Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering

Harsha Ravishankar; Martin Nors Pedersen; Mattias Eklund; Aljona Sitsel; Chenge Li; Annette Duelli; Matteo Levantino; Michael Wulff; Andreas Barth; Claus Olesen; Poul Nissen; Magnus Andersson

doi:10.1126/sciadv.aaz0981

Tracking Ca²⁺ ATPase intermediates in real time by x-ray solution scattering

Harsha Ravishankar, Martin Nors Pedersen^*, Mattias Eklund, Aljona Sitsel, Chenge Li, Annette Duelli, Matteo Levantino, Michael Wulff, Andreas Barth, Claus Olesen, Poul Nissen, Magnus Andersson

^*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

34 Citations (Scopus)

Abstract

Sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca₂] E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca²⁺ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment.

Original language	English
Article number	eaaz0981
Journal	Science Advances
Volume	6
Issue	12
Number of pages	10
ISSN	2375-2548
DOIs	https://doi.org/10.1126/sciadv.aaz0981
Publication status	Published - Mar 2020

Keywords

CA2+-ATPASE
CALCIUM-TRANSPORT
CONFORMATIONAL-CHANGES
MEMBRANE
MOLECULAR-DYNAMICS
PHOSPHOENZYME
PUMP
SARCOPLASMIC-RETICULUM
SKELETAL-MUSCLE
STRUCTURAL DYNAMICS

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title = "Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering",

abstract = "Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca2] E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca2+ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment.",

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author = "Harsha Ravishankar and Pedersen, {Martin Nors} and Mattias Eklund and Aljona Sitsel and Chenge Li and Annette Duelli and Matteo Levantino and Michael Wulff and Andreas Barth and Claus Olesen and Poul Nissen and Magnus Andersson",

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AU - Ravishankar, Harsha

AU - Pedersen, Martin Nors

AU - Eklund, Mattias

AU - Sitsel, Aljona

AU - Li, Chenge

AU - Duelli, Annette

AU - Levantino, Matteo

AU - Wulff, Michael

AU - Barth, Andreas

AU - Olesen, Claus

AU - Nissen, Poul

AU - Andersson, Magnus

PY - 2020/3

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N2 - Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca2] E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca2+ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment.

AB - Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca2] E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca2+ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment.

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KW - PHOSPHOENZYME

KW - PUMP

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JO - Science Advances

JF - Science Advances

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ER -

Tracking Ca²⁺ ATPase intermediates in real time by x-ray solution scattering

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