title: Conformational fluctuations of the Ca2+-ATPase in the native membrane environment. Effects of pH, temperature, catalytic substrates, and thapsigargin.
abstract: Digestion with proteinase K or trypsin yields complementary information on conformational transitions of the Ca(2+)-ATPase (SERCA) in the native membrane environment. Distinct digestion patterns are obtained with proteinase K, revealing interconversion of E1 and E2 or E1 approximately P and E2-P states. The pH dependence of digestion patterns shows that, in the presence of Mg(2+), conversion of E2 to E1 pattern occurs (even when Ca(2+) is absent) as H(+) dissociates from acidic residues. Mutational analysis demonstrates that the Glu(309) and Glu(771) acidic residues (empty Ca(2+)-binding sites I and II) are required for stabilization of E2. Glu(309) ionization is most important to yield E1. However, a further transition produced by Ca(2+) binding to E1 (i.e. E1.2Ca(2+)) is still needed for catalytic activation. Following ATP utilization, H(+)/Ca(2+) exchange is involved in the transition from the E1 approximately P.2Ca(2+) to the E2-P pattern, whereby alkaline pH will limit this conformational transition. Complementary experiments on digestion with trypsin exhibit high temperature dependence, indicating that, in the E1 and E2 ground states, the ATPase conformation undergoes strong fluctuations related to internal protein dynamics. The fluctuations are tightly constrained by ATP binding and phosphoenzyme formation, and this constraint must be overcome by thermal activation and substrate-free energy to allow enzyme turnover. In fact, a substantial portion of ATP free energy is utilized for conformational work related to the E1 approximately P.2Ca(2+) to E2-P transition, thereby disrupting high affinity binding and allowing luminal diffusion of Ca(2+). The E2 state and luminal path closure follow removal of conformational constraint by phosphate.
authors.affiliation: California Pacific Medical Center Research Institute, 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: Chikashi
authors.initials: C
authors.lastname: Toyoshima
authors.forename: Ayami
authors.initials: A
authors.lastname: Hirata
authors.forename: Leopoldo
authors.initials: L
authors.lastname: de Meis
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D018360:Crystallography, X-Ray; D019286:Endopeptidase K / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000201:enzymology; D011487:Protein Conformation; D011817:Rabbits; D013379:Substrate Specificity; D019284:Thapsigargin / Q000494:pharmacology*; D013816:Thermodynamics; D014357:Trypsin / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D014357:Trypsin; D019286:Endopeptidase K; D000252:Calcium-Transporting ATPases