FIGURE 12.

Schematic for E2P processing and Ca²⁺ handling. Top panel, structures of E1Ca₂·AlF₄⁻·ADP (a structural analog for the transition state in phosphorylation, E1PCa₂·ADP^‡, PDB code 1T5T (Ref. 10)), E2·BeF₃⁻ (PDB code 2ZBE (Ref. 12)), E2·BeF₃⁻(TG) (PDB code 2ZBF (Ref. 12)), and E2·AlF₄⁻(TG) ((a structural analog for the transition state in hydrolysis, E2P^‡, PDB code 1XP5 (Ref. 11)) are shown as a schematic. In these structures, the residues involved in the formation of the Tyr¹²² hydrophobic cluster (Y122-HC) are depicted with van der Waals spheres and are colored as in Fig. 10 (see the residue numbers in Fig. 10). The cytoplasmic part of M2 and the TGES¹⁸⁴ loop are colored in purple and blue, respectively, and the A, P, and N domains are colored in yellow, cyan, and pink, respectively. The open or closed state of the Ca²⁺ path (luminal gate) and the assembling state of the Tyr¹²² hydrophobic cluster are indicated above the structures. Bottom panel, schematic of the structural changes for Tyr¹²² hydrophobic cluster formation and for the property of the Ca²⁺ transport sites and release gate during E2P processing with Ca²⁺ release (E2PCa₂ to E2P). In this model, the main body of Ca²⁺-ATPase is shown in red, orange, and yellow to indicate the open high-affinity, open low-affinity, and closed states, respectively, for the property of the Ca²⁺ transport sites and release gate. The green semicircle indicates the part of Tyr¹²² hydrophobic cluster formed in E2PCa₂ upon EP isomerization (E1PCa₂ → E2PCa₂) and composed of Ile¹⁷⁹/Leu¹⁸⁰ on the A domain, Val⁷⁰⁵/Val⁷²⁶ on the P domain, and Ile²³² on the A/M3 linker. The pink structure indicates the cytoplasmic part of M2, including Leu¹¹⁹ and Tyr¹²². During E2P processing, Tyr¹²²/Leu¹¹⁹ gathers to Ile¹⁷⁹ and then to the assembled five residues, completing the Tyr¹²²-hydrophobic cluster, which is coupled with affinity reduction. Mutation of Leu¹¹⁹, Tyr¹²², or Ile¹⁷⁹ probably destabilizes the hydrophobic cluster, thereby retarding affinity reduction. Also note that our previous detailed kinetic study has revealed (18) that the E2P ground state structure has a closed luminal gate and that the Tyr¹²² hydrophobic cluster is tightly fixed (as described under “Discussion”), which is depicted here with a yellow body.