Interactions of calcineurin A, calcineurin B, and Ca2+.
Calcineurin B (CN-B) is the Ca(2+)-binding, regulatory subunit of the phosphatase calcineurin. Point mutations to Ca(2+)-binding sites in CN-B were generated to disable individual Ca(2+)-binding sites and evaluate contributions from each site to calcineurin heterodimer formation. Ca(2+)-binding properties of four CN-B mutants and wild-type CN-B were analyzed by flow dialysis confirming that each CN-B mutant binds three Ca2+ and that wild-type CN-B binds four Ca2+. Macroscopic dissociation constants indicate that N-terminal Ca(2+)-binding sites have lower affinity for Ca2+ than the C-terminal sites. Each CN-B mutant was coexpressed with the catalytic subunit of calcineurin, CN-A, to produce heterodimers with specific disruption of one Ca(2+)-binding site. Enzymes containing CN-B with a mutation in Ca(2+)-binding sites 1 or 2 have a lower ratio of CN-B to CN-A and a lower phosphatase activity than those containing wild-type CN-B or mutants in sites 3 or 4. Effects of heterodimer formation on Ca2+ binding were assessed by monitoring (45)Ca2+ exchange by flow dialysis. Enzymes containing wild-type CN-B and mutants in sites 1 and 2 exchange (45)Ca2+ slowly from two sites whereas mutants in sites 3 and 4 exchange (45)Ca2+ slowly from a single site. These data indicate that the Ca2+ bound to sites 1 and 2 is likely to vary with Ca2+ concentration and may act in dynamic modulation of enzyme function, whereas Ca(2+)-binding sites 3 and 4 are saturated at all times and that Ca2+ bound to these sites is structural.
Feng
B
B
Department of Pediatrics, College of Medicine, University of Nebraska Medical Center, Omaha 68198-6055, USA.
Stemmer
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D019703:Calcineurin / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D019281:Dimerization; D004789:Enzyme Activation / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism
D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D018698:Glutamic Acid; D019703:Calcineurin; D008239:Lysine; D002118:Calcium