microscopy imaging of cells or tissue, protein-protein interaction studies, protein stability, and aggregation studies.

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Concerted but Noncooperative Activation of Nucleotide and Actuator Domains of the Ca-ATPase upon Calcium Binding

Cited by 20 publications

References 52 publications

2-Color Calcium Pump Reveals Closure of the Cytoplasmic Headpiece with Calcium Binding

2-Color Calcium Pump Reveals Closure of the Cytoplasmic Headpiece with Calcium Binding

Structural Changes of Sarco/Endoplasmic Reticulum Ca2+-ATPase Induced by Rutin Arachidonate: A Molecular Dynamics Study

Exploration of Biarsenical Chemistry—Challenges in Protein Research

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