1.6.3
The Sarcoplasmic Reticulum Calcium Pump
Primary ATPase transporters, in which transport is directly coupled to the hydrolysis of ATP, are
categorized as P-, F-, and V-type ATPases and ABC transporters (Pedersen 2007). Among these,
P-type ATPases comprise a family of cation transporters where the formation of an aspartylphosphorylated intermediate drives the active transport of cations. SERCA1a, the SR Ca
2+ -ATPase, is the
prototype of P-type ATPases and the first primary transporter whose 3D structure was solved by X-ray
diffraction (Toyoshima et al. 2000; Toyoshima and Nomura 2002). Comparison of its stability and
functionality in a detergent vs. an APol environment has played a key role in understanding the
mechanisms of stabilization of MPs by APols (Chap. 5). SERCA1a is present in large amounts in
skeletal muscle, particularly in the longitudinal tubules of fast-twitch muscle SR. It is responsible for
terminating muscle contraction by pumping back Ca
2+ ions from the cytosol into the lumen of the SR,
from which they had been released when the muscle fiber was excited. Various other isoforms of
SERCA are present in other tissues and other cell compartments, including the plasma membrane
and the Golgi apparatus. Ca
2+ -ATPases are involved in maintaining the low cytosolic concentration of
Ca
2+ that is essential to many signaling processes other than muscle contraction, such as neurotransmission, neurosecretion, egg fertilization, etc. (Carafoli 2002). All P-type ATPases are structurally
alike, and their transport cycle is driven by the formation and hydrolysis of an “energy-rich”
aspartylphosphorylated intermediate formed by reaction with ATP (hence their name; for a review,
see Møller et al. 2010).
SERCA1a is a large, monomeric MP, with ten TM helices; three extramembrane (cytosolic)
domains, called A (for “actuator”), N (“nucleotide binding”), and P (“phosphorylation”); and an
overall MW of 110 kDa. The functional cycle – schematized in Fig. 1.34 – involves large relative
movements between the cytosolic domains, coupled to extensive displacements of the TM helices
relative to one another. Pumping is actuated by the phosphorylation of Asp351, in Domain P, at the
expense of ATP, followed by hydrolysis. Each molecule of ATP consumed results in the thermodynamically uphill pumping of two Ca
2+ ions from the cytosol into the SR lumen and the downhill
release of 2–3 luminal H
+ ions into the cytosol.
During its functional cycle, SERCA1a transits between State E1 (on the right in Fig. 1.34), with
two Ca
2+ ions bound in the core of its TM region, and State E2 (on the left), in which they are replaced
by protons. The following description of the main steps in the cycle is adapted from Møller et al.
(2010). At the onset (upper left cartoon in Fig. 1.34), the H n E2:ATP state carries an ATP molecule,
bound to domain N (in red in Figs. 1.34 and 1.36). In the transition to state Ca 2 E1-ATP (step ① in
Fig. 1.34), two cytosolic Ca
2+ ions become strongly coordinated to TM sites formed by side-chain and
main-chain groups carried by helices M4, M5, M6, and M8 (Fig. 1.35), releasing n H
+ ions (n ¼ 2–3)
in the cytosol. The complexation of calcium ions forces rotational and translational changes in
the position of these helices. In particular, helix M4, which carries Glu309, one of the residues forming
Ca
2+ -binding site II, is pushed by ~6 Å toward the cytosol (Toyoshima and Nomura 2002; Jensen et al.
2006; Sonntag et al. 2011). As a consequence, the cytosolic A-domain (in yellow in Figs. 1.34 and
1.36) is dislodged from its contacts with the N- and P-domains and moves to a new binding site on the
N-domain. This permits the ATP molecule bound to N to closely approach Asp351, on the P-domain
(in blue in Figs. 1.34 and 1.36), leading to the formation (step ②) of the [Ca 2 ]E1~P state, where the
bound Ca
2+ ions become occluded as a result of closure of the cytosolic entrance (Fig. 1.36). In
the following step, where the P- and N-domain interactions are loosened because of the transfer of the
γ-phosphate of ATP to Asp351, ADP is exchanged for ATP ③. Attracted by the phosphorylated
P-domain, the A-domain pulls on its links with the M1, M2, and M3 helices, which opens a luminal
channel. This leads to the release in the lumen of the two Ca
2+ ions, in exchange for n luminal protons,
44
1 Membrane Proteins and Their Natural Environment
The Sarcoplasmic Reticulum Calcium Pump
Primary ATPase transporters, in which transport is directly coupled to the hydrolysis of ATP, are
categorized as P-, F-, and V-type ATPases and ABC transporters (Pedersen 2007). Among these,
P-type ATPases comprise a family of cation transporters where the formation of an aspartylphosphorylated intermediate drives the active transport of cations. SERCA1a, the SR Ca
2+ -ATPase, is the
prototype of P-type ATPases and the first primary transporter whose 3D structure was solved by X-ray
diffraction (Toyoshima et al. 2000; Toyoshima and Nomura 2002). Comparison of its stability and
functionality in a detergent vs. an APol environment has played a key role in understanding the
mechanisms of stabilization of MPs by APols (Chap. 5). SERCA1a is present in large amounts in
skeletal muscle, particularly in the longitudinal tubules of fast-twitch muscle SR. It is responsible for
terminating muscle contraction by pumping back Ca
2+ ions from the cytosol into the lumen of the SR,
from which they had been released when the muscle fiber was excited. Various other isoforms of
SERCA are present in other tissues and other cell compartments, including the plasma membrane
and the Golgi apparatus. Ca
2+ -ATPases are involved in maintaining the low cytosolic concentration of
Ca
2+ that is essential to many signaling processes other than muscle contraction, such as neurotransmission, neurosecretion, egg fertilization, etc. (Carafoli 2002). All P-type ATPases are structurally
alike, and their transport cycle is driven by the formation and hydrolysis of an “energy-rich”
aspartylphosphorylated intermediate formed by reaction with ATP (hence their name; for a review,
see Møller et al. 2010).
SERCA1a is a large, monomeric MP, with ten TM helices; three extramembrane (cytosolic)
domains, called A (for “actuator”), N (“nucleotide binding”), and P (“phosphorylation”); and an
overall MW of 110 kDa. The functional cycle – schematized in Fig. 1.34 – involves large relative
movements between the cytosolic domains, coupled to extensive displacements of the TM helices
relative to one another. Pumping is actuated by the phosphorylation of Asp351, in Domain P, at the
expense of ATP, followed by hydrolysis. Each molecule of ATP consumed results in the thermodynamically uphill pumping of two Ca
2+ ions from the cytosol into the SR lumen and the downhill
release of 2–3 luminal H
+ ions into the cytosol.
During its functional cycle, SERCA1a transits between State E1 (on the right in Fig. 1.34), with
two Ca
2+ ions bound in the core of its TM region, and State E2 (on the left), in which they are replaced
by protons. The following description of the main steps in the cycle is adapted from Møller et al.
(2010). At the onset (upper left cartoon in Fig. 1.34), the H n E2:ATP state carries an ATP molecule,
bound to domain N (in red in Figs. 1.34 and 1.36). In the transition to state Ca 2 E1-ATP (step ① in
Fig. 1.34), two cytosolic Ca
2+ ions become strongly coordinated to TM sites formed by side-chain and
main-chain groups carried by helices M4, M5, M6, and M8 (Fig. 1.35), releasing n H
+ ions (n ¼ 2–3)
in the cytosol. The complexation of calcium ions forces rotational and translational changes in
the position of these helices. In particular, helix M4, which carries Glu309, one of the residues forming
Ca
2+ -binding site II, is pushed by ~6 Å toward the cytosol (Toyoshima and Nomura 2002; Jensen et al.
2006; Sonntag et al. 2011). As a consequence, the cytosolic A-domain (in yellow in Figs. 1.34 and
1.36) is dislodged from its contacts with the N- and P-domains and moves to a new binding site on the
N-domain. This permits the ATP molecule bound to N to closely approach Asp351, on the P-domain
(in blue in Figs. 1.34 and 1.36), leading to the formation (step ②) of the [Ca 2 ]E1~P state, where the
bound Ca
2+ ions become occluded as a result of closure of the cytosolic entrance (Fig. 1.36). In
the following step, where the P- and N-domain interactions are loosened because of the transfer of the
γ-phosphate of ATP to Asp351, ADP is exchanged for ATP ③. Attracted by the phosphorylated
P-domain, the A-domain pulls on its links with the M1, M2, and M3 helices, which opens a luminal
channel. This leads to the release in the lumen of the two Ca
2+ ions, in exchange for n luminal protons,
44
1 Membrane Proteins and Their Natural Environment