Homo sapiens sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) is a P-type ATPase that facilitates the movement of calcium ions (Ca2+) from the cytosol into the sarcoplasmic reticulum (SR) in cardiac myocytes. The membrane protein is critical for maintaining a Ca2+ reservoir in the SR at concentrations 10,000 times higher than cytosolic levels. When depolarization events along the cardiac myocyte membrane open Ca2+ channels, Ca2+ flows into the cytosol and initiates contraction. Relaxation is induced when cytosolic Ca2+ is removed via the pumping of the ions back into the SR by SERCA2a. This movement of Ca2+ against its electrochemical gradient requires a conformational change of the protein that is facilitated by ATP hydrolysis. Thus, SERCA2a is critical for excitation-contraction coupling in cardiac muscle, the fundamental process that underlies heart function.
SERCA2a performs its ion pump function by alternating between two states. In E1, the is exposed to the cytoplasm and has a high affinity for Ca2+. Following the translocation of Ca2+ into the sarcoplasmic reticulum, SERCA2a alternates into E2, where acidic residues of the Ca2+ binding site are protonated, giving the site a low affinity to Ca2+. In both E1 and E2, the lies in the cytoplasmic domain of the protein and has high affinity for the substrate. Thus, this model shows SERCA2a bound to ATP but unfavorable to Ca2+ binding within the transmembrane domain.
Fig. 1. Simplified reaction diagram of transition states of Ca2+-ATPase (SERCA) from Kabashima et al. (2020). Analogs of ATP and Pi used for fixing the reaction intermediates are shown. TG stands for thapsigargin, a very potent inhibitor that fixes SERCA in E2. The SERCA2a state we focus on here is E2.ATP (yellow box).
SERCA2a is a 997 residue SR membrane protein that is structurally organized into 3 cytoplasmic domains , 10 transmembrane helices, and 9 short luminal loops. The cytoplasmic domains facilitate 1) ATP-binding , 2) phosphorylation , and 3) dephosphorylation , These domains are crucial for the phosphoryl transfer reactions that induce the conformational changes allowing Ca2+ transportation. Interestingly, this crystal structure shows the E2.ATP state, which highlights the prevention of phosphoryl transfer despite ATP binding due to the absence of Ca2+ (Figure 1). On the other hand, the transmembrane helices (M1-M10) regulate Ca2+ binding and translocation into the lumen. Ca2+ transportation by SERCA2a is also controlled via protein-protein interactions with phospholamban (PLN), which is nested between of SERCA2a and engages in transient, phosphorylation state-dependent interactions with the cytoplasmic domains.
On the other hand, the transmembrane helices (M1-M10) regulate Ca2+ binding and translocation into the lumen. Ca2+ transportation by SERCA2a is also controlled via protein-protein interactions with phospholamban (PLN), which is nested between of SERCA2a and engages in transient, phosphorylation state-dependent interactions with the cytoplasmic domains.
The lies within the transmembrane domain, where two Ca2+ ions interact with residues of the adjacent transmembrane alpha-helices , In particular, Glu309, Glu770, Asp799, and Glu907 , as well as Ser766, Asn767, Asn795, and Thr798 facilitate Ca2+ transport through SERCA2a. However, the E2.ATP state represents a conformation of SERCA2a that is not suitable for Ca2+ binding. This is indicated by the arrangement of the Ca2+ binding site and the protonation of its acidic residues, especially glutamate. The protonation state of the Ca2+ binding residues is expected to stabilize the empty binding site (non-Ca2+-bound) by engaging in . The carboxyl group of the Glu770 side chain acts as a hydrogen donor and acceptor to the carbonyl and amide, respectively, of Asn795. Nearby, additional hydrogen bonds are formed between the backbone carbonyl of Val304 and the hydroxyl of the protonated Glu309.
However, the E2.ATP state represents a conformation of SERCA2a that is not suitable for Ca2+ binding. This is indicated by the arrangement of the Ca2+ binding site and the protonation of its acidic residues, especially glutamate. The protonation state of the Ca2+ binding residues is expected to stabilize the empty binding site (non-Ca2+-bound) by engaging in . The carboxyl group of the Glu770 side chain acts as a hydrogen donor and acceptor to the carbonyl and amide, respectively, of Asn795. Nearby, additional hydrogen bonds are formed between the backbone carbonyl of Val304 and the hydroxyl of the protonated Glu309.
In the E2.ATP state, the lies between the N and P domains. This interaction is facilitated through interactions between the adenine ring and Phe487 and hydrogen bonds between the ATP phosphates and the protein. Hydrogen bonds are formed through water-mediated interactions between the and the direct interactions between the . Both of these arginines are within the N domain. The , however, forms hydrogen bonds with the hydroxyl side chains of Thr624 and Thr353 and the backbone amides of Thr353, Gly625, and Lys352, all in the P domain. This binding of alpha- and beta-phosphates to the N domain and gamma-phosphate to the P domain is indicative of the delivery of ATP to the phosphorylation site, even in the absence of Ca2+ binding. The ATP crosslink of the N and P domains in the E2 states suggests a conformation that may allow phosphoryl transfer.
The , however, forms hydrogen bonds with the hydroxyl side chains of Thr624 and Thr353 and the backbone amides of Thr353, Gly625, and Lys352, all in the P domain. This binding of alpha- and beta-phosphates to the N domain and gamma-phosphate to the P domain is indicative of the delivery of ATP to the phosphorylation site, even in the absence of Ca2+ binding. The ATP crosslink of the N and P domains in the E2 states suggests a conformation that may allow phosphoryl transfer.
The core of the P domain is characterized by a Rossmann fold, where 7 parallel beta-strands are linked at each end by flanking alpha-helices to form a . Beta-strands 1-4 (Pn domain) are directly involved in ATP binding, including in its sequence the Thr625 and Gly626 that interact with the gamma-phosphate. Beta strands 5-7 (Pc domain) coordinate phosphoryl transfer and stabilization of the Asp351 when it is phosphorylated. Despite the crosslink of the N and P domains, the structural change of the P domain as SERCA2a transitions from the E2 to E2.ATP state is limited to its Pn domain. ATP binding only induces a small bending of the P domain, suggesting a direct coupling between phosphoryl transfer, not ATP binding, and P domain bending. The central beta-sheet remains in a staggered form due to the between beta-strands 1 (Pn domain) and 5 (Pc domain) being a hydrogen bond between the backbone amide of Asp351 and the side chain hydroxyl of Thr700, instead of a backbone carbonyl. In this conformation, forms hydrogen bonds with Asp706, the backbone carbonyl of Thr700, and the side chain carbonyl of Asp351 to stabilize Asp351 away from the gamma-phosphate of ATP, contributing to the prevention of phosphoryl transfer in the absence of Ca2+ (Video 1). Video 1. Staggering/aligning of the central β-sheet in the P-domain from Kabashima et al (2020). The video shows the different alignments of Asp351 in E2.ATP and E1.ATP.2Ca2+ states that affect its interaction with ATP (or ACP). K684, T701, and D707 shown here correspond with Lys683, Thr700, and Asp706, respectively, of the SERCA2a isotype. Pink broken lines represent hydrogen bonds, and light green ones coordination of the divalent cation (green sphere).
Despite the crosslink of the N and P domains, the structural change of the P domain as SERCA2a transitions from the E2 to E2.ATP state is limited to its Pn domain. ATP binding only induces a small bending of the P domain, suggesting a direct coupling between phosphoryl transfer, not ATP binding, and P domain bending. The central beta-sheet remains in a staggered form due to the between beta-strands 1 (Pn domain) and 5 (Pc domain) being a hydrogen bond between the backbone amide of Asp351 and the side chain hydroxyl of Thr700, instead of a backbone carbonyl. In this conformation, forms hydrogen bonds with Asp706, the backbone carbonyl of Thr700, and the side chain carbonyl of Asp351 to stabilize Asp351 away from the gamma-phosphate of ATP, contributing to the prevention of phosphoryl transfer in the absence of Ca2+ (Video 1).
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Inoue, M., Sakuta, N., Watanabe, S., Zhang, Y., Yoshikaie, K., Tanaka, Y., Ushioda, R., Kato, Y., Takagi, J., Tsukazaki, T., Nagata, K., Inaba, K. (2019).Cell Reports, 27(4), 1221-1230.
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