Sarcoplasmic Reticulum Ca2+-ATPase

Josh McGee

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 Ca2+-ATPase also known as SERCA

• SERCA pump activity lowers the concentration of Ca2+ 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

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

• Transmembrane Domains = Blue

• N-Domain = Yellow• P-Domain = Green• A-Domain = Red

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 N768, E771 on M5, T799, D800 on M6, and E908 on M8

• The second calcium binding site has contributions from main chain carbonyl oxygens on M4 and side chain oxygens from E309 on M4, and N796, D800 on M6

• The M4 sequence PEGL311 lies at the heart of this second site.

N-Domain• Known as nucleotide domain• Contains the ATP binding site K492 • 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 (D351) for phosphorylation transfer

• It is believed that the N-domain is very flexible and mobile too make up for this distance

P-Domain

• Known as the phosphorylation domain• Phosphorylation occurs on Asp351

• 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 D351

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 TGES184 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

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 TGES184 which interacts with the P-domain in the absence of calcium

Pump Mechanism

• In the E1 conformation, Ca2+-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 D351 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

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 (D351) and returns the pump to the beginning of the cycle or the E1 conformation

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

ReferencesModelling sarcoplasmic reticulum calcium ATPase and its regulation in cardiac myocytesJ. T. Koivumaki, J. Takalo, T. Korhonen, P. Tavi, M. WeckstromPhilosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 367(1896):2181-

2202 (2009)

Structure and function of the calcium pump.Stokes DL, Green NM. Annu Rev Biophys Biomol Struct. 2003;32:445-68. Epub 2003 Feb 19. Calcium activation of the Ca-ATPase enhances conformational heterogeneity between nucleotide binding and

phosphorylation domains.Chen B, Squier TC, Bigelow DJ. Biochemistry. 2004 Apr 13;43(14):4366-74 Transport mechanism of the sarcoplasmic reticulum Ca2+ -ATPase pump.Møller JV, Nissen P, Sørensen TL, le Maire M. Curr Opin Struct Biol. 2005 Aug;15(4):387-93. Structural basis of ion pumping by Ca2+-ATPase of the sarcoplasmic reticulum.Toyoshima C, Inesi G. Annu Rev Biochem. 2004;73:269-92. Phosphoryl transfer and calcium ion occlusion in the calcium pump.Sørensen TL, Møller JV, Nissen P. Science. 2004 Jun 11;304(5677):1672-5.