Sacroplasmic reticulum Ca2+-adenosine triphosphatase Ca2-E1-AMPPCP from (PDB: 1T5S), or SERCA, from the fast twitch muscle of Oryctolagus cuniculus is a P class ATPase that primarily functions to pump calcium ions back into the sarcoplasmic reticulum (SR) lumen from the cytoplasm to relax muscles after contraction. The pump changes between two conformations, named E1 and E2, in order to ensure that the calcium ions can bind the transmembrane channel, have restricted access back into the cytoplasm, and be released into the SR lumen. SERCA is composed of one A chain, 994 residues in length (1). The molecular weight of SERCA is 109489.69 Da and has an isoelectronic point estimated at pH= 5.15 (2).

          Muscle fibers are coated in the sarcolemma and are composed of multiple myofibrils. The SR forms a network around each myofibril and is a reserve of calcium ions. During muscle contraction, a neuron innervating the muscle fiber is stimulated to release acetylcholine, which binds to receptors in the neuromuscular junction and opens Na+ and K+ channels so that Na+ can diffuse into the muscle fiber and K+ to diffuse out. This causes a rapid change in membrane potential, inducing an action potential that propagates down T tubules, which are sarcolemma infoldings that penetrate through the muscle fiber (3). This process opens voltage gated channels of the SR to release calcium ions into the cytosol. Calcium ions bind to troponin on tropomyosin, changing the conformation of actin myofilaments to expose their active sites. Adenosine triphosphate (ATP) powers myosin myofilaments that bind to actin to pull the thin actin myofilaments causing a contraction. The sum of myofilament contractions causes the muscle fiber to contract and the contraction of multiple muscle fibers allows organisms to move skeletal muscles. Muscle relaxation results when the signal to contract stops, closing the voltage gated channels and allowing for SERCA to reabsorb calcium ions into the SR lumen (3).

          The mechanism by which SERCA pumps calcium ions into the SR has been extensively studied. SERCA in an unbounded, E1, state binds two calcium ions to two high-affinity binding sites accessible from the cytosolic side (4). ATP then binds to the pump and hydrolyzes ATP to transfer a phosphate group to a specific aspartate group, forming a high energy acyl phosphate bond called E1~P. This transfer is followed by a conformational change, a state called E2, that decreases the affinity of the binding sites and exposes the channel with calcium ions intact to the SR lumen (4). The calcium ions then dissociate into the SR lumen and dephosphorylation resets the conformation to the E1 state, ready to pump another two calcium ions. What is thought to drive the conformation change between E1 and E2 is that the free energy of hydrolysis of the aspartyl-phosphate bond in E1~P is greater than in E2-P, and this reduction in free energy of the aspartyl-phosphate drives the conformation to a more thermodynamically stable state (4). The binding of the calcium ions is representative of how channels attract and bind ions for transfer in cells. In solution, the calcium ions have a hydration shell of water molecules oriented geometrically in a characteristic way. The amino acids that constitute the high affinity binding sites are oriented such that carboxyl oxygen atoms of aspartates and glutamates side chains and the backbone carboxyl oxygen atoms of other amino acids replicate the geometry of the hydration shell around the incoming calcium ion. In contrast, the E2 state configuration change from E1 alters the orientation of the amino acids that constitute the binding sites and can no longer mimic the hydration shell as effectively, causing a decrease in affinity at the binding sites for the calcium ions (4). This mechanism by which SERCA transports calcium ions back to the SR using ATP serves as a model for understanding how other P class ATPases function.

          SERCA is structurally characterized by two domains: a transmembrane domain (M) consisting of ten alpha-helical segments, and a large cytoplasmic domain consisting of a nucleotide binding domain (N), a phosphorylation domain (P), and an actuator domain (A) (5). Each step in the mechanism previously outlined is accompanied by conformation changes of these domains. ATP binding leads to a 90° rotation of N and a 30° rotation of A, pulling transmembrane helices 1 and 2, thereby closing the cytosolic entrance and preventing backflow of calcium ions (5,6). The E1-P to E2-P sate change after phosphorylation of Asp-351 and disassociation of ADP induces a 110° rotation of A and rearranges M so that M opens facing the SR lumen (5). Nucleophilic attack by water on E2-P releases the phosphate and is guided by a TGES sequence in A (residues 181- 184) and reverts the conformation to E1 (5).

          The E1-P state with calcium ions bound in M can be further studied by using non hydrolysable variants of ATP, such as AMPPCP and ADP:AlF4- , which stop further conformation changes that would normally occur. Crystallographic analysis of E1 with calcium ions bound show no evident relation between N and the phosphorylation site (Asp-351) in P which poses unsolved questions about the structural basis of ATP binding and the phosphoryl transfer coupled to calcium ion occlusion in the E1-P state (6). After nucleotide binding, the M2 helix is elongated by four residues, drawn into the membrane at the N-terminal (luminal) end, and the M1-M2 segment results in the formation of a kink that permits Phe-57 to engage in hydrophobic interactions with Val-63, Leu-65, and Leu-98 (6). The charged residues at the N-terminal (cytosolic) end of M1 are exposed to a more polar and thermodynamically stable state as a result (6). The nucleotide binding pocket at the interface of N and P is highly conserved among P class cation pumps.Arg-489, Arg-560, and Arg-687 stabilize the bent conformation of the nucleotide that manifests a hydrogen bond between the 3’OH and the beta-phosphate group (6). Magnesium ion and Lys-684 side chain interact with the gamma-phosphate of AMPPCP, which also receives hydrogen bonds from the side chains ofThr-353, Thr-625, and the main chain of Gly-626 (6). An orientation that facilitates phosphoryl transfer is further supported by magnesium ion coordinating with the carboxyl of Thr-353 and the side chains of Asp-703 and Asp-351 at the phosphorylation site. Positive Lys-684 is in close proximity with the negative phosphate, nucleotide, and Asp-351 to stabilize the negative charges (6). This arrangement is further stabilized by two magnesium ions that coordinate with the alpha and beta phosphates of the would-be ADP leaving group (6). Transition state and product separation of E1-P is stabilized by main chain amides of Gly-626 and Thr-353 (6).

          PSI-Blast searches through a database to find proteins with similar primary structure to a protein of interest and the Dali server searches through a database to find proteins with similar tertiary structure. PSI-Blast searches through a protein query subtracting points from an E score when there is homlogy between prtoein primary structures and adding to the E score when there are gaps between protein primary structures. The Dali server uses a sum of pairs method that compares intermolecular distances to determine tertiary structre and fold similarities which factor into a Z score.  In searching for good proteins for comparison, theNa+, K+ ATPase in the Na+ bound state (PDB: 4HQJ) protein from Sus scrofa is suitable as the protein was rated with an E value of 0.0 by PSI-Blast and a Z-score of 38.4 (7,8). An E value below 0.05 means the comparison protein has a very similar primary structure and a Z score above 2 indicates high similarity in tertiary structure. Na+, K+ ATPases are also P class cation pumps and this particular ATPase similarly uses magnesium ion to stabilize phosphryl transfer and bonded ADP. The ATPase, as its name suggests, uses the energy of ATP to force Na+ out of the cell and K+ into the cell, both against their concentration gradients. SERCA and the Na+, K+ ATPase differ primarily in tertiary structure where SERCA has one alpha subunit in its total structure while Na+, K+ ATPase has an alpha subunit and two beta subunits (7,8). The M region likewise must have differences between SERCA and Na+, K+ ATPase because the channels have specific binding sites for Na+, K+, and Ca2+ which means the channels must be able to mimic the hydration shells specific to each cation. Although SERCA does not have two beta subunits, the alpha subunits of both proteins are similar in that the M domains of both consist of 10 transmembrane alpha helices and both alpha subnunits have an A, P, and N domain on the cytosolic side (5). One key difference in the primary structures of the two proteins is that Asp-369 is phosphorylated in the case of the Na+, K+ ATPase whereas SERCA is phosporylated at Asp-351 (5).

          The Na+, K+ ATPase performs an important function in heartbeat. If sodium ions are not pumped out of the cell and potassium is not brought in, the electrochemical and concentration gradients in the cell will not return to pre-contraction levels which indirectly inhibits the Na+, Ca2+ antiporter and calcium levels remain high. This increases the duration and intensity of a heart contraction. Cardiac glycosides like digitalis inhibit the dephosphorylation and conformation change of Na+, K+ ATPase to produce this effect, which helps individuals suffering from congestive heart failure (5). SERCA continues to work during this process to reabsorb calcium into the SR lumen and helps continue the contraction, relaxation cycle that keeps the heart beating. The importance of studying SERCA and by extension P class ATPases is evident in advancing life-saving medical treatments.