1. Sarcoplasmic Reticulum Ca 2+ -ATPase Josh McGee

2. Introduction Ca2+-ATPase is grouped into a large family of ATP dependent ion pumps known as P-type ATPases In skeletal muscle, the dominant P-type ATPase is sarco(endo)plasmic reticulum Ca 2+ -ATPase also known as SERCA SERCA pump activity lowers the concentration of Ca 2+ in the cytoplasm while at the same time raising that of the sarcoplasmic/endoplasmic reticulum Two key events in the functional cycle of SERCA are ATP hydrolysis and the formation of an acid-stable aspartyl phosphate

3. Structure 994 amino acids Two conformations called E1 and E2 Ten transmembrane domains known as M1- M10 Three cytoplasmic loop domains – N-Domain – P-Domain – A-Domain

4. Transmembrane Domains = Blue N-Domain = Yellow P-Domain = Green A-Domain = Red

5. Transmembrane Domains Consists of ten alpha helices and contains the calcium binding sites E1 conformation the binding of two calcium ions takes place and is required for the phosphorylation from ATP to occur The first calcium ion is bound by N 768, E 771 on M5, T 799, D 800 on M6, and E 908 on M8 The second calcium binding site has contributions from main chain carbonyl oxygens on M4 and side chain oxygens from E 309 on M4, and N 796, D 800 on M6 The M4 sequence PEGL 311 lies at the heart of this second site.

6. N-Domain Known as nucleotide domain Contains the ATP binding site K 492 It contains seven stranded anti- parallel β-sheets sandwiched between two α-helix bundles and the binding site is located under a flap created by one of the α-helices There is a large distance between this site and the target Aspartate (D 351 ) for phosphorylation transfer It is believed that the N-domain is very flexible and mobile too make up for this distance

7. P-Domain Known as the phosphorylation domain Phosphorylation occurs on Asp 351 P-domain is characterized by six stranded parallel β-sheets flanked by 3 α-helices on each side Firmly connected to the transmembrane domain by extensions of M4 and M5 When both calciums are bound to the transmembrane domain then phosphorylation is available for the aspartate D 351

8. P-Domain In the presence of calcium the P-domain is available for phosphate transfer from the ATP bound within the N- domain In the absence of calcium it interacts with TGES 184 loop of the A-domain Represents a kind of switch under the control of calcium binding that selects between the N-domain and the A-domain

9. A-Domain Known as the transduction or actuator domain Composed of primarily β- strands which form a distorted jelly roll β-stands are tethered to the M2 and M3 transmembrane domains Contains the sequence loop TGES 184 which interacts with the P-domain in the absence of calcium

11. Pump Mechanism In the E1 conformation, Ca 2+ -ATPase exposes two high affinity calcium binding sites to the cytoplasm. Binding of the first calcium induces a reorientation of the transmembrane to form a second calcium binding site Upon occupancy of the second site phosphorylation on D 351 by Mg-ATP precedes a transition to the E2 conformation In the E2 conformation the three cytoplasmic domains all undergo large, rigid body movements. The A-domain rotates 110°, the P-domain rotates 30°, and the N-domain rotates 50° The conformational changes that accompany the reaction with ATP pull the transmembrane helices and close a cytosolic entrance for calcium which prevents backflow before the calcium is released into the lumen

12. Pump Mechanism In the E2 conformation the calcium binding sites have been transformed into low affinity sites and are exposed to the lumen of the sarcoplasmic reticulum Upon the release of calcium into the lumen of sarcoplasmic reticulum the transmembrane binding sites signal the hydrolysis of the aspartyl phosphate group (D 351 ) and returns the pump to the beginning of the cycle or the E1 conformation

14. Diseases Associated with SERCA Brodie's disease, which is manifested as a defect in skeletal muscle relaxation, is associated with mutations from SERCA Darier's disease, an autosomal-dominant skin disorder, has been shown to result from mutations from SERCA Changes in SERCA expression have been associated with heart failure in humans and with animal models of heart disease, hypertension, diabetes, and aging

15. References Modelling sarcoplasmic reticulum calcium ATPase and its regulation in cardiac myocytes J. T. KoivumakiJ. T. Koivumaki, J. Takalo, T. Korhonen, P. Tavi, M. WeckstromJ. TakaloT. KorhonenP. TaviM. Weckstrom Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 367(1896):2181- 2202 (2009) Structure and function of the calcium pump. Stokes DLStokes DL, Green NM. Annu Rev Biophys Biomol Struct. 2003;32:445-68. Epub 2003 Feb 19.Green NM Calcium activation of the Ca-ATPase enhances conformational heterogeneity between nucleotide binding and phosphorylation domains. Chen BChen B, Squier TC, Bigelow DJ. Biochemistry. 2004 Apr 13;43(14):4366-74Squier TCBigelow DJ Transport mechanism of the sarcoplasmic reticulum Ca2+ -ATPase pump. Møller JVMøller JV, Nissen P, Sørensen TL, le Maire M. Curr Opin Struct Biol. 2005 Aug;15(4):387-93.Nissen PSørensen TLle Maire M Structural basis of ion pumping by Ca2+-ATPase of the sarcoplasmic reticulum. Toyoshima CToyoshima C, Inesi G. Annu Rev Biochem. 2004;73:269-92.Inesi G Phosphoryl transfer and calcium ion occlusion in the calcium pump. Sørensen TLSørensen TL, Møller JV, Nissen P. Science. 2004 Jun 11;304(5677):1672-5.Møller JVNissen P