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

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.

An intracellular pathway controlled by the N-terminus of the pump subunit inhibits the bacterial KdpFABC ion pump in high K+conditions

“…An intracellular pathway between helices M1, M2 and M4 is also postulated for the sarcoplasmic reticulum Ca 2+ -ATPase [15], and was observed in the first crystal structure of SERCA [16], as well as in inhibitor-bound structures of SERCA [17]. The M1 helix probably plays a role in the occlusion of the bound Ca 2+ in SERCA [18, 19]. Palytoxin, which converts the Na+, K + ATPase to a channel-like protein, opens a continuous ion-pathway which also passes between M1, M2, M4 and M6 on the cytoplasmic side [20].…”

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.…”

“…In Saccharomyces cerevisiae, release of the autoinhibition upon glucose sensing has been linked to phosphorylation of Ser 899 (Ser 901 in N. crassa) by Ptk2 (34) and tandem phosphorylation of Ser 911 /Thr 912 (Ser 913 /Thr 914 in N. crassa) by an unknown kinase (35). Ser 899 -P leads to an increase in K m (Michaelis constant), and Ser 911 -P/Thr 912 -P to an increase in V max (9,(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). Notably, neither of these residues is located at the binding interface of the cis-acting R domain (Fig.…”

Structure of the hexameric fungal plasma membrane proton pump in its auto-inhibited state

“…To develop a model for the catalytic cycle of Pma1, we generated a series of SERCA-based homology models. The transition state of phosphorylation, E1~P, is linked to ion occlusion within the M domain 30,31 . Interestingly, in this state Asp730 moves away from Asn154, implying a break of the suggested hydrogen bond (Fig.…”