Transcript Sarcoplasmic Reticulum Ca2+

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 M1M10
• 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 antiparallel 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 Ndomain
• 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 βstrands 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
References
Modelling sarcoplasmic reticulum calcium ATPase and its regulation in cardiac myocytes
J. T. Koivumaki, J. Takalo, T. Korhonen, P. Tavi, M. Weckstrom
Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 367(1896):21812202 (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.