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.

Ravishankar H., Pedersen M.N., Eklund M., Sitsel A., Li C., Duelli A., et al. (2020). Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering. SCIENCE ADVANCES, 6(12), eaaz0981 [10.1126/sciadv.aaz0981].

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

Levantino M.
Methodology
;
2020

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.
Settore FIS/07 - Fisica Applicata(Beni Culturali, Ambientali, Biol.e Medicin)
Ravishankar H., Pedersen M.N., Eklund M., Sitsel A., Li C., Duelli A., et al. (2020). Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering. SCIENCE ADVANCES, 6(12), eaaz0981 [10.1126/sciadv.aaz0981].
File in questo prodotto:
File Dimensione Formato  
eaaz0981.full.pdf

accesso aperto

Tipologia: Versione Editoriale
Dimensione 1.83 MB
Formato Adobe PDF
1.83 MB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10447/424083
Citazioni
  • ???jsp.display-item.citation.pmc??? 12
  • Scopus 18
  • ???jsp.display-item.citation.isi??? 18
social impact