Because the calcium pump constitutes more than 80 percent of the integral membrane protein in muscle SR membranes, it is easily purified and has been studied extensively. Determination of the three-dimensional structure of this protein in several conformational states, representing different steps in the pumping process, has revealed much about its mechanism of action, which serves as a paradigm for understanding many P-class ATPases.

The current model for the mechanism of action of the Ca²⁺ ATPase in the SR membrane involves multiple conformational states. For simplicity, we group these into E1 states, in which the two binding sites for Ca²⁺, located in the center of the membrane-spanning domain, face the cytosol, and E2 states, in which these binding sites face the exoplasmic face of the membrane, pointing into the lumen of the SR. The coupling of ATP hydrolysis with ion pumping involves several conformational changes in the protein that must occur in a defined order, as shown in Figure 11-10. When the protein is in the E1 conformation, two Ca²⁺ ions bind to two high-affinity binding sites accessible from the cytosolic side; even though the cytosolic Ca²⁺ concentration is low (see Table 11-2), calcium ions still fill these sites.

Next an ATP binds to a site on the cytosolic surface (step 1). The bound ATP is hydrolyzed to ADP in a reaction that requires Mg²⁺, and the liberated phosphate is transferred to a specific aspartate residue in the protein, forming the high-energy acyl phosphate bond denoted by E1~P (step 2). The protein then undergoes a conformational change that generates E2, in which the affinity of the two Ca²⁺-binding sites is reduced (shown in detail in the next figure) and in which these sites are now accessible to the SR lumen (step 3). The free energy of hydrolysis of the aspartyl-phosphate bond in E1~P is greater than that in E2–P, and this reduction in the free energy of the aspartyl-phosphate bond can be said to power the E1 → E2 conformational change.

The Ca²⁺ ions spontaneously dissociate from the binding sites to enter the SR lumen because even though the Ca²⁺ concentration there is higher than in the cytosol, it is lower than the K_d for Ca²⁺ binding in the low-affinity state (step 4). Finally, the aspartyl-phosphate bond is hydrolyzed (step 5). This dephosphorylation, coupled with subsequent binding of cytosolic Ca²⁺ to the high-affinity E1 Ca²⁺ binding sites, stabilizes the E1 conformational state relative to E2; furthermore, it can be said to power the E2 → E1 conformational change (step 6). Now E1 is ready to transport two more Ca²⁺ ions. Thus the cycle is complete, and hydrolysis of one phosphoanhydride bond in ATP has powered the pumping of two Ca²⁺ ions against a concentration gradient into the SR lumen.

Much structural and biophysical evidence supports the model depicted in Figure 11-10. For instance, the muscle calcium pump has been isolated with phosphate linked to the key aspartate residue, and spectroscopic studies have detected slight alterations in protein conformation during the E1 → E2 conversion. The two phosphorylated states can also be distinguished biochemically; addition of ADP to phosphorylated E1 results in synthesis of ATP, the reverse of step 2 in Figure 11-10, whereas addition of ADP to phosphorylated E2 does not. Each principal conformational state of the reaction cycle can also be characterized by a different susceptibility to various proteolytic enzymes such as trypsin.

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Figure 11-11 shows the three-dimensional structure of the Ca²⁺ pump in the E1 state. As can be seen in Figure 11-11c, the 10 membrane-spanning α helices in the catalytic subunit form the passageway through which Ca²⁺ ions move. Amino acids in four of these helices form the two high-affinity E1 Ca²⁺-binding sites (Figure 11-11a, left). One site is formed from negatively charged oxygen atoms from the carboxyl groups (COO⁻) of glutamate and aspartate side chains, as well as from bound water molecules. The other site is formed from side- and main-chain oxygen atoms. Thus as Ca²⁺ ions bind to the Ca²⁺ pump, they lose the water molecules that normally surround a Ca²⁺ ion in aqueous solution (see Figure 2-7), but these waters are replaced by oxygen atoms with a similar geometry that are part of the transport protein. In contrast, in the E2 state (Figure 11-11a, right), several of these binding side chains have moved fractions of a nanometer and are unable to interact with bound Ca²⁺ ions, accounting for the low affinity of the E2 state for Ca²⁺ ions.

The binding of Ca²⁺ ions to the Ca²⁺ pump illustrates a general principle of ion binding to membrane transport proteins that we will encounter repeatedly in this chapter: as ions bind, they lose most of their waters of hydration, but interact with oxygen atoms in the transport protein that have a geometry similar to that of the water oxygens that are bound to them in aqueous solution. This reduces the thermodynamic barrier for ion binding to the protein and allows tight binding of the ion even from solutions of relatively low concentrations.

The cytosolic region of the Ca²⁺ pump consists of three domains that are well separated from one another in the E1 state (Figure 11-11b). Each of these domains is connected to the membrane-spanning helices by short segments of amino acids. Movements of these cytosolic domains during the pumping cycle cause movements of the connecting segments, which are transmitted into movements of the attached membrane-spanning α helices. For example, the phosphorylated residue, Asp 351, is located in the phosphorylation (P) domain. The adenosine moiety of ATP binds to the nucleotide-binding (N) domain, but the γ phosphate of ATP binds to specific residues on the P domain, requiring movements of both the N and P domains. Thus, following ATP and Ca²⁺ binding, the γ phosphate of the bound ATP sits adjacent to the aspartate on the P domain that is to receive the phosphate. Although the precise details of these and other protein conformational changes are not yet clear, the movements of the N and P domains are transmitted by lever-like motions of the connecting segments into rearrangements of several membrane-spanning α helices. These changes are especially apparent in the four helices that contain the two Ca²⁺-binding sites: the changes prevent the bound Ca²⁺ ions from moving back into the cytosol when released, but enable them to dissociate into the exoplasmic space (lumen).

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All P-class ATP-powered pumps, regardless of which ion they transport, are phosphorylated on a highly conserved aspartate residue during the transport process. As deduced from cDNA sequences, the catalytic α subunits of all the P-class pumps examined to date have similar amino acid sequences and thus are presumed to have similar arrangements of transmembrane α helices and cytosol-facing A (actuator), P, and N domains (see Figure 11-11). These findings strongly suggest that all such proteins evolved from a common precursor, although they now transport different ions. This suggestion is borne out by the similarities of the three-dimensional structures of the membrane-spanning segments of the Na⁺/K⁺ ATPase and the Ca²⁺ pump (Figure 11-12); the molecular structures of the three cytoplasmic domains are also very similar. Thus the operational model in Figure 11-11 is generally applicable to all of the P-class ATP-powered pumps.

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