title: Concerted conformational effects of Ca2+ and ATP are required for activation of sequential reactions in the Ca2+ ATPase (SERCA) catalytic cycle.
abstract: 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 adenosine 5'-(beta,gamma-methylene) triphosphate (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 gamma-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 because of electrostatic repulsion at the phosphorylation site. However, this conformation is subsequently stabilized by covalent engagement of the -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., E1-P to E2-P transition). Finally, we measured low-affinity ATP binding to stable phosphoenzyme analogues, 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.
authors.affiliation: California Pacific Medical Center Research Institute, 475 Brannan Street, San Francisco, California 94107, USA. ginesi@cpmcri.com
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Inesi
authors.forename: David
authors.initials: D
authors.lastname: Lewis
authors.forename: Hailun
authors.initials: H
authors.lastname: Ma
authors.forename: Anand
authors.initials: A
authors.lastname: Prasad
authors.forename: Chikashi
authors.initials: C
authors.lastname: Toyoshima
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D008958:Models, Molecular; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D055672:Static Electricity
chemical_list: D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium