Calcium ATPase
Created by Natalie Zuffi
Calcium ions are used as signals in a variety of cellular responses. The flood of calcium ions in the cytoplasm triggers these mechanisms. However, the signal would be no good if it were always present so the calcium ions have to be removed from the cytoplasm when the signal response is not desired. The calcium pump returns the calcium ion level in the cytoplasm to sub-micromolecular levels as it functions as an ATP-driven pump (3). The calcium pumps pump these calcium ions back into the sarcoplasmic or endoplasmic reticulum (2).
In striated muscle, calcium ions are thought to be primarily stored in the sarcoplasmic reticulum while in smooth muscle and non-muscle tissue the calcium ions are thought to be stored in the endoplasmic reticulum. In muscle contraction, calcium is released into the cytoplasm my calcium release channels or ryanodine receptors to initiate the contraction. The calcium pump then recovers these ions to promote relaxation of the muscle. Calcium binds to the calcium pump in a cooperative manner (2). Two calcium ions bind as well as phosphorylation by ATP occur on the cytosilic side of the transmembrane protein (4). A conformational change occurs and the calcium ions are released into the lumen. During dephosphorylation of the pump, hydrogen ions bind to the lumen site and are transported to the cytosol (4).
The sodium-potassium pump is also an ATP pump and has similar primary and tertiary structure to the calcium pump. The sodium-potassium pump with PBD ID 3B8E gives an E-value of 8.2012E-92 and a score of 860 in sequence similarity on the RCSB Protein Data Bank. The sodium-potassium pump with PBD ID 2ZXE gives a score of 2196.87 in tertiary structure similarity on the RCSB Protein Data Bank. The sodium-potassium pump uses an ATP molecule to transport three sodium ions out of the cell and two potassium ions into the cell. This maintains electrochemical gradients important in animal cells. One use for this electrochemical gradient is generating action potentials (5). Both the calcium pump and the sodium-potassium pumps are ATPase transmembrane proteins that create a concentration gradient for different ions so that the loss of the gradient will signal some cellular response.
The potassium-transporting ATPase (3IXE) has sequence similarity to the calcium pump with an E-value of 2.66034E-90 and a score of 847 on the RCSB Protein Data Bank. The potassium-transporting proton is also an ATPase transmembrane proton. It pumps protons across the gastric membrane to create a low pH in the stomach by generating a million-fold proton gradient (1). A similar in function protein, the plasma membrane proton pump (3B8C) has a score of 1423.34 in tertiary structure similarity to the calcium pump according to the RCSB Protein Data Bank. This plasma membrane protein is a type III P-type ATPase, whereas the sodium-potassium pump and the calcium pumps are type II (6).
The Copper Pump (2VOY) has an E-value of 1.85061E-18 and a score of 227 in primary structure similarity and the Copper Pump (3AID) has a score of 508.59 in tertiary structure similarity to the calcium pump according to RCIS Protein Data Bank. This protein is a type I P-type ATPase. It maintains copper homeostasis to provide sufficient copper for metalloenzyme biosynthesis, since copper often acts as a coenzyme, while preventing oxidative damage and free radicals in the cell (10).
The Calcium ATPase consists of 994 amino acids. The protein includes a head in the cytoplasmic region and a transmembrane region consisting of ten helices (M1 - M10). Four stalk leading from helices M2, M3, M4, and M5 connect the transmembrane section to the cytoplasmic headpiece. The headpiece looks split with one extension consisting of ß-strands and the other extension consisting of an alternation between strands and helices. This second extension is where the phosphorylation and ATP sites are located (McIntosh 2000) The cytoplasmic headpiece consists of three domains, N, A, and P (8). The nucleotide-binding domain (N) and the phosphorylation domain (P) are part of the catalytic site. The A is the actuator domain and appears to be involved in the transmission of conformational changes that occur in the protein (3).
When there is a rise in calcium levels in the cytoplasm, the two high-affinity Ca2+-binding sites become saturated (3). Negatively charged residues on M4, M5, M6, and M8 are suspected to cause these high-affinity binding sites (8). When calcium first binds the N and P domain are in an "open jaw" conformation. TNP-AMP, an analog of ATP, binds to the N domain (3). The binding pocket for the ATP analog is positively charged while the region around the phosphorylation site is negatively charged (8). Amino acid, Asp 351 in the P domain needs to be phosphorylated, but in the "open jaw" conformation, the ATP analog in the N domain is still too far from Asp 351 (3). The binding of the ATP analog and a metal ion such as Mg2+ brings the headpieces together. The adenine ring of the ATP analog binds to the N-domain around Phe 487 and the γ-phosphate binds to the Asp-351 of the P domain (7).
When bound, the calcium ions are positioned side by side on one side of M5 (4). M4, M5, M6, and M8 form a circle and M5 is located at the center of the molecule extending from the luminal surface to the center of the P domain (4 and 8). The portions of M4 and M6 that contribute to calcium binding are unwound (4). The disruption of this secondary structure allows more side-chain oxygen atoms to contribute to calcium coordination since they provide a hydrophilic pathway leading to the calcium binding sites. The side-chain oxygen atoms of Asn-768, Glu-771, Thr-799, Asp-800, and Glu-908 contribute to the binding of the first calcium ion. The main-chain oxygen atoms of Val 304, Ala 305, Ile307 and the side-chain oxygen atoms of Asn 796, Asp 800, and Glu 309 contribute to the binding of the second calcium ion. The rows of exposed oxygen atoms constrict at the binding sites and also trap a water molecule. Hydrogen-bond networks stabilize the two calcium-binding sites. These networks are influential in the cooperative binding of calcium ions in the Calcium-ATP pump. The trapped water molecule also participates in hydrogen bonding and allows this water molecule to be displaced (8).
After the phosphorylation of Asp-351, the gap between the A domain and the P and N domains close. This leads to the occlusion of the calcium ions between the cytoplasmic and luminal sides (3). The protein then loses its ability to re-phosphorylate ADP and loses its high affinity for calcium. The protein opens up towards the luminal side (3). As calcium dissociates, the transmembrane helices rearrange. M5 bends, which causes the P domain to incline since M5 extends into the P domain, and then M3 - M6 incline. The incline of the P domain causes M3 - M6 to incline by van der Waals interactions between the P domain and the helices. The P domain also conducts the movements of M1 and M2 although they are not directly connected. The movement of these two helices is associated with calcium dissociation from the ATPase (9).
The binding site for the first calcium ion is called site I and the site for the second calcium ion is site II. Site I contains many exposed carbonyl oxygen atoms from M5, M6, and M8 as mentioned above. Site II is practically on the M4 helix, with three residues' carbonyl groups contributing and Glu-309 capping the bound calcium ion from above. For calcium dissociation, residues Asn 796, Thr 799, and Asp 800 rotate about 90º. This rotation results in Asp 800 replacing Thr 799 to face the molecules center and results in Asn 768 facing Site II. The result of the inclination of M5 is less oxygen atoms free at Site I, which means these oxygen atoms are no longer available for calcium binding (9).
Once the calcium ions dissociate into the luminal space, water enters the catalytic site and hydrolyses the phosporylated aspartic acid residue to return the calcium-ATPase to its original dephosphorylated conformation. It is thought that the release of the calcium ions induces movement in the A domain to open up the protein and allow water molecules into the phosphoenzyme (3).
The secondary structure of the calcium pump is very important in creating the domains and the tertiary structure links them through hydrogen bonding. The P domain is assembled into a seven-stranded parallel ß -sheet and eight short helices. The phosphorylation site, Asp 351 is located on the C-terminal portion of central ß -strand. The N domain contains seven antiparallel ß -sheets in between two helix bundles. The A domain consists of about 110 residues between M2 and M3 and the 40 residues of the N-terminal that form two short helices. This domain is connected to the transmembrane region by long loops. A loop ( L67) connects M6 and M7. This loop can mediate interactions between the P domain and transmembrane domain. Therefore, when calcium binds, the induced changes in M6 will be transmitted to the P domain through L67 (8). The middle of M5 is linked to L67 through Arg-751 on M5 (9).
More Hydrogen bonding between residues help create the domains and help them function as a unit. Asp-703 and Asp-707 are hydrogen-bonded and seem to be involved in the coordination of the catalytic magnesium ion for the process of phosphorylation (4). The P domain is connected from its P1 helix to the top part of M3 through hydrogen bonds involving Glu-340 on the P1 helix and the NH group of Leu-249 and the OH group Tyr-247 on M3 (8 & 9). Other critical hydrogen bonds are between Gly-354, Arg-604 and Asp-737, Asn-739, which function in linking the movement of the hinge between the N and P domains to M5. This linkage transmits the phosphorylation signal to the calcium binding sites resulting in the release of calcium (8).
Primary structure influences secondary structure, which influences tertiary structure. The domains of the Ca2+-ATPase have various functions contributing to the overall goal to pump calcium from the cell into the sarcoplasmic reticulum against a concentration gradient. This transmembrane protein has its domains linked in such a way that changes in one domain will influence the other domains so the protein functions properly as a unit due to hydrogen bonds between different helices, ß-strands, and loops.