The SERCA residue Glu340 mediates inter-domain communication that guides Ca²⁺transport

Abstract

The sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) transports Ca 2+ ions from the cytosol into the SR/ER lumen. SERCA's activity is dependent on a tight coupling between movements of the transmembrane helices that comprise the two Ca 2+ binding sites and the cytosolic ATPase headpiece. We have addressed the molecular basis for this intramolecular communication by analysing the structure and functional properties of the SERCA mutant E340A. The conserved residue Glu340 is located at a strategic position in the ATPase, at the interface between the phosphorylation domain and the cytosolic ends of six out of SERCA's ten transmembrane helices. The mutant displays a reduced rate of binding Ca 2+ ions from the cytosol. The structure of E340A reveals a rotated headpiece, altered connectivity between the cytosolic domains and an altered interaction pattern around the mutated residue Glu340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilisation of SERCA in a more occluded state, causing slowed dynamics in the ion binding region. This finding underpins the crucial role of Glu340 in the interdomain communication of SERCA.

Structural basis of the conformational and functional regulation of human SERCA2b, the ubiquitous endoplasmic reticulum calcium pump

“…In this context, mutation of E340 located at the P1 helix causes a severe functional defect due to the loss of interdomain communications. [ 34 ] The crystal structure of the SERCA1a E340A mutant in the E1∙2Ca 2+ ‐AMPPCP state showed a marked downward shift of the cytosolic headpiece cluster toward the membrane, as was observed for the SERCA2b T1032stop mutant. In addition, the SERCA1a E340A mutant displayed a slower Ca 2+ ‐binding rate at the E2‐to‐E1∙2Ca 2+ transition step.…”

“…[30] Previous mutational, biophysical, and computational studies, indeed, indicate that the cytosolic-domain rearrangements induced by ATP binding, hydrolysis, and ADP release are coupled to TM-domain movements to promote the entry of two Ca 2+ ions from the cytosol and their release into the ER lumen. [31][32][33][34] It is thus well-supported that the interplay between the Ca 2+ binding sites in the TM domain and the phosphorylation site in the cytosolic domain is key to the ATP-driven Ca 2+ transport function of SERCAs. [30,35,36] Considering the above-described situation of this field, we herein focus on recent findings of unique structural and functional features of…”

“…ATPases. [29][30][31][32][33][34][35] MD simulations have provided a number of proposals by tracking the trajectories of conformational changes, with high-resolution structures typically being used as starting/ending models. [57] Single-molecule FRET (smFRET) detects biological interactions as well as intramolecular motions, along with reaction coordinates represented by changes in the distance between individual particles or domains to which donor and acceptor fluorophores are attached.…”

“…In addition, the SERCA1a E340A mutant displayed a slower Ca 2+ ‐binding rate at the E2‐to‐E1∙2Ca 2+ transition step. [ 34 ] Similarly, the SERCA2b T1032stop mutant also showed a slower E2‐to‐E1 transition than SERCA2b WT. [ 39 ] These findings suggest that the LE plays a significant role in regulating the overall conformation and Ca 2+ binding kinetics of SERCA2b.…”

Structure of the hexameric fungal plasma membrane proton pump in its autoinhibited state

“…S11), we generated a series of homology models based on SERCA in the transition of phosphorylation ( E 1~P), open-to-outside ( E 2P), transition of dephosphorylation ( E 2~P), and dephosphorylated ( E 2) states. The transition state of phosphorylation, E 1~P, is linked to ion occlusion within the binding site ( 30 , 31 ). In this state, Asp 730 moves away from Asn 154 , implying a break of the suggested hydrogen bond (Fig.…”