The SERCA1a calcium pump (PDB ID: 3w5A) is vital to normal muscle function as it establishes a calcium ion concentration gradient that relaxes muscles after contraction. This calcium pump is usually associated with fast twitch muscles, which generate short bursts of energy and fatigue quickly (1). SERCA pumps, the main regulators of cytoplasmic Ca²⁺, are located in the golgi membranes and sarcoplasmic reticulum. Normally, the pump transports two calcium ions for each hydrolyzed ATP and cotransports hydrogen ion with calcium ion. This pump belongs to the P-type ATPase family as it is associated with temporary ATP energy conservation. Scientists are extensively studying SERCA1a, as the proper function of this protein is critical to muscle movement. Particularly, there are multiple states of this pump and each state needs to be understood thoroughly in order to identify potential defects. Malfunctions of this pump can lead to many illnesses including Brody’s disease and Darier’s disease. Brody’s disease is caused by reduced activity in quadriceps muscles and decreased SERCA1a expression in twitch muscles. Darier’s disease is an autosomal dominant disorder caused by mutations in SERCA1a (2).

Structure and Function

Toyoshima et al. obtained the crystralline structure of SERCA1a by isolating and purifying the molecule from rabbit hind muscle. Subsequently, the investigators crystalized the calcium pump by vapor diffusion in 40mM magnesium sulfate and magnesium chloride, avoiding the presence of any calcium. The authors used molecular replacement techniques to determine the crystal structure. Furthermore, the study investigated the interaction between the calcium pump and sarcolipin by creating a mutant form of the protein in HEK 293 cells. The mutant form included marked Cys residues and flag tag attached to the N-terminus of sarcolipin and copper phenaltroline induced crosslinking. The authors then probed the molecule with anti-flag antibodies (3). 

This calcium pump has three major states, the E2 state without bound ions, Mg²⁺ bound E1 state and the 2Ca²⁺ bound E1 state (Figure 1). A complete representation of the  2Ca²⁺ bound E1 state in the same organism could not be found.  The present analysis discusses the structure and function of the Mg²⁺ bound E1 state in rabbits, which occurs when there is a lack of calcium ions. E1-Mg²⁺ bound SERCA1a in rabbit has a molecular weight of  223 kDa and an isoelectric point of 5.17 (6). The sequence of SERCA1a consists of 995 amino acid residues. This protein is made up of two major subunits composed of alpha helices, beta sheets, and random coils, and there are no known drug complexes associated with the structure. Each subunit is embedded in the cell membrane in order to facilitate ion transport (7).

There are four ligands associated with this calcium pump: (1) Phosphatidylethanalamine (PTY), (2) 2',3'-O-[(1r)-2,4,6-trinitrocyclohexa-2,5- diene-1,1-diyl]adenosine 5'-(dihydrogen phosphate) (TM1),  (3) Mg²⁺, and (4) Na²⁺.  None of these ligands induce crystallization. Mg²⁺  occupies Ca²⁺ binding sites, preventing the phosphorylation of the protein without adequate Ca²⁺ present. This expedites the reaction cycle and causes shorter intervals between muscle contractions.  Na²⁺ is usually transported across the plasma membrane and encourages movement of other ions. Tm1 attracts positively charged ions, such as Mg²⁺ and Na²⁺ in the absence of calcium (7). This occupies binding sites until there is an adequate calcium concentration. PTY regulates the activation of the calcium pump by modulating Ca²⁺ affinity (8).

 The E1-Mg²⁺ bound form is very similar to the E1-2Ca²⁺ form; however, a significant difference is the presence of an opening in the E1-2Ca²⁺ form that exposes the two major transmembrane Ca²⁺ binding sites. In the Mg²⁺ bound E1 state, Mg²⁺ take the place of high-affinity calcium binding sites with a binding affinity of around 10^-3 M. Mg²⁺ does not allow phosphorylation without prior Ca²⁺ binding, thus expediting the reaction. Once Ca²⁺  is bound to the two major binding sites, the protein undergoes a conformation change that exposes Asp 381,the phosphorylation site.  Glu 486, Thr 484, and Thr 171 block the phosphoryl transfer in the Mg²⁺ bound E1 state. Inhibiting untimely phosphorylation is especially important for fast twitch skeletal muscles, which rely on short intervals between muscle contractions. 

The interaction of alpha and beta subunits of SERCA1a creates binding sites primarily for Ca²⁺ that are critical to the protein function. Ca2+ binding site I is composed of a couple amino acids, including Glu 771, Asp 800, Ala 305, and two water molecules. These molecules regulate Mg²⁺ binding through ionic interactions by the carboxyl groups on Asp 800 and Glu 771 along with two water molecules. The negative carboxyl groups attract positive divalent ions. Ala 305, located at the C-terminus of the alpha subunit, and is responsible for Mg²⁺ binding in the E1-Mg²⁺ bound form and Ca²⁺ binding in the E1-Ca²⁺ bound form. Although, Glu 771 and Asp 800 form Ca²⁺ binding site I, this site is vacant as these two amino acid residues do not bind to Ca²⁺ (3) . 

The area within the binding sites cannot fit both Ca²⁺ and Mg²⁺, so once the Ca²⁺ concentration is large enough, Ca²⁺ will displace Mg²⁺. Successful binding of Ca²⁺ to Ca²⁺ binding site I induces the formation of Ca²⁺ binding site II, which is closed in the E1-Mg²⁺ bound form. Compared to Ca²⁺ binding site I , Ca²⁺ binding site II is located closer to the cytoplasmic surface of the membrane (3). Glu 771 and Asp 800 move closer to binding site I which provides room for Arg 768 and Glu 309 to form binding site II. Binding site II includes Ala 305, Ile 307, Glu 309, and Arg 796. In the E1-Mg²⁺ bound form, the closed binding site II is located directly adjacent to binding site I.  Therefore, the interaction between the two binding sites promotes transition from from the E1-Mg²⁺ bound state to the E1-Ca²⁺ bound state (Figure 2). Ultimately this prevents premature phosphorylation of SERCA1a.

 Sacrolipin (PDB ID: 3W5A), a protein with similar structure and function as phospholamban, has an inhibitory role on SERCA1a function. Sacrolipin most likely binds to SERCA1a in the E1-Mg²⁺ bound state and decreases Ca2+ binding affinity (3).  Sarcolipin binds in the interface of the alpha and beta subnits of SERCA1a .  The Val 49 residue of sarcolipin crosslinks with the Val 89 residue of the main calcium pump. The interaction of sarcolipin with SERCA1a prolongs the E1-Mg²⁺ bound state while suppressing the transition to the E1-Ca²⁺ bound state. This stabilization effect decreases the energy level of the protein and decreases the affinity for Ca²⁺. Furthermore, sarcolipin increases the energy required to rotate the A-domain of SERCA1a in order to discourage the transition to the E1-Ca²⁺ bound state. Phosphorylation of sarcolipin at Thr 5 decreases its inhibitory effects (3).

Related Proteins

There are many proteins that have similar structure and function as the SERCA1a calcium pump. The authors found similar proteins by running a series of PSI-BLAST and Dali queries. PSI-BLAST compares protein primary structure and identifies proteins with overlaps in sequence. The PSI-BLAST querie outputs an E-value which attempts to quantify the difference between two proteins. The lower the E-value the better the match; generally a threshold of less than 0.005 suggests significant similarity. The Dali Server compares tertiary structure of proteins and performs statistical tests that output z-scores. Significant similarities have higher z-scores; generally statistically significant z-scores are higher than 2.

In particular, E2p form of Sodium Potassium pump from the pig (PDB ID: 3N23) has nearly the same sequence and tertiary structure with a PSI-BLAST E-value of approximately 10^-85 and a Dali Z-score of 27.8 (9,10). Other similar structures include the copper transporting pib-type ATPase (PDB ID: 3RFU, E-value of 2x10^-72 and Z-score of 14.5) and the copa ATP binding domain (PDB ID: 2B8E, E-value of 9x10^-40 and Z-score of 16.6). All three of these proteins have similar functions to SERCA1a, primarily consisting of active ion transport (9,10). The sodium-potassium ATP-ase also actively transports ions as the protein moves three Na+ out of the cell and two K+ into the cell (11). The copper transporting pib-type ATPase and the copa ATP binding domain move heavy metals and copper ions out of the cytoplasm (9-13). 

The E2p form of Sodium Potassium pump is made up of three different subunits whereas SERCA1 is only made up of two. The sodium potassium pump is more globular, and it is hard to distinguish between the subunits. SERCA1a is characterized as T-shaped. These two proteins are both bound to Mg²⁺. SERCA1a has four side chain oxygens that regulate Mg²⁺ binding approximately 800 residues into the sequence while sodium-potassium ATPase binds approximately 200 residues into the sequence.  Unlike SERCA1a, the E2p form of sodium potassium pump binds to ouabain, which stabilizes the phosphorylated form of the protein (11).

Conclusion:

            SERCA1a is vital in controlling calcium concentrations during muscle contractions. Research on this calcium pump sheds light on movement disorders. The E1- Mg²⁺ state of SERCA1a is critical as, among other functions, it ensures that the protein does not become phosphorylated until there is an adequate Ca²⁺ concentration. Mg²⁺ blocks the first binding site and the second one is closed off until Ca²⁺ is bound to the first one. SERCA1a makes mobility possible through this involved interaction of amino acids and ligands.