Tracking Ca2+ATPase Intermediates in Real-time by X-Ray Solution Scattering

Abstract

Sarco/endoplasmic reticulum Ca 2+ 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 [Ca 2 ] 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 Ca 2+ 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 E1P to the E2P state is associated with a large rotation of the A domain, which causes a distortion of the coordination geometry at the highaffinity 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 analysis provides new mechanistic insight into ATP binding to Ca 2+ ‐ATPase SERCA2b

“…The presence of four similar cryo-EM maps (Fig EV1A ) suggests that the closed form is less diverse in cytosolic domain arrangement or fluctuates to a lesser extent than the open form. In this context, a pre-ATP-bound form had been identified by unrestrained MD simulations from the "open-form" crystal structure of SERCA1a (Ravishankar et al, 2020), during which the A and N domains were calculated to approach each other, with the distance of 45 A between Thr171 (A domain) and Lys515 (N domain) decreasing to 29 A. Eventually, these domains were settled at the intermediate positions between those in the present "closed-form" cryo-EM structure of SERCA2b and those in the "open-form" crystal structure of SERCA1a.…”

“…While it is believed that the Ca 2+ transport cycle of SERCA is initiated by the coordinated binding of two Ca 2+ ions and one ATP molecule to the transmembrane (TM) and nucleotide-binding (N) domains, respectively (Mueller et al, 2004;Inesi et al, 2006;Toyoshima, 2009;Møller et al, 2010), one of the most discussed issues is the functional significance of the widely opened cytosolic domain arrangement observed in the crystal structure of SERCA1a in the E1Á2Ca 2+ state (Liu & Barth, 2003;Dyla et al, 2019b). Thus, it is still under debate as to whether such a gate opening accompanied by large domain movements actually takes place when the ATP molecule enters the ATP-binding pocket (Ravishankar et al, 2020). Based on the highly mobile nature of the actuator (A) and N domains in this state, and the possible bias caused by crystal packing, the crystal structure may represent only one structural aspect of the calcium-bound but ATP-unbound state.…”

“…Based on the highly mobile nature of the actuator (A) and N domains in this state, and the possible bias caused by crystal packing, the crystal structure may represent only one structural aspect of the calcium-bound but ATP-unbound state. In this context, scientists have been seeking genuine and as-yet-unidentified SERCA intermediates, and visualizing their structures by employing various spectroscopic and computational methods, including fluorescence resonance energy transfer (FRET) (Dyla et al, 2017;Raguimova et al, 2018), molecular dynamics (MD) simulation (Mueller et al, 2004;Huang et al, 2009;Kekenes-Huskey et al, 2012;Das et al, 2014), and time-resolved X-ray solution scattering (TR-XSS) (Ravishankar et al, 2020). Recent TR-XSS analysis combined with MD simulations identified three transient states of SERCA1a during the transition to the ATP-bound state, named the pre-pulse (i.e., pre-ATP-bound), intermediate, and late states, with respect to the cytosolic domain arrangements (Ravishankar et al, 2020), although this approach did not provide high-resolution structures due to its inherent technical limitations.…”