title: Effects of modifying individual amino or carboxyl groups on the affinity of calmodulin for calcineurin.
abstract: The effects of modifying individual lysyl, aspartyl, or glutamyl residues in calmodulin on its ability to bind to the neural phosphatase calcineurin have been investigated using a competitive binding method termed "label selection." Samples of calmodulin were radiochemically labeled at a low level (0.03-0.6 group/molecule) by acetylation of amino groups or coupling carboxyl groups with ethanolamine to produce preparations containing predominantly single-site modified and unmodified molecules. These preparations were incubated in a 5-10-fold molar excess with bovine calcineurin under conditions appropriate for complex formation. The bound population was isolated, and the level of modification of each reactive residue was compared with the level in the corresponding group in the intial unselected preparation to determine if molecules modified at specific sites had been selected for or against during the competition for complex formation. Significant selection was observed against molecules modified at Lys21, Asp64, Glu67, Lys75, Glu84, Glu114, Asp118, or Lys148, whereas modification of Glu83 increased binding. The modification of other groups, including components of the four Ca2+-binding sites, had no effect on the interaction. Glu67, a Ca2+-liganding residue in Ca2+-binding site II that may regulate the orientation of this site in relation to the central helix, had the strongest influence on complex formation. Most of the residues identified form a nearly linear array in the three-dimensional structure of calmodulin and indicate the location of an extended surface for interaction with calcineurin and other enzymes.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Miami, Florida 33136.
authors.forename: D
authors.initials: D
authors.lastname: Chin
authors.forename: K
authors.initials: K
authors.lastname: Brew
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D019703:Calcineurin; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D008239:Lysine; D008297:Male; D008433:Mathematics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013552:Swine; D013737:Testis / Q000378:metabolism
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D005971:Glutamates; D001224:Aspartic Acid; D018698:Glutamic Acid; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D008239:Lysine