title: Roles of long-range electrostatic domain interactions and K+ in phosphoenzyme transition of Ca2+-ATPase.
abstract: Sarcoplasmic reticulum Ca(2+)-ATPase couples the motions and rearrangements of three cytoplasmic domains (A, P, and N) with Ca(2+) transport. We explored the role of electrostatic force in the domain dynamics in a rate-limiting phosphoenzyme (EP) transition by a systematic approach combining electrostatic screening with salts, computer analysis of electric fields in crystal structures, and mutations. Low KCl concentration activated and increasing salt above 0.1 m inhibited the EP transition. A plot of the logarithm of the transition rate versus the square of the mean activity coefficient of the protein gave a linear relationship allowing division of the activation energy into an electrostatic component and a non-electrostatic component in which the screenable electrostatic forces are shielded by salt. Results show that the structural change in the transition is sterically restricted, but that strong electrostatic forces, when K(+) is specifically bound at the P domain, come into play to accelerate the reaction. Electric field analysis revealed long-range electrostatic interactions between the N and P domains around their hinge. Mutations of the residues directly involved and other charged residues at the hinge disrupted in parallel the electric field and the structural transition. Favorable electrostatics evidently provides a low energy path for the critical N domain motion toward the P domain, overcoming steric restriction. The systematic approach employed here is, in general, a powerful tool for understanding the structural mechanisms of enzymes.
authors.affiliation: Department of Biochemistry, Asahikawa Medical University, Midorigaoka-Higashi, Asahikawa 078-8510, Japan. kyamasak@asahikawa-med.ac.jp
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yamasaki
authors.forename: Takashi
authors.initials: T
authors.lastname: Daiho
authors.forename: Stefania
authors.initials: S
authors.lastname: Danko
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Suzuki
mesh_terms: D000465:Algorithms; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002522:Chlorocebus aethiops; D017136:Ion Transport; D007700:Kinetics; D018021:Lithium Chloride / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011189:Potassium Chloride / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D013997:Time Factors
chemical_list: D010750:Phosphoproteins; D011189:Potassium Chloride; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D018021:Lithium Chloride; D002118:Calcium
keywords: Bioenergetics; Calcium ATPase; Domain Motion; Electrostatic Interaction; Enzyme Mechanisms; Enzyme Structure; Enzyme Structure and Function; Kinetics; Molecular Modeling; Mutagenesis Site Specific