CONCERTED CONFORMATIONAL EFFECTS OF Ca 2+ AND ATP ARE REQUIRED FOR ACTIVATION OF SEQUENTIAL REACTIONS IN THE Ca2+ ATPase (SERCA) CATALYTIC CYCLE

摘要

We relate solution behavior to the crystal structure of the Ca²⁺ ATPase (SERCA). We find that nucleotide binding occurs with high affinity through interaction of the adenosine moiety with the N domain, even in the absence of Ca²⁺ and Mg²⁺, or to the closed conformation stabilized by thapsigargin (TG). Why then is Ca²⁺ crucial for ATP utilization? The influence of AMPPCP, Ca²⁺ and Mg²⁺ on proteolytic digestion patterns, interpreted in the light of known crystal structures, indicates that a Ca²⁺ dependent conformation of the ATPase headpiece is required for a further transition induced by nucleotide binding. This includes opening of the headpiece, which in turn allows inclination of the “A” domain and bending of the “P” domain. Thereby, the phosphate chain of bound ATP acquires an extended configuration allowing the γ-phosphate to reach Asp351 to form a complex including Mg²⁺. We demonstrate by Asp351 mutation that this “productive” conformation of the substrate-enzyme complex is unstable due to electrostatic repulsion at the phosphorylation site. However, this conformation is subsequently stabilized by covalent engagement of the γ-phosphate yielding the phosphoenzyme intermediate. We also demonstrate that the ADP product remains bound with high affinity to the transition state complex, but dissociates with lower affinity as the phosphoenzyme undergoes a further conformational change (i.e., E1P to E2-P transition). Finally, we measured low affinity ATP binding to stable phosphoenzyme analogs, demonstrating that the E1-P to E2-P transition and the enzyme turnover are accelerated by ATP binding to the phosphoenzyme in exchange for ADP.