Tracking Ca ²⁺ 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.

The SERCA residue Glu340 mediates interdomain communication that guides Ca 2+ transport

“…The exact functional implication of this effect is not entirely clear. Crystal structures, X-ray solution scattering, and our MD simulations show that the A domain undergoes the largest and most diverse movements during the catalytic cycle ( 9 , 10 , 20 , 26 ), and through its direct linkage to TM helices M1–M3 it directly affects the geometry of the Ca 2+ -binding sites. On the other hand, its position relative to the N and P domains dictates whether the site of ATP hydrolysis can adopt a catalytically competent conformation or not.…”

“…This goes along with a smaller shift of M3 relative to M5 ( 7 , 8 ). The transition from the E 1P to the E 2P state is associated with a large rotation of the A domain, which causes a distortion of the coordination geometry at the high-affinity Ca 2+ -binding sites and a concomitant loss of Ca 2+ affinity, along with ADP release ( 9 ) and the formation of a luminal exit pathway for Ca 2+ release ( 10 ).…”

Cryo-EM structures of SERCA2b reveal the mechanism of regulation by the luminal extension tail

“…Although further studies are required to fully elucidate how the LE controls each step of conformational transitions in SERCA2b throughout the catalytic cycle, the present structural data in combination with previous biochemical and biophysical evidence suggest a possible link between the LE-mediated conformational stabilization in SERCA2b and the different kinetic properties of the enzyme from other isoforms lacking the LE. Time-resolved x-ray solution scattering experiments combined with molecular dynamic simulation recently accomplished for SERCA1a ( 25 ) will reveal yet unidentified mechanisms of action of SERCA2b. In conclusion, the regulatory role of the LE revealed in this study provides a framework for understanding the mechanism underlying the ER Ca 2+ homeostasis ensured by the regulated cytosol-to-ER Ca 2+ transport by SERCA2b.…”

Tracking Membrane Protein Dynamics in Real Time

“…In the first TR-XSS experiment on a non-light sensitive membrane protein, structural dynamics were recorded in sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle from 20 μs to 200 ms following laser-activation of caged ATP (Ravishankar et al 2020 ) (Fig. 5 b).…”

“…The ATP and ADP displayed as four and three purple pentagons, the TGES motif is represented by a green rectangle, and phosphorylated aspartic acid in yellow, and the calcium ions are depicted as green circles. Adapted from Ravishankar et al ( 2020 ) …”