Contractions of cardiac and skeletal muscle cells are triggered by the rapid, voltage-dependent release of Ca2+ into the cell's cytoplasm,mainly achieved by emptying the internal Ca2+ stores of the sarcoplasmic reticulum. The increase in cytoplasmic Ca2+concentrations is opposed by several outward transport systems that remove Ca2+ from the cytoplasm. In order to keep calcium-dependent systems running, muscle cells must maintain tight control of Ca2+homeostasis. The sarcoplasmic reticulum Ca2+-ATPase (SERCA) is one such controller of Ca2+ homeostasis. This ATP-driven pump returns a significant portion of the cytoplasmic Ca2+ to the sarcoplasmic reticulum. But how exactly does this enzyme fulfill this vital physiological role?

The first few glimpses of this unique ion pump structure were taken in two high-resolution atomic models published a few years ago by Chikashi Toyoshima and colleagues. In a recent Nature paper, Toyoshima and Tatsuaki Mizutani now add new color to the palette of available crystal structures,thereby providing fascinating insights into the enzyme's allosteric mode of action.

SERCA is a member of the P-type ion translocating ATPase superfamily, which form a phosphorylated intermediate during the reaction cycle. SERCA is so far the only pump of this superfamily for which high-resolution structures have been determined and hence it is a structural `blue print' for other P-type ATPases. The enzyme is an integral membrane protein of about 110 kDa consisting of 10 transmembrane helices and three cytoplasmic domains: the actuator (A), the nucleotide binding domain (N) and the phosphorylation domain(P). The classical model describing the reaction cycle of P-type ATPases is based on the protein assuming two alternate conformations, known as E1 and E2. For SERCA it is thought that Ca2+ enters the enzyme in the E1 state via two high affinity-binding sites exposed to the cytoplasm. When the pump binds magnesium and ATP, the enzyme becomes auto-phosphorylated and undergoes several conformational changes, first occluding the Ca2+ions and then releasing them from the low affinity-binding sites in the E2 state through a lumenal gate. After Ca2+ release, the enzyme dephosphorylates and is recycled to its initial state. Although the E1–E2 model does not enjoy unanimous acceptance, many basic features appear to have structural equivalents in the atomic models deduced by Toyoshima and coworkers.

To zoom in on the reaction cycle, Toyoshima and Mizutani grew crystals in the presence of a non-hydrolysable ATP analogue, Mg2+ and Ca2+, trapping the protein in a transition state between the E1 and E2 states just before the phosphoryl transfer. The comparison of this crystal structure with previous ones revealed major allosteric changes within the molecule. Binding the ATP analogue leads to the rearrangement of the cytoplasmic A, N and P domains, which are widely separated in the nucleotide-free state, but form a compact headpiece in the trapped state. The bound nucleotide also seems to bridge the N and P domains and may function as a cleavable cross linker. Moreover, the structure of the P domain itself is altered, also affecting in turn the orientation of the N domain. Collectively,these positional alterations tilt the A domain, locking the cytoplasmic gate by moving one of the transmembrane helixes to occlude the bound Ca2+ ions. At the same time, structural strain seems to be generated within the molecule, which may open the lumenal gate in a later step of the reaction cycle.

Although the pathway of Ca2+ across the membrane is still not completely elucidated, a detailed structural framework is now available for integrating other data about the pump's function. By providing a snapshot of the Ca2+ pump frozen in one of the reaction cycle's transition states, Toyoshima and his colleagues have once again applied their genius to the fascinating machinations of P-type ATPases in general, and SERCA in particular.

References

Toyoshima, C. and Mizutani, T. (
2004
). Crystal structure of the calcium pump with a bound ATP analogue.
Nature
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