Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer

Blackwell, Daniel J.; Zak, Taylor J.; Robia, Seth L.

doi:10.1016/j.bpj.2016.08.005

Cited by 28 publications

(46 citation statements)

References 63 publications

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“…However, the E2‐AlF 4 ‐CPA crystal form is marked by an unusual parallel packing of SERCA2a molecules involving contact points between the A‐ and N‐domains, between the A‐ and P‐domains, as well as N‐domain and L7/8 (Fig EV1). These SERCA2a‐specific regions may play a role in the formation of SERCA2a dimers, which also have been reported in cardiomyocytes (Blackwell et al , ). No such parallel packing modes have been observed for SERCA1a crystal forms, and many of the involved residues differ for SERCA2a and SERCA1a, and are conserved within one isoform.…”

Section: Resultssupporting

confidence: 69%

Structures of the heart specific SERCA 2a Ca ²⁺ ‐ ATP ase

et al. 2019

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The sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase 2a (SERCA2a) performs active reuptake of cytoplasmic Ca 2+ and is a major regulator of cardiac muscle contractility. Dysfunction or dysregulation of SERCA2a is associated with heart failure, while restoring its function is considered as a therapeutic strategy to restore cardiac performance. However, its structure has not yet been determined. Based on native, active protein purified from pig ventricular muscle, we present the first crystal structures of SERCA2a, determined in the CPA-stabilized E2ÀAlF À 4 form (3.3 Å) and the Ca 2+occluded [Ca 2 ]E1-AMPPCP form (4.0 Å). The structures are similar to the skeletal muscle isoform SERCA1a pointing to a conserved mechanism. We seek to explain the kinetic differences between SERCA1a and SERCA2a. We find that several isoform-specific residues are acceptor sites for post-translational modifications. In addition, molecular dynamics simulations predict that isoformspecific residues support distinct intramolecular interactions in SERCA2a and SERCA1a. Our experimental observations further indicate that isoform-specific intramolecular interactions are functionally relevant, and may explain the kinetic differences between SERCA2a and SERCA1a.

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Section: Resultssupporting

confidence: 69%

Structures of the heart specific SERCA 2a Ca ²⁺ ‐ ATP ase

et al. 2019

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“…No such parallel packing modes have been observed for SERCA1a crystal forms, and many of the involved residues differ for SERCA2a and SERCA1a and are conserved within one isoform. These SERCA2a specific regions may possibly play a role in the dimerization of SERCA2a in cardiomyocytes (Blackwell et al, 2016). Note that due to an amino acid deletion in SERCA2a at position 509, the SERCA2a residue numbering from position 509 is shifted by -1 as compared to SERCA1a.…”

Section: E2-alf4-cpa and [Ca2]e1-amppcp Structures Of Serca2a Closelymentioning

confidence: 99%

Structures of the heart specific SERCA2a Ca²⁺-ATPase

Nissen

Raeymaecker

Kotsubei

et al. 2018

Preprint

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The sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase 2a (SERCA2a) performs active reuptake of cytoplasmic Ca2+ and is a major regulator of cardiac muscle contractility. Dysfunction or dysregulation of SERCA2a is associated with heart failure, while restoring its function is considered as a therapeutic strategy to restore cardiac performance. However, its structure has not yet been determined. Based on native, active protein purified from pig ventricular muscle, we present the first crystal structures of SERCA2a, determined in the CPA‐stabilized E2−AlF4− form (3.3 Å) and the Ca2+‐occluded [Ca2]E1‐AMPPCP form (4.0 Å). The structures are similar to the skeletal muscle isoform SERCA1a pointing to a conserved mechanism. We seek to explain the kinetic differences between SERCA1a and SERCA2a. We find that several isoform‐specific residues are acceptor sites for post‐translational modifications. In addition, molecular dynamics simulations predict that isoform‐specific residues support distinct intramolecular interactions in SERCA2a and SERCA1a. Our experimental observations further indicate that isoform‐specific intramolecular interactions are functionally relevant, and may explain the kinetic differences between SERCA2a and SERCA1a.

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“…We have previously quantified FRET in cardiac myocytes (15,39,40), but motion artifacts of actively contracting cells make quantification of dynamic changes in fluorescence challenging. As an alternative, we reconstituted aspects of muscle cell Ca 2ϩ handling with co-expression of RyR2 and SERCA2a in live HEK-293 cells, and subjected the proteins to confocal microscopy.…”

Section: Serca Structural Dynamics In Response To Changes In Intracelmentioning

confidence: 99%

Redistribution of SERCA calcium pump conformers during intracellular calcium signaling

Raguimova

Smolin

Bovo

et al. 2018

Journal of Biological Chemistry

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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 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.

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Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer

Cited by 28 publications

References 63 publications

Structures of the heart specific SERCA 2a Ca ²⁺ ‐ ATP ase

Structures of the heart specific SERCA 2a Ca ²⁺ ‐ ATP ase

Structures of the heart specific SERCA2a Ca²⁺-ATPase

Redistribution of SERCA calcium pump conformers during intracellular calcium signaling

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Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer

Cited by 28 publications

References 63 publications

Structures of the heart specific SERCA 2a Ca 2+ ‐ ATP ase

Structures of the heart specific SERCA 2a Ca 2+ ‐ ATP ase

Structures of the heart specific SERCA2a Ca2+-ATPase

Redistribution of SERCA calcium pump conformers during intracellular calcium signaling

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Product

Resources

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Structures of the heart specific SERCA 2a Ca ²⁺ ‐ ATP ase

Structures of the heart specific SERCA 2a Ca ²⁺ ‐ ATP ase

Structures of the heart specific SERCA2a Ca²⁺-ATPase