Abstract
The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+ from the cytosol into the sarco(endo)plasmic reticulum (SR/ER) lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca2+-binding sites and the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of 5 of SERCA's 10 transmembrane helices. The mutant displays a marked slowing of the Ca2+-binding kinetics, and its crystal structure in the presence of Ca2+ and ATP analog reveals a rotated headpiece, altered connectivity between the cytosolic domains, and an altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca2+ sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in interdomain communication between the headpiece and the Ca2+-binding transmembrane region.
Highlights
The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+ from the cytosol into the sarco(endo) plasmic reticulum (SR/ER) lumen, driven by ATP
The E2 to Ca2+-bound E1 form (Ca2E1) transition of E340A is significantly slower than that of the WT, with an increase of t1/2 of about threefold (Fig. 1 D–F and SI Appendix, Fig. S1). This suggests that the effect of the E340A mutation on the overall turnover of SERCA may be caused by a delay in the conformational change associated with Ca2+ binding, i.e., the HnE2 → E1 → Ca2E1 transition
In order to assess whether the differences we found between the crystal structures of WT SERCA and E340A might reflect stable conformations occurring in the native membrane environment, and to find further explanations for the observed functional impairment of the mutant, we carried out 3 × 200 ns of all-atom molecular dynamics (MD) simulations of each structure reconstituted in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer
Summary
E340A mutant (red) in an enzyme-coupled assay. WT: 3.45 ± 0.26 (n = 9); E340A: 0.86 ± 0.22 (n = 9); background (dashes): 0.18 ± 0.06 (n = 18). M4 is linked to the so-called P1 helix (Pro337-Cys344), a short α-helix that runs roughly parallel to the membrane surface, at the membrane-facing side of the P domain In such a location, P1 may be a key element of interdomain communication: it connects directly to a β-strand in the P domain ending with the phosphorylated aspartate residue, and it makes contact with the cytosolic end of M3 and to the loop between M6 and M7 (L6-7), which has been shown to play an important role in SERCA catalysis [11,12,13,14,15,16,17] (Fig. 1A). Our data link the structure to Glu340’s functional importance and provide insight into the interdomain communication that guides Ca2+ transport by SERCA
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